P-R-O-C-E-E-D-I-N-G-S

8:03 a.m.

SECRETARY FREAS: Dr. Priola, members of the public, invited guests and public participants, I would like to welcome all of you to this our 14^th meeting of the Transmissible Spongiform Encephalopathies Advisory Committee. I am Bill Freas. I am the executive secretary for this Committee. At this time, I would like to go around and introduce to you the members at the head table, starting on the right hand side of the room.

The first chair will soon be occupied very shortly by Dr. Pierluigi Gambetti. He is a professor and director Division of Neuropathology Case, Western Reserve University. Okay. Then the second chair will soon be occupied by Dr. Richard Johnson, professor of neurology at Johns Hopkins University. And then going around the table, the people who are here, Dr. Arthur Bracey, associate chief Department of Pathology, Saint Lukes Episcopal Hospital. Next is Dr. Lisa Ferguson, a senior staff veterinarian, U.S. Department of Agriculture.

Next is Dr. Nick Hogan, associate professor of ophthalmology, University of Texas, Southwestern Medical School. Next is Dr. Rima Khabbaz, associate director for Epidemiologic Science, National Center for Infectious Diseases, Atlanta, Georgia. Around the corner of the table is a gentleman, whom I'm going to ask to join us at lunch time, if that is okay, Dr. Nelson. Could I ask you to join us at lunch time instead of in the morning?

DR. NELSON: Certainly.

SECRETARY FREAS: This is my mistake. I apologize. Dr. Nelson will be a temporary voting member, and he will join us right at lunch time, and if you could just sit over in the FDA section up until Topic 1 is over. And when I read the Conflict of Interest statement, hopefully, that will be explained. My apologies for not checking before I started. Okay.

Next is our Chair, Dr. Suzette Priola. She is an investigator of Laboratory of Persistent and Viral Diseases of the Rocky Mountain Laboratories. Next is our consumer representative, Ms. Shirley Walker, vice president of the Health and Human Services, Urban League of Greater Dallas in north central Texas. Next is Dr. Sidney Wolfe, director of Public Citizen Health Research Group, Washington, D.C.

Next is Dr. John Bailar, professor in America's University of Chicago. Next is our non-voting industry representative, Dr. Stephen Petteway, director of Pathogen Safety and Research, Bayer Corporation. Three Committee members in addition to the two that are joining us shortly could not be with us at all for this meeting. They are Mr. Val Bias, consumer representative, Lynn Creekmore, staff veterinarian and Dr. Stephen DeArmond from the University of California.

I would now like to read into the public record the Conflict of Interest statement for this meeting. "The following announcement is made part of the public record to preclude even the appearance of a Conflict of Interest at this meeting. Pursuant to the authority granted under the Committee Charter, the Director Center for Biologics Evaluation and Research has appointed Mr. Terry Rice and Drs. Kenrad Nelson, who I just asked to leave the table, and David Stroncek as temporary voting members for Topics 2, 3 and 4 of this meeting.

In addition, the associate commissioner of External Relations of FDA has appointed Dr. Charles Edmiston as a temporary voting member for Topics 2, 3 and 4 of this meeting. Based on the agenda, it has been determined that the Committee will not be providing advice on specific firms or products at this meeting. The topics deemed discussed by the Committee in open session are considered general matters issues.

To determine if Conflicts of Interest exist, the Agency reviewed the agenda and all relevant reported financial interests from meeting participants. The Food and Drug Administration prepared general matters waivers for special Government employees, who required a waiver under 18 U.S. Code 208. Because general matters topics impact on so many entities, it is not prudent to recite all potential Conflicts of Interest as they apply to each member.

FDA acknowledges that there may be potential Conflicts of Interest, but because of the general nature of the discussion before the Committee, these potential conflicts are mitigated. We would like to note for the record that Dr. Stephen Petteway is serving as a non-voting industry representative member for this Committee. He is employed by Bayer and thus has interests in his employer and other similar firms.

Listed on the agenda are speakers making industry presentations and/or updates. These speakers have financial interests associated with their employer and with other regulated firms. These speakers were not screened for these Conflicts of Interests. With regard to FDA's invited guest speakers, that's all other speakers, except those from industry, the Agency has determined that the services of these speakers are essential.

The following interests are being made public to allow the meeting participants to objectively evaluate their presentations and comments that they may make. Dr. Robert Rohwer has disclosed he has financial interest with various firms that could be affected by the Committee discussions. Dr. William Rutala receives consultant fees from several firms that could be affected by the Committee discussions. Dr. Robert Somerville has research supported by the Gelatin Manufacturers of Europe. His expenses to this meeting were also paid by the Gelatin Manufacturers of Europe. Dr. Charles Weissmann holds patents related to Prion Disease work.

Members and consultants are aware of the need to exclude themselves from the discussions involving specific products or firms which they have not been screened for the Conflict of Interest. Their exclusion will be noted in the public record. With respect to all other meeting participants, we ask, in the interest of fairness, that they address any current or previous financial involvement with any firm whose product they may wish to comment upon. Waivers may be available by written request to the Freedom of Information Office."

That's the end of the Conflict of Interest statement. I do ask that throughout this meeting before it starts if you would check your cell phone or your pager and, please, put it in the silent mode, so it won't disrupt those people sitting next to you.

Next, the FDA is continually trying to improve its Advisory Committee Program and to reduce any perceived Conflicts of Interest. It has asked Dr. Katherine McComas from the University of Maryland to conduct a survey of this program, and I would like to give her the opportunity to tell us how we can help her with this survey and how the survey is being conducted. Dr. McComas, either place. Keep talking and they'll turn the mike volume up.

DR. MCCOMAS: Okay. Good morning and thank you. I'm Katherine McComas and I'm a faculty member at the University of Maryland, and I'm here today to conduct a study of what people know and understand about the Conflict of Interest procedures that the FDA uses to monitor real or potential Conflicts of Interest of its Advisory Committee members. This is a study that is being conducted across multiple meetings. This is the 11^th meeting I have attended across the centers at FDA, including CBER.

For those of you in the audience, I've distributed a questionnaire on your chair and I have also distributed a different questionnaire to the Advisory Committee members. If you have an opportunity today to complete this questionnaire or tomorrow, there is a box on the registration desk where you can drop it. Otherwise, there is a business reply envelope that you can just drop it in the mail as soon as you can. Your participation is voluntary, but it is important. The more responses we get, the better we are to provide feedback to the FDA about what people know and understand about the Conflict of Interest procedures, and what may be done to improve satisfaction, if necessary, with the Advisory Committee process.

Again, I appreciate your participation and if you have any questions, my contact information is included in the letter, in the questionnaire and I would be happy to provide any answers. Also, when the study is done, the responses will be available in summary form to everyone who is interested. So thank you very much for your time and have a great day.

SECRETARY FREAS: So if you got here early and did not receive a questionnaire, the questionnaires are on the table outside and, please, everybody on the FDA staff will be more than glad to help you if you have any questions with this questionnaire.

Dr. Priola, I turn the microphone over to you to start the meeting. Thank you.

DR. PRIOLA: So since we have a very full agenda today, we will just get started with the first speaker, who is Dr. Potter, who will give us background on Topic 1.

DR. POTTER: Good morning. FDA has been considering the safety of gelatin with regard to BSE for a number of years, and has come to this Committee on a number of occasions to get its recommendations on FDA's guidance to gelatin manufacturers and users. The safety of gelatin is determined as you've told us before by the safety, the source materials in the degree to which the gelatin manufacturing process destroys prions that enter the system.

Questions to the Committee have dealt with these two factors and how well knowledge about TSE's was reflected in FDA guidance for assuring the safety of gelatin for food and cosmetic use. Before 1996, FDA did not include gelatin within its recommended restrictions concerning bovine ingredients in FDA regulated products. In 1996, FDA began to review its position on gelatin in light of new information that associated BSE exposure with Variant CJD in humans and new data from a study on the effect on infectivity of gelatin processing that suggested only partial effectiveness.

In 1997, this Committee met to consider the safety of gelatin and to provide an assessment on the overall risk to humans associated with imported gelatin. This Committee made the following observations: First, that the scientific information available no longer justified excepting gelatin from restrictions recommended by FDA for other bovine derived materials from BSE countries. Second, that bovine gelatin injected or implanted forms posed a higher risk of transmitting BSE to humans than gelatin that is ingested. Third, that brains and spinal cords from cattle from BSE countries should be excluded from raw materials used to produce gelatin for human consumption. Fourth, alkaline or acid processing in gelatin manufacturer may only reduce rather than eliminate BSE infectivity, and the Committee called for better validation studies, particularly to investigate the other steps of gelatin manufacture. And finally, that porcine gelatins appear to pose no known risk of transmitting TSEs to humans.

After the 1997 TSEAC meeting, FDA issued its gelatin guidance document which remains the current FDA position in policy on the production of gelatin. In this guidance, FDA proposed the following recommendations concerning the acceptability of gelatin in FDA regulated products intended for human use. First, that importers, manufacturers and suppliers should determine the tissue, species and country source of all materials used in processing gelatin for human use.

Second, that bone and hides from cattle from any source country that show signs of neurologic disease should not be used as raw materials. Third, gelatin production from bones and hides obtained from cattle that reside in BSE countries or countries that do not meet the latest BSE related OIE standards should not be used in injectable, ophthalmic or implanted FDA regulated products or in their manufacture, but may be used in FDA regulated products for oral consumption and cosmetic use by humans if the cattle come from BSE-free herds and if the slaughter house removes heads, spines and spinal cords directly after slaughter.

Fourth, gelatin produced from bovine hides from any source country may be used in FDA regulated products for oral consumption and cosmetic use by humans if processors insure that the hides have not been contaminated with brain, spinal cord or ocular tissues of cattle residing in or originating from BSE countries. Fifth, gelatin produced from bovine hides and bones may be used in FDA regulated products for human use if the gelatin is produced from raw materials from countries like the United States that observe OIE standards and have not diagnosed BSE in their national cattle herd, that is RBSE-free. And finally, gelatin produced from porcine skins from any source country may be used in FDA regulated products for human use.

In 1998, this Committee met again to discuss gelatin among other issues. FDA's guidance, based on the 1997 TSEAC recommendations, was presented to the Committee to consider several new pieces of relevant information. For example, the infectivity of dorsal root ganglia and low level infectivity in bone marrow and the growing number of BSE cases being discovered in Europe. The Committee considered this new information and decided gelatin could be safely sourced from bones and hides of cattle in BSE countries as long as the recommendations in the guidance were met. That is that the cattle came from BSE-free herds and the high-risk materials were removed after slaughter.

And this is at present the status of the safe source factor for gelatin. Continuing on with the other key factor, that of validated effectiveness in the manufacturing process, in June 2001, the Committee was given an update from the Gelatin Manufacturers of Europe on the interim validation study results on the inactivation of BSE through the gelatin manufacturing process. This was an information sharing meeting only and no questions were posed to the Committee.

The Committee reviewed the study design and the preliminary data and requested a presentation of the final results as soon as they were available. The Committee is now about to get its wish as GME will present their completed studies, and we will hear other marketing and manufacturing information on gelatin in North America and Europe. After you have heard this new information, we would like you to comment on the studies and to consider the current gelatin guidance in light of these completed studies and other relevant information.

And I think, according to my schedule, Yuan-Yuan will now charge up the Committee. Thank you.

CHAIR PRIOLA: Thank you, Dr. Potter. Dr. Chiu will now present the questions for Committee.

DR. CHIU: Good morning. First, I would like to thank Dr. Priola and the Committee members to take the time to come out and also we have sent you a huge package, gelatin studies protocols and procedures and the results. We appreciate how much time you need to really review those studies. In the early days, in the 1998 year, when the Agency and the Committee together made a decision for the Agency's recommendation on gelatin was based on previous study which the Committee thought was somewhat flawed.

So generally, this reason follows the advice of the Committee to then redesign the studies and then today, you know, we have the new study results. You did not review the interim results, but today we have the final results off of five studies. We're hoping, you know, with the presentation today and the background information you have you will be able to help the Agency to answer two questions.

Next slide, the first question is "Do those results of these new studies demonstrate a reduction in infectivity that is sufficient to protect human health?" And we are only limited to hear the question to bovine bone gelatin is consumed by humans through oral or topical administration. The question is not for gelatin of other administrations, such as the injection, you know, implantable. We would like the Committee to focus on oral and topical administration.

Next slide, now, the first question, you know, the answer could be yes or no or in between regardless, you know, the answer we also would like you to answer the second question. There are two parts. The first part is "Do the scientific data and the information available support the current FDA recommendations on bovine bone gelatin for oral and topical administration?"

The current recommendations, next slide, is on this slide. The general policy of FDA is for FDA regulated products, the bovine derived material should come from cattle not bone residing as slaughtered in BSE countries, but the Agency also provides some exemptions. The exemption could be a total exemption unconditional, such as milk, dairy products and the milk derived product. But some of the substances, you know, the Agency provide conditional exemptions, and the gelatin for oral and topical use are giving conditional exemptions.

So if the cattle actually is coming from BSE countries, then that condition is the cattle must be from a BSE-free herd and also at the slaughter house the head, the spine and spinal cord should be removed. And this is from BSE countries. Now, some countries may not have BSE cases, but there is consider of high-risk of BSE. Then the recommendation is the heads, the spine and the spinal cord should be removed as the first step in the slaughter house. So the first question is whether this current recommendation still is valid, based on the scientific information we have today.

Next slide, if the answer is yes, then that's the end of it. If the answer is no, then we would like to know what changes the Committee would like to recommend to our current policy. The changes can be in all different directions. You may consider we can actually grant a total exemption to the gelatin for oral and topical use or you may consider to modify the current recommendation the FDA has, either by strengthening or by relaxing the conditions. So we are anxious and grateful you will give Agency your deliberation. Thank you.

CHAIR PRIOLA: Okay. Thank you, Dr. Chiu. Our next speaker is Mr. Masson, who will discuss market trends in the U.S.

DR. MASSON: Yes, good morning everybody. Madam Chairman, I would just like to thank the Committee and the FDA, in particular, about the opportunity to address the Committee. As we have heard from Drs. Potter and Chiu, it has been a long and winding road, the saga of gelatin, and we hope today that we can reach a satisfactory conclusion and see gelatin taken off the file, so to speak, having reassured you of its safety.

My first slide, please. Can I have the first slide, please? Okay. Thank you. Well, just an introduction of I'm currently the president of our industry association, the GMIA, and also president and CEO of one of its members at Russelot. The next slide? A bit of history as to GMIA and credentials, so to speak. Our association was formed in 1956. We have six members, all NAFTA based, four from the U.S., one in Mexico and one in Canada. And we've listed here the typical working committees by which we run the institute. There is no particular order of precedence, but the technical and regulation committees as you can imagine, indeed, are the primary focus of most of our work, I guess.

Next, in terms of what we represent, we, as you see, represent roughly 22 percent of the global gelatin production, and almost 100 percent of all the gelatin made in North America. And, indeed, three of our members are also affiliates of the Gelatin Manufacturers of Europe. And I should have added actually that one of the other members is an affiliate of the Japanese Gelatin Manufacturing Group.

Next, please. This lists our objectives. As you see, we try to monitor and inform our members of any and all regulations which can impact gelatin. We are the liaison with FDA, USDA and other regulatory authorities, and we gather and distribute technical information to our members, endeavor to promote a broader knowledge of gelatin and encourage its wider consumption. And we provided the forum as you've seen from our committee information on all of the major aspects concerning technical, environmental and safety issues.

And as time has gone by and as other industry associations have been formed around the world in Japan, South America and so on, a major function which has emerged has been to liaise with them to ensure that technical information and regulation information, etcetera, is shared with the other associations around the world. And I just participated, for instance, in the Japanese meeting or the Asia Pacific meeting, which was held in Japan, just last month, as an example of the increasing international corporation among the industry associations.

Next, please. This slide lists the primary uses of the concentrated, obviously for today's purposes, bovine bone gelatin and, as you see here, this is a list of the major uses for bovine bone gelatin in the United States. It is listed in the standing order of use with photographic still being the largest consumer going on down to food. There's less and less bovine bone gelatin used in food products, by food we mean confectionery and marshmallows or whatever else.

It is being, I guess, more replaced there by pigskin porcine gelatins. But anyway, those are the primary uses and, as you see, no matter what the end use, all the gelatins are produced through the same manufacturing processes and my colleagues will be describing those in some detail in a few minutes.

Next, please. To give you an idea of the scale of the gelatin business globally, and in particular the bovine part of that production, we have listed here the various theaters, so to speak. Europe is still the biggest gelatin producer. Significantly so with 117,000 tons out of a total of some 270,000 tons around the world. Above that, I would have to say, over 25 percent or so is actually bone gelatin. The U.S. in total we make something like 60,000 tons. And again, these are all gelatins, whether bovine or porcine or bovine hide or bovine bone, porcine skin.

And as you see, of the 60,000, about 17,000 tons is actually bone gelatin. Other covers is Asia Pacific, the Asia Pacific regions and South America, and you see about a third of their gelatin is of bovine bone origin. So totally, bovine bone represents almost 80,000 out of the total of 270,000 tons. And to give you an idea again of this international value industry total globally is not that big. It's 1.5 billion dollars equivalent.

Next, please. What we tried to do here is to put to ourselves a few questions, the elimination of which, I think, will be helpful to the committee in looking at bovine bone gelatin, in particular, in the U.S. The first question, as you can see is, "Can the U.S. gelatin industry supply total U.S. capsule industry's needs?" The answer is no. And this illustrates how that is the case.

As you saw earlier, the U.S. bovine bone gelatin production totals some 17,000 tons, but of that 11,500 are needed for photographic and other non-capsule uses. So you see that that remains, there remains only about 5,500 tons which can be used by the capsule industry, but the total needs, in fact, are 10,000 tons, and this means that the shortfall roughly 4,500 tons of bovine bone gelatin has to be imported and they come primarily from Europe, also from Japan and India, and immediately derived nevertheless from U.S. bones or from bones from other countries.

Next, just continuing that theme, of those 4,500 tons of which we need to import, "Can they be derived solely from U.S. bones, even if it's not actually manufactured in the U.S.?" Again, the answer is no. As you see here, the total amount of U.S. bones which are made available to the gelatin business is roughly 130,000 tons and because of its use in photographic production, whether in Europe or in the U.S., and also for manufacturing bovine bone gelatin by other companies outside of the U.S., the amount remaining available for pharmaceutical gelatin production here is only 28,000 tons.

The next line which is in bold print illustrates that we need roughly 6 tons of bones to make a ton of gelatin. So that the 10,000 tons of gelatin, which the capsule industry needs, is actually equivalent to 60,000 tons of bovine bones and, consequently, you see the shortfall here is roughly 32,000 tons, so to speak, to be able to make all of the capsule industry requirements strictly from U.S. bones. So in other words, the deficit has to be sourced from bone suppliers outside of the United States.

Next, so then I apologize, this is a little bit of a busy slide, but the bottom line is that there are, indeed, other sources outside of the United States, but even though the quantity is maybe available for various reasons in terms of surveillance, the inspection procedures and so on, it's not so obvious that the quantities, the tonnages, which are listed in the second line or the second section are, indeed, available and because of the various restrictions and so on, you see, in fact, that the bone and that those numbers diminished to rather smaller numbers.

And this really drives to the heart of the matter. This is the crux really of what we want to get at today and my colleagues will be addressing this individually and then in the public comment session later, the question of how we can determine the BSE status adherents and also the question as Dr. Chiu referred to of just when they have to be removed in the gelatin bone process. Again, we'll be traveling to that in some much more detail in later presentations.

Next, please. I guess that concludes my presentation, unless there are any questions.

CHAIR PRIOLA: Yes, are there any questions for Mr. Masson?

DR. MASSON: Thank you.

CHAIR PRIOLA: Oh, Dr. Hogan?

BOARD MEMBER HOGAN: I have one question.

CHAIR PRIOLA: Just a second, Mr. Masson, there's a question.

BOARD MEMBER HOGAN: Sorry. I had one question, perhaps it is contained in this information you provided us, which is quite huge. In terms of the amount of gelatin that is derived from Europe, could you tell us something about the country breakdown, that is it's most from the UK, France, Switzerland, etcetera?

DR. MASSON: Yes, I think you'll find in the information packet there is a detailed breakdown of the various imports. The consumption in the U.S., actually the total marketing, is closer to 80,000 tons. And as you saw, we make 60,000. There is a net import/export situation. The U.S. does export gelatin, but basically to get to the 80,000 that we need, we need effectively a net import of 20,000 tons.

Those 20,000 tons come from quite a variety of countries and, indeed, they are listed in the information packet. We didn't go into the detail of it here, because it's somewhat difficult to differentiate, certainly differentiate country by country. It's a little bit more difficult to differentiate within certain countries whether it is bovine gelatin or porcine gelatin, which is actually being imported. But basically the primary countries who do export into the States would be France, Germany, not so much any more from UK, for obvious reasons, Brazil, Argentina, Japan, India. Those would represent the large majority of the total import picture.

And again, the variety of gelatins some of that is bovine bone, for sure, but also a lot of bovine hide gelatin comes, for instance, from South America, and bovine bone also from India. It's quite a variety of types from those principle countries.

CHAIR PRIOLA: Dr. Khabbaz?

BOARD MEMBER KHABBAZ: Yes, I didn't hear you well and I apologize. When you said in foods increasingly, there's less bovine gelatin and an increased used of porcine gelatin. Was that porcine skin?

DR. MASSON: Yes, one can make and, indeed, one does use porcine bones, but the large majority of porcine gelatin made around the world is from porcine skins. And again just to elaborate on that point, the food industry, the present manufacturing process of bone gelatin, which we'll hear much more about in a few minutes, is a very long process. It's a very costly process. Whereas porcine gelatin and hide gelatin, certain portions, is a much sorter process. And economically, therefore, it's much more viable to utilize porcine gelatin, in particular, in the food industry compared to bone, you know.

CHAIR PRIOLA: Dr. Bailar?

BOARD MEMBER BAILAR: The numbers went by pretty rapidly, but it looked to me like the proportionate shortfall from U.S. production is about the same as the proportionate shortfall when you add production from U.S. bones processed elsewhere. Is that correct? I'm looking at the second and third from the last slides.

DR. MASSON: And again, could you just repeat that?

BOARD MEMBER BAILAR: Well, in the answer here to question 1, the third from last slide.

DR. MASSON: Yes.

BOARD MEMBER BAILAR: There was a shortfall of 4,500 tons and a need of 10,000. And in the next one, it was a shortfall of 32,000 tons and a total need of, was it, 60,000, maybe I have misread this. Yes, 60,000. It's about the same proportions, but I understand why these includes other production and the other does not.

DR. MASSON: The shortfall with the U.S., as you see, makes 17,000 tons, that is equivalent to over 100,000 tons of bones, and as we said, basically the cattle industry needs 10,000 tons of gelatin and only half of that effectively is made here in the States. The other half, because of lack of availability of bones and lack of capacity in the States for bovine bone production, has to come from outside of the States, and that's, as you mention, roughly the same proportion. It's almost 50/50. Does that help?

BOARD MEMBER BAILAR: If I understand correctly then, adding the U.S. bone processed elsewhere doesn't help much at present?

DR. MASSON: Excuse me, adding?

BOARD MEMBER BAILAR: Adding gelatin from U.S. bones processed elsewhere does not, at present, help very much.

DR. MASSON: No, because again the total demand for U.S. bones, because of the other applications, particularly for photographic and other European and other countries utilization of U.S. bones, they don't always end up as pharmaceutical gelatin. The end up more often as photographic gelatin, so there's just not the amount of U.S. bones going overseas which can come back to the U.S. as pharmaceutical gelatin for capsule production.

BOARD MEMBER BAILAR: At what point is the distinction made regarding the ultimate use of the gelatin?

DR. MASSON: Regarding what, sir?

BOARD MEMBER BAILAR: Regarding the ultimate use of the gelatin. Is it all processed? I thought it was all processed in the same way.

DR. MASSON: Well, my colleagues will describe that in a great deal of detail, and it is more or less, yes.

CHAIR PRIOLA: And Dr. Wolfe?

BOARD MEMBER WOLFE: This is sort of a follow-up on John's question. You mentioned two factors that are rate limited, so to speak, in terms of the use of U.S. bones. One was the capacity, presumably, to convert U.S. bones into gelatin, and secondly, was the unavailability or the shortage of U.S. bones. I can't believe that the second one is really a problem. It is likely that only a small fraction of U.S. bones are currently being exported to other countries for reprocessing. I mean, is that correct or not? I mean, it must be a limitation on production, not a limitation on U.S. bones, and that gets to the issue of why there couldn't be an increase. If the capacity is the problem, why there couldn't be an increased export of U.S. bones to European countries to use them, preferentially, in favor of bones from BSE countries.

DR. MASSON: Yeah, your point is well- taken. The problem, however, is that the largest consumer of bovine bone, as you see, is the photographic industry. Out of the 130,000 tons, which is produced in the states, over 100,000 or approximately 100,000 goes to the photographic industry. And by definition, therefore, the remainder simply isn't satisfactory, and we can't drive -- the gelatin industry is at the bottom of the totem pole, so to speak, in terms of creating greater availability of bones. The different industries sell so much bone that's made available to us basically, and there is only so much.

BOARD MEMBER WOLFE: Are you saying that the bone either goes to photographic industry or elsewhere and that there isn't, at the present time, bone from U.S. beef that is not being converted into gelatin? I mean, what percentage of the, theoretically, available bone from U.S. beef is, in fact, being converted to some kind of gelatin? Because my question is sort of getting to the issue of whether or not it is possible to divert or not to divert, but just to increase the use of bone from U.S. beef, even though you want to -- you said there's a tug between photographic gelatin and other gelatins if the total amount of bone was available, you could satisfy both of them. So just, specifically, how much of U.S. bone is, in fact, getting converted into some kind of gelatin? Half of it, two thirds of it, all of it? What?

DR. MASSON: Well, the bone that is available is being converted. Again, there are only a few bone producers of the major beef players, but only a few of them actually make gelatin bone at some of their facilities. And again, it's supply and demand. We can't. Basically, there's not enough demand from our side that would force them or encourage them, let's say, to produce still more bone. It's that simple.

BOARD MEMBER WOLFE: Okay.

CHAIR PRIOLA: Dr. Johnson?

BOARD MEMBER JOHNSON: Yeah, I may understand the way this is processed. But it seems to me that the ready solution would be that U.S. bone would be used for all consumables, whether they be dietary supplements and then you could use the foreign bone for photographic materials. It's about even.

DR. MASSON: On paper that's true, but that's --

BOARD MEMBER JOHNSON: That's what I'm looking at.

DR. MASSON: Yes. But it's rather simplistic, because again we can't make that determination. It's those industries who make that determination. The photographic industry has determined that they will use bovine bone, and that's their prerogative that we can't influence it.

BOARD MEMBER JOHNSON: So a solution would be if we deregulated photographic bovine bone, and that would be a possibility. Does FDA regulate photographic gelatin?

DR. MASSON: No.

BOARD MEMBER BAILAR: No, they can use whatever they want.

DR. MASSON: Yes.

BOARD MEMBER JOHNSON: So you could split it up.

DR. MASSON: I'm sorry?

BOARD MEMBER JOHNSON: If there's no regulation on photographic gelatin, you're subtracting it out to produce all this shortfall, why not make the photographic gelatin from British bones?

DR. MASSON: I'm sure some is, but again, we as an industry can't make that determination. It's the photographic people who make that determination.

CHAIR PRIOLA: Dr. Bracey?

BOARD MEMBER BRACEY: Yes, in the information that you present, the majority of the gelatin is used for photographic purposes. It seems to me that there has been a major move away from film based photography towards digital. Have you seen a reduction in the demand and, in essence, your picture is a static picture, but what does it look like really as far as the demand for photographic gelatin in the future?

DR. MASSON: That's a very good question. As you rightly observed, digital photography is here in a big way and will continue to grow. But there is some complimentality between silver halide, the traditional silver halide process, which does utilize photographic gelatin and the digital business. So that the two things, digital is growing certainly at a much more rapid rate, but photographic traditional silver halide photography is still very much en vogue and, indeed, you know, the last photographic companies, Kodak, Fuji and so on still continue to invest quite significantly in the traditional side of the business as well. So the two things, I'm not sure of --

BOARD MEMBER BRACEY: Well, I guess, what I'm wondering is over the years the data in terms of total demand has been static or has it been actually declining?

DR. MASSON: I would say it is fairly static. There has been a diminution for sure in some sectors of the traditional silver halide, photographic side of the graphic arts, for instance, probably uses any photographic gels any more. That has gone totally, more or less totally, to the digital side. But the traditional film that you or I shoot, the amateur film, medical x-ray and other types of cinema, film photography for movies, those are still the traditional situation, and that demand is still very much there.

CHAIR PRIOLA: Yes, is there a question from this side or answer?

MR. SCHRIEBER: Thank you, Madam Chairman. I would like to make -- Reinhard Schrieber.

CHAIR PRIOLA: Could you identify yourself?

MR. SCHRIEBER: From GME, and I would like to make a remark about potential replacement for the photographic industry of domestic bones and imported bones. The following situation is the biggest manufacturer of photographic gelatin is Eastman-Kodak sitting here in the United States. They are forced to use domestic bones, because as a ban on import of bones from out of the U.S. into U.S., because the risk of bringing in bones from maybe BSE risk countries is tremendously high to bring in just in case by the bones BSE into the United States.

So gelatin is safe to be imported, but importing bones from other countries, I think, is of high-risk for this community here, so therefore it would really replace and most probably negligible risk with gelatin by a big risk by importing bones, degreased bones from other countries, who therefore is a replacement in this way, I think, is not a good idea for the U.S. On the other hand, I think it is really impossible to force Eastman-Kodak just out of using bones from the U.S. I don't know how their reply would be in this case.

And maybe one more question, answer to your question about from which European countries is sourced in Europe has no bone at all coming for the last 20 years from UK, so the European gelatin industry did not source bone from UK. We do not source bone from Ireland. We do not source bone from Switzerland, Portugal, the so-called higher risk countries in Europe. All the bones, proven bones used by the European industry are coming from either Germany, France, Belgium, Netherlands or Austria. These are the source countries. Thank you.

SECRETARY FREAS: Because our meetings are being transcribed, we're asking everybody who uses a microphone other than at the table to identify themselves. That was Mr. Schrieber, the chief manufacturing officer of the Gelatin Group.

CHAIR PRIOLA: Okay. I think we'll move on to our next speaker. Thank you, Mr. Masson.

DR. MASSON: Thank you.

CHAIR PRIOLA: Our next speaker is Dr. Dunn, who is going to explain some of the manufacturing processes for gelatin in the U.S. and that might address some of the questions that have arisen.

DR. DUNN: I also would like to thank the FDA and the Committee for the opportunity to come in and speak with you today about the practices of the U.S. gelatin manufacturers. My name is again Michael Dunn. I'm currently vice president of Gelita North America, and I also serve as the chairman of the Regulatory Committee for GMIA. As you can tell on this slide, there are two current manufacturers of bone gelatin here in the United States, Eastman Gelatin, who provides to Kodak, they are primarily producing photographic gelatin, and GELITA USA, who is primarily a pharmaceutical producer.

When we put those together, though, the majority of this gelatin goes to the photographic applications, although there is a substantial quantity that does go to the pharmaceutical sector as well. The limed share of the gelatin that we produce is limed bone gelatin. We do, however, produce a small amount of what we call Type A or acid bone gelatin, but this is a very small quantity.

I also wanted to note that the practices that I'm going to be talking about today, as well as the processes, apply to both GELITA USA as well as Eastman Gelatin. Could I have the next slide? So just to set the overall objectives, they basically are two-fold today. I want to adequately describe for you today what our current sourcing practices are, as well as the processing conditions that we use to manufacture bone gelatin in the United States.

I also want to clearly confirm that the bone gelatin processing conditions that we employ here are virtually the same that are currently used in Europe. And more importantly, they meet or exceed the minimum processing requirements that were spelled out in the GME TSE Inactivation Study Protocol. This I want to make clear, because we want to make sure that any of the results, we want to make sure that they are applicable to what we are producing here in the United States, as well as what is being produced in Europe.

Could I have the next slide? So when we get to sourcing, in the U.S. degreased gelatin bone is sourced exclusively from USDA inspected beef processing facilities in the United States, and this raw material is derived solely from healthy cattle that have been deemed fit for human consumption based upon both anti and postmortem inspections.

Could I have the next slide, please? When it comes to SRMs, the U.S. gelatin bone suppliers have been removing SRMs with the exception of vertebrae since as early as 1998. And right now, limited quantities of vertebrae-free gelatin bone have been available from as early as fall of 2002. Currently, there are no FDA or USDA requirements for the removal of SRMs in the United States. We primarily do the two above bullet points primarily because of EU regulations and we supply a large number of customers that have business in Europe that must comply with those kinds of regulations.

Could I have the next slide, please? So let's go on to the process. What I have outlined here is an overview of what happens in a daily gelatin production. The major input, of course, to this is the degreased gel bone. We're on the order of about 100,000 pounds of gel bone per a production day. And we have an equivalent amount of hydrochloric acid, so another 100,000 pounds of hydrochloric acid would go into this next. We use at least a half a million gallons of water in the production and, of course, there is a lot of labor and energy that goes into this as well.

What I'll be talking about primarily today is what goes on in this blue box here, in terms of the DTL processing conditions. The output we're looking for, of course, is gelatin. On a base of 100,000, you get out about 25,000 pounds of gelatin, and then about 50,000 pounds of dicalcium phosphate, which is the primary byproduct of this process.

Could I have the next slide? So overall, what we're trying to achieve here, we're starting with the protein we call collagen, which is an extremely fibrous insoluble protein and we're going to transform that into a protein that is fragmented and soluble, but has a variety of very interesting functionalities, which makes gelatin such an interesting business. So there are three major things we are trying to achieve here.

Initially, we need to hydrolyze the collagen. We do this by breaking, there is intra and inter molecular cross links between the adjacent chains. We start to break up peptide bonds, so that we're able to water extract this material from the ossein that we're producing. Subsequent to that, we spend a lot of time purifying and concentrating the gelatin. When we do that initial extraction, it's a very dilute solution about 5 percent, so we have to take a lot of water back out of that and then we purify the material from both a chemical, physical and micrological point of view.

If I could take the next slide, please? So the incoming gel bone comes to us. It's delivered by a truck or rail car and these are just simply typical characteristics of that material, and we would use the same material to make either the Type B or the Type A gelatin. So the fat content ranges from 1 to 2.5 percent. The size of these chips is an 1/8^th inch to 5/8^th inch. The mineral protein ratio is about 2 to 1. And the moisture content is about 6 to 9 percent. And in contrast, it's worth mentioning in the EU all of the producers there have their own degreasing facilities, which is different than the way things are done here in the United States. The big meat producers have their own gel bone processing facilities, and they supply us with this finished bone chip.

Next slide, please. The first pretreatment step is what we call acidulation. But what is happening here is the demineralization of the bone. This is where all that hydrochloric acid comes into place. What we're trying to achieve here is the production of what we call ossein, which is this demineralized bone material. There's a number of washings, hydrochloric acid washings during this process. We also remove a lot of non-collagen impurities that come in with the raw bone.

The concentrations that we're looking at here, maximum, 4 to 6 percent. The way this works is it's a counter-current distribution process. We start out with a dilute hydrochloric acid concentration, that's what the initial bone is exposed to, and it's gradually raised up over this 4 to 5 days. It's a very exothermic reaction, and this is why it takes to long to carry this out to dissolve out all of this material. The typical ambient range as far as temperature after this process is done, the residual acid, is washed out for about a 24 hour period before we go on to the next step, which is on the alkaloid side of things.

Could I have the next slide? So if we choose to lime, at this point, this is the breaking point we choose to make either lime bone or acid bone, at this point. In the case of liming, this is a lime pit that you're seeing up there in the picture. Again, we being, this is where we continue to hydrolyze the collagen molecules and there's a lot of washing that goes on here with the refreshing of the lime solution, so we're moving impurities.

There is also something important that happens here chemically that is different than porcine gelatin. You hydrolyze away the asparagine and glutamine. You deanimate those and form their respective acids which drops the iso-electric point of that molecule from about 9 down to about 5. So electrically, the porcine and the bovine gelatins are quite different. We use a saturated lime slurry to do this. The pH is approximately 12.5. The liming time is 25 to 70 days that we're tying up this material in production for a long period of time before we can make gelatin out of it. Again, and the temperatures, these lime pits are agitated on a daily basis. We're there to make sure we're getting proper exposure to the alkaline material to the bone chips that are in the pit. And these lime slurries are completely refreshed on a weekly basis.

Next slide, please. After that, there is a washing and acidification step. We want to neutralize the excess lime, again remove, wash out additional non-collagen impurities, and we want to adjust the pH of the ossein slurry, so we can prepare it for extraction. So this wash out period under alkaline conditions is 24 to 48 hours under vigorous agitation, temperatures from 45 to 70 degrees. The neutralizing or souring of acids in this case are either hydrochloric or sulfuric acid, and our target pH for this part of the process range between 5 to 7.

Could I have the next slide? In lieu of liming or alkaline which is what we do most of the time, we're only talking a few percent of the time we do this process. We can do an acid treatment and produce Type A or acid bone gelatin. So the purpose of this process here is to condition and ready the ossein material for an extraction at a very low pH. In the traditional process, we use a sulfuric acid and we expose the ossein to a pH of about in the range of 1 to 2 for about 6 hours, and then we rinse that back to a pH ranging from 2.8 to 3.2. And this is where we will extract the gelatin. This is, I mean, usually pH to extract gelatin. Most gelatin is extracted at much higher, more neutral pH.

We also have an alkaline pretreatment option that we're looking at, that some of our customers are looking at, because of all the discussion around sodium hydroxide pretreatment. In this situation, you would do this alkaline pretreatment prior to the ossein treatment. And in this case, you are able to maintain the pH at 13 or greater with sodium hydroxide for a period of three hours.

Next slide, please. Okay. Now, we've finished with the pretreatment, whether it be for acid bone gelatin or lime bone gelatin and the rest of this will be common to both of these types of gelatins. Now, we extract the gelatin. This is where we've wetted the gelatin, we've hydrolyzed it, now we're going to actually pull this, extract this out of that ossein particle to produce the gelatin.

We use demineralized water. What you're seeing up there is a typical gelatin extractor. We do a series of extractions. I said 4 to 6 depending on the plant and the company, the way they do that. But the initial extracts are done at a lower temperature, and what you will get out is a material that typically has a higher molecular weight, a higher viscosity, a higher bloom strength.

As you go to subsequent extracts, that material will become more degraded. It will have a longer profile of treatment with time and temperature. And those ending extracts will conversely have higher collagens, lower molecular rates, lower viscosities and so on and so forth. So the temperature range is from about 120 to 200 as you go through that series of separate gelatin extracts that you are pulling out. The conditioning time for each extraction ranges from 1 to 6 hours and it's 4 to 6 extracts.

Next slide, please. When that extract comes off, it's a typical, very dilute solution somewhere in the range of about 4 to 6 percent. So you're saying to get to a dry product, we got to pull a lot of water out of here as well. So we have initial filtration, this is a U.S. type filter, vertical leaf type filter. It's precoated with diatomaceous earth and cellulose. And that basically is to give us initial and improvement in the clarity. The solution will also go on to ion exchange. We want to protect these ion exchange collagens.

Could I have the next slide, please? So you're looking here at an ion exchange battery. You see three columns in the forefront and three in the background. Those are batteries of cation in that exchange columns. Of course, the objective here is to deionize this material, depending on whether it is pharmaceutical or photographic. It gets more exposure to those columns depending on what is needed.

Primarily, the cations we're removing are calcium magnesium and iron. On the anion side, it would depend on the acid that we were souring the material with before we extracted it. And sometimes we use hydrochloric and sometimes sulfuric. So those would be the primary anions that would be removed under those conditions. And the finished product from an ash standpoint would be somewhere between .1 and 1 percent, depending on the product that we're making.

Could I have the next slide, please? Now, we begin to remove water, and we do this by using evaporative means initially. So we have this 5 percent solution that we're going to drive up to a 15 to 25 percent concentration. The evaporator you see there in the picture is a triple effect plate and frame type evaporator. The output temperature is not too high. It usually runs about 125 to 130 degrees on the average. Basically, a temperature that will just make sure the gelatin doesn't gel up in the production plant.

Could I have the next slide? Then we have another filtration. We heat it again. We've concentrated that material, so there is more particulate becoming apparent, in certain cases, and then there is a chance that you may get some coagulated protein, so we have another clarification step here. The medium we use are exactly the same in the prior filtration cellulose and diatomaceous earth, but we use a plate and frame pressure filter. The viscosity of this solution is increasing now as we move along in the process, and this is what requires a completely different configuration for filtration.

Could I have the next slide, please? Then we take the opportunity to adjust the pH, at this point. The final pH targets of the finished product are usually in the range of 5 to 7. At this point, it's usually just a fine adjustment and most typically it's done with sodium hydroxide.

Could I have the next slide? Then we do our final concentration with evaporative means. Again, this solution is becoming quite viscus, so we're concentrating our thick, what we call at this point, our thick gelatin liquor. This is an example of a double effect plate and frame type evaporator as well. And the concentration here will be a fairly broad range here from 25 to 50 percent, and this is because, I talked earlier about your initial extracts are much higher viscosity, so you only will be able to drive those up to about a 25 percent. However, the latter extracts, which have a much lower viscosity, you're able to drive those up to a much higher concentration level, and that's what is done.

Next slide, please. Then we go through a sterilization step at the end of the liquid phase. After this, we're going to be going into a more solid mode with the gelatin production, so this is our last opportunity to do something with the liquid phase. So we use direct steam injection. We use a temperature that ranges anywhere from 138 to 149 C for 8 to 16 seconds, and this is primarily to ensure the product, hygiene of the product.

Next slide, please. Then we're taking another tack here in terms of drying the gelatin. We're beyond evaporative means, so what we do is to increase the surface area, then able to dry this material, we cool it down from about 120 down to about 70 degrees where the gelatin actually sets, starts to set, and this is down with a glycol cooled heat exchanger. Then it is extruded out through these perforated heads to form these noodles, which will range in size from under 2 feet long and about an 1/8^th inch thick, and they are deposited on the front end of a dryer, which is in the next slide.

These dryers are typically about 12 feet wide and about 150 feet long. The air quality we use is heated, dehumidified and filtered air. The object is to produce a stable product. It has very low water activity. Typically, it has 10 to 12 discrete zones with different temperatures. There's a gradient that ranges from about 80 to 160 degrees fahrenheit that goes across that entire dryer. It takes about like 2 to 3 hours to get through this system, and the final moisture content of the gelatin product is about 10 to 12 percent.

It's a very touchy process. It's very easy to melt the gelatin. If you try to dry it too fast, you know, with too much water, the melting point is lower and it is going to melt down or you can get case hardening. It's a very delicate process drying this gelatin effectively.

Next slide, please. Then we do a milling after the drying and our size is typically 8 mesh. That's our kind of working mesh size. We can do a variety of mesh sizes in the finished product, but most of our intermediate products we're producing these intermediate extracts that we use to do our final finished blending, and it's typically about 8 mesh.

Next slide, please. So as these individual extracts, whether it be 4 or 6, come off there, they are separated on the dryer as discrete extracts. Those individual extracts from daily production are individually blended to make sure that there is no lack of homogeneity as that material is processed across that dryer. So we blend those with homogeneity. We sample those materials, as intermediate product, and those that go in the dryers are weighed and go into storage as intermediate product for future blending and mixing.

Next slide, please. So there is our inventory that we're building up with our daily production, and then based on the specifications of our customers, we build mixes and we formulate mixes with these individual extracts that we have been producing. These are much larger blends. Some of these are 10, 20, 40,000 pound mixes, so now we have a high capacity blender that allows us to put those together.

Many times we'll make a much smaller small scale mix to make sure that we can blend it properly, particularly if it's a new product. We can hit the specification before we go to the large scale blend. So sometimes there is a series of analysis that we've done it two or three times before we finish off the finished product.

Next slide, please. And then we provide that product once we are ensured that it meets the specifications of customers. We'll package that up using drums, FIBCs or small bags and then it is off to the customer.

I hope that has given you a quick -- I had to go through that rapidly. There is a lot of information to cover there, but you've got that in your handouts there. So I hope that was useful and I would be glad to entertain any questions you have. And I also would like to invite you to come out to see our facility in Sioux City, Iowa if you would like to see first hand how we make gelatin.

CHAIR PRIOLA: Yes, Dr. Bracey?

BOARD MEMBER BRACEY: Yes, I have one question. You said in the cation exchange process that you treat the product in a different manner depending upon the end use, i.e., photographic versus other. So, in essence, that suggests that there is the potential for control.

DR. DUNN: That's right. That's right. I mean, there are certain types of food products where you may not go through the columns at all. I mean, it depends on the ash content. Typically, the ash if it was unprocessed, it could be as high as 2 percent, okay. In some cases, there would be no need. And it would get very sophisticated with the photographic realm whether you are interested in anions and cations, you go through a cation and bypass an anion or you may go through a secondary column.

You know, we have a battery with three columns of each type. Usually, one is a lead column, lag column and then there is a regenerate one under regeneration. So there is a variety of ways to go through that ion exchange system, depending on what the specifications of the customer are. You might have a food customer who says well, ash is less than 2 or you might have a photo customer and it has got to be between .1 and 2.5 or .1 and .25 or something like this. There is all kinds of variations on the thing in terms of exposure to ion exchange.

CHAIR PRIOLA: Dr. Bailar?

BOARD MEMBER BAILAR: I understand from Dr. Chiu that it is the processors who are responsible for the safety of supplies. How is that monitored or enforced here and abroad?

DR. DUNN: You're talking about the supply of our gel bone?

BOARD MEMBER BAILAR: Right.

DR. DUNN: Okay. We audit our suppliers. One of the things that makes it a little bit easier here in the States is we only have a few. We basically have -- it depends on the company. Between the two companies, I think, we have five or at most six different suppliers. So it's not an unmanageable deal to go in and audit these customers on a regular basis. We also know that USDA is in these plants. They help us with this. As a partnership, they are in there auditing all the time.

For example, when we worked with the USDA because of these European regulations to start taking our SRMs, back in 1998, they worked with us to do that, to go in and validate those procedures and so on. So we have an ongoing program in that respect and we work with the USDA sometimes to do various things as well.

BOARD MEMBER BAILAR: What about foreign supplies?

DR. DUNN: All of our suppliers here in the United States, everything we source is here in the United States right now.

CHAIR PRIOLA: Can you remind me, you said there were Type B and Type A --

DR. DUNN: That's right.

CHAIR PRIOLA: -- process and the Type A is acid?

DR. DUNN: Type A is the acid. Type B means base.

CHAIR PRIOLA: Right. And why do you choose one of those others?

DR. DUNN: Like I said, we do very little Type A. I mean, very little. We're talking probably less than a couple percent, something like 2 to 3 percent, and that's all directed to the pharmaceutical capsule industry, and there are reasons for that. Because of the way we process this material, the ratio of viscosity bloom and the ratio of viscosity to concentration is very different. We can acquire a very low viscosity concentration ratio with this process for acid bone.

And sometimes those customers who make the capsules require that they have a higher concentration. And the limit usually is viscosity. So if they can get a gelatin that has a lower viscosity to concentration ratio, that allows them to bring more gelatin into that capsule, and sometimes in the soft gel, it depends on the drug fill and what is going on there, that can be very important. So it's very important for a number of applications in the soft gel area.

CHAIR PRIOLA: All right. So even though it's a small percent of the time you do this process, most of it goes to the pharmaceutical industry?

DR. DUNN: That's right.

CHAIR PRIOLA: Then the sodium hydroxide option, the base treatment, you said that's under review. Is that to see how that might effect --

DR. DUNN: That's right. That's under review for acid bone. The most important thing it's under review by our customers, and they are currently evaluating that to see if there is not any other shortcomings of the fact that the sodium is there as opposed to the calcium from the lime.

CHAIR PRIOLA: Does it seem to change the end product at all?

DR. DUNN: From our prospective, it doesn't, but that's why we're relying on the capsule manufacturers to do their full evaluation and that's what we're looking for. Okay. So we can do it. It's easy for us to do. It's not a problem for us to do that.

CHAIR PRIOLA: Dr. Khabbaz?

BOARD MEMBER KHABBAZ: Yeah, I have a question regarding the bovine bone sourcing practices. You said since 1998, you have been removing the specified risk materials, except for vertebrae.

DR. DUNN: Yes.

BOARD MEMBER KHABBAZ: Why that exception and is it still practiced?

DR. DUNN: That's a very difficult thing to do, and there is really up until recent times there has been no requirement. There are EU regulations now developing and that's why there is concern there that that may be a requirement coming into place as early as the end of this year. We're not sure how this is going to roll out, so we're looking at this strategically. Right now, there is not a requirement, but there is a big hurdle there in terms of industry's ability to do this.

This will cost us more money. It will reduce the amount of bone available. Right now, if you take the vertebrae and take it somewhere else, you reduce the quantity right there by 25 to 50 percent. And then there will be certain facilities that will just not be able to do this with the equipment they have. They won't be able to make this change without investing new capital. But anyway, the prices we are seeing now, you can get this material, small masses of this material now, but it is going to cost you 50 to 100 percent more than the traditional. So, I mean, nobody wants to go there unless we have to. It's going to be very costly for us, our suppliers and our customers.

CHAIR PRIOLA: Okay. Thank you very much, Dr. Dunn.

DR. DUNN: Thank you.

CHAIR PRIOLA: I think we'll move on to the next speaker. It will be Mr. Schrieber, who will describe the European manufacturing processes for gelatin.

MR. SCHRIEBER: First slide, please. I would like to thank you, Dr. Priola, this Committee and the FDA for the opportunity of presenting on behalf of the Gelatin Manufacturers Association, GME. Again, details about raw materials sourcing and the bone gelatin manufacturing practices in Europe. My name is Reinhard Schrieber. I'm the chief manufacturing officer operating GELITA Gelatin Group. I'm 36 years in the gelatin business, and I have served at European Gelatin Association for many years as president, chairman of the regulatory committee and the chairman of our BSE Steering Committee.

After my American colleague, Mr. Dunn, has already substantially presented the details of the bone gelatin manufacturing process, I would like to go only relatively shortly into this issue. The manufacturing processes in general and although the bone gelatin manufacturing processes in particular are very similar to each other, not only in the U.S. and Europe but all over the world. The main differences which can be noticed between the continents are related to the safety status of the raw material and the sourcing systems in place.

This is why I like to focus more on these topics, whereas I would like to try as well to connect the connections and the conditions of our study to those existing in reality.

Next slide, please. GME members have taken several voluntary steps to ensure the safety of the raw materials. Long before the emergence of BSE, the European gelatin industry has decided to use, and this applies for all types of raw materials, only raw material coming from healthy slaughtered animals and released for human consumption, regardless of whether this was mandatory or not in different member states. So we don't use any materials from fallen or sick animals.

So traditionally, no material from fallen animals have been used by European manufacturers. The three bones gelatin manufactures in Europe have never used UK bones, but when BSE in the UK became evident, they confirmed immediately in writing not to use UK bones. After the condition of BSE to humans was detected, the GME members committed themselves to stop the use of skull bones, the target which was reached in 1997. This was further followed by the complete removal of spinal cord by European meat packers only on request of the European gelatin industry.

In parallel, our industry started to replace European bones to a certain extent by imported bone chips, mainly from the United States, but also from other countries outside Europe. In 1999, the European gelatin industry was able to convince its suppliers to remove vertebrae from bovine bones of all ages, which again was more than European law required.

Next slide, please. As I stated before, on top of our European sourcing of our demand for bovine bones can only be covered with additional imports from different countries. So we always force our suppliers in GBR II countries to voluntarily take measures in order to increase the safety of our raw materials. GBR II country means that there are so far no BSE case detected and the European has assessed that it is unlikely that there will be a case, but it cannot be excluded.

The U.S. is and Canada has been until recently GBR II countries. Together with our American colleagues, we implemented the removal of spinal cord, also in the U.S., and one year before we succeeded in doing so in Europe, we had forced our suppliers in India, Pakistan, Nigeria to remove the vertebrae as a precautionary measure.

Next slide, please. Most of the measures which we had already implemented became mandatory by regulation in Europe some years later. On top came the postmortem rapid testing of all cattle older than 30 months. Furthermore, the removal of vertebrae as requested now by law only for animals older than 12 months, but again in the bones we use in Europe, there are no vertebrae in at all. So in practice, the vertebrae is removed from all cattle in the European Union if the bones are intended to be supplied to the gelatin industry.

I assume that you are aware of all those regulations presented to you, I think, by Dr. David Asher in February of this year. With gelatin regulations, the EU fixed raw material sourcing conditions and certain safety relevant procedures to all kinds of food grade gelatin. This has been presented to this Committee two years ago by my colleague, Dr. Scheigas. Those requirements are in line with the new study conditions, and our regular intervals controlled by public veterinarians responsible for the supervision of our plants, although the FDA has made audits to the gelatin bone manufacturers in Europe two years ago, they went to all plants.

Next slide, please. Because of the steps taken by the industry, there was always only a very little chance that BSE infectivity could be present in the raw materials used to produce bovine-origin. To date, due to additional more recently implemented controls like the postmortem BSE testing and the careful removal of all SRM, it is almost impossible for highly infected material to enter our supply chain.

Next slide, please. As with any process and systems, there is a certain possibility of error. What could happen, for example, animals with very low infectivity might not be detected by the rapid BSE test. But they are considered today as to posting no risk to human health. The surveillance systems in place might not be adequate in all countries. The removal of SRM may not be done perfectly. The infectivity of bone marrow has not been finally clarified. Based on our experience, we believe that those risks are low, but they are not negligible. They will be quantified by the Scientific Steering Committee of the European Union and then used in the coagulation of the quantitative risk assessment, which is currently under development.

Next slide, please. Last year, more than 9 million normal slaughtered animals were tested on BSE within the whole European Union, including the UK. And 287 positive cases were found, which gives a ratio of 1 to 50,000. But our tests which had been done and our study has assumed that all animals used were clinically infective. Supposing that the removal of SRM is not effected perfectly and that those impurities may not be detected by the gelatin industry when inspecting the incoming fresh bones, some might enter the process. Again, our tests and our study have assumed that the bones from all animals contained the food quantity of infective spinal cord and dorsal root ganglia. Well, this gives a huge safety margin between the study conditions and reality.

Next slide, please. Here again, the major production steps applied during the commercial and the study manufacturing processes, most of them have already been described by Dr. Dunn. All plants in Europe are ISO 9000 certified for the quality management and they apply the HACCP principles. The combination of those is about equivalent to GMP. FDA audits have been successfully conducted in all European bone gelatin operations two years ago. And a further round of audits is scheduled for the end of August and early September this year.

It has to be noted that SGS and independent institute specialized in quality certification carried out a validation audit. And each of the bone gelatin plants of GME in Europe and there are no known GME bone gelatin plants in Europe, and by these inspections all processed parameters of our study design have been validated against minimum production conditions in place in those plants.

Just to clarify what this means, minimum conditions. In certain plants, for example, a higher concentration of the hydrochloric acid or a longer liming time might be applied by one or the other manufacturer compared to the conditions of this study. But we used in our study the minimum conditions applied at least by all manufacturers.

Next slide, please. One of the differences in Europe compared with the rest of the world is the fact that in Europe bone gelatin manufacturers have their own bone degreasing plants. In other countries, like the U.S., degreasing is part of the meat packers work. In the Far East, for example, it is effected by independent specialized companies. As mentioned before, only bones from healthy slaughtered animals released for human consumption following audit and postmortem inspection are collected from the meat processors, who do then later the deboning of the carcasses.

In the U.S., slaughtering and deboning is done normally at the same premises. In Europe, we have very often different locations. So this means that the carcasses of the animals are transported to a sausage manufacturer, to a meat packer at a different place and during this transport, the bones are still with the carcass. Only the SRM, the spinal cord, the heads are gone, spinal cord is out, but the bones are still with the meat.

The incoming uncrushed bones are then inspected by the gelatin industry on sorting belts for extraneous materials, including potential SRM contamination. Then the bones are crushed to small chips of about 5/8^ths of an inch, this fingernail size. Then the bones -- this means after crushing that we have a big surface. And for example, with the hollow long bones the inside would as well become an outside.

These small bone particles are then degreased by hot water in a continuous flow process at approximately 185 degree fahrenheit of an average period of about 20 minutes in equipment with high education. This mix of water, temperature and movement separates fat and soft tissue from the solid bone particles. The little ones are then separated by sieves and cyclones, dried with hot air, but the surface temperature of the bone particles will stay below 150 degrees fahrenheit to avoid degradation. Then they are sieved to remove fine particles and stored in silos.

Next slide, please. Demineralization to remove the phosphates from the bones is carried out at the same conditions like in the U.S. in a conduct current system. The total treatment is about 4 days with hydrochloric acid of 4 percent. The remaining protein matrix of the bones is called ossein.

Next slide, please. To cut the cross veins of the collagen acid or alkaline can be applied. This was addressed just before. For a small portion of the total bovine bone gelatin production, it is about 2 to 3 percent for special pharmaceutical soft gel capsules. The ossein is treated again for 24 hours with sulfuric acid at the low pH and after some washes, the gelatin can be extracted at a pH between 2 and 3.

So standard bovine bone gelatin is normally extracted at a pH between 6 and 7. And the ossein is treated before the saturated or over saturated lime solution for at least 20 days. As you have heard, the pH of this lime solution, which is replaced several times during the process, is around pH 12.5.

Next slide, please. To make sure that acid bone will be as safe as lime bone, our industry looked into an alternative process which would include an alkaline pretreatment, but without working the special physical and chemical properties of this pharmaceutical as in bone gelatin. Based on the knowledge that after the bones are crushed potential infectivity would sit on the surface of the bones and not inside the bone matrix, we assumed that a short time treatment of about 2 hours with .3 molar sodium hydroxide solution should be enough to inactivate infectivity if this pH is kept at 13 for this time.

Our study results have shown that this treatment is very effective. But our study has also shown that gelatin made by the traditional acid bone process did not show any detectable remaining infectivity, which means there is a demand for this type of gelatin is still very rare. You've heard that we are depending, of course, on our suppliers to do it or not to do it.

Next slide, please. During extraction of the pretreated raw material, several single extracts are collected, each with different physical properties due to an ongoing hydrolysis during the extraction. It has to be stated that due to the different requirements of the gelatin using industry, quite often photographic, pharmaceutical and food grade gelatins manufactured from the same raw material batch in sequence. Also, Eastman-Kodak is manufacturing some pharmaceutical and some food grade gelatin.

This means that all gelatin of one production day, including the photographic gelatin, have to comply with the regulatory requirements for food and pharma. When talking about food and pharma, one has to keep in mind, as well, that the same capsules might be filled today with nutritional products, being food, and tomorrow with Rx drugs.

Next slide, please. For further clarification, the diluted gelatin solution is filtered by different types of equipment and filter media in the ossein and ion-exchange columns and concentrated in the apparatus.

Next slide, please. So final concentrated gelatin solution is sterilized by direct steam injection. The temperature is at 4 bar. The pressure in the liquid phase, which is very important, is a minimum of 280 degree fahrenheit and the temperature stays for at least 4 seconds.

Next slide, please. Finally, the sterilized gelatin solution is chilled to set and then dried with purified and conditioned air on belt dryers. Each production batch, which is a single extract, is then tested on physical, chemical and bacteriological properties. According to customer specification, different production batches are then dry blended. The final blends are again tested under compliance with regulatory and customer requirements and then released for shipment. These are the common processes applied by the European industry.

Next slide, please. There is one special process which is done by only one company in Europe to manufacture gelatin with low gelling strength for limited applications. The degreasing is done of the bones in the common way, but then the bone chips are autoclaved for at least 20 minutes under 3 bar pressure and 270 degree fahrenheit. After the autoclaving, bone chips are rinsed with salt water. A certain quantity of gelatin goes into solution.

After this gelatin solution is taken out, autoclaving at lower temperature and shorter time is repeated several times. Then these different extracts are collected, flocculated, ion-exchanged and evaporated, drying, testing, blending, retesting and shipping is effected, like with all other gelatins. Low gelling strengths, the gelatin is used only for certain applications, and the Committee members might remember that two years ago at this meeting, we already explained that the main application is a confectionery licorice, although this process has been successfully simulated during our study.

Next slide, please. What are the conclusions which we have drawn from this review presented here? So commercial mineral manufacturing conditions are reflected by the GME study conditions. The GME plants and process parameters have been validated for conformity against the study design. The inactivation results of the study, which will be presented next, are therefore fully applicable to the practical gelatin manufacturing processes. The study demonstrates the ability of the gelatin manufacturing process to remove and inactivate infectivity even under conditions in which raw material contain unrealistically high infectivity levels.

Last slide, please. So safety of European bone bovine gelatin is established on two principles. The safety of the raw material as required by GME practices and EU law and the safety of our manufacturing processes as demonstrated by the GME study. The Scientific Steering Committee of the European Union has concluded based on all these principles, in it's opinion, on the safety of gelatin that the risk is close to zero.

Madam Chairman, Committee, that concludes my presentation. I would like to thank you and the Committee for your attention. Thank you.

CHAIR PRIOLA: Okay. Thank you, Mr. Schrieber. Are there any questions? Okay. Thank you very much. Our schedule says there is a break, I believe, after Mr. Schrieber, so we can adjourn and return at 10:00, so that's about 15 minutes from now. All right. Thank you.

(Whereupon, at 9:42 a.m. a recess until 10:03 a.m.)

SECRETARY FREAS: We're going to go ahead and resume the meeting.

CHAIR PRIOLA: Okay. I would like to go ahead and get started. Dr. Hogan had a question for our last speaker, Dr. Schrieber, that he would like the Committee to hear the answer to. So, Dr. Hogan, do you want to?

BOARD MEMBER HOGAN: Mr. Schrieber, I asked just after our last talk about how the meat processors were audited, in terms of providing safety of the raw materials to the gelatin manufacturers. Could you address that, Mr. Schrieber, please?

MR. SCHRIEBER: Surely. The standard procedure in Europe is that in every slaughter house, every meat packing operation there is a public vet present all time, every day as long as this operation works to supervise that regulation is followed, removal of SRM is done and so on. And besides this, the gelatin manufacturer are auditing their suppliers on a regular basis, normally once a month or every two months, again inspecting as well the commercial documents about where the animals have been sourced, because commercial document which is required as well by law. So there's a double-fold. But the main thing is that the public vet is present all day, all the time. Thank you.

CHAIR PRIOLA: Okay. Thank you, Mr. Schrieber. We'll go on to our first speaker for this later morning session. That's Dr. Robert Somerville, who is going to discuss the GME validation studies on bone gelatin.

DR. SOMERVILLE: Okay. Thank you, Madam Chairman. It's a pleasure to be back in the USA where I've spent quite a few happy years working a couple of decades ago. My task is to describe to you the actual validation studies that were performed in three labs actually over several years. There were several people involved and I want to mention them. First, Ad Grobben is perhaps the most important one of them all, because he, as an employee of gelatin, which was a member of GME, actually performed or was participating in all three studies in Edinburgh, in Holland and here in the USA in Baltimore.

Phil Steele, actually I should say that, I know a consultant to GME and is present in the audience and I hope will assist in any different questions you might ask me later on. Phil Steele is a technician in my group and he assisted at not only in the work he did in Edinburgh, but also in Holland to assist in the experiments there.

David Taylor was my predecessor in running the inactivation group, and he initiated the studies that we're about to describe and collaborated in setting up the whole thing. He again is here in the audience. I inherited the work from David and responsibility for the work when David retired in 2000, so it's my duty to report the results, but all the hard work was done before that.

The work I'm specifically going to concentrate is on the Neuropathogenesis Unit, which is part of the Institute for Animal Health in Edinburgh. It was funded by GME with further support from the European Union. I should also say that at the end of my presentation, I suggested that Bob Rohwer, who performed the Baltimore studies, spend a few minutes describing the work that was done in Baltimore.

The next slide, please. I thought it would be helpful to describe the basic mechanisms of TSE inactivation first, and there are three ways in which inactivation or removal can take place. The first is through some form of destruction through combustion, incineration, oxidation with hypochlorite, hydrolysis of extreme pHs or with very high concentrations of highly effected proteases and radiation can have an affect at very high doses.

Next section, please. What possibly concerns us mainly today is denaturation type of processes where materials hydrated, in particular, will have a degree of inactivation effect and exposure to chemicals, such as strong detergents or chaotropes, can also have an effect.

Next. And we have to look at treatment variables. There are several biological parameters that we must consider. The strain of the TSE agent is particularly important and I will illustrate that in a couple of slides time. The PrP genotype may well be important, and we have to consider that. The tissue and the state in which the tissue is presented in the experiment are also important. We have to consider physicochemical parameters such as heat temperature, pH and the kind of chemicals that one uses. And finally, the dynamics and kinetics of the reaction have to be considered, the time, concentration of any chemicals involved and the temperature are particularly critical.

Next. Okay. This shows a slide of some data that was originally published in 1983 by Kimberlin, et al, where TSE infectivity from two TSE strains was heated for various lengths of time shown on the axis. On the Y axis is the titre that was recovered after these treatments. Two strains were used, as I say the 22A strains and the 139A strain, and you can see that there is a lot of rapid reduction in the amount of infectivity present, first. Then a plateau. So the reaction is biphasic with respect to time, and there is little effect of time after initial exposure.

The second point to notice is that was a strain difference, so the 22A strain on this particular example is much more resistent to an activation than the 139A strain.

Next. Now, in this slide, we're looking at what happens when we heat at a constant time, 30 minutes, with a range of temperatures, and what we can see here is that there is little reduction in infectivity to start off with until we reach an inflection point, and then the amount of infectivity drops rather rapidly, and that happens for both the TSE strains that we're looking at here. But you can see that the inflection point for these two strains differs, so that for 22C, it's rather thermolabile, which might be a surprise to some. In fact, this temperature which starts to inactivate is only about 70 to 75 degrees centigrade.

With 22A it's higher, about 97 degrees. But we haven't specifically done experiments comparable to this BSE or BSE derived strains, although I'm hoping to do them in the near future. But from the data that we have available, we think that BSE derived strains are even more resistent to inactivation than the 22A strain here in red, which is the more thermostable of the ones we have seen. So we can say the inactivation process is biphasic with respect to temperature and dependent on temperature. TSE strain, and I mentioned the hydration state, and I'll come on to that in one moment.

Next slide. Okay. This slide shows the effect of hitting again at 126 degrees centigrade for 30 minutes autoclave, three strains of TSE, and you can see with the 22C strain that all infectivity shown in red has been destroyed. The blue shows the starting titres. The two different blue bands are indicative of two different PrP genotypes that the TSE strains were passaged in, and there is no effective PrP genotype in this experiment available.

With ME7, we cover both the types, a little infectivity, but with 301V, we cover a lot more. Now, 301V is important to the rest of this talk. 301V is the most thermostable TSE strain that has been derived from the passage of BSE through a particular strain of mice, the VM strain of mice. And it has certain advantages to these studies. Notably, it is very high thermostability, and that makes it a greater challenge to the studies that we are performing.

On the right hand side of the panel, you can see a different experiment where material was heated in a dry oven to 200 degrees centigrade for either 20 minutes or 60 minutes. And I think the contrast between what happened in the autoclave and the dry oven is really quite remarkable. We get much less reduction in infectivity and we've lost our strain differentiation. So there's no strain difference in the results. And also material survives the dry oven much better than it does in the autoclave. So that emphasized the point about hydration status. I think if we dry out infectivity, we make it much more resistent to inactivation.

Can I have the next one? Okay. This is an experiment where we have combined temperature treatments with a range of pHs. I don't suppose you can read this, but each line represents a different pH from pH 7 up to pH 12 with three strains of TSE again, 301V, ME7 and 22C. And the point is to say that with 301V, in particular, we got very little reduction in the amount of infectivity up to 100 degrees. Certainly up to pH of 11.

We didn't measure what happened at pHs greater than 11, whether we were getting any reduction infectivity at pH 12 up to 100 degrees, but at 60 and below there was very little reduction in the amount of infectivity recovered there. You do start to see, in effect, that pH 12 with the more thermolabile strains ME7 and 22C. So the suggestion is that high pH acts synergistically with temperature when TSEs are inactivated.

Can I have the next slide, please? So on the left you can see a list of the things that I have been showing on all previous, three or four slides, and results and conclusions. Thermostability is an intrinsic property of TSE agents developed to kinetic mother which I'm not going into today, and so forget about the rest. Thank you.

Next slide. Okay. I want to move on now to reduction of risk of TSE infectivity in gelatin. The challenges that we face, there is very high resistent to inactivation, and the resistance increases on drying up infectivity. There are several available approaches. We can remove by filtration, for example. We can denature with heat and high pH or we can use at very high pH, we can get hydrolysis of infectivity.

Looking specifically at the risk reduction steps that are available in gelatin manufacturing, the sourcing of bones, which Mr. Schrieber has just described, is important as practices in precleaning the raw materials. I'm not going to discuss this. The standard gelatin extraction methods are thought, were thought to be effective, and that is what the valid study of it we've been involved in is designed to test. And then the sterilization steps are steps which may specifically move TSE infectivity and, of course, other contaminants.

Next slide. Now, this slide shows the results from the very first studies that were performed in the gelatin manufacturing process. What was done by Inveresk was to take any 7 grain homogenate and look at two components of the process, either treating with hydrochloric acid, the liming step or the combination of the two. The reduction in titre after exposure to the hydrochloric acid was about 1 log, 1.2 was measured. So that's 10-fold roughly.

Exposure to lime for 20, 45 or 60 days resulted in a reduction in titre of about 2 to 2.3 logs. And you can see that even after 60 days, these values are very similar. So there was a small reduction of about 100-fold, but the time of exposure had new extra effects. And the combined treatment results in the reduction of nearly 3 logs, but you can see that adding these two values together does not come to 2.8. So there isn't complimentary effect, but the treatments are not completely out of it.

Can you give me that slide, please? So there is a reduction in infectivity titre measured by the acid and alkali in combined treatment of any 7 homogenate. The combined treatment is more effective than either single treatment, but they are not titre, and time of exposure to costs in hydroxide does not effect infectivity titre. And these processes were not representative of the actual process involved in the plant.

There is another study by Manske, et al, which showed that there was removal of proteins under industrial degreasing conditions. These initial studies led to the desire for more systematic studies to be performed.

Next slide, please. As I've already indicated, there was several experiments performed. In Edinburgh we performed four experiments, two alkaline treatments using two TSE strains, the 301V strain, which I have described, also the 263K strain, which is a hamster-facade strain, which we believe is reasonably thermostable, but may not be quite as thermostable as 301V.

We also looked at an acid process and we tested the addition of an NaOH treatment in the acid process. Mr. Schrieber has described the Dutch heat and pressure method and an experiment was performed in the Netherlands to look at that process. And as I say, Bob Rohwer will describe the sterilization filtration experiments later.

Next slide. Okay. The rational of the experimental design. The TSE source is a high titre BSE derived model. It's thermostable. It's readily assayed in experimental mice. We feel that the total titre is a likely BSE contamination event during industrial processing the gelatin, as Mr. Schrieber has already suggested. Short incubation periods, but we have to be aware that we occasionally see very extended incubation periods after heat treatments. And so we kept the mice under observation for up to 600 days.

The limits of detection depend on concentration of the sample and the toxicity of the sample. We cannot inject material that is toxic, obviously, to the mice. So sometimes dilution factors had to be included so that we could inject the mice and not limit the clearance levels that we can measure. However, we feel that near optimum demonstrate clearance levels are demonstrated from this model.

The scaled down to simulate typical gelatin manufacturing conditions was performed by Ad Grobben from earlier and that was reviewed by an international panel prior to initiation, and as already indicated, Ad Grobben is here to answer specific questions on that matter. And the quality of gelatin was checked as the experiments proceeded and again I can address those questions.

Next slide. Okay. So this is what was done. The raw materials were 1.5 kilograms of fresh crushed bones and 500 grams of intact calf backbone, spiked with approximately 10 grams of TSE infected grain homogenate. Half this back was injected into the spinal column and the remainder smeared onto bones and dried onto the surface, and the backbone was then sawed into pieces. There was a degreasing process where the bone chips were washing at 85 degrees centigrade to remove soft tissue and fat after the spike had been added, and then dried in the hot air at 120 degrees centigrade.

Then the demineralization step was performed. The bones were soaked in hydrochloric acids of increasing concentrations. The ossein, of course, remains as already described. Then the liming process, the ossein was exposed to saturated calcium hydroxide of pH 12.5 for the minimum of three weeks, and then neutralized. On the acid treatment left over night, also a pH 3 and then washed in water. The NaOH treatment, which is included in the acid experiment, one acid experiment, the ossein was exposed to .3 molars of sodium hydroxide pH 13 for two hours.

Then the extraction process ossein was stirred gently with water at temperatures from 60 to 90 degrees to a final gelatin concentration of 2.8 percent. And then purification steps were performed, depth filtration, ion-exchange, heat sterilization and drying, and all steps were designed to accurately represent the conditions of the industrial process.

And it should be pointed out that in the larger process we used indirect heating, but in the industrial skill process, of course, direct steam injection is used.

Could I have the next slide? Okay. The spike, as I've already indicated, we use the 301V strain in three of the four experiments, and we use it because it is the most thermostable TSE strain tested so far, and it also is BSE derived. We actually titrated the spike on three separate occasions. We actually had two spikes, Pool 1 and Pool 2, and you can see the values are very similar in all three titrations that we performed with a value of about 7.7 in each case.

And as I've already mentioned, all clinically negative animals were observed for at least 600 days, and then we examined the brains for any evidence of pathological lesions of TSE infection afterwards. And all positive clinical cases were confirmed by pathological examination.

Next slide. Okay. Some results. So this is the first experiment where the bone was spiked with 301V. The steps were performed degreasing, demineralization and DCP, the dicalcium phosphate, which is a byproduct of the gelatin manufacturing process, was also tested for residual infectivity and we find little. The extract sample after the liming initialization extraction had a little bit of infectivity here, and you can see the individual numbers on the left, and that calculates, according to the Carver Method, to titre of less than or equal to 10^1.8 ID₅₀ per mil.

I say less than or equal to, because if you use the Carver Method, you have to make it -- and you've got incomplete groups at either end of your dilution series, you have to make assumptions about what happened in that group. So we don't have a 10⁺¹ group, but we assume to get the number 1.8 that that value, all the mice would have gone down the 10⁺¹ dilution. So the number over here is the total recovery calculated against the 10 gram spike that was used. So we got from total infective load to 10^8.7 to total recovery of 10.5.

Then the sample was taken through the filtration ion-exchange in concentration steps and the sterilization steps, and a sample was then also measured for infectivity, and no infectivity at all was recovered. And in this case, what these data say is that we couldn't detect anything. We don't know what would have happened if we had been able to inject a more concentrated sample again. So again, we can only say this is the limit of the clearance that we have achieved. So the total recovery is less than 10^3.8 starting with the 10^8.7.

Okay, next slide, please. Okay. So this is the second experiment in the alkaline process where we used the other strain, 263K, and I will go through this a bit quicker. The total infective load is 10⁹. We recovered a little infectivity again and the DCP, the dicalcium phosphate, and we also recovered a little bit of infectivity in the extract sample totaling out to a total recovery of less than or equal to 10^4.3.

Next slide. The acid process here we again had a spike which had a total infectivity of 10^8.8 and following the steps of degreasing, demineralization, then the acid treatment and extraction we had a recovery of infectivity of 10^6.2. In this case, we got a clear end point to the experiment, because the neat fraction, all the animals came down to 10^-2, none did, so a nice, neat Carver calculation of 10^6.2.

After the filtration, ionization, concentration, sterilization steps no infectivity was recovered and we can say that is less than or equal to 10⁴ logs of infectivity were recovered.

Next slide. Okay. Now, this is the variation on the acid treatment where the sodium hydroxide step was included after the acid treatment had been performed. And when this was done, we find that no infectivity at all was recovered in the titration and we can calculate total recovery of less than or equal to the 10^3.3.

Next slide. Okay. Looking at the data across the way, that's the first alkaline process and you can see it went from 8.7 to less than 5 down to 3.8 with no positives.

Next. Next? Oh, there we go. Thank you. And this is the alkaline process with 263K, and now you can compare the numbers directly with each other. So we start off with slightly higher spiked titre and slightly lower recovery of infectivity at this point on the crude gelatin extract.

Next. And next again. And with the two acid process experiments, we start off with the spike of 8.8, 8.7. We recover a little bit more infectivity than in the alkaline process at the crude gelatin extract, but again when we look at the purified material, no infectivity is recovered and we can, as I already said, indicate the clearance values from that part of the process. And you can see now that the acid process with the included NaOH treatment in here resulted in no infectivity being recovered.

Next. So this summarizes the data and now I have included on the right hand side the clearance factors that have been obtained from the experiment. So we can say that the alkaline process, the crude gelatin extracts have a clearance of greater than 3.7, logs of infectivity and for the 263K experiment it was greater than or equal to 4.7. The finishing, the purification and sterilization steps have additional clearance factors that we have demonstrated of greater than 1.2 and that totals over the two parts of the process to greater than or equal to 4.9.

In the acid process, we got a clearance from the -- in the crude gelatin extract of 2.6. The sterilized gelatin after finishing has got an additional demonstrated clearance of greater than 2.2 and adding that together, we've demonstrated a clearance for greater than or equal to 4.8. And then in the acid process with the additional NaOH treatment, the overall clearance demonstrated is a value of greater than or equal to 5.4 logs of infectivity.

Next. Okay. So that summarizes what we have. From the acid bone process, we have got substantial infectivity measured before purification in the third experiment, but complete appearance after -- complete clearance after purification, including sterilization. Complete clearance, no infectivity detected before purification if an additional sodium hydroxide step is included.

With the alkaline process, there is greater removal of infectivity than after equivalent acid hydrolysis procedure and there was complete clearance. No infectivity detected after purification, including sterilization.

Next. So our conclusions are that the gelatin manufacturing procedure was successfully scaled down and normal bone gelatin was produced. Both degreasing and the standard acid and alkaline treatments alone remove most, but not all, of the implied infectivity before final purification of gelatin. The liming or alkaline procedure was more effective and the additional sodium hydroxide step in the acid procedure inactivates a residual detectable infectivity before purification. After purification, all samples do not show any detectable infectivity. And again, this was pointing out the removal of infectivity is cumulative, but not necessarily additive.

Okay. I want to move on to report the data obtained by the Dutch experiment where they applied pressure treatment to produce their gelatin. They started off with titre of their spike, total titre of 9.2. They went through the standard procedures of degreasing and preheating, then did the pressure treatment at 3 bar, 20 minutes, 130 degrees centigrade, then extracted the gelatin. In the crude gelatin extract, they showed that no infectivity could be recovered, and the volume that comes to is less than or equal to 0.2.

So they record a clearance factor of greater than or equal to 6.8 with this process. They did nothing -- well, they didn't follow this, the purification steps. They didn't test that, but there was no infectivity in the gelatin solution that would have come through this procedure anyway, so it would have been a waste of time.

Next. So risk reduction and gelatin. We've had several descriptions of this already in the earlier session about sourcing using only animals passed fit for human consumption, omission of head bones and vertebrae from source material in BSE infected countries, and we have shown the removal or inactivation steps or removal of TSE infectivity during the gelatin extraction and purification process.

It's also worth noting that the species barrier would reduce the effect of titre or BSE being -- if humans were exposed to BSE from the source. It is worth also noting that the acids were performed by injection intracerebrally, and this is by far the most efficient route of infection, other routes of infection are less efficient.

Next. I'll skip that. That's it. Okay. Thank you very much.

CHAIR PRIOLA: Are there any questions for Dr. Somerville? Dr. Bailar?

BOARD MEMBER BAILAR: I have a couple of related questions. First, I find the time deactivation curves somewhat troubling. They suggest that some of the infected agent is being protected somehow. What is your take on that?

MR. SOMERVILLE: Exactly that. That there is -- I didn't want to get too much into the fundamental thoughts that I'm having at the moment about that, but I think we're getting a dissociation reaction and a protective reaction occurring when inactivation, heat inactivation is attempted. And the protected species that is formed or the stabilized species is much more difficult to inactivate. It may be similar to the dried material that I was showing in some of the earlier slides, too, and that we know is much more difficult to inactivate.

BOARD MEMBER BAILAR: Well, there are at least a couple of other possible explanations.

MR. SOMERVILLE: Sure.

BOARD MEMBER BAILAR: One is that some of the agents being protected inside little particles. There many be subtle differences in the chemical structure of the ones that survive versus those that don't. Which leads me to my second question. In the intact animal, the infection occurs while the animal is alive. It gets circulated and I would presume gets distributed throughout all the tissues and whatever titre is appropriate for that. In the experiment, the infective agent was added to the bone chips, that is at a considerably later stage of things, where it might be more on the surface of any particles that remain or it might stay on particles and so forth. So I'm asking if you have looked into this, and if there is any reason for concern about this difference in the sequence of when the infection is added to the materials that you are processing.

MR. SOMERVILLE: Well, let me answer this, your question this way. I don't know if it actually addresses what you are saying. But the reason for doing the experiment the way we did it was to try and maximize the exposure in the experiment. So the thinking was that the greatest risk of BSE contaminating bones was that during the slaughter process, and that spinal cord, for example, would get spread down the vertebrae column included with it and dry onto it. So that was what was attempted to be mimicked in the experiment.

I'm not -- I suppose the other side of the question is how much infectivity in living animals associated with bone and bone related tissue? Our primary concerns in that respect again is to do with spinal cord in BSE infected cattle with spinal cord and ganglia and related nerves and, of course, the brain in the skull. These should be removed, and again we're asking the question what happens if they don't, and we've tried to include that kind of thought in the design of the spiking of the experiment.

BOARD MEMBER BAILAR: Have you tried to grow out the infective agent that survives the steps for 20, 40, 60 minutes to see if it remains highly resistent?

DR. SOMERVILLE: No, not formally. It's an experiment I want to do, obviously, but I haven't formed it. I don't think -- David Taylor whether he has actually done that experiment, either. My prediction is that it would not be in the protected form after passage through an animal, but we have to do the experiment. Thank you.

CHAIR PRIOLA: Dr. Petteway?

DR. PETTEWAY: Thanks. I just have a couple of questions about the process of doing the studies and setting them up. Just to make sure I understand, these were scaled down, coupled steps, so that the spike was at the initial step and then removal was monitored throughout the process without respiking it each additional step, correct?

DR. SOMERVILLE: That's right. Yes, that's correct.

DR. PETTEWAY: Okay.

DR. SOMERVILLE: I think the experiments that Dr. Rohwer will describe are looking at process of the final steps in the process with spiking at the beginning of those individual steps.

DR. PETTEWAY: Exactly. So that your final removal shows the cumulative effect of the process to remove the input spike.

DR. SOMERVILLE: Yes, yes.

DR. PETTEWAY: I have one other question and that's with the magnitude of the clearance numbers.

DR. SOMERVILLE: Yes.

DR. PETTEWAY: And the less than or equal to or greater than or equal to. The magnitude reflects the limit of detection of the assay.

DR. SOMERVILLE: Precisely.

DR. PETTEWAY: As opposed to what may actually be the magnitude of removal. The magnitude of removal is likely to be much greater than the numbers reflect, because of the limit of detection of the assay, right?

DR. SOMERVILLE: Basically, yes. We can only report what we observe.

DR. PETTEWAY: Right.

DR. SOMERVILLE: But we can also make some predictions about what we know from other parts of the process.

DR. PETTEWAY: Right.

DR. SOMERVILLE: And that is why, as you've said it before, it's important not only to look at the overall process, but to look at individual steps and evaluate what they may be contributing to the inactivation process or removal process. However, as the study illustrated, for example, we also have to be aware that individual steps will not be additive and that one part of the process may remove the same thing as a later part, later stage might also remove, so you have to be very careful when you're doing that.

DR. PETTEWAY: But we can be confident in the linking of these studies that based on the input spike that there was no detectable infectivity based on the limited detection of the assay at the end of the process?

DR. SOMERVILLE: Yes, yes.

DR. PETTEWAY: And then the last question I have is the additional step with the sodium hydroxide. That was evaluated independently?

DR. SOMERVILLE: What? It was a separate experiment, if that's what you mean.

DR. PETTEWAY: Yes, that was a separate experiment?

DR. SOMERVILLE: Yes.

DR. PETTEWAY: Evaluated independently. Okay.

DR. SOMERVILLE: Right.

DR. PETTEWAY: Thanks.

CHAIR PRIOLA: Dr. Hogan?

BOARD MEMBER HOGAN: Very nice studies, Bob. I had a question on when you are calculating the clearance factor here, you've listed an equation that says clearance factor is equal to gram spike times 10 the log titre spike divided by milliliters of gelatin times correct factor times 10 to the log titre reduction or gelatin. For somebody that can't balance their checkbook, what do you mean by correction factor in the denominator and why was that entered?

DR. SOMERVILLE: Okay. The correction factors are to account for the inherent losses in the process by taking a sample out for intermediate titration or other evaluations. So there is natural loss in the amounts going through the process. Does that deal with that?

BOARD MEMBER HOGAN: Yes, that's great and it makes good sense. The second question is did you look at any place in the process where titre might have accumulated or concentrated, such as inner vessels or on any of the columns or anything like that?

DR. SOMERVILLE: I think the short answer is no. Unless Dr. Grobben would like to comment on that. But as far as I'm aware, there was no specific attempt to evaluate that.

MR. GROBBEN: I do want to. I would like to comment to that, I think. No attempt was done to try to measure the infectivity which remains in the equipment, especially for filtration and ion-exchange, because of the problem to extract that infectivity from that equipment, so that was not done. We just measured what was left in the gelatin.

CHAIR PRIOLA: Go ahead.

BOARD MEMBER HOGAN: That's what I presumed. It's just very difficult to get that stuff off to measure it regardless. Now, am I to understand that in the gelatin processing process that these filters would be reused batch after batch or are new filters introduced in the manufacturing process either in Europe or the United States?

MR. SCHRIEBER: May I answer this. There is no reuse. It's a one time use. It may be the answer as well as with the ion-exchange columns. They are regenerated with either alkaline or assay to purify for the next round of ion-exchange. So there is a constant chemical treatment after the gelatin has passed those columns.

CHAIR PRIOLA: Okay. If there are no further questions, we'll move on. Thank you very much, Dr. Somerville, and Dr. Rohwer is going to present some data.

DR. ROHWER: Can we go to the next slide, the first slide here or do I control it? Are you controlling it or am I? Where is it here? Oh. Yes, please, go to the first slide. Thanks. The gelatin manufacturing process is a diverse one. It has many generic features like the contractionation for plasma, at least I see it that way having worked in both areas. So they needed a protocol representing as much of their collective production as possible. And the steps that we were asked to validate in our laboratory were on bone gelatin.

Next. And we used the process parameters that were selected by GME, their scale down and this took a lot of time setting this up. Ad Grobben deserves a lot of credit for this, as was mentioned. And from our end, our major concern was about hazard control, and we spent quite a bit of time on this as well. We did this study at a scale that was much larger than we typically use in the laboratory. We were using meters and liters instead of 100 mls at a time and some of these steps did not fit easily into the valid safety cabinets and that type of thing, so we had to figure out other ways to do them. But in the end, we were successful and it worked without a hitch when we finally got down to doing it.

Next. The filtrations, the way this was done is we tested several different types of filtrations that are used across the industry and then pulled the filtrates, and that's what was actually titred. I'll show you that in a moment. And getting them all done though it took quite a lot of time, because of the scale and the precautions we had to take to do it safely.

Also, all of the work that is done with gelatin has this complication that it is only liquid above 50 degrees centigrade, so you have to keep things warm. You have to keep them warm on a large scale, and so we developed a lot of technologies for doing that, which the tempering beaker turned out to be one of our best tools, but circulating baths and hot pads were also useful.

Next. Next, please. About hazard control, we used safety cabinets, bags to cover everything up during the actual processes. All joints between chromatography column unions and filtration things were -- transfers were covered with plastic sleeves in case they leaked, put things in large pots when we could. We poured nothing. Everything was done by pumping from one vessel to another in a safety cabinet.

Next. We are also concerned about cross-contamination simply because of the scale that we were doing this on and also because of the sensitivity of the results. And as a consequence, all new dedicated equipment was used for these steps. Everything was disposed -- most things were disposable. The only things that weren't were the stainless steel filtration vessels and a couple of other things which could be autoclaved under sodium hydroxide for reuse.

Next. Next, please. We had a question about -- some discussion about the spike earlier, and I think this was a very gratifying experiment for me. We've been trying to figure out whether our spikes are relevant in our plasma studies and that type of thing. But in the case of bone gelatin, the most likely source of infectivity is CNS tissue. And as a consequence, in this particular case, at least we can say that the brain derived spike is probably the most appropriate spike for testing removal from this type of study.

And personally, I think this is the relevant tissue and we can use it with confidence. There are issues about whether 263K or the less adapted BSE strain is more relevant. My feeling is that there are advantages to both. Actually, clinically, hamster 263K looks a lot more like BSE than the less adaptive 301V strain. On the other hand, this is a strain that was devised from BSE and so we use that as well. The important thing is when you do two different strains, is what you're looking for is the point of convergence between the two to give you some confidence that the result you are getting are generalized more.

Next. The continuous process was done at the Institute for Animal Health, and we are only working on the end stage process right here. Robert has discussed the rest of this. The continuity was maintained by Ad Grobben, who took copious notes, and we also have a lot of further documentation, which I'll share some of that with you in a moment.

Next. So here is the process we have been looking at. This is the part that Robert has been describing right here. Well, actually, they carried it through this stage as well, but the only part of this process that we're going to be dealing with is this part right here at the bottom. The so-called purification steps, the filtration, ion-exchange and UHT sterilization, and we're going to look at step wise removals.

Actually, we're going to gang these two together in one experiment. We're going to look at them independently as well, and we're going to compare the cumulative versus the serial with the individual testing of these two steps. This was done just individually.

Next. Here is the basic layout of these experiments. We have the infectivity spike. It goes into the crude gelatin, which is taken directly from production at the same stage of production. It's passed through the filtration device. And in the filtration experiments, on one arm, we took the filtrates and took it straight through the ion-exchange columns and then titrated it. On another arm, we took it over here and respiked it, figuring that we may have -- hopefully, we had removed something in the filtration.

This spike, at the most, would only double the titre that we started out with here by respiking. If we got any kind of removal here, we're just starting over at this point. And then testing the ion-exchange by itself. We wanted to use this so that we had filtrated material to test the ion-exchange process with.

Next. The filtration steps, in fact, involved five different protocols with various compositions of cellulose, sources of cellulose and formulations depending on different manufacturing setups across the industry. The filtrates from those were all pooled and then they were titrated by themselves before being passed to the ion-exchange column over here or respiked on this arm and passed through the ion-exchange columns here. The ion-exchange consisted of two columns, the cation exchange followed by an anion exchange, and what we assayed was the eluate from both, the final eluate from both.

Next. And then in terms of the UHT sterilization, the ultra high temperature sterilization, we again started with gelatin from production, infected that, spiked that with infectivity and then did the UHT test and titred that. So it's a much simpler pathway.

Next. Next, please. Here is a picture of Ad Grobben setting up the filtration experiments. This is the filter apparatus over here. We're transferring, I believe at this step, we're preheating the filter with hot water that has been heated over here, and I'm not sure that's what we're doing there, but that's what this is doing. This is the hot water. It had to be preheated so that it was warm enough to keep the gelatin melted once we put it in there. We've got another bath heating up the gelatin to dry through the filter.

Next. Here's the filter being assembled. It's quite a large apparatus, compared to what we're used to, but we were able to do all of this within the hood, though the transfers had to be through this pump on the outside. There's the filter A being added.

Next. Here is the filter A being stirred in the filter and then it's drained to form the filter cake in the bottom.

Next. Here is the filtration apparatus setup being done. Here is the vent in case, because you have to vent some air out of it in the early stages, and through HEPA filter here. And here is the assembly after the filtration is over.

Next. This was a keeper in that experiment, and this was a failure. We always inspected the filter cakes after the filtrations to make sure they were intact. There were no possibilities of leaks and that kind of thing before we would keep the filtration as a successful one. And so the only thing that goes into this study were successful filtrations.

Next. Here is a picture of the column apparatus, the ion-exchange apparatus. These were gigantic by our standards. We set them up on a mobile cart on a chromatography rack that we can roll, so that once we got everything setup and ready to go, we could roll the cart over a very large plastic bag and then cover the whole thing in this plastic bag and seal it up during the actual experiment in case there were leaks. Thank God there were none.

Next. And here is the apparatus that we used for the UHT Inactivation Experiment, and I can make that a little clearer in the next slide, which is diagrammatic.

Next. The principles that we are trying to employ in the UHT study that we were trying to mimic from the actual production environment is, from my prospective in studies that I'll talk about later this afternoon, infectivity is not intrinsically resistent. The problem is delivery of the inactivant and the inactivant finding sanctuaries to hide from the steam, and drying is -- drying into a film is one of the biggest problems.

And one of the nice features of the UHT process is the gelatin is being pumped through a pipe in which live steam is being injected. There is no head space. There are no sanctuaries. There is no place for this stuff to dry. There is no place for it to escape from the hydrolytic environment. We wanted to duplicate that as best as we could.

Next. Next, please. So we did that with this apparatus where we filled this stainless steel capillary and we used this capillary so that we could affect a very rapid heating and cooling, because the whole process, the UHT process, is a 4 second exposure to 140 degrees centigrade. So how do you do that in 4 seconds? Well, you have to get the heat to it in a hurry. We didn't try to do it dynamically. We did it statically. But we did it in this way.

So we have this chromatography capillary here. We have a thermocouple, which is embedded in the tube. The probe is right about here. We have another thermocouple on the outside to track what is happening in the bath. And then to relieve any over pressure in the device, we have a water column going here to a back pressure gauge, which ultimately if it were to leak, it would go into this tube right here. And this relieves the hydrostatic pressure that is developed by the fact that we're heating this gelatin up in here. But we've got within the gelatin column itself, we have no head space.

Next. We'll take that and the way we get our rapid heating is through trial and error. We set up a protocol where we can dip this thing, hooked to its various thermocouples, up to this recording device into our 160 degree oil bath and then as we see the temperature hit our target transfer temperature, which was about 80 degrees, we quickly dump it into the 140 degree bath and it comes to equilibrium in the successive period. We then take it from there after 4 seconds has elapsed and dump it into the other temperature. We're tracking this whole thing on the computer. We're watching it in real time as we're doing it.

Next. So we get curves like this. This is seconds down here. This is degrees over here. This is the outside of the capillary tube. So we're seeing the capillary dipped into the 160 degree bath, and here we're seeing the transfer into the 140 degree bath, and we're seeing it come to -- and this is the internal thermocouple and it is coming to temperature very quickly thereafter, and then at 4 seconds we plunge it into the water bath and that's the way there.

Now, what I showed you first was the hamster experiment. We have now advanced to the mouse experiment. And there is one important difference. As we got more and more experienced with this, we were able to get this ramp time down to shorter and shorter periods. We had about 4 seconds on the first one and about 1 second here. We did four or five trials, actually, three in the end we focused on once we got the method working. And then we picked the best of those trials. And what I have shown you is the temperature records for the two best trials for mouse and hamster and that's what got titrated.

Next. Next, please. This is the results of all these experiments. The pooled filtrates gave a very disappointing clearance. I was expecting much higher than that. The respiked column gave this, only about a half log removal, and remember for these types of input titrations, we got about a .3 log error associated with these numbers. The successive filtration and ion-exchange gave about 1.8, which very interestingly, but probably somewhat randomly, is exactly the same as the added values between these two.

But I think it gives us some confidence that putting these things together, even though the removal at each step is low, we are getting some significant removal here of about 1.5 to 2 logs. The UHT sterilization by comparison gave a much better result. Even that 4 second exposure is giving us about 4 logs of removal. Attached to this, we have about 6 logs cumulative. And I think it is legitimate to attach these, because these are quite different methods of removal versus inactivation.

Next. And this is just a comment on that, these things were showing independent removal to the extent that we could detect it with the lower levels that we saw there. But in terms of looking at the total, at what was actually going on there, the serial experiment is actually the best one to use, and that's the one I think we should focus on. But both of these steps were much less effective than I had expected. And I don't know whether it is because of the matrix, the apparatus, the gelatin itself, but in the next slide I'll show you some data.

Next, please. In our experience, this is about half these experiments were done by us. The other half were done by other folks, but they were compiled for a former presentation of the FDA and a TSE Advisory Committee meeting in October 7, 1997. And typically, and we've done more of these since then, especially these depth filtrations, and they are typically removing 3 logs or better.

So there's something different about gelatin. And it's either the way we did the experiment or it could be that the gelatin is so overwhelming in terms of a competitive binder for the matrix that we're not getting removal because of that. But anyway, it doesn't fall into expectation. There is a warning in this though, which says that you have to check these things. You can't extrapolate from this cumulative experience and presume that it's going to work in all cases.

Next. I've already dealt with this. Let's go on. Next, please. I just want to point out that this UHT result is the worst case result. We did it under static conditions. It is heated from the outside instead of the inside. 4 seconds is a minimum exposure that is seen in the industry. And we're using crude brain homogenate instead of material that has been already refined by the process. And my guess is that the stuff that has been through the process may be even more susceptible, but that's a guess.

Next. I want to make one final point and that is that the total exposure that these samples got really begins, at least for sure, with the 263K case, with the 80 degree exposure. I mean, somewhere between 80 and 100 degrees. We have a series of experiments which are actually on the next slide that I did in the '80s showing that we start to see affect around 100 degrees, and we get total killing in a few seconds at 121.

So this ramp temperature is also contributing to the inactivation here. And if we take these ramp temperatures and add it from 100, the ramp exposure plus the exposure temperature for the 263K case and the 301V case and plot them on the same curve, which I'm going to do next.

Next, please. This is just showing you that there is an effect at 100 degrees and above for the 263K case, at least.

Next. These are the ramp times for those former experiments.

Next. Let's go on. Next. I just want to show you this last slide. If you plot the data from the 301V case and the 263K case on the same time axis down here, including these earlier studies out here which were done at 121 versus 140, and plot it back to the origin, you get a straight line through these things. Well, I first draw the line through them. And what that is telling me is two things.

One, there isn't really any significant difference between the sensitivity of these two agents to this process. And two, it gives me some confidence in saying that if you are to extend this process to 10 or 12 minutes, you get another 4 logs or so removal. This is something that should be checked with actual kinetic experiment and kinetic measurements, but it seems to me that this being a minimum is a very -- this 4 seconds being a minimum exposure is a very encouraging feature of this experiment.

Next. In conclusion, the purification steps are removing 4 to 6 logs and the UHT step, in particular, provides a potentially very secure inactivation step. Thank you.

CHAIR PRIOLA: Dr. Bailar?

BOARD MEMBER BAILAR: The next to the last slide you showed the susceptibility to heat over time, and what you had you mentioned the straight line fit, but I didn't see any intermediate points there that could really detect curvature in the line.

DR. ROHWER: No, there isn't. What I'm saying is we're working with the data that I have. And I think I also said at the same time that it would be very nice to do a complete kinetic study on this.

BOARD MEMBER BAILAR: Yes, but I would not conclude from that that it's a straight line.

DR. ROHWER: Oh, I see what you're saying. It may not be. You're right. From here to here, well, from here to here, extrapolation, I don't know. I mean, it's hard. I guess, what would you say? You could have something like that, I guess.

BOARD MEMBER BAILAR: I would say you do not have the evidence on which to detect whether there is any curvature.

DR. ROHWER: Okay. Well, I'll grant you that. And all I'm saying is that this is -- let's put it this way, this data is consistent with a first order process here, with these two samples behaving very, very similarly.

BOARD MEMBER BAILAR: Okay. That's all.

CHAIR PRIOLA: Dr. Petteway?

DR. PETTEWAY: That's a very impressive set of experiments, Bob, especially dealing with the scale down, handling it all. That's an extremely difficult thing to do. But the 4.2 logs, was that the magnitude with some residual infectivity found?

DR. ROHWER: Oh, yes.

DR. PETTEWAY: Okay.

DR. ROHWER: Yes, I mean, we started with 7.5 logs.

DR. PETTEWAY: Yes.

DR. ROHWER: So there's still 3 or 4 logs left.

DR. PETTEWAY: And that was at 4 seconds which is worst case?

DR. ROHWER: Yes.

DR. PETTEWAY: And what you're saying, I mean, even given other points that would show a change in that curve, the likelihood is 8, 10, 12 seconds, there would be nothing left is the point?

DR. ROHWER: I was very interested this morning when Michael Dunn pointed out in his presentation that in North America anyway the typical time is 8 to 16 seconds, as opposed to 4 seconds, and apparently gelatin can tolerate that quite well. If you would like to say something about that? Well, that's up to Sue. Sorry.

CHAIR PRIOLA: Dr. Dunn, do you want to comment on that?

DR. DUNN: Could you say it again?

DR. ROHWER: Yes, if I could repeat that, what I just heard here is that there is apparently no problem extending that time for 8 to 16 seconds.

CHAIR PRIOLA: And Dr. Hogan?

BOARD MEMBER HOGAN: Well, the question is why does the European process use 4 seconds and is there a ramp up time to that or is it just the batch is brought in, zap 4 seconds and then it is taken out?

DR. ROHWER: I would like to defer to Mr. Schrieber, if I could.

CHAIR PRIOLA: Yes, Mr. Schrieber?

MR. SCHRIEBER: What I explained in my presentation already is that we used the softest condition we have found in one of the gelatin plants in Europe. So it's not uncommon to have like in the States a longer temperature or even a somewhat higher, longer time or even somewhat higher temperature, but we had to choose the minimum conditions founded in the three or four studies, and that's what it is. You are right if the time would be expanded to 6 seconds or the temperature would go up to 140 instead of 138, this would not really harm the quality of the gelatin.

CHAIR PRIOLA: All right. If there are no other questions, thank you very much, Bob.

(Applause)

CHAIR PRIOLA: I would just like to say having gone through the bulk of this, these infectivity studies in our rather thick handout, that it is very impressive the work that Drs. Taylor, Somerville, Rohwer, Ad Grobben and Schrieber have done studying inactivation of TSE infectivity through the gelatin processes. It's a lot of real nice work.

I would like now to ask Dr. Morris to come up and explain to us the USDA's gelatin policy.

DR. MORRIS: Okay. Good morning and thank you for the opportunity to speak with your Committee regarding APHIS's policies regarding the importation of gelatin.

COURT REPORTER: Dr. Morris, hit the volume button.

DR. MORRIS: Thank you.

CHAIR PRIOLA: I'm sorry. My apologies. It was supposed to be Dr. Rogers. I'm very sorry. That's my error. Can we just go with that?

DR. ROGERS: Yes.

CHAIR PRIOLA: Okay. My apologies. I'm sorry. You should have told me. I'm misaligned in the agenda. Okay. So, in fact, we're not going to hear from Dr. Morris yet. It's Dr. Rogers who is going to give us a risk analysis of infectivity.

DR. ROGERS: Well, I guess the slide has disappeared for a minute there, so don't start the thing until -- the timer until it shows up. Is the mike on? Okay. Thanks for inviting us down here today from Canada. I'm from Health Canada.

CHAIR PRIOLA: We can't hear.

DR. ROGERS: So is the mike on? Oh, it's on now? Oh, closer. Taller and closer. How's that? Okay. At Health Canada we have been doing a number of quantitative risk assessments and part of our topic today that we will be covering is what's on your agenda. But I did want to say that what we're really looking at is the varying-CJD risk to consumers eating foods containing small amounts of processed ruminant products. And I want to talk about some of our modeling functions.

Next slide, please. We have just completed a quantitative risk assessment for basically products that contain beef extracts and the beef extract industry certainly has a lot of similarities to the gelatin industry, so some of the information I'm going to provide today will certainly with some understanding of the overall picture. I do want to present today like the quantitative model parameters for the evaluation on pairing CJD risks. I want to focus on the front end parameters for risk analysis, and I want to provide some information on evaluating uncertainty in the parameters and provide information on variability that we're using in our models.

Next slide, please. The purpose of our risk assessments are really to provide information on two risk outcomes of the probability of individuals acquiring varying-CJD through the consumption of a product and the annual number of varying-CJD infections that could be predicted.

Next slide, please. The approach that we're using, basically, the first thing that we look at is the length from BSE agent to varying-CJD. To date, there is no direct evidence linking the acquiring of varying-CJD to particular products. And I want to emphasize that certainly the only information we had previously was the work of Simon Cozens of the UK for food products which had some implicated meat pies, sausages, these types of things in his work, but he has, in fact, recalculated his and reevaluated some of that publications and in Edinburgh last year he has, in fact, shown there has been no statistical significance to particular food products and varying-CJD. So that's an important picture.

The presence of the BSE agent in the product of concern are not measurable by our current techniques. The only thing that we can actually still seem to have some type of laboratory analysis for is the presence of CNS materials through IHCGFP and some neuro analyst techniques. The hazard identification basically has established that there is a route from BSE to consumption exists. And so that's the reason for the presentation of the modeling.

Next, please. In Canada, we are using the model that has basically been setup by the Kodak element. We have an issued statement. We do hazard identification, hazard characterization, exposure assessment and risk characterization, but nothing goes forward until you have hazard identification.

Next slide, please. Our structure in our risk characterization is depicted here. Basically, we are looking for these probability statements in the middle, which are outcomes, but we are looking at the infectivity consumed, which really comes through our exposure assessments and the consumption frequencies from the exposure assessment, and then the dose response models that we have been developing, which are in the hazard characterization.

Next slide, please. Our structure in hazard characterization, basically, the main things, variables that we would be looking at are the susceptibility in human population. We can say that certainly the we in our risk assessment are looking at worst case assumptions. In fact, with the human population, we are not looking at divergence, for instance, because of met type of codons, we say that all humans are susceptible. We're not looking at immuno-compromised or younger children. There's no infant instances of that, so like our population characteristics say that all populations are susceptible.

Infectivity accumulation is one of the things we are looking at particularly with dose response type of modeling. Our species barrier from bovines to humans, I guess, what I would say there again is that we are looking at the worst case. We're saying that there is no species barriers. It's a 1 to 1 ratio, but certainly when we're looking at the advice that we get from the Scientific Steering Committee over in Europe that they say that we should use the range of 1 to 10,000.

And, in fact, the latest publications do say for oral transmission to food products, you probably should be looking at 10,000. And we are looking at risk assessments to be tried and say, for instance, they do want things to be practical and realistic, but then we are going with due caution. And so some of these products that we're looking at to start with, we are emphasizing generally worst case assumptions and seeing what those numbers generated look like, and so that's what we have been doing.

Next slide, please. In particular, it's the dose response area for varying-CJD. And in our models we are using a threshold dose response as well as an accumulation dose response. I have a lot of slides here, so I'll have to hurry along with this. We'll talk about that a little further along.

Next slide, please. The structure and our exposure assessment just basically analyze the model that we use.

Next slide, please. And particularly, which is of interest to this particular audience is the fact that we are looking for the presence of BSE in cattle populations. Our models are setup in such a way that we do know that the disease status of a country changes and I think we have that from our own experience, but we have them working along the lines, for instance, that food products and gelatin products are produced over periods of time, so the BSE status and the amount of BSE possibly infected cattle in the country change, and so we really want to be able to adapt that to the different lots and processes.

The tissue infectivity information that we use, basically, a lot of it from the oral pathogenesis studies to start with from Dr. Wells group and that continues on in the UK. Our source of infectivity in the slaughtering areas, our sources of infectivity certainly depend on the tissues that are used in the products, and I think you've been discussing a lot of those today with specified risk materials, for instance.

And this area here I put the word gelatin for the commercial product of what we're looking at. In fact, this model was developed particularly for beef extracts, but it is the front end that I wanted to talk about today. Because what we've been doing as well as we do look at consumption, the actual final products, the amount of material that are in final products, and then the amount that go to consumers and consumer individual servings.

Next slide, please. So our quantitative model prevalence of BSE into -- the BSE infected bovines in populations in the screening procedures. The inference from the countries of BSE surveillance, first of all, I want to say one thing and that is that the products we were looking at were generally ones that were coming from the European Union or could have been coming from other places. But when we were looking at prevalence data itself, because of the wide enhanced surveillance targets that have been going on in the EU that we particular have some good observational data there to work backwards from.

And I must say that certainly the EU has also been doing a lot of missions out to their member states to go for audit and compliance, and they have been doing a number of good reports on that. That's why we get some excellent data to sort of give some parameters around to put in models. However, I'll tell you that particularly our concerns are detected diagnosed cases are removed from -- are diverted from food chains.

But the incubating cattle are a question in the amount of infectivity in incubating cattle, certainly one of the major things that we have been struggling with. But in our particular assessments, we're using 4 incubating cattle per adult cattle diagnosed and we've done that, basically, from talking to experts in Europe. And the other thing that we're doing, though, is that we're talking about the infectivity of tissues.

We started off with giving them exactly the same infectivity as the clinical animals to run through the worst case numbers, and then we scaled that backwards. But I'll explain a little bit later. But what we've done is basically we try to group countries into low, medium and high prevalence rates and so then I do have numbers on that, but again this presentation is going to be a little small for that, but we'll get to that.

As far as the abattoir screening itself it's concerned that certainly now there are rapid tests involved, and we've seen that they were talking about 100 percent sensitivity, 100 percent specificity. There's a number of rapid tests out there. We have done an evaluation of them and we've used the worst case sensitivity for one particular rapid test, because we cannot tell because there are varied tests that have been used in similar countries and so you don't necessarily have all that information.

But for modeling purposes, again, we're using the worst case. And for the ante-mortem, postmortem inspections only for diverting BSE infected cattle from the food chain, we're using a 2.5 percent removal from ante-mortem. And I think most people know how very difficult it is to diagnose TSE diseases and that they are very complicated.

However, they have gone back and it is a requirement in the European Union to, in fact, state where you are during your diagnoses, are they ante-mortem or are they rapid test. And so that the range of variability in ante-mortem depends on the country and in the awareness, the education and in a lot of infrastructure elements. However, I can tell you that for what we've done, I've only seen six reports so far from mission compliance audit states and the lowest amount of divergence is 3 percent out of Belgium from ante-mortem and up to in the high 30 percents in Germany. So there's a lot of variability in the amount of BSE infected animals that are removed postmortem. And this goes into the models as well.

Next, please. Next, please. Oh, sorry, I couldn't see it right. Okay. So now, I'm looking at tissue infectivity. I just want to give a brief run down here that we are using .1 gram of raw unprocessed brain tissue from a clinically infected bovine as the minimal or as the threshold dose in our models, at this time. I think that most of you are aware that that is the amount of unprocessed tissue now that has been orally given to a cow that has come down with BSE in the UK in the latest pathogenesis studies. That animal was 52 months. Again, like a very low dose, but it is our starting point.

However, we do put uncertainty around these things, again, up to, per program, 10¹ to 10³ infectious doses. And then the infectivity that we are assumingly using the same infectivity scaling standardization to the trigeminal root ganglia, the dorsal root ganglia, the spinal cord and emboli could possibly go into this slaughtering and stunning procedures. And I have mentioned already we are looking at the incubating bovine, and particularly the sensitivity issue around that, and so we have a scale at different levels in our final results.

Next, please. Our sources of infectivity, particularly, when we are looking at raw materials, it could be going into things like gelatin. Our CNS emboli in the blood, possibly spinal column cross-contaminations, blood itself, edible fat contaminations, bone marrow, spinal column, and trigeminal ganglia.

Next, please. And, in fact, this is the way that we have started in our beef extract risk assessment that we sort of look at in terms of tissue restrictions and no tissue restrictions, and particularly, although I guess this was the top line here that would be very much parallel to what could, in fact, be going on in the gelatin industry, because in beef extract, we do have some productions that only use muscles only.

Next, please. One of the things that we see are really the number of bovines that are, in fact, going into batches and lot production and silo storage in beef extract and this is very similar to some of the things you are seeing in the gelatin production. So that we do have, in fact, calculated the probability of a batch contamination, lot contaminations by the prevalence rates and by -- well, it's a little complicated here for me just to go over that quickly, but it's those calculations that we were looking for to say that there's a probability that the consumer product is made from a contaminated product at that end.

And so we are looking at the number of infected bovine tissues that go into the batch or lots based on those country prevalence ratios as well. We also are looking at the infectivity reductions, because in beef extract production, as well, you get a lot of heating, wet heat, filtering, decanting and denaturation and, in fact, we have tried to mix estimations on the log reductions there.

Next, please. In terms of defining our concerns and characteristics for these products, and I suppose this is one of the difficulties that we do have with food products that contain small amounts of a ruminant product ingredient, not always on the labels and not always necessarily going to the ingredients. In the beef extract production business, for instance, there is no GME type organization.

We, in fact, had to go to every country, major country, around the world that does have beef extract production and do our individual investigations by companies to find out the capacity of their equipment, the number of animals, for instance, that -- first of all, one animal contributes so much tissue per lot, and so there is a range of animals that go into lots or batches. And so the probabilities are all derived from that type of information.

And so like that's something I'm -- actually, I skipped over that a little bit, but it's very important for this type of estimation of, for instance, that even if you did have a BSE infected cow going to gelatin production, for instance, you have to know the capacities of the equipment and the type of equipment and the different processes and certainly that the gelatin industry have indicated that there basically are very similar processes, a little different in the other areas.

I see I'm at stop time already, but I just need to go to the next slide, please. Basically, these were the variabilities and the components that we've been looking at, and I'm probably way over time here. But can I just kind of continue on just real quick? Okay? Because certainly like our particular interests is really in the production methods themselves, the production practices, the sources of infectivity, all of these that we've quite clearly documented in our written reports, which we would be glad to share with this Committee at a later time.

And the consumer product characteristics themselves, because there is differences amongst the groups of products and within the groups of products. And so we have gone through actual analysis of the amounts of materials that go in there, and then the consumption characteristics themselves, because each product has a different consumption characteristic and so we've tried to work that through with a point estimate of the maximum values.

Next, please. And the uncertainty issues that we really weren't looking at in our reports and reporting them as sensitivity, you really have to do a tissue infectivity incubating bovines or species bearing the dose response.

Next, please. And if you want to -- next, please. Because this is basically the charts that we prepare, and we are providing like product groups within our report.

Next, please. The BSE prevalence is basically put into our charts.

Next. Our abattoir screening techniques.

Next. For divergence of BSE animals.

Next. Other production methods that we have been going through with all information we collect and we can provide that as a range.

Next. And then we've done production parameters which are a range of ranges depending on the type of processing, etcetera, and the types of tissues that are added.

Next, please. And I think I'll skip this one right now. Next, please. Next. And this basically is just giving us some information on if you're using rapid test and ante-mortem tests.

Next, please. Because it was this type of scatter diagram that we're trying to provide to sort of show or give the information on the probability of the consumer batches themselves. If you've got large batches, small batches, high prevalence, low prevalence, and so we tried to do some diagrammatic information in our reports to give some idea of the dilution of tissues with no infectivity or with infectivity.

Next, please. And the difficulties I have talked to, and next, please. Bottom line is that these are the individual outcomes that we have been trying to quantify in our risk assessments, and particularly, though that we -- you will find that you can have a lot of problems with surveys, nutrition surveys, etcetera, for the types of details that you would be looking for for trying to get estimates on consumption values.

I'm sorry to have gone over.

CHAIR PRIOLA: Okay. Well, thank you. Are there any questions for Dr. Rogers? Yes?

BOARD MEMBER WOLFE: You mentioned earlier on that your assumption of the ratio of incubating cows to infectiveness is 4 to 1. What is the basis for that?

DR. ROGERS: Excuse me, well, that's basically expert opinion from Europe, because like we had talked to people that had the experience with BSE for a number of years, and so that that is just strictly an expert opinion. There's no rationale for that except there is a range of incubating cattle that we do use, but I can tell you the reason why we're using 4, at this time, is because we have implied such harsh assumptions to the fact that there is, it has the same amount of infectivity as the clinical animal.

BOARD MEMBER WOLFE: Can you tell me what the range is that you said that you use it for? What is the range or ratio?

DR. ROGERS: 4 to 10.

BOARD MEMBER WOLFE: 4 to 10. Thank you.

CHAIR PRIOLA: Okay. Thank you again, Dr. Rogers. After carefully checking my agenda, now, we're going to hear from Dr. Morris from the USDA. I apologize again for putting you on the spot earlier.

DR. MORRIS: Good morning. Thank you again for the opportunity to share our Agency's policy on gelatin. Next slide, please.

I am Dr. Terry Morris with the National Center for Import/Export. I am representing the United States Department of Agriculture, Animal Implant Health Inspection Service, Veterinary Services.

Next slide, please. We are headquartered out of Riverdale, Maryland.

Next slide. And we are under the direction of Dr. Karen James-Preston.

Next slide, please. Title 9, Code of Federal Regulations, Part 94, 95, 121 and 122 gives APHIS the authority to regulate animal products. Part 94 gives us the authority to regulate gelatin.

Next slide, please. We regulate gelatin based on the presence or absence of BSE and the association with the BSE affected region, BSE being Bovine Spongiform Encephalopathy. We have lumped gelatin into one of three categories. One category would be gelatin that is derived from non-ruminant species. A second category would include ruminant gelatin that is derived from cattle that have no association with a BSE affected region. And the third category is ruminant gelatin that has been derived and has an association with a BSE affected region. For the gelatin that has an association with a BSE affected region, those regulations are found in Part 94, Section 18(c).

Next slide, please. And pretty much to summarize, 94.18(c), the gelatin that is derived from ruminants and the ruminants are from a BSE affected region, that gelatin is prohibited entry, unless the gelatin is imported for human food purposes, pharmaceutical products, photography or any other similar uses that would not result in the gelatin coming in contact with ruminants in the United States.

Next slide, please. 94.19 addresses gelatin derived from non-ruminant species. This would include your pig, horse, poultry and fish gelatin. On May 27, 2003, an interim rule was signed that modified the current verbiage in 94.19.

Currently, next slide, please, the gelatin that is imported that is derived from pigs, horses, birds and fish species must be accompanied by an original official certificate endorsed by the full-time salaried veterinarian responsible for animal health of the exporting country, and it must state the animal species of origin.

Next slide, please. 94.19 also deals with gelatin derived from ruminants, provided those ruminants have not been in a BSE affected region.

Next slide, please. This part of the regulation requires that each shipment should be accompanied by an official original certificate endorsed by the full-time salaried veterinarian responsible for animal health of the exporting government, and that certificate must state four things. The first thing it must state the animal species from which the material is derived. The second statement must include the region in which the facility where the material was processed is located. The third statement would include a statement that the material was derived only from ruminants that have never been in a BSE affected region. And the fourth statement must address dedicated facility conditions, meaning the facility cannot receive, store or process any ruminant material from any BSE affected region.

Next slide, please. The last category deals with ruminant gelatin that has been associated with a BSE affected region.

Next slide, please. Ruminant gelatin that has been associated with a BSE affected region must be accompanied by a veterinarian import permit. A permit is a legal document that authorizes the importation of controlled materials or organisms or vectors into the United States. For ruminant gelatin associated with a BSE affected region, the permit would address the country of origin. It would address the animal tissue species, meaning hide or bone. It would address the exporting and the processing country of origin. Again, we're looking at BSE-free versus BSE affected.

Next slide, please. The next few slides depict scenarios that address how APHIS would regulate the importation of ruminant material under certain circumstances. In this scenario, the ruminant material whether it be hide or bone is derived of ruminants from a BSE-free country, but it is processed and exported in a BSE affected country. In this case, we would issue a permit for this material.

The permit would require that the government certify the country of origin of the raw animal materials and the government would also have to certify specific conditions that exist within that facility and the BSE affected region. Again, that facility would have to be a dedicated facility, meaning it cannot store, receive or process any ruminant material from any BSE affected region, with the exception of milk and hides.

Next slide, please. In the second scenario, we address high derived gelatin only, sourced from ruminants. In this case, whether the hide is derived from ruminants from a BSE-free or a BSE affected region, the fact that it is processed in a BSE affected region requires the need for the permit. The permit, when issued, would require that the government certify that the gelatin is hide derived only, and again because the facility is in a BSE affected region, the facility would have to be dedicated.

Next slide, please. The last scenario addresses bone derived gelatin. For bone derived gelatin, and in this case, the ruminants are from a BSE affected region. This material is allowed entry, provided the individual obtains a permit. And when we issue the permit, the permit would require that the individual maintain affidavits that they obtained from individuals who they distribute this gelatin to. The affidavits would require that the individual certifies that the material will not be used as livestock feed ingredient.

The material cannot be incorporated into veterinary pharmaceutical uses or the material cannot be incorporated into veterinary biologic products. And this goes back to 94.18(c), which says that the material can be imported, provided it is imported for human food, pharmaceutical products and other uses, photography, and other uses that does not result in the material being introduced to U.S. ruminants.

Next slide, please. To complete the process for obtaining an import permit, you have to submit an application, which is VS form 16-3. It takes about 2 to 3 weeks between the time that we can process the application and turn around a permit to you. The application can be submitted electronically through our website. It can be submitted by fax or my mailing it into our office. The permit is good for one year, and the permit will only allow for the specific commodity requested from the specific exporters, and it would have to go to the importer that requested the permit.

Next slide, please. This is my contact information, in the event that you need to contact our office.

Next slide, please. And again, I wanted to thank the Committee for the opportunity to share APHIS policies regarding the importation of gelatin. I'm happy to answer any questions you may have.

CHAIR PRIOLA: Dr. Wolfe?

BOARD MEMBER WOLFE: This is not meant to put you on the spot, but as you know, the Department of Agriculture is seriously considering, we have heard from others, on the verge of, lifting the ban on importation of cattle, beef, from Canada to this country. You've outlined a thoughtful and, I think, careful permit process that affects gelatin, for instance, which would come from a BSE affected region, such as Canada.

Do you not think that there is somewhat of a contradiction between being so tight properly and restrictive about allowing gelatin to come in from there, but seriously considering lifting the ban on meat from what would be the first time the United States would ever have lifted a ban that previously existed from a BSE affected country?

DR. MORRIS: Yes, sir, the APHIS TSE working group has devised a list of low-risk commodities and a list of mitigation factors under which those low-risk commodities can be imported, the specific criteria under which we would consider accepting these low-risk commodities. That list has been presented through channels to the White House and it is our understanding that the White House has disseminated that list to the trading partners, so it is in negotiation to make sure that all of our trading partners are aware of what the potential actions would be.

BOARD MEMBER WOLFE: So you're saying that beef is presumably on a list as a low-risk commodity? Is that what I interpreter you're saying?

DR. MORRIS: And I would have to look at the list, but it's specific categories and it's specific ages. And, Lisa, if you want to help me out here? Thank you.

BOARD MEMBER FERGUSON: Yes, I'll try and help you out. Speaking for the Department, first of all, I would like to reiterate the point that you probably shouldn't necessarily believe all the rumors that are in the press and everything that you hear. There are lots of things under consideration, not only at USDA, but through the entire Administration, so all of the departments are contributing to these discussions.

And the discussions are centered around, you know, is there a science-based way to look at the situation? Are there things that we can do, that are based on known science to address the situation with Canada? And as Terry has described, at least, we, at APHIS, have provided some recommendations for certain products that perhaps could be considered low-risk and could initially be allowed for import under certain conditions.

I'm not at liberty necessarily to say what is on that list, what might not be on that list, but we have tried to address it. Okay. First of all, products that are accepted internationally not to present a risk of transmission, obviously, are not affected already. But we are looking at a range of things to say these could be considered lower risk than other things. And really it's a wide ranging list that goes over a lot of issues.

BOARD MEMBER WOLFE: Thank you.

DR. MORRIS: Thank you.

CHAIR PRIOLA: Okay. Thank you very much, Dr. Morris. We'll now move on to the open public hearing portion of the morning. So, Dr. Freas?

SECRETARY FREAS: As part of the Advisory Committee program, we hold open public hearings, so that members of the public may wish to make comments to the Advisory Committee will have the opportunity to do so. At this time, I have received two requests. One is a written request. This written request was run off for the Committee members, posted in the viewing notebook out on the table and some copies were provided for the public if you were here early.

The second request is from Mr. David Bieging and he is at the microphone right now. Welcome.

MR. DWYER: Actually, I'm Dan Dwyer. Dave Bieging made the request and I'm going to speak. I'm Dan Dwyer. I represent the Gelatin Manufacturers of Europe, and I'm also speaking today on behalf of the Gelatin Manufacturers Institute of America. You've already heard from representative of these two associations this morning. These associations represent virtually all of the gelatin produced in Europe and in the United States.

These two associations have been working for many years, as you know, to ensure that gelatin is safe and we've been pleased to be able to do so in cooperation with the FDA. As we've discussed with FDA previously, we would like, at this time, to comment on the questions that FDA has asked this Committee to address today.

Specifically, FDA's Question 1 currently reads "Do the results of these new studies demonstrate a reduction in infectivity that is sufficient to protect human health?" This question must be interpreted in light of the normal circumstances surrounding gelatin production. In particular, the question focuses only on the manufacturing processes that were studied, but in practice, as you've heard today, the safety of gelatin is based on two principles.

The first principle is the use of raw materials. As you know, in Europe this involves controls on raw materials imposed by the European Union and by GME members. The second principle is the use of manufacturing processes that can eliminate any potential infectivity that might theoretically be present in the raw materials. In Europe, this involves the use of the processes that you've already heard discussed today and that have been studied by GME.

These two principles of gelatin safety apply as well to all bone gelatin regardless of geographic origin. Therefore, we request that when the Committee considers FDA's Question 1 it take these two principles into consideration, that is we would recommend that the question be revised to read "Based on the use of raw material sources and gelatin manufacturing processes, as described in the information presented to the Committee today, do the results of these new studies demonstrate a reduction in infectivity that is sufficient to protect human health?"

FDA's Question 2 addresses the Agency's guidance on gelatin. As you have heard already today from Dr. Potter, in 1997, FDA issued a guidance document that established certain parameters for the sourcing and processing of gelatin in order to avoid BSE risk. At that time, the effect of the gelatin manufacturing process on in infectivity had not been proven. The data discussed with the Committee today, however, in our view, provides a basis for concluding that FDA's guidance is no longer necessary.

Indeed, as Dr. Chiu mentioned to you earlier, you may decide that gelatin should be exempt that gelatin should be exempt from any FDA restrictions. At a minimum, we believe that the guidance should be modified so as to improve the opportunity for European raw materials to be brought into compliance with the guidance while at the same time maintaining appropriate controls on the use of European raw materials and, as Mr. Masson expressed before, ensuring a continued adequate supply of gelatin for pharmaceutical use.

If the Committee takes the approach of modifying the guidance in this way, we request that the Committee consider two potential modifications to the guidance. The current text of the guidance has been provided already to you by FDA and, indeed, our recommended changes to the text have also already been provided to you for your consideration.

First, FDA's guidance currently requires that "cattle come from BSE-free herds." As a practical matter, the term BSE-free herd refers to a herd in which there has not been a single animal identified with BSE. In Europe, it is mandatory, as you've heard, that animals over 30 months of age be tested for BSE, whereas animals under that age are normally not tested, because they have not been defined, at this time, to pose a risk to human health.

Thus, in practice, a BSE-free herd is a herd in which BSE has not been detected in tested animals. FDA's guidance in this regard would be clearer if it were to include a brief explanation of the term BSE-free herd by stating "BSE-free herd as determined by generally accepted testing procedures."

The second modification to the guidance that we would ask the Committee to consider is one that this Committee has considered before. FDA's guidance currently requires that heads, spines and spinal cords be removed from gelatin raw materials "directly after slaughter." In 1998, this Committee recommended that the removal of spines may be done at any time during the deboning process. Indeed, the removal of heads and spinal cords is not an issue as you heard, because they are already removed before or at the time of slaughter.

Therefore, it continues to be appropriate for FDA's guidance to be modified to permit the removal of spines at any time during the deboning process. As the Committee considers FDA's Question 2B then, we request that these proposed modifications to the guidance be taken into consideration. A copy of our recommended changes to the guidance has been distributed to you already for your consideration, and it also has been made available to the public.

Thank you very much. We appreciate the opportunity to appear before you today.

SECRETARY FREAS: Thank you for your comments. Is there anyone else in the audience who would like to address the Committee, at this time? If not, Dr. Priola, I would like to state that we all have three more open public hearings throughout this meeting, and we do encourage the public participation. Thank you.

CHAIR PRIOLA: Okay. So the questions put to us by the FDA are now open for discussion and voting. Do we have the questions to put up? So the first question, while they're getting it up there, is simply, well, hopefully simply, do the results of these new studies demonstrate a reduction in infectivity that is sufficient to protect human health? Are there any comments or any discussion from the Committee? Yes, Dr. Hogan?

BOARD MEMBER HOGAN: Since nobody else is biting, let me take this opportunity to say that when I reviewed your article and when I started reading this voluminous amount of material, I sort of looked with the same sort of skeptical eye that I do when I accept papers for publication, and I initially had, when I started reading, several questions about processing and scale-down issues and residual infectivity, etcetera. But as I got deeper and deeper in this, we concur that those had been addressed.

So the initial questions that I have asked today, I am extremely personally pleased with the results of these studies. And while no study can be absolutely perfect, and I think that all the questions that the original Committee in 1997 had regarding the data, in my mind, have been answered.

CHAIR PRIOLA: Dr. Bailar?

BOARD MEMBER BAILAR: I agree that these are very important experiments. They were very well done. I read the reports also as somebody who has done a lot of reviewing. I do have one remaining question or set of questions. I'm not sure that we know enough about the time course of deactivation and why some of the infective agents seem to be so resistent.

CHAIR PRIOLA: Well, maybe Dr. Rohwer would like to address that more specifically, but, well, would you, Bob, would you like to, since this is your day. I don't want to speak for you.

DR. ROHWER: You're asking a fundamental question of TSE science, actually. And it's something that is going to get a lot of discussion this afternoon. And so I don't know, I mean, I have another talk that I'll be giving and it goes directly to that question, and Robert Somerville has given his perspective on it, and we're going to hear from David Taylor as well. And I think, is there anybody else? I honestly can't remember. Well, and David Asher has some new data on this area as well.

And you, yourself, put your finger, I think, on the central issues in your first question to the panel, I mean, to the speakers earlier this morning about the biphasic nature of these inactivations and what is behind them. And we don't know for sure. My own bias is quite different from Robert's. I mean, I don't think there is any intrinsic difference between these agents. What we're talking about is sanctuaries and an inability to actually reach all of the agent. But there are other interpretations.

You pointed at one which is a genetic one and there are different ways that you can look at these kinds of protections. We don't have the answers to that. And I think it is a residual question that haunts every single validation study, inactivation study that is done, is to know just how far you can extrapolate this data to zero.

I would like to point out that this is not a new issue. It's something that has bedeviled the vaccine industry, water purification, virtually any area in which you want to assure that something is sterile, but you have no way of measuring the entire production lot to find out whether it is or not. And we're kind of in the same boat here.

BOARD MEMBER BAILAR: Yes, but I'm not concerned about extrapolating down past the last data point. You have data showing that the curve flattens out, at least, to a considerable extent.

DR. ROHWER: Yes, and the point that I'll be making this afternoon is that the place where that flattening out occurs is very context dependent. And you can force it down or up depending on what kind of mixture you are inactivating, what the conditions are and that type of thing. And so the one thing I can say about these studies is that the knowledge that that occurs was part of the design of the study.

And your other question about intrinsic versus extrinsic infectivity, the idea that you have to introduce the spike into this spine preparation at the beginning, and you can't know for sure whether you have really mimicked the invito situation in which you would find the infectivity in a BSE infected cow is a very appropriate one. However, in this particular case, I feel more comfortable with it than practically any other study like this that I have done, because it is the spinal cord and the ganglia that we feel are the threat. They are extrinsic to the bone.

And what was done here is the stuff was actually injected into the spinal cord and smeared on the bone, actually given an opportunity to dry on the bone, which is something that probably actually happens, and is something that is very, in my opinion, probably very dangerous to do with TSE infectivity. And so the original for the total process experiment, which by the way I wasn't part of the experiment, but nevertheless, my perspective on that is that that was probably just about as good a spike as you could devise. And I mean, I can't think of anything better.

You could always argue with the downstream position of spiking homogenate into these things, but even there I think we're talking about a worst case spike in the sense that the homogenate is completely unrefined. And having taken this through the process, you're liable to have stripped away some of the fats and things like that that may be protective to these agents in pure brain type associations. But that's speculation on my part. I can't satisfy your basic underlying concern there, because we don't have data on that point.

BOARD MEMBER BAILAR: Well, I remain a little bit concerned, because I recall reading basically in the Daily Press that in the usual method of slaughter, bits of CNS material do get into the peripheral tissues. Is that correct?

DR. ROHWER: With penetrating concussive slaughter, I think, it is without a question that that happens. And that's -- I don't want to comment on that. There are people here from the USDA who can probably tell us just whether that practice still occurs there or not. I'm not sure.

BOARD MEMBER FERGUSON: I'll answer that. Actually, the issue is with air injected stunning, where you've got a captive bolt and then you've got holes drilled at the end of it, and you inject a bolt, a blast of air, and that type of nomadic air injected stunning is not used in the U.S. industry any more. Our colleagues at the Food Safety Inspection Service are actually in the process of promulgating regulations that officially prohibit that, but based on our understanding of slaughter practices, it is not used in the U.S. Is it used elsewhere?

BOARD MEMBER WOLFE: Well, like in the countries where we're talking about, the European countries?

BOARD MEMBER FERGUSON: In Europe it also prohibited by regulation within Europe.

BOARD MEMBER WOLFE: Within all of Europe?

BOARD MEMBER FERGUSON: Yes, yes. Actually, well, within the EU. EU regulations prohibit it. So within the community, I think, you can probably also then assume that any of those countries that are exceeding to the community, the same thing applies.

CHAIR PRIOLA: Dr. Somerville, I think, also wanted to address part of your question, Dr. Bailar. Thanks, Bob.

DR. SOMERVILLE: Can I just -- is this on? Okay. Just to add to what Bob was saying and to emphasize what I said at the beginning of my talk, was that in processes that were considering its denaturation reaction which is, I suggest, leading to the stabilization of the aging, past the drying processes that Barbara has just mentioned. There are other processes involved in the gelatin extraction procedure which may assist in its destruction or removal.

I suggest that possibly there is a degree of hydrolysis of infectivity which would not necessarily depend on the stability of the agent in terms of its denaturation properties, and also, of course, the filtration properties described are of importance in removing, in the totality of the removal of infectivity from the process.

CHAIR PRIOLA: Yes, I think it is also worth remembering that having sat through many of these Committee meetings and always asking for data, I now have before me 2 inches of data, all of which point to the same thing. That in the worst case scenario you can still inactivate these huge doses of infectivity. And then in the real world we're talking about starting material that doesn't even have, at least from the European point of view, as we've heard, since they are now removing the vertebrae, it doesn't even have that material there to start.

So whatever contamination may be present is going to be significantly lower than anything that has been discussed here today. So at every step of the process, precautions are being taken that should also be taken into consideration when you're thinking about these things about total inactivation and sequestering evasion.

Are we ready to vote, dare I ask? Does anyone else have anything they would like to say now? Shall we call for a vote then?

SECRETARY FREAS: There are currently nine voting members at the table. I will go around the table starting with Dr. Johnson over there. Dr. Johnson, how would you like to vote?

BOARD MEMBER JOHNSON: I vote yes.

SECRETARY FREAS: Dr. Bracey?

BOARD MEMBER BRACEY: I vote yes.

SECRETARY FREAS: Dr. Ferguson?

BOARD MEMBER FERGUSON: Yes.

SECRETARY FREAS: Dr. Hogan?

BOARD MEMBER HOGAN: Yes.

SECRETARY FREAS: Dr. Khabbaz?

BOARD MEMBER KHABBAZ: Yes.

SECRETARY FREAS: Dr. Priola?

CHAIR PRIOLA: Yes.

SECRETARY FREAS: Ms. Walker?

MS. WALKER: Yes.

SECRETARY FREAS: Dr. Wolfe?

BOARD MEMBER WOLFE: Abstain.

SECRETARY FREAS: Dr. Bailar?

BOARD MEMBER BAILAR: No.

SECRETARY FREAS: The tally is 7 yes votes, 1 abstain vote, and 1 no vote.

CHAIR PRIOLA: Okay. So we can move on to Part A of the second question, which is due to scientific data and information available support the following current FDA recommendation on bone gelatin. And we can keep in mind that we can modify as the FDA has said we can modify this question if we think it is necessary for this recommendation. So that's open for discussion. Dr. Bailar?

BOARD MEMBER BAILAR: Before we vote on this, could we have somebody from FDA say whether the modifications suggested are acceptable?

CHAIR PRIOLA: I'm sorry, the modifications suggested by the gelatin manufacturers?

BOARD MEMBER BAILAR: Yes.

CHAIR PRIOLA: Yes. Would someone from FDA, yes, Dr. Chiu.

DR. CHIU: I would put the question back to the Committee. If the Committee think, you know, the modification suggested by industry is acceptable, then we will take that recommendation back to the Agency and then have internal discussion.

CHAIR PRIOLA: Comments from the Committee? I would like to read through the gelatin manufacturers recommendations. Is there any overwriting reason that anyone can see here to alter what the FDA already has down, which seems to cover what it should in terms of removing risk materials? Dr. Hogan?

BOARD MEMBER HOGAN: No, I don't think it should ever go under non-exempt. I think that this is good. The question is from the industry, why is it important to -- when you say BSE-free herds, that covers it. I guess what you're not allowed to use then are herds which contain animals that are younger than 30 months, and you would like to be able to do that. Is that the sense of why you want the modification? Since animals that are less than 30 months are already assumed to be BSE-free.

CHAIR PRIOLA: Dr. Schrieber, Mr. Schrieber?

MR. SCHRIEBER: This request for modification is based of an opinion expressed by the USDA. USDA has stated to FDA we do not consider any herd in Europe being BSE-free. So this means the current text, the way this is written, would exclude altogether all European bones to be used for gelatin manufacturing and then exported into the U.S. So therefore we need the clarifications that under certain circumstances, and that's what we have described, that the animals are tested according to current procedures in Europe, that this would be, let's call it, equivalent to the BSE-free herds. So that's one point.

And the other request for the modification is what I said before. Due to the transport of the carcasses from a slaughter house to a meat packer to sausage companies, with bone in, if the request will stay, removal of spine, I'm not talking about spinal cord, this is directly removed after slaughter. But removal of spine has to be taking place directly after slaughter, this would as well totally exclude the use of European bones, because this is not the standard procedure.

So we need some time frame during the further processing, because deboning is done somewhere else and transport of carcasses without the bones is not possible. This is the ratio behind our request.

BOARD MEMBER HOGAN: Well, then I would ask Lisa, is that true the USDA considers no herds in Europe BSE-free, despite testing?

BOARD MEMBER FERGUSON: Well, I think what we're dealing here with is the way our regulations are written. And our regs prohibit the entry of ruminant from any country that is on the BSE restricted list. So, you know, since our regs are clearly prohibiting all these animals, we can't necessarily make an exemption and say yes, okay, something is free, something is not free.

CHAIR PRIOLA: I'm sorry. Dick, go ahead.

BOARD MEMBER JOHNSON: Yes. If this were modified by this Committee, that would not affect the FDA regulations, and then you would have two conflicting rules, right? Is that right?

CHAIR PRIOLA: Well, you're not necessarily going to have two conflicting rules. You know, the way our regs are written, we prohibit gelatin from entering, as Dr. Morris has described, unless it can be demonstrated that it is not going to go for animal use. Okay.

BOARD MEMBER FERGUSON: So we don't make this type of an exemption, you know, for stuff going for animal use, if that's clear.

BOARD MEMBER JOHNSON: I thought it was all products derived from cattle that were from BSE positive countries that you don't permit. But as long as we eat it, it's all right? As long as humans eat it.

BOARD MEMBER FERGUSON: APHIS' authority is related to animal health issues. APHIS' authority is not related to public health issues, so our regs are written based on that authority.

BOARD MEMBER JOHNSON: But doesn't your animal health issue say that products derived from, cattle products derived from BSE positive countries cannot be brought into the country?

BOARD MEMBER FERGUSON: Correct. Our regs in general prohibit not just bovine products, but most ruminant products.

BOARD MEMBER JOHNSON: Yes.

BOARD MEMBER FERGUSON: From countries on our BSE restricted list. However, I think, as Dr. Morris explained in her presentation, there are certain things in the regs that can be allowed entry and one of those things is gelatin under certain conditions that is not going for animal use.

BOARD MEMBER JOHNSON: That's in your exemptions at FDA?

BOARD MEMBER FERGUSON: Correct.

CHAIR PRIOLA: This is USDA, Dick, so, yes. They are USDA.

BOARD MEMBER JOHNSON: USDA, that's okay.

CHAIR PRIOLA: Yes, the FDA is strictly concerned with oral or topical applications in humans of gelatin, so the USDA regs aren't our concern. It's the FDA. It's this specific recommendation by the FDA.

BOARD MEMBER JOHNSON: Except it isn't -- wouldn't it be a regulation, for instance, that you couldn't bring in cattle hides from Europe under the safety of animals?

BOARD MEMBER FERGUSON: No, hides and skins are exempted from our regs.

BOARD MEMBER JOHNSON: They are exempted? Okay.

BOARD MEMBER FERGUSON: They are considered, yes, not to present a risk of transmission.

BOARD MEMBER JOHNSON: Okay.

CHAIR PRIOLA: Dr. Bailar?

BOARD MEMBER BAILAR: How is herd defined? Is that all the animals on a single farm or ranch?

BOARD MEMBER FERGUSON: I don't know why you guys are looking at me, because these aren't our regs. Actually, I have to admit, I mean, these are the types of things that you always run into when you put that type of a thing in a reg. It's very difficult to define that. When we look at it from an animal health point of view, it's a group of animals that is housed together. And if a premise has, you know, two separate groups of animals that never come into contact with each other and are managed completely differently, those could, technically, be considered two different herds. But essentially, it's a group of animals that are managed together and handled together.

BOARD MEMBER BAILAR: Okay.

CHAIR PRIOLA: Mr. Dwyer has been standing there for a couple of minutes. Would you like to make a comment?

MR. DWYER: Yes, thank you. As you've explained, there is a complete distinction between the FDA's guidance and the USDA's regulations. The USDA's regulations are intended only to protect animals and not to deal with anything that the FDA has going on here. If you go back and look at the early meetings and transcripts of this Advisory Committee when FDA was discussing with the Committee the formulation of this guidance, you'll see that the requirement for BSE-free herd restriction was put in as one of a series of restrictions in FDA's guidance that were intended to protect the safety of gelatin.

There wasn't much discussion, at that time, of what a BSE-free herd meant or how that would be defined. Because it is obviously possible to define it in many different ways, what the industry was looking for is a way of defining it in a logical, rational way that is consistent with current practice in Europe. And that's basically it.

BOARD MEMBER BRACEY: Although --

CHAIR PRIOLA: Go ahead.

BOARD MEMBER BRACEY: Although I think the safety of gelatin has been certainly demonstrated to be rather robust today, what bothers me is, in essence, a disconnect between two levels of animals. One is the human where we are considering saying that well, it's okay, based on the data that we see, to allow humans to ingest these materials. Whereas, on the other hand, another arm of the Government says that another animal, which some of us think would be on a lower level perhaps than the human, that it is not acceptable.

And, you know, I really feel that we need to have some sort of harmonization, because the message, I think, that -- if I were the public, I would be somewhat concerned about the message that we would be issuing.

BOARD MEMBER FERGUSON: Yeah, that's a valid point. I would ask everybody, however, to keep in mind that, you know, it's one thing to talk about an agent that is coming from cattle and going directly back into cattle versus an agent that is coming from cattle and is going into a different species. Granted, it has been demonstrated that there is that transmission, but you do still have somewhat of a species barrier there.

CHAIR PRIOLA: Dr. Khabbaz?

BOARD MEMBER KHABBAZ: Yeah, and actually, listening to the USDA presentation, I have that same reaction saying there is an apparent contradiction here between conditions for animals and humans. But in thinking about it, I mean, you have a potential amplification. I mean, these are some different issues that go into place with animals and that's why I didn't comment. But there is an apparent contradiction. I agree.

CHAIR PRIOLA: Yes, Lisa?

BOARD MEMBER FERGUSON: Can I go back to the term BSE-free herd? That's very difficult to define and I don't want to necessarily sound too harsh, but in some ways it is sort of meaningless. I know we have struggled with those types of definitions as we tried to setup our scrapie or CWD eradication programs. And, you know, specifically, as we're doing our CWD program, we don't necessarily define under the auspices of that program as herd as free until they have gone through a 5 year period with extensive surveillance and a lot of that. So it is a bit difficult to define.

I guess I'm not quite sure exactly what level of risk mitigation it's necessarily adding in this guidance. Probably more of the risk mitigation is coming from removing those tissues that are at highest risk and also just through the inactivation of the process itself. So perhaps what we should consider is that specific point even necessary in there or does it just cause more confusion than it is really worth?

CHAIR PRIOLA: Dr. Bailar?

BOARD MEMBER BAILAR: I feel like I just don't know enough about all this. And I am concerned about the definition of a herd. Does this include animals that come from the same source, prior to the time they are parceled out into different farms? Does it include any element of time? That is, you know, if all those animals there today are gone and you bring in new ones, is that part of the same herd? What about overlap in time, which I understand is common in the industry, that there is a continuing flow of young ones in and older ones out. I just don't know enough about it.

CHAIR PRIOLA: Dr. Chiu, do you want to comment on what the FDA means by BSE-free herd or is there something more specific you can tell us?

DR. CHIU: Well, I will try. If I didn't get the picture across right, then I will ask Dr. David Asher to add it. I think in our original discussion we were thinking a herd is a group of animals managed, you know, by the same people and also physically they are together, so they are sort of separated from another group of animals. And also we think when we say BSE-free means, you know, that group of animals in the past there was never, you know, a BSE case among that group. In addition, we were also thinking, you know, that group of animals were never fed with meat and bone marrow, so therefore they don't have that kind of risk to contact BSE.

CHAIR PRIOLA: Dr. Wolfe, did you want to say something?

BOARD MEMBER WOLFE: I just wanted to ask Lisa, just from your perspective, what do you think the difference is between this guidance or recommendation as it now exists and the way that the industry would like to redefine it? I mean, the reason I'm asking you is (a) you're from the USDA, but (b) you have just gotten done saying you don't think the phrase herd has any meaning at all. So if it doesn't have any meaning, then what is the difference between our current version and what they propose?

BOARD MEMBER FERGUSON: That's a good point, and actually I don't really see a whole lot of difference in true meaning between what the industry has proposed and what currently exists. My sense of what industry has proposed is trying to make it more realistic and to make it more meaningful in what fits with industry practices, which is a very valid point, especially this one about the removal of tissues and where.

BOARD MEMBER WOLFE: I'm specifically just talking about the herd definition.

BOARD MEMBER FERGUSON: Actually, I mean, after what Dr. Chiu has just said, you know, if those are the specific issues that FDA is intending to address with that point, then I guess my suggestion would be to put that in there as the guidance to say that these animals have not been fed meat and bone meal, those types of things. That is a more accurate definition of the risk mitigation measure and is more easily understandable and leads to less confusion.

CHAIR PRIOLA: Looking though this, I don't have any trouble. I think that's an excellent suggestion actually for the FDA to modify it according to what they mean by BSE-free herd. The other suggested modification by industry down there at the bottom, I'm somewhat uncomfortable with, but you had mentioned that you weren't as uncomfortable. Why exactly is that?

BOARD MEMBER FERGUSON: Well, I think that's probably just because of my understanding of slaughterhouse practices. And if this is saying, you know, as it currently says, let me find it, "and if the slaughterhouse removes the heads, spines and spinal cords directly after slaughter," that lends itself to a lot of interpretation.

First of all, talking about spine directly after slaughter, does that mean right after the animal is stunned and, you know, hung up on the rail and bled out? if so, that's not necessarily common practice. You need that vertebral column there to give some structure to the carcass that's moving through the plant. You know, and I think the point is that those tissues are removed at some point in time during the process. Although they are not going into the start of the gelatin manufacturing process. It's not as much a point as specifically when are they removed, it's that they are removed.

CHAIR PRIOLA: Which the current guidance says anyway. I mean, I don't see where the industry modification makes that much of a difference if, in fact, they take that out at the level of the slaughterhouse they take out that requirement. The way I read it.

BOARD MEMBER FERGUSON: Well, I guess from an interesting point of view and actually let me rephrase that. From a Government point of view, as a federally employed Government veterinarian that might be put in a position to certify to this, I probably couldn't. And it is just because of the way that that is worded, where this stuff is removed directly after slaughter.

CHAIR PRIOLA: Where does it say directly after slaughter?

BOARD MEMBER FERGUSON: Right in the text, yes. If the slaughterhouse removes the head, spines and spinal cords directly after slaughter.

CHAIR PRIOLA: I just have after slaughter. Do I have the wrong one?

BOARD MEMBER FERGUSON: Can we put it up?

CHAIR PRIOLA: Oh, I see. You're looking in the -- I see. It says directly after slaughter if it's from a BSE herd. Later in the recommendation it says if the slaughterhouse removes after slaughter. So there is two different ones.

BOARD MEMBER FERGUSON: Yes, but even, I mean, the later one remove head, spines and spinal cords as a first procedure following slaughter, that just leaves open a lot of ambiguity and, you know, there are some of our folks who are very literal, you know, when they would read that and say oh, no, they didn't, you know, stun this animal, bleed her out and then immediately remove things, therefore, I can't attest to that type of certification.

CHAIR PRIOLA: I guess again, could we ask FDA, is there -- since that's a USDA interpretation of this recommendation, does the FDA have the same sort of reservations or are they concerned about those same sort of reservations as to when exactly the tissue is taken after slaughter or is the discussion enough?

DR. ASHER: No, I think the discussion is very useful. My recollection of the intent of the FDA with both those issues was that the reason why BSE-free herds was specified but not defined was just to put the industry on notice that under no circumstances did we consider material from a herd recognized to have BSE as being an acceptable source for any kind of gelatin entering the United States. No effort at the time was made to define a BSE-free herd.

If one were to try to define an acceptable BSE-free source, I would certainly agree with Dr. Chiu that it would not simply be all tests of 30 month old animals going to slaughter are negative. The herd would have to have a certified history of never using food supplements containing prohibited proteins. There would have to be an adequate surveyance program, not just 30 months slaughter animals.

And my personal opinion would have to include a sufficient number of older sentinel animals and, of course, careful veterinary surveyance to make sure that all sick animals were recognized. My personal opinion also is that this Committee not entertain an assertion that an animal that tests negative at 30 months poses no threat to the public health. I say both those things without attempting to influence the discussions of the Committee. Thank you.

CHAIR PRIOLA: Would it be sufficient to say something like a BSE-free herd is defined by the FDA, if that is in fact defined somewhere, clearly?

DR. CHIU: No, we have not put in writing. And regarding the slaughter, you know, the first procedure are directly after slaughter, I remember our discussion in the past, was because if you remove spinal cord, it is not possible, you know, to make sure entire cord, everything is removed. You might have residual, you know, tissues. And if you carry that to somewhere else and then remove the spine, then create contamination of other tissues, in the bones of other tissues. So we thought, you know, it would be better to remove, you know, the spine, the vertebrae at the slaughterhouse. That was the thought at that time.

CHAIR PRIOLA: I guess the other thing to consider is, again, given all the data we have seen showing inactivation of infectivity following the gelatin extraction process, the issue of contamination, cross-contamination by a spinal cord being removed at a different part of the slaughter process may not be as major an issue given the fact that now there are these five individual studies, all of which saying that the gelatin process itself, as you get to the end, can remove extremely high levels of infectivity under worst case conditions. So it's possible that this discussion as to when things are removed and may not, given that data, be as critical as it might have been before we had access to this data. Dr. Bailar?

BOARD MEMBER BAILAR: Dr. Priola, we have had questions about some of the wording in this recommendation, this draft recommendation. I have a question about the last sentence that the processors are responsible for the safety of what comes into them. Without offering any guidance about that, would it be appropriate? I don't want to vote against this. On the other hand, I'm not very comfortable about voting for it.

Would it be appropriate to defer action until the next meeting with a request that FDA consider revising the wording? I think the intent is fine. I have no particular quarrel with the intent of the changes proposed by the industry, but I think it needs some tightening up.

CHAIR PRIOLA: Well, I think in a way that's what the FDA is actually asking us to discuss. Given what we have heard today and the current discussion, how can we modify this or should we modify it in a way that addresses the concerns of the Committee? So this, I would think would be an opportunity to make that known, how you would want to do that.

BOARD MEMBER BAILAR: I'm not sure we can modify it on the fly this way. That's why I would like to allow a little bit of time for people who know a lot more about the process, the problems, than some of us on the Committee, and time for some reflection about the implications of any changes.

BOARD MEMBER WOLFE: I would agree with John, because I think based on what Lisa has said, which, I think, amplifies the understanding of the process somewhat and what other people are saying, that the FDA has gotten some input from us, which is what Question 2B is, and it would make most sense for us to get at the next meeting the new version of the recommendation to vote on.

CHAIR PRIOLA: Do you have suggestions for changes that we can make? I mean, we still have to actually vote on Question 2A, but would you have suggested for recommendations?

BOARD MEMBER WOLFE: I mean, defining, as Lisa suggested, what a BSE-free herd is, sorting out the differences between directly after, immediately after, first process or just after. I mean, there are three different ways of describing in the current recommendation the timing between slaughter and removal of spine, spinal cord and so forth, so I think, I mean, those are, I think, two areas that need to be neatened or tightened up.

CHAIR PRIOLA: Yes. Dr. Khabbaz?

BOARD MEMBER KHABBAZ: Yes, it's a question to the FDA. Can we vote on this recommendation and then leave to the FDA to wordsmith the BSE-free herd and the timing of removal based on the discussion that they heard?

DR. CHIU: I think we definitely can go back to before BSE-free herd, you know. We have some idea, you know, over the years, you know, we have in mind. We would like to get advised whether to remove the spinal, the spine, the vertebras in the slaughterhouse is needed or not because of the results you have seen, you know, from the validation studies.

CHAIR PRIOLA: Dr. Hogan?

BOARD MEMBER HOGAN: Well, it seemed, the validation studies suggested if you can start with really high titre material, that you get rid of almost, I mean, virtually totally. So I think what you start with in the real world is sort of irrelevant, because it's never going to be as high as what they are starting with in these validation studies.

Now, I am a little concerned that if you leave the spinal cord in and then you drive, you know, 200 miles to have the spinal cord removed, it is going to dry during that time period. Is that going to sequester some agent that might be more difficult to remove later? But as I just said, I think the titres will be much less than the validation studies. So I personally wouldn't have a problem with that.

CHAIR PRIOLA: Mr. Dwyer, do you have a brief comment?

MR. DWYER: Thank you. Just as a reminder, this Committee voted in 1998 to agree with or recommend to FDA the removal of the spines in the manner that we have suggested in our draft modifications to the guidance. We have attempted to craft our draft modifications to the guidance with respect to this issue, that is spine removal in a way that is consistent with what this Committee recommended in 1998 as reflected in the transcript of the meeting from April 1998. Thank you.

CHAIR PRIOLA: Dr. Johnson and then Dr. Ferguson.

BOARD MEMBER JOHNSON: Well, I think it's very impressive how much this does decrease the infectivity. On the other hand, we should go back to remember that there is that inactivated tail or whatever you want to call it, so there is inactivated particles, and think back to the Committee hearings after the Cutter episode with Jonas Salk where they forgot, they neglected looking at the unneutralized tail, which caused the whole Cutter episode.

Is it there and I think we should consider that. I don't think that's enough to change the rules, but I don't think it's enough to say well, let's not worry about splashing a little spinal cord around. I think we still ought to keep that as tight as absolutely possible to keep the contamination membranes of the spinal cord down.

So I would agree that I don't think we need to change. I voted yes on 1, but on this I would not want to see it made more permissive for the possibility of contamination despite the good inactivation studies.

BOARD MEMBER FERGUSON: I guess I would like to briefly run through sort of the standard slaughter practice at least in the U.S., and ask everybody to sort of think about the possibilities for contamination. You know, an animal comes in. It is stunned, rendered unconscious, then, essentially, hooks are applied to the rear legs and it is bled out. The animal is skinned. The head is removed and then, generally, the carcass is split, at that point in time.

The standard practice is to go ahead and remove the spinal cord, at that point in time. That is the easiest time to do it. But the issue is not necessarily the removal of the cord. The issue is the removal of the spine and that, you know, vertebral structure that allows the carcass to sort of hold together and it's going through the rest of the processing process.

So if concerns are about cross-contamination from removal of the vertebral column later in the process, I'm not quite sure where that cross-contamination is going to come from, especially on bones and bone chips that are going into a gelatin derived process, because even if you assume okay, you can get some contamination when you split that carcass in half or if you have a missplit, you're getting aerosolized cord that is going on the surface of that carcass, and the bones aren't on the surface of that carcass. The meat on those bones is removed elsewhere in the meat cuts, and the contamination isn't necessarily going to be on those bones, per se, which is what is going into the gelatin process.

CHAIR PRIOLA: I think we could -- at least, the sense I'm getting is the recommendation as it stands needs some tightening up in terms of clarifying definitions of BSE herd and when after slaughter things need to be removed. The primary question prior to this is do the scientific data and information available support the following FDA recommendation? If the answer is no, what changes? Are there changes other than tightening up these definitions that anyone would like to recommend?

For myself, the removal of the vertebral column, I think, is a big issue for European countries because of the European BSE, but given that here in the United States there is as yet no BSE and they haven't yet moved, if I remember correctly from this morning, to removal of the entire vertebral column, is that right, that has happened. There is a significantly different level of risk, if I understand correctly. So these rules seem to apply to really primarily European BSE countries.

Should we call for a vote on Part 2A and, if necessary, move on to Part B with specifics? Are there any objections to that? If the FDA has gotten what they need from the discussion, which I think they have, we can move on to a vote for 2A.

SECRETARY FREAS: I will go around and poll the table exactly as last time. Dr. Johnson?

BOARD MEMBER JOHNSON: With the likely changes made by FDA, do we vote yes or no?

CHAIR PRIOLA: I think you vote no.

BOARD MEMBER JOHNSON: You vote?

CHAIR PRIOLA: And then we ask what changes for 2B. Isn't that right? Well, actually, I think yes. Well, because I think that the

scientific --

BOARD MEMBER JOHNSON: I would vote.

CHAIR PRIOLA: Yes.

BOARD MEMBER JOHNSON: If I looked at that just as it says, which states there on the board, my answer would be yes.

CHAIR PRIOLA: Yes. I think you can vote yes or no and we can still make modifications in 2B, because this Committee has never hesitated to make modifications.

BOARD MEMBER JOHNSON: Then my vote stands, Sue.

CHAIR PRIOLA: So I'm sorry, so what is it again, Dick, officially?

BOARD MEMBER JOHNSON: It's a yes.

SECRETARY FREAS: Dr. Bracey?

BOARD MEMBER BRACEY: I would second that yes.

SECRETARY FREAS: Dr. Ferguson?

BOARD MEMBER FERGUSON: Yes.

SECRETARY FREAS: Dr. Hogan?

BOARD MEMBER HOGAN: Yes, but we need modification.

SECRETARY FREAS: Dr. Khabbaz?

BOARD MEMBER KHABBAZ: Yes.

SECRETARY FREAS: Dr. Priola?

CHAIR PRIOLA: Yes.

SECRETARY FREAS: Ms. Walker?

MS. WALKER: Abstain.

SECRETARY FREAS: Dr. Wolfe?

BOARD MEMBER WOLFE: No.

SECRETARY FREAS: Dr. Bailar?

BOARD MEMBER BAILAR: No.

SECRETARY FREAS: The industry, would you, please, express your comments on this? Okay. Out of the nine voting members at the table, we have 2 nos, 1 abstention and 6 yeses.

CHAIR PRIOLA: Okay. Under the part of 2B, even though we answered yes, if I tallied right, there are three specific things that we would ask the FDA to clarify. And that would be the definition of a BSE-free herd, to make the recommendations at slaughter, directly after slaughter, you know, just after slaughter, if they could be more specific as to when the vertebral column should be removed, and also, Dr. Chiu had asked specifically about whether removal of the vertebral column is necessary. They wanted some clarification on that, too, I believe. Are there any comments, Dr. Bailar?

BOARD MEMBER BAILAR: I would add a point also about some clarification about the insurance by the processors that their supplies are adequately protected.

CHAIR PRIOLA: Well, I think that -- isn't that in the last? That is in the last sentence, right, gelatin processes should ensure?

BOARD MEMBER BAILAR: It says the processors should ensure, and I would like to know more about that.

BOARD MEMBER HOGAN: Well, unfortunately, Dr. Gambetti isn't here, but from my experience in removing spinal cords, there can be left dorsal root ganglia and other nervous tissues depending on how you do it. So I think the issue of vertebral column if you're asking just for some comments, may be important if you want to reduce that last little bit.

CHAIR PRIOLA: Does anybody want to recommend any specific language if we can, I don't know if we can, to give the FDA some further guidance? You know, for example, industry recommendation for BSE-free herd. Is that an appropriate way to qualify it, to introduce the concept of testing according to standard procedures? Lisa?

BOARD MEMBER FERGUSON: I guess I'm uncomfortable with having testing in there as the only thing that's qualifying the herd. You know, I don't think that testing is necessarily the critical thing to hang your hat on. I think the point is lack of exposure, and that should probably be where that definition heads.

CHAIR PRIOLA: Are there any other comments as to specificity as to modifications of the recommendation? Okay. Sidney, do you have anything in terms of the slaughterhouse issue directly after? I mean, how specific should specific be, given again all the data we have heard this morning?

BOARD MEMBER WOLFE: No, I understand that, but, I mean, we heard that there is a vastly different slaughtering process in Europe versus here, so what I thought I heard this morning from the Europeans was that since it's done in a different place, it's not even within the slaughterhouse. I mean, is that correct? I mean, in Europe, you just repeat what you said is the main difference between European slaughtering techniques in terms of bone, in terms of getting the bone for gel, as opposed to this country?

MR. SCHRIEBER: The difference is only the place of the removal of the bones.

BOARD MEMBER WOLFE: The place, right.

MR. SCHRIEBER: Just the place.

BOARD MEMBER WOLFE: So it's not --

MR. SCHRIEBER: Slaughtering practice is exactly the same, I think.

BOARD MEMBER WOLFE: But in one case, in this country, the removal of the bones is in the slaughterhouse and there, somewhere else? That's the difference. So it's the issue of transport and whatever. So it's beyond just where in the slaughterhouse. It's in the slaughterhouse or not. The issue is whether we think that it's okay for -- which is the issue the industry raised, whether we think it's okay for the bone removal to be somewhere else with at least risks to workers and others that are different than they would be if it were all done within the slaughterhouse.

CHAIR PRIOLA: Go ahead.

MR. SCHRIEBER: In addition to this, the places of the removal, which other meat processes are, are exactly under the same supervision of the authorities of the public like the slaughterhouse itself. They are meat processors, so they have to follow the same rules. It's just a question of distance. It's not a question of how procedures are done, whether they are inspected, whether they are controlled. That's all the same whether it's here or there.

CHAIR PRIOLA: Are there any other comments? I guess I should ask the FDA. Do you have sufficient information in terms of what the Committee is asking for, for modification to the recommendation based on the discussion and what was just said?

DR. CHIU: Well, in my mind, I'm still not quite clear, you know. We have read and heard, you know, the study results and as many of you expressed, it's quite impressive. So I am not quite clear, you know, whether we get any advice. Is it necessary to remove spine and if it's necessary, then when it should be done? So if we can, you know, have a little more discussion whether the spine, the vertebrae, actually needs to be removed.

CHAIR PRIOLA: Lisa?

BOARD MEMBER FERGUSON: Yes, I guess I'll throw my two cents worth in here and everybody else can have at it. I guess, I think it's important to essentially limit the use of vertebral column in the production of gelatin or say, you know, you are not using vertebral column in the production of gelatin. I don't think it makes any difference where or when that is removed, but to say yes, it's not included, it's not going into the gel balm is important.

CHAIR PRIOLA: Yes, I would actually agree with that, that it's important that it is being removed given the data where heard. Where exactly it's removed may not be that big of an issue since you can activate, apparently, quite effectively quite a bit of infectivity that might be residual on the bone surface after removal of the spinal cord.

I am actually comfortable if the FDA does tighten up the definition of BSE-free herd. I am comfortable for myself with the recommendation, how it sits, with just some tightening up of those definitions, BSE-free herd, as well as being careful when you describe when the vertebral column should be removed after slaughter. I think in Europe, all the vertebral columns are removed anyway, so that is moot. It's just where the removal is, and that is not a primary concern for myself.

Would anybody else like to contribute? Is it just too near to lunch? Are we running out of steam? Well, if anyone has any -- I mean, so I guess we have addressed the questions and if anyone else would like to say anything after lunch, feel free to do that. When we restart the session at 1:30, 1:40?

SECRETARY FREAS: Let's try 1:30.

CHAIR PRIOLA: Okay. Let's go for 1:30.

(Whereupon, the hearing was recessed at 12:45 p.m. to reconvene at 1:37 p.m. this same day.)

A-F-T-E-R-N-O-O-N S-E-S-S-I-O-N

1:37 p.m.

SECRETARY FREAS: Okay. Thank you very much for rushing through lunch. In the afternoon, we are very fortunate. We will be joined by four new temporary voting members. I would like to go around and introduce them. I won't introduce the whole table, just the four new temporary voting members. Well, I will introduce at least three of the new temporary voting members.

On the far side of the table is Mr. Terry Rice, Board of Directors, Committee of 10,000 from Windham, Maine. Would you raise your hand, Mr. Rice? At the corner of the table right in front of the screen is a new voting temporary member for today, Dr. Charles Edmiston. He is associate professor of surgery, Medical College of Wisconsin, and he is also chair of the CDRH General Hospital in Personal Use Device Panel, and he will be taking these issues from today to his center's panels.

And we will be very shortly joined by Dr. Kenrad Nelson, who will be sitting next to Dr. Priola, our Chair, and Dr. Nelson is a professor, Department of Epidemiology, Johns Hopkins University School of Hygiene and Public Health, and he is chair of the Center of Biologics Blood Products Advisory Committee. And there is one more person, and that is also from the Blood Products Advisory Committee. That is Dr. David Stroncek, Chief Lab Service Section, Department of Transfusion Medicine, NIH. And to all four of you, I would like to welcome you to the table and thank you.

CHAIR PRIOLA: Okay. We'll go on to starting with Topic 2, which is BSE in Canada, and I want to let the Committee know that this is an informational topic only. It's for our benefit. We're here to listen, and there are no questions being posed to us. This is an informational topic only. The first speaker is Dr. Jay Epstein from FDA.

DR. EPSTEIN: Thank you, Dr. Priola. Before Mr. Hills makes a presentation on what is known about the reported case of BSE in Canada, I would just like to take a moment to make a brief statement about FDA's current thinking in regard to potential implications of that case report for blood safety policy.

FDA is undertaking an assessment of the BSE exposure risk to blood donors in the U.S. and Canada in light of the single BSE case that has recently been reported in Canada. Although, it is premature for the FDA to present any results of this assessment now, we believe that the likelihood of exposure to the BSE agent for both Canada and the U.S. is and has been very small.

The exact magnitude of BSE risks for Canada and the U.S. will be difficult to quantify because of methodological limitations. However, preliminary considerations suggest first that the risk of exposure of blood donors in North America to the BSE agent has been extremely low and is even lower now than it was several years ago. And then secondly, in particular, implementation of the feed ban of 1997 both in the U.S. and in Canada significantly reduced the likelihood of human exposure to the BSE agent for both countries.

FDA does not believe that there are sufficient data, at this time, to warrant changing our blood donor deferral guidance. However, we will continue to study this issue and will take further action as appropriate. Thank you very much.

BOARD MEMBER WOLFE: I have a question.

CHAIR PRIOLA: Dr. Wolfe?

BOARD MEMBER WOLFE: Given that the spectrum of countries for which there are limitations on blood donation go from UK, lots of cases, to EU with some countries with very small numbers of cases in just cattle, and that Canada is still a "moderate risk country" that has had a case, the benefit risk equation is always important, and have you at least tried to get some data, so that this question can be answered better later as to what fraction of the blood supply in this country would be affected if there was some sort of constriction on the ability of people who have spent whatever amount of time in Canada?

Is there at least some effort to collect that, because otherwise, as we learn more about the possible risk, small though it may be, and the benefit, which is having a blood supply that is not impaired some comes up to it? So just a simple question. Is someone trying to get a hold of those kinds of data?

DR. EPSTEIN: Yes, Dr. Wolfe. Thank you for that question. We are mindful of the need to try to assess the impact on the blood supply of any potential change to our donor exclusion policy and, indeed, we have already had dialogue with major blood organizations on both the feasibility and scope of surveys that could establish the impact of any candidate deferral policy related to Canadian exposure on the U.S. blood supply.

And more broadly speaking also, we are thinking about similar questions as they might pertain to say exposure in Japan or other countries that have had case reports of BSE in cattle. So that enterprise is recognized and is ongoing.

BOARD MEMBER WOLFE: So you are going to be collecting data on what this impact would be?

DR. EPSTEIN: Well, blood organizations have been asked --

BOARD MEMBER WOLFE: Right.

DR. EPSTEIN: -- if they would collect such data, and we have had preliminary statements of agreement.

BOARD MEMBER WOLFE: Okay. Thank you.

CHAIR PRIOLA: Any other questions for Dr. Epstein? Okay. Thank you very much.

DR. EPSTEIN: Thank you.

CHAIR PRIOLA: Our next speaker will be Dr. Robert Hills from Health Canada Ottawa who will discuss the review of Bovine Spongiform Encephalopathies in Canada.

DR. HILLS: All right. First, thanks very much for inviting me to give you a little update of what the situation is in Canada right now with respect to BSE. I will just wait for the slides to come up. There is a fair bit of information on the slides, so what I will try to do is go through it relatively quickly. All right. Thanks a lot.

First of all, I thought what I would do is give you a little bit of background to what Canada has been doing with respect to BSE before we found a case in May of this year. First of all, there was a prohibition of the importation of products assessed to have a high-risk of introducing BSE in Canada. There was importation of meat and meat products only from countries that Canada recognized as being BSE-free.

In 1990, there was a designation of BSE as being a reportable disease in Canada, and any suspect case of BSE would be reported to a federal veterinarian. In 1992, there was the creation of the National BSE Surveillance Program. In 1997, the same year as the U.S. did, as well, there was the implementation of the feed ban of feeding rendered protein products from ruminant animals to other ruminants. In the year 2001, there was the creation of the Canadian Cattle Identification Program for Cattle and Bison making it possible to trace individual animal movements from the herd of origin to the slaughter.

Next slide, please. This is just a quick pictorial of sort of how Canada has approached it. Canada has adhered to the OIE guidelines on TSE risk management. Up until our finding of the case, we were considered to be provisionally free, and we have also done a risk assessment that was completed in December of 2002 with respect to Bovine BSE cattle in Canada, and in that risk assessment, we actually determined that the likelihood of finding BSE in Canada would be remote. That has changed, but still remote.

Next slide, please. Before we go on, what I would like to do though is just bring you back a little bit in time, because we did have a case previously. In 1993, we did diagnose a case of BSE in a beef cow that was imported from the UK in 1987. The exposure of this animal to BSE occurred prior to its arrival to Canada. The index herd and all the UK animal imports were destroyed, at the time, and it was subsequently determined that the UK herd, which was a source cow for this particular animal, did have other infected animals, as well.

Next slide, please. This graph here is, again, a pictorial showing the importation of animals into Canada, particularly, and we're it the North American Disposition of Imported UK Cohort Members prior to the index case discovery in 1993. So there was importation of animals prior to our taking action in 1993 to eliminate those animals.

Next slide, please. So getting on to our first indigenous case. January 31, 2003, a 6 to 8 year-old downer beef cow from northern Alberta went to slaughter to a provincially licensed meat facility. Alberta Agricultural Food and Rural Development meat inspector condemns the carcass as being unfit for human consumption.

At that time, the head was collected and submitted as part of our National Surveillance Program, the Federal Provincial Surveillance Program for BSE. And the carcass, at that time, because it was condemned, was sent to inedible rendering.

Next slide, please. On May 16^th, the testing was completed with a tentative diagnosis of BSE by the Alberta Ministry of Agriculture. The sample was then sent immediately to Canadian Food Inspection Agency's National Center for Foreign Disease in Winnipeg, Manitoba where they also confirmed BSE, and then the sample was subsequently sent to the Veterinary Laboratory Agency in Weybridge, England, which is the OIE Reference Center for BSE. And on May 20^th, they actually confirmed our diagnosis of it being an actual case of BSE. Immediately upon notification, we notified the OIE, in fact, that we did have a case of BSE.

Next slide, please. So what did we do from there? So we had a start in epidemiological investigation. We basically broke it down into three phases. The first phase, we're calling that the case itself, which we'll call the animal trace back, its immediate management, which we'll call the animal trace forward, and the most probably origins, which is where did the animal get the exposure from?

Next slide, please. This is a well used graph. It is done in colors for a particular reason. The red is the case herd where the index animal was. The blue line is considered the primary line of inquiry where we think the source animal came from, which is in Saskatchewan. The yellow line is considered the secondary line of inquiry. That was an Alberta line, and the green one in the middle is, in fact, that we did discover that there was some commingling with another herd. So those are the areas, which we were tracing out as part of this investigation.

Next slide, please. As I said earlier, the index case was a 6 to 8 year-old Angus. It was a member of a herd that was recently established within a two year period between 2001 and 2002, and the animals that made up that herd were from two farms. What we believe initially from the age of the animal was that the expression of the clinical BSE at this age offers the first epidemiological insight, which would probably mean it was a low level BSE exposure given the age of the animal.

As I mentioned earlier in a previous slide, the Saskatchewan blue line of inquiry was the most probable avenue for which the positive animal moved to the Alberta farm. That particular line of inquiry, the animals were culled and depopulated and tested, and all tests were negative by Prionics Western Blot and immunohistochemistry.

Next slide, please. At the same time as we were culling and depopulating and testing, we were also trying to confirm through DNA testing the origin of the particular index animal. Unfortunately, the DNA testing did not come back with a clear definitive result, and as a result, we needed to then move down the second line of inquiry.

So we proceeded with the depopulation and testing of animals in the Alberta line of inquiry, which was that yellow line in the previous pictorial that I showed you. We culled those animals and tested those animals and all tests came back negative again from Western Blot and immunohistochemistry. Even though we didn't have a confirmed definitive answer for the DNA, it is still a probably line of inquiry, and most probable one, is the Saskatchewan blue line for the introduction of the animal to this farm.

Next slide, please. The next phase, which was the Animal Trace Forward Investigation, was to determine what would happen with the animals that left the farm. So there was movement. We looked for the movement of the cattle from the index herd. We looked at, as I mentioned earlier there was a green box, where there was some commingling. We traced out those animals. We culled them. We depopulated them. We tested them. We found that they were all negative by Western Blot and immunohistochemistry.

Next slide, please. So to summarize, the Trace Forward and Trace Back Investigations, we had 15 premises that were quarantined, an additional 25 herds were scrutinized and the tracing-out of single animals or cohorts from the Saskatchewan line of inquiry.

The trace out also included the identification and notification of the export of five animals to the United States, which you all should be aware of, we did let you know as soon as we found that out. And in all, we had a culling of more than 2,700 animals, 2,000 of which were 24 months of age and older, and all, as I mentioned earlier, have been found negative by Prionics Western Blot and immunohistochemistry.

Next slide, please. This is just a pictorial or a graphic of the disposition of the carcass of the particular BSE index case. It shows the yellow line, which shows the line of investigation with respect to the use of the products for laboratory testing. We have the lighter blue line, which shows what happened with the processing of the hide. And then, again, we have the mauve or the purple, which is moving the carcass to inedible rendering and what happened to it from there. And as you can see, it did get rendered into some meat and bone meal.

Next slide, please. So we then looked at the Feed Investigation. Since the index cow was condemned unfit for human consumption, its carcass was sent to an inedible rendering. And I would want to reemphasize because it was sent to inedible rendering, there was no part of the animal that actually went into the human food chain.

Next slide, please. The carcass of the index as I showed you in the previous pictorial, the carcass of the index case was traced via Canadian Food Inspection Agency from the abattoir, the renderer, the feed mill, the producer continuum to its direct allocation into pet food. And in the pet food case, we did find that actually there was some pet food that actually was exported to the U.S. to which we notified the U.S. when we found that out, and there was pet food in Canada, as well. And there was the production of meat and bone meal.

What is important to take out of this though is the visit to the renderer and the feed mills confirmed adherence to our feed ban or meat and bone meal feed ban legislation on the product receipt, segregation, labeling and distribution. So there was no breach in compliance, at that point, at the renderer. So it was not labeled to be fed back to ruminants.

Next slide, please. Further investigation was the trace out of the feed to individual farms, and we did find that three additional farms were quarantined when the investigation could not preclude exposure of 63 head of cattle to the feed destined for poultry feed. In that case, it was evidenced that the farm itself had allowed poultry feed to be fed to ruminants. The animals were culled and tested by Prionics Western Blot and immunohistochemistry and, again, all those animals came back negative.

Next slide, please. This is just a pictorial, again, of what I was describing before from the inedible material from the index case going to the renderer. It went up to pet food. It went into poultry and some pet food, and there was the feed mills that we traced out afterwards. So this is sort of managing the risk of the disposition of the material from the index animal.

Next slide, please. There were other considerations that we wanted to take into account as we proceeded in with the exposure investigation. We did look at maternal transmission. We looked at contaminated meat and bone meal used in feed products, particularly early risk factors, any UK imports slaughtered prior to 1993 or other European imports. It was figured into our investigation.

We looked at TSEs resident in other animals, CWD and scrapie as examples, and we did look at the possibility of it being a spontaneous case. Our investigation right now is at the point now, we are looking at feed products that are considered the most probable root of exposure.

Next slide, please. Again, this is just a graphic again to illustrate the hypothetical foreign domestic exposures of the index case, and it's just groups of, you know, what the possibilities might be for the exposure of this particular case.

Next slide, please. What we did find out in the investigation is there was two potential meat and bone meal epidemiological exposure roots that were identified. The first was a feed concentrate and the second was a high energy feed block. Both have incorporated meat and bone meal, at some point in time. The investigation did find though that the feed mill records and compounding formulae confirmed that the meat and bone meal incorporated in both of these products was curtailed in 1997 upon implementation of the meat and bone meal feed ban.

Next slide, please. So what can we conclude? What we can conclude so far is that discovery of BSE in Canada proves that the active surveillance and the diagnostic programs were working, because we did find the case. Epidemiological evidence supports the probability that BSE in this case animal was associated to exposure to infected material through the feeding system, at some point, early in the animal's life.

Next slide, please. What we felt that we needed to do was to ensure that what we were doing was accepted and would be recognized, so it was decided that what we would do is convene an expert panel to actually go over our procedures and how we did it and what we were doing and what actions we were going to be taking, and get their recommendations back to us.

That particular panel comprised of Ullie Kihm from Switzerland, Will Hueston from the USA, Dagmar Heim from Switzerland, and we did have contact with Stuart MacDiarmid from New Zealand, as well. The first three met on June 7^th to 9^th and met with the members of the Canadian Food Inspection Agency and Health Canada to which the team was provided with an overview of the epidemiological investigation. All actions taken to date and the scope of the options and the measures being considered to adjust domestic policies.

Subsequently to that meeting, the team went back and did actually do a report, and what the panel did find was that the -- they found that the risk management measures put in place in Canada achieved the desired outcome. The surveillance did detect the case with BSE. The animal did not enter the food chain and the measures in place have reduced the spread and amplification of BSE in Canada.

Next slide, please. They did come back with some specific recommendations, however, for us to strengthen our current situation. They did say that there should be a prohibition on Specified Risk Materials in human food and animal feed, including advanced recovery meat products, tighter controls on non-ruminant feed, enhanced audit and compliance, strengthening the existing cattle identification tracking and tracing systems that are existing in Canada, enhanced disease testing and surveillance by increasing the coverage of fallen and dead stock, downer and diseased animals, and to work at efforts to improve the awareness among producers, veterinarians and the general public with respect to BSE.

Next slide, please. So what will Canada do? Well, the government of Canada will be responding to the recommendations of the International Team. We'll respond through our consultation process with our provinces or territories, the Canadian industry, our U.S. counterparts and other trading partners, and there will be a new policy measure for Specified Risk Materials as being the first step.

Next slide, please. I felt before we sort of should go a little bit further, I would just give you a little bit of background about -- because I'm going to be talking about Specific Risk Materials Ban right now, which is the first step, that in Canada, 95 percent of the slaughters is in federally registered establishments and the majority of those animals that are slaughtered are between the age of 18 and 24 months.

5 percent of the slaughter is in provincial abattoirs and the majority of those being over 30 months of age. Only animals slaughtered in registered establishments can be exported. The provincial slaughtered animals can only be traded within provinces and sold within provinces. If they are to leave provinces or to leave the country, they have to be at a registered establishment. Removing SRMs at the point of slaughter and disposing of them, we estimate removes about 99 percent of the human exposure to potentially infected material.

Next slide, please. The immediate objective of the SRM policy is to establish a requirement that the SRMs be removed at the time of slaughter, and that they be removed from human use, human food and human use. The new policy will define Specified Risk Materials and require removal, as I mentioned earlier, at slaughter.

The list that I have right here are some of the things that we have been considering as being the most probable. They will likely include the brain, spinal cord, dorsal root ganglia, eyes, tonsils, skull and distal ileum.

Next slide, please. Following our first step, as was recommended in the expert panel report, there will be other measures that will be taken. There will be areas that will be looked at with restrictions on animal feed and process and protect human and animal health, that should be, expanded surveillance, as was mentioned earlier, expanded food safety plans, comprehensive tracking and tracing systems and national standards and approaches will be implemented in Canada.

Next slide, please. And that concludes a very quick overview of what we did. Hopefully, it did give you an idea of the scope in which we reacted and what we looked at. I have listed here a number of different sites that you can look at for updates. We're trying to be as open and as transparent about our investigation or actions as we possibly can, and I would encourage you to go to these sites and go to this to get the most up to date information. That's it.

CHAIR PRIOLA: All right. Thank you, Dr. Hills. Dr. Wolfe?

BOARD MEMBER WOLFE: This morning, we sent a letter to the Secretary of Agriculture, Veneman, strongly urging them not to lift the ban on meat coming from Canada to this country, and one of the questions we have was, and I will just read you three sentences, because it's really the form of the questions.

Public information regarding the enforcement of the Canadian Feed Ban, and we know it went into effect in '97, but we also know the data from the United States show very spotty and uneven enforcement, particularly, the first few years of the ban, which is very similar to the U.S. ban and was enacted about the same time. It's available, it's data unenforcement. It's not on the website of the Canadian Food Inspection Agency and a telephone call to the agency requesting these data has not produced any information. Most tellingly, the report from the team of international experts, which is, I assume, the one you just referred to.

DR. HILLS: Yes.

BOARD MEMBER WOLFE: That investigated the Canadian government's response to the outbreak makes no mention of compliance with the feed ban. It mentions the feed ban, of course, but data incompliance.

DR. HILLS: Yes.

BOARD MEMBER WOLFE: It is simply impossible to assess the wisdom of lifting the ban you wisely put in place, you, Secretary Veneman, in this case, on an emergency basis without these data.

So my question to you is what is your knowledge? I thought your presentation was excellent. What is your knowledge of looking backward how effective the enforcement of the feed ban has been from 1997 when it was imposed to now?

DR. HILLS: Unfortunately, I don't have the history to go back in time from '97 backwards or forwards. I know it has come up in our discussions numerous times, being able to put some sort of quantifiable number to it and to try to do that. I have not yet seen that myself. We are trying to determine that now, because we have had investigations. We have looked at it. We have found that our plants themselves have been in compliance.

As I noted in our Feed Investigation, we did find though that there was a possibility that if a farm was coproducing, that there was a possibility of cross-contamination, if you want to call it that, and those are things that we're trying to address now to improve.

BOARD MEMBER WOLFE: Yes, I mean, needless to say, it's the essential issue, because you have admitted that that's the most likely place that the cow that got infected got infected from, and given --

DR. HILLS: Well, no, that's not quite what I said.

BOARD MEMBER WOLFE: Well, I think it's all on your slides, for the most part.

DR. HILLS: No, I think what I said was we found that there were three slides as part of the investigation from the index case that actually found that there were three farms in B.C. that actually were not in compliance, because they inadvertently had fed or that we couldn't definitively tell whether or not the feed that was destined for poultry did not end up having inadvertently been fed to ruminants. What I said was that our most likely possibility would be --

BOARD MEMBER WOLFE: The feed.

DR. HILLS: -- exposure before the feed ban.

BOARD MEMBER WOLFE: For that particular cow.

DR. HILLS: Yes.

BOARD MEMBER WOLFE: But, you know, again, are there going to be some data on enforcement? I mean, I assume that once it went into effect, there was some kind of government effort.

DR. HILLS: Yes.

BOARD MEMBER WOLFE: To check on enforcement.

DR. HILLS: Yes.

BOARD MEMBER WOLFE: When can we expect to see those data?

DR. HILLS: I can't give you a date on that. I can certainly find out for you, but I cannot give you that, but I am not aware of what date they are going to be able to make that information available.

BOARD MEMBER WOLFE: Okay. Thank you again, very good presentation.

CHAIR PRIOLA: Dr. Johnson?

BOARD MEMBER JOHNSON: Yes. Dr. Hills, a question that was brought up early on was the possible U.S. origin of that cow, that it might be a North Dakota or Montana cow, and it may have been our feed ban was the problem. Is there any further data on that original origin of it, and has the U.S. origin been ruled out?

DR. HILLS: The data we have right now suggests that the line of inquiry was the source was the Saskatchewan farm right now. We have no definitive evidence that would say that it was an animal that was imported from the U.S.

CHAIR PRIOLA: Dr. Gambetti?

BOARD MEMBER GAMBETTI: Can you describe the procedure that Health Canada uses to diagnose this particular animal and suspected animal in general? You listed Prionics Western Blot and the immunohistochemistry. Is that done in more than one area and in that particular animal that turned out to be positive, were both positive? Can you, in other words, amplify a little bit on how the animal or another animal are studied in Canada?

DR. HILLS: If I understood your question correctly, Canada has a national TSE laboratory network. The gold standard, the immunohistochemistry test, is a test that is used, was used in all these laboratories, and so there was, as I mentioned earlier, the National Surveillance Program.

The Prionics test for Western Blot was brought in and we were evaluating it, at the time, but it was brought in for use mainly because we had so many animals to do, at the time, that we needed to find some mechanism by which we could up the volumes, and at the same time, we feel that it was a mechanism by which we could do validation testing on the Prionics test itself.

So in doing that, the work was done in the Alberta and Winnipeg labs for the Prionics test, but we're in the process now of looking at and getting that test now distributed across the TSE laboratory network.

BOARD MEMBER GAMBETTI: How many tissue samples or brain area were examined, only one, the lower medulla or more than one and were all of them, if there were more than one, were they all positive?

DR. HILLS: I believe there was more than one and yes, they were all positive, but I can't give you the exact number.

CHAIR PRIOLA: Dr. Bracey?

BOARD MEMBER BRACEY: Perhaps along that line, I guess a question that comes up is the issue of with any test, there is always the chance of having a false positive the more times you do an assay. How are you all, in essence, getting at that and is there a plan to actually look at infectivity in some of these animals or do you feel, in essence, confident enough with the assay system in terms of eliminating that rare false positive?

DR. HILLS: Well, that was one of our concerns, was to go to tests other than the immunohistochemistry analysis, but we felt that the information that we have generated now based on the culling exercise that we have gone through with the animals that we have right now, because each one of those animals was testing in parallel with the gold standard test, that we felt that we're starting to now get the numbers that would indicate whether or not the test is, in fact, what the manufacturer suggests, which is 100 percent no false negatives. So we are generating the information now, and that is the only way that we can do it.

BOARD MEMBER BRACEY: What about the issue of false positives though?

DR. HILLS: Well, in doing the animals that we're doing now, we have found no false positives, but that, as I said, was only 2,700 animals. That is what we have done. That is the claim of the Prionics, I believe, is what they are suggesting is it is being 100 percent accurate.

I did mention that the animals that we were talking about were -- we were targeting 24 months and older animals, because we still believe that there is some possibility that the test will not work sufficiently well for animals below that age, and so the testing system itself is probably a little questionable for younger animals.

CHAIR PRIOLA: Dr. Nelson?

DR. NELSON: You mentioned that there were 2,700, I think, animals that were tested and found not to be -- not found any positives, but how many were of the similar age to this animal? In other words, the infection in this animal could have occurred six, seven years ago and not shown up if the animals examined where younger.

DR. HILLS: Yes, that's a very good question. That's why when we were looking at the culling and the depopulating, we were looking at the specific herds, so we were getting an age distribution. I can't give you the exact number of animals that were there, but because we were targeting the animals older than 30 months, we would then take into account some of those animals. And some of those herds on the Saskatchewan side were breeding animals, so many of them were older. I just can't give you the exact number.

CHAIR PRIOLA: Dr. Taylor, did you have a comment?

DR. TAYLOR: David Taylor from Edinburgh. You mentioned how you concluded that because clinical disease emerged somewhere between when the animal was somewhere between 6 and 8 years-old, that this may be reasonably construed as evidence of low level challenge. Now, certainly, it is true in the UK that incubation period as we have taken to broadly equate with age, because meat and bone meal was usually and often only fed in calf food.

But if the animal, in fact, received meat and bone meal A for the first time or subsequently on several occasions after it was born, you can't really pin down the incubation period. So it could still be a high level dose if it got its meat and bone meal at a later stage.

DR. HILLS: Yes, you are correct. I have no trouble with what you're saying, but I will go back to our feed ban that we have in place right now. The fact that the feeding of animals back and forth, the feeding of ruminant material back to ruminants is prohibited in Canada, so the likelihood of that would be, in our estimation, remote, not nil, but it would be remote. So yes, so that's what we're saying is we do think it's preceding the feed ban, so that would make it then '95, '96, somewhere around in there, and it is a possibility, yes, at that time.

CHAIR PRIOLA: Dr. Bailar? I'm sorry, Dr. Bailar, excuse me.

BOARD MEMBER BAILAR: You mentioned the Canadian system for identification, tracking and tracing of animals. How much help was that to you in your investigation?

DR. HILLS: It was significantly helpful for us. Unfortunately, because we instituted it in the year 2001, it really only was successful for animals that were within that age group. Even though all animals are tagged for movement now, what we can't do is really definitively say, for example, the age of the animal, where the entire history of the animal was, but for the younger animals, yes, we can do that. So after 2001, we can certainly trace it.

BOARD MEMBER BAILAR: Can anyone tell us about the present status of moves to have such a system in the U.S.? I'm sorry Dr. Ferguson isn't here now. I'm concerned, of course, about the possibility that there might be a single case here sometime.

CHAIR PRIOLA: Yes, and I'm not sure anyone here can answer that. That is sort of a USDA issue, not an FDA one, I think. We're about to proceed to the open public hearing portion. Do you gentlemen have a couple of quick questions? You have been standing very patiently. Do you have a couple of quick questions for the speaker?

MR. HAFFENDEN: Yes, Paul Haffenden from TerraCell. I would just like you to comment. Several years ago, the European Scientific Steering Committee assessed Canada and the U.S. as category 2 countries, equivalent risk, given the -- maybe you could comment on the movement, high incidence of movement of animals between the two countries in both directions, high incidence of movement of animal feed products between the two countries in both directions, and then any comment on how you think that might affect the adjustment and category risk now with this case in Canada?

DR. HILLS: I don't think I can discuss anything about how that is going to affect the categorization. I think that is something that somebody else will determine, not me. But what I can say is that given the trade between our two countries, there is movement of both animals and feed across the border.

CHAIR PRIOLA: If it's a very quick question.

MR. BROOKLANG: Yes, Nelson Brooklang, Ortech International, New York. You made a distinction between federally registered and provincially registered abattoirs and the age of cattle that are processed in those, and the fact that provincial cattle don't get exported. I wanted to ask whether blood byproducts used in the biotech industry, what I am from, like Bovine Serum Albumin transferred, purified from bovine blood could be collected from provincial abattoirs in Canada and sold in the U.S.?

DR. HILLS: Good question. My recollection is that the material itself is not. From the provincial licensed establishments, the provincial government looks after that, and I do not believe it then goes to the federally registered renderers, but I would have to reverify that. I'm not 100 percent sure.

CHAIR PRIOLA: Okay. Thank you very much for your presentation. I will move on to the open public hearing portion.

SECRETARY FREAS: In response to our Federal Register announcement, I have received two requests to speak at today's open public hearing for this afternoon, and the first one is Mr. Wayne Vaz. Would you, please, come to the microphone and make your presentation?

MR. VAZ: Good afternoon. My name is Wayne Vaz. I am representing Serologicals Corporation. We are a leading supplier of animal-based products of the global health care industry. We are based in Atlanta, Georgia with more than 800 employees worldwide. We greatly appreciate the opportunity to be here today to talk about the TSE safety of our bovine products and their critical importance in global health care.

Next, please. Our goal is to raise the level of awareness regarding the pervasive use of bovine products in the production of life-saving drugs and other essential health care products. We want to present the facts according to high safety and qualify of Serologicals' bovine-based products, and we would like to work with the TSE Advisory Committee and regulators to further develop industry guidelines to assure the continued availability of bovine products.

Next, please. Serologicals is a global provider of biological products and enabling technologies, which are essential for the research, development and manufacturing of biologically-based life science products. Some examples of our products include antibodies, cell culture supplements, such as bovine albumin and other products for diagnostic and research.

Next, please. This is a listing of our bovine-based products. We're focusing on this, because these are the products that are believed to offer a theoretical TSE risk. At present, we have two manufacturing facilities, one in Toronto, Canada, the other in Kankakee, Illinois. We have a third facility under construction in Lawrence, Kansas.

Next, please. Our bovine-based products are used in the development and production of life-saving FDA-approved drugs, FDA licensed diagnostics, medical devices and animal vaccines. Our bovine-based products are used in the development and production of FDA approved biologics for treatment of cancer, arthritis, Crohn's Disease, psoriasis, blood clotting disorders, spondylitis, RSV and at least one genetically predisposed orphan disease.

In the diagnostic area, our bovine products are used in the screening of U.S. blood for key viruses, such as HIV and HCV, for screening of cancer markers and in serological testing as a potentiator of blood typing prior to transfusions. In medical devices, our bovine products are used in the production of a medical device that is used during surgery, and in animal vaccine, our products are used for the cultivation of Leptospira, which is used to produce animal vaccines for the treatment of Leptospirosis, which is a worldwide problem in livestock. Also, these bovine products are used pervasively in life science research as reagents in the lab for protein assays and other lab assays like chromatography and electrophoresis.

Next, please. So in April of 2000, we received an update, which was issued to manufacturers of biological products from Kathryn Zoon, the former director of CBER, that essentially says avoid using ruminant origin products derived from BSE-affected countries in the production of FDA-regulated products that are intended for humans.

Make sure you identify all the ruminant materials used in production of the regulated products, and document the country of origin, and make sure you maintain traceability records for each lot. Of course, the purpose of this guidance is to minimize the TSE threat to the public.

Next, please. So this creates some regulatory uncertainty regarding the products under development today that are made using bovine ingredients. Also, there is a risk of current production of approved drugs, which use bovine ingredients. This may lead to a possible interruption to the supply of these biotech drugs if BSE occurs in the U.S. Bovine-based products provide unparalleled performance. There has been a few attempts to replace these products in cell culture, but they typically result in lower productivity and higher costs.

Next, please. I would like to switch gears for a minute and talk about the prion infectivity clearance studies that Serologicals has conducted. We scaled down our manufacturing processes and we used a hamster-adapted strain of scrapie agent as a model to emulate the BSE. Like many presentations before, we used a 263K agent. We spiked known titres of infectivity prior to key process steps, and using serum tenfold dilutions we titrated the infectivity downstream to measure the ability of the intervening steps to reduce infectivity, looking at that in-vivo using hamsters and looking at the clinical signs, abnormal gait, tremors, ataxia or incoordination, also looking at a histopathological examination of the brain tissue to confirm the clinical diagnosis, and the characteristic protease resistance of the transformed prions.

The conclusion of these studies, if you look at our Bovine Serum Albumin product line, our HS product line, looking at four process steps achieving a total clearance of 16 log₁₀, Bovine Aprotinin, a total of 17 logs and EX-CYTE completing one manufacturing step to date, achieving 3.7 log₁₀.

Next, please. So we feel that these prion clearance studies offer some objective evidence that these products are very safe from a TSE risk. In summary, the high safety and quality of our bovine-based products are summarized by the following.

One, many are manufactured from bovine blood, which is recognized as being low-risk of TSE infectivity, according to the World Health Organization and the EC. Moreover, Serologicals use either plasma or serum for added safety. One of the theories is that it is believed that prions may reside in the cellular fraction of blood, for example, leucocytes. We only use bovines that are less than 30 months of age, and they are typically less than 20 months of age.

According to the DEFRA statistics in Great Britain, no BSE reported in cattle that is less than 20 months. It is uncommon for it to happen in less than 30 months. We use only USDA-approved raw materials collected in USDA-licensed establishments. All these products are manufactured within an ISO 9002 registered GMP environment.

We have completed and published prion and viral clearance studies, and this compounded with the similar clearance studies that our customers have completed that are producing these biologics, we also maintain EDQM certificates of suitability, which again is an assessment of low TSE risk. We have a proven track record of safety in global health care.

Next, please. So this is an example of one of the viral clearance studies that we have completed on our bovine product line. Due to time constraints, I won't get into this other than to say that we have demonstrated more that 6 log₁₀ of bovine viruses.

Next, please. So in summary, one, bovine-based products are critical to the production of life-saving health care products. Secondly, manufacturers of FDA-regulated products cannot replace bovine ingredients quickly, easily or economically. The high safety and quality of Serologicals' products is supported by the low TSE risk raw materials that we use, the controlled production and the research studies that we have conducted that demonstrates robust virus and prion clearance ability of the manufacturing process, and our track record of safety and success. We're pleased to work with the TSE Advisory Committee to further develop the TSE risk guidelines covering these important products to permit their continued use.

Next, please. Some considerations. In addition to using low TSE risk raw materials, why not recognize the value of prion clearance studies and let's establish minimum acceptance criteria. Let's have suppliers perform prion clearance studies to provide objective evidence supporting the product safety. Also, why not consider prohibiting the sourcing from countries with a high incidence of BSE, rather than just one or two cases?

And finally, when it comes to setting policy, we would request that the FDA and the USDA carefully weigh the impact to the end consumers, i.e., the patients, producers of biomedical products, which are our customers and supply chain producers like Serologicals.

Next, please. Finally, we would like to leave you with two contacts at Serologicals Corporation. If any of you wish to discuss this further, we would be happy to do that. Our email addresses are listed. Thank you very much.

SECRETARY FREAS: Thank you for your comments. Our next speaker for this open public presentation is Dr. Merlyn Sayers.

DR. SAYERS: Excuse me. Thanks for this opportunity to talk to you. See if you can rustle up my first illustration. It's a brave blood bank of the talks in the immediate shadow of the regulators, so I am indebted to Dr. Hills and to Mr. Vaz for giving me some narrative separation from Dr. Epstein.

No, let's have the earlier slide, please. I'm speaking to you as the CEO of Carter BloodCare, and that's the community independent blood program that meets the transfusion needs of something like 150 hospitals in the Dallas, Fort Worth area. I am also speaking to you as a former chairman of the Blood Products Advisory Committee and as a consultant to this group, and I only make those two comments to emphasize how keenly I appreciate the challenges that the regulators confront and also advisory groups like yours have to confront, as well.

By way of a preface, let me have the first illustration. Something like 40,000 Americans donate every day and their health history, their screening for markers of diseases that are potentially transmissible by transfusion, their subsequent counseling if that counseling is indicated, these elements constitute what is perhaps the largest public health exercise in the country and might even be the largest public health exercise in the world.

Now, bear in mind that those 40,000 individuals that donate originally were some 50,000 individuals, close to 50,000. Of course, many get deferred during the history and examination even before any serological testing is done on those folk. So what happens at a local level?

At Carter BloodCare, at our blood program, last year we registered something like 270,000 individuals, 40,000 were deferred and they were deferred either permanently or temporarily, as I say, even before testing. The majority of these deferrals certainly are temporary deferrals, and they are attributable to medications that those individuals might have been on. They might have a low hematocrit. They might have traveled to a malaria area.

But those temporarily deferred individuals are of particular interest to us as blood bankers, because potentially those individuals are individuals that we could get back to continue their donation to the community's needs. We have been considering what has happened to temporarily deferred blood donors for a long period of time.

May I have the next illustration, please? What is their subsequent conduct? We looked to 500 temporarily deferred donors and followed them for two years, and you can see from this illustration that 58.5 percent returned to donate successfully. 8 percent returned only to be deferred again, but 33.5 percent of that original starting temporarily deferred group did not return.

So for one third of temporarily deferred donors, that temporary deferral is so discouraging an experience that those individuals resist all our entreaties for them to come back and donate again. Now, the likelihood of deferral is obviously proportional to the amount of scrutiny that these individuals are subjected to.

So let's have the next illustration. And certainly, the amount of scrutiny that donors are being subjected to has increased dramatically. You can see the number of questions that donors were asked in 1988 and the number of questions donors are asked in 2003. For those of you that are donors, the donor history questionnaire does not list 160 separate questions. It's probably closer to 40, but each question has become so complex. There are multiple questions. There are questions within questions. There are nested questions. So what we now want are 160 pieces of information from donors.

So where do these increasing scrutinies relate to our consideration for deferring donors who have some geographic risk and our need to potentially exclude them from the donor base? Let's show in the next illustration. The number of donors that we have deferred since the year 2000 now that we have introduced additional scrutiny with regard to deferral for attempts to decrease the likelihood of transfusion transmitted CJD.

You can see that at our blood program in the Dallas, Fort Worth area, we have now deferred something like 3,500 donors, and this sad tally is a significant underestimate of the actual number of donors that have been deferred, and it is an underestimate, because many of these donors are individuals who have paid attention, taken heed of our broadcasts, our announcements, our publications urging them about the new restrictions. We have no idea of what that number is. This number reflects only those individuals who fail to appreciate the new restrictions that we are publishing and who came to donate anyway.

So what sort of contributions might these individuals have made? Let's have the next illustration. The next illustration. Thanks. How many donations have these 3,500 individuals made? Well, they have made something like 13,000 previous donations, and these are individuals. These citizens are now people who are indefinitely deferred. They have obviously made important contributions to the community blood program, and it is interesting that in that conflict of interest questionnaire that was handed out this morning, you were asked if you regarded it as important that citizens affected by decisions are directly involved in the Advisory Committee process. And certainly, I believe that citizens, in this case donors, are important and should be involved, but they frequently do not get that opportunity.

If you were to ask them how they respond to their temporary or permanent deferral, let me show you some of the questions that have been posed to us during counseling sessions when we have spoken to individuals deferred as a result of some geographic exclusion.

Let's have the next illustration. This is what we get posed. What should I tell my wife, my husband, children, my dentist? What should I tell my family practitioner? Where can I get tested? Where can I get treated? Will this affect my medical insurance, my disability insurance, my life insurance? Will this count as a preexisting condition? Should I reconsider having a family?

Let's have the next illustration. Why do the criteria change? Other donors have asked questions along these lines. So if I had been in the UK for one day short of three months, I would be safe? If you're telling me that I can't donate anymore, what are you telling patients who got my blood? Why didn't I hear about this from the military? How many patients have got this disease from my blood? How many patients have got this disease from a blood transfusion anywhere?

Now, these are tough questions and deferral criteria can be debated in the relatively academic climate of these meetings, but justifications for deferral that are acceptable here do not sit well when they are explained to the donor community at a lay level. The donor deferral process is essentially a contributor and an important contributor to all those layers of transfusion safety that we recognize as being valuable, but the process is also responsible for increasing numbers of former numbers whose experience is marked by a sense of frustration and alienation.

And this next illustration shows what has happened to the rate of permanent deferrals amongst blood donors in our community. You can see that between 1999 and 2003, there has been a threefold increase in the risk of permanent deferral of individuals in the community. So just as we are concerned about individuals who are temporarily lost and our difficulty with getting them back, we are concerned, too, about the fact that the rate of permanent deferrals significantly contributed to geographic exclusion is increasing so dramatically.

So in closing then, a few comments. For many deferred donors, there is a credibility gap that our explanations just do not bridge. And to avoid the risk of transfusion safety being achieved at the expense of availability of blood for transfusion, CBER's Blood Action Plan that was promulgated in '97, which addresses increasing the blood supply and removing restrictions to safe donation really needs our enthusiastic support and endorsement.

And then lastly, and this sincerely is not meant as a plea for less safety or a plea for less regulation, let me just say that it is easy to add eligibility restrictions, and there are many recent examples, smallpox, SARS, West Nile Virus, but the more difficult task of lifting restrictions that no longer serve a purpose is a task that also needs to be addressed. Thanks.

SECRETARY FREAS: Thank you, Dr. Sayers. Is there anyone else in the audience, at this time, who would like to address the Committee on this topic before the Committee?

MR. FILLBURN: Charles Fillburn, Nutramax Laboratories. I would like to ask Dr. Hills, does he exclude the possibility that the lone BSE animal that was observed is not due to a mutation? Have you sequenced the gene?

SECRETARY FREAS: Could you come to a microphone, so our transcriber can record the comments, please?

DR. HILLS: Bob Hills. Yes, we did look at the spontaneous possibility, in other words the mutation of PrP gene. What I can say is that we did look at it. We have excluded it right now and for other reasons, I really can't comment. There are some publications coming out shortly with respect to that.

MR. FILLBURN: Do you think it's possible that it could arise again either here or in the United States simply as a consequence of mutation?

DR. HILLS: I --

MR. FILLBURN: The reason I ask is we seem to assume that the only way this can occur is through feed. If that's not the case, then we need to be more aware that some of these restrictions that we're putting on may be overkill.

DR. HILLS: Well, I think there are ways that we can look at the PrP gene to determine whether or not it is spontaneous mutation based on that. Now, whether or not you can determine whether or not you have sufficient testing power to ensure that that one case you found is spontaneous or not, I don't know.

MR. FILLBURN: Okay. I would just like to echo the comments of Mr. Vaz that in how we react to the situation in Canada and our importation of any products really has a dramatic -- can have a dramatic effect on health care in the United States and it may be an extreme overkill. I would like to see the USDA and the FDA be on the same page in how they try to treat this, that they be more realistic about it, and demand more clearance work by processors who may be dealing with these types of products.

SECRETARY FREAS: Thank you for your comments. Do you have a quick comment?

MR. HAFFENDEN: I'll try to keep it really quick. I would like to echo the same comments, the same that was expressed by Mr. Vaz. We do collect, have up until May 24^th collected Canadian origin animal-derived blood products that are sold into the veterinary and pharmaceutical industry. We have collections in Australia and in the United States. These are critical supply raw materials, and I believe that we do need harmony between USDA and FDA on guidance. We also do have an isolated herd, isolated BSE-free herd in Canada and would like to volunteer to participate in a committee than can help to set those guidelines and give some examples of what is really there.

I understand why the USDA in particular and the FDA have to react quickly and close doors, but I believe we need to put the resources shortly to analyzing products on a product by product basis, not a global product entity, and reopen those doors for products that are needed and critical.

SECRETARY FREAS: Thank you for your comments. There will be two more open public hearings tomorrow, and at that time, we will be more than glad to welcome your comments.

CHAIR PRIOLA: This topic is open for discussion by the Committee if anyone would like to make a comment or have any additional questions. I know I have one question that I actually forgot to ask Dr. Hills. You said you ruled out the possibility of this case having originating in Saskatchewan or no, sorry, excuse me.

As a consequence of exposure to CWD or scrapie, as well as as a consequence of a spontaneous event, did you do that based on purely pathological assessment or how did you come to the conclusion that this was not a case of a cow coming into contact with a CWD infected deer or elk, a scrapie infected sheep, especially since Saskatchewan is where the CWD is, right?

DR. HILLS: Yes. That actually was a concern, which is why what we did when we sent the sample over to Weybridge, we actually were asking them to look specifically at the strain that they had in hand and to compare that with the strains that they had and they saw no differences.

DR. ROGERS: Ron Rogers, a little bit more.

CHAIR PRIOLA: Okay.

DR. ROGERS: I just wanted to say that some samples have been sent over to a stacks group in the UK, and like they are doing a differentiation on the glyco-civilization patterns, and so the CWD -- we had already previously been involved with them in some research activities to look at CWD profiling, I guess you have to say at this stage. So this did have some positive material over there already, and so this material also was brought over to sort of see if, in fact, we can get those kinds of patterns.

CHAIR PRIOLA: Okay.

DR. ROGERS: So it's purely at the research level that this was ruled out.

CHAIR PRIOLA: So basically, right now, pathological assessment, and you're doing the molecular assessment of the PrP. Okay. Any other questions or comments? Yes, Shirley?

MS. WALKER: I find that it is interesting that Canada is now moving to provide the restriction to add SRMs after reviewing their new case, and we have just been asked to look at that language in our present policy, so we might be cognitive of looking at and changing our policy too quickly.

CHAIR PRIOLA: Okay. If there are no other questions or comments from the Committee, then I think we can move on to Topics 3 and 4, so the remainder of the afternoon is going to be a general introduction to TSEs and decontamination of medical equipment and facilities. The first speaker will be Dr. David Asher from the FDA.

DR. ASHER: Thank you. Well, it's a great pleasure to open this session on decontamination of TSE agents, which has been developed jointly by the FDA Centers for Biologics and Devices. This topic is presented, next slide, please, in response to a request from this Committee last year for more specific information before members felt comfortable advising the Agency concerning appropriate decontamination of tissue establishments where the TSE agents might be encountered.

Next slide, please. FDA, of course, as part of its mission, is responsible for helping industry to keep regulated products safe and that includes keeping products free of pathogens, today's pathogen of interest, of course, the TSE agent. Properties of the TSE agents complicate those efforts. Thank you. You have already heard some discussion about the context-dependency of inactivation of TSE agents. I expect you will hear more.

Scrapie, of course, scrapie agent has not been completely inactivated after exposure or after drying and then exposure to steam heat. Fortunately, TSE agents are substantially inactivated in solutions of sodium hydroxide, sodium hypochlorite and probably other chemicals. Hence, the World Health Organization consultants have recommended decontamination in health care environments using combined sodium hydroxide or sodium hypochlorite and moist heat. Some other authorities have doubted the need for such harsh chemical treatments, and we would like the Committee today to consider these different points of view.

The situations for which we are soliciting advice today are for products regulated by the Center for Devices, that is instruments and by the Center for Biologics Instruments and Surfaces used in the production of tissue products and plasma derivatives.

There are, of course, somewhat similar though not identical situations that would involve other centers, our Center for Drugs is particularly interested in today's discussion, because, of course, some drugs have components of human or animal origin. We are aware that some of the problems involved in the production of food or feeds are similar, but the contexts are really quite different, for example, of course, sodium hydroxide poses certain problems in proximity to food products.

We would certainly logically expect that some issues of the U.S. Department of Agriculture, which regulates animal slaughter and meat production in interstate commerce and the Environmental Protection Agency, which regulates water affluence, including affluence from autopsy rooms, that they would have relative issues, but this Committee is not advisory to those agencies, and we do not solicit advice for those problems.

Next slide, please. There is no question that contamination of classes of products regulated by the Food and Drug Administration have transmitted Creutzfeldt-Jakob Disease to human beings, fortunately few, relatively few such cases have been recognized in the United States.

Next. One such class of contaminated products is reusable surgical instruments of which a contaminated cortical electrode shown here in pieces is the best known example.

Next slide, please. I am aware of only six cases summarized here in which transmission of CJD has been plausibly attributable to contaminated surgical instruments, and I would note that in none of those was modern cleaning or steam water used to decontaminate the instruments involved.

In addition, at least two epidemiological studies of which I am aware have claimed to demonstrate some association with previous surgery, though most studies have not found that and the association is not particularly impressive.

Next slide, please. You have heard that inactivation by heating of scrapie agent is very much context-dependent. This is a slide of data from Bob Rohwer some 20 years ago demonstrating that scrapie infectivity in aqueous suspension was reduced to the level of detection in less than five minutes at 121 celsius. That was carefully suspended in aqueous solution. Although, at boiling temperatures, although, there was also prompt reduction in infectivity, a reduction, a resistant fraction, that's the term of art for Dr. Johnson, a resistant fraction remained.

Next slide, please. But when dried onto surfaces, infectivity was readily detected even after autoclaving for an hour at 134 degrees celsius.

Next slide, please. Dr. Rohwer, who will be our second speaker in this session, has confirmed that apparent-- or rather, Dr. Taylor who will follow Dr. Rohwer has confirmed that apparent paradox, and found that different strains of TSE agent in similar preparations, at least, appeared to have differences in thermal stability. The conclusion of those studies were that in worst case scenarios, autoclaving has not been validated to decontaminate all TSE agents completely.

Next slide. A number of factors listed here must be considered in deciding whether there is a significant risk that a contaminated instrument might transmit Creutzfeldt-Jakob Disease, including infectivity of the contaminating material, the reduction in activity achieved by cleaning and decontamination, and the root by which a susceptible individual is exposed and Martha O'Lone will talk more about those things tomorrow. Our speakers later this afternoon and tomorrow will address those and other issues.

Next slide, please. Central nervous system tissues for humans as for animals have been consistently demonstrated to be infectious when assayed in susceptible animals.

Next slide. But there is also -- next slide, please. There is also a significant though smaller likelihood that tissues of patients with Creutzfeldt-Jakob Disease outside the central nervous system, and that's not just Variant Creutzfeldt-Jakob Disease, that is typical sporadic Creutzfeldt-Jakob Disease, will have some infectivity found and here are positive tissues listed.

Next slide, please. Of course, fortunately, most human tissues, fluids, excreta have never been found to be infectious.

Next slide. Although, confidence in the negative results is somewhat tempered because of the very small number of samples studied, and the fact that the assays used were animal transmissions, and there does appear to be some species barrier even between human beings and primates, which might raise the level of the limit of detection.

Next slide. Just a couple of examples of really how small the number of tissues successfully studied has been.

Next slide, and the next slide. In human CNS tissues, the mean content of infectivity measured in the NIH series was estimated to be about 10⁵ primate intracerebral lethal doses per gram, but note that one brain was found to be infectious at a dilution of 10^-8, and considering both that and the species barrier, it might be prudent to assume such high levels of infectivity for high-risk human tissues, at least in an occasional patient.

Next slide. Because of the substantial uncertainties attendant to the biology of the TSEs and the properties of the agent effects of cleaning and decontamination, advice offered to public health authorities in the United Kingdom and the USA concerning surgical instruments has not been consistent, and I won't read these. But the UK CJD Incidence Panel has advised incinerating instruments exposed to brains of patients with known CJD where our respected authority, Bill Rutala, whose is going to speak later, has felt that cleanable critical or even semi critical devices in contact with high-risk tissues of CJD patients can be cleaned and sterilized by autoclaving either at 134 celsius or 121 celsius, etcetera. Our proponents of both points of view are present here today, and we really would encourage a discussion of these conflicting points or, at least, apparently conflicting points of view.

Next slide, please. The two FDA centers who developed today's program have generally relied on advice concerning decontamination that came from a consultation convened at the World Health Organization in 1999 published the following year. That consultation was chaired by Paul Brown, who was then the chair of this Committee, and our next two speakers, who are among the most influential of the participants, are two of this Committee's former CDC members, both of whom are in attendance today, were also in attendance at that meeting. The consultation identified recipients of potentially contaminated products as being the group of persons at the greatest risk of iatrogenic CJD.

Next slide, please. And they offered the following general advice. They acknowledged that decontamination is context-dependent and that one method may not be completely effective in all circumstances. Cleaning facilitates decontamination using the best validated methods available, essentially meaning based on actual pilot studies. And then they advised using what we call an orthogonal strategy, that is using two different methods, methods based on different physical chemical principles whenever that is possible.

FDA staff might add that in choosing those orthogonal methods, that a method that inactivates the agent is generally considered more reliable than one that simply removes it, because when an agent is removed, there is always the danger that it can be reintroduced back into the product of interest.

Next slide, please. They recommended single-use to instruments, destroying reusable instruments wherever possible, but they acknowledged that there are obvious situations in which instruments simply cannot be discarded, and that less effective methods than destruction may have to be used.

Next slide, please. The consultation recommended a series of decontamination methods in order of decreasing probable effectiveness.

Next slide. I expect that they recommended six such methods. I won't go through them all here. I expect that they will be discussed by several of the speakers later today and tomorrow, but note that the first four all include the use of either sodium hydroxide or sodium hypochlorite either with or followed by steam autoclaving.

Next slide. The last of the six was to autoclave 134 celsius 18 minutes with the caveat that in worst case scenarios, that is where brain tissue has been baked, dried under surfaces, the infectivity will be largely, but not completely removed.

Next slide, please. And for surfaces or heat sensitive instruments, they recommended sodium hydroxide or sodium hypochlorite at room temperature.

Next slide. A number of other treatments listed here were dismissed as being inadequate.

Next slide. We tend to think of the decisions regarding effective decontamination in these four general categories, and I suggest that it might be useful if the Committee addressed them in this way, as well. The surface of these situations are surfaces or instruments potentially contaminated with either high-risk tissue or lower-risk tissue from a subject with definite or probable TSE, and then the same categories for someone where TSE is not suspected.

Next slide. We're fortunate to have with us today Bob Rohwer, who has already spoken, David Taylor to review the general principles of TSE decontamination and the basis for the WHO recommendations. Unfortunately, Dr. Philippa Edwards of the UK, CJD Incidence Panel, is ill, but she kindly emailed a talk for us, and that will be delivered by Pedro Piccardo, who is an alumnus of this Committee and a most welcome recent addition to our CBER staff, dealing with TSE issues.

Bill Rutala, who is consultant to the CDC on hospital infection control, will share his extensive experience here in the USA. Ed Rau will then report on interesting studies of incineration that he and Paul Brown have been conducting. And finally, today we'll end with Stan Brown of our Center for Devices, and I, who will report some early results of models that we have been studying based in part on the work of Charles Weissmann, who will speak to us tomorrow.

Next slide. Then tomorrow the issue for discussion will be presented for CDRH by Lillian Gill, Martha O'Lone and Charles Durfor, and for CBER by Ruth Solomon and Dorothy Scott. Ellen Heck will review the needs of Eye Bank. Christoph Kempf and Andrew Bailey will represent the Plasma Proteins Therapeutics Association in discussing the needs of plasma processes, including a study that they will propose.

Please, note that, again, although the discussions will doubtlessly be of great interest to other agencies of the U.S. Government, and especially to our Center for Drugs, we do not solicit advice for these other agencies, only for FDA-regulated devices, tissue products and blood products.

Last slide, please. With that, I hope that you find the program that we have assembled informative, and we anticipate having useful comments during our open public hearing, and discussions and votes by the Committee. Thank you very much.

CHAIR PRIOLA: Okay. Thank you, Dr. Asher. Our next speaker will be Dr. Bob Rohwer.

DR. ROHWER: Thank you, and let me begin with an apology to those people who have heard this talk before. It is one that I have given to this Committee actually in an earlier form of it in the past, and have given it fairly frequently over the last few years, but I was asked to do it again just because David felt that the place needed revisiting. And so we'll begin with the first slide. The main points I'm going to make in this talk are that the susceptibility to inactivation of TSEs infectivity is within the normal range for viruses and spores, but that the TSE infectivity is resistant to disinfection or sterilization. That may seem like incompatible statements, but I will try to show you what I mean in just a minute.

The susceptibility to inactivation is an intrinsic property of the agent, and this susceptibility is normal. That's what I'm saying, but the resistance is context-dependent and a property of the environment of the infectivity.

Next slide. The best sources for this at the current time are these old papers of mine in Science and Nature, which have the complete experiments behind the kinetics that I'm going to show you here and this review, which puts it into a larger context.

Next. And then the WHO report, which Dr. Asher just reviewed is also a very excellent source.

Next. I am going to begin by just talking about the inactivation process itself. And this is actual data taken from an activation process with the scrapie agent. This is was a hypochlorite experiment, I believe. I have forgotten now. I put this together so long ago, but the main points are the following.

If I can have the next. We're going to build this slide as we go through it. The inactivation process, one way to think about this is if we think of this is surviving fraction on this axis over here where we start with 100 percent survival, no killing. At 10^-1 survival, we have destroyed 90 percent of the population.

Next slide. So for example here, I mean, here we're starting. If we convert this to a 100 individuals, what we have got by the time we're here on this inactivation curve is only 10 percent of the population left. We have killed 90 percent of the population in these very first moments of exposure, and by the time we get to the second log of inactivation, we're down to one out of 100 survivors. This is just by way of review of things that you probably had in your elementary chemistry class, but we sometimes forget this with time.

Next. And then if we look down here, we notice that 90 percent of the kill occurs during this first tiny interval. Only 10 percent of the kill occurs during this next interval. Only 1 percent during this, a 10^th of a percent, a 100^th of a percent during this interval right here. We're getting less and less effect as we go along. The vast majority of what is happening to this population is occurring right here in the very first moments of exposure.

Next. And this is reflected by this component of the inactivation, which is reflected by this line right here, and the inactivation rate constant for this line is the inactivation rate constant, which is defining the behavior of the vast majority of the individuals in this population.

Next. Next. This line describes a second component, and it is describing, next, a much smaller proportion of the population. About one in 10,000 of the individuals behaves like this.

Next. So what is going on here? We have susceptibility to inactivation as defined by this initial rate of inactivation, is intrinsic to the agent. It is actually less complex and there are fewer controlling parameters. Whereas, over here, this population that is being inactivated at a different rate could be gaining those properties in a number of context-dependent ways, and these are different for each environment and they are much more complex.

It could be due to the container. It could be a factor. I mean, it could be any combination of these factors, as well, but among the things that we have to consider are the container, rough or smooth surfaces, reactive surfaces, porous surfaces, cofactors like fats, proteins, oxidants, reductants, water, air, in the case of autoclaving, buffers, pH, temperature can all affect the inactivation suspension, whether what the source of the tissue is and its composition, the procedure for making it, how it was homogenized, the dose rate, various transfers.

And then we have these kind of procedural problems with making these measurements themselves, which are the accuracy limitations of the assay and its reproduce-ability. This is a real issue in the case of end point dilution titration of TSE infectivity, which is only good to about .3 logs. And cross-contamination is an issue, and it becomes an especially important issue when we're talking about very low levels of survival at the very end of these inactivation curves.

How do we know that this survival isn't due to something that got transferred from here, which has almost 100,000 times more infectivity in it? The way we know is we're very careful when we do these experiments, but it's something that you have to be very careful about.

Next. Next, please, and next. Let's go to the next slide. Click through to the next slide. Thanks. In comparing agent properties, the properties that are intrinsic to the agent are reflected in the initial rate of inactivation. That is when the vast majority is being inactivated, and the interpretation is less complex. The size of the residual fraction is a complex function of environmental parameters, and cannot be used to compare the intrinsic sensitivities of agent strains.

This is where I differ with Robert's perspective that he gave this morning. In other words, I consider these plateaus to be a very important public health and agricultural problem in terms of infection control, but they are not telling us that much about the intrinsic properties of the agent. They are telling us about the context of the agent. They are telling us something about the context of the agent, and it has been very difficult for us to figure out what that is.

Next. So let's consider an example from chemical inactivation.

Next. This is a hypochlorite inactivation in which we can see that. In the scrapie curve right here, we have -- this is surviving fraction, the same kind of curve I just showed you minutes ago down here, that on contact with hypochlorite, .5 percent, this is a normal concentration, which bleaches use, and we get an initial very rapid killing down to about 3 logs, but then we hit a plateau and there is at the level of 1 part per 1,000 or a 10^th of a percent, we have got something in this population that is protected from hypochlorite. It's not seeing it.

By the way, we checked. The available chlorine did not change significantly over the course of this infection. We did this same experiment with some controlled viruses. These are bacteria phages. They are non-involute viruses. They are very easy to assay and they are reasonably robust in some ways and not others. But here is PhiX 174 showing the exact same phenomenon. It plateaus at a lower level. FD and M13 like phages doing the same thing over here.

Here are these two. These two viruses were spiked into the same kind of normal brain homogenate that the scrapie brain was in, and they exhibited this behavior in a purified form in PDS. They were inactivated to the limit of detection almost instantly on contact with bleach. Another example of context.

Next. Next, please. Well, two of the things that we are going to discuss here are the things that work best for TSE agents, and bleach is one of them, and I hope that David will be sharing. He has a lot more data on bleach than I do, and I hope he will be sharing that with us. But sodium hydroxide is something that I have been pursuing for a long time, and this was an experiment a long time ago with Paul Brown, one of our initial experiments, comparing CJD and 263 scrapie.

Again, in a very highly dispersed 10 percent brain homogenate of these two infectious agents, and adding sodium hydroxide at these concentrations, and I would just ask you to concentrate on these first three lines here. At 60 minutes with one normal, we had limited detection killing here and here for both CJD and scrapie. By 15 minutes, we had almost as much inactivation. A 10^th normal did almost as well as one normal. It's a very effective method.

On the other hand, next slide, please, this is a table of -- it's now out of date. There are more experiments would could be put on here, but at the time I made this, these were all the sodium hydroxide experiments that were in the literature, and we got very, very good inactivation by sodium hydroxide, but there are examples here. These are the experiments that we had done, at that time, but there are examples here where there is some activity left after considerable amount of exposure, and that always was very puzzling to me, but my guess is that it has to do with how the stuff is presented and homogenized.

Next. I went back and revisited. We have revisited this subject with a kinetic experiment on sodium hydroxide, which is presented here. And in this case, the black circles indicate infectivity, and this is time of exposure, and we're seeing something that is very similar to the sodium hypochlorite effect, except much more dramatic even. We're getting a huge reduction on contact, essentially, with sodium hydroxide. This is the point that was taken in the shortest amount of time, interval, that we could effect between adding the sodium hydroxide and then adding the acid to neutralize it, and then taking the points. So it's right around 30 seconds. This is two minutes, etcetera.

But on this same curve, I have got two other plots. One is a plot, which I am labeling denaturation in one hydrolysis. And what do I mean by that? Well, we went back later and used a Western Blot on these samples to see whether we could recover Western Blot signal or not from these various fractions. And the Western Blot, especially at the time that we did this, was not as sensitive as the infectivity assay, so we couldn't detect it over as long a range, but it was very clear that upon contact with sodium hydroxide, we destroyed the pk resistance of PrP. It was gone. It was showing the same inactivation kinetics, essentially, as the infectivity.

Whereas, if we didn't pk digest and we just put the stuff on the gel to see whether there was anything left, it also disappeared, and this is a disappearance by hydrolysis. The protein is being hydrolyzed. It no longer shows up on the gel, and it is showing quite different kinetics. So one of the points that we can take home from this is that to the extent that infectivity and PrP are related and the prion protein are related, and I am not entirely convinced of that, but nevertheless, to the extent that they are, it's denaturation that is the correlate with inactivation of infectivity not hydrolysis. This is basically good news, because it's much easier to denature something than hydrolyze it.

Next. Heat inactivation will be the next topic.

Next. This is a -- I have just taken the 121 degree autoclave experiment out of that family of curves that Dave just showed you and that I showed you earlier in the day, because it makes the points best in my opinion. Here is a case where, at the time we did this experiment, the story was that you couldn't kill this stuff with autoclaving, you know, that 121 degrees was not sufficient to destroy the infectivity from 263K hamster scrapie.

This is a kinetic experiment showing that, and this was done not in an autoclave. It was done in an oil bath. The samples were sealed in ampoules. They were plunged into the oil bath, so that we could control their -- and I was using thermistors, at that time. The temperature was being recorded, so I knew when they got to 121 degrees. I knew what the ramp time was. I had that on my recorder, and we could control the actual time of exposure within very narrow limits.

So this first point on this curve was taken after the 58 second ramp time to 121 degrees, so it had just got there. By the time it got to 121 degrees, we had already destroyed 99.9999 percent of the infectivity in that sample. On the other hand, it took another 10 or 15 minutes to get to the limit of detection of the infectivity. There was a residual population that took longer.

And this is a concern, and this was a very highly dispersed sample again of infectivity, and you get quite a different story, next, please, if you do this type of experiment. Now, I think David is going to be showing a lot more of this in a few minutes, but later on, David Taylor started doing these experiments using brain macerates. Now, this is not a homogenate. This is a mush of brain. It is not dispersed in fluid. It's a paste, basically, and it is being exposed at these various temperatures in the autoclave, and this is the untreated sample, and these samples are getting incomplete killing even after these very extreme treatments. I mean, this is quite extreme for steam inactivation.

Well, you definitely have to say that this poses -- you wouldn't want this on your scissors when they go back into the next patient, for example. This is an extremely important public health result. On the other hand, what does it tell us about the agent in what we're dealing with? Well, you can get a titre out of this, because you're at limiting dilution here, and we can do something we call a parson, we get a parson titre out of this type of sample. This is how we make our measurements in low titre blood samples. And I have done this on the next slide and just put these figures on next. This is where these samples would fall on this curve that I just showed you. There has still been an extremely high level of inactivation associated with these, but you have got survival going all the way out to 134 degrees here, at 134 all the way out to 60 minutes for some of these samples that were done in these macerates.

Personally, I think we're talking about the same story here. It is just a matter of what we're talking about, and the context has been ramped up in the case of macerates versus homogenates, and the survival lingers for longer periods of time.

Next. Next, please. So what are we dealing with here? These could be intrinsic differences, and that was a question that came earlier in the day from Dr. Bailar and it's a legitimate one. I think it needs more study. A lot of us have this on our books. Robert said he has been planning to do this. I have been doing it. I have got these samples in the freezer. I just have not gone back and redone this experiment, redone the kinetics on these, but it is on the books. Someday, it will get done.

But when you talk about these heritable differences, the point that I want to emphasize is that I feel that they have to be discussed on the basis of inactivation rate, not residual infectivity. And my own prejudice is that the rates will be exactly the same, because what we're dealing with here is context, not intrinsic differences.

Aggregation is another issue. This is something that I was very interested in early on in my career, but I think we have this under control at the moment with the way we are homogenizing and dispersing things, and aggregation would give you a recognizable difference in the inactivation kinetics. It would not look like first order. It would be first order with a delay. There would be a delay in something like that contributing to that.

The most likely reason for this, in my opinion, is compartmentalization. The inactivant is not actually reaching the infectivity, and our challenge before us is to find ways to open and destroy this compartment to get at the infectivity.

Next. I just have a couple more here. So if we compare these two moduses of investigation, what we are using is 10 percent homogenate sonicated highly dispersed versus whole brain macerate. This is sealed in a serum bottle. I can't remember, David. Do I have this wrong? I think David will correct me if I have got this not exactly correct here on how he has got these set up. We are using an oil bath versus an autoclave. Our samples were being constantly stirred while we were inactivating them versus static. And, in fact, this is kind of an idealized type of inactivation to get at the properties of the phenomenon. Whereas, this is a worst case scenario, which gets at the worst case problems that might be confronted in the public health or agricultural context.

Next. Okay. Now, we had some dry heat data earlier in the day from Robert Somerville, so I'm not going to go over this, except to say that if you dry this material onto a surface, the inactivation properties become completely different. It becomes much, much more resistant to inactivation. However, this isn't a completely unfamiliar phenomenon. It happens with spores and it happens with other microbes, as well.

Next. And so, in fact, my own interpretation of this in a nutshell is that what is happening in these experiments and where the source of residual infectivity may be coming from in our ampoule type of experiments is that as we stick our ampoule into the oil bath, it boils and flashes off immediately, and we throw things up on the walls and they dry. We get little specks drying on the walls. I was very religious about trying to recover everything when I went back to reanalyze this material, so I scraped the walls and got everything back into the test tube.

And what if what is happening is we have the infectivity in a form in which it is basically anhydrous. We have little drips and drops here that end up in little droplets of fat. Fat when it is oxidized becomes a varnish, which is, essentially, a plastic. And so, basically, what we're subjecting this to is a dry heat sterilization at the rate of parts per million in our case. It's not something that's happening very often, but we create a dry heat environment for a very small part of this infectivity, and that is what is escaping. If the reagent can't kill it, if you can't reach it, you can't kill it.

The other example I like to give is that if you put brain homogenate in a Zip Lock bag and throw it into one normal sodium hydroxide, nothing will happen to that either. And so it has to be available.

Next. Next, please. Next. Not that one. That's not supposed to be there. So the point I want to make here is that 132 degrees uses a significantly higher temperature than 121 for steam sterilization where the inactivation takes place in minutes or even seconds, but 132 degrees is only incrementally more effective than a 121 degree centigrade environment for dry heat sterilization where the inactivation takes hours to days at those temperatures depending on what you're talking about.

So this does form, I think, a rationalization for what we're seeing in this situation, and it also tells us -- and this was the rationalization for trying to remove all headspace from those devices in which we did the gelatin inactivations that I showed you this morning. We didn't want any opportunity, any place for drying to occur.

Next. I think there is just two more. Steam sterilization, the agent is not intrinsically resistant to steam sterilization. There are problems with delivery.

Next. And for effective delivery, we recommend surfactants, homogenization, high levels of dispersion, eliminate sanctuaries, agitation is helpful. My guess is that a refinant will also reduce the potential for protective associations and will improve the ability to inactivate.

Next. Prevent drying, immerse in water prior to enduring steam sterilization and combine two or more methods. And the processing details can be critical. Adhere closely to validated approaches, and this is referring to this stainless steel result we'll hear more about tomorrow.

And where we should go with this. We need to know more about the underlying principles of resistance, and we definitely need more robust methods for sterilization, which will actually get at these last little bits of infectivity.

Why don't you end right there, and let me just end by saying that the way we inactivate in the laboratory, our own instruments, is for stainless steel and things that can take it and things that are recycled and go back into animals, immediately after use they go into one normal sodium hydroxide. They are immersed in one normal sodium hydroxide for at least an hour, and then if they can take it, they are put through the autoclave under one normal sodium hydroxide. They are cleaned after decontamination under those conditions, and then they are reprocessed in sterile packs back into the facility for further use.

CHAIR PRIOLA: Okay. Thank you very much, Dr. Rohwer. Are there any questions before we move on to Dr. Taylor? Okay. If there are none, we'll go on. Oh, I'm sorry. David, go ahead.

DR. ASHER: Can you comment on aluminum vessels, please?

DR. ROHWER: I didn't hear that.

DR. ASHER: Aluminum vessels.

DR. ROHWER: I still didn't hear it.

DR. ASHER: Can you comment on the use of aluminum vessels?

DR. ROHWER: Oh, yes, right. We use a lot of sodium hydroxide in our environment, and we learned early on that you don't mix sodium hydroxide with aluminum. And, in fact, aluminum and sodium hydroxide in an autoclave can explode and can be quite dangerous, so you have to be very careful about that. So we, essentially, have no aluminum in our BL3.

CHAIR PRIOLA: I think I'll check our BL3. I'm not sure if we have aluminum. Dr. Taylor, if you would? Our next speaker is Dr. Taylor, and he is going to talk about decontamination of TSE agents and the WHO recommendations.

DR. TAYLOR: Thank you very much. Well, thank you for the invitation to speak this afternoon. As you can see, coming from the UK, I'm using thumb roll technologies, slides and overheads. I was warned there could be problems with the electronic system, so I didn't bother with the front-line. I just brought the backup.

As has already been discussed and as this group will appreciate, there has been accumulating evidence over decades that TSE type agents are remarkably resistant to a wide variety of decontamination methods, which are quite effective with conventional microorganisms. This does not mean to say that these methods have no effect, but rather that they are impractical for usage in medical settings, etcetera. These include things like strong oxidizing agents, phenolic disinfectants and even ionizing radiation.

Because of this general resistance, there have been some known examples of iatrogenic transmission where instruments or devices that were in contact with the brains of CJD infected individuals went on to cause accidental transmission in subsequent patients despite having been processed in some fashion or another.

Now, I use the phrase in some fashion or another advisably, because the methods that were used would not, in fact, be used nowadays, but David Asher showed you the x-ray of implantation electrodes, which would be put into a marmoset to look for infectivity after this was suspected of causing this disease in humans through accidental transmission. In this case, the electrodes were washed in benzine and in a well meaning exercise to try and sterilize them, they were then exposed to alcohol and formaldehyde, which we now know is not terribly good as far as TSE agents are concerned.

The second example, which David Asher also listed, was instruments used on a suspect case of CJD, neurosurgical instruments, I should say, were exposed to hot air, 180 degrees centigrade, for two hours before reuse, and there was transmission from patient to patient.

Now, as I said, and as David Asher referred to, there is actually no convincing evidence that we have seen accidental transmissions through neurosurgical instruments, but some data suggests epidemiologically that there is perhaps some evidence of this, but there is no hard and fast evidence.

Nevertheless, with such dreadful diseases that are incurable, untreatable, there has been a constant nagging doubt about transmission of CJD. To some extent, this was aggravated when Bob Will reported in the UK the emergence of Variant CJD. As you know, the number of cases has risen into the hundreds now and is still mainly confined to the UK. The worrying aspect of that, of course, was that the work of Moira Bruce clearly demonstrated that the agent causing Variant CJD was identical to the BSE agent in cattle and quite dissimilar to any other TSE agent that had ever been discovered.

Concerns regarding accidental transmission of Variant CJD between patients was elevated by the finding that New Variant CJD lymphoreticular system tissues in the patient examined, infectivity or at least positive PrP was detected with 100 percent of these samples compared with nil percent of the iatrogenic sporadic cases that were examined or in other controls. That was a study here.

We also know that in a limited number of studies, if you have archival tissue from patients who end up with Variant CJD, in this case, appendix. You can find PrP in the appendix at the time when the patient had no clinical signs of disease. So the potential for accidental transmission through surgery is, at least, in theory enhanced by the fact that surgeons compared with neurosurgical would much more commonly be invading lymphoreticular tissues either deliberately or incidentally.

Now, I would like to just show a few overheads if I may. Both David Asher and Bob Rohwer referred to the WHO meeting in 1999, which resulted in guidelines being issued. It was related to not only clinical aspects of CJD-like diseases, but also to concerns for the practical issues, such as protection of laboratory staff, pathologists, surgeons, etcetera.

Now, within the guidelines, there is this table here, which you may not all be able to see, which is almost a short form of what I started with, talking about ineffective methods. And the only thing I would say here is that I will go in to talk a little about this procedure here, which is regarded as variably or partially effectively boiling in 3 percent sodium dodecyl sulfate, SDS, because this has been commonly banded around as a probably relative effective procedure.

In terms of the actual processes recommended, and David Asher did show you a summary of this, incineration, I will say nothing about, because there will be something said about that coming up shortly. These procedures, they were based on what was known from the literature on TSE inactivation at the time of the meeting. To my knowledge, not much has happened since then to alter the views and recommendations in these guidelines, and they are listed in their perceived order of effectiveness.

So we start with emersion in sodium hydroxide and heating in an autoclave, as opposed to going on here to immersing in hydroxide then transferring into water and going on to autoclaving. Also, here, the alternative is to immerse in sodium hypochlorite, and then going on to autoclave.

Here, we have emersion in hydroxide or hypochlorite, and then going into an open pan and then autoclaving. This is because one of the options here is the 134 degree centigrade porous load cycle in which you cannot put fluids. So if you're putting instruments through these after the fluid treatment, you must remove them from the fluid.

We then go on to suggestions for boiling. These are listed in order of decreasing perceived effectiveness, bearing in mind that WHO recommendations are, essentially, for the health community worldwide, and that facilities and equipment availability will vary tremendously, especially in some more deprived areas of the world.

Finally, we go on to talk about autoclaving at 134 for 18 minutes. And then when it comes to things like surfaces, we revisit procedures like sodium hydroxide and sodium hypochlorite. Then you can just do thorough cleaning if you can't do anything else. And then there are some questions about dry goods and autoclaving.

So I have been asked to address or discuss with you the data, in a sense, that we use to back up these recommendations, which I will do and finish with one or two bits of additional, perhaps anecdotal information.

Will you go back to the slides now, please? I did mention that I would talk briefly about SDS, because simply boiling in sodium dodecyl sulfate or concentrations as low as 3 percent has been widely, well, fairly widely recommended as a very effective procedure. However, in our own experiments where we used 5 percent of this compound and even went on to autoclave at 121 degrees centigrade, we certainly did not completely inactivate.

Now, we did get down to almost a limiting dilution. In other words, we have reduced infectivity probably in the region of 10,000 fold or something like that, but within a medical care context to have surviving infectivity at this level would be a concern. And so I present this simply to discuss an idea that hot SDS is a universal panacea.

Now, Bob Rohwer discussed with you his hydroxide data that he co-published with Paul Brown in 1986, I think, and he also showed a list of, if you like, some contradictory data. These are publications, which are all saying much of the same thing, and that is that sodium hydroxide looks to be pretty effective, but not completely so. A suggestion is that you are knocking down infectivity, because this is at room temperature, by the way.

And the one comment I would make to Bob about certainly our experiments compared to his, I can't talk for many of the others, but clearly, you found complete inactivation, but we didn't know it. It's acknowledged in your paper that the sensitivity of your assays were slightly reduced because of the toxicity of the hydroxide to the examples. In other words, you diluted these to make them so that they could be tolerated by the hamsters.

In our own experiments, what we found is that if we fiddled around considerably, we could actually neutralize, get the pH down to neutral in the end products, and provided they were injected very quickly into the brains of mice, we didn't need to dilute. So there is a slight difference in sensitivity between the tests. I'm not saying that is necessarily the explanation, but it is possibly so, because we do have a solid bank of data saying cooled hydroxide is not completely effective.

Right. In our own studies, what we found was that after exposure and, again, room temperature, this is a hamster agent, one molar hydroxide, two molars for two hours. We brought the infectivity level something down, certainly, but we were left with about 4 logs of infectivity.

Now, if we combine the hydroxide treatment with heat as has been recommended, then, in fact, we find complete inactivation either when you add hydroxide to the samples and immediately autoclave or when you hold in hydroxide for an hour and then go and autoclave. And in other studies, such as those from the Rocky Mountain Lab, they found that if you held in hydroxide, then neutralized the pH and went on to autoclave, you still got inactivation.

And these are the various publications, which all come to the same viewpoint, somewhat unusual in TSE studies to have so many publications saying the same thing, that hot hydroxide is effective, whether this is a sequential process or whether the hydroxide treatment is at the same time as your autoclaving.

Now, in terms of sodium hypochlorite, which is one of the recommended procedures, we did some studies quite some time ago with two strains of mouse agent exposed to sodium hypochlorite containing various concentrations of available chlorine, and we found that once you got up to about 8,250 parts per million of available chlorine, you had a complete effect.

Now, the data here, and these were studies that were done on behalf of the Department of Health in the UK some time ago, and being extremely conservative, the Department of Health accepted the data, but said well, to play it safe, we'll make the recommendation that you should use sodium hypochlorite containing 20,000 parts per million of available chlorine, which considerably exceeds the lowest levels of efficiency here, but that's where the recommendation came from to use 20,000 parts per million.

Somewhat later, using two sources of BSE infected cow brain, we tested sodium hypochlorite once again. Alongside it, we also tested sodium dichloroisocyanurate, which is another chlorine releasing compound, which is generally considered to have a comparable efficiency compared with hypochlorite when compared at the same levels of available chlorine.

In these studies of these various concentrations of available chlorine, there was no infectivity detected in any of the BSE cow brain samples treated with hypochlorite. But when you looked at the samples treated with the dichloroisocyanurate at comparable levels of available chlorine, there were, in fact, a significant number of positives.

This came as rather a surprise, but we found then by doing assays on the chlorine content left after the exposure periods, that the sodium hypochlorite compared with the dichloroisocyanurate, if you look at the starting and finishing concentrations of chlorine, the hypochlorite much more readily gave up its available chlorine during these decontamination procedures compared with this compound. It may be that longer exposures might be effective, but we are already up to two hours, which it's getting a bit impractical to extend things beyond that.

Mention was made of boiling. Well, we certainly do have data, which have only actually ever appeared in an abstract sort of meeting. They have never been formally published, but we did find with 301V, that if you boiled for one minute, that we have no detectable infectivity left compared with material exposed to hydroxide at room temperature or microwaved for one minute.

Bob mentioned the data produced based on 134 to 138 degrees centigrade porous load autoclaving. This was in either BSE infected cow brain, scrapie infected sheep brain or scrapie infected hamster brain, and we had survival rates as shown here, which, as Bob suggested from this graph, fall pretty far down on his survival curve. And, indeed, when we titrated, the starting titre here again was 9 and a half logs. It came down to about 2 logs or less. So substantial inactivation, but, in fact, in terms of health care, still a worrying amount of infectivity left.

In terms of more recent studies using 301V, we had really surprising data for this experiment where we autoclaved either at 134 or 138 degrees centigrade for these periods of time with these weights of tissue. Now, the norm is, of course, as you increase autoclaving time and/or temperature, you expect the efficiency of decontamination to increase.

In these studies, the reverse was true. In fact, we had more cases of TSE in the case injected with the samples from the 138 compared with the 134 process, which was statistically significant. If done on a one off basis, I would have had severe doubts about the technical quality of our experiments here, but, in fact, we had other experiments running at the same time, which showed the same trends, perhaps not so impressively as here, but definitely showed the same trends.

Also, there were studies being carried out on behalf of the Department of Health who insisted quite correctly that all of the equipment and the processes should be independently monitored. And so we had a third party monitoring the progress of these experiments, thermocoupling of blanks for every single stage of the process. And there are some of you that know there is still, I think, a T-chest full of trace-outs for all these experiments. So I have no doubt that we're seeing a genuine trend here.

I mentioned that we're using 301V and we do know, as Robert Somerville mentioned this morning, that 301V within the spectrum of the agents that we have tested is certainly far more thermostable than others. These strains here are all most precise scrapie agents. 301V is our most precise BSE agent. There is a survival after autoclaving and the blue bars are the untreated samples. And you can compare the titre losses with the different agents after the autoclaving process.

My take on what was happening in these experiments was that in the past where we found much more efficient inactivation or in some cases, complete inactivation, we often used intact pieces of brain tissue. In the more recent experiments, as Bob Rohwer said, we were using brain macerate. This is undiluted brain, which is just mixed up, so it's a homogenous sample to give you a blancmange like material for autoclaving.

Now, in putting these samples into what Ron described as long neck tubes, not terribly long neck, but there is almost inevitably some smearing and drying of the infectivity onto the tubes before you get to the autoclaving stage.

My concept, my take of what is happening and what explains the results is that during the porous load autoclaving process, which I must tell you, unlike the gravity displacement system where there is usually a slow buildup of steam, the porous load system involves a huge and rapid admission of steam into the chambered autoclave, which, in my simple hypothetical structure here, is able to fix any proteinaceous material in these fringes, and that paradoxically, if that protein is PrP protein, the actual fixation process, which occurs early and rapidly at the beginning of the steam process actually protects that infectivity from the subsequent sterilization of the steam effect.

If that was so, that would explain why the 138 degree samples were more positive than the 134 since you would expect the rapidity and efficiency of that heat fixation to be greater at 138 compared to 134. I hope to show you in the next few slides that this is not all quite cuckoo land.

We do know that if you fix infectivity or fix infected tissues with formaldehyde, you make that infectivity colossally more resistant to inactivation by autoclaving. Here, we have 50 milligram fragments or whole mouse brains that are infected with the strain called 22A, fixes in formalin and then autoclaved. And, in fact, 100 percent of the recipient animals have gone down in disease. Whereas, in these experiments, we were completely able to inactivate infectivity in these samples if they were simply emerged in saline.

Similarly, if you immerse infected mouse brains in ethanol, another protein fixative, and then autoclave, you get remarkable survival of infectivity even though ethanol fixed in autoclave, 100 percent recipient animals going down. So there is clear evidence that if you fix the PrP protein by whatever means, you, in fact, stabilize it to the extent that it is not normally taken out by the standard autoclaving procedures that we're looking at.

And to test the hypothesis a bit further, we picked up on the experiments of David Asher's going back, I think, to the 1980s. I think he was among the first to observe that with scrapie-like agents, if the materials are dried onto surfaces, they become extremely difficult to inactivate.

Here, we have an infected brain homogenate simply autoclaved and then injected into mice, and in this case, one of the eight animals went down. If on the other hand, we took the homogenate, dried it onto a slide, autoclaved it and then reconstituted it, scrape the top of the infectivity again and try to challenge animals, 100 percent of these recipient animals went down.

That, again, would be compatible with the idea that this thin sheet of material on a microscope slide in autoclave would be subject to very rapid and efficient heat fixation. And there is one more experiment that we carried out with this in mind where we knew that dry heat at 160 for an hour would not inactivate the agent, but that autoclaving without any other processing was effective. We dry heated, which would heat fix, and then autoclaved and, again, we had substantially more survival of infectivity.

So my interpretation is that the effects that we're seeing of smearing and drying of tissue in tubes in autoclaving experiments may well be down to heat fixation. I make no apology for the fact that many of the experiments that I have done have used brain macerate and not brain homogenate, that these have all been more scarce conditions, because many have been driven by public health concerns funded by the Department of Health who do actually want to know what happens under worst case circumstances that could reflect conditions relating to tissues dried on instruments, etcetera.

In terms of concern over instruments, the Department of Health has funded quite a number of studies relating to decontamination, disinfection. They are quite interested in the combined hydroxide and heating effect, and one of the concerns is what effect does this have on stainless steel instruments and devices?

So one of the studies being carried out in Edinburgh is to look at test pieces made of stainless steel before and after various hydroxide treatments. It is mainly facilitated by collaboration with the engineering department who have a scanning white light interferometer where you can compare the roughness indexes of surfaces before and after various treatments. It will print out different graphs giving you the roughness indexes.

And here, just to the naked eye are test pieces, which on the left hand side are all untreated. These are different grades of stainless steel. On the right hand side are pieces that have been subjected to autoclaving at 121 centigrade for 24 hours. And as you can see, in some cases, there is hardly any difference, but in some cases, there is a darkening of the testing piece.

As I understand it, this is due to precipitation of chromium salts and what I'm unaware of is whether you can clean these chromium salts off and start again with a pristine surface. This is what was started before I left the unit, so I'm not quite sure of the current state of play.

I will finish off with just three slides containing anecdotal information, which may be of some interest. One is that a low formalin fixed tissue is incredibly difficult to inactivate, and one would recourse usually to incineration for its disposal. We did find that the hot hydroxide process, when applied to infected brain tissue fixed in formalin was, in fact, effective at removing that.

And I will finish up with two more overheads, if I may. One goes back to the GME study, and the figures may have changed slightly, but the principles are nevertheless the same. It was discussed how the superimposing of our sodium hydroxide step, especially to the ossein material that remains after the acidic extraction process where there was a significant amount of infectivity surviving, at that point. If you then applied -- I'm sorry, this should be hydrochloric acid up here. If you then apply .3 molar sodium hydroxide for two hours at ambient temperature, there was no infectivity detectable in the resulting gelatin.

This clearly suggests that earlier studies using infected brain suggested that one more sodium hydroxide is quite effective and not completely so, these studies suggest that when you get the circumstances in an environment such as ossein where you are largely devoid of any extraneous lipids or proteins, that the hydroxide process is much more effective.

And I will leave you with some recent data from a commercial study, which I have some sketch information for you from. This involves a process where raw materials exposed to saturated lime calcium hydroxide, and then it goes on to hot lime at 80 degrees centigrade, here we are, sorry, and thereafter onto even hotter lime at greater than 140 degrees centigrade under pressure, much of the same conditions if not higher conditions than those described that are completely effective.

Now, the thing to be on your mind here is that the pH of the lime, the maximum pH of lime is significantly lower than that of one molar sodium hydroxide. What we seem to be finding here is that after the exposure to saturated lime for three hours at 80, we do have some titre loss. The expectation then might be that when you go on to this very high pressure, high temperature process, that you might lose all the infectivity, but, in fact, you do not.

To me, this demonstrates potentially two things. One is that molarities of hydroxide lower than one molar may not be truly effective under the high pressure conditions and/or separately than any surviving infectivity from this stage, which is carried out at 80 degrees centigrade, the heat fixation, which goes on in here, all the surviving infectivity may, in fact, render it more resistant to inactivation at this level. So these are speculative comments, but they all contribute to the general arguments about heat and hydroxide. And I will leave it there. Thank you.

CHAIR PRIOLA: Are there any questions? Oh, please, Dr. Edmiston.

DR. EDMISTON: I have a comment, which I want to direct to the speakers, the previous speaker and Dr. Taylor, and also a general comment to the members of the panel in terms of how this applies to surgical instruments in the operating room.

I am not surprised that you haven't achieved complete inactivation, because as a rule, it's a general trend we sort of adhere to, is as long as there is biological material present or, I should say, as long as the organic component is still there, it's unlikely you're going to see complete inactivation.

From a surgical perspective, one needs to recognize, and I'm not quite clear on what my colleagues are doing in Europe, but at least from the U.S. perspective, we just don't take surgical instruments and put them into an autoclave. There is a pretreatment facility, which reduces organic comment, and I know the next speakers will address that probably in some detail.

Actually, I am heartened by some of the data you have shown in terms of inactivation, especially in the presence of high organic content. The fact that in these high carbon environments, you are able to reduce the number of viable particles, so I think we need to think about this two step process as we procedure through the next day and a half in that we're just not talking about instruments being directly sterilized. We're talking about a process in which instruments are being rendered sterile by virtue of not only a sterilization process itself, but also the removal of organic material prior to sterilization.

DR. TAYLOR: Yes. Could I make one comment now? There is, at least in the UK, what I consider to be a worrying trend, and that is that traditionally in a very common sense fashion, it was common in wards and even theaters for certain instruments to be washed in the sink before they went on for washing in the Central Sterilization Department.

That process is increasingly being discouraged for health and safety reasons. It's resulting in an increasing number of instruments reaching the Central Sterilization Department with absolutely dried on blood, tissue and whatever, and this is the problem that, I think, you are referring to. The washing processes as they exist at the moment, at least in the UK, are largely incapable of dealing with the situation where you have material that is absolutely dried or baked on.

DR. EDMISTON: And I think the recommendations and this Committee needs to anticipate the fact that that is a problem. Therefore, the recommendations not only from this Committee, but from other professional organizations such as APEC and others would suggest that pretreatment of these instruments is mandatory.

CHAIR PRIOLA: Yes, Bob?

DR. ROHWER: Yes. I am aware that that's how it's done in the hospital setting. I work in a hospital, but the problem that we have with that is the potential for cross-contamination at the level of the cleaning, and especially in a laboratory environment at least, that would be a disaster for us to spread this stuff around in our sinks and cleaning stations before it ever got to the autoclave.

So we want to make absolutely sure that we know that our instruments are contaminated. You don't necessarily know that yours are, and so we want to make absolutely sure that everything is gone before we even handle them, and we do that by going to these extreme measures.

On the other hand, the only thing that I find encouraging about what you do is what standard practice is in the hospital, is that slide that Dr. Asher showed in his introduction. There really isn't any evidence that instruments cleaned and sterilized in the way that is specified by current practice are causing CJD infections, and I think we have to give a lot of weight to that.

On the other hand, I think it's also very important to think about the cleaning step and what kind of potential that poses for having a major accident if you don't contain that particular environment, as well, because I consider that a high-risk environment.

DR. EDMISTON: Well, you need to know that most of us have had a high threshold interest in this for several years, and more and more of neurosurgical instruments are being triaged and actually being treated separately in separate kits. And for the most part, and I will say for the most part, because there are exceptions, are not getting into the main surgical instrument stream.

And we're spending a lot of time and effort with our neurosurgical colleagues to first of all identify potential patients or suspected patients, but overall, I can tell you most surgical departments, most hospitals, will have unique surgical, neurosurgical kits, and this is becoming more and more common for the reason that you just mentioned.

DR. ROHWER: I guess the thing is I would like to know more about how that segregation takes place, because it's not particularly comforting to me to know that the neurosurgical instruments are being segregated. Neurosurgery is potentially the biggest hazard in terms of passaging the disease, so you run through a set of CJD exposed instruments, and then that is followed by a set of cleaned instruments, you know, coming from a normal patient or something like that. How do you assure yourself that you're not getting cross-contamination at the level of neurosurgical instruments in that type of environment?

DR. EDMISTON: I won't go into a lot of detail on this, because I know my colleague over here will discuss it, but when patients are identified, those instruments are quarantined and sequestered, so that they are treated entirely separate from the rest of the general instruments. So that is the policy that most of us have developed over the years in dealing with these suspected patients.

Now, the other issue is well, how about all of the other neurosurgical patients, which you find out about anecdotally? Now, that is an important process to discuss, but in terms of those that we identify or we suspect, those instruments are quarantined and they are triaged and segregated out of the system.

DR. ROHWER: Okay. Can I say anything more? Are you tired of hearing me? I guess my rebuttal to that would be that my guess is that the greatest part of the risk comes from people that you will never, ever identify as even carrying the disease, and that's the greatest part of your exposure. You will never know about it, and what I see a need for is some way to actually effectively sterilize the cleaning environment between uses.

CHAIR PRIOLA: Are there other questions for the speakers? I have one quick one for Dr. Taylor about the experiment you showed where you exposed material to dry heat at 160 degrees, and you had complete transmission. And then you took the material, exposed it to dry heat and then, if I remember, you autoclaved following the dry heat, and that dropped to almost 50 percent survival or you get 50 percent survivors.

What implications do you think that has or does it have any implications for multiple rounds of autoclaving, say wet autoclaving or multiple rounds of sterilization and getting rid of that residual activity?

DR. TAYLOR: It's difficult to answer your question within the context of infectivity dried onto surfaces. All I can say is that I have done one experiment where I didn't make any attempt to smear and dry, but just using standard samples with the hamster agent where after one round of sterilization, and I am quoting figures very crudely here.

In the first round of a standard autoclaving procedure, I lost something like 4 logs, somewhere about there. And when that material was taken and just reautoclaved, the loss on the second round was about 1.7 logs. So the second autoclaving, even under these conditions, was certainly not very efficient, and I suspect would have been even poorer if this had been agent that partially survived after smearing and drying.

CHAIR PRIOLA: Dr. Gambetti?

BOARD MEMBER GAMBETTI: Listening to all these presentations, of course, are very informative. One, however, wish that experiments were available in which decontamination of surgical instruments is monitored under more realistic conditions. For example, one wished that there would be some data on decontamination of surgical instruments used experimentally in a more surgical operation on a CJD brain, and then see how this level of contamination that is a classic level of contamination that you may expect from a CJD brain in surgery, how the decontamination is effective on those particular conditions.

Vice versa, one would like to know how much decontamination is achieved on contaminated surgical instruments after the routine sterilization that the surgical instruments undergo, as I said, under routine conditions. Those are the data that I would like to know whether they are available at all. I have never seen, so I think those would be very useful data to have for this discussion.

CHAIR PRIOLA: Dr. Taylor, do you have a response to that?

DR. TAYLOR: Yes, I have an experiment that I started before I retired, and I'm going to throw the buck right over to Robert Somerville here as my successor. In this experiment, the very question that you're asking was asked. In other words, how realistic or how appropriate are the inactivation we're achieving to real life situations?

Now, we weren't doing neurosurgery on human patients, but we were daily doing surgical interventions within the brains of infected animals. So we took deliberately infected instruments that had been deliberately traumatized into animal brain, subjected them to routine washing procedures, and then proceeded to reuse these instruments again neurosurgically or in subsequent animals.

My take on things before I left, and I haven't looked at the data since, was that even the washing processes in the lab, which were not anything up to the Central Sterilization Department were having a useful, if not complete effect. But by the time we got to reuse of these instruments on animals, they weren't, as measured at that time, producing any significant levels of infections in the animals. I don't know if Robert can add anything to these data or are they still lying buried?

DR. SOMERVILLE: I think they are still lying buried, David. I don't have access to the data at present. What I would say to Professor Gambetti though is that attempts, which I think Professor Weissmann is addressing the Committee about tomorrow, I think model the kind of situation that you are trying to -- the kind of question that you're asking, and that is the implantation of contaminated surgical instruments, stainless steel. Professor Weissmann has already done some studies with contaminated wares and our lab is also hoping to initiate this kind of system with different grades of stainless steel.

One of the problems that you have to appreciate is that surgical instruments made out of various kinds of stainless steel, and that is one of the challenges of actually set up these kinds of experiments, is how you model the different kinds of surfaces that will be involved in real life. But to summarize, I think the best way of testing your question is through this kind of model.

CHAIR PRIOLA: Okay. I think we'll move on to the next speaker who, as Dr. Asher mentioned, was supposed to be Dr. Philippa Edwards, but she has taken ill and is unable to attend, so Dr. Pedro Piccardo from the FDA has graciously agreed at the very last minute to give her presentation. Dr. Piccardo?

DR. PICCARDO: Thank you. Well, obviously, Dr. Edwards could not attend, and yesterday I was given somehow the daunting task of presenting the information that she provided. I will try to do this as objectively as I can.

Next one. Okay. It has been settled already iatrogenic transmission of Transmissible Spongiform Encephalopathies from person to person has occurred in non-Variant CJD, and this has instated already, and here are the numbers that were provided by a publication called Brown and Neurology in the Year 2000. And as you see, the bulk goes to growth hormone treatment and dura mater grafting.

However, there are here five cases implicating which neurosurgery, meaning contaminated instruments, have been implicated. On top of that, we have a few cases following treatment with gonadotrophin, chromium transplants and, of course, electrodes here.

The next, please. However, one of the big problems came when in 1986, Bovine Spongiform Encephalopathy was described in the UK, and the problem became humongous when in 1996, vCJD was described in humans. As you see here, I mean, obviously, there are a numbers of barriers that have been established to try to prevent the transmission of vCJD, the agent, from animals to humans. However, the big question here is humans are being exposed. Humans died with vCJD, and the question is we don't know how many people has been exposed, how many people could be infected.

The next one, please. And, of course, we don't know how many people may be asymptomatic, at this time and carriers.

The next one, please, the next one, the next one. Okay. The next one, please. The next one, please, the next one, the next one. So due to great uncertainties, risk assessment has been considered. What happened? Oh, okay. Here we go. Due to great uncertainties, risk assessment has been considered. The risk assessment has considered a wide range of scenarios.

And why the risk assessment was done? Basically, for two reasons. One was to determine the risk of transmission of vCJD through surgical instruments, and the second one to indicate what measures could be the most effective to reduce the risk.

The next one, please, the next one, next one, next one, next one. The guidance follows the assumption that an average of 10 milligrams of material could remain in instruments, and this information I gather from the document that was provided by the CJD Incidence Panel.

Next one, please. Go ahead again, again. The risk could be calculated for different scenarios, and the effect of different actions could be estimated.

Next, please. Next, please. Okay. Improving the standards of decontamination is one of the main objectives of the UK policy, and single-use instruments have been considered, for example for extraction of CSF, and the idea was to use as much as possible single-use instruments without compromising the clinical standards, of course. And a pilot program was established to use single-use instruments for tonsillectomies.

Next one, please. Go ahead. Why tonsillectomies? Why was this chosen? Because infectivity is present in vCJD in tonsils. I mean, PrP has been found in tonsils from patients with vCJD. The other thing was the relatively large number of operations and the other thing is the young patients usually with long life expectancy go through this type of surgery, and these are instruments that can be identified.

Next one, please. Okay. But there were some adverse reactions. I mean, why there were problems? One was you cannot probably think that the problems raised from the quality of the sets, the surgeon preferences and the other problems were unrelated to the use of single-use instruments. So at this time, there is an audit on this situation.

Next one, please. So what Dr. Edwards tried to convey, the message that she tried to convey with this cartoon is that while trying to solve one problem, you create another.

The next one, please, again. The best decontamination available cannot be guaranteed to remove all sorts of infectivity, and single-use instruments definitely is a situation that is not possible for all kinds of surgery, so we must bear that in mind.

Next one, please. Okay. Here, we have on this panel, a tissue forceps, the tip of a forceps that has been routinely decontaminated. Here, we have electromicroscopy, and this what we can see here in green is material that remains on the tip of that forceps. This is the kind of material that remains. This is florescent staining for protein, and this is the superimposition of these two images gave this image. So, obviously, there is a significant amount of material that remains, a lot of which is protein following routine decontamination.

The next one, please. So to reduce the risk of transmission of TSE from person to person, the Department of Health seek guidance from the Advisory Committee.

Next, please. Next, please. And a first version was done in 1998 and now, there is a revised version, June 2003.

Next, please. This presentation concentrates mainly on the risk arising from the care of patients.

Next, please. Yes, okay. So well, one of the issues is dealing with symptomatic patients. I mean, when dealing with patients with CJD, there are three types of definition. One is a definite case. By definite we mean something that has been clinical and pathologically confirmed. Probable case, which is has clinical, but on top of that usually, there is electron encephalographic analysis and there is MRI imaging analysis, and possible CJD when usually is by clinical presentation.

Next, please. Now, when we are dealing with asymptomatic patients, when we talk about risk in the case of -- when we talk about asymptomatic patients and we talk about risk, we have to consider two situations. One is in the case of inherited diseases, and by inherited diseases, we consider that there are two or more blood relatives are affected by a prion disease or one or more blood relative showed genetic testing, show a mutation in the prion protein gene. Usually what is done is PCR sequence, the open reading frame of the protein, and then from there you can detect mutations.

Next, please. Now, the other is the iatrogenic risk, and this case already was mentioned treatment with hormones, dura mater grafts, and that is why, I mean, obviously, the Department of Health seeks advice.

Next, please. This table is based on what we currently understand about the distribution of infectivity in sporadic CJD or in non-Variant CJD, and obviously, when we talk about tissue infectivity as it has been said already many times, the highest amount of infectivity is here in the CNS or retina and low medium type of infectivity in the eye and olfactory epithelium.

Next, please. Now, when we talk about risk of different tissues in Variant CJD, the situation varies, because we have to introduce into the medium risk tissue lymphoid tissues. The rest remains the same.

Next, please. So we don't have a problem here and we don't have a problem here, because this is by genetic testing or what was said already, is we can know who these people are, and we understand who these people are. But the problem is when we deal with sporadic or when we deal with variant, people that are asymptomatic, at this time, but might have infectivity.

The next, please. Yes, okay. So the problem comes or starts when, obviously, a CJD patient is diagnosed, and immediately the question should be has that patient had surgery or donated blood, etcetera, and then try to assess what is the risk to other patients that have been exposed to instruments that have been used on this patient.

Next, please. And the risk, basically, will depend on the type of tissue that we are talking about, because we said already that there are tissues with high levels of infectivity and in this case of vCJD, the lymphoid tissue corresponds with tissues with medium levels or medium risk.

Next, please. So this graph is an estimate that comes from animal studies, so this is an estimate that comes from animal studies. And, obviously, the paren of tissue infectivity in vCJD probably could follow this, and this is the onset of clinical symptoms. So if we go, let's say that the surgery was done way before the development of clinical symptoms, probably the amount of infectivity will be very low, and because we are dealing with vCJD in this graph, we have two parameters or two tissues to consider. One is the CNS and the other is lymphoid tissues.

Next, please. So, as I said already, the risk depends basically on the type of -- I mean, depends once again on the type of tissue where the surgery is performed, and if there is variable time between surgery and onset of disease. Well, this basically refers to the previous graph.

Next, please. Okay. One of the issues here is that the risk depends -- let me see. In the document provided by the CJD Incidence Panel, it is stated that the first washing and autoclaving would achieve at least a 10^5.4 reduction of infectivity, and this was already mentioned by Dr. Taylor before, and that subsequent cycles of decontamination reduce the infectivity, but it's much less effective.

The next one, please. So at this time, the Department of Health is in discussion with manufacturers of surgical instruments. I mean, the discussion is based on what is the probability of using single-use instruments or to replace parts or to provide instruments that could be easily decontaminated.

Next, please. So what are the aims of the CJD Incidence Panel? Well, it seems to be quite obvious, which is to protect the patients. Let me see.

Next, please. The aims. Go ahead again, again. Next, please. No, yes, yes, here we go. Obviously, the aims are to protect the patients and to inform potentially exposed patients, and to inform the public and to increase the knowledge.

Next, please. So in management of risk, I mean, what is being done is quarantine the instruments during the risk assessment, and instruments that have undergone less than 10 cycles of decontamination should be incinerated.

Next, please. Again, next, please. Okay. So the patients will be contacted to alert them of their possible exposure and to take health protective actions. So these are the patients that should be contacted under these circumstances. If the index patient goes through, I mean, the material went through high-risk procedures, the first six patients that follow that first surgery should be contacted. This is for tissues with less amounts of infectivity, and these are the amount of patients that should be contacted.

Next, please. So this is sort of the kind of data that has been gathered during the last two years of experience, and this is the incidence reported to August of 2002, and definitely we have 39 cases implicated in Variant CJD, 39 cases implicated in sporadic CJD, and that there are few that correspond to familial or non CJD or unclear, cases that could not be determined.

The next one, please. The type of surgery is 131. So obviously, before were 87, I believe. Yes, 87, and now, we are talking about 131 surgeries, and the reason is that some patients went through more than one surgical procedure. And what we see here is that the GI surgery takes the bulk followed by obstetric and gynecology and here we have neurology, neurosurgery.

Next one, please. So in 76 incidents, tracing was sought. Some or all were traceable, that means in 34. In 18, it was not possible to trace them. And in 24, there is incomplete information, at this time.

The next one, please. So instruments that have been quarantined are 48 and that have been not quarantined are 39.

The next one, please. The fate of the quarantined instruments, we have 21 that have been returned to use, because it was assessed that the risk was not higher than the usual risk for the UK population. Here, we have four that have been completely destroyed, the whole panel was destroyed, because it was not possible to identify exactly which of the instruments was involved. And in 23 cases, the hospital directly decided to take care of the instruments and destroy them.

The next one, please. So, obviously, this is a very difficult task and there are a number of dilemmas and difficulties, and there are a number of scientific uncertainties, and it is very difficult to trace back instruments and sometimes patients and, of course, there are ethical issues that are involved.

The next one, please. Go ahead, yes. And these are the websites that Dr. Edwards suggested consulting for further information. I think this is it. Thank you.

CHAIR PRIOLA: Dr. Khabbaz?

DR. PICCARDO: Oh, before -- excuse me, sorry. Yesterday, after I learned that I had to give this presentation, I called the UK immediately, right away, and the first question I asked before you ask me the question was what do I do with -- how do I handle the questions?

So the thing is we will make clear note of your questions, and then we will forward the questions to the UK, and Dr. Edwards has been kind enough to review them and, hopefully, if she feels well enough, to provide the answer tomorrow. So we are in business. Anyhow, if you want to ask the question, go ahead.

CHAIR PRIOLA: So that gets you off the hook, doesn't it, Pedro? Yes.

BOARD MEMBER KHABBAZ: You may know the answer. I had actually a couple of questions. One has to do with the adverse events related to single-use of instruments for tonsillectomy. Do you have any idea what types of adverse events would occur?

DR. PICCARDO: Yes, the answer is bleeding, bleeding. That is the answer to that.

BOARD MEMBER KHABBAZ: Thanks. The second question is I don't think -- I may not have understood you correctly. I think when you talked about the various types of CJD, you mentioned for inherited and iatrogenic not concerned for infectivity?

DR. PICCARDO: Sorry, come again. I mentioned --

BOARD MEMBER KHABBAZ: No infectivity for inherited and iatrogenic versus sporadic and Variant CJD. Is that in terms of how they got it or for peripheral tissues?

DR. PICCARDO: Let me see. Can you pose the question again? I have a problem hearing, too.

BOARD MEMBER KHABBAZ: Okay.

DR. PICCARDO: Listening to the question. Yes, go ahead.

CHAIR PRIOLA: Yes. I think you're referring to the slide you showed where you had sporadic patients, iatrogenic and inheritable. And are you asking if there was infectivity associated with those patients?

BOARD MEMBER KHABBAZ: I didn't understand the statement that there is no infectivity related to inherited and iatrogenic.

CHAIR PRIOLA: Oh, I don't think that's what you said.

DR. PICCARDO: No, well, I would be happy to review Dr. Edwards' slide. However, my answer to that is that there is no difference between inherited and sporadic. We probably put them in the same box. We will do a difference when we deal with Variant CJD, because that is when we have tonsils and we have the lymphoreticular system involved that we tend not to have in sporadic or other forms of CJD.

CHAIR PRIOLA: Right, and I think that was one to identify, patients at risk, right, prior to use of instruments. If someone has inheritable mutation, then that is a patient that you identify as being at risk of possibly transmitting to somebody else. I think that's what that --

DR. PICCARDO: Right, in terms of risk. I mean, if you have a patient that, obviously, comes from a family and has the mutation, etcetera, etcetera, you know that patient is at risk already, so it's very easy to recognize that patient. It's also very easy to recognize a patient that, let me see, that went through surgery that has a dura mater graft. It could be a patient at risk. However, if you say well, let's take sporadic CJD, maybe I am incubating sporadic CJD and I don't know and no one will know.

CHAIR PRIOLA: Dr. Nelson?

DR. NELSON: How would you classify cerebrospinal fluid that, let's say, has lymphocytes or is an inflammatory cerebrospinal fluid? Would that be the same as blood being low-risk or would it be closer to CNS tissue?

DR. PICCARDO: Well, I would like someone else, probably Dr. Asher, to attend that. Before we go ahead with that, Dr. Edwards made clear that they provide disposable instruments for CSF extractions, so now, it's single-use. Go ahead.

DR. ASHER: Yes. In the NIH series, about 15 percent of spinal fluids from subjects with mostly sporadic CJD did transmit disease to primates. So the risk of spinal fluid is comparable to the risk of some non CNS solid tissues, lymphoid tissue, liver, kidney, spleen, lung.

DR. NELSON: But it's definitely higher than blood?

DR. ASHER: Higher, definitely higher than blood.

CHAIR PRIOLA: Yes, Dr. Gambetti?

BOARD MEMBER GAMBETTI: I believe that the experiment that, David, you are quoting included not only sporadic, but also Kuru patients, and three of 37 or so, spinal fluid tested transmitted the disease. Do you know whether some of the CSF that transmitted the disease were actually from Kuru patients, rather than sporadic case? Do you know that?

DR. ASHER: I think it's in the '93 article, but I don't remember.

CHAIR PRIOLA: Okay. I think we'll move on. Thank you, Dr. Piccardo. We'll move on to our last speaker before the break if there are no other questions, and that is Dr. Bill Rutala who is going to discuss TSE agents and infection control in U.S. hospitals.

DR. RUTALA: Thank you very much and good afternoon. What I would like to do very quickly, and certainly by looking at the next slide, review the recommendations on and the practices in U.S hospitals as it pertains to the prevention of cross transmission from medical devices contaminated with prions and, hopefully, have a few minutes to discuss how important methodology is, and how important methodology is from the standpoint that we can fail to inactivate even easy to kill microorganisms like bacteria with FDA cleared sterilization processes dependent upon the methodology that is employed to include the absence of cleaning.

Next slide. Let's begin with the rationale for the U.S. recommendations, and these recommendations have existed for decades, the recommendations in infection control literature, surgical literature, certainly, essential processing literature and so forth.

But let's look at the next slide. As we know as far as the epidemiology of prion transmission, we know that it's not spread by contact. It is not spread by airborne. It is not spread by environment, but we are concerned about the iatrogenic spread.

Next slide. We can see here that contaminated medical instruments have been implicated in disease transmission, and we'll discuss that in just a minute.

Next slide. Well, let's look at this issue of prion transmission via surgical instruments.

Looking at the next slide, we see, essentially, the two confirmed cases that have already been mentioned. Those two confirmed cases, of course, were reprocessed by a method that we never use in U.S. hospitals, a combination of benzine, alcohol and formaldehyde vapor, and then we also have four suspected cases.

Those four suspected cases are involved with CJD that has occurred in persons following brain surgery. However, only one of the four had an index CJD case identified. These cases occurred before 1980 and there has been no known failure of steam sterilization to date.

Next slide. How about the infectivity of human tissue as we discuss this rationale? As we already know by looking at the next several slides, we used epidemiology data and, of course, we used experimental data and infectivity data. We know that there is evidence of transmission via eye and brain from an epidemiology standpoint, and we know that experimentally we can inoculate animals, susceptible animals, and demonstrate that certain body fluids and tissues transmit CJD.

And we have already discussed the contents of the next slide, which is that there are certain tissues that are considered high-risk, certain tissues are considered low-risk and, of course, some tissues that are considered no risk.

Next, we see the issue of removing microbes by cleaning, something that, certainly, we need to discuss a little bit more as it pertains to the methodology and how methodology is so important in this issue of prion inactivation.

In the next slide, we will see, essentially, something that has already been mentioned by one of the panelists. The issue that effectiveness should not consider only the effectiveness of the disinfection of sterilization procedure, but also has to consider the effectiveness of removal by cleaning. And, of course, the probability of a device remaining capable of transmitting disease is related to not only the initial concentration of that prion on the surgical instrument, but also it is related to the effectiveness not only of disinfection and sterilization, but also cleaning.

And there are literally dozens of studies in the literature, which show how effective cleaning is. Cleaning will reduce anywhere from 4 to 6 logs of microorganisms by a manual or a mechanical cleaning procedure. We don't have as much data regarding protein reduction, but there are a few papers in the literature that demonstrates there is, approximately, a 2 log reduction of protein by the various cleaning procedures.

In the next slide, we see the prion inactivation studies. We don't need to go over this very much. We're just going to very quickly go through a few slides. We could possibly put a question mark up here with prions. The question mark I would put up there is related to the fact that maybe the studies that have been done are artifactual in nature and, essentially, a reflection of the methodologies that are employed, and I think I can show you data that would be supportive of that.

And then also, we would see here that other microorganisms fall below possibly prions and spores and it pertains to the susceptibility to disinfection and sterilization procedures. And in just a minute, I'm going to show you some data where bacteria will survive FDA cleared sterilization processes, because precleaning did not precede the sterilization process.

Next slide. We know, of course, there are many procedures that are ineffective or partially effective.

Next slide. We know also that there are some gaseous sterilization procedures and, of course, physical procedures that are also ineffective or partially effective processes.

Next slide. We can see, of course, that there are some effective disinfectants and, of course, by effective here, we're saying a 4 log reduction decrease in the ID₅₀ within one hour and, certainly, among them include sodium hydroxide and sodium hypochlorite.

The next slide will just tell us what the effective processes are as it pertains to sterilization, and this is what we use in U.S. hospitals. We, of course, use sterilization primarily by steam sterilization with a prevacuum sterilizer at 134 for 18 minutes. Sometimes, the combination of sodium hydroxide and steam sterilization is employed, but it's not widely employed because of some of the deleterious issues associated with the combination of sodium hydroxide and steam, deleterious, of course, to the instruments, deleterious to the sterilizer and, of course, the vaporization of sodium hydroxide to staff. But we certainly recognize the effectiveness, and that is an option for hospitals to choose.

Next slide. As it pertains to risk associated with instruments, let's see what we have with that.

Next slide. I just wanted to mention, essentially, that there are certain categories of instruments in every health care institution, not only in the United States, but in the world. There are certain instruments that we consider must be sterile. They are instruments that have contact with sterile tissue or the vascular system. We consider, of course, then to be very critical.

There are other instruments like in endoscopes that have contact with mucous membranes or skin that is not intact, and we have a very high level of disinfection associated with those instruments. And the other instruments are noncritical, only have contact with intact skin and, essentially, are not involved in disease transmission.

The reason for mentioning that is seen in the next slide and that is, essentially, in a minute we're going to develop, essentially, the scheme for how we disinfect and sterilize instruments in health care setting in the United States.

As it pertains to surgical instruments, a question was just asked. What is the microbial load associated with surgical instruments? Actually, we do know the microbial load associated with surgical instruments. A few studies have actually evaluated the microbial load. Of course, it's not for prions. It's for other microorganisms, and microbial load 80 percent of the time is less than 100 organisms. Rarely does it exceed 1,000 organisms. Many surgeries, many different investigators have made that observation.

Next slide. Well, this is how we decide how to, essentially, employ special prion precautions in U.S. hospitals. We, essentially, assess the patient, assess the tissue and assess the device. Of course, we consider whether it's a high-risk patient, a high-risk tissue and a high-risk medical device, again, those critical and semicritical devices.

Next slide. As far as that is concerned, most U.S. hospitals then would do special prion reprocessing, and that would be those higher temperatures or a combination of sodium hydroxide with steam, special prion reprocessing when it's a high-risk tissue, a high-risk patient and a high-risk medical device, and for all other situations with one possible exception, it would just be conventional disinfection and sterilization.

The one possible exception would be a high-risk patient, a critical and semicritical device and low-risk tissue. Some hospitals do treat low-risk tissues from a high-risk patient, critical and semicritical device as instruments requiring special prion reprocessing, so possibly this would go into that category.

Next slide. So the conclusions of this, of course, is that from an epidemiology standpoint, we have two cases of disease transmission that are definitive, possibly four other cases. The guidelines that we have discussed and are used in the U.S. are based upon epidemiological evidence, tissue infectivity, the risk of disease associated with certain medical devices and, of course, inactivation data, and the risk assessment is based again on patient, tissue and device. And only when there is critical and semicritical devices, contacting high-risk tissue and possible low-risk tissue from high-risk patients do we require, essentially, special prion reprocessing.

Now, the next slide, we see, essentially, what those special reprocessing procedures are. We have already mentioned that this is the preferred procedure that many hospitals use, the 134 at 18 and a prevacuum sterilizer. There is no low temperature sterilization technology that is recommended and we know, essentially, that there are some disinfectants that have activity against CJD.

Next slide. So this is what we're talking about here. We're talking about used instruments, and this is true for all used surgical instruments. They are kept wet. They are not allowed to dry. They are, essentially, cleaned before they are sent to central processing. We don't let tissue and fluid dry on them. When they get to central processing area, an area where all instruments are received for a quality control standpoint, the instruments generally go into a mechanical washer disinfector. In the case of special prion reprocessing, there would be a special steam sterilization cycle, and that instrument would be returned to health care.

Now, we have already mentioned, essentially, the rationale. The last thing I want to do is very quickly look at methodology and how methodology affects results. And I am going to show you some slides, and let's begin with the next slide where, essentially, we can fail to kill easy to kill microorganisms by methodological manipulations, and I will call them manipulations, because all we do is we don't add cleaning to, essentially, the process. And, of course, we have already seen how important cleaning is.

Next slide. This is the issue. I don't want to get too involved in this, but, essentially, the point needs to be made that there are a number of studies that have been done, and most of those studies are done in a worst case scenario, of course, and, of course, we try to achieve sterilization by using appropriate reprocessing procedures.

And there are no studies, including the studies that have been published involving cleaning, that reflect the reprocessing procedures in a clinical setting. We use enzymatic cleaners. We use mechanical sterilizers in a closed system and we use mechanical washer disinfectors in a closed system.

Next slide. Now, I want to talk briefly how factors affected sterilization, and many factors affect sterilization, but I am only going to choose a couple. First, let's look at protein and salt.

Next slide. If we just put, essentially, some microorganisms on a penicylinder like this right here, next slide, and then we let it dry for 30 minutes, next slide, and then we put those penicylinders that are inoculated with easy to kill microorganisms, such as E. coli and pseudomonas and enterococcus faecalis in a FDA cleared sterilization process like ethylene oxide or hydrogen peroxide gas plasma, in the absence of serum or salt, you get 100 percent kill.

In the presence of serum or salt, you get 40 percent failure, as well as in this case, 63 percent failure, a significant amount of failure, because cleaning did not precede the sterilization procedure. Now, the amount of salt and serum is not really that high. The amount of salt is .65 percent. The amount of serum is 10 percent, but failure in the absence of any cleaning.

Next slide. If we use, essentially, a lumen device and do the same experiment, next slide, we see, essentially, 60 percent failure. Again, we're failing to sterilize instruments that are contaminated with easy to kill microorganisms, because we failed to clean them. And it's not really the lumen device that is causing the problem, because we see here in the absence of serum or salt, those organisms are killed. So the failure to clean allows the survival of easy to kill microorganisms in a sterilization process, such as low temperature sterilization.

Next slide. So all technologies have limitations. Salt and serum provide protections for spores and bacteria, and salt and serum with a lumen carrier even provides extraordinary protection.

Next slide. Now, let's look at the issue of cleaning and let's look at spores.

Next slide. Let's just put some spores on a stainless steel scalpel and we'll see on the next slide. We're going to put about 10⁶ geobacillus stearothermophilus spores on this stainless steel scalpel, and then we're going to put that scalpel in a low temperature sterilization technology, such as hydrogen peroxide, gas plasma in the absence of cleaning, and we can see complete failure here. 60 out of 60 positive stainless steel scalpels.

Now, let's look at the next slide where the only thing we did is we again put the spores on the stainless steel scalpel.

And then the next slide. All we did was place the stainless steel scalpel into either distilled or tap water for 60 seconds, just placing it there for 60 seconds, taking it out and then putting it into low temperature sterilization technology.

Next slide. You can see that there is complete success, a complete ability to kill microorganisms to include spores just because of a static soak. Of course, you can see here a very light rinse also was successful.

Next slide. Here, we are going to, essentially, try to identify why this is happening, and what we're really looking at is what is going on as far as chloride, protein and spore concentration by just doing that static soak.

In the next slide, we see, essentially, in a matter of seconds we see the salt, protein and spores released from the fetal bovine serum dried on stainless steel blades and placed into deionized water at room temperature. In a matter of seconds, essentially, you get significant reductions in salt, proteins and spores.

Next slide. Now, so what we found is, essentially, inorganic, organic and microbial contaminants on the device are dramatically reduced during washing and, of course, there is a significant reduction of spores.

Next slide. Well, let's see if that is effective for steam sterilization. Right now, we're really talking about these low temperature sterilization technologies. Does the same thing happen with steam sterilization? This is a study from Doyle and Ernst in 1967 where, essentially, all they did was monitor the effect of spore occlusion and calcium carbonate crystals in inactivation in steam dry heat and ethylene oxide sterilization processes.

They were just inoculating 10³ or 8 times 10³, bacillus subtilis spores, and let's see what happened because of the spore occlusion and calcium carbonate. Here, we see steam at 121 degree centigrade in the unoccluded spores, the biological challenge. It only takes 10 seconds. You can kill 10⁴ in 10 seconds, no time. But in the presence of the calcium carbonate, to kill that 10⁴ took 150 minutes. For dry heat, it's three and a half hours. In the presence of the calcium carbonate, it's 50 hours.

Next slide. So, essentially, a number of things have been found, that is contact with water or cleaning for just a short period of time rapidly leads to the dissolution of crystals, of course, removed microorganisms and, of course, also has an effect on protein elimination. And, of course, minimal cleaning eliminates the effects of these salts, which effect the effectiveness of sterilization processes. And simulated use tests that do not include washing would not represent conditions that exist in clinical use situations.

Next slide. And this is, essentially, what you see in electron micrograph. If you look at, essentially, .75 sodium chloride in the presence of spores, you see the salt crystal, essentially, occluding the microorganisms from exposure and, essentially, cleaning dramatically effected those results.

Next slide. The point that we need to make for all these studies that have involved prion inactivation is that you can clean without sterilization, but you never can sterilize without cleaning. That is a point, a principle, that is known to every professional in health care, certainly, every professional that is involved in reprocessing instruments.

Next slide. So the conclusions would be all sterilization processes are effective in killing spores. Salts favor crystal formation and impairs sterilization not only for low temperature sterilization, but also high temperature sterilization. Cleaning removes salts and proteins and must precede sterilization. Failure to clean or ensure exposure of microorganisms to the sterilant could affect the effectiveness of the sterilization process. We say repeatedly if the organism does not have exposure to the germicide or the sterilant, inactivation will not occur. And, of course, these salts and protein materials and possibly other environmental conditions to include surfaces affect that exposure. And lastly, CJD inactivation studies should be consistent with actual clinical practice.

I think we have done what we said we were going to do. We have looked at the recommendations from the U.S. We have talked a little bit about methodology and how methodology affects results not only for hard to kill organisms such as spores, but easy to kill microorganisms such as bacteria.

Next slide. I thank you very much for your attention.

CHAIR PRIOLA: Okay. Thank you, Dr. Rutala. Are there are questions from the Committee or from Dr. Taylor? Would you like to make a comment?

DR. TAYLOR: Just a couple of quick comments. A very nice talk and very much to the point, I think. Two comments, one is mentioned that generally, the washing procedure is usually pretty effective in taking off bacteria and spores, etcetera. The one comment here is that one might anticipate that TSE infectivity might, nevertheless, be somewhat more adherent to instruments because of the hydrophobicity of the PrP protein.

The other comment is that there are concerns about damaged autoclaves by hydroxide, but that is certainly not inevitable, because it depends on the grade. The commercial company that we asked where do you dispose of animal carcasses, in reactor vessels with hot hydroxide, have had vessels running for many years now and they have certainly subjected these to x-ray analysis, etcetera, and they are absolutely fine.

DR. RUTALA: To your two points, I certainly agree with the first point. Certainly, the data that I presented, of course, are non-prion proteins, as well as microorganisms and, certainly, the same type of analysis needs to be done with prion proteins, and I support that work.

In regard to the second point, the effect of sodium hydroxide on sterilizers, certainly some sterilizer manufacturers have threatened the owner of the sterilizer that in the hospital, if they use sodium hydroxide in the sterilizer, they will nullify the warranty, which, of course, affects the utilization of sodium hydroxide.

But there are ways, as you probably know better than I do, to limit that vaporization and contain the vaporization with, for example, containers that have lids. And certainly, Dr. Asher knows more about that, and possibly that can be discussed.

CHAIR PRIOLA: I have one very quick question before Dr. Rohwer makes a comment, and that is you stress very strongly that instruments are always kept wet, so that you don't have this problem of material drying on the instrument and then perhaps adversely affecting its ability to be sterilized.

During a surgery -- I mean, I know that when I do my little surgeries on mice that as you're doing it, stuff does dry on the instrument just as you're poking around, so how is that dealt with?

DR. RUTALA: Well, many times -- it is dealt with in different ways. Many hospitals, essentially, don't even take the surgical instrument and put it on a dry tray. They very commonly place it in a basin, which has, for example, saline or water or possibly even a germicidal agent, so it doesn't go into a setting, which is going to allow dry fluids and tissues to be achieved.

And then, of course, the other issue is that there is sometimes precleaning before it is sent to central processing. Central processing does not want instruments that are contaminated with tissue and blood. They won't accept instruments that are contaminated with tissue and blood. Sometimes, there is also a washer sterilizer that, essentially, is a precleaning procedure before it goes to central sterilization.

So different hospitals do different things, but the one thing in common is there is an effort to keep it wet, and there is an effort to keep it clean, because most central processing areas won't accept surgical instruments that have dried tissue on them or bloody instruments.

CHAIR PRIOLA: You know, I understand that. I guess my point was more during the procedure as you use the instrument. Just as you're using it, it's going to air dry, because it is exposed to the environment, and so you can't keep it wet the entire time. I mean, you're just going to have some dried material that will probably be taken care of possibly by the cleaning and whatnot.

DR. RUTALA: Yes.

CHAIR PRIOLA: But some drying will occur no matter what you do.

DR. RUTALA: And by immersion in the bath, but some drying, depending upon the level, of course, will occur.

CHAIR PRIOLA: Okay. Dr. Rohwer, what was your comment?

DR. ROHWER: Yes, I have a couple of comments and also would like to get Dr. Rutala's opinion on something, and that is first, I would highly advise not autoclaving with the lid on the vessel, and I am also mystified by this concern about sodium hydroxide vapors. As far as I know, sodium hydroxide has no measurable vapor pressure, and a properly operating autoclave should not be aerosolizing it either, because it shouldn't boil on the way down. That is my first point.

But what I would like to have you address is this issue of cross-contamination at the level of washing, because that is our major objection with that approach. We're talking about an agent that is very difficult to get rid of. If it gets spread around the laboratory and the environment then, we just couldn't tolerate that. You know, we don't want it in our sinks. We don't want it on our surfaces, etcetera. And so how is it that you deal with the eluates and the washers that come off of a set of instruments, which you know have been exposed to a Creutzfeldt-Jakob Disease patient, for example? How are the washers sterilized? How do you dispose of that?

It seems to me you create a cascading level of problems that have to be dealt with, and I am absolutely willing to concede all the points that you are making, except that in the case of this particular agent, it is very difficult for me to accommodate this idea of spreading this stuff around, exposing myself to it willingly before it has actually been decontaminated.

DR. RUTALA: Well, to your point, certainly, this practice has been employed for a number of years, and I think it has been practiced for the following reasons. One, of course, in a decontamination area in central processing, the persons in that area wear personal protective equipment. They wear gowns, gloves, protective masks. Second is that nearly every hospital where decontamination takes place in central processing, it's a closed unit. It's a washer disinfector completely closed. That is there is no aerosolization of droplets, of fluid that are related to the washing procedure. It's a closed procedure.

The closed procedure, of course, has many steps to it. Some of them are the use of enzymatic detergents, high temperatures, rinses and so forth. And I guess the third point related to that, protective apparel, a closed system, is the issue that while there is a recognition that some prion proteins may go down the drain connected to a sanitary sewer, we don't believe that that's the only source of prions reaching, essentially, the sanitary sewer system.

And to that point also, prions, of course, are unlike many other infectious diseases, are not transmitted by direct contact, indirect contact, droplets spread, airborne or the environment. So I am not sure what the level of concern is as far as transmission. It certainly wouldn't be, as I look at the issue, transmission to health care workers, it could possibly be contamination of the environment, but I believe that there is other forms of contamination in the environment outside that setting.

CHAIR PRIOLA: Dr. Bailar?

BOARD MEMBER BAILAR: Very nice talk. I do have a question about this very simple straightforward slide you had on the decreasing order of resistance of microorganisms disinfected in sterilants. You didn't go into the details of that, but I suspect it's ranked on the basis of things we know are effective at the susceptible end, that is the bacteria and the enveloped viruses.

Is anybody looking at other kinds of agents, you might call them unconventional agents, that may not be very effective at that end, but might be pretty good with prions?

DR. RUTALA: As far as that slide is concerned, you know, it is a general slide. There is an exception to that slide as it pertains to germicidal agents and particular groups of microorganisms, but it is a general slide. As your question intimates, most of the data, and there is literally hundreds of papers that support that slide, and most of the data is with the types of germicides that you have seen today and in my presentation and in other presentations. That is the conventional, not the nonconventional germicides and sterilization processes, products such as the alcohols, the phenols, the ethylene oxide, the steam sterilization and dry heat and so forth. Does that answer your question?

BOARD MEMBER BAILAR: Yes, it leaves me wondering whether it might be worthwhile for somebody to try some of these things, but I am no expert in this field.

DR. RUTALA: Well, to your point, I think that there are people interested in that.

CHAIR PRIOLA: If there are no more questions, we have two more speakers, but I think we should take maybe a 10 minute break. We're about 15 minutes behind, but let's take a 10 minute break and reconvene at 5:10.

(Whereupon, at 5:06 p.m. a recess until 5:18 p.m.)

CHAIR PRIOLA: If I could have the Committee members take their seats, so we can conclude this session. Okay. Our next speaker is Captain Edward Rau, Environmental Health Officer for NIH, and he is going to discuss infectivity of air emissions and the incineration of scrapie tissue.

CAPTAIN RAU: Thank you very much. Unfortunately, our only TSE expert in our group, Paul Brown, couldn't be here today. He is on some kind of a hardship assignment in southern France at the beach right now, so he has left that up to me to take care of.

The other disclaimer is that the results that I am going to present here are really very preliminary. Our experiments are still in progress. The paper is not written. None of the data is published. So with that, we'll go ahead here.

Could I have the next slide, please? I don't need to elaborate on all of the difficulties there are in inactivating the prion agents, and that the resistance to thermal inactivation is, of course, highest under conditions of dry heating. And that has led some concerns about even incineration being an effective technology to dispose of certain TSE waste.

Sitting here this afternoon, I think I have a new definition for incineration. It's that process, which incorporates all of the things that make inactivation difficult to do. We start out with a material that has been smeared and mashed around by all the handling of the medical waste process. It has not been precleaned. In fact, it is, in some cases, pure dirt. Then we're going to take that into a process, which begins by a drying and probably fixation step, and maybe melt a few varnish like materials over it before we really get into the combustion process.

Next slide, please. As you are aware, incineration is still the technology of choice for disposing of most medical waste that contain TSEs, and it is also being used to dispose of large volumes of animal products, contaminated carcasses and so forth, some of which are still in large quantities in storage. The potential for TSEs being in emissions from combustion processes is of public concern, and has received very little investigation so far.

Next slide, please. We have published some previous experiments documenting the unprecedented level of resistance to thermal inactivation, both crude brain tissue and purified PrP from the 263 scrapie infected hamsters. That included a partial inactivation after heating for 300 degrees for 15 minutes, and several transmissions after actually ashing brain material at 600 degrees C. And at those higher temperatures, there were similar patterns of resistance in both formalin fixed and non- fixed tissues.

Next. The objectives of our experiments in progress are first to confirm the results from our previous study. Some people were a little bit skeptical about the transmission after 600 degrees C. Others might have even labeled it science fiction. And secondly, we wanted to investigate the potential for transmission via the air emission that might come from a medical waste incinerator.

Our previous experiments were rather primitive in that we merely headed brain tissue macerate in vented crucibles. The new experiments, we are actually going to simulate the conditions of humidity and the gas mixtures and so forth that occur in two types of incinerators.

The first are the reducing environment or starved air incinerators. These are the most commonly used type of incinerator in the United States. A synonym for that is the controlled air incinerator. The other situation we wanted to look at was an oxidizing environment or referred to as a normal or excess air incinerator. And in this study, we repeated the temperatures that were used in the previous study, 600 degrees and 1,000 degrees C.

Next. Materials, our tissues samples were cooled, hamster brain tissue macerates from terminal animals with the 263 scrapie strain, about 10 logs of infectivity per gram, and for controls we had tissue from normal animals. The incineration situation was referred to as a Lindberg Furnace in a quartz reactor tube, the removal of the specimen crucible and holder. The gas supply coming into the incineration unit was normal air or nitrogen with flow and humidity controls. And then coming out of the unit was an impinger train and terminal filter for collection of the air emissions.

Next slide. This is a photograph of the main part of the simulator. It all fit in a large chemical fume hood. The incoming gases come into the furnace here. The quartz tube is contained inside of the Lindberg Furnace. This is a pyrometer. And then the outflow coming out of the combustion process goes into this impinger system, a series of collectors, the first one being ice water bath. The second one, dry ice, and then out through a terminal filter.

Next. This schematic gives an idea of the inside of the reactor tube, inside of the furnace. The tube is, approximately, one inch diameter, all quartz construction. At this end, we can remove the plug and insert into a thermocouple to directly measure the temperature or we can insert the sample on a glass rod, which is housed right here.

The gas flow comes from this direction in the top. It passes the sample. Exists through a ball joint and then on to the impinger train. In designing this, we tried to ensure that all the components in the system were inert. We used quartz and teflon joints as the materials.

Next. This schematic shows a little bit more information about the impinger system. It's a rather challenging design, because we really didn't know what we were trying to trap coming out of this process. Again, we have the ice water bath and the crushed dry ice bath following into a cartridge filter and exhausting into the hood.

Next. The methods began by introducing a one gram sample of the brain tissue into the reactor tube. We incinerated that for 15 minutes at either 600 or 1,000 degrees in either normal air or starved air conditions. Following the process, we collected the air and air emission samples separately from each run, and then replaced the impinger train between each run. We didn't have enough reactor tubes to use one for each experiment, so we disinfected those with bleach after each test.

Next. So each experimental run gave us three different samples. We had the ash residue that was collected in the crucible. The small amount of residue that formed in the reactor tubing as it exited the quartz reactor and came out cooled down at the border of the furnace, and then the emissions collected in the impinger traps.

Next. This gives you the array of samples that we collected. We ran both normal and infected tissues in the two different gases, two different temperatures, and for the infected material, that gave us three different samples. We combined samples for two of the normals and we did not run some of the exit tubes and traps on those. The reason for that is simply economics. We're dealing with about 500 animals to be maintained here, a great cost and time.

Next slide, please. The bioassay method, we concentrated the samples of the ash and emissions from each test into, approximately, a one milliliter volume in saline, and that was intracerebrally injected into, approximately, 30 Wingling hamsters. That is about 3 hundredths of a milliliter per animal. So the entire emission from each test was injected. We're not taking a sub sample out of those emissions. The entire emission from each burn was injected into animals.

Animals, of course, were segregated by test group. We observed them over 12 months for symptoms and then examined all of the brains for the presence of prion proteins by Western Blot testing. That testing is still in progress on the negative appearing animals.

Next. Results, we had no transmissions from the controls. There was some possibility, my commentor thought, we might be able to get some kind of symptoms as artifacts of this trauma and injecting into the animals, this residue. We just wanted to rule that out. We had no transmissions from any of the materials collected at the 1,000 degree C burn, and we had no positives from any of the residues collected in the impingers or the end of the reactor tube.

However, we did get two transmissions, and these were after very long incubation time from the ash from the crucible from the 600 degree group in normal air. The asymptomatic animals, again, we're still testing those for silent infections. We're not finished with that, so we have to call our results preliminary, at this point.

Next. Conclusions from the experiment. First, that the results were very similar from the previous study showing that there is, apparently, a threshold transmission from tissues at about 600 degrees C. The low transmission rate and very long onset time for the symptoms suggest, again, that that is the extinction temperature or very near it. We found no evidence of infectivity in the air emission samples.

Next. Speculate a little bit about what the environmental implications of this are. First, we did not see any evidence of transmission in the air emissions, so it's probably unlikely that will have a possible emission to the air from a properly operated medical waste incinerator. It is possible that some survival of the agent could occur in ash if there is not enough penetration of the temperature and time of exposure in the ash bed.

I wanted to kind of put this in perspective a little bit though, because I think it's a very low potential for transmission. First off, as Dr. Taylor said, we don't see environmental sources in transmission going on. Secondly, what we are seeing these two positives on is a simulated burn load that is pure material with 10 logs of infectivity per gram, and we're collecting virtually of the residue from that and injecting it into, approximately, 100 animals. So there is a very low level of infectivity in that material that is coming out.

Probability of survival in ash not only depends on a lot of factors, the load density, the turbulence, the type of equipment, other operational factors. And as we heard earlier today about the importance of context, I really can't imagine a more complicated context than a medical waste incinerator and all the combustion and mixing and reactions and things that are going on inside of that process.

Next. Our colleagues at EPA felt that these factors probably would be the most important in increasing the potential for prion survival in ash from medical waste incinerators. Under normal conditions, there are certain design factors, particularly in the grate oriented designs that might allow some of the material to not be treated for 15 minutes. It falls through the grates or it somehow gets passed on through the system faster than the nominal residence time for the solids.

Particularly, as things are just inserted into the incinerator, you tend to get a boil off of some of the material, a flash burn. That can be carried over very quickly into the second chamber. The other factor is that the ash bed temperatures often may run 100 degrees C lower than the actual air temperature.

Reported temperatures for incinerators refer to the air. That is what is being monitored, and not the actual temperature in the ash. Under abnormal conditions, a lot of things can really go wrong, cold start up conditions, overloading, inadequate control of the under fire air flow.

Next. We tried to compare what our experimental conditions were with the conditions in actual medical waste incinerators and some other types of processors used for bone meal products, and the most common incinerator in the U.S., the controlled air or starved air type of incinerator, in that primary chamber, you have temperatures of about 760 to 980. If you subtract about 100 degrees from that, allowing for some cooler temperatures in the ash, we're right on that threshold of survival that we saw in our experiments.

The secondary chamber, which mostly sees the pyrolysis products and not the ash is usually well up into that 1,000 degree temperature. I don't have much information on the temperatures in the excess air incinerators. They are probably quite variable because of the way that process is run. The secondary chamber is, again, quite hot, sufficient probably to inactivate prions.

There is some information on the other types of burn units that are being used in Europe for disposal of the meat by-product material. One of the articles had indication that there is actually a measurement of temperature at the ash grate in this 800 to 1,000 degree range, which looks pretty good for inactivation.

Next. There are other possible incineration options. If we get into a situation where we have a large amount of material to dispose of, the mass burn municipal waste incinerator in the United States operates at about 1,000 degrees, so that would likely inactivate prions.

Western Europe is looking at a variety of other types of industrial incinerators, fuel burners and so forth. Again, they have some high temperature and residence times. In one system, a holding time of 30 to 40 minutes, which is very encouraging.

I think that's the last slide. Next. I believe that is the last slide. Again, our results are very preliminary. So far, all of the testing on the negative appearing animals is confirming that's the case, but we're not quite finished with that yet. Any questions?

CHAIR PRIOLA: Any questions from the Committee? All right. Thank you very much, Captain Rau.

CAPTAIN RAU: Thank you.

CHAIR PRIOLA: Our final talk of the -- oh, I'm sorry. You had a question? Oh, sorry. Yes, go ahead.

DR. ROHWER: One issue I had with the original study was that the thermocouples were not actually in the sample, and the sample was loaded wet, and it wasn't clear here whether you're starting with wet tissue or dry tissue, and where the temperature measurement is actually being made, vis-a-vis, the sample. And the reason I bring this up is that a wet sample will not spend as much time at 600 degrees as a dry one, because you got to boil off the water first, and that could actually take some time.

CAPTAIN RAU: We did start with wet samples, wet tissue samples. The burn time is 15 minutes, however, in here, so I think we're probably spending most of that time at temperature. With regard to the thermocouple, before each sample was inserted, it was measured, then the thermocouple was withdrawn. We still have a pyrometer on the outside of the tube that we're confirming temperatures with, and that is really the best we could do. There is just not a way to have the thermocouple in there and be able to insert and withdraw a sample out of there. I agree with your boil off concern, but that is also real world, what's happening in the incinerator.

DR. ROHWER: In the original experiment, there was a thermocouple between the crucibles, so I took that to mean that there are thermocouples that can survive those kinds of temperatures. Is that incorrect?

CAPTAIN RAU: Yes, the problem is getting the output out of the burn chamber. It was a design issue. But in the first experiment, the thermocouple was right adjacent to the crucible and we were able to measure that in the muffle furnace.

CHAIR PRIOLA: Okay. Thank you. I think we'll move on to our last speaker who is Dr. David Asher from the FDA, as well as Dr. Brown, Dr. Stanley Brown. Oh, actually, you're going to start.

DR. BROWN: Actually, the last team is -- I am the rigger coming in from CDRH. Could I have my first slide, because it tells the whole story? Let's see, it worked on my computer. It was created on Terry's computer.

Well, anyway, I'll start. I'm Stan Brown. I am an engineer from the Center for Devices. I will present the first half of the team effort between myself and David Asher's group, which was funded by the FDA Office of Science, and these data, some have been published. Some are preliminary. Some are in manuscripts in preparation, and this is not good news on the screen.

Basically, what we were doing in my side of town was to look at four questions. The first question is can you safely autoclave in sodium hydroxide without wrecking your autoclave? The second question is what are the effects of the WHO protocols on surgical instruments? The third part of that was to develop an experimental instrument that could be used in a simulated instruments contamination study that would be compatible with the hamster model that David Asher has. And the fourth was to do some -- there we go. Okay. Let's click through here.

First of all, instruments from CDRH, we were thinking about primarily reuse, as you have heard, or reusables, but there is also growing concern about these things called SUDs or single-use devices, and with the law we are now reevaluating how we assess the reprocessing and validation of some of the reusables, particularly those that are neurological or other type of tissue contact. From the CBER point of view, as you know, we are talking about contact of instruments that you use for tissue preparation.

Next. Disclaimer. We developed these methods, because they fit within the financial constraints and the laboratory constraints. In our laboratories, these presentations do not constitute a regulatory endorsement for these methods. They are simply methods we thought would get answers that we're after.

Next. You know all about this story and we're primarily interested in the sodium hydroxide autoclaving phase and the soaking in sodium hydroxide or bleach.

Next. So if you go to the CDC website, you will see that there are a couple of warnings. One is that if you autoclave in sodium hydroxide, you wreck your autoclave and two, if you soak in bleach, you will wreck your instruments. And these are based on the studies that we started a few years ago, and I think CDC may actually have now inserted the reference from our work on that.

Next. Again, there are the four questions that we are trying to answer.

Next. The first one has just been published in the American Journal of Infection Control with Kathy and myself.

Next. She did the work and I wrote the paper, so I got to be first author. And again, the question is the autoclave manufacturer said if you autoclave in sodium hydroxide, you don't have a warranty on your autoclave. Knowing, of course, this must be done in a gravity displacement, so it doesn't fit in the standard central storage big prevacuum type autoclave. It has got to be controlled with a liquid cycle.

Next. Two approaches. One was we would put a liter of sodium hydroxide in a pan and cover it. Two, we would put some sodium hydroxide in a beaker and put the beaker in a pan and cover it. And then we put it in an autoclave. We did repeat one hour sterilizations, and we did these at 134C just to be a little more extreme, and we did them for an hour, up to five cycles, and we put pH paper inside, outside, everywhere. We put pH meters inside and outside, and it is a closed system for the little tabletop with six liters of water reservoir. We put it through five cycles to see what happens to the water in the reservoir to see, again, what happens to the autoclave.

Next. And we got thinking about pans and lids. This one probably, the condensate will get on the top and drip down and wreck your autoclave. This one, it might wreck your autoclave and it might drip inside. Some of them have little nipples or construction bars. To me, that is a Black Iron Dutch Oven where these drips, so that they roast while you're doing it. And then you have got some that actually have gutters that contain the lid within the pan. So part of this is what kind of pan and lid design you have.

Next. The two that we used successfully was a Nalgene Instrument tray shown here. This was filled with a liter of sodium hydroxide and closed.

Next slide. And if you look here, you can see this is a drain on that gutter, so the condensate goes back into the pan. It doesn't go out.

Next. And there you see the lid that is fully contained within the gutter and, of course, this has been used for years. It's for control of human waste and biohazards.

Next. The other type of pan was a Lid (D), which has a lip on the lid. It also has crossbars that act as condensate drip spots.

Next. And the results of this were no pH changes outside the containment. Inside the lids were very caustic. The bottom of the pans were very caustic. There was lots of vaporization, condensation going inside, but it was all contained within the containment vessel. So we conclude that if you use this kind of -- if you use the right kind of pans and lid, you can do it without wrecking your autoclave. And, of course, those of you who have been doing this in the lab for years know that. Obviously, hot caustic is hot and you have to be careful. It probably cannot be done in a standard central storage autoclave and it may require larger approved type pans.

Next question. What do these things do to your instruments? Next. What we did was we bought surgical instruments from Roboz, which is a medical device supplier in Rockville, and we bought lab stuff from VWR. Some of them are labeled Germany with CE marks and some of them were labeled Pakistan, and there are some members in the audience who will appreciate this. We put them through repeat cycles of the WHO including autoclave and water.

Next. And here you see some carbide tipped needle holders. This one has been through five times autoclave and sodium hydroxide, five hours in sodium hydroxide. There is a little bit of blackening in the box joint. This one was soaked for one hour in Clorox, and you can see there is a tremendous amount of corrosion going on at the box joint and around the carbides.

Next. These had beautiful gold handles, high quality instruments, and that is one hour in bleach. So if you got gold handles, don't bleach them. This is five times gold handles autoclaved in sodium hydroxide. It looks fine.

Next. This is a German pair of scissors versus a Pakistani pair of scissors, five times in sodium hydroxide. You can't photograph shiny, but this is shiny. It just looked great, and this looked really dark and dingy.

Next. This is Germany versus Pakistan, and you notice the Roboz label on this thing as stainless steel, and this tubing clamp around this weld really took it with the Clorox. This tubing clamp after five hours in bleach, this is the 6 percent, which is what, 2,800 parts per million. It looked fine. So some go, some are fine.

Next. So the conclusion of this, and I didn't show you any pictures. Titanium really does not like sodium hydroxide, and this is well-known in the material science literature, as well. Soaking in sodium hydroxide, they couldn't care less, none of them. Soaking in bleach, some did fine, some didn't. The problems were the gold handles and the welds. But the important message here is if it's going to corrode, it's going to do it first time. So you don't have to do a long experiment. If you put it in Clorox and it comes out rusty, you know it's going to rust and you go on and find a better instrument.

Next. So Part 3. We wanted pins and this is part to lead into an animal model of the simulated instrument for David's studies. He was using a 25 gauge needle on a half cc syringe in his animal work for injection, and we wanted to make pins instruments that were like that, but we also wanted to be able to suspend them over 96 weld plates, so we could do serial dilutions of bacteria, viruses in brain homogenate, and the system needed to be autoclavable.

Next. So there is the syringe needle that he was using. That is a good old copper penny, and what we did was we took Eppendorf tips. My wife is a microbiologist, and so is Kathy. We took half millimeter stainless steel pins. We took some epoxy. We used the Eppendorf to draw the epoxy up into the tip, stuck the metal pin into it, hung it in a rack, put it in an oven to cure and now, you have things that can be autoclaved, and it's the same size as the needle that is used. So from an ergonomics point of view, it's something that he would feel comfortable with, I think.

Next. And here, you see the setup. This is your standard Eppendorf rack with modified stainless screws to adjust the height, and the pins were sitting in the wells of a 96 weld plate, and Kitty, that after a little bit of practice, she could actually get all the needles into all the holes, right?

Next. Finally, we wanted to do some preliminary of adhesion of both blood and tissue and looking at WHO protocols, and one question was what about damage and adherence? So we were using stainless steel pins and we also made pins out of piano wire, which really did not have a good time in Clorox.

Next. Pins are placed in a rack and stuck into a slab of liver for an hour, and then we left to dry as a worst case. The pins were stuck in a 96 weld plate in sheep's blood for an hour, and then they were left to dry. We went through ultrasonic cleaning, which the standard protocol is 60 degrees C with an enzyme cleaner. We autoclaved in sodium hydroxide. We soaked in bleach and we got unclean controls.

The results were the unclean ones, that the protein was more adherent from liver than it was from blood, and the amount was using Bradfords reagent. It's about the equivalent of -- our minimum was one microliter detection limit. Damaged pins did not seem to be more adherent, so that the blackening in the box from autoclaving and sodium hydroxide is probably not a problem. Repeat exposure did not show accumulative effect.

Next. So then Kathy wanted to do some bacteriology, and she soaked them in a solution of staph epi. for 24 hours, let them dry and did the same kind of cleaning things, and then stuck them into an agar in a test tube and incubated for 24 hours.

Next. Lo and behold, autoclaving in bleach killed everything. So we had to try some modified WHO. So we dropped the sodium hydroxide autoclave and the ultrasonic cleaning was done at room temperature.

Next. So what we found was that only the pins and bleach showed no growth, but, of course, we don't know if we cleaned it or we just killed them. The other produce showed fewer protocol than the untreated control, but bacteria was still present. And then the question is are we cleaning or are we just killing?

Next. And we tried some SEM work, and you can see a little small column of staph epi. here on the uncleaned tip. It was very unconvincing in terms of whether we really were cleaning or we were just killing.

Next. So our conclusions were, first of all, yes, you can safely autoclave in sodium hydroxide with the right pan and lid. Some WHO protocols can damage some instruments. Discoloration does not seem to impair function or cleaning. The bacteria leave a lot of unanswered questions, and the questions for prions, of course, we don't touch them in CDRH, that is David's role.

So next, and I will turn the podium over to David to talk about his hamster studies.

DR. ASHER: Thanks. You can go right to the next slide, please. Thank you. I can't believe that it's almost 6:00 and we're still giving talks. Quite a few people in CBER participated in TSE related activities. These studies are really involved only people in CBER in my own little group, and especially Kitty Pomeroy who I think is still here in the back. Without her holding the whole enterprise together, there is no way that we could have done this.

Next slide, please. And among the staff at CDRH, of course, Kathy Merritt and Stan Brown, who has just spoken, and I don't believe that we would have gotten the funding to do what we have been able to do without Stan's efforts.

Next slide, please. We have developed two simple methods to evaluate methods for decontamination of TSE agents dried on surfaces. In this talk, I am going to concentrate on two regimens that more or less replicate recommendations of the World Health Organization consultation. We used two general models for both of which 263K scrapie was the agent.

The first model was a modification of a method for evaluating virucides that was described years ago by John Chen of the Environmental Protection Agency. He dried viral agents onto glass cover slips, treated them and then assayed residual virus.

The second method was stimulated by two reports from Charles Weissmann's group, and Professor Weissmann will speak here tomorrow morning. They dried scrapie agent onto steel wires implanted into mouse brains. We didn't do that, but as you saw from the devices that Stan showed you, our model was very similar.

Years ago, we had done a couple of experiments with model squirting scrapie through actual hypodermic needles, but it was very cumbersome, and we never followed up on it. We have used the first method.

Next slide, please. The first method for many years, simply dropping suspensions of scrapie infected hamster brains onto glass cover slips.

Next slide, please. Then they are dried in a petri dish in a hood.

Next slide. And then they can be exposed to any number of disinfectant or decontamination regimens, here potassium permanganate solution that can be autoclaved, that can be soaked. You can do all kinds of things with it.

Next slide, please. Then they can be ground up and supernatant fluid assayed. Now, we used plastic pipettes and tubes for the whole procedure, because they are disposable. We don't have to worry about potential carryover of infectivity. Although, the original method described by Chen used 10 brack tissue grinders of the kind seen here. So the slips are ground up in an ml of diluent.

Next slide, please. The glass is allowed to settle out, and then the supernatant fluids are assayed for infectivity by intracerebral injection of hamsters.

Next slide. If the hamsters get sick, their brains are removed and then they are checked for protease resistant prion protein as evidence that scrapie agent was present and was not eliminated by the decontamination regimen.

Next slide, please. For reasons that may become clear at the end of this talk, I have about six minutes left, we think that it might be useful to do immunohistochemistry on some of these brains, as well. Although, we haven't done that yet.

Next slide, please. One advantage of the method is that you can rid of residual toxic disinfectants, Robert Somerville talked about that problem this morning, by simply rinsing the cover slips in distilled water to get rid of things like Clorox, which is really terrible for assay animals.

As you see here, this is sort of an upside down dose response curve. It's hamster survival times plotted against the dilution of supernatant fluid. And here are three curves, one for unsoaked or dipped slides, one that has been soaked in water, and the other that was soaked in water and then dipped in water a second time. And you can see that they are virtually superimposable. None of the infectivity appeared to come off on this exposure to distilled water.

Next slide, please. And it was using this method that we first demonstrated the resistant fraction of infectivity that survived drying on glass and then prolonged exposures to steam autoclaving at elevated temperatures.

Next slide. To investigate some of the performance characteristics of the model, you can imagine doing many, many assays in hamsters is extremely cumbersome. We tried drying specimen samples of polio virus and two other viruses suspended in brain onto glass, and then titrating multiple samples. The results summarized here suggest that the agreement from test to test and day to day was reasonably good, but that there was enough variability, so that a controlled titration really should be done in each test. And it might even be reasonable to consider putting in a test decontamination, a reference decontamination treatment. Although, we have never had enough resources to support that.

Next slide, please. When we began to work with the scrapie brain suspensions dried onto the steel needles that Stan Brown has just showed you, we decided first to do some similar preliminary studies with conventional virus suspended in saline containing 10 percent brain extracts to get some idea of how the scrapie agent might be expected to behave, so we wouldn't waste months and months on a model that wouldn't get enough infectivity on.

But we were surprised when neither polio virus nor porcine parvo virus suspended in brain appeared to stick to the steel needles at all or at least we couldn't detect any of them in cell culture assays. We had no trouble getting them to stick to glass, but we couldn't find detectable porcine parvo virus or polio virus dried onto steel needles.

So now wanting to take a chance that we would set up our test with the limited funding available and the find that we hadn't had enough challenge agent stuck to the steel needle, so we decided to suspend the 263K scrapie hamster brain in a normal brain paste, and we used a calf brain as the source of the normal brain material.

Next slide, please. I must say we decided to check out our concern. We had enough hamsters to do a rough titre of 263K scrapie diluted in phosphate-buffered saline without any additional brain material, and it appears that the scrapie infectivity in the saline suspension did stick to the steel wires. You will notice we get positives out to a dilution of 10^-5, so that the behavior of the polio virus and the porcine parvo virus does not appear to have predicted the behavior of the scrapie agent.

But the studies that I am going to summarize in the next few slides use scrapie infected brains as a paste to charge the needles. The glass was charged in the way that I described previously with saline suspensions.

Next slide, please. So let me summarize for you the general design of the efforts. We looked at two variations of two kinds of decontamination regimens that generally resemble those recommended by the WHO consultation, and then after that, I will add some other results that we thought you might find of some interest.

First, as we have mentioned, infected brain is dried on the objects, either a saline suspension on glass slides or tissue paste onto steel needles. Then come the decontamination steps, which are either a chemical soak in one normal sodium hydroxide in the autoclave for 30 minutes or a soak in sodium hypochlorite, full strength chlorine bleach from the grocery store at room temperature for 60 minutes followed by an autoclaving at 121 celsius for 30 minutes or at 134 celsius for 90 minutes. The autoclaving with sodium hydroxide is in the sodium hydroxide. The autoclaving with bleach is after it is in water.

Following that, all these materials were cleaned in an ultrasonic cleanser using a laboratory proprietary detergent with a pH of 9.45. All of them got this, because, as pointed out by Dr. Rutala, it is important to try and replicate the conditions under which these things would be done in a hospital. The sonic cleaner was cranked up the highest temperature that it would take, which was over 60 degrees, although, somewhat variable, and for the longest time the timer kept, which was for 90 minutes.

This was done by putting each object into a separate tube filled with the cleaner, and then the tubes were immersed in more cleaner in the chamber. Following that, there was a water rinse and then, finally, a terminal sterilization in the autoclave at 121 celsius for 20 minutes in order to model what we took to be standard hospital practice.

Next slide, please. As I mentioned, the sonicator was set at maximum temperature and time. We only did the one set of conditions. We made no effort to select a better cleaning solution. I am sure there are many others, that many others are available.

Okay. Next slide, please. First, the assay technique for residual infectivity on the glass slips. Each experiment, a positive control consisting of 10 slips each holding .1 ml of dried on scrapie infected, 10 percent hamster brain dried down, not exposed to any decontamination regimen, ground to a powder and a ml of PBS glass allowed to settle. Fluids were then pooled, tenfold dilutions performed in phosphate-buffered saline, each dilution assayed in four hamsters, the same volumes that Dr. Rau showed you, .03 ml each intracerebrally into the left frontal lobe. That is simply so that we would know in each test how much infectivity had been used.

Next. Hang on a second. Let me finish. Each of 10 slips then was exposed to some decontamination regimen, and then also ground to a powder in phosphate-buffered saline, the fluid assayed as for the controls, so that each experiment on glass involved 10 slips and 40 hamsters.

As David Taylor had mentioned to you earlier, we deducted incidental deaths. We took 45 days as the cutoff between considering it a death incidental. Perhaps we shouldn't have done that.

Next slide, please. For the steel needles, the positive controls were tenfold, dilutions of infected hamster brain as a 10 percent paste in normal calf brain, and then serial dilutions were done with normal calf brain paste. Four needles were charged for each dilution, dried and then a separate hamster was assayed for each needle. For the actual test, 40 needles charged with 10 percent hamster brain and normal calf brain, dried, tested and then assayed as for the control above. Less incidental deaths occurring before four days.

Next slide, please. This is just to show you what a titration on glass looked like, the interim score here at eight and a half months. Notice that the last positive animal so far as those inoculated with a dilution of 10^-8 calculated from the original brain tissue.

Next slide, please. And a similar titration for scrapie dried onto steel needles, also positive to a reasonably high dilution. Actually, somewhat higher than we got with the saline suspension, so we weren't sorry that we had used the brain paste.

And you might notice that there is one negative at the lowest dilution. That was an animal that died at 55 days. Brain was negative, and that is why we're wondering whether 45 days might have been the best date to estimate incidental deaths.

Next slide, please. Before I move on to the actual results, we were interested to see what ultrasonic cleaning in hot alkaline detergent alone without any other treatment would do, so we did a titration from that and found a substantial reduction, both of the infectivity on glass and on steel needles from the hot ultrasonic cleaning alone. The log reduction factor is slightly over 5 logs. Although, for both models there was some residual infectivity left on the surface.

Again, we made no effort to optimize, to modify or optimize the procedure. We presume that most of the infectivity probably went into the liquid, but we haven't made any attempt to find out whether that is true.

Next slide, please. So here are the WHO studies. After exposure of glass slips, there are the glass slips, to sodium hydroxide or to sodium hypochlorite with autoclaving at either 121 celsius or at 134 celsius, there was obviously a dramatic removal of infectivity, but darn, one of the animals assaying material exposed to one normal sodium hydroxide at 134 autoclave, 134 celsius for 90 minutes has come down positive.

Next slide, please. And similar experiments with steel yielded relatively similar results, at least two, maybe three of the assay animals have had positive Western Blots. We are going to have to check those out. Obviously, these stray positives have been seen before, and we have to convince ourselves as to whether they are really positives or whether it's inadequately digested PrP in the Western Blot or whether it's real.

So the methods are, obviously, highly effective. They saved almost all the hamsters and removed so much infectivity that most of the objects assayed didn't show evidence of contamination. Remember that each of these objects was charged with at least a million lethal doses of scrapie agent, but we can't say that they are perfect.

Next slide, please. We have seen similar results in the past using single chemical soaks. These are all done with the Chen glass test. And, again, we have frequently seen, these are sodium hydroxide soaks at various temperatures, an occasional stray positive.

Next slide. Some tests have found no positives at all, but remember with the Chen glass test, we sample only about 12 percent of the supernatant fluid from each slip, so that these results are not necessarily different from the ones that show single positives. There is a substantial sampling problem when you're dealing with very small amounts of infectivity.

Next slide, please. Here is another result with sodium hypochlorite where we had no positive animals. I marked these all as interim, because we haven't finished all the Western Blots even though some of these are not new experiments.

Next slide, please. And finally, I would like to say that some other chemical agents are probably also very effective. Here are some results using concentrated formic acid, which is used to treat tissues for histology and immunohistochemistry. Note that there is only a single positive animal out of 37 tested. Reports of a commercial phenolic disinfection, at least temporarily unavailable here in the United States is reported to be very effective, and we have heard that there are other decontamination regimens in development not yet ready to share with the FDA or the public that are showing promise.

Let me conclude now by, next slide, please, just summarizing that methods developed to evaluate the effects of virucides are adaptable to evaluate decontamination of TSE agents. Studies with two models both suggested that exposure to 263K scrapie agent dried on surfaces to solutions of sodium hydroxide, sodium hypochlorite with simultaneous or sequential autoclaving and ultrasonic cleaning in hot alkaline detergent markedly reduced amounts of infectivity, and the risk that any object would retain detectable amounts of agent.

Other chemical treatments may also be effective, but uncertainties remain. One, the reliability of the decontamination procedures, not only the fact that we see stray positives, but also there is a theoretical concern that the predictive value of these results, the results from such models, may not adequately predict the behavior of decontamination regimens in the actual health care or manufacturing setting, concern that there may be sanctuaries of the kind that Bob Rohwer and David Taylor have discussed that might occur in manufacturing processes or health care setting that would impair the ability of otherwise effective decontamination regimens to act.

It is quite late, but I am happy to answer questions for anybody who has got the energy still to ask them. Thank you.

CHAIR PRIOLA: Are there any questions for Dr. Asher or Dr. Brown? All right. If not, I -- okay, Dr. Somerville has one for you.

DR. SOMERVILLE: I'm just going to make a brief comment about the first part of the talk, and that is that in our experience, in our survey, we find that the various different grades of stainless steel are used from the manufacturer of surgical instruments and with various different finishings, and they have different responses to the kinds of treatment that Stan Brown was trying on the instruments.

The one brief question I have is have you tried anything other than visual inspection to see what the degree of damage is being done to the instruments?

CHAIR PRIOLA: Can Dr. -- oh, he is coming up there. Dr. Brown can answer that.

DR. BROWN: The answer, at this point, is we have done nothing other than visual, and part of the next generation of study is to be looking at some of the different alloys, some of the different corrosion test methods. Some of these effects are so blatant that why both to -- I mean, I cut up the gold handles and put them in the SEM just to make sure it really was gold.

And, in fact, there was gold on those handles, but no, we haven't gone any further. But one of the questions is are the different grades, you know -- in talking with the instrument manufacturers and the people who do chemical analysis of instruments, there are a whole wide range of grades of stainless steels, but the manufacturers will tell you what probably is the most important is actually the mechanical treatment in terms of how they make them, coworking, etcetera. And it may not be a matter of chemistry, but it's a matter of mechanical parts.

The finger rings very typically are attacked by Clorox, and that is an area where there has been a lot of mechanical cowork to form the rings. Whereas, elsewhere on the same instrument, the surface looks fine. So it's not just the chemistry, but it's actually the mechanical processes used in the forming or fabricating. And again, if it's going to go, it's going to go the first time you throw it in bleach.

BOARD MEMBER HOGAN: Dr. Brown, before you leave, I have one more question. Dr. Brown, could you get rid of the black deposit that formed on the sodium hydroxide instruments?

DR. BROWN: First of all, we didn't do any other cleaning. We just over and over and over, autoclave and bleach. We didn't use what do they call it, milk, the cleaning milk that is used in standard central storage.

BOARD MEMBER HOGAN: So you didn't try? Is that it?

DR. BROWN: So what I did actually on some of them is I did a bit of gentle scrubbing in the box joints to see if it would come off. It wouldn't come off much by general scrubbing. Actually, if you reuse them, you can begin to wear off the blackening. But it's really a very superficial kind of blackening, and then the thing with the protein adherence with the piano wire, they really did corrode and at least the serum protein stuff we did didn't show any difference.

CHAIR PRIOLA: Dr. Edmiston?

DR. EDMISTON: I know it's late and I don't want to hold anybody up, but I really want to commend Dr. Asher and Dr. Brown. You are heading in the right direction. The question that I have is do you contemplate looking at this in devices that have larger bores in terms of if you're looking at a hollow device, are you looking at other devices that may have a larger bore where the cleaning process may be expedited, normal cleaning process may be expedited on the basis of having a larger internal diameter?

DR. BROWN: These are solid pins.

DR. EDMISTON: These are solid pins?

DR. BROWN: These are solid pins. They were not needles.

DR. EDMISTON: Okay.

DR. BROWN: So the idea was that David had been using a needle. He was used to the feel of that size needle, and I made solid pins to match. So these were not hollow.

DR. EDMISTON: So you don't know what would happen with a hollow bore device?

DR. BROWN: No. One can sort of guess, but I think --

DR. EDMISTON: Right.

DR. BROWN: You know, this term of, you know, the nooks and crannies and the hiding places, I think that's the next generation of the studies. Polymer coated, we have got some that, apparently, are even teflon coated that are part of the next step in the study.

DR. ASHER: Yes. As I mentioned, years ago I did some standard hypodermic needles, just squirting suspensions of scrapie through and letting the needles dry and autoclave. You know, you're not surprised to hear that they were not sterilized.

DR. EDMISTON: I think our experiences have been that, especially in the case of neurosurgery, that those patients who fall into that risk category, a lot of us are moving towards the use of disposable biopsy needles.

CHAIR PRIOLA: Dick, did you have a question? I'm sorry, can you what?

BOARD MEMBER JOHNSON: Can we leave our papers behind?

CHAIR PRIOLA: I think you --

SECRETARY FREAS: If you want it tomorrow morning, I would really recommend you take it to your room. I do have a couple of quick announcements. This morning, we passed out about 200 Conflict of Interest questionnaires and we got about five of them back. I would like to encourage you to look at the questionnaires and if you could drop them off on your way out, we'll pass out another 100 tomorrow and, hopefully, we'll got some back.

Also, somebody left behind a Palm Pilot. It looks like it's a very expensive Palm Pilot, and if you can identify it, it's yours. Tomorrow morning, we'll be seeing you at 8:00 sharp.

CHAIR PRIOLA: Okay. I would like to thank all of the speakers for presenting published and unpublished data to the Committee, and we're adjourned until 8:00 a.m. Thank you.

(Whereupon, at 6:18 p.m. the meeting was adjourned.)