[Federal Register: January 7, 2009 (Volume 74, Number 4)]
[Notices]
[Page 712-719]
From the Federal Register Online via GPO Access [wais.access.gpo.gov]
[DOCID:fr07ja09-47]
=======================================================================
-----------------------------------------------------------------------
DEPARTMENT OF HEALTH AND HUMAN SERVICES
Centers for Disease Control and Prevention
Notice Regarding Revisions to the Laboratory Protocol To Measure
the Quantity of Nicotine Contained in Smokeless Tobacco Products
Manufactured, Imported, or Packaged in the United States
AGENCY: Centers for Disease Control and Prevention (CDC), Department of
Health and Human Services.
ACTION: Notice and Summary of Public Comments.
-----------------------------------------------------------------------
SUMMARY: This notice amends the uniform protocol for the analysis of
nicotine, total moisture, and pH in smokeless tobacco products
(``Protocol''). The Protocol, originally published in the Federal
Register in 1999 (64 FR 14086) and revised in the Federal Register on
March 14, 2008 (73 FR 13903), implements the requirement of the
Comprehensive Smokeless Tobacco Health Education Act (CSTHEA) of 1986
(15 U.S.C. 4401 et seq., Pub. L. 99-252) that each person
manufacturing, packaging, or importing smokeless tobacco products shall
annually provide the Secretary of Health and Human Services (HHS) with
a specification of the quantity of nicotine contained in each smokeless
tobacco product. CDC re-published the notice in the Federal Register on
June 23, 2008 (73 FR 35395) concerning the revision of the Protocol (1)
To make a technical change to correct the date when the first report of
information under the revised Protocol is due and (2) to solicit public
comments concerning a change in the Protocol that increased the volume
of water in the pH determination from 10 mL to 20 mL, and (3) to
solicit public comments concerning the addition of the following
commercial smokeless tobacco product categories: dry snuff portion
packs, snus, snus portion packs, and pellet or compressed. This Notice
also includes a summary of public comments and CDC's response to them.
The Protocol as published in the Federal Register on March 14, 2008
(73 FR 13903), remains in effect with the technical correction to the
date as described in the Federal Register notice published on June 23,
2008 (73 FR 35395).
DATES: First report of information due June 30, 2009, with subsequent
submissions due by March 31 of each year.
FOR FURTHER INFORMATION, CONTACT: Matthew McKenna, M.D., Director,
Office on Smoking and Health, Centers for Disease Control and
Prevention, Telephone: (770) 488-5701.
SUPPLEMENTARY INFORMATION: Since the implementation of the Protocol in
1999, several smokeless tobacco product categories have entered the
U.S. smokeless tobacco market including snus, low moisture snuff sold
in portion pouches, and smokeless tobacco sold in a compressed, pellet
form. Some of the new smokeless tobacco product categories differ
physically from previous smokeless tobacco categories, prompting a
revision to the Protocol to reflect the current state of the
marketplace.
Through its review of the Protocol, CDC also determined that an
increase in volume of deionized, distilled water would facilitate
measurements of pH values. After evaluating information that was
brought to the attention of CDC regarding low moisture smokeless
tobacco products packaged in portion pouches, CDC conducted an
independent comparison of pH measurements in a wide variety of low and
high moisture smokeless tobacco products. The results of the comparison
indicated an acceptable (less than 2%) level of change in pH values
when measurements were taken with 20 mL deionized, distilled water
compared to the volume of deionized, distilled water specified in the
previous Protocol. Increasing the volume of water in the mixture
ensured that the matrix was sufficiently fluid to facilitate ease of
measure. Thus, it is anticipated that the change in the volume of
liquid for pH determination will facilitate the ease of measure of
smokeless tobacco pH for all currently marketed smokeless tobacco
categories (i.e., plug, twist, moist snuff, dry snuff, snus, loose
leaf, chew, moist snuff in portion pouches, smokeless tobacco
compressed into a pellet, and dry snuff in portion pouches).
Summary of Public Comments and CDC's Response: On June 23, 2008, a
notice (73 FR 35395) was published reflecting the above discussed
revisions to the Protocol and to solicit public comment on these
specific changes. Six comments were received by the CDC, a majority of
which suggested alternative approaches. A summary of the
[[Page 713]]
comments received and CDC's response follows.
One commenter expressed a concern for the Federal funding and
overall direction of the ``smokeless tobacco program.''
The issues raised in this comment were beyond the scope of the
Protocol and solicitation of public comment.
One commenter, on behalf of several smokeless tobacco
manufacturers, agreed with the proposed revision of Section IV(B) (see
below for Protocol) of the Protocol to increase the volume of
deionized, distilled water to be used in pH measurements from 10mL to
20mL.
One commenter, on behalf of several smokeless tobacco
manufacturers, suggested that ``some flexibility be incorporated into
Section IV(B) of the Protocol by providing that, as long as a minimum
of 20 mL of liquid and 2 grams of sample are utilized, then larger
amounts of liquid and sample may be utilized provided they are in a 10
to 1 ratio.''
CDC appreciated the suggestion that there be flexibility in
adjusting the quantity of liquid and sample so long as the ratio of
liquid to sample is 10 to 1. In evaluating this suggestion, CDC
determined that adopting such a change would deviate from principles of
good scientific practice as it does not promote protocol consistency,
contrary to the aims of a uniform analytical protocol. According to the
Cooperative Centre for Scientific Research Relative to Tobacco
(CORESTA), a central organization responsible for promoting tobacco-
related cooperative research, ``[t]he development of standard methods
is critically important in ensuring consistency and comparability of
data reported by the association members and as part of regulatory
reporting of data.'' [Further details on CORESTA's viewpoint and its
objectives are available online at http://www.coresta.org/Home_Page/
PresentationCORESTA(Oct08).pdf.] As the fundamental purpose of the
Protocol is to implement a multi-site testing protocol, CDC concluded
that the development of a uniform analytical protocol is paramount to
ensuring sound scientific efforts.
One commenter, on behalf of several smokeless tobacco
manufacturers, raised the following point regarding the categorization
of smokeless tobacco products in Section I(F) of the Protocol:
``* * * many of these separate product `categories' are
essentially identical smokeless tobacco products for the purposes of
sample preparation (e.g., Moist snuff and snus; Moist snuff portion
packs and snus portion packs) * * * since a number of smokeless
tobacco manufacturers have stated that they are developing new or
`innovative' smokeless tobacco products, an approach that creates a
new `category' and sample preparation instruction every time a
smokeless tobacco product is introduced with a different name or
description will result in a proliferation of smokeless tobacco
product `categories' and a need to constantly revise the Protocol to
add new sample preparation instructions. Such revisions would
trigger a notice and comment process under the Administrative
Procedure Act.''
CDC made the determination to include the four newly listed
categories after having reviewed the number and types of smokeless
tobacco products that had entered the market since 1999. In this
review, CDC concluded that several new products would benefit from a
separate categorization to not only better aid manufacturers in
distinguishing their products in this protocol, but also reflect the
variety of products being sold to and recognized by consumers. This
review also determined that in the years since the implementation of
the Protocol in 1999, the quantities of new products introduced to
market requiring separate categorization had been fairly limited; thus,
CDC did not believe that constant revisions to the Protocol would be
necessary. However, CDC will continue to monitor the introduction of
new smokeless tobacco products and provide assistance to reporting
entities on the application of the Protocol as needed.
One commenter, on behalf of several smokeless tobacco
manufacturers, suggested an alternative approach that would
``eliminate, or at the least minimize, the need for new `categories'
and sample preparation instructions.''
This alternative proposal suggested that:
``The alternative approach would be to define the smokeless
tobacco product categories based on physical characteristics
relevant to sample preparation (essentially tobacco particle size
and whether tobacco particles are in a pouch), rather than on a
manufacturer's package label statement or description * * * ''
Three product categories were thus proposed.
If any products did not fall into the three categories, the
proposal suggested that:
``* * * in the event that a smokeless tobacco manufacturer or
importer believes that a newly marketed smokeless tobacco product
does not fit within any of the above categories, then samples should
be prepared in a manner compatible with the above sample preparation
instructions and the manufacturer or importer should describe the
sample preparation procedures used when making its submissions to
CDC.''
After an evaluation of this alternative approach, CDC concluded
that the current method of categorization is more appropriate for
several reasons. First, the current method has been in place since
1999, with no noted difficulties associated with this product
categorization. Second, CDC noted that other Federal agencies, such as
the Federal Trade Commission (FTC) and United States Department of
Agriculture (USDA), receive and review information on smokeless
tobacco, not on the basis of physical size characteristics, but on
these commonly accepted types of categories. Examples can be found in
the FTC's ``Federal Trade Commission Smokeless Tobacco Report for the
Years 2002-2005,'' available online at http://www.ftc.gov/reports/
tobacco/02-05smokeless0623105.pdf, or in the USDA Economic Research
Service's ``Tobacco Situation and Outlook Yearbook'', available online
at http://usda.mannlib.cornell.edu/usda/ers/TBS-yearbook//2000s/2007/
TBS-yearbook-01-12-2007.pdf/.
Furthermore, CDC viewed the existing categorization of products by
traditional ``consumer-oriented'' descriptions as useful in easily
identifying issues that concern the general consumer and the overall
public's health. In contrast, adopting a method of categorization based
solely on physical product characteristics would not be beneficial
towards that goal.
Finally, during its review of this alternate approach, CDC noted
that there are only three existing methods to prepare smokeless tobacco
products for analysis in this protocol, despite the varied physical
characteristics of currently marketed smokeless tobacco products.
One commenter, on behalf of several smokeless tobacco
manufacturers, suggested that ``the reporting provision of the FRN be
amended to provide the following: (i) The revised Protocol shall take
effect January 1, 2009, and (ii) the first report of information
pursuant to the revised Protocol is due March 31, 2010, with subsequent
submissions due by March 31 of each year. This amendment would afford
smokeless tobacco manufacturers a reasonable amount of time to prepare
for the implementation of the revised Protocol, and would continue the
current practice of manufacturers submitting a full year of data based
on a consistent methodology.''
For the purposes of this comment, CDC took into consideration a
Federal Register Notice published in March 2008 (73 FR 13903), which
served as public notice about the changes in the Protocol. CDC regarded
this duration of notice as sufficient for the first report of
information to be due June 30, 2009,
[[Page 714]]
with subsequent submissions due by March 31 of each year, as laid out
in the June 23, 2008 Federal Register (73 FR 35395).
Collection of Information
This proposed amendment does not call for any new collection of
information under the Paperwork Reduction Act of 1995 (44 U.S.C. 3501-
3520).
Dated: December 29, 2008.
James D. Seligman,
Chief Information Officer, Centers for Disease Control and Prevention.
Revised Protocol for Analysis of Nicotine, Total Moisture, and pH in
Smokeless Tobacco Products
I. Requirements \1\ \2\
A. Reagents \3\
1. Sodium hydroxide (NaOH), 2N
2. Methyl t-butyl ether (MTBE)
3. (-)-Nicotine (Fluka 72290) >99% purity \4\ \5\
4. Quinoline (Aldrich)
5. Standard pH buffers; 4.01, 7.00, and 10.00
6. Deionized distilled water
B. Glassware and Supplies
1. Volumetric flasks, class A
2. Culture tubes, 25 mm x 200 mm, with Teflon-lined screw caps
3. Pasteur pipettes
4. Repipettors (10 mL and 50 mL)
5. Linear shaker (configured to hold tubes in horizontal position) \6\
\7\
6. Weighing dishes, aluminum
7. Teflon-coated magnetic stirring bars
8. Polypropylene containers, 50 mL
C. Instrumentation
1. Robot Coupe Model RSI 2V Scientific Batch Processor
2. Capillary gas chromatograph, Hewlett Packard, Model 6890, with
split/splitless injector capability, flame ionization detector, and a
capillary column (Hewlett Packard HP-5, Crosslinked 5% PH ME Siloxane,
30 m length x 0.32 mm ID, film thickness 0.25 or 0.52 [mu]m)
3. Orion Model EA 940 pH meter equipped with Orion 8103 Ross
combination pH electrode
D. Additional Equipment
Forced-air oven, Fisher Isotemp[reg], regulated to 99
1.0[deg]C. Suggested dimensions: 18 x 18 x 20inches.
E. Chromatographic Conditions \8\ \9\
1. Detector temperature: 250[deg]C
2. Injector temperature: 250[deg]C
3. Flow rate at 100[deg]C--1.7 mL/min; with split ratio of 40:1 \10\
4. Injection volume: 2 [mu]l
5. Column conditions: 110-185[deg]C at 10[deg]C min-\1\;
185-240[deg]C at 6[deg]C min-\1\, hold at final temperature
for 10 min.
F. Sample Preparation \11\
There are ten different categories of commercial smokeless tobacco
products:
1. Dry snuff;
2. Moist (wet) snuff;
3. Moist (wet) snuff portion packs;
4. Plug;
5. Twist;
6. Loose leaf;
7. Dry snuff portion packs;
8. Snus;
9. Snus portion packs; and
10. Pellet or Compressed.
Because of their physical characteristics, some of the ten product
categories must be ground (whole or in part) before nicotine, total
moisture, and pH analyses can be conducted. The objective of grinding
the samples is to obtain a homogeneous sample with particles measuring
approximately 4 mm. Grinding to achieve this particle size should take
no more than 3 minutes. To ensure proper grinding and an adequate
amount of the ground sample for analysis, the minimum sample size of
all commercial products to be ground should not be less than 100 grams.
To ensure precision of analyses for nicotine, total moisture, and
pH, the samples that require grinding should be ground using a Robot
Coupe Model RSI 2V Scientific Batch Processor or its equivalent. This
is a variable speed (0 to 3000 RPM) processor. The variable speed motor
is required to ensure proper grinding of the tobacco tissues (and in
the case of pH determination, the portion pack). Elevated temperatures
can result in moisture loss and an underestimated value for moisture
content. Hence, care must be taken during grinding to avoid elevated
temperatures. The bowl should be cleaned after each grinding to obtain
accurate results. Freeze- or cryo-grinding is also an acceptable
grinding method.
1. Dry snuff: Dry snuff samples do not need to be ground since the
product is a powder. The sample must be thoroughly mixed before
weighing for nicotine, total moisture, and pH analysis.
2. Moist (wet) snuff: Moist (wet) snuff samples do not need to be
ground. The sample must be thoroughly mixed before weighing for
nicotine, total moisture, and pH analysis.
3. Moist (wet) snuff portion packs: The tobacco contents of the
moist (wet) snuff portion packs do not need to be ground for nicotine,
total moisture, or pH analysis. The tobacco packaging material (the
``pouch'') should be separated from the tobacco and ground to obtain
particles measuring approximately 4 mm for pH analysis. The tobacco of
the moist (wet) snuff portion pack and the ground pouch are combined
and thoroughly mixed before pH analysis.
4. Plug tobacco: Break or cut apart plugs and add in portions to
grinder at 2000 RPM. Reduce RPM or stop grinding if sample bowl becomes
warm. Pulse the Robot Coupe, when needed, to complete grinding. Grind
samples until approximately 4 mm in size. The total grinding time
should be no more than 3 minutes.
5. Twist tobacco: Separate twists, add to grinder and grind at 2000
RPM. Reduce RPM or stop grinding if sample bowl becomes warm. Continue
grinding until sample particles are approximately 4 mm in size. The
total time for grinding should be no more than 3 minutes.
6. Loose leaf: Grind in the same manner as described in 4 and 5 to
obtain product with particle size of approximately 4 mm.
7. Dry snuff portion packs: The tobacco contents of the dry snuff
portion packs do not need to be ground for nicotine, total moisture, or
pH analysis. The tobacco packaging material (the ``pouch'') should be
separated from the tobacco and ground to obtain particles measuring
approximately 4 mm for pH analysis. The tobacco of the dry snuff
portion pack and the ground pouch are combined and thoroughly mixed
before pH analysis.
8. Snus: Snus samples do not need to be ground since the product is
a powder. The sample must be thoroughly mixed before weighing for
nicotine, total moisture, and pH analysis.
9. Snus portion packs: The tobacco contents of the snus portion
packs do not need to be ground for nicotine, total moisture, or pH
analysis. The tobacco packaging material (the ``pouch'') should be
separated from the tobacco and ground to obtain particles measuring
approximately 4 mm for pH analysis. The tobacco of the snus portion
pack and the ground pouch are combined and thoroughly mixed before pH
analysis.
10. Pellet or compressed: Break apart compressed tobacco pellets
and add in portions to grinder at 2000 RPM. Reduce RPM or stop grinding
if sample bowl becomes warm. Pulse the Robot Coupe, when needed, to
complete grinding. Grind samples until approximately 4 mm in size. The
total grinding time should be no more than 3 minutes.
[[Page 715]]
II. Nicotine Analysis \12\
A. Calibration Standards
1. Internal Standard (IS)
Weigh 10.00 grams of quinoline, transfer to a 250 mL volumetric
flask and dilute to volume with MTBE. This solution will be used for
calibration of the instrument for the nicotine calibration curve
(II.A.2), for the standards addition assay (II.B), and for preparation
of the extracting solution (II.D).
2. Nicotine Calibration Curve
a. Weigh 1.0000 gram of nicotine into a clean, dry 100 mL
volumetric flask and dilute to volume with MTBE. This gives a nicotine
concentration of 10 mg/mL for the stock solution.
b. Accurately pipette 0.5 mL of IS from stock solution (II.A.1) to
five clean, dry 50 mL volumetric flasks. To prepare a nicotine standard
corresponding to a concentration of 0.8 mg/mL, pipette exactly 4.0 mL
of the nicotine standard (II.A.2.a) to a 50 mL volumetric flask
containing the internal standard and dilute to volume with MTBE. To
obtain nicotine concentrations equivalent to 0.6, 0.4, 0.2, and 0.1 mg/
mL, pipette precisely 3.0, 2.0, 1.0, and 0.5 mL, respectively, of the
nicotine standard into the four remaining flasks and dilute to volume
with MTBE.
c. Transfer aliquots of the five standards to auto sampler vials
and determine the detector response for each standard using gas
chromatographic conditions described in I.E.
d. Calculate least squares line for linear equation from these
standards by obtaining the ratio of Areanicotine/
AreaIS. This ratio will be the Y value and the concentration
of nicotine will be the X value for determining the linear equation of
the line (Equation 1):
[GRAPHIC] [TIFF OMITTED] TN07JA09.000
Where:
X = Concentration of nicotine in mg
Y = Areanicotine/ AreaIS
a = intercept on the ordinate (y axis)
b = slope of the curve
The final result will be reported in the following units:
Concentration of nicotine = mg of nicotine/gram of tobacco sample.
e. Determine the recovery of nicotine by pipetting 10 mL of the 0.4
mg/mL nicotine standard to a screw capped tube containing 1.0 mL of 2 N
NaOH. Cap the tube. Shake the contents vigorously and allow the phases
to separate. Transfer an aliquot of the organic phase to an injection
vial and inject. Calculate the concentration of nicotine using the
equation of the line in II.A.2.d above. This should be repeated two
more times to obtain an average of the three values. The recovery of
nicotine can be obtained by using the following equation:
[GRAPHIC] [TIFF OMITTED] TN07JA09.001
B. Standards Addition Assay
Prior to analyzing a smokeless tobacco product for nicotine
content, the testing facility must validate the system to verify that
matrix bias is not occurring during nicotine extraction. This is done
by analyzing the nicotine calibration standards in the same vegetable
matrix as the smokeless tobacco. The first time each smokeless tobacco
product is tested and whenever a change is made to the product
formulation (including a change to the tobacco blend or cultivar), the
Standards Addition Assay will be performed, and documentation of its
performance and of the nicotine concentrations selected for the
standard curve (II.B.2) will be submitted to the Centers for Disease
Control and Prevention.
1. Using an analytical balance, accurately weigh 1.000
0.020 gram of the homogeneous, prepared tobacco sample into a culture
tube. Repeat this five times for a total of 6 culture tubes containing
the smokeless tobacco product. Record the weight of each sample.
2. Prepare a five-point standard curve for the Standards Addition
Assay. The standard curve must consist of nicotine concentrations that
encompass the range of values expected from adding known concentrations
of the nicotine standard (II.A.2.a) to a measured quantity of the
smokeless tobacco product (1.000 0.020 gram, described in
II.B.1). The sixth culture tube is not supplemented with nicotine and
serves as an analytical blank. Allow the samples to equilibrate for 10
minutes.
3. Pipette 5 mL of 2 N NaOH into each tube. Cap each tube. Swirl to
wet sample and allow to stand 15 minutes.\13\
4. Pipette 50 mL of extraction solution (II.D.1) into each tube.
Cap each tube and tighten.\14\
5. Place tubes in rack(s), place racks in linear shaker in
horizontal position and shake for two hours.
6. Remove rack(s) from shaker and place in vertical position to
allow the phases to separate.
7. Allow the solvent and nicotine supplemented samples and the
blank to separate (maximum 2 hours).
8. Transfer aliquots of the five standards and the blank from the
extraction tubes to sample vials and determine the detector response
for each using gas chromatographic conditions described in I.E.
9. Subtract the Areanicotine/AreaIS of the
blank from the Areanicotine/AreaIS of each of the
standards.
10. Calculate least squares line for linear equation from the
corrected standards as described above (Equation 1) in II.A.2.d. The
final corrected result will be reported in the following units:
Concentration of nicotine = mg of nicotine/gram of tobacco sample.
11. Determine the recovery of nicotine by pipetting 10 mL of the
0.4 mg/mL nicotine standard to a screw capped tube containing 1.0 mL of
2 N NaOH and 10 mL of extraction solution (II.D.1). Cap the tube and
tighten. Shake the contents vigorously and allow the phases to
separate. Transfer an aliquot of the organic phase to an injection vial
and inject. Calculate the concentration of nicotine using the equation
of the line above in II.A.2.d. This should be repeated two more times
to obtain an average of the three values. The recovery of nicotine can
be obtained by using Equation 2: Recovery =
Nicotinecalculated/Nicotineactual.
12. Compare the results of steps II.A.2 and II.B. If they differ by
a factor of 10% or more, the recovery of nicotine from the aqueous
matrix is not equivalent to recovery from the vegetable matrix of the
smokeless tobacco product. In this instance, the nicotine concentration
of the smokeless tobacco product must be determined from a nicotine
calibration curve prepared from nicotine standards in a vegetable-based
matrix.
C. Quality Control Pools
At least two quality control pools at the high and low ends of the
expected nicotine values are recommended to be included in each
analytical run. The pools should be analyzed in duplicate in every run.
The quality control pools should be available in sufficient quantity to
last for all analyses of a product.
D. Sample Extraction Procedure \12\
1. Extraction solution is prepared by pipetting 10 mL of the IS
from the stock solution (II.A.1) to a 1000 mL volumetric flask and
diluting to volume with MTBE.
2. Using an analytical balance, accurately weigh 1.000
0.020 gram of prepared tobacco sample into culture tube and record
weight.\15\ Sample each smokeless tobacco brand name according to the
provided testing frequency schedule.\19\ The number of
[[Page 716]]
products sampled should reflect an acceptable level of precision.\16\
The test material is to be representative of the product that is sold
to the public and therefore should consist of sealed, packaged samples
of finished product that is ready for commercial distribution. Samples
are to be analyzed in duplicate.
3. Pipette 5 mL of 2 N NaOH into the tube. Cap the tube. Swirl to
wet sample and allow to stand 15 minutes.\13\
4. Pipette 50 mL of extraction solution into tube, cap tube and
tighten.\14\
5. Place tubes in rack(s), place racks in linear shaker in
horizontal position and shake for two hours.
6. Remove rack(s) from shaker and place in vertical position to
allow the phases to separate.
7. Allow the solvent and sample to separate (maximum 2 hours).
Transfer an aliquot from the extraction tube to a sample vial and cap.
8. Analyze the extract using GC conditions as described above (I.E)
and calculate the concentration of nicotine using the linear
calibration equation. Correct percent nicotine values for both recovery
and weight of sample by using Equation 3.\17\
[GRAPHIC] [TIFF OMITTED] TN07JA09.002
9. Report the final nicotine determination as mg of nicotine per
gram of the tobacco product (mg nicotine/gram), to an accuracy level of
two decimal places for each brand name (e.g., Skoal Bandits
Wintergreen, Skoal Long Cut Cherry, Skoal Long Cut Wintergreen, etc.).
All data should include the mean value with a 95% confidence interval,
the range of values, the number of samples tested, the number of lots
per brand name, and the estimated precision of the mean. Information
will be reported for each manufacturer and variety (including brand
families and brand variations) and brand name (e.g., Skoal Bandits
Wintergreen, Skoal Long Cut Cherry, Skoal Long Cut Wintergreen, etc.).
III. Total Moisture Determination
A. This procedure is a modification of AOAC Method 966.02 (1990)
and is referred to as ``Total Moisture Determination'' because it
determines water and tobacco constituents that are volatile at
temperatures of 99 1.0[deg]C.
B. Accurately weigh 5.00 grams of the sample (ground to pass <= 4
mm screen) \20\ into a weighed moisture dish and place uncovered dish
in oven.\21\ Sample each smokeless tobacco brand name according to the
provided testing frequency schedule.\19\ The number of products sampled
should reflect an acceptable level of precision.\16\ The test material
is to be representative of the product that is sold to the public and
therefore should consist of sealed, packaged samples of finished
product that is ready for commercial distribution. Samples are to be
analyzed in duplicate.
C. Do not exceed 1 sample/10 sq in (650 sq cm) shelf space, and use
only 1 shelf. Dry 3 hr at 99 1.0[deg]C. Remove from oven,
cover, and cool in desiccator to room temperature (about 30 min).
Reweigh and calculate percent moisture.
D. Report the final moisture determination as a percentage (%), to
an accuracy level of one decimal place for each brand name (e.g., Skoal
Bandits Wintergreen, Skoal Long Cut Cherry, Skoal Long Cut Wintergreen,
etc.). All data should include the mean value with a 95% confidence
interval, the range of values, the number of samples tested, the number
of lots per brand name, and the estimated precision of the mean.
Information will be reported for each manufacturer and variety
(including brand families and brand variations) and brand name (e.g.,
Skoal Bandits Wintergreen, Skoal Long Cut Cherry, Skoal Long Cut
Wintergreen, etc.).
IV. pH Measurement \12\ \22\
A. Test samples as soon as possible after they are received. Sample
each smokeless tobacco brand name according to the provided testing
frequency schedule.\19\ The number of products sampled should reflect
an acceptable level of precision.\16\ The test material is to be
representative of the product that is sold to the public and therefore
should consist of sealed, packaged samples of finished product that is
ready for commercial distribution. Samples are to be analyzed in
duplicate.
B. Accurately weigh 2.00 grams of the sample. Place in a 50 mL
polypropylene container with 20 mL deionized distilled water.
C. Place Teflon-coated magnetic stirring bar in container and stir
mixture continuously throughout testing.
D. Measure pH of sample after a two-point calibration of the pH
meter to an accuracy of two decimal places using standard pH buffers
(4.01 and 7.00 or 7.00 and 10.00) that will encompass the expected pH
value of the smokeless tobacco product.
E. The first time pH values are determined for a smokeless tobacco
product, measure the pH of the smokeless tobacco product at 5, 15, and
30 minutes. If there is no systematic variation in pH values with time,
all subsequent pH determinations are made at 5 minutes. If there is
systematic variation in pH values, continue to measure the pH of the
smokeless tobacco product until the pH value is stable and does not
vary more than 10% over 15 minutes. Report the final pH value.
F. Report the final pH determination to an accuracy level of two
decimal places for each brand name (e.g., Skoal Bandits Wintergreen,
Skoal Long Cut Cherry, Skoal Long Cut Wintergreen, etc.). All data
should include the mean value with a 95% confidence interval, the range
of values, the number of samples tested, the number of lots per brand
name, and the estimated precision of the mean. Information will be
reported for each manufacturer and variety (including brand families
and brand variations) and brand name (e.g., Skoal Bandits Wintergreen,
Skoal Long Cut Cherry, Skoal Long Cut Wintergreen, etc.).
G. Estimate the un-ionized (free) nicotine content with the
Henderson-Hassel Balch equation (Equation 4), based on measured pH and
nicotine content.
[[Page 717]]
[GRAPHIC] [TIFF OMITTED] TN07JA09.003
H. Report the final estimated un-ionized (free) nicotine as a
percentage (%) of the total nicotine content, to an accuracy level of
two decimal places and as mg of un-ionized (free) nicotine per gram of
the tobacco product (mg un-ionized (free) nicotine/gram), to an
accuracy level of two decimal places for each brand name (e.g., Skoal
Bandits Wintergreen, Skoal Long Cut Cherry, Skoal Long Cut Wintergreen,
etc.). All data should include the mean value with a 95% confidence
interval, the range of values, the number of samples tested, the number
of lots per brand name, and the estimated precision of the mean.
Information will be reported for each manufacturer and variety
(including brand families and brand variations) and brand name (e.g.,
Skoal Bandits Wintergreen, Skoal Long Cut Cherry, Skoal Long Cut
Wintergreen, etc.).
Sample calculation:
[GRAPHIC] [TIFF OMITTED] TN07JA09.004
[[Page 718]]
V. Assay Criteria for Quality Assurance
A. Establishing Limits for Quality Control Parameters
All quality control parameters must be determined within the
laboratory in which they are to be used. At least 10 within-laboratory
runs must be performed to establish temporary confidence intervals for
the quality control parameters. Permanent limits should be established
after 20 runs and should be reestablished after each additional 20
runs.
B. Exclusion of Outliers from the Calibration Curve \18\
The coefficient of determination between Areanicotine/
AreaIS and nicotine concentration should be equal to 0.99 or
higher. Any calibration standard having an estimated concentration
computed from the regression equation (Equation 1) which is different
from its actual concentration by a factor of 10% can be excluded from
the calibration curve. Up to two concentrations may be excluded, but
caution should be used in eliminating values, since bias may be
increased in the calibration curve. If an outlier value is eliminated,
its duplicate value must also be discarded to avoid producing a new
bias. All unknowns must fall within the calibration curve; therefore,
duplicate values excluded at either end of the calibration curve will
restrict the useful range of the assay.
C. Quality Control Pools and Run Rejection Rules
The mean estimated nicotine concentration in a pool should be
compared with the established limits for that pool based on at least 20
consecutive runs. An analytical run should be accepted or rejected
based upon the following set of rules adapted from Westgard et al.
(1981).
1. When the mean of one QC pool exceeds the limit of x
3 standard deviations (SD), then the run is rejected as out of control.
Here, x and SD represent the overall mean and standard deviation of all
estimated nicotine concentrations for a particular pool in the runs
which were used to establish the control limits.
2. When the mean nicotine concentrations in two QC pools in the
same run exceed the same direction, then the run must be rejected. The
same direction is the condition in which both pools exceed either the x
+ 2 SD or the x -2 SD limits.
3. When the mean nicotine concentrations in one or two QC pools
exceed their x 2 SD limits in the same direction in two
consecutive runs, then both runs must be rejected.
4. When the mean nicotine concentrations in two QC pools are
different by more than a total of 4 SD, then the run must be rejected.
This condition may occur, for example, when one QC pool is 2 SD greater
than the mean, and another is 2 SD less than the mean.
Endnotes
The comments and notes listed below can be described as Good
Laboratory Practice guidelines; they are described in detail in this
protocol to ensure minimal interlaboratory variability in the
determination of nicotine, total moisture, and pH in smokeless tobacco.
\1\ This protocol assumes that the testing facility will implement
and maintain a stringent Quality Assurance/Quality Control program to
include, but not be limited to, regular interlaboratory comparisons,
determination of the quality and purity of purchased products, and
proper storage and handling of all reagents and samples.
\2\ When a specific product or instrument is listed, it is the
product or instrument that was used in the development of this method.
Equivalent products or instruments may also be used. Use of trade names
is for identification only and does not constitute endorsement by the
Public Health Service or the U.S. Department of Health and Human
Services.
\3\ All chemicals, solvents, and gases are to be of the highest
purity.
\4\ Companies must ensure that the purity of the nicotine base is
certified by the vendor and that the chemical is properly stored.
However, nicotine base oxidizes with storage, as reflected by the
liquid turning brown. If oxidation has occurred, the nicotine base
should be distilled prior to use in making a standard solution.
\5\ A suggested method for the determination of nicotine purity is
CORESTA Recommended Method No. 39.
\6\ Horizontal shaking will allow more intimate contact of this
three phase extraction. There is a minimal dead volume in the tube due
to the large sample size and extraction volume. This necessitates
horizontal shaking.
\7\ If a linear shaker is not available, a wrist action shaker
using 250 mL stoppered Erlenmeyer flasks can be substituted. Values for
nicotine are equivalent to those obtained from the linear shaker.
\8\ After installing a new column, condition the column by
injecting a tobacco sample extract on the column, using the described
column conditions. Injections should be repeated until areas of IS and
nicotine are reproducible. This will require approximately four
injections. Recondition column when instrument has been used
infrequently and after replacing glass liner.
\9\ Glass liner and septum should be replaced after every 100
injections.
\10\ Most older instruments operate at constant pressure. To reduce
confusion, it is suggested that the carrier gas flow through the column
be measured at the initial column temperature.
\11\ The testing facility must ensure that samples are obtained
through the use of a survey design protocol for sampling ``at one point
in time'' at the factory or warehouse. The survey design protocol must
address short-, medium-, and long-term smokeless tobacco product
variability (e.g., variability over time and from container to
container of the tobacco product) in a manner equivalent to that
described for cigarette sampling in Annex C of ISO Protocol 8243.
Information accompanying results for each sample should include, but
not be limited to:
For each product--manufacturer and variety (including brand
families and brand variations) and brand name (e.g., Skoal Bandits,
Skoal Long Cut Cherry, Skoal Long Cut Wintergreen, etc.):
1. Product ``category,'' e.g., loose leaf, plug, twist, dry snuff,
moist (wet) snuff, etc.
2. Lot number.
3. Lot size.
4. Number of randomly sampled, sealed, packaged (so as to be
representative of the product that is sold to the public) smokeless
tobacco products selected (sampling fraction) for nicotine, moisture,
and pH determination.
5. Documentation of method used for random sample selection.
6. `''Age'' of product when received by testing facility and
storage conditions prior to analysis.
\12\ Extraction of nicotine and pH determination must be performed
with reagents and samples at a room temperature of 22-25[deg]C. Room
temperature should not vary more than 1[deg]C during extraction of
nicotine or pH determination.
\13\ Use non-glass 10 mL repipette for transferring NaOH solution.
\14\ Use 50 mL repipette for transferring MTBE.
\15\ For dry snuff, use 0.500 0.010 gram sample.
\16\ The testing facility is referred to ISO Procedure 8243 for a
discussion of sample size and the effect of variability on the
precision of the mean of the sample (ISO 8243, 1991).
\17\ When analyzing new smokeless tobacco products, extract product
without IS to determine if any
[[Page 719]]
components co-elute with the IS or impurities in the IS. This
interference could artificially lower calculated values for nicotine.
\18\ The calculated nicotine values for all samples must fall
within the low and high nicotine values used for the calibration curve.
If not, prepare a fresh nicotine standard solution and an appropriate
series of standard nicotine dilutions. Determine the detector response
for each standard using chromatographic conditions described in I.E.
\19\ The testing frequency for each smokeless tobacco brand name
(e.g., Skoal Bandits Wintergreen, Skoal Long Cut Cherry, Skoal Long Cut
Wintergreen, etc.) is based on the manufacturing duration (refer to
table below). Each smokeless tobacco brand name will be sampled and
tested for nicotine, total moisture, and pH no fewer than twice and no
more than four times during a calendar year.
------------------------------------------------------------------------
Test
Manufacturing duration in weeks frequency*
------------------------------------------------------------------------
up to and including 4...................................... 2
up to and including 28..................................... 3
up to and including 52..................................... 4
------------------------------------------------------------------------
*Use a statistical program to determine random sampling dates based on
the total manufacturing duration during a calendar year. Sampling
dates should fall on actual manufacturing days for the product when
test material that is representative of the product that is sold to
the public (consisting of sealed, packaged samples) is available. If a
statistically determined sampling date falls on a day that does not
meet this criterion, sample the product on the next date that does
meet the criteria.
For smokeless tobacco brand names with episodic production during a
calendar year, the total number of sampling dates is determined by the
sum of the individual test frequencies, not to exceed four. For the
purpose of the Protocol, episodic production is defined as
manufacturing intervals separated by periods of 30 or more days when
the smokeless tobacco brand name is not manufactured.
Example 1: Within a single calendar year a smokeless tobacco
brand name is manufactured from January 1 to March 31 and from
September 1 to December 15. The testing frequency for the first
manufacturing interval is 3 and for the second manufacturing
interval is 3. The Protocol allows that each smokeless tobacco brand
name be tested for nicotine, total moisture, and pH no more than
four times during a calendar year. Therefore, 4 random sampling
dates, as described in the footnote to the above table, are
determined for the smokeless tobacco brand name. The values for
nicotine, moisture, and pH determinations, and unionized (free)
nicotine calculations and the mean of the 4 data points for that
smokeless tobacco brand name are reported.
Example 2: Within a single calendar year a smokeless tobacco
brand name is manufactured from April 5 to May 3 and from September
1 to December 15. The testing frequency for the first manufacturing
interval is 2 and for the second manufacturing interval is 3. The
values for nicotine, moisture, and pH determinations, and unionized
(free) nicotine calculations and the mean of the 4 data points for
that smokeless tobacco brand name are reported.
Example 3: Within a single calendar year a smokeless tobacco
brand name is manufactured from January 1 to January 15 and from
September 1 to September 22. The testing frequency for the first
manufacturing interval is 2 and for the second manufacturing
interval is 2. Four random sampling dates are selected to fall
within the 6 weeks of manufacturing for the smokeless tobacco brand
name. The values for nicotine, moisture, and pH determinations, and
unionized (free) nicotine calculations and the mean of the 4 data
points for that smokeless tobacco brand name are reported.
\20\ The method is a modification of AOAC Method 966.02 (1990) in
that the ground tobacco passes through a 4 mm screen rather than a 1 mm
screen.
\21\ When drying samples, do not dry different products (e.g.,
moist (wet) snuff, dry snuff, loose leaf) in the oven at the same time
since this will produce errors in the moisture determinations.
\22\ The method is a modification of a method published by
Henningfield et al. (1995).
References
AOAC (Association of Official Analytical Chemists). Official
Methods of Analysis. 966.02: Moisture in Tobacco. (1990) Fifth Edition.
K. Helrich (ed). Association of Official Analytical Chemists, Inc.,
Suite 400, 2200 Wilson Boulevard, Arlington, Virginia 22201 USA.
CORESTA (Centre de Coop[eacute]ration pour les Recherches
Scientifiques relatives au Tabac). Recommended Method No. 39:
Determination of the purity of nicotine and nicotine salts by
gravimetric analysis--Tungstosilic acid method. November, 1994. 87-90.
CRC Handbook of Chemistry and Physics. R.C. Weast, D.R. Lide, M.J.
Astle, and WH. Beyer (eds). 70th ed. Boca Raton, Florida: CRC Press
(1989-1990) D-162.
Henningfield, J.E., Radzius A., Cone E.J. (1995). Estimation of
available nicotine content of six smokeless tobacco products. Tobacco
Control 4:57-61.
ISO (International Organization for Standardization). IOS 8243:
Cigarettes--Sampling. (1991). Second Edition. Prepared by Technical
Committee ISO/TC 126, Tobacco and tobacco products. International
Organization for Standardization, Case Postale 56, CH-1211 Genve 20,
Switzerland.
Westgard J.O., Barry P., Hunt M., and Groth T. (1981). A multi-rule
Shewhart chart for quality control in clinical chemistry. Clinical
Chemistry 27:493.
[FR Doc. E9-19 Filed 1-6-09; 8:45 am]
BILLING CODE 4163-18-P