Press Center
NTP Liaison Office Update - March 1997
March, 1997
- Electric and Magnetic Fields (EMF) Research and Public Information Dissemination (Rapid) Program
- EMF Science Review Symposium March 24-27, 1997 Durham, North Carolina
- NTP Board of Scientific Counselors' Meetings for 1996
- Preliminary Agenda Topics for May 14 Meeting of the NTP Board of Scientific Counselors'
- NTP Board of Scientific Counselors' Technical Reports Review Subcommittee Draft Technical Reports Tentatively Scheduled for Review in 1997
- NIEHS Conference "Estrogens in the Environment IV" July 20-23, 1997, Arlington, Virginia
- Call for Nominations of Naturally Occurring Chemicals for NTP Study
- Chemicals Reviewed for Listing in the 8th Biennial Report on Carcinogens (BRC)
- Call for Nominations for Listing or Delisting Substances in the Biennial Report on Carcinogens
- "What's New" on the NTP Website
- Validation and Regulatory Acceptance of Toxicological Test Methods: A Report of the ad hoc Interagency Coordinating Committee on Validation of Alternative Methods (ICCVAM) Now Available
- NTP Technical Reports Review Subcommittee Board of Scientific Counselors'
EMFRAPIDProgram
BACKGROUND
The Electric and Magnetic Fields Research and Public Information Dissemination (EMFRAPID) Program was established by Congress in the Energy Policy Act of 1992. The Department of Energy and The National Institute of Environmental Health Sciences (NIEHS) are partners in the EMFRAPID Program's effort to address the question of whether extremely low frequency electric and magnetic fields (EMF) produced by the generation, transmission, and use of electric energy pose a risk to human health, and if so, to determine the significance of the risk and to develop mitigation technologies.
REPORT TO CONGRESS
In addition to the establishment
of a research program on EMF health effects, the NIEHS director
must provide a report to Congress on the extent to which exposure
to EMF affects human health. The report will be an objective scientific
judgment based upon a synthesis of all available knowledge of
both EMF and the biological systems they may be perturbing. This
assessment will combine both a critical evaluation of the scientific
literature as well as an evaluation of the weight and strength
of the evidence concerning human health effects associated with
EMF exposures.
SCIENCE REVIEW SYMPOSIA AND WORKING GROUP MEETING
To instigate the scientific
review process, the NIEHS will convene a series of three science
review symposia for these study areas: theoretical and in vitro
research findings, epidemiological results, and in vivo
and clinical laboratory findings. At each symposium the participants
will discuss and evaluate the quality and reproducibility of the
research findings and the degree to which the scientific evidence
can support a causal linkage between EMF and biological and/or
health effects. A working group meeting of a select group of principal
authors will follow the symposia. Using information from the symposia's
discussions as well as performing an overall critical review of
the literature, the working group will produce a report that will
draw conclusions on the strength and robustness of the data and
its implications for human health and disease etiology.
PUBLIC ACCESS AND COMMENT
The science symposia will
be public, open meetings and the symposia's discussion reports
and the working group document will be widely circulated for comment.
When summarizing findings from the symposia and working group
document as part of its report to Congress, the NIEHS will solicit
input and review from the public, advisory groups, and the scientific
community.
Additional information about EMFRAPID
is available at its world-wide-website: http://www.niehs.nih.gov/emfrapid/home.htm,
by e-mail request to EMF@pbpk.niehs.nih.gov, or by mail to LCBRA,
NIEHS, P.O. Box 12233, MD A3-06, Research Triangle Park, NC 27709.
EMF SCIENCE REVIEW SYMPOSIUM
The first EMF Science Review
Symposium is scheduled for March 24-27, 1997 at the Regal University
Hotel, Durham, NC. This symposium will focus on a discussion and
evaluation of theoretical mechanisms and in vitro research
findings related to extremely low frequency EMF. A topic outline
for this symposium is as follows:
March 24 | Biophysical Mechanisms |
March 25 | Cellular Growth, Differentiation, and Control of Gene Expression |
March 26 | Enzymes, Intracellular Pathways, and Signal Transduction |
March 27 | Implications of Theoretical Mechanisms and In Vitro Research Findings for Human Health Risk |
General overviews of the selected
areas will be presented in plenary talks and breakout group sessions
will provide time for in-depth, small group discussions and evaluations
of experimental findings and biophysical observations. On the
fourth day, an expert, scientific panel will debate the scientific
evidence based upon theoretical mechanisms and in vitro
research findings for whether exposure to EMF is a human health
risk. The symposium is open to all interested parties and a nominal
registration fee will be charged.
To obtain additional information
about registration call 919-541-7539 or send a message by fax
to 919-541-1479 or by e-mail to emf@pbpk.niehs.nih.gov.
NTP Board of Scientific Counselors' Meeting, December 13, 1996
The NTP Board of Scientific Counselors'
met in public session at the NIEHS on December 13, 1996. The primary
agenda topic was a comprehensive discussion and review of the
NTP nomination and selection process. Background on the process
was provided by NIEHS staff and presentations were made by staff
from NTP participating agencies including FDA, NIOSH, NCI, EPA,
and OSHA about their contributions to the nomination and selection
process. The Board reviewed and approved concept proposals for
a support contract for an Interagency Center for the Evaluation
of Alternative Toxicological Methods and for expanded support
for the preparation of the Biennial Report on Carcinogens.
The Board was informed of recent activities of its subcommittees
-- the BRC Subcommittee and the Technical Reports Review Subcommittee.
Scheduled presentations by NTP agencies on their ongoing and planned
research activities on endocrine disruptors were deferred to the
first 1997 meeting due to insufficient time.
For copies of the summary minutes
from the meetings of the Biennial Report on Carcinogens Subcommittee,
November 18-19, 1996; the Technical Reports Review Subcommittee,
December 11-12, 1996; and the Board of Scientific Counselors',
December 13, 1996, please contact:
Central Data Management (CDM)
P. O. Box 12233,
Mail Drop E1-02
Research Triangle Park, NC 27709
(Tel): (919) 541-3419
(Fax): (919) 541-3687
Internet at CDM@niehs.nih.gov
Preliminary Agenda Topics for May 14 Meeting of the NTP Board of Scientific Counselors'
Upcoming Board and Subcommittee
Meetings in 1997
The next meeting of the NTP Board
of Scientific Counselors' will be held at the NIEHS on May 14,
1997. Primary agenda topics will include (1) a discussion of nomination
and selection strategies for reproductive and developmental toxicology
efforts in the NTP, and (2) presentations by NTP agencies on their
ongoing and planned research activities on endocrine disruptors.
The next meeting of the Board's
Biennial Report on Carcinogens (BRC) Subcommittee to review chemicals
nominated for listing in or delisting from the 9th Biennial
Report on Carcinogens will be held at the NIEHS in the Fall
of 1997. The next meeting of the Board's Technical Reports Review
Subcommittee to peer review draft Technical Reports of NTP long-term
toxicology and carcinogenesis studies also will be held at the
NIEHS in the Fall of 1997. A listing of the chemicals for which
draft Reports are tentatively scheduled for review is provided
in a separate following section of this Update.
For more information closer to any of these meetings, please contact
Dr. Larry Hart, (919)541-3971; FAX (919) 541-0295; or e-mail
hart@niehs.nih.gov.
NTP Board of Scientific Counselors' Technical Reports Review Subcommittee Draft Technical Reports Tentatively Scheduled for Review in 1997
Tentatively scheduled for peer review in 1997 are draft technical reports for 12 chemicals:
2-Butoxyethanol, 1-Chloro-2-propanol, Coconut Oil Diethanolamine, Diethanolamine, Furfuryl Alcohol, Isoprene, Isobutene, Lauric Acid Diethanolamine, Oleic Acid Diethanolamine, Oxymetholone, Pentachlorophenol, Pyridine
NIEHS CONFERENCE:
"ESTROGENS IN THE ENVIRONMENT IV"
JULY 20-23, 1997
CRYSTAL GATEWAY MARRIOTT
Arlington, Virginia
NIEHS held the first Conference on Estrogens in the Environment in 1979 which was a key factor in establishing this area as a public health concern. This fourth conference will continue the tradition of the previous three conferences by evaluating links between fundamental knowledge, toxicology, and risk assessment.
Conference Topics Will Include:
- Overviews of Human Health Effects
- Trends and Health Effects
- Sources of Human Exposure
- Experimental Models
- Mechanisms of Action
- Mixtures/ Interactions
- Sensitive Sub-Populations
- Risk Assessment Implications
- Emerging Issues
To receive further information, including Registration information, please contact:
Ms. Alma Britton at the National Toxicology Program Liaison Office, P.O. Box 12233, A3-01, Research Triangle Park, North Carolina 27709, USA.
Tel: (919) 541-0530, Fax: (919) 541-0295, e-mail: britton@niehs.nih.gov.
Call for Nominations of Naturally
Occurring Chemicals for NTP Study
The NTP and its predecessor, the
National Cancer Institute
Bioassay Program, have in the past 25 years addressed the toxicity
of thousands of chemicals in one or more short- or long-term assays.
Together these Programs have studied and reported the results
of two-year carcinogenicity bioassay tests
of approximately 470 chemicals,
both synthetic and naturally occurring. However there are still
thousands of chemicals that have not
been studied. Thus, the NTP has an active program to identify
and select the best candidates
for our studies. We also depend heavily on nominations from organizations
and individuals outside the Federal government. For example, numerous
excellent nominations have been received from the general public
which, over the past 15 years, has been the third most prolific
contributor of nominations for NTP studies.
In every case, we want selections
for NTP studies to be as relevant as possible. Relevance is based
primarily on the relation to human health risks. One measure of
risk is the number of people exposed to the respective chemical.
It is natural to assume that human exposure is related to the
volume of an industrial chemical produced. This may or may not
be the case. Many chemicals are produced in high volume, but used
primarily or exclusively in closed systems so there is no appreciable
exposure. Other chemicals are not produced commercially, but occur
naturally and may be consumed or encountered by a large portion
of the population. Most naturally occurring chemicals are innocuous
or actually nourishing, however some are quite toxic and others
are known to be carcinogenic.
Since the NTP has a responsibility
to identify risks associated with all types of exposures, we are
increasing our emphasis on naturally occurring chemicals. To this
end, the NTP is making a special request for nominations of chemicals
that 1) occur naturally, 2) that have not been adequately tested
previously and 3) that may represent human health risks.
This request for nominations of
naturally occurring chemicals does not mean that we are no longer
interested in nominations of commercially produced chemicals.
In the past, primary emphasis has been on commercially produced
chemicals, and we would like to place increased emphasis on chemicals
for which primary human exposure is from natural sources. As with
all nominations, new nominations should be accompanied by a statement
supporting the need for study and any available data relating
to chemical identity, toxicity, carcinogenicity or other toxicities,
as well as possible sources and levels of human exposure. All
nominations should be directed to Dr. Errol Zeiger, NIEHS, P.O.
Box 12233, Research Triangle Park, NC 27709.
Chemicals Reviewed for Listing
in the 8th Biennial Report on Carcinogens (BRC)
The NIEHS BRC Review Committee
(RG1), the NTP Executive Committee's Working Group for the BRC
(RG2), and the NTP Board's Subcommittee for the BRC (the Outside
Peer Review Subcommittee) have reviewed and made recommendations
for substances nominated for listing in the 8th BRC. Based on
the recommendations of the three review committees, the NTP is
proposing the addition of 14 agents, substances or mixtures to
the 8th BRC, one of which is to be listed as a known human
carcinogen. The 13 remaining agents, substances or mixtures
are being added as reasonably anticipated to be a human carcinogen.
In addition, it is proposed that thiotepa, which is currently
listed as reasonably anticipated to be a human carcinogen be
moved to the known human carcinogen list. These 15 substances
are listed alphabetically along with supporting information in
the attached table. The review committees used the revised criteria
for listing or delisting substances in the BRC, which were approved
by the Secretary of DHHS on September 13, 1996, and also public
comment received on individual substances in their review.
The review committees were also
asked to advise the Program on the appropriateness of continuing
to reference in the BRC certain manufacturing processes and occupations
which have been considered by the International Agency for Research
on Cancer (IARC) and classified by IARC as sources which are associated
with increased incidences of cancer in workers. There was also
public input received on this issue. Formal comments were submitted
by the Nickel Development Institute (NiDI) and by the Footwear
Industries of America, Inc. (FIA), whose representatives also
made a public statement at the BRC Subcommittee meeting. Both
organizations opposed any reference of their respective "occupation"
or "manufacturing process" in the 8th BRC for legal
as well as scientific reasons. It was the consensus opinion of
all three review committees that, in the interest of public health,
it is appropriate that the BRC contain a reference somewhere in
the report to "Manufacturing Processes and Occupations"
as classified by IARC as sources which are known to be carcinogenic
to humans. The review committees stated that there should be a
disclaimer which indicates that these "Manufacturing Processes
and Occupations" have not been formally reviewed by the NTP
and are provided for information only in the interest of public
health. The disclaimer should refer the reader to the original
IARC references and emphasize that manufacturing processes and
occupations are different throughout the world and that the manufacturing
processes and occupations reviewed by IARC in their determinations
may differ greatly from what has been or is used in the United
States. A copy of the Manufacturing Processes, Occupations, And
Exposure Circumstances Classified by the International Agency
for Research on Cancer (IARC) is attached.
Preparation of the final draft
report for submission to the DHHS Secretary will consider the
input received from the three review groups, the public and the
Federal agency representatives on the NTP Executive Committee.
The final draft of the Report will be completed in the next two
months and submitted by the Director, NTP, to the Secretary, requesting
final approval. It is anticipated that the Report will be released
to Congress and the public during the summer, 1997. Questions
concerning the 8th Biennial Report on Carcinogens should be sent
to Dr. C.W. Jameson at the address shown below:
National Toxicology Program, Biennial Report on Carcinogens, MD WC-05, P.O. Box 12233, Research Triangle Park, NC 27709: (919)541-4096, fax: (919) 541-2242,
email: jameson@niehs.nih.gov
CALL FOR NOMINATIONS FOR LISTING OR DELISTING SUBSTANCES IN THE BIENNIAL REPORT ON CARCINOGENS
NTP encourages the nomination
of agents, substances or mixtures for listing or delisting in
the BRC. The procedures and recently revised criteria for listing
or delisting agents, substances or mixtures in the BRC can be
obtained from Dr. C.W. Jameson at the address shown below.
The most recent report, the 7th Annual Report on Carcinogens (now changed to the Biennial Report on Carcinogens) was published in 1994. The 8th BRC is nearing completion and will be published in 1997. All nominations received in 1997 are expected to receive consideration and review in 1997 and 1998 and any new listings or delistings would be included in the 9th BRC to be published in 1999. Copies of the 7th Annual Report on Carcinogens may be obtained by contacting:
NTP Central Data Management
P.O. Box 12233
MD E1-02
Research Triangle Park, NC 27709
phone: 919-541-3419
fax: 919-541-4714
e-mail: cdm@niehs.nih.gov.
Petitions for listing or delisting
an agent, substance or mixture in the BRC may be submitted by
any interested party and should be sent to:
Dr. C. W. Jameson
NTP, Biennial Report on Carcinogens
P.O. Box 12233
MD WC-05
Research Triangle Park, NC 27709
phone: 919-541-4096
fax: 919-541-2243
e-mail: jameson@niehs.nih.gov.
Petitions will be reviewed as expeditiously as possible. Any questions regarding the
process, procedures, or the information contained herein may also be directed
to Dr. Jameson.
"What's New" on the NTP website:
The EMF Rapid Home Page - Electric
and Magnetic Fields Research and Public Information Dissemination
Program.
Transgenic Mouse Study Data -
Information about the evaluation of Tg.AC (v-Ha-ras) and
the Heterozygous p53-deficient (+/-) mouse as models for rapid
identification of potential carcinogens.
Structural Descriptors of NTP Chemicals - Eighty-two structural descriptors for approximately
240 NTP chemicals.
NTP Shape Database - This database
contains the shape and slope of the dose response curve for approximately
450 NTP chemicals.
Actions on NTP Draft Technical
Reports: Summary Data and Levels of Evidence for Technical Reports
Reviewed at the Meeting of the Board of Scientific Counselor's
Technical Reports Review Subcommittee, December 11-12, 1996.
Correction to the 1996 Annual Plan Summary
The URL for the NTP homepage given
in the NTP 1996 Annual Plan Summary (http://ntp.niehs.nih.gov/ntp)
is incorrect "nig" should be "nih"
Environmental Health Information Service to be Available in 1997
An "Environmental Health Information Service" is expected to be implemented in the
latter half of 1997 by the NIEHS, headquarters of the NTP. This
will be a new website (http://ehis.niehs.nih.gov)
and will be a fee-based service that will include hard copies
and internet access to NTP Technical Reports and the Biennial
Report on Carcinogens, as well as the NIEHS journal, ENVIRONMENTAL
HEALTH PERSPECTIVES, the monthly issues and the supplements. To
receive information on fees and a description of services, contact
EHP at (919) 541-3406, FAX (919) 541-0273, or e-mail at roberts10@niehs.nih.gov.
Validation and Regulatory Acceptance
of Toxicological Test Methods: A Report of the ad hoc
Interagency Coordinating Committee on the Validation of Alternative
Methods Now Available
The report totals 105 pages and
consists of four chapters as well as an Executive Summary. Chapter
one is an introduction that provides a general overview of the
need for toxicological test methods, how they are used, and the
driving forces for the development and validation of new methods.
Chapter two discusses the concept of validation and the criteria
that should be met for a new or revised test method to be considered
for regulatory risk assessment purposes. Chapter three discusses
the criteria that should be used in considering the acceptability
of a test method proposed for regulatory use. It also discusses
the processes involved in achieving regulatory acceptance of a
test method. A series of recommendations for developing a consistent
and efficient process for evaluating new methods for regulatory
acceptance is provided. Recommendations address development and
validation, regulatory review of new methods, intra- and interagency
coordination and harmonization, communication, and international
harmonization. Chapter four discusses an implementation plan to
facilitate the review and consideration of new test methods proposed
for regulatory acceptance.
A standing interagency committee will be established to coordinate the development, validation, acceptance, and national/international harmonization of toxicological test methods. The committee will be designated as the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), and will replace the ad hoc ICCVAM. The ICCVAM will seek to promote sound toxicological test methods that (1) enhance agencies' ability to assess risks and make decisions, and (2) reduce animal use, refine procedures involving animals to make them less stressful, and replace animals in toxicological tests, where scientifically feasible and practical. The Committee anticipates that this effort will help to better evaluate risks to human and animal health and the environment, reduce costs necessary to establish the safety of agents in commerce, and facilitate international trade.
Obtaining the Report
An electronic version of the report,
A Report of the ad hoc Interagency Coordinating Committee on
the Validation of Alternative Methods, is now available on the internet.
To receive a copy of the document, please contact:
NTP Liaison and Scientific Review Office
NIEHS
P.O. Box 12233
MD: A3-01
Research Triangle Park, NC 27709
Fax: (919) 541-0295.
For further information about the Report, please contact one of the ICCVAM co-chairs:
Dr. William Stokes
NIEHS
P.O. Box 12233
Research Triangle Park,
NC 27709
Tel: (919) 541-7997
Fax: (919) 541-0947
e-mail: stokes@niehs.nih.gov
Dr. Richard Hill
US EPA
Mail Code: 7101
401 M Street, S.W.
Washington, D.C. 20460
Tel: (202) 260-2897
Fax: (202) 260-1847
e-mail hill.richard@epamail.epa.gov
NTP Technical Reports Review Subcommittee
Board of Scientific Counselors'
NTP Presents Bioassay Results
The National Toxicology Program
presented ten Technical Reports in the carcinogenesis bioassay
series for public review by the NTP's Board of Scientific Counselors
on December 11 and 12, 1996 at NIEHS. Each report involves a series
of long-term studies in which rodents were given a range of doses
of test chemical followed by extensive histopathologic examination.
Also consider during the review meeting was a series of presentations
on the presence of Helicobacter hepaticus, an organism
that has been associated with liver lesions in mice in some of
the NTP study animals. In some cases liver disease associated
with Helicobacter infection complicated interpretation
of the significance of neoplasms in the liver of male mice; interpretation
of effects at other sites was not adversely affected in these
studies. Female mice are considerably less affected by the organism.
Chloroprene
is the monomeric component in the synthesis of polychloroprene
elastomer (neoprene), and nearly a billion pounds of chloroprene
are used annually worldwide. Rats and mice exposed by inhalation
to atmospheres containing 12.8 to 80 ppm of chloroprene developed
tumors at a variety of sites, including the oral cavity, kidney
and thyroid gland in both male and female rats and lung in male
rats and in both sexes of mice. Mice also had increased incidences
of neoplasms of the circulatory system, Harderian gland, skin,
kidney and liver, and both female rats and female mice had increased
incidences of mammary tumors. Chloroprene was judged to show clear
evidence of carcinogenic activity in all four studies in male
and female rats and mice.
Ethylbenzene
is another high volume chemical, with over 10 billion pounds consumed
annually, mainly in the production of styrene and cellulose acetate.
Male and female rats exposed to atmospheres containing from 75
to 750 ppm ethylbenzene had increased incidences of kidney hyperplasia
and neoplasms, while male mice exhibited higher incidences of
lung tumors, and female mice had increased incidences of liver
tumors. These chemical-related neoplasms constituted clear evidence
of carcinogenicity in male rats and some evidence of carcinogenic
activity in female rats and both sexes of mice.
Oxazepam
is one of the family of benzodiazepine drugs used in the treatment
of anxiety. In earlier NTP studies, oxazepam was shown to cause
liver tumors in both males and females of two strains of mice;
the chemical was tested again, this time in rats. In the present
study oxazepam caused a marginal increase of kidney neoplasms
in male rats, which was judged equivocal evidence of carcinogenic
activity and increased kidney nephropathy in both males and females.
No neoplasm increases were associated with the chemical in female
rats.
Cobalt sulfate
is widely used in the electroplating industry, and occupational
exposure to cobalt compounds occurs mainly by inhalation of dusts
or fumes in refining processes and the production of alloys. Exposure
of rats and mice to atmospheres containing 0.3 to 3 ppm cobalt
sulfate heptahydrate caused lung adenomas and carcinomas in males
and females of both species. In addition, extensive non-neoplastic
lesions in the nose (olfactory epithelial atrophy and metaplasia)
and larynx (squamous metaplasia) were seen in most exposed animals
in all four studies.
Primidone,
a desoxybarbiturate which metabolizes to phenobarbital, is used
as an anticonvulsant in the control of seizures. In the NTP studies
primidone was given in the feed to rats (concentrations up to
2,500 ppm) and to mice (up to 1,300 ppm) for two years. Male rats
had marginal increases in thyroid and kidney tumors, which were
judged equivocal evidence of carcinogenic activity. No tumor increases
were seen in female rats. Male and female mice had marked increases
in incidences of a variety of liver neoplasms, which constituted
clear evidence of carcinogenic activity in both sexes.
AZT
(3'-azido-3'-deoxythymidine) is the most widely used chemotherapeutic
agent for the treatment of persons with AIDS or sero-positive
for HIV. AZT was administered to male and female mice by gavage
in a methylcellulose solution both with and without accompanying
doses of a-interferon A/D (to mimic some therapeutic regimens).
Male mice had slight increases in kidney and Harderian gland neoplasms,
which were judged equivocal evidence of carcinogenic activity.
Female mice had increased incidences of squamous cell carcinomas
of the vagina, which constituted clear evidence of carcinogenic
activity.
Tetrahydrofuran
has a variety of industrial uses as a reaction medium, an intermediate
in chemical synthesis, and as a solvent, and human exposure occurs
primarily in occupational settings. When rodents were exposed
to atmospheres containing up to 1,800 ppm tetrahydrofuran, male
rats experienced increased incidences of kidney tumors and female
mice had increased incidences of liver tumors. Female rats and
male mice did not have increases in neoplasms at any sites.
Theophylline,
an alakaloid structurally similar to caffeine, is found in tea
and chocolate and is used as a bronchodilator and heart stimulant.
When administered daily by oral gavage for two years it showed
no evidence of carcinogenic activity in male or female rats or
mice.
Isobutyraldehyde
is used as a chemical intermediate and flavoring agent and occurs
in many food products. Annual industrial consumption of isobutyraldehyde
in the United States is approximately 500 million pounds. Exposure
of rats and mice to atmospheres containing up to 2,000 ppm isobutyraldehyde
produced inflammation and degeneration of the olfactory epithelium
but no evidence of carcinogenicity in males or females of either
species.
Polyvinyl alcohol, a synthetic polymer with a wide range of chain lengths, has a variety of uses including textile sizing, paper coatings, cosmetics, and as an ingredient in intravaginal contraceptive foams and films. Because of the latter use, the chemical was studied by intravaginal instillation in female mice. No neoplasms or other lesions were observed.
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