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Return to main Five-Year Plan Page
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Plan Overview |
Preface |
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Executive Summary |
Introduction |
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CHAPTER 1 - Research, Development, Translation, and Validation Activities for Priority Test Methods
to Reduce, Refine, and Replace Animals in Regulatory Testing |
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CHAPTER 2 - Incorporating New Science and Technology |
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CHAPTER 3 - Fostering Acceptance and Appropriate Use of Alternative Test Methods |
CHAPTER 4 - Developing Partnerships and Strengthening Interactions with ICCVAM Stakeholders |
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References - Acronyms and Abbreviations - Glossary |
Appendices |
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Acknowledgements - ICCVAM Roster - About NICEATM and ICCVAM
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Chapter 1: Research, Development, Translation, and Validation Activities for Priority Test Methods to Reduce, Refine, and Replace Animals in
Regulatory Testing
ICCVAM’s priorities are based on agency priorities1 as well as other criteria that include:
- The potential impact that alternative test methods may have on reducing, refining, or replacing the use of animals for testing, taking into consideration the severity of pain and distress and numbers of animals involved
- The potential for the proposed test method(s) to provide improved prediction of adverse health or environmental effects
- The applicability of testing alternatives across agencies
ICCVAM uses these criteria to prioritize test method nominations and submissions for evaluation.
Priority Activities
This chapter describes ICCVAM's priority areas based on these criteria. Currently, the four highest priorities are ocular toxicity, dermal toxicity,
acute toxicity, and biologics. Other priority areas include immunotoxicity, endocrine disruption, pyrogen testing, reproductive/developmental toxicity,
and chronic toxicity/carcinogenicity. These priorities will likely evolve over time in response to new testing needs and advances in science and technology.
The inherent complexity of human and animal responses to toxicants means that it is unlikely that any single alternative test method will be able to
serve all regulatory needs for a specific testing area. Rather, integrated approaches using batteries of two or more alternative test methods combined
with other information about the properties of a test substance will likely be needed to significantly reduce or replace the use of animals for each
type of testing. As outlined below, these integrated approaches are being investigated for a number of different toxicity testing areas. Such approaches
may be critical to the development of successful hazard assessment methodologies for complex endpoints such as carcinogenicity or reproductive/developmental
toxicity, which can result from effects on many different pathways. A priority for each of the areas identified below is the collection and use of available
human, animal, and ecological data to assess the performance of existing and new test methods for protecting human and animal health and the environment.
Ocular Toxicity Testing
The evaluation of alternative methods for ocular (eye) safety testing is one of ICCVAM’s four highest
priorities because it is required by multiple agencies as one of the four most commonly required product
safety tests and can therefore involve large numbers of animals, and because rabbits used in tests to
identify ocular hazards can experience significant pain and distress when eye injuries occur. Regulatory
agencies require identification of potential ocular hazards to warn consumers and workers when exposure to a
chemical or product may cause blinding or other kinds of eye damage. Two critically important goals are the
replacement of the rabbit eye test with one or more alternative assay(s) that can provide equal or greater
prediction of these types of hazards, and the implementation of procedures to avoid pain and distress
where animals must still be used. NICEATM and ICCVAM recently evaluated and recommended two in vitro test
methods that can be used to identify certain types of substances that cause permanent and severe eye damage
and that do not use animals. NICEATM and ICCVAM will carry out activities to improve the usefulness and
applicability of these test methods. In addition, in collaboration with the European Centre for the Validation
of Alternative Methods (ECVAM), NICEATM and ICCVAM will evaluate the use of these and other in vitro
test methods for accurately identifying substances that cause reversible eye damage or that do not damage
the eye. NICEATM and ICCVAM will also evaluate in vitro approaches for determining the ocular irritation
potential of antimicrobial cleaning product formulations, and will facilitate the submission of in vivo
reference data to be added to a database for use in expanding the development and applicability of new
alternative ocular test methods.
NICEATM and ICCVAM recently organized scientific symposia on “Mechanisms of Chemically-Induced Ocular
Injury and Recovery,” and “Minimizing Pain and Distress in Ocular Toxicity Testing.” Symposia recommendations
for relevant research, development, and validation studies have been provided to the scientific and regulatory
communities for consideration. NICEATM and ICCVAM will encourage stakeholders to carry out the recommended
studies, and will evaluate new methods or combinations of in vitro test methods that are developed to reduce
or replace animal use for corrosivity and irritation testing. In addition, a comprehensive review of the use
of topical anesthetics and systemic analgesics for reducing pain and distress will be conducted to determine
their applicability in ocular testing.
Biologics Testing
Biological products (commonly referred to as biologics) include vaccines, blood and blood components,
allergenics, somatic cells, gene therapy, tissues, monoclonal antibodies, and recombinant therapeutic proteins
that are used to treat or protect humans or animals. Biologics testing is also one of ICCVAM’s four highest
priorities because it can require large numbers of animals that may experience significant pain and distress
during testing, and it is required by multiple agencies. As such, it is important to identify in vitro
alternatives to the current in vivo tests that provide equal or greater protection of human or animal health,
and to identify procedures that can be used to reduce or avoid pain and distress where animals must still be
used. Alternative test methods are under development that target reduction and replacement of animal testing
with in vitro test methods, as well as refinement of animal testing through modifications to the current animal
tests. To facilitate the development of these types of alternatives, ICCVAM, NICEATM, and ECVAM recently
co-sponsored a workshop that identified activities needed to further reduce, refine, and replace the use
of mice for determining the effectiveness of a biologic product. NICEATM and ICCVAM will evaluate alternative
test methods and testing strategies for vaccine potency testing and will facilitate the acceptance of
adequately validated test methods and humane endpoints found to be sufficiently accurate and reliable.
A priority for evaluation will be an in vitro vaccine potency test being developed by the USDA to reduce the
numbers of animals required to evaluate the potency of a common veterinary bacterial vaccine for Leptospirosis.
Dermal Toxicity Testing
The evaluation and development of alternatives for dermal (skin) safety testing is also one of ICCVAM’s
four highest priorities because it is required by multiple agencies as one of the four most commonly required
product safety tests and therefore can involve large numbers of animals, and because rabbits used in tests
to identify dermal hazards can experience significant pain and distress. Regulatory agencies require
identification of dermal hazards to warn consumers and workers when exposure to a chemical or product
may cause skin corrosivity (permanent scarring/burns) or irritation. This information is used to determine
appropriate precautions needed to avoid such injury. Test results are also used to determine appropriate
packaging to minimize dangerous spills during transport. ICCVAM’s ultimate goal in this area is the
replacement of the rabbit skin test for both corrosivity and irritation with alternative methods that
meet the requirements of U.S. regulators.
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Working Towards Replacement
ICCVAM has organized independent scientific peer reviews of the usefulness and limitations
of in vitro corrosivity test methods for use as alternatives to the in vivo
rabbit skin and eye tests. By using these alternative methods, animal testing of substances
can be avoided that would otherwise cause corrosive injuries to the skin or eyes of rabbits.
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In vitro alternatives for dermal corrosivity have been developed, and several of these test methods
have been recommended and accepted for regulatory use as screening methods. In appropriate circumstances,
substances yielding positive results can be classified and labeled as corrosives without the use of animals.
NICEATM and ICCVAM will evaluate alternative dermal irritation test methods for their usefulness and
limitations in U.S. regulatory testing. This will include an evaluation of the use of a combination of
in vitro test methods for both corrosivity and irritation to reduce or replace animals. NICEATM and ICCVAM
will also evaluate non-animal methods and approaches for determining the skin irritation potential of
antimicrobial cleaning products.
Acute Toxicity Testing
Acute toxicity testing is the most commonly conducted product safety test worldwide. It is also one of
ICCVAM’s four highest priorities because it is required by multiple agencies and therefore can involve large
numbers of animals, and because it can result in significant pain and distress to test animals. Until the
recent adoption of alternative rodent tests that use significantly fewer animals and international guidance
on humane endpoints, a rodent acute oral test using a large number of animals with death as an endpoint was
used to satisfy the requirements of agencies to appropriately label products that cause acute toxicity
(poisoning). ICCVAM has contributed significantly to recent progress in reducing and refining acute
toxicity testing. For example, ICCVAM evaluated and recommended an alternative animal test method that has
now been accepted by regulatory agencies as a replacement for the traditional acute oral toxicity test.
This alternative test method can reduce the use of animals for this purpose by up to 70%. NICEATM and ICCVAM
were also involved in the development of international guidance for humane endpoints that can be used as
criteria to euthanize animals rather than allowing them to die during the study. The ultimate goal is to
find ways to conduct acute oral toxicity testing without animals. In support of this goal, ICCVAM evaluated
and recommended two cell culture test methods that, while not sufficiently accurate to replace animals,
can be used to estimate the starting doses for animal studies, and thereby further reduce the number of
animals needed for each test.
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Reduction Alternative: An ICCVAM Success Story
In 2002, ICCVAM recommended the revised Up-and-Down Procedure (UDP) as a replacement for the
conventional acute oral systemic toxicity test. The UDP can reduce the use of animals for this
type of testing by up to 70%. All Federal regulatory agencies that require acute oral toxicity
testing have accepted the revised UDP. In 2007, ICCVAM recommended that, prior to testing in
animals, in vitro cytotoxicity test methods should be considered as one way to estimate the
starting dose for the revised UDP. This approach is expected to further reduce the number of
animals required for an acute toxicity test by up to 20%.
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An independent peer review panel made several recommendations to ICCVAM for future studies to advance the
use of in vitro methods for assessing acute oral toxicity. In response to these recommendations, ICCVAM plans
to carry out several related activities to promote further development of non-animal replacements, to expand
the use of the in vitro test methods to further reduce animal use, and to further reduce the potential pain
and distress associated with acute toxicity testing. NICEATM and ICCVAM will organize an international
workshop to (1) identify standardized procedures for collecting mechanistic information from acute oral
toxicity testing to aid in developing batteries of predictive in vitro test methods that can further reduce
and eventually replace animals for acute toxicity testing, and (2) seek more predictive and more humane
endpoints that may be used to terminate studies earlier in order to further reduce pain and distress.
In addition, NICEATM will conduct a study to determine how the two cell culture test methods can be used
to set the starting dose for mixtures, which represent a significant percentage of acute testing studies.
NICEATM will also assemble high quality rodent acute oral toxicity data (either from previous studies or,
in cooperation with industry, from future required regulatory studies) and make this reference database
available for the development and validation of other new in vitro tests (or batteries of tests) to
more accurately predict oral acute systemic toxicity. NICEATM and ICCVAM will look for opportunities
to collaborate with the ECVAM ACuteTox Project, which seeks to
develop an in vitro testing strategy to predict human acute oral toxicity in order to replace the animal
acute oral toxicity tests currently used for regulatory purposes. This could include evaluating the state
of the science to better understand the key pathways involved in acute oral toxicity. NICEATM and ICCVAM will
also consider potential alternative methods for acute dermal systemic toxicity and acute inhalation toxicity.
The ATSDR, DOI, EPA, FDA, and NIH also have ongoing or planned activities relevant to the 3Rs for testing
chemicals for acute toxicity. These activities include consideration of modifications to current animal tests
to reduce the number of animals used where possible, as well as evaluations of in vitro test methods
to be used independently or in combination with other tests as possible replacements for animal tests.
NICEATM and ICCVAM will work with these agencies to assist in characterizing the usefulness and
limitations of these methods and to foster their appropriate use among the regulated community.
Immunotoxicity Testing
Immunotoxicity testing is an ICCVAM priority because it can result in significant pain and distress to
test animals, can involve large numbers of animals, and is required by multiple Federal agencies. Regulators
use skin sensitization tests to identify substances that might cause this response in humans following
repeated skin exposure. The Murine Local Lymph Node Assay (LLNA) is an alternative test method used for
skin sensitization testing that reduces the number of animals needed, reduces the time required for testing,
and can substantially reduce or minimize the pain and distress associated with the traditional testing method.
The LLNA was the first alternative test method evaluated and recommended by ICCVAM, and
it has been accepted by regulatory agencies. Based on this evaluation ICCVAM prepared a test guideline
for the LLNA that has been accepted by the Organisation for Economic Co-operation and Development (OECD).
NICEATM and ICCVAM will evaluate whether or not the LLNA can be used as a stand-alone method for the
determination of potency (including severity) and evaluate the possible expansion of the scope of substances
and mixtures for which the LLNA may be used. NICEATM and ICCVAM will also evaluate a number of modifications
to the LLNA that may further reduce the number of animals used, or that may eliminate the need to use
radioactive materials as part of the protocol.
Additional assays are under development that may reduce, refine, or replace the use of animals in skin
and respiratory sensitization testing. Where appropriate, NICEATM and ICCVAM will review and foster
approaches to incorporate valid computational and in vitro methods into laboratory testing strategies.
This will include closely following the ECVAM Sens-It-IV Project that seeks to develop in vitro
alternatives for identifying potential skin or lung sensitizers.
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Refinement Alternative: Minimizing Pain and Distress
ICCVAM recommended the Murine Local Lymph Node Assay (LLNA) as a valid substitute for the
guinea pig maximization test in many testing situations. The LLNA can substantially reduce or minimize
the pain and distress in treated animals that can result from sensitizing chemicals, and also requires
fewer animals. Based on the recommendations of ICCVAM and an independent scientific peer review panel,
the LLNA is accepted as an alternative to the guinea pig test for assessing allergic contact dermatitis
by U.S. regulatory agencies. Following an implementation workshop co-sponsored by ICCVAM and the
International Life Sciences Institute (ILSI), the LLNA was incorporated into an international test
guideline by the thirty member countries of the Organisation for Economic Co-operation and Development
(OECD). ICCVAM is now evaluating the validation status of modifications to the LLNA that may further
reduce the number of animals used, expand the usefulness of the LLNA, and eliminate the need to use
radioactive materials. ICCVAM is also evaluating the usefulness of the LLNA for assessments of
sensitization potency (strength of response). Successful validation in these areas could broaden
the use of the LLNA and thus significantly increase the impact of the LLNA as a refinement alternative.
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Endocrine Disruptor Testing
Endocrine disruptor testing is an ICCVAM priority because some types of testing can involve large numbers of
animals, may involve significant pain and distress, and may be of use to multiple agencies. A variety of
substances have been shown to affect hormones or processes involving hormones, sometimes resulting in
developmental or reproductive problems for humans or other species; these substances are called endocrine
disruptors. Laws passed in 1996 mandate the development and implementation of a screening program for
endocrine disruptors. Programs are being developed throughout the world, including the United States,
to screen for chemicals that might interfere with the endocrine systems of humans or wildlife. These programs
could result in the use of large numbers of animals if valid alternatives to the current animal tests are not
identified. NICEATM and ICCVAM recently reviewed
a number of in vitro tests designed to detect chemicals that
might act as, or interfere with, male and/or female hormones. Based on this review, ICCVAM provided
recommendations for
future test method development and validation activities that are being implemented
in studies by the EPA and NICEATM. Related test methods for detecting chemicals that might act like or
inhibit estrogen have recently been nominated for evaluation.
NICEATM will lead a joint international study with ECVAM and the Japanese Center for the Validation of
Alternative Methods (JaCVAM) to evaluate the usefulness and limitations of an in vitro test method to
identify estrogen-like chemicals that does not require the use of animals as donors for test components.
NICEATM and ICCVAM, working primarily through the U.S. National Coordinator for the OECD Test
Guidelines Program, are also increasing their involvement in OECD test guideline activities related to
endocrine disruptors. This includes an early exchange of information concerning test method validation.
It also includes working together with international stakeholders, where possible, to best utilize
existing resources to maximize the efficiency of evaluation/validation efforts towards the goal of
facilitating national and international recognition, acceptance, and implementation of scientifically
valid test methods.
Pyrogen Testing
Products injected or implanted into the body must be appropriately shown to be free of pyrogens (substances
that could cause fever) and other adverse health effects prior to their use in humans and animals. Although
these types of pyrogen tests are of primary concern to several programs in one agency (that is, the FDA),
they can require large numbers of animals that might experience pain and distress. Therefore, pyrogen testing
is considered an ICCVAM priority. Recently, alternative pyrogenicity test methods based on the activation of
cultured human blood cells have been developed that take advantage of the role of these cells in the fever
response. ICCVAM recently evaluated five
such in vitro test methods proposed as potential replacements
for the current rabbit test. ICCVAM will issue recommendations on the current usefulness of these test
methods and recommendations for future studies that may support their expanded use. Once additional
studies have been completed, ICCVAM will re-evaluate the validation status of these test methods.
Reproductive and Developmental Toxicity Testing
Reproductive and developmental toxicity testing is an ICCVAM priority because it is required by
multiple agencies, uses large numbers of animals, and can involve pain and distress to test animals.
ICCVAM evaluated the usefulness and limitations of the Frog Embryo Teratogenesis Assay - Xenopus (FETAX)
to measure the effects of chemicals on mortality, malformation, and growth inhibition.
This assay was proposed as a screen to identify potential developmental toxicants. Although FETAX was not
considered sufficiently reproducible for regulatory use, ICCVAM endorsed the recommendations of an
independent expert peer review panel that further studies should be conducted to improve test method
performance. In addition, the panel and ICCVAM recommended a list of reference substances that can be
used for validation studies of this and other developmental toxicity methods.
Renewed emphasis will be placed on identifying mechanism-based tests that could be useful in understanding
one or more of the many different pathways involved in reproductive or developmental toxicity. For example,
the FDA is involved in development of in vitro tests that could reduce the number of animals
used in developmental toxicity testing. However, the complexity of these endpoints means that it is
unlikely that any single alternative test method will be able to serve all regulatory needs. NICEATM and
ICCVAM will closely follow the ECVAM ReProTect project, a consortium
of European partners working towards the development of in vitro testing batteries that will provide
detailed information on the hazards of compounds to the reproductive cycle. ICCVAM will also explore,
and promote, where appropriate, possible revisions to existing in vivo testing protocols to reduce the
overall number of animals required without compromising assay performance.
Chronic Toxicity and Carcinogenicity Testing
Chronic toxicity and carcinogenicity testing is an ICCVAM priority because these methods are required by
multiple agencies, use large numbers of animals, and may involve significant pain and distress from resulting
systemic effects and cancers. Two-year studies approximating lifetime exposure in rats and mice remain the
primary method by which chemicals are tested for their potential to cause cancer and chronic disease in humans.
NIEHS and FDA are involved in the research and development of alternative models that could reduce the
number of animals used and shorten the duration of these tests. The development and validation of the
battery of alternative test methods needed to serve all regulatory needs in this area will likely take
longer than the five-year time frame for this strategic plan. However, ICCVAM and NICEATM will facilitate
efforts towards developing alternative models for one or more of the multiple mechanisms associated with
these endpoints, and that better simulate living organisms.
Federal regulatory agencies also typically require the use of tests that evaluate genetic toxicity, the
ability of chemical or physical agents to damage the DNA and/or chromosomes of cells. Genetic toxicity can
potentially contribute to the cancer-causing or developmental toxicity potential of a chemical. Although
genetic toxicity testing is not currently considered a substitute for carcinogenicity testing, the FDA is
studying the usefulness and limitations of various human primary cells and cell lines for use in genetic
toxicity testing. NICEATM and ICCVAM are participating in a JaCVAM-sponsored international validation study
(which also includes ECVAM) of an alternative animal test (that is, the alkaline Comet assay) to determine
the induction of DNA damage in cells of multiple organs. If the JaCVAM validation study is successful, there
are plans for the possible validation of an in vitro Comet assay that might be incorporated into the battery
of genetic toxicity assays.
Other Toxicity Areas of Interest
NICEATM and ICCVAM recognize that there are other areas of toxicity testing for which alternative test
methods are needed. Identifying alternative test methods for potential neurotoxins (chemicals that affect the
nervous system) is a priority of the FDA and the NIH. Both agencies are involved in the development of in vitro
methods to identify biomarkers of neurotoxicity. NICEATM and ICCVAM will closely follow ongoing efforts
in these areas and will work to identify the most useful tests and to facilitate their review and acceptance.
Footnotes
1 Testing priorities of individual Federal agencies may
differ because of the different statutory mandates under which they operate (Appendix B).
2 ICCVAM considers test method translation activities as those that are carried out
to characterize if there is evidence of relevance and applicability of a test method for a specific testing purpose. If so,
then the test method may be considered for further evaluation in a formal validation study.
Note: Some of the links on this page, which are the same as
URLs in footnotes in the PDF/hard copy version of this report, lead to pages outside the NICEATM-ICCVAM website. These links are for the convenience
of the readers of this document. NICEATM and ICCVAM are not responsible for the availability or content of
these external sites, nor do we endorse, warrant or guarantee the products, services or information described
or offered at these other sites.
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