General Information About ICCVAM Test Method Evaluation Areas
View a summary of test methods evaluated by ICCVAM
View a summary of the current status of ongoing and completed ICCVAM
alternative test method evaluation projects
View a summary of all ICCVAM test method recommendations
View ICCVAM-recommended Test Method Protocols
Please scroll down to read an overview on each area
In the United States, accidental poisonings are second only to motor vehicle crashes as the
leading cause of accidental deaths. According to the
Centers for Disease Control
 , more than 80
people a day die in the United States from accidental poisonings and over 2000 are treated in
emergency rooms. Most poisonings in the United States are due to accidental drug overdoses; however,
household chemicals, pesticides, and environmental agents such as lead and carbon monoxide also
present poisoning risks.
Potential poisons must be accurately identified and characterized in order to adequately protect
human and animal health and to determine the appropriate use of child-resistant packaging. Acute
systemic toxicity test results enable appropriate hazard classification of products. This
information is used to develop labeling to alert handlers and consumers to potential toxicity
hazards.
Acute systemic toxicity tests are the most commonly performed type of safety test worldwide.
Acute systemic toxicity tests may be oral tests (measuring toxicity of a substance when swallowed),
dermal tests (measuring toxicity of a substance when absorbed through the skin), or inhalation tests
(measuring the toxicity of a substance when inhaled). Traditional toxicity tests yield an LD50
value, referring to the dose that produces lethality in 50% of the animals tested. The LD50 value is
used to place substances in various toxicity categories that determine the hazard phrases that are
used on product labels.
NICEATM and ICCVAM have evaluated alternatives to the LD50 for
assessment of
acute oral systemic toxicity. The Up-And-Down Procedure (UDP)
is an in vivo acute oral systemic toxicity test that reduces and refines animal
use. ICCVAM has recommended the revised oral UDP;
acceptance of the revised oral UDP method via national and international test guidelines has resulted in
reduced animal use for acute oral systemic toxicity testing.
A Validation Study of In Vitro
Cytotoxicity Test Methods
coordinated by NICEATM and its European counterpart generated in vitro
toxicity data to predict rodent in vivo LD50
values and starting doses for acute oral systemic toxicity test methods.
ICCVAM has recommended that in vitro cytotoxicity test methods be considered
as part of a weight-of-evidence approach before
using animals for acute oral systemic toxicity testing, and that the methods should be used where determined
appropriate
for estimating starting doses for acute oral systemic toxicity tests with rodents.
A 2008 workshop
on “Acute Chemical Safety
Testing: Advancing In Vitro Approaches and Humane Endpoints for Systemic Toxicity Evaluations”
was organized by NICEATM, ICCVAM, and their international partners to further evaluate alternatives
to existing acute systemic toxicity test methods. International
experts in the fields of toxicology and human and veterinary medicine considered the identification of key toxicity pathways in order to
apply mechanistically-based in vitro approaches and humane endpoints for systemic toxicity
evaluations.
Biological products, commonly referred to as biologics, are products intended for therapeutic use
that are
derived from biological sources. They include viruses, therapeutic sera, toxins, antitoxins and
vaccines, and
a variety of other substances that have therapeutic use in humans or animals (e.g., insulin, alpha1
proteinase
inhibitor, anti-hemophilic factor). Current regulatory requirements include testing in animals for
identity or
potency for labeling and lot release purposes. ICCVAM welcomes nominations and submissions of
alternative test
methods that will refine, reduce, or replace animal testing for these products.
In response to a test method nomination from the Humane Society of the United States,
ICCVAM, in collaboration with ECVAM, sponsored a November 2006 workshop entitled
Scientific Workshop on Alternative Methods to Refine, Reduce, and Replace the Mouse LD50
Assay for Botulinum Toxin Testing. Over 100 scientists from nine countries reviewed methods
with the potential to refine, reduce, and replace current botulinum toxin test methods. A report on the
workshop was published in 2008.
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In 2008, the American Association of Poison Control Centers reported nearly 140,000 injuries
due to chemical burns from agents such as acids, alkalis, peroxides, bleach, and phenols or
phenol products (source, "Chemical Burns",
eMedicine website). Many of these injuries arose from the use of household products.
Injuries to the skin, also called dermal injuries, fall into two categories. Skin
corrosion
is permanent damage to the skin that occurs when contact with a substance kills or destroys
cells in the epidermis and dermis. Skin irritation is reversible damage that occurs
when a
chemical causes injury to skin cells. Regulatory agencies test substances to determine
whether they are likely to present dermal corrosion or irritation hazards. The agencies
then use test results to classify and label corrosive or irritant chemicals so that
consumers and workers can take appropriate precautions to prevent injury. Test results
are also used to determine appropriate packaging that will minimize hazardous spills during
transport.
Traditionally, dermal corrosion and irritation testing is done by applying a test substance
to the skin of a laboratory animal. In 1999 and 2002, ICCVAM conducted independent scientific peer
reviews of the usefulness and limitations of four non-animal (in vitro) corrosivity test
methods
for use as alternatives to the in vivo rabbit skin test. Based on these reviews, ICCVAM
recommended that all four methods (Corrositex®, EPISKIN™, EpiDerm™, and the rat skin
transcutaneous electrical resistance assay) could be used as part of weight-of-evidence
approach in an integrated testing scheme for dermal corrosion/irritation. In this approach,
positive in vitro corrosivity responses do not generally require further testing and can
be used for classification and labeling without the need for animal testing. Use of this
approach has since been adopted internationally via test guidelines developed by the
Organisation for Economic Co-operation and Development. ICCVAM is currently
participating in development of OECD test guidelines for the use of in vitro
human skin model systems such as EPISKIN and EpiDerm for the identification of
substances with the potential to cause skin irritation.
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Current Federal regulations require determination of the developmental toxicity
(i.e., toxicity to the developing fetus) potential of many chemicals and products
(including pesticides, food additives, industrial chemicals, and pharmaceuticals)
marketed in the United States. Most developmental toxicity test protocols use rats,
rabbits, or other mammalian species. ICCVAM sponsored an evaluation of the utility
of the Frog Embryo Teratogenesis Assay -Xenopus
(FETAX) to determine the developmental
toxicity potential of chemicals.
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Endocrine disruptors are substances that interfere with the normal function of hormones in the
endocrine system. These interferences can lead to abnormal growth, development, or reproduction. A
number of studies have been published indicating that animal populations exposed to high levels of
these substances have an increased incidence of reproductive and developmental abnormalities.
Exposure of humans to endocrine disruptors has also been linked to adverse health outcomes.
In response to these concerns, the U.S. Environmental
Protection Agency (EPA) initiated the Endocrine Disruptor Screening Program (EDSP) to screen
pesticides and environmental contaminants for their potential to affect the endocrine systems of
humans and wildlife. An ICCVAM review of the validation status of in vitro estrogen receptor (ER)
and androgen receptor (AR) binding and transcriptional activation (TA) test methods indicated
that there were no adequately validated test methods.
Consequently, ICCVAM invited nominations of such test methods for which there were standardized
protocols, prevalidation data, and proposed validation
study designs.
Two ER test methods for the detection of estrogen receptor agonists and antagonists were nominated for
validation studies in response to this request. One of these methods, the BG1Luc ER TA (LUMI-CELL®)
test method developed by Xenobiotic Detection Systems, Inc.,
was the subject of a NICEATM-sponsored international interlaboratory validation study. Data from this
validation study were the basis of ICCVAM recommendations that the BG1Luc ER TA assays could be used
as screening tests to identify substances with in vitro estrogen receptor agonist or antagonist
activity. U.S. Federal agencies, including the EPA, agreed with this recommendation.
The other test method nominated to ICCVAM was the MCF-7 cell proliferation test method
developed by CertiChem, Inc. (CertiChem). A NICEATM-sponsored international interlaboratory
validation study of the CertiChem MCF-7 cell proliferation test method was recently completed.
Evaluation of data from this study is in progress.
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Genetic toxicology is the study of compounds or physical agents that have the ability to damage
the
DNA and/or chromosomes of cells. Such damage can lead to mutations that increase the likelihood of
certain
diseases, such as cancer and birth defects. The EPA, the U.S. Food and
Drug Administration (FDA), and the U.S. Consumer Product Safety Commission (CPSC) have testing
requirements and
guidelines in place for assessing the genotoxicity of regulated products. ICCVAM and
its Genetic Toxicity Working Group review and provide comments to sponsors on proposed validation
studies, provide recommendations on test method nominations and submissions for alternative test
methods related to genetic toxicity, and evaluate proposed OECD test guidelines and activities
relevant
to genetic toxicity.
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According to the U.S. Bureau of Labor
Statistics,
occupational skin diseases are the most common type of occupational illness. Many of these
cases arise from exposure to
skin-sensitizing substances, which can lead to allergic contact dermatitis (ACD), an
immunologically-mediated hypersensitivity reaction. Studies have shown
(Hutchings et al. 2001
,
Skoet et al. 2003
) that ACD
has a significant impact on quality of life in
affected individuals.
There is a growing international need for non-animal test methods that can identify substances
with the potential to cause ACD in humans. Working with U.S. and international collaborators,
NICEATM and ICCVAM are evaluating the application of the electrophilic allergen screening assay and
other in vitro test methods as part of integrated decision strategies for the regulatory safety
assessment of ACD hazards.
Chemicals and products have traditionally been tested for ACD hazard potential using
guinea pig tests such as the guinea pig maximization test or the Buehler test. In 1999, ICCVAM evaluated the
murine local lymph node assay (LLNA) and recommended it as a valid stand-alone alternative
test method to currently accepted guinea pig tests for assessing ACD hazard potential. In comparison
to guinea pig tests, the LLNA uses fewer animals, avoids animal pain and distress
associated with an allergic reaction, requires less time to perform, and
provides dose-response information.
ICCVAM and its interagency Immunotoxicity Working Group drafted OECD Test Guideline 429 for the LLNA,
which was adopted by the OECD in 2002. OECD Test Guideline 429 was updated in 2010 in response
to an ICCVAM proposal based on subsequent evaluations.
In 2007, the U.S. Consumer Product Safety Commission requested that NICEATM and ICCVAM assess the
validation status of new modifications and applications of the LLNA. In response to this request,
ICCVAM conducted the following evaluations and activities:
- ICCVAM evaluated the reduced LLNA (rLLNA), a modification of the multi-dose LLNA that
uses 40% fewer animals for each test. ICCVAM concluded that the rLLNA could be used
routinely to determine the ACD hazard potential of chemicals and products. Use of the rLLNA is
recommended before conducting the multidose LLNA in cases that do not require dose-response
information or if the test substance is expected to have no ACD hazard potential.
- ICCVAM developed internationally harmonized LLNA performance standards that can be used as
the basis for determining the acceptability of new versions of the LLNA. The performance standards
include an updated test method protocol that reduces animal use by 20% compared to the original ICCVAM-recommended test
method protocol by decreasing the minimum number of animals per dose group from five to four.
- ICCVAM evaluated an updated database for the use of the LLNA for testing pesticide
formulations, metals, substances in aqueous solutions and other products (applicability domain for
the LLNA). ICCVAM recommended that the LLNA may be used to test any chemical or product for ACD
hazard potential unless the chemical or product to be tested has properties that
may interfere with the ability of the LLNA to detect sensitizing substances.
- ICCVAM recommended that two nonradioactive versions of the LLNA (the LLNA: DA and LLNA: BrdU-ELISA) can
be used to identify
substances as potential skin sensitizers or nonsensitizers, with certain limitations. The
availability of LLNA methods that do not use radioactivity is expected to allow more institutions to
take advantage of the animal welfare benefits of the LLNA and provides environmental benefits as
well.
- ICCVAM, in collaboration with the Japanese Center for the Validation of Alternative Methods,
submitted draft test guidelines for the LLNA: DA and LLNA: BrdU-ELISA to OECD. The final OECD Test
Guidelines 442A and 442B, which are based on the ICCVAM-recommended LLNA: DA and LLNA: BrdU-ELISA test
method protocols, respectively, were adopted in July 2010.
- ICCVAM evaluated the use of the LLNA for potency categorization of chemicals causing
ACD in humans. ICCVAM concluded that the LLNA can be used to categorize substances as strong
sensitizers (Globally Harmonized System of Classification and Labelling of Chemicals Subcategory
1A). However, the LLNA cannot be used as a stand-alone assay to categorize substances as other
sensitizers (Globally Harmonized System of Classification and Labelling of Chemicals Subcategory
1B).
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Nanomaterials Testing
Nanotechnology is being applied in many fields in the physical and biological sciences to create
improved materials, devices, and systems. Because an understanding of the toxicity associated with
these types of materials continues to evolve, the applicability of current toxicity tests to
nanomaterials will have to be evaluated and new tests may be needed for regulatory use. NICEATM and
ICCVAM are closely following progress in this area and will work with regulators and stakeholders to
identify test methods that reduce, refine, and replace the use of animals for such testing
requirements.
Each year, an estimated 2 million eye injuries occur in the United States. Of these, more than
40,000 cause permanent visual impairment. Chemicals and compounds are the third most common cause
of eye injuries, with many eye injuries in consumers associated with the use of household cleaning products.
To warn consumers and workers of the potential for chemicals and products to cause eye injuries, eye safety
testing is performed to determine if substances may cause temporary or permanent eye damage. Test
results are then used for hazard classification of chemicals and products using appropriate national
and/or international hazard classification systems.
Nearly all eye safety testing, also referred to as ocular safety testing,
has been conducted using the rabbit eye test. Evaluation
of scientifically valid eye safety testing methods that reduce, refine, or replace animal use is a high
priority for NICEATM and ICCVAM. To refine animal use for eye safety testing,
ICCVAM recommended (2010) a balanced preemptive pain management plan
that should always be used when it is necessary to conduct
the rabbit eye test for regulatory safety testing purposes. These procedures include the
routine use of topical anesthetics, systemic analgesics, and humane endpoints.
To reduce animal use for eye safety testing,
ICCVAM has made recommendations to Federal agencies that
will enable eye safety testing to be conducted using fewer animals while maintaining current hazard
classification criteria. These recommendations have been forwarded to Federal agencies, with agency responses expected in 2013.
Following a 2010 evaluation of non-animal eye safety testing methods, ICCVAM recommended
that the
Cytosensor microphysiometer (CM) test method can be used
as a screening test to identify some types of substances that may cause permanent or severe eye
injuries without using animals. ICCVAM also recommended that the CM test method
can be used to determine if a limited type of substances will not cause sufficient injury to require
hazard labeling for eye irritation.
In a prior evaluation (2006),
ICCVAM recommended the bovine corneal opacity and permeability
(BCOP) and isolated chicken eye (ICE) test methods for use in a tiered testing strategy to identify
ocular corrosives and severe irritants, with specific limitations for certain chemical classes
and/or physical properties. These recommendations were subsequently adopted by the Organisation for
Economic Co-operation and Development (OECD). These test methods may now be used for regulatory
classification and labeling in the 34 member countries of the OECD.
ICCVAM is accepting data
to support evaluation of in vitro test methods
and testing strategies with the potential to replace animal use for eye safety testing.
Further details on ICCVAM evaluations of test method to identify ocular hazards can be
found in the ocular test methods evaluation section of the website.
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Pyrogens are substances (such as Gram-negative and Gram-positive bacteria, fungi, and viruses)
that can produce a rise in body temperature (in other words, fever). Pyrogenic substances cause an increase
body temperature by inducing leukocytes to release pro-inflammatory cytokines (such as interleukin
[IL]-1, IL-6, and tumor necrosis factor-α) that can act as endogenous pyrogens. Pharmaceutical
products (such as fluids for
injection, medical devices, and human biological products) intended for parenteral administration
must be properly and accurately evaluated for the presence of pyrogenic substances and shown to be
free of contamination prior to their clinical or veterinary use.
The U.S., European, and Japanese Pharmacopoeias
currently recognize two test methods for pyrogen testing. The rabbit pyrogen test
(USP28[151]) involves measuring the rise in temperature of
rabbits following intravenous injection of a test solution. The bacterial endotoxin test
(USP28[85]) is an in vitro assay based on the coagulation of Limulus amoebocyte
lysate following exposure to endotoxin. An important distinction between these two tests is
that the bacterial endotoxin test detects only endotoxin pyrogens, whereas the rabbit pyrogen test is capable of
also detecting non-endotoxin pyrogens.
Recently, alternative test systems based on the activation of human monocytes or
monocytoid cell lines in vitro have been developed that take advantage of
the role of these cells in the fever response. ICCVAM evaluated the validation status
of five in vitro pyrogen test methods proposed as
potential replacements for the rabbit pyrogen test. In their recommendations to Federal agencies,
ICCVAM stated that, although none of these test methods could be considered a complete replacement
for the rabbit pyrogen test, they can be considered
for use to detect Gram-negative endotoxin in human parenteral drugs on a case-by-case basis,
subject to validation for each specific product to demonstrate equivalence to the RPT,
in accordance with applicable U.S. Federal regulations. All applicable Federal agencies,
including the U.S. Food and Drug Administration, accepted or endorsed the ICCVAM recommendations in 2009.
ICCVAM has evaluated the validation status of five in vitro test methods proposed for assessing the
potential pyrogenicity (i.e., ability to induce fever) of pharmaceuticals and other products,
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Additional Information
Scientific Advisory Committee on Alternative Toxicological Methods (SACATM)
Chartered on December 18, 2001, the Scientific Advisory Committee on Alternative Toxicological
Methods
(SACATM) was established as a replacement
for the NTP Federal Advisory Committee on Alternative Toxicological
Methods (ACATM). This committee provides advice on the activities and
priorities of NICEATM and ICCVAM, and recommends ways to foster
partnership activities and productive interactions among all
stakeholders. SACATM will typically meet twice yearly, and meeting
materials and minutes will be posted.
Partnership Opportunities
Opportunities for organizations and
agencies to partner with NICEATM to support the development, validation,
and review of new alternative testing methods are available. Interested
individuals should contact Dr. Warren Casey, NICEATM Acting Director, for
further information (see below).
Additional Information
Additional information can be found
elsewhere in this website and in the
publication: Validation and Regulatory Acceptance of Toxicological
Test Methods, a Report of the ad hoc Interagency Coordinating Committee
on the Validation of Alternative Methods (NIH Publication 97-3981)
or you may contact NICEATM at 919-541-3398 (telephone), or at
iccvam@niehs.nih.gov (e-mail).
Specific questions about ICCVAM and NICEATM can be directed to
NICEATM Acting Director Dr. Warren Casey,
NIEHS, EC-17, P.O. Box 12233, Research Triangle Park, NC, 27709
Phone: 919-316-4729 - Email niceatm@niehs.nih.gov
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