General Information About NICEATM-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 NICEATM-ICCVAM
alternative test method evaluation projects
View ICCVAM-recommended Test Method Protocols
Please scroll down to read an overview on each area
To reduce the risk for accidental poisonings, U.S. Federal regulatory agencies
require
the testing of marketed products for acute oral systemic toxicity in rodents. Acute oral systemic toxicity test
results are
used to determine the hazard classification and labeling of products, which alert handlers
and consumers to potential toxicity hazards. The LD50 values (dose that produces
lethality in 50% of the
animals tested) determined from acute oral systemic toxicity tests are used to place substances in various
toxicity
categories that determine the hazard phrases that are used on product labels.
In addition to classification and labeling, acute oral systemic toxicity test results may also be used
for:
- Establishing dosing levels for repeated dose toxicity studies or other toxicity studies
- Identifying potential target organs
- Providing information related to the mode of toxic action
- Aiding in the diagnosis and treatment of toxic reactions
- Providing information for comparison of toxicity and dose response among substances in a
specific chemical or product class
- Aiding in the standardization of biological products
- Aiding in judging the consequences of single, high accidental exposures in the workplace,
home, or from accidental release
- Serving as a standard for evaluating alternatives to animal tests
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. U.S. regulatory agencies have adopted the ICCVAM
recommendations
and now accept the UPD method (OECD Test Guideline 425; OPPTS Harmonized Test Guideline 870.1100)
which has replaced the conventional acute oral systemic toxicity test method.
A Validation Study of In Vitro
Cytotoxicity Test Methods 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 these 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 joint NICEATM-ICCVAM/ECVAM/JaCVAM Workshop
entitled “Acute Chemical Safety
Testing: Advancing In Vitro Approaches and Humane Endpoints for Systemic Toxicity Evaluations” was
held in 2008 to further evaluate alternative 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,
NICEATM-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.
Return to the top of the page
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.
Return to the top of the page
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.
Return to the top of the page
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. ICCVAM
concludes that the results of this validation study support use of the BG1Luc ER TA test method as a
screening test to identify substances with in vitro estrogen receptor agonist and/or antagonist
activity. This conclusion concurred with the views of an international independent peer review panel
that reviewed the results of the validation study in March 2011.
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.
Return to the top of the page
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.
Return to the top of the page
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* that ACD
has a significant impact on quality of life in
affected individuals.
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 in most testing situations,
requires less time to perform, and
provides dose-response information.
ICCVAM and its interagency Immunotoxicity Working Group drafted an OECD test guideline for the LLNA that
was adopted by the OECD in 2002 (OECD Test Guideline 429).
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. The rLLNA lowers the number of animals by using only a single high-dose group
along with concurrent
negative and positive control groups.
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 multi-dose 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 a 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. The
performance standards also specify a minimum list of reference substances to evaluate the accuracy
and reliability of the modified test method, and the accuracy and reliability values that must be
achieved in order for the modified test method to be considered equal to or better than the
LLNA.
- 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.
- NICEATM and ICCVAM proposed revisions to OECD Test Guideline 429, which incorporate the
performance standards and updated test method protocol. The updated Test Guideline 429 also provides
for the use of the rLLNA where appropriate and considers the updated applicability domain for
the LLNA. OECD adopted the revised Test Guideline 429 in 2010.
- 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 B, 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).
* Hutchings et al., Contact Dermatitis 45:17-20 (2001);
Skoet et al., Br J Dermatol 149:452-456 (2003).
Return to the top of the page
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.
Eye injury is a leading cause of visual impairment in the United States, with 40,000 to 50,000
new cases of impaired vision reported each year. Many eye injuries occur due to contact with
products or chemicals used in the home or workplace. Accidents involving common household products
such as oven cleaner or bleach cause about 125,000 eye injuries each year (source, American Academy of
Ophthalmology). To protect workers and consumers, regulatory agencies require testing to determine if chemicals
and products may cause eye injuries. These tests represent one of the four most commonly
conducted types of product safety tests. Testing results are used to classify the ocular hazard and determine
appropriate labeling to warn consumers and workers of the potential hazard for temporary or
permanent eye injuries from chemicals and products.
Nearly all ocular safety testing has been conducted using the Draize rabbit eye test. Evaluation
of test methods that identify such hazards and also reduce, refine, or replace animal use is a high
priority for NICEATM and ICCVAM. To refine animal use for this purpose, ICCVAM recommended (2010) a
balanced preemptive pain management plan
that should always be used when it is necessary to conduct
the Draize rabbit eye test for regulatory safety testing purposes. These procedures include the
routine use of topical anesthetics, systemic analgesics, and humane endpoints.
ICCVAM also recommended (2010)
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, replacing animal use for this purpose. 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 32 member countries of the OECD.
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.
Return to the top of the page
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,
Return to the top of the page
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. William S. Stokes, NICEATM 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 the
ICCVAM Executive Director:
Dr. William S. Stokes,
NIEHS, EC-17, P.O. Box 12233
Research Triangle Park, NC, 27709
919-541-7997
stokes@niehs.nih.gov
|
