Test Method Evaluations
All chemicals, drugs, and natural substances are potentially poisonous, or toxic, at high enough doses. Testing substances to characterize their toxicity provides data that can be used to develop product protective packaging and warning labels, personal protective equipment requirements, and environmental release guidelines.
Acute systemic toxicity tests are commonly used to evaluate the toxicity of chemicals, drugs, and other substances via three routes of exposure:
- Swallowed (oral toxicity tests)
- Absorbed through the skin (dermal toxicity tests)
- Inhaled (inhalation toxicity tests)
These tests provide an LD50 value, representing the dose that would be expected to produce lethality in 50% of the animals tested. The LD50 value is used to assign substances to toxicity categories that determine hazard phrases used on product labels to indicate precautions to be taken while handling.
NICEATM and ICCVAM evaluated alternatives that reduce and/or refine animal use for assessing acute oral systemic toxicity. ICCVAM recommended:
- A revised oral up-and-down procedure as an approach to assess acute oral systemic toxicity that reduces and refines animal use
- Consideration and use of in vitro cytotoxicity test methods as part of a weight-of-evidence approach before using animals for acute oral systemic toxicity testing, and use of these methods where appropriate for estimating starting doses for acute oral systemic toxicity tests with rodents
NICEATM and ICCVAM agencies are working to develop, validate, and implement approaches with the potential to replace animal use for acute toxicity testing. To that end, NICEATM published a Federal Register notice requesting available data and information on approaches and technologies currently used to identify substances with the potential to cause acute systemic toxicity. Other related activities include:
- A 2016 webinar series on Alternative Approaches for Acute Inhalation Toxicity to Address Global Regulatory and Non-regulatory Data Requirements, which explored alternatives to animal use for acute inhalation toxicity testing (co-sponsors: NICEATM and the PETA International Science Consortium [PISC])
- A 2015 workshop on Alternative Approaches for Identifying Acute Systemic Toxicity: Moving from Research to Regulatory Testing; attendees developed plans to advance methods for product safety testing that meet the needs of regulatory agencies while reducing the use of rodents (co-sponsors: NICEATM, PISC, and the Physicians Committee for Responsible Medicine)
- Collaborations with the U.S. Environmental Protection Agency (EPA) to determine if acute oral toxicity data can be used to reliably assign EPA acute dermal hazard classifications (NICEATM)
- Participation in an international validation study of an in vitro assay for evaluating human liver metabolism and toxicity (NICEATM and ICCVAM)
- A 2008 workshop on Acute Chemical Safety Testing: Advancing In Vitro Approaches and Humane Endpoints for Systemic Toxicity Evaluations to evaluate alternatives to existing acute systemic toxicity test methods (organized by NICEATM, ICCVAM, and international partners)
Biologics are medical products derived from biological sources and include viruses, toxins, antitoxins, vaccines, and other substances such as insulin. Many of these products require safety and potency testing in animals for package labeling and lot release purposes. ICCVAM member regulatory agencies have evaluated and accepted alternative test methods that replace, reduce, or refine animal use for specific testing purposes.
NICEATM and ICCVAM cosponsored workshops that allowed regulators and manufacturers of biologics to discuss how to facilitate appropriate implementation and use of available alternatives for animal tests.
- NICEATM, the U.S. Food and Drug Administration (FDA), and regulators from Europe and Canada convened a series of workshops to identify alternatives to the animal test that is currently required for pertussis vaccine lot release. These workshops produced a proposal for international adoption of a non-animal replacement for this test.
- Participants in a 2012 workshop considered alternatives for potency testing of Leptospira vaccines and recommended steps to encourage their use. The U.S. Department of Agriculture (USDA) is monitoring the change in hamster use to maintain Leptospira challenge cultures; a 25–30% reduction in hamsters used for this purpose is anticipated.
- Recommendations from a 2011 workshop on alternatives for rabies vaccine testing were reflected in guidances issued in 2012 and 2013 by the USDA Center for Veterinary Biologics.
- In response to a test method nomination from the Humane Society of the United States, NICEATM and ICCVAM, in collaboration with international partners, sponsored a Scientific Workshop on Alternative Methods to Refine, Reduce, and Replace the Mouse LD50 Assay for Botulinum Toxin Testing (2006). An in vitro method for potency testing of botulinum neurotoxin discussed at this workshop was later accepted by the FDA.
Computational toxicology studies use mathematics, informatics, and computer models to better understand toxicity mechanisms and predict toxic effects.
Traditional toxicity testing methods, which involve using laboratory animals, are time- and resource-intensive. As a result, many thousands of chemicals currently in commerce lack basic safety information. Computational toxicology methods can help address these needs through the development and use of computer models of chemical activity or conceptual models of toxicity mechanisms. NICEATM has been exploring development of computational models for predicting the potential of chemicals to cause skin sensitization and developmental toxicity and to interact with the endocrine system.
Dermal irritation is a reversible injury to the skin caused by contact with a chemical and is typically characterized by redness and swelling. Dermal corrosion is permanent damage to the skin that occurs when contact with a chemical kills cells beneath the skin surface. Dermal corrosion differs from dermal irritation in that corrosion of the skin is not reversible. (Chemicals can also cause an allergic reaction in the skin. This is called allergic contact dermatitis and is described below.)
Regulatory agencies require manufacturers to test chemicals to identify those likely to cause dermal irritation or corrosion. Results from these tests are used to classify and label chemicals so that consumers and workers can take precautions to prevent injury. Test results are also used to determine appropriate packaging that will minimize hazardous spills during transport.
In traditional dermal corrosion and irritation testing, a test substance is applied to the skin of a laboratory animal. Four non-animal (in vitro) corrosivity test methods proposed as alternatives to animal tests were evaluated in ICCVAM-sponsored independent scientific peer reviews. 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 a weight-of-evidence approach in an integrated testing scheme for dermal corrosion/irritation. Using this approach, chemicals classified as corrosive by these tests do not generally require further animal tests. This approach has since been adopted internationally via test guidelines developed by the Organisation for Economic Co-operation and Development (OECD).
Developmental toxicity tests evaluate the extent to which exposures to a chemical can interfere with normal development and cause adverse effects in the offspring. Current federal regulations require developmental toxicity tests for a variety of products including pesticides, food additives, industrial chemicals, and pharmaceuticals.
Most developmental toxicity test protocols use rats, rabbits, or other mammalian species. To support development of alternative methods:
- NICEATM published a request for information (RFI) on approaches and/or technologies used to identify potential developmental toxicants. This information will be used to assess the state of the science and determine technical needs for non-animal test methods to evaluate the potential of chemicals for their potential to induce adverse effects in offspring.
- A subsequent RFI sought information specifically on protocols using zebrafish embryos for chemical screening assays. These assays may be useful for identifying potential developmental toxicants.
- NICEATM is participating in the NTP’s Systematic Evaluation of the Application of Zebrafish in Toxicology (SEAZIT) program. SEAZIT’s goal is to enable the broader adoption of zebrafish for toxicological screening by promoting harmonization of key protocol components.
Endocrine disruptors interfere with the normal function of hormones, often through interactions with hormone receptors. Animals exposed to endocrine disruptors in the environment may exhibit reproductive and developmental abnormalities. The effects of endocrine disruptors in humans are less clear, but there is evidence suggesting endocrine disruptors may cause human health problems.
NICEATM Support of the Endocrine Disruptor Screening Program
NICEATM is working with federal agencies to develop non-animal test methods and approaches to identify potential endocrine disruptors. NICEATM and EPA scientists developed and validated an approach that combines data from 18 high throughput screening assays to identify endocrine disrupting chemicals with the potential to interact with the estrogen receptor (ER). This approach has been accepted by the EPA as an alternative to three assays used in its Endocrine Disruptor Screening Program Tier 1 battery, one of which is the rodent uterotrophic assay. NICEATM and EPA also developed and validated a similar approach to identify chemicals with the potential to interact with the androgen receptor (AR).
NICEATM activities contributing to these projects include:
- Creation of a database of high-quality in vivo uterotrophic testing data to support validation of alternative test methods used to identify chemicals with ER pathway interaction potential
- Curation of literature reviews to support development of a reference chemical list for AR pathway activity
- Development of computational approaches to predict potential ER and AR pathway activities
- Development of models to correlate in vitro and in vivo dosimetry for endocrine-active substances
Validation Study of Test Method to Identify Chemicals with Estrogenic Activity
ICCVAM reviewed the validation status of in vitro test methods to identify substances with ER and AR interaction potential in 2003 and concluded that no adequately validated test methods were available. Subsequently, ICCVAM invited test method nominations for these purposes, and two methods were nominated in response. One method, the LUMI-CELL® assay, was the subject of a NICEATM-sponsored international inter-laboratory validation study. Data from this validation study supported the ICCVAM recommendation that the LUMI-CELL agonist and antagonist assays could be used as screening tests to identify substances with in vitro ER interaction potential. The LUMI-CELL assay was later successfully adapted to a high throughput format and is now used in the Tox21 testing program.
Please note: In June 2016, the generic name of the LUMI-CELL assay changed from the BG1Luc ER transactivation assay to the VM7Luc ER transactivation assay. This reflects new information regarding the identity of the cell line used in the assay. Refer to the webpage summarizing the assay validation for more details.
Manufacturers test cosmetics, household cleaners, and other chemical products before marketing to identify skin sensitizers, substances with the ability to cause allergic contact dermatitis (ACD). ACD can develop after repeated contact with a sensitizing substance, and can have a significant impact on quality of life in affected individuals.
There is a growing international need for non-animal test methods that can identify skin sensitizers. ICCVAM has developed a strategy for evaluating test methods to identify potential skin sensitizers without the use of intact animals.
Chemicals and products were traditionally tested for ACD hazard potential using guinea pig tests. In 1999, ICCVAM evaluated the murine local lymph node assay (LLNA) and recommended it as a valid alternative test method for assessing ACD hazard potential. Compared 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. In 2002, the LLNA was accepted internationally via OECD adoption of Test Guideline 429, which was drafted by ICCVAM.
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:
- Evaluated and recommended the reduced LLNA (rLLNA), a modification of the LLNA that uses 40% fewer animals for each test
- Evaluated the LLNA for testing pesticide formulations, metals, substances in aqueous solutions, and other products, and recommended a broader applicability domain for the LLNA
- Evaluated and recommended two nonradioactive versions of the LLNA (the LLNA: DA and LLNA: BrdU-ELISA); these methods allow more institutions to take advantage of the animal welfare and environmental benefits of the LLNA
- Contributed to development of an updated OECD Test Guideline 429 in 2010, which incorporated the reduced LLNA, the expanded LLNA applicability domain, and an updated ICCVAM-recommended protocol for the LLNA
- Evaluated the use of the LLNA for potency categorization of chemicals causing ACD in humans, concluding that the LLNA can be used to distinguish between strong sensitizers and other sensitizers, but not as a stand-alone assay to discriminate among other classes of sensitizers
Nanotechnology is being applied in many fields to create improved materials, devices, and systems. Because the unique characteristics of nanomaterials can affect their metabolism and toxicity, the applicability of current toxicity tests to nanomaterials will need to be evaluated, and new tests may be required for regulatory use.
NICEATM and ICCVAM are working with regulators and stakeholders to identify test methods that minimize or avoid animal use for nanomaterials testing. In February 2015, NICEATM staff joined experts from industry, academia, government, and other sectors to discuss technical details for a proposed validation study of an in vitro test to assess the effects of inhaled carbon nanotubes. Reports from the meeting were published in the scientific literature in 2016.
Chemicals and substances such as personal care products and cleaning supplies are tested to determine if they present eye injury hazards and to classify them for appropriate labeling and packaging. Nearly all of this testing, referred to as ocular toxicity testing, has been conducted using the rabbit eye test. Evaluation of alternatives to the rabbit eye test that replace, reduce, or refine animal use is a high priority for NICEATM and ICCVAM. Activities to support this goal include:
- ICCVAM is providing guidance on validation activities to the developer of the in vitro OptiSafe test method.
- NICEATM prepared a summary document on the in vitro short time exposure test that was included with a recent international proposal for its adoption.
- ICCVAM recommended the use of the in vitro Cytosensor microphysiometer (CM) test method as a screening test to identify products that cause permanent or severe eye injuries without using animals. ICCVAM also recommended use of the CM test method with specific types of substances to determine if they will not cause sufficient injury to require hazard labeling for eye irritation.
- ICCVAM recommended the bovine corneal opacity and permeability and isolated chicken eye test methods for use, with specific limitations, to identify substances that may cause permanent or severe eye injuries.
Furthermore, ICCVAM recommended
- Protocols requiring fewer animals for the rabbit eye test while maintaining current hazard classification criteria
- Routine use of topical anesthetics, systemic analgesics, and humane endpoints when it is necessary to conduct the rabbit eye test for regulatory safety testing purposes
Pyrogens are substances (such as Gram-negative and Gram-positive bacteria, fungi, and viruses) that can produce fever. Pharmaceutical products (such as fluids for injection, medical devices, and human biological products) intended for implantation or 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) involves measuring the rise in temperature of rabbits following intravenous injection of a test solution. The bacterial endotoxin test (USP28) is an in vitro assay based on the coagulation of Limulus amoebocyte lysate following exposure to endotoxin, a pyrogenic substance that is produced by certain bacteria. 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 FDA, accepted or endorsed the ICCVAM recommendations in 2009. In 2012, the FDA issued guidance on the use of alternatives to standard pyrogen tests that was consistent with the ICCVAM recommendations.