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Toxicity Assessment

Opportunities to end or significantly reduce the use of animals for the toxicity assessment of substances in the context of regulatory toxicity requirements are detailed below. Also described are areas in which greater support is required to develop innovative methods that are relevant for the assessment of human health and environmental endpoints.

Please note that when tests are required for regulatory purposes, direct sources—such as the websites of the Organisation for Economic Co-operation and Development (OECD), the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH), and the U.S. Environmental Protection Agency (EPA)—should be consulted for the most recent versions of test guidelines and guidance documents.

Approaches to Toxicity Assessment

Regulatory decision-making is facilitated by using all the relevant information available on a substance. One way to evaluate all the lines of evidence is to use an integrated approach to testing and assessment (IATA)1 that considers multiple types of information to conclude the toxicity of a substance in a weight of evidence (WoE) approach. Information to determine includes existing data on the substance (e.g., from in chemico, in vitro, in vivo human, or in vivo animal studies), the physiochemical properties of the substance, data from non-testing approaches (e.g., QSARs and read-across), newly generated data (preferably from reliable and relevant non-animal methods), omics technologies (e.g., toxicogenomics), and use patterns or exposure scenarios. Data considered more reliable, relevant, and/or useful for the regulatory question have a greater influence on the final conclusion of the assessment. By assessing the available data together, it may be possible to conduct a robust risk assessment of a substance without generating new data through additional in vivo studies. (For example, see the “Carcinogenicity” section.) Additionally, a holistic data assessment will ensure that existing in vivo studies are not duplicated.

IATAs and WoE assessments often require expert judgment when integrating the results from combined approaches to make an informed conclusion for decision-making purposes. The methods, technologies, and frameworks that may be included in such approaches are accessible to those with the appropriate technological knowledge, and there are various guidance documents and case studies to help in developing an IATA. For example, the OECD has published guidance on using defined approaches within an IATA.2 Defined approaches consist of a fixed data interpretation procedure (e.g., a mathematical model or a rule-based approach) applied to data generated with a defined set of information sources to derive a prediction without the need for expert judgment.3 For examples of defined approaches, see the Skin Sensitization section.

Adverse outcome pathways (AOP) offer an additional framework for organizing data collected from various methods and biological levels to assess the connections between key events and adverse effects. Unlike tests on animals, non-animal methods can reflect human-relevant biology and mechanisms of toxicity. AOPs comprise causally linked key events that connect chemical exposure to an adverse outcome. Non-animal tests that query specific key events in an AOP allow for a mechanistic understanding of whether an adverse outcome will occur following chemical exposure in humans. The OECD’s AOP Development Programme supports the structured design of AOPs and provides guidance for using them within an IATA, as outlined in its Guidance Document for the Use of Adverse Outcome Pathways in Developing Integrated Approaches to Testing and Assessment. This initiative promotes the practical application of AOPs in regulatory settings.4

As mentioned above, consideration of exposure may also be part of an integrated approach. When human and environmental exposure to a substance is low or when the physicochemical properties of a substance dictate that specific routes of exposure are not relevant, it may not be scientifically justified (or possible) to conduct toxicity tests for certain data requirements. When exposure is considered, the focus of regulatory decision-making can shift from a hazard-based “tick box” approach to a risk-centric approach that allows for the minimization of tests on animals.5

However, a systematic framework is needed to evaluate individual methods’ biological and toxicological relevance while also considering different exposure scenarios. Consolidating these approaches, the International Cooperation on Cosmetics Regulation (ICCR) has outlined key principles for integrating non-animal methods into a strategy for next-generation risk assessment (NGRA),6 which is an exposure-led, hypothesis-driven risk assessment approach that integrates non-animal methods to ensure that chemical exposure does not cause harm.7 The Partnership for the Assessment of Risks from Chemicals(PARC), an EU-funded initiative to modernize chemical safety assessments, also aims to make NGRA the default approach to chemical risk assessment in EU chemicals legislation.8

In addition to minimizing animal testing, IATAs can leverage data and use high-throughput methods to assess a large number of chemicals more efficiently than tests on animals. They have the potential to fundamentally transform the current regulatory landscape by allowing more human-relevant decision-making based on both hazard and exposure assessments. Furthermore, with a concerted effort between relevant stakeholders, it is expected that similar gains will be made with respect to integrated approaches for environmental protection.9

Cosmetics

Legislation prohibiting either tests on animals for cosmetics purposes and/or the sale of cosmetics products containing ingredients tested on animals has been enacted in numerous regions, including Australia, Canada, the EU, India, South Korea, Switzerland, Taiwan, and the U.K. In other countries, such as the U.S., legislation limiting the use of animal testing data has been brought in at a state level rather than on a national basis. This global shift away from tests on animals for cosmetics means that the sector has often been at the forefront of innovative safety assessment methods that have the potential to be applied more broadly.

However, despite the groundbreaking nature of these bans, certain regulatory requirements have undermined their full implementation. For example, companies can sell products in the EU and the U.K., even if they are tested on animals elsewhere, such as in China, provided that the results of these tests are not used for meeting regulatory requirements under the relevant cosmetics regulations. Companies may pay for tests on animals required in other markets while using data from non-animal methods to meet EU or U.K. regulations. In the U.S., although there is no specific requirement to test cosmetics products or their ingredients on animals, in some instances, the FDA calls for such tests after products have been approved for market due to differing regional approaches to the classification of products.10 Sunscreens, for example, are regulated as cosmetics in the EU but as over-the-counter pharmaceuticals in the U.S. The FDA has announced its intention to require that new tests on animals be conducted to keep sunscreens on the market if they contain any of the 12 specific active ingredients specified in a 2021 order. No similar request for new data on the same products has been made in the EU or elsewhere.

Conflicts also exist in some areas between industrial chemicals and separate cosmetics legislation. The European Chemicals Agency (ECHA), backed by the European Commission, can mandate animal testing under the Registration, Evaluation, Authorisation, and Restriction of Chemicals (REACH) regulation to assess worker exposure and environmental risks for substances used exclusively in cosmetics products. For the assessment of substances used in cosmetics and other types of products, the REACH regulatory requirements apply to all human and environmental health endpoints regardless of worker exposure.11 Similar legislative conflicts also exist in Australia and Canada.

Since an array of non-animal methods and frameworks are now in use for the assessment of cosmetics, it’s possible to eliminate the use of tests on animals while enhancing scientific rigor. For example, NGRA is a progressive approach involving hypothesis-driven, exposure-led evaluation combining in silico, in chemico, and in vitro methods to enable more accurate risk prediction and ensure the reliability of safety assessments.12131415 NGRA frameworks can be adapted to make decisions on the safety of workers exposed to chemicals during product manufacture.16 Likewise, OECD case studies demonstrate how tiered, flexible approaches to testing and assessment can be used to address safety concerns across different regulatory scenarios, from skin sensitization to systemic and reproductive toxicity.17 The Scientific Committee on Consumer Safety guidance on cosmetic safety assessments offers insights into how these innovative approaches can be applied effectively.18 In addition, the International Collaboration on Cosmetics Safety, a coalition of cosmetics and personal-care companies, ingredient manufacturers, trade associations, and NGOs,19 is developing standardized best practice guidance on the use and understanding of new approach methodologies and NGRAs to further their regulatory acceptance.20

The mismatch between policy and scientific development for the assessment of cosmetics underscores the urgent need to take effective action in order to ensure that non-animal methods are used to protect consumers, workers, and the environment. In May 2023, the U.K. government took a significant step by halting the issuance of new licenses for animal testing on ingredients used exclusively in cosmetics.21 By November 2023, the Home Office confirmed that such tests had also ceased under all remaining legacy licenses.22 This move marks the U.K.’s progress toward completely ending animal testing for cosmetics. However, ingredients that are also used in other household products continue to undergo animal testing, as they are not fully exempt from testing requirements.

Full transparency is essential to foster informed consumer choices, ensure public trust, and address the erosion of legislation and policy designed to ensure that animals are not used to assess cosmetics products or their ingredients for all regulatory purposes.

Ecotoxicity

Aquatic Toxicity and Bioaccumulation

Aquatic tests are conducted to measure the effects of chemicals on the environment and wildlife. In 2022, over 122,000 fish were used for regulatory purposes in the EU and Norway.23 Since assessment of bioaccumulation and aquatic toxicity is required in various regulatory frameworks, strategies to replace testing using aquatic animals are urgently needed.

A promising cytotoxicity assay using the RTgill-W1 cell line has been developed for assessments,24 and the respective OECD test guideline was adopted in 2021.25 This in vitro assay can potentially reduce or even replace the use of fish in the acute fish toxicity test.26

To enhance the prediction of acute fish toxicity, project 2.54 in the OECD Test Guidelines Programme work plan is developing a guidance document on IATAs for acute fish toxicity. This project is co-led by Austria and the International Council on Animal Protection in OECD Programmes (ICAPO), represented by PETA Science Consortium International.

When testing on animals is still required, the number of animals used and the need to repeat studies can be reduced by careful application of OECD Guidance Document 23 on Aquatic Toxicity Testing of Difficult Substances and Mixtures.27 This document was updated in 2019 to provide information on approaches to aquatic toxicity testing of difficult-to-test chemicals. Particular attention was paid to updating the methods available for testing poorly water-soluble test chemicals while avoiding using solvents. Thus, the need for a solvent control group is eliminated, reducing the number of animals used for testing. In addition, the U.S. and ICAPO (represented by PETA Science Consortium International) are co-leading project 2.55 in the OECD Test Guidelines Programme work plan on the use and analysis of control fish in toxicity studies. In this project, statistical analyses of existing data and simulations are being used to investigate whether it’s possible to conduct aquatic toxicity studies with only one control when a solvent is involved, further reducing the number of animals exploited for this purpose.

Several non-animal methods are now available to reduce the number of fish used in bioaccumulation testing. In 2018, the OECD adopted two assays for the assessment of in vitro intrinsic clearance using cryopreserved rainbow trout hepatocytes28 and rainbow trout liver S9 subcellular fraction29 and an associated guidance document.30 Liver intrinsic clearance values can be used either for physiologically based toxicokinetic models for fish bioaccumulation or for extrapolation to an in vivo biotransformation rate. The latter can be used with in silico models for the prediction of bioconcentration factors. Thus, although these test guidelines require the use of fish to obtain primary cells, they can contribute to replacing the use of live fish in OECD Test No. 305 on bioaccumulation in fish.31

Avian Toxicity

Most regulatory authorities currently require avian toxicity tests to assess the potential ecological effects of chemicals on terrestrial birds. Three avian toxicity tests, including acute oral, dietary, and reproduction tests, are commonly required to fulfill regulatory requirements. In the acute oral and dietary tests, up to 120 birds are used. In the oral test, they are dosed with a chemical through gavage for one day, followed by a 14-day observation period. In the dietary test, they are fed the chemical for five days, followed by a three-day observation period. In the reproduction test, more than 120 adult birds are fed the chemical for eight to 10 weeks and several hundreds to thousands of offspring are killed in order to examine potential adverse reproductive outcomes.

Scientists have raised concerns about the utility of avian tests to protect terrestrial species. The results of these tests, often conducted on two species, are used to extrapolate the potential effects on thousands of regional bird species. Additionally, food avoidance, regurgitation, and other issues caused by the methods used for dosing the birds have led to inaccurate toxicity estimates.

To address these concerns, PETA Science Consortium International collaborated with the EPA to assess the use of avian oral and dietary tests in risk management decision-making.32 The retrospective review examined 20 years of risk assessment data and found that the dietary test is generally not used for risk management. This study was used to support the EPA’s 2020 policy titled “Final Guidance for Waiving Sub-Acute Avian Dietary Tests for Pesticide Registration and Supporting Retrospective Analysis,” which can prevent more than 700 birds from being subjected to toxicity tests each year and save resources that would be better spent developing fit-for-purpose non-animal methods for terrestrial toxicity testing.33

PETA Science Consortium International is undertaking a similar initiative to examine the use of two species in avian reproduction tests. This retrospective review will examine differences in avian species sensitivities to hundreds of active ingredients in pesticides to analyze trends concerning how toxicity responses are used in regulatory decision-making. The initiative aims to identify information that is not being used in regulatory decision-making. In addition to these projects, initiatives such as Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS) aim to modernize ecological testing using predictive computational methods with the potential to reduce testing on terrestrial animals while improving ecological protection.34

A lack of global alignment results in increased testing to meet unique regional requirements. For example, the European Commission and the Central Insecticides Board and Registration Committee (CIB&RC) in India require using a single test species for the avian reproduction test, yet the EPA in the U.S. and Canada’s Pest Management Regulatory Agency require two test species. Furthermore, the EPA allows waivers for the avian dietary test, and that test is not required by the European Commission or in Japan but is still required by the CIB&RC and in China. Alignment is necessary to end globally the requirement for tests that have been shown not to provide useful information or that are affecting the quality of regulatory decision-making.

Endocrine Disruption

Endocrine disruptors are natural or synthetic chemicals that interfere with the body’s endocrine system,35 influencing various responses in biological pathways responsible for regulating fundamental biological functions, such as growth, development, reproduction, energy balance, metabolism, or regulation of body weight. From a regulatory chemical safety perspective, the most investigated endocrine pathways are the estrogen, androgen, thyroid, and steroidogenesis (EATS) systems and, to a lesser degree, non-EATS systems, such as the retinoid pathway.36

Much is understood about the complex mechanisms through which chemicals can interfere with endocrine pathways in humans37 and wildlife.38 Numerous AOPs related to endocrine disruption are included in the AOP Wiki,39 and the OECD has published several case studies on IATAs.40 In vivo tests assessing endocrine disruption demonstrate high variability (e.g., stress experienced by an animal can significantly influence a study’s outcome) and low sensitivity, and they are unlikely to detect relevant endocrine disrupting events.41 Classical endpoint studies are not appropriate in this area and need to be replaced by in vitro studies in which the multiple factors that could affect test results can be more effectively controlled.

From 2019 to 2024, eight projects under the European Cluster to Improve Identification of Endocrine Disruptors (EURION), with €50 million of funding from the European Commission, focused on the development of tools aiming to improve regulatory assessment of endocrine-related effects (thyroid hormone system disruption, metabolic disorders, developmental neurotoxicity, and female fertility) and reduce the reliance on animal testing. A concluding policy brief for the EURION project concluded that support is needed for faster implementation of scientific findings into test methods as well as for the update of test requirements in chemical regulations to include newly developed tests.42

The EPA’s Office of Research and Development (ORD) is developing in silico and in vitro assays as well as AOPs to support the robust assessment of chemicals for effects on the endocrine system. For example, the EPA’s Toxicity Forecaster (ToxCast) ranks and prioritizes chemicals using more than 700 high-throughput screening assays and computational toxicology approaches, which cover a variety of relevant cellular responses and signaling pathways.

Following a comparative study of ToxCast estrogen pathway assay results and uterotrophic assay results,43 the EPA announced that it will accept the data from the ToxCast ER Bioactivity Model as an alternative to at least one animal test4445—the uterotrophic assay—which screens for effects on the estrogen pathway.46 In the EU, the ToxCast ER Bioactivity Model is currently accepted as a source of the in vitro mechanistic mode of action information required as part of the identification of substances as endocrine disruptors under the current regulatory framework for biocides and plant protection products.

In collaboration with other organizations, the EU Joint Research Centre and the EPA ORD are developing and assessing the validity of sets of relevant assays based on the thyroid AOP.47 In 2024, the OECD added two of these assays, targeting different molecular initiating events related to thyroid pathway disruption, to its workplan for test guideline development.

Eye Irritation/Corrosion

To assess eye irritation and corrosion using the Draize test, a chemical substance is applied to rabbits’ eyes and the degree of damage is monitored over a 14-day period. Rabbits may endure eye swelling, discharge, ulceration, hemorrhaging, cloudiness, or blindness. The Draize test was developed in 1944, and advanced replacement methods have since been developed and shown to be as or more reliable and relevant than the rabbit test. For example, an analysis of 491 chemicals with at least two rabbit eye tests showed that the probability of obtaining the same UN Globally Harmonized System of Classification and Labelling of Chemicals (GHS) classification more than once was 73% for GHS category 1 (causes serious eye damage), 32.9% for GHS category 2A (irritant), 15.5% for GHS category 2B (mild irritant), and 93.9% for no category (non-irritating).48 Importantly, these results showed that there was a 10.4% chance that a chemical once identified as causing serious irreversible damage (category 1) would later be identified as non-irritating (no category).

Robust and defined non-animal methods are available to fully replace the Draize test without the need for expert judgment or a WoE assessment.

  • OECD Test No. 492B: Reconstructed Human Cornea-Like Epithelium (RhCE) Test Method for Eye Hazard Identification, which may be used to identify chemicals not requiring classification (GHS no category) and those requiring eye irritation classification (GHS category 2) and serious eye damage classification (GHS category 1)
  • OECD Test No. 467: Defined Approaches for Serious Eye Damage and Eye Irritation. The defined approaches in OECD Test No. 467 are based on the following:
    • Physicochemical properties and in vitro data from OECD Test No. 492: Reconstructed Human Cornea-Like Epithelium (RhCE) Test Method and OECD Test No. 437: Bovine Corneal Opacity and Permeability (BCOP) Test Method for neat non-surfactant liquids
    • In vitro data from OECD Test No. 491: Short Time Exposure (STE) in Vitro Test Method and OECD Test No. 437 for neat and/or diluted non-surfactant liquids or solids dissolved in water
    • In vitro data from OECD Test No. 437 and OECD Test No. 492 for neat solids

The defined approaches may be used to identify chemicals not requiring classification (GHS no category) and those requiring eye irritation classification (GHS category 2) and serious eye damage classification (GHS category 1).

Other in vitro methods may be combined—as outlined in the OECD guidance document on an IATA of serious eye damage and irritation49—to fully replace the Draize test.

  • OECD Test No. 494: Vitrigel-Eye Irritancy Test Method—This test may be used to identify chemicals not classified for eye irritation or causing serious eye damage (GHS no category).
  • OECD Test No. 496: In Vitro Macromolecular Test Method—This test may be used to identify chemicals causing serious eye damage (GHS category 1) and/or not requiring classification.
  • OECD Test No. 460: Fluorescein Leakage Test Method—This test may be used to identify chemicals causing serious eye damage (GHS category 1). It is recommended as an initial step within a top-down approach to identify ocular corrosives or severe irritants.
  • OECD Test No. 438: Isolated Chicken Eye Test Method—This test may be used to identify chemicals causing serious eye damage (GHS category 1) or not requiring classification. It is recommended as the first step within a top-down or bottom-up testing strategy.

These methods are generally validated for use with cosmetics and industrial chemicals. Certain methods will be more appropriate than others, depending on the applicability domain of the method, the purpose of testing, and the type of test chemical (e.g., surfactants or solids).

The EPA currently accepts the use of in vitro and ex vivo methods for determining eye irritation and corrosion when classifying industrial chemicals, antimicrobial cleaning products, and, on a case-by-case basis, other pesticide products. The EPA Office of Pollution Prevention and Toxics published a decision framework in 2024 that discourages prospective Draize tests for new chemical products,50 and in 2015, the Office of Pesticides Programs (OPP) published a guidance document describing the testing framework that industry can use for this endpoint.51 OPP also published on its webpage52 a paper in which the authors proposed defined approaches combining in vitro and ex vivo methods to assess the eye irritation/corrosion potential of agrochemical formulations.53 The paper was coauthored with PETA Science Consortium International, the National Toxicology Program (NTP) Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM), and others.

Genotoxicity and Carcinogenicity

Genotoxicity

The major genotoxicity endpoints to be evaluated for regulatory purposes are gene mutation, structural chromosomal aberrations (clastogenicity), and numerical chromosomal aberrations (aneuploidy). OECD test guidelines for assessing genotoxicity in vitro cover one or two endpoints simultaneously.

  • OECD Test No. 471: Bacterial Reverse Mutation Test—This test, commonly known as the Ames test, uses amino acid, requiring Salmonella typhimurium and Escherichia coli to detect point mutations by base substitutions or frameshifts.
  • OECD Test No. 487: In Vitro Micronucleus Test—This test can be used to detect micronuclei in the cytoplasm of interphase cells that have undergone cell division during or after exposure to a test substance. This assay detects structural and numerical chromosomal aberrations.
  • OECD Test No. 490: In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene—Two distinct assays can be used to detect gene mutations induced by chemical substances.
  • OECD Test No. 473: In Vitro Mammalian Chromosomal Aberration Test—This test identifies chemical substances that cause structural chromosomal aberrations.
  • OECD Test No. 476: In Vitro Mammalian Cell Gene Mutation Test Using the Hrpt and xrpt Genes—These tests can detect gene mutations induced by chemicals.

The assessment of genotoxicity for regulatory purposes typically follows a stepwise approach, starting with a core battery of in vitro tests (e.g., the Ames test, the micronucleus test, and the chromosome aberration test). The need to follow up in vitro tests with in vivo tests depends on the results and regulatory requirements. For example, in the case of the EU’s industrial chemicals and biocides regulations, a positive result in any of the required in vitro tests must be followed up with an in vivo test.5455 However, if a substance produces negative results in the in vitro tests, it can be categorized as having no genotoxic potential and no further genotoxicity testing would be required. Conversely, for some chemical classes, in vivo testing is required regardless of the in vitro test results (e.g., plant protection products and pharmaceuticals).5657

Appropriate data from in silico studies (e.g., QSARs and read-across) can help reduce the requirement to conduct in vivo tests. The EURL ECVAM–consolidated genotoxicity and carcinogenicity database published in the EURL ECVAM collection of the Joint Research Centre (JRC) data catalog, for example, provides substantial resources for read-across.58

Furthermore, advanced in vitro methods can provide follow-up and de-risking options for use in a WoE approach. For example, the in vitro transcriptomic biomarker responsive to DNA-damage-inducing (DDI) agents, TGx-DDI,5960 and the ToxTracker assay616263can provide information on the mode of action of potential genotoxicants and have been submitted to formal regulatory “qualification” programs.6465 Data generated using the ToxTracker assay and read-across have been used in the EU’s REACH dossiers.66

The three-dimensional reconstructed skin micronucleus and comet assays are additional non-animal methods that can be used to follow up positive results from standard in vitro genotoxicity assays for dermally applied compounds. They present an important opportunity to avoid the use of animals for genotoxicity testing.6768 The information requirements for genotoxicity assessment of cosmetics69 may already invoke the micronucleus test using three-dimensional reconstructed human skin or a comet test using either mammalian cells or three-dimensional reconstructed human skin. Rapid progress in the development of three-dimensional liver and airway models holds the prospect of assessing the genotoxic potential of compounds administered by the oral or inhalation route in the near future without using animals.70

Non-animal methods are gaining ground internationally. Generating comprehensive data based on these methods and developing case studies, such as that on coumarin used in cosmetics products, is an important component of supporting the adoption of next-generation risk assessment.7172

The genotoxicity73 and mutagenicity74 case studies on IATA, under the OECD IATA case studies project,75 illustrate feasible approaches to developing adequate safety assessment guidelines for systemic genotoxicity risk assessment without animal testing.

Carcinogenicity

The assessment of carcinogenicity often requires testing on rats and/or mice for the majority of their lives (up to two years). The test requires a minimum of 400 rats and/or mice per chemical assessment (OECD Test Nos. 451, 452, and 453).

While carcinogenicity studies in animals are still routinely conducted, the test has been under scientific scrutiny since the early 1970s for its lack of reproducibility76 and its inability to predict human outcomes.77 Notably, there are two flawed assumptions that underlie these bioassays: (1) rodent carcinogens are human carcinogens, and (2) high-dose chemical exposure in rodents indicates an environmentally relevant dose. Both have been proved incorrect by 50 years’ worth of carcinogenicity data. Decades of scientific reviews highlight the overall lack of reliability in the rodent cancer bioassays to predict cancer in humans.7879808182

For example, an assessment of 202 pesticide evaluations from a review program conducted by the EU demonstrated that the mouse carcinogenicity study contributed little or nothing to either derivation of an acceptable daily intake for assessment of chronic risk to humans or hazard classification for labeling purposes.83 In terms of pesticide approvals, the authors showed that the mouse study did not influence a single outcome. An additional study reported that data collected from 182 pharmaceutical chemicals show that little value is gained from the carcinogenicity study when compounds lack certain histopathologic risk factors, hormonal perturbation, and positive genetic toxicity results.84 This study was used to support an international collaboration that developed a WoE approach to fulfill some of the carcinogenicity test requirements without the two-year test on rats.8586 The collaboration resulted in an addendum to the guideline for carcinogenicity assessment of pharmaceuticals (ICH S1B)—thus providing an opportunity to spare 400 animals per pharmaceutical regulatory evaluation.87 A similar effort called Rethinking Chronic Toxicity and Carcinogenicity Assessment for Agrochemicals Project (ReCAAP), led by PETA Science Consortium International, developed a framework to support a WoE-based assessment of agrochemicals without long-term carcinogenicity testing on rats and mice.88 The ReCAAP framework has since been accepted for publication by the OECD Working Party for Hazard Assessment (WPHA), whereby eight global regulatory bodies endorsed the WoE-based approach to fulfilling safety assessment needs—without conducting lifetime tests on rats and mice.89

Additional activities are ongoing to develop a framework to provide a modular strategy for assessing carcinogenicity in non-genotoxic chemicals, including efforts from the OECD Working Party for the Test Guideline Program (WNT) expert group on non-genotoxic carcinogens (NGTxC). This framework offers a modular approach to evaluating and integrating in vitro and in silico data into an AOP-style for assessing bioactivity that could potentially lead to carcinogenicity.90

The OECD WNT is also assessing the in vitro cell transformation assays (CTA) for their ability to recapitulate a multistage process that models some aspects of in vivo carcinogenesis. The CTA has the potential to detect both genotoxic and non-genotoxic carcinogens.91 In its recommendation on the CTA based on the Bhas 42 cell line, EURL ECVAM notes that information on the transforming potential of substances generated by CTAs may be sufficient for decision-making.92 Following a study in which the Bhas 42 CTA was tested with 98 substances—including known human carcinogens—the OECD has recommended that this assay be used as part of a testing strategy to help assess potentially cancer-causing substances.9394 When combined with other information, such as genotoxicity data, structure-activity analysis, and toxicokinetic information, CTAs in general—and the Bhas 42 CTA specifically—can contribute to the assessment of carcinogenic potential and may provide an alternative to in vivo testing.9596

Several computational tools and models further help to assess carcinogenicity potential. Structural alerts flagging potential non-genotoxic carcinogens have been incorporated into the OECD QSAR Toolbox.97 Additionally, the EPA has published a computer model, OncoLogic™, to evaluate chemicals for carcinogenic potential,98 and commercial options are also available, such as those from Lhasa Limited, MultiCASE, UL Cheminformatics, and Instem. Ultimately, identifying DNA-reactive chemicals with the Ames test or genotoxic structural alerts can potentially be combined with identifying non-genotoxic carcinogens using structural alerts, leaving CTAs to model most of what is left unexplained in a WoE approach.

Given the complexity of carcinogenesis, experts recognize that there needs to be an integration of new approaches (e.g., in silico or in vitro) to support a fit-for-purpose WoE-based safety assessment.99 Fortunately, initiatives are underway to facilitate the integration of methods to ultimately achieve an animal-free, rapid, and human-relevant carcinogenicity assessment for chemical and pharmaceutical regulation.100101102103

Phototoxicity

Substances that absorb light in the UV and visible range (290 to 700 nm) and can reach the skin or eyes may require testing for potential phototoxicity, which is the toxic response to a topically or systemically administered substance that occurs after exposure to light. It can cause symptoms ranging from first-degree burns (redness, itching, and pain) to full-thickness third-degree burns. Phototoxicity, often also called photosensitivity, is a well-known adverse effect of many drugs, including antimicrobials, nonsteroidal anti-inflammatory drugs, diuretics, and chemotherapeutic agents.104

Phototoxicity testing for systemically or topically administered compounds has been conducted in various species, including guinea pigs, mice, and rats. However, no validated or standardized in vivo study design has been established.105106 By contrast, so far, three OECD test guidelines have been developed using in chemico and in vitro methods to assess phototoxicity.

  • OECD Test No. 495: Ros (Reactive Oxygen Species) Assay for Photoreactivity—This in chemico method measures the capacity of a substance to create reactive oxygen species under exposure to artificial sunlight.
  • OECD Test No. 432: In Vitro 3T3 NRU Phototoxicity Test—This test measures the viability of a mouse cell line incubated with a potential phototoxicant and exposed to light.
  • OECD Test No. 498: In Vitro Phototoxicity—Reconstructed Human Epidermis Phototoxicity Test Method—In this test, a three-dimensional reconstructed human epidermis model is incubated with the potential phototoxicant and exposed to light.

OECD Test No. 498 is based on a principle similar to that of OECD Test No. 432 but uses a three-dimensional reconstructed human skin model instead of the mouse cell line. This expands the applicability domain to a wider selection of substances, including final formulations, complex mixtures, or dermatological patches.107 Substances with an extreme pH can also be tested using three-dimensional skin models.

These OECD test guidelines can be combined with other physico-chemical assessments andin vitro and in silico approaches—as outlined in the OECD’s Guidance Document on Integrated Approaches to Testing and Assessment (IATA) for Phototoxicity Testing—without animal testing to assess a substance’s phototoxic potential.108

Pyrogenicity

Regulators require testing to demonstrate that specific drugs and medical devices are not contaminated with substances that trigger a fever response. These substances, collectively termed pyrogens, are chemically and structurally diverse but generally prompt fever in humans through a common mechanism: Peripheral blood monocytes and macrophages detect pyrogens and release pro-inflammatory cytokines that induce a rise in body temperature. Two in vitro methods are available that detect pyrogens:

  • Monocyte activation test (MAT), defined in European Pharmacopoeia (Ph Eur) general chapter 2.6.30 and permitted in United States Pharmacopeia (USP) general chapter 151
  • Recombinant Factor C (rFC) assay, defined in Ph Eur general chapter 2.6.32 and, beginning in May 2025, in USP general chapter 86

Even though the human fever response mechanism is well understood, most global regulators still commonly require two animal-based tests to assess pyrogen contamination. In the rabbit pyrogen test (RPT), rabbits are injected with a test substance and subsequently restrained for three hours, during which time changes in their body temperature are monitored rectally. In the EU and Norway alone, more than 125,000 rabbits were used between 2018 and 2022 in the RPT.109 Although some countries appear to have ceased using the RPT, others like France and Spain still used over 6,000 animals each in 2022—even though it has never been formally validated for its relevance to humans and its results can vary depending on an animal’s stress level. There are also differences in pyrogen sensitivity among species, and the test is incompatible with certain drug classes.110

The limulus amoebocyte lysate test (LAL) requires the use of hemolymph from captured horseshoe crabs and detects only bacterial endotoxins and no other pyrogens. After the bleeding process, up to 30% of the crabs die. Those who recover are less likely to survive in nature.111 A synthetic version of the LAL, in which the hemolymph is replaced by a recombinant reagent (the rFC assay), is available to test for bacterial endotoxins. The rFC assay is a reliable and animal-friendly test with performance equal or superior to that of the LAL.112

Since 2010, the in vitro monocyte activation test (MAT), capable of detecting both endotoxin and non-endotoxin pyrogens, has been validated and included in the Ph Eur.113 In the MAT, drugs and medical devices are incubated with human whole blood or isolated human monocytes. After this exposure period, tests measure pro-inflammatory cytokines released by monocytes.114 It avoids the aforementioned problems with the RPT and LAL tests, and case studies document instances in which the MAT detected pyrogen contamination in products that had passed the RPT and LAL but caused fever in human patients.115

Regulators in the EU, India, the U.K., and the U.S.—as well as the pharmacopeias used in these regions—all allow the use of the MAT and rFC following product-specific validation. Nevertheless, tests on animals are still being used despite their well-documented limitations.116 To eliminate the use of animals in pyrogen tests, regulatory authorities and standards organizations must make an increased effort to integrate and harmonize a preference for non-animal tests in international testing requirements and to encourage drug and device manufacturers to use and submit data from these tests in their product dossiers. In September 2018, participants at a workshop organized by PETA Science Consortium International and NICEATM discussed non-animal approaches to medical device pyrogen testing and called for more opportunities for training and education to increase the use of the MAT for regulatory purposes.117

Following a survey of pyrogen test users, the European Directorate for the Quality of Medicines & HealthCare (EDQM) revised the Ph Eur general chapter on the MAT to improve the usability of the method and to emphasize that it is considered a replacement for pyrogen tests using animals.118119 This endorsement is repeated in statements from the European Medicines Agency,120 and the Ph Eur Commission has announced that sanction of the RPT will be officially removed from the Ph Eur in 2025.121 The International Organization for Standardization (ISO) is revising its guidance to allow use of the MAT when evaluating medical device pyrogen contamination, but the revision process has moved slowly.122 In the 8th edition of Indian Pharmacopoeia, the Indian Pharmacopeia Commission revised the pyrogen testing general chapter, introduced the monograph on the MAT, and replaced the RPT with LAL.123 However, due to unclear guidance and regulatory ambiguity about the applicability of the MAT as a stand-alone pyrogen test, the RPT and LAL continue to be used.

Reproductive and Developmental Toxicity

Reproductive toxicity studies measure the effect of a chemical on reproductive organs and fertility, while developmental toxicity studies measure a chemical’s effect on developing offspring during pregnancy.

Developmental toxicity studies for chemical and pharmaceutical human safety assessment are primarily performed using rats. However, many regulatory frameworks—including the biocidal products and plant protection product regulations and, in some circumstances, REACH in the EU—require registrants to submit test results using a second species, usually rabbits, under the assumption of interspecies differences in sensitivity to developmental effects. These studies use a large number of animals. For example, a study estimated the total number of animals used for reproductive and developmental endpoints in existing registration dossiers from the public ECHA database (as of December 2022) to be approximately 2.7 million.124

None of the in vivo methods used for testing reproductive and developmental toxicity have been formally validated for their relevance to humans,125 and retrospective evaluations demonstrate their significant limitations and the subjectivity of data interpretation.126127 Therefore, significant investment is required to develop human-relevant non-animal methods. Recently, 42 AOPs from the AOP-wiki, relevant for mammalian reproductive toxicity, were included in an AOP network for estrogen-, androgen- and steroidogenesis-mediated reproductive toxicity, covering effects on hormone levels or hormone activity, cancer outcomes, male and female reproductive systems, and overall effects on fertility and reproduction.128

Due to the extensive knowledge about key events of reproductive and developmental toxicity, many promising assays and test batteries have been developed. The EU ReProTect project, which aimed to develop innovative methods of assessing reproductive toxicity, demonstrated that a battery of several in vitro and in silico tests, including the embryonic stem cell test, could be used to provide valuable information on adverse effects during embryonic development.129 A novel human stem cell–based biomarker assay, ReproTracker®, identifies the teratogenicity potential of chemicals.130 Additionally, a battery of diverse assays was developed, including the CALUX transcriptional activation assay (for steroidogenic activity), ReProGlo assay (for body axis patterning and cell fate specification), embryonic stem cell test (for differentiation into cardiomyocytes), and zebrafish embryotoxicity assay.131

In addition, the EU-ToxRisk project integrates advances in cell biology, “omic” technology, systems biology, and computational modeling to define the complex chains of events that link chemical exposure to toxic outcomes. The project focuses on repeat-dose systemic toxicity and developmental and reproductive toxicity. The EPA’s National Center for Computational Toxicology is also exploring the potential for chemicals to disrupt prenatal development through the use of its virtual embryo model, v-Embryo™, which integrates in vitro and in silico modeling approaches.132 The OECD, JRC, European Food Safety Authority (EFSA), and the EPA have developed recommendations to demonstrate how the integration of a battery of in vitro assays can be used to determine the potential of chemical developmental neurotoxicity, and the partner agencies are working on case studies that apply to different chemical classes.133134135 A study compared in vitro bioactivity-based points of departure (PODBioactivity) with points of departure from oral repeat-dose, developmental, and reproductive studies (PODTraditional) used in risk assessment. For 43 out of 46 of the examined chemicals, PODBioactivity was more conservative than the lowest PODTraditional, demonstrating confidence in using in vitro bioactivity as a surrogate lower bound estimate of in vivo adverse effect levels—a strong indication that using PODBioactivity would be at least as protective as using PODTraditional.136

While the field is gradually moving toward a range of integrative strategies in order to cover the majority of possible mechanisms, much more research is required.

Skin Irritation/Corrosion

Skin irritation and corrosion tests for chemicals are required or recommended by several regulatory agencies. In the animal test, a test substance is applied to the shaved skin of a rabbit, and they are observed for up to 14 days to assess the degree of skin damage. The tests can cause permanent skin damage, ulcers, bleeding, scabs, and scarring.

Skin irritation studies involving animals have been used for years, even though they have been shown to be generally poor predictors of human skin reactions and highly variable.137 For example, a comparison of data from rabbit tests and four-hour human skin patch tests for 65 substances found that 45% of classifications of chemical irritation potential based on tests on animals were incorrect.138

There are opportunities to avoid the animal test based on criteria described in OECD guidance document no. 237.139 Furthermore, the OECD has developed an IATA for skin irritation using in vitro skin irritation and corrosion methods that avoid or minimize animal use.140

  • OECD Test No. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis (RHE) Test Method—This test may be used for the hazard identification of irritant chemicals (substances and mixtures), in accordance with the GHS, as category 2, or unclassified chemicals. It may be used as a stand-alone test or in a tiered testing strategy.
  • OECD Test No. 431: In Vitro Skin Corrosion: RHE Test Method—This test may be used to identify corrosive chemical substances and mixtures. It may also distinguish between severe and less severe skin corrosives.
  • OECD Test No. 435: In Vitro Membrane Barrier Test Method for Skin Corrosion—This test allows corrosive chemicals to be categorized as one of the three GHS corrosivity subcategories.

Recently, OECD Test Guideline No. 439 was validated for use in assessing the ability of medical device extracts to cause skin irritation, and the ISO 10993 guidance has been updated to include this test.141

Skin Sensitization

The assessment of skin sensitization involves measuring the likelihood that a substance will cause an allergic reaction if applied to the skin. In animals, such assessments have previously been based on injecting a test substance into—or applying a test substance to the shaved skin of—guinea pigs in the guinea pig maximization test or applying it to the ears of mice in the local lymph node assay.

The regulatory requirement to test for skin sensitization can be met with a defined approach, as described in OECD Test No. 497: Defined Approaches on Skin Sensitisation, using a combination of in chemico and in vitro assays that each addresses a different key event in the AOP.142 The “2 out of 3” defined approach provides sufficient information for hazard identification, and the integrated testing strategies (ITSv1 and ITSv2) collate information from two of the in vitro assays included in the guidelines listed below, along with in silico predictions, to predict hazard and potency.

  • OECD Test No. 442C: Key Event-Based Test Guideline for In Chemico Skin Sensitisation Assays Addressing the Adverse Outcome Pathway Key Event on Covalent Binding to Proteins—This test addresses the molecular initiating event of the skin sensitization AOP.
  • OECD Test No. 442D: In Vitro Skin Sensitisation Assays Addressing the AOP Key Event on Keratinocyte Activation—This test addresses the second key event of the skin sensitization AOP.
  • OECD Test No. 442E: In Vitro Skin Sensitisation Assays Addressing Key Event on Activation of Dendritic Cells—This test addresses the third key event of the skin sensitization AOP.

When compared to human data, the non-animal approaches to predicting skin sensitization are as good as or better than the local lymph node assay.143

Systemic Toxicity

Acute Systemic Toxicity

To determine the danger of exposure to a product or chemical, a substance is administered to animals through the oral, dermal, or inhalation routes. Acute toxicity refers to adverse effects observed following one high level of exposure to a substance for a short duration (up to 24 hours). In these tests, the dose at which half the animals would be killed—called the lethal dose 50 (LD50) or lethal concentration 50 (LC50) for inhalation testing—is determined. The LD50 test and its adaptations have never been scientifically validated, and their accuracy in predicting chemical effects in humans remains in question. An analysis of the variability of the acute oral toxicity animal test showed that there is 78% or 74% accuracy in obtaining the same EPA or GHS classification, respectively, if the same chemical is tested more than once,144 while another analysis of existing acute oral LD50 data demonstrated that replicate studies result in the same hazard categorization on average 60% of the time.145 This second study demonstrated that inherent biological or protocol variability most likely underlies the variance in the results.

When scientific justification is provided, regulatory authorities may allow acute toxicity assessment without testing on animals. The OECD has published guidance for waiving or bridging acute toxicity testing,146 and the EPA has published similar guidance for pesticides and pesticide products.147 This includes the use of existing data for read-across and the consideration of the physicochemical properties of the test substance.148149

Repeat-Dose Systemic Toxicity

In repeat-dose toxicity studies, animals are exposed repeatedly to substances for up to one month (sub-acute), up to three months (sub-chronic), or up to several years (chronic) in order to measure the effects of multiple chemical exposures. Chemicals are usually administered to animals using oral gavage unless humans are more likely to be exposed via another route. As with other endpoints, there is evidence that regulatory studies using animals to assess repeat-dose toxicity are not fit for the purpose, and there is a clear need to develop new approaches. In 2020, Pham and colleagues evaluated the sources of variability in the values used to derive safe exposure levels from a variety of repeat-dose studies in rodents and found that approximately one-third of the total variance could not be accounted for through considerations of study differences, e.g., administration route or study type.150151

The assessment of repeat-dose toxicity is a standard requirement in human safety evaluations, and while read-across approaches are accepted for regulatory purposes, other non-animal methods have yet to gain full acceptance. To address this gap in the use of non-animal methods, various projects across academia, industry, and regulatory bodies have proposed diverse sets of high-content, high-throughput, and “omic” technology-based in vitro and in silico assays. These initiatives focus on developing non-animal testing methods to derive in vitro points of departure, predict maximal plasma concentrations, or calculate bioactivity exposure ratios.152153154155 An OECD case study on the use of an IATA for systemic toxicity demonstrates the application of such advanced methodologies.156

While the development and regulatory implementation of in vitro testing systems advances, the number of animals used for repeat-dose toxicity testing under various regulatory frameworks may be immediately reduced by the extrapolation of points of departure from sub-chronic to chronic studies.2 A review of points of departure (NOAELs or LOAELs) determined from in vivo studies with food additives showed that the chronic values may be extrapolated with high confidence from sub-chronic studies, supporting previous analyses of other types of substances, including industrial chemicals and pesticides. The risk assessment and derivation of health-based guidance values may be further strengthened by a precautionary application of an additional uncertainty factor of 2 to account for any outlying values—an approach recommended by EFSA and supported by data from a number of recent studies.157

Oral Route

NICEATM and the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) carried out a project to develop predictive models for acute oral systemic toxicity.158 The outcome was the Collaborative Acute Toxicity Modelling Suite (CATMoS) tool for predicting acute oral toxicity to meet various regulatory needs, which were presented at an April 2018 workshop.159 It generated 139 predictive models using data from about 12,000 chemicals. A consensus model was built, combining the individual models after weighing their individual performance. CATMoS is implemented through Open Structure-Activity/Property Relationship App (OPERA), a freely available and open-source QSAR tool.160 This model is routinely optimized and further evaluated,161 and updates are available on the NICEATM Integrated Chemical Environment (ICE) and EPA websites.162 PETA Science Consortium International, the Physicians Committee for Responsible Medicine, and the EPA developed webinars to provide overviews of both the CATMoS tool and the ICE database (ThePSCI.eu/training-videos-webinars).

EURL ECVAM recommends using the in vitro 3T3 neutral red uptake (NRU) cytotoxicity assay, which can be used in a WoE approach to support the identification of non-classified substances.163 EURL ECVAM additionally investigated how to increase confidence in the 3T3 NRU through the use of QSARs and by accounting for target organ information and the lack of metabolism in 3T3 cells.164165166

In its “Guidance on Information Requirements and Chemical Safety Assessment,” ECHA advises that an in vivo acute oral toxicity study can potentially be avoided if a registrant has relevant data, which are used in a WoE approach.167 In cases in which the WoE adaptation leads to the assumption of low/no expected acute oral toxicity (>2000 mg/kg bw/d), the registrant can avoid animal testing pursuant to REACH Articles 13(1) and 25(1).168 More information about ways to reduce the number of animals used to assess acute oral toxicity for REACH can be found at ThePSCI.eu/training-videos-webinars.

Dermal Route

The EPA and NICEATM analyzed the relative contributions of data from acute oral and dermal toxicity tests to pesticide hazard classification and labeling. Finding that the dermal data provided little to no added value in regulatory decision-making, the EPA published guidance allowing registrants to submit scientifically sound justification for why the acute oral test results are protective for potential acute dermal effects.169170 In addition, dermal studies are not required for substances that are non-classified by the oral route and not absorbed dermally.171 Furthermore, substances not classified by the oral route do not require dermal data under REACH Annex VIII.

Inhalation Route

Testing by the inhalation route can be avoided based on physicochemical parameters (e.g., low volatility) or if exposure through inhalation is unlikely (e.g., in cases in which the substance is not aerosolized or otherwise made respirable under conditions of use). When testing is required, non-animal methods can be applied to fulfill the informational requirements. For example, to fulfill an informational need, the EPA accepted the use of an in chemico biosolubility test, which showed that a polymer, initially classified as a poorly soluble, low toxicity substance, was soluble in simulated epithelial lung fluid and, therefore, was not a hazard concern from lung overload.172 In another example, the EPA accepted data from in silico computational fluid dynamic modeling and in vitro testing using three-dimensional reconstructed human lung tissues to fulfill the re-registration requirements for a pesticide instead of a 90-day rat inhalation study.173174 Several other promising research efforts are underway to develop non-animal methods for inhalation toxicity.175

PETA Science Consortium International has hosted numerous webinars (ThePSCI.eu/inhalation-webinars) and workshops, at which several approaches were presented that could eventually replace animal testing for this endpoint.176177 Additionally, the Science Consortium has funded method development and organized several awards to provide researchers with equipment and in vitro respiratory tissues to conduct inhalation toxicity studies.178 More information on inhalation toxicity testing can be found at ThePSCI.eu/our-work/inhalation.

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