Pesticide Testing

Pesticides (also known as biocides, agrochemicals, and plant-protection products) undergo a battery of tests to fulfil government regulatory agencies’ data requirements for health and safety. These include tests for eye and skin irritation, skin sensitisation, and acute oral, inhalation, and dermal toxicities – the “six-pack” of acute toxicity tests – as well as studies on reproduction and development, neurotoxicity, immunotoxicity, carcinogenicity, mutagenicity, and ecological effects. Many of the required tests use animals, and in the US, more than 7,000 rats, mice, rabbits, birds, fish, and dogs are used to test a single active ingredient and additional animals are used to test various formulations. However, there is global interest in moving away from traditional animal tests that have been increasingly demonstrated to be unreliable or not relevant to humans1–11 and towards non-animal toxicology testing approaches that better protect human health and the environment.

Eye Irritation/Corrosion

The use of non-animal methods of assessing the eye irritation potential of pesticides may be allowed and even encouraged, depending on the regulatory region. For example, in the UK, guidance from the Health and Safety Executive requires the use of non-animal methods and warns that data from tests using animals may be rejected.

In the US, the Environmental Protection Agency (EPA) Office of Pesticide Programs (OPP) accepts the use of an alternate framework for evaluating eye irritation for antimicrobial cleaning products and, on a case-by-case basis, for other classes of pesticides and pesticide products (policy established in 2013; updated in 2015). To broaden the use of reliable testing methods, the Science Consortium collaborated with the EPA OPP, the European Commission’s Joint Research Centre, and others to review the available test methods (the in vitro, ex vivo, and rabbit tests) and show that the in vitro and ex vivo methods are less variable and as or more human-relevant than the currently used rabbit test. The review also states that the rabbit test is not a reliable reference standard and that new methods should not be compared to the rabbit test to show their validity. Working with the NTP Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM), the Science Consortium has also coordinated and funded the testing of agrochemical formulations in various in vitro and ex vivo methods to better understand how the different methods compare (publication forthcoming).

Avian Dietary Toxicity

For decades, the EPA has required data from avian acute oral toxicity (OCSPP 850.2100) and avian dietary toxicity (OCSPP 850.2200) tests for the registration of pesticides. However, trends over the past 20 years have suggested that the avian sub-acute dietary test generally does not drive risk management decisions, and scientists from the Science Consortium and EPA conducted a detailed retrospective analysis to confirm this hypothesis.

One-hundred-and-nineteen pesticides registered between 1998 and 2017 were evaluated, and risk quotient (RQ) data from the avian dietary toxicity tests showed that no pesticide surpassed the level of concern set by the EPA that was also identified in the acute oral test. These results demonstrate that the sub-acute dietary test conducted on birds is not used in risk management and can be removed without causing harm to the environment.

This analysis was used to support the EPA’s guidance for replacing the avian sub-acute dietary test for pesticide registration with a science-based integrated assessment (see here). The EPA now joins the EU, Canada, and Australia in avoiding the use of this test in favour of reliance on other scientific information.


The Science Consortium, government, and industry are working to modernise carcinogenicity testing through the Rethinking Carcinogenicity Assessment for Agrochemicals Project (ReCAAP). As a next step, efforts are now underway to bridge ongoing initiatives towards achieving a science-based paradigm for agrochemical safety assessment. More information on this project and carcinogenicity testing can be found here.

Acute Systemic Toxicity

The Science Consortium partnered with NICEATM, the EPA, and others on the implementation of alternative approaches for acute systemic toxicity testing, including a publication outlining current regulatory requirements, data obtained from the currently required tests, data actually used and needed by regulators, and opportunities for the use of non-animal approaches to meet regulatory needs. The publication of this paper was an action item proposed at a 2015 workshop co-organised by the Science Consortium and was followed, in 2016, by a workshop focused on acute inhalation toxicity testing. A summary of the Science Consortium’s extensive work on respiratory toxicity testing can be found here.

One-Year Dog Toxicity Test

Many countries used to require both a three-month (sub-chronic) study and one-year (chronic) study in dogs as part of the data requirements for registering pesticide active ingredients. In these tests, beagles are exposed to pesticides orally or via inhalation. Because the two studies have been required for more than 30 years, there is an extensive database of results. Beginning in the late 1990s, numerous scientific articles12–21 have been published comparing the results of the two studies and supporting the conclusion that a one-year test in addition to the three-month test is of little value and that its elimination would not compromise human safety or protection.

In the US, following urging from Science Consortium member PETA US and other groups, the EPA conducted its own comparison of data from the two tests and, after reaching the same conclusion as previous investigators, removed the one-year study from its requirements in 2007.22 The EU passed legislation in March 2013 also eliminating the requirement for the one-year dog study. Consortium scientists subsequently began contacting regulatory agencies in other parts of the world, including Canada, Australia, Japan, South Korea, India, and Brazil, urging them to follow suit.

Following discussions between the Science Consortium and Health Canada staff, Canada eliminated its requirement in March 2016. Japan and South Korea eliminated their requirements in April 2018, and Brazil eliminated its requirement in July 2019. Australia, China, and India have indicated that they can waive or do not require the one-year test.

Advancing the Use of Non-Animal Methods

On 17 March 2016, in a letter to stakeholders, the EPA OPP announced its commitment to transitioning towards the use of non-animal testing approaches that will enhance the quality of risk assessment and ensure better protection of human health and the environment. This announcement was followed in June 2020 by a work plan (updated in December 2021) to prioritise agency efforts towards meeting this goal and with a metrics webpage to help gauge progress and identify priorities for future work. The EPA OPP has taken numerous steps to reduce the use of animals and advance the use of non-animal methods, including the following:

  • Issuing guidance for waiving acute dermal toxicity testing of pesticide formulations (2016) and active ingredients (2020), based on a holistic assessment of other toxicity tests
  • Instituting a policy accepting the use of in vitro defined approaches for skin sensitisation testing of single chemicals (2018)
  • Instituting a policy to reduce the number of test concentrations – and therefore fish – used in fish bioconcentration tests (2020)
  • Conducting a risk assessment for the re-registration of six isothiazolinones using in vitro tests and an artificial neural network–based defined approach (2020)
  • Investigating the use of the GHS additivity formula (2021), which uses toxicity data from the individual ingredients in formulations to estimate the toxicity of end products without having to conduct additional testing

The Science Consortium participates in various workgroups of the EPA, pesticide industry members, and other stakeholders to discuss efforts to reduce the number of animals used in pesticide toxicity testing. This includes participation on the Pesticide Program Dialogue Committee (PPDC), which is a federal advisory committee for the EPA OPP with members representing industry, growers, and the animal protection, environmental, and farmworker communities.

The Science Consortium organises training opportunities to familiarise regulatory staff with non-animal test methods. These sessions are led by in silico and in vitro method experts, such as the Institute for In Vitro Sciences and the Laboratory of Mathematical Chemistry. The Science Consortium co-organises a webinar series on the use of new approach methods in risk assessment with the agency.

In the EU, the Science Consortium participates in member state competent authority meetings for biocides, in which data requirements are discussed and updated. The Science Consortium works with the European Commission, member state authorities, animal protection organisations, and industry representatives on a number of far-reaching regulatory initiatives, such as the Chemicals Strategy for Sustainability, to replace tests on animals with robust non-animal approaches.

Similarly, in India, the Science Consortium works with the Central Insecticides Board and Registration Committee and industry members, providing scientific recommendations on the implementation of effective, non-animal methods.

  • References

    1Luechtefeld T, Maertens A, Russo DP, Rovida C, Zhu H, Hartung T. Analysis of Draize eye irritation testing and its prediction by mining publicly available 2008-2014 REACH data. ALTEX. 2016;33:123-134.

    2Clippinger AJ, Raabe HA, Allen DG, et al. Human-relevant approaches to assess eye corrosion/irritation potential of agrochemical formulations. Cutan Ocul Toxicol. 2021;40(2):145-167.

    3Pham LL, Watford SM, Pradeep P et al. Variability in in vivo studies: defining the upper limit of performance for predictions of systemic effect levels. Comput Toxicol. 2020;15:100126.

    4Paparella M, Colacci A, Jacobs MN. Uncertainties of testing methods: what do we (want to) know about carcinogenicity? ALTEX. 2017;34:235-252.

    5Allen DG, Rooney J, Kleinstreuer NC, Lowit AB, Perron M. Retrospective analysis of dermal absorption triple pack data. ALTEX. 2021;38(3):463-476.

    6Kleinstreuer et al. Non-animal methods to predict skin sensitization (II): an assessment of defined approaches. Crit Rev Toxicol. 2018;48(5):359-374.

    7Rooney JP, Choksi NY, Ceger P et al. Analysis of variability in the rabbit skin irritation assay. Regul Toxicol Pharmacol. 2021;122:104920.

    8Karmaus AL, Mansouri K, To KT, et al. Evaluation of variability across rat acute oral systemic toxicity studies. Toxicol Sci. 2022;188(1):34-47.

    9Gottmann E, Kramer S, Pfahringer B, Helma C. Data quality in predictive toxicology: reproducibility of rodent carcinogenicity experiments. Environ Health Perspect. 2001;109(5):509-514.

    10Kleinstreuer NC, Ceger PC, Allen DG et al. A curated database of rodent uterotrophic bioactivity. Environ Health Perspect. 2016;124:556-562.

    11Browne P, Kleinstreuer NC, Ceger P et al. Development of a curated Hershberger database. Reprod Toxicol. 2018;81:259-271.

    12Gerbracht U, Spielmann H. The use of dogs as second species in regulatory testing of pesticides. I. Interspecies comparison. Arch Toxicol. 1998;72(6):319-329.

    13Spielmann H, Gerbracht U. The use of dogs as second species in regulatory testing of pesticides. Part II: Subacute, subchronic and chronic studies in the dog. Arch Toxicol. 2001;75(1):1-21.

    14Baetcke KP, Phang W, Dellarco V. A comparison of the results of studies on pesticides from 12- or 24-month dog studies with dog studies of shorter duration. Health Effects Division, Office of Pesticide Programs. US Environmental Protection Agency. Available at Published 2005.

    15Box RJ, Spielmann H. Use of the dog as non-rodent species in the safety testing schedule associated with the registration of crop and plant protection products (pesticides): present status. Arch Toxicol. 2005;79(11):615-626.

    16Doe JE, Boobis AR, Blacker A, et al. A tiered approach to systemic toxicity testing for agricultural chemical safety assessment. Crit Rev Toxicol. 2006;36(1):37-68.

    17US Environmental Protection Agency. Length of dog toxicity study(ies) that is appropriate for chronic RfD determinations of pesticide chemicals. Health Effects Division, Office of Pesticide Programs. Available at (Docket # EPA-HQ-OPP-2004-0387-0179). Published 2006.

    18van Ravenzwaay B. Initiatives to decrease redundancy in animal testing of pesticides. ALTEX. 2010;27(3):112-114.

    19Dellarco VL, Rowland J, May B. A retrospective analysis of toxicity studies in dogs and impact on the chronic reference dose for conventional pesticide chemicals. Crit Rev Toxicol. 2010;40(1):16-23.

    20Kobel W, Fegert I, Billington R, et al. A 1-year toxicity study in dogs is no longer a scientifically justifiable core data requirement for the safety assessment of pesticides. Crit Rev Toxicol. 2010;40(1):1-15.

    21Kobel W, Fegert I, Billington R, et al. Relevance of the 1-year dog study in assessing human health risks for registration of pesticides. An update to include pesticides registered in Japan. Crit Rev Toxicol. 2014;44(10):842-848.

    22US Environmental Protection Agency. Pesticides: data requirements for conventional chemicals, technical amendments, and data requirements for biochemical and microbial pesticides; final rules. Federal Register Notice 72(207):60934-60988. Available at Published 2007.

For a list of our publications, see here.