Wings of Change

Background

Environmental risk assessment evaluates the likelihood that organisms in the environment will be affected due to exposure to chemicals. For pesticides, the potential for exposure to a chemical to cause toxicity in avian species must be assessed to meet regulatory requirements. The studies are carried out in vivo to determine the effects of acute and chronic exposure to the test chemical. The testing is conducted for the registration of pesticides in adherence to the Organisation for Economic Co-operation and Development (OECD) and the United States Environmental Protection Agency (US EPA) Office of Chemical Safety and Pollution (OCSPP) guidelines. Modified versions of the studies described in the guidelines are also conducted by companies for internal screening to prioritise candidates in development. In vivo avian studies are increasingly required for substances covered by the Toxic Substances Control Act that has been implemented following reforms that mandate US EPA review of the safety of the thousands of chemicals produced or imported into the USA. 

The guidelines require the use of large numbers of birds and there are ongoing efforts to reduce the number of in vivo studies required. The US EPA published guidance in 2020 for waiving the sub-chronic avian dietary study [1, 2] in cases where it would not provide additional value to the risk assessments, such as where sufficient toxicity data is already available from other studies (e.g. for the acute oral toxicity study) [3, 4]. This study is also no longer required in the European Union (EU) for the registration of new pesticide active ingredients [5, 6]. A collaboration between the US EPA and the animal protection group the People for the Ethical Treatment of Animals is evaluating whether a protective risk assessment can be calculated using chronic testing data from a single bird species instead of two, as is currently required in the USA and Canada. These programmes have the potential to reduce animal use in environmental risk assessments, nevertheless a significant amount of avian in vivo testing will continue to be required.

The increasing global interest in the development and use of NAMs, defined as full and partial replacement approaches for assessing chemical or drug toxicity, provide opportunities to move away from in vivo studies. For example, avian NAMs that use chicken and quail embryos for the assessment of endocrine disruption have recently been proposed for consideration within the OECD framework. Other NAMs in development incorporate in silico and in vitro approaches and include:

• Quantitative Structure Activity Relationship (QSAR) models for the assessment of acute toxicity [7, 8].
   
• A dynamic energy budget – toxicokinetic-toxicodynamic (DEB-TKTD) model supported by bobwhite quail embryo injection data to predict key reproductive endpoints for the assessment of chronic toxicity [9].
   
• EcoToxChips [10] that use quantitative PCR technology to assess the effects of chemical exposure on the transcription of around 380 toxicologically-relevant genes. EcoToxChips are available for two avian species – the Japanese quail and the double-crested cormorant, although neither of these species are regularly used for regulatory testing.
   
• Immortalised cell lines are available for some avian species, including chicken, the Japanese quail and the double-crested cormorant [11, 12], and have been used for toxicity testing of chemicals such as flame retardants, but not pesticides [13]. 

All NAMs currently in development require further development and validation for use in a regulatory context and this is likely to be a lengthy process before they are able to replace the use of animals. In the meantime, there is also the potential for NAMs to be used in Integrated Approaches to Testing and Assessment (IATA) [14]. IATAs are frameworks in which multiple lines of evidence including data from in vitro assays and in silico models are combined to guide custom testing strategies that use as few animals as possible. 

3Rs benefits

The avian acute oral [15, 16] and reproduction [17, 18] toxicity tests are required for the regulatory assessment of acute and chronic avian toxicity respectively. 

Acute toxicity assessments typically use either an upland game bird or a waterfowl (usually the bobwhite quail) and a passerine (usually the canary or zebra finch) with the number of birds used depending on the guideline. In OCSPP 850.2100, ten birds per dose with five doses plus controls (60 birds in total) or a limit test of a single dose plus control (20 birds in total) are used [16]. OECD test guideline 223 is an “up-and-down test”, with results from the previous stage used to determine the next phase of testing. It typically uses between ten and 24 birds but can use up to 49 animals [15]. Effects include mortality and there are significant animal welfare concerns because of the potential suffering involved. The test substance is administered either by capsule or gavage and is followed by a 14-day observation period which can be extended if required.

Chronic toxicity assessments require the dietary exposure of test chemicals to young adult animals for approximately 20 weeks. Depending on the countries in which a pesticide is registered, testing in one or two species is required, in most cases an upland game bird and a waterfowl (usually the bobwhite quail and mallard duck). These studies use around 128 adult animals (16 pairs (male and female) per dose level, three dose levels plus controls) and 2,560 chicks (at an average of 40 chicks per pair) per study. Effects most often seen on reproductive endpoints, include the numbers of eggs laid, egg viability, embryo survival, hatching success, hatchling survival, and eggshell thickness.

 Delivery of this Challenge will enable scientists (and regulators) to: 

• Replace the use of animals for internal decision making by providing predictive approaches that can be used for early screening to rank or prioritise candidate chemicals for further development and prevent unsafe chemicals progressing into regulatory animal studies.
   
• Reduce the number of animals used for regulatory risk assessment by integrating NAMs into an IATA.

Learnings from this Challenge could also have the potential to further the development of NAMs for other taxonomically relevant species such as reptiles. New knowledge on avian toxicokinetics and/or metabolism could also be applied to predict the metabolism of chemicals, where currently in vivo studies are required for the purposes of human safety assessment for the consumption of poultry products from hens exposed to pesticides. 

References

1. EPA (2012). OCSPP 850.2200: Avian Dietary Toxicity Test, Ecological Effects Test Guidelines, OPPT, USEPA. EPA-HQ-OPPT-2009-0154-0011 
2. OECD (1984). Test No. 205: Avian Dietary Toxicity Test, OECD Guidelines for the Testing of Chemicals, Section 2. doi: 0.1787/9789264070004-en
3. Hilton M et al. (2019). Evaluation of the avian acute oral and sub-acute dietary toxicity test for pesticide registration. Regulatory Toxicology and Pharmacology 105, 30-35. doi: 10.1016/j.yrtph.2019.03.013
4. US Environmental Protection Agency Office of Pesticide Programs (2020). Final Guidance for Waiving Sub-Acute Dietary Tests for Pesticide Registration and Supporting Retrospective Analysis, EPA, USA. epa.gov/sites/default/files/2020-02/documents/final-waiver-guidance-avian-sub-acute-dietary.pdf
5. European Commission (2013) Commission Regulation No 283/2013 of 1 March 2013 setting out the data requirements for active substances, in accordance with Regulation (EC) No 1107/2009 of the European Parliament and of the Council concerning the placing of plant protection products on the marketText with EEA relevance, Official Journal of the European Union, Luxembourg.  data.europa.eu/eli/reg/2013/283/oj
6. European Food Safety Authority (2009) EFSA Annual Report, EFSA, Italy. doi: 10.2805/42846
7. Basant N et al. (2015) Predicting toxicities of diverse chemical pesticides in multiple avian species using tree-based QSAR approaches for regulatory purposes. Journal of Chemical Information and Modeling 55 (7), 1337-1348. doi: 10.1021/acs.jcim.5b00139
8. Mukherjee RK et al. (2022) Ecotoxicological QSTR and QSTTR modeling for the prediction of acute oral toxicity of pesticides against multiple avian species. Environmental Science & Technology 2022 56 (1), 335-348. doi: 10.1021/acs.est.1c05732
9. Martin T et al. (2024) Reproductive toxicity in birds predicted by physiologically-based kinetics and bioenergetics modelling. Science of The Total Environment, 912, 169096. doi: org/10.1016/j.scitotenv.2023.169096.
10. Basu N et al. (2016) ecotoxchip.ca
11. Ball AL and Lavado R (2021). Ecotoxicology assessments in avian species using cell-based models: A review. Avian Biology Research 14(4):105-112. doi:10.1177/17581559211030850
12. Sharin T (2021). Development and Use of Avian In Vitro and In Vivo Models for Toxicological Screening and Prioritization of Five Bisphenol A Replacement Compounds: Bisphenol F, TGSH, DD-70, Bisphenol AF, and BPSIP. University of Ottawa. hdl.handle.net/10393/42326
13. Sharin T et al. (2022). Toxicity screening of bisphenol A replacement compounds: cytotoxicity and mRNA expression in LMH 3D spheroids. Environ Sci Pollut Res Int. 29 (29). doi: 10.1007/s11356-022-18812-z
14. OECD (2024) oecd.org/en/topics/sub-issues/assessment-of-chemicals/integrated-approaches-to-testing-and-assessment.html
15. OECD (2016). Test No. 223: Avian Acute Oral Toxicity Test, OECD Guidelines for the Testing of Chemicals, Section 2. doi: 10.1787/9789264264519-en
16. EPA (2012) OCSPP 850.2100: Avian Acute Oral Toxicity Test, Ecological Effects Test Guidelines, OPPT, USEPA. EPA-HQ-2009-0576
17. OECD (1984) Test No. 206: Avian Reproduction Test, OECD Guidelines for the Testing of Chemicals, Section 2. doi: 10.1787/9789264070028-en
18. EPA (2012). OCSPP 850.2300: Avian Reproduction Test, Ecological Effects Test Guidelines, OPPT, USEPA. EPA-HQ-OPPT-2009-0154-0012

Assessment information

Review Panel membership

This Challenge is now closed for applications. 

NC3Rs CRACK IT Challenges are run using Contracts for Innovation. Innovate UK are the custodians of Contracts for Innovation and more information about the mechanism can be found here.

Wings of Change key documents:

Wings of Change key dates: