Alternatives to the use of vertebrate animals in ecotoxicity testing – Progress so far

There is a growing need for alternatives to the use of vertebrate animals in the hazard assessment of chemicals. Many regulatory frameworks, including REACH (e.g., the European Union’s regulation on the Registration, Evaluation, Authorisation and Restriction of Chemicals), stipulate that vertebrate tests should be conducted only as a last resort. Nevertheless, there is still a lot to be done to achieve regulatory acceptance of alternative methods to vertebrate testing. In recent years, there has been significant progress on alternative methods covering human health endpoints but ecotoxicology lags behind. 

An article by Lillicrap et al. (2016) presents an overview of the different approaches that are now available for ecotoxicologists and risk assessors to evaluate potentially hazardous chemicals while minimizing the use of vertebrates in ecotoxicity testing. The challenge for regulatory acceptance of any new strategies and methods to reduce, replace and refine animal tests is that these should also include consideration of an additional “3Rs”— namely, reproducibility, reliability and ecological relevance. According to Lillicrap and colleagues, alternative methods already in use for ecotoxicity testing include:

1. Quantitative Structure–Activity Relationship (QSAR) models 

• Physical parameters such as the log octanol–water partition coefficient (Kow) or knowledge of the 2-dimensional or 3-dimensional structure can be used to predict (eco)toxicological endpoints, i.e., bioaccumulation, short- and long-term toxicity in fish, persistent, bio-accumulative and toxic (PBT) properties.

2. Thresholds of Toxicological Concern (TTC)

• An extension of the human safety TTC concept for application in environmental situations, termed the “ecological TTC” or “eco-TTC”” (Belanger et al., 2015), useful in providing hazard perspective on chemicals that lack QSAR models, guiding product development discussions and assisting read-across or category justifications.

3. Progress and development of in vitro assays for predicting the acute toxicity of chemicals in fish

• Cytotoxicity assays with fish cell lines, i.e., fish gill cell line help to distinguish effluents that are nontoxic to fish from those that are acutely toxic.
• Evaluated as a new potential standard method in the International Organization for Standardization (ISO) due to its robustness and interlaboratory reproducibility.

4. The use of fish embryos as a surrogate life stage

• Fish embryos are considered in many jurisdictions to be a non-protected life stage (i.e., not subject to the same animal welfare considerations as juvenile and adult fish). 
• OECD Guideline 236 (Fish Embryo Acute Toxicity (FET) Test): A promising alternative test to predict acute lethality with similar sensitivity to the acute fish lethality test.

5. Alternative approaches to chronic ecotoxicity testing

• The early life stages of fish are recognized as a robust model for toxicological studies and predictive of chemical effects in full–life cycle tests, i.e., use of embryos and newly hatched fish for sub-chronic and chronic testing due to sensitive life stages representation (OECD Guideline 210 – Fish early-life stage toxicity test).
• Environment Canada test protocol using salmonids with 3 test options: embryo tests for frequent or routine monitoring, an embryo/alevin test for measuring effects on multiple phases of development, and an embryo/alevin/fry test for definitive investigations.
• Weight of evidence using a fish cell line, combined with mechanism-based computational models, could replace tests using juvenile fish. 

6. Animal alternatives to fish bioaccumulation testing

• QSAR models for predicting bioaccumulation potential based on the Log Kow approach. 
• Biotransformation rate estimates for different trophic levels of fish using Arnot and Gobas bioaccumulation model. 
• Use of molecular size (and some molecular properties) as an indicator for reduced bioaccumulation potential and tiered assessment strategies.

7. Alternative methods and study design refinements for wildlife (amphibians, reptiles, birds, and mammals) toxicity tests

• The use of in vitro test systems to predict toxicity and risk of contaminants to wildlife lags well behind methods currently employed for aquatic and mammalian species. 
• More efforts in developing scaling and extrapolation factors derived from traditional avian and mammalian test species for mortality related endpoints.
• OECD Guideline 223 (avian acute oral test) refinement – derivation of limit dose, lethal dose and dose response curve for chemical with reduced number of animal use from earlier 40 to now less than 15.

8. Amphibian ecotoxicology and recent advances

• The frog embryo teratogenesis assay (Xenopus laevis) – an alternative to the use of mammalian species for the assessment of developmental toxicity. 
• OECD Guideline 231 (Amphibian Metamorphosis Assay) and OECD Guideline 241 (The Larval Amphibian Growth and Development Assay (LAGDA)) – for the identification of endocrine disruptors.

9. Transgenic Aquatic Models for Ecotoxicological Assessment of Endocrine Disruptors

• Development of transgenic medaka strain (ChgH-GFP) harboring the green fluorescence protein (GFP) gene, which enables the activity of the estrogen axis to be visualized in vivo and development of Spiggin-GFP medaka fish line and embryo to identify disruption of the androgen axis.
• OECD Guideline 248 (the Xenopus Eleutheroembryo Thyroid Assay (XETA)) – an amphibian screen for the hazard classification of chemicals into potentially thyroid active or inactive.
• OECD guideline development based on The EASZY assay (detection of endocrine active substances, acting though the estrogen receptor, using transgenic cyp19a1b-GFP zebrafish embryos) – Adoption of the guideline is expected in April 2020.

10. AOPs as a unifying framework

• Adverse outcome pathways (AOP) provide a conceptual framework for summarizing knowledge about linkages between key events at various levels of biological organization while providing an adverse outcome relevant to ecological risk assessment. 
• Lack of funding for research on developing AOPs for environmental science compared to the human safety systems biology (e.g., USEPA ToxCast program). 
• AOPs along with biologically based extrapolation tools (e.g., toxicokinetic and toxicodynamic models, concentration–duration–response models, species extrapolation models) may help gradually replace whole-animal toxicity testing as the primary source of data generation to support ecological risk assessment and environmental decision-making.

Considerable advances have been made towards alternative approaches for ecotoxicity assessments over the last few decades, but more research and funding are needed to come close to the advances made for human health endpoints. More focus has been recently given to investigate potential ecotoxicology hazard and environmental endocrine disruption effects of chemicals and it will be interesting to follow further updates in this field.