Animal Alternatives: SETAC Europe 2021 Session Summary

Adam Lillicrap, NIVA; Teresa Norberg-King, USEPA; and Sarah Hughes, Shell

This was the third virtual animal alternatives session hosted by the Animal Alternatives Interest Group (AAIG) aiming to attract presentations on new and novel alternative approaches to vertebrate ecotoxicity tests. The live discussion for the session served as a forum to discuss the latest challenges in adopting animal alternatives into environmental hazard and risk assessments of chemicals and foster new collaboration and research in addressing the uncertainties and challenges in this space. The AAIG are particularly interested in research supporting the integration of animal alternatives into regulatory frameworks. Numerous technical and regulatory challenges exist for integrating traditional 3Rs of animal alternatives – Reduction, Refinement and Replacement of animal tests – plus the additional 3Rs: Reproducibility, Relevance and Regulatory acceptance that have emerged for the adoption of alternative approach methods. For SETAC Europe 2021, we had 14 posters and talks with topics ranging from the latest findings of fish and frog cell lines and embryos as replacement of whole organism tests to development of QSARs for biomagnification studies, non-lethal biomonitoring, and statistical refinement of avian tests. The live session drew 164 participants in total. In the Animal Alternatives live discussion session, we wanted to first get a feel for where session participants were coming from; both in what they think of with regards to animal alternatives and also whether their perspectives differed depending on what employment sector they are from.

Figure 1. This word cloud depicts the session participants' top terms in describing animal alternatives.

To support our efforts, we led session participants through a series of live Mentimeter polls and questions. To get our session participants “warmed up,” we had everyone contribute the first three words that came to their mind for animal alternatives to generate a word cloud. Not surprisingly “in vitro,” “QSAR,” “in silico,” “cell lines” and “modeling” came out on top (Figure 1). We also performed a poll to better understand who was participating in the session by polling participants on what sector they represented. We had representation of the traditional tripartite sectors that SETAC represents (academia, government and business), but we created a bit more granularity by also adding categories of students, research institutes, NGOs and Consultants and Contract Research Organizations (CROs) (Figure 2).

From there, we asked participants to identify which “R” for animal alternatives was of most interest or importance to them (i.e., Reduce, Refine, Replace, Reproducible or Reliable, Relevance, and Regulatory acceptance). Overall, the Rs with the most votes and interest were “regulatory acceptance,” “reproducible or reliable,” and “replace” (Figure 3). Notably, it was very interesting to see how each sector responded to this question. Industry respondents (and perhaps not surprising) were most interested in “regulatory acceptance” of alternative methods with over 41% of the votes in this category. In contrast, those in academia were most interested in “replacement” (55%) as well as “reproducibility or reliability” (33% of respondents within category but equal number of votes as “replacement”). This is also not surprising where scientific flexibility is common and experimenting with new methods is fostered. What was surprising was to see government respondents rank “relevance” higher at 33% than “regulatory acceptance” (9%) and “reproducible or reliable” (9%). These responses clearly highlight the different needs and experiences the sectors have. It was interesting to see our future research body, students, having interest in all 6 of these Rs with almost equal importance. Looking at all these interests and keeping these at the forefront of our mind when trying to support alternative approach methods may help find common ground and foster overall progress towards adoption of animal alternatives.

Figure 2. Session participants by sector.

After setting these contexts, we went through a discussion of three main questions to distill down the key perspectives of session participants. The first was to frame things in a positive light and highlight what are the latest successes in the space of animal alternatives. For this, we encouraged session participants (including presenters in the AAIG session) to report their latest findings. We then shifted to discussing identifying remaining research gaps and had a final discussion identifying and polling what were the greatest barriers to adoption of animal alternatives.

Animal Alternatives Successes

There were over a hundred research “successes” shared in the animal alternatives space by session participants. Some of the successes were more anecdotal, such as identifying that there is a critical mass of researchers now working on alternative test methods, and funding for research on alternative methods is increasing. Others were more specific, like the development of the EcoTTC tool or the development of the bioconcentration tests in the freshwater amphipod Hyalella Azteca (HYBIT).

Figure 3. Bar graph describes the session participants' answer to the question “What ‘R’ is of greatest interest or importance to you?”

Overall, the successes that had the most submissions in the session included:

1. The adoption of the Xenopus Eleutheroembryo Thyroid Assay (XETA) by OECD (OECD 248) in June of 2020;
2. The fish embryo test (FET) test may be used within weight-of-evidence approaches together with other independent relevant and reliable sources of information under the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) regulation (no. 1907/2006) of the European Union (Sobanska, 2018); and
3. The recent acceptance of the Rainbow trout gill cell line cytotoxicity assay as an OECD test guideline (OECD 249)

Remaining Research Gaps

There were over 70 research “gaps” suggested in the session and, like the identification of successes, some were broader, more generic statements of gaps like “uncertainty,” while others were more specific statements, such as the need to understand how alternative test methods can predict behavior changes or incorporate metabolism into alternative methods. Notably, some of the topics that were considered successes were also identified as remaining research gaps such as in vitro to in vivo extrapolation (IVIVE) or using animal alternatives for testing difficult-to-test substances and mixtures. This is perhaps a reflection of the nature of science: more research drives more questions and identifies more things to explore and test. The research gaps that generated the most submissions were around the testing of complex mixtures, difficult-to-test substances, and substances that are unknown or variable composition, complex reaction products or biological materials (UVCBs). The other major gap was around developing chronic toxicity alternative tests (e.g., alternative approaches to the OECD 210 chronic fish test) and gaps related to the ability to ensure an animal alternative method is adopted by regulators.

Barriers to the Adoption of Animal Alternatives

There were over 38 contributions of barriers to animal alternatives with 230 total votes. The top barriers identified were grouped together as follows:

1. Applicable to real world scenarios and ecological relevance (67 votes)
2. Regulatory acceptance and clear guidance on interpretation for a weight -of-evidence (55 votes)
3. In vitro to in vivo extrapolation or accurate extrapolation (42 votes)
4. The need to compare animal alternative methods to traditional animal-based methods (26 votes)
5. Confidence and understanding in what tests can provide (21 votes)
6. How do we agree when we have enough data to accept a new method? (18 votes)

During this section on barriers to the adoption of animal alternatives, it was evident that challenges to problems emerge when there is a disconnect between priorities within different stakeholders. This perception is based on the implication from some industry representatives who perceive regulators as not understanding how alternative approaches can be used. Confounding this is that some regulators do not always trust industry about the approaches that are being proposed. Until this barrier is broken it remains difficult to imagine that the adoption of alternative approaches to the use of vertebrates for ecotoxicity testing will gain support and approval.

Another point that was discussed within the AAIG session, and perhaps should be considered wider by the scientific community, was the use of terminology, particularly in this case towards animal alternatives. An example being the use of the term NAMS (new approach methodologies) to represent alternative approaches to vertebrate ecotoxicity test methods. If the term NAMS was “Non-Animal Methods,” then it could be applicable. However, the current acronym covers a wide spectrum of different scientific disciplines, and alternative approaches to vertebrate ecotoxicity tests are often not “new.” A better acronym for animal alternatives should be more specific and encompass all the different “R’s” to ecotoxicity testing (e.g., 6R alternative methods, or 6RAMs) however, the jury is still out on this suggestion.

Moving forward in 2021 and into 2022, we call all researchers to consider these successes, gaps and barriers in their work towards animal alternatives for environmental risk assessment. We hope these discussions will help us further focus our study on the uncertainties and challenges as well as potential limitations, as well as encourage the adoption of animal alternatives into regulatory frameworks. Stay tuned to this topic for the SETAC North America Annual Meeting where we have platform and poster presentations that will provide updates and insights to animal alternatives plus a discussion session where we can continue the discussion from May. Please help us continue to move the science forward with various approaches. And, please consider joining the Animal Alternatives Interest Group; there’s no cost. We can share progress and papers, and more.

Authors’ contact information: [email protected], [email protected] and [email protected]