SETAC Globe - Environmental Quality Through Science
9 October 2014
Volume 15 Issue 10
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A Report from the SETAC Global Animal Alternatives Advisory Group

Adam Lillicrap, NIVA, Norway and Teresa Norberg-King, USEPA

During the 2014 SETAC Europe annual meeting in Basel, an Animal Alternatives Advisory Group (AAAG) meeting was held to discuss progress in the state of the science within animal alternatives for environmental toxicity testing. During the meeting, five ongoing projects were presented from members of academia, regulatory authorities and industry to highlight progress to date. A brief overview of these presentations is below.

Updated Fish Early Life Stage Test
Gerd Maack (UBA, Germany) gave an update to the OECD Fish Testing Framework Project regarding the proposed update of the Fish Early Life Stage Test (OECD 210). Aftin er more than 2z ye in use, the guideline is widely recognised of need for an update to (1) incorporate 20 years of experience; (2) to remove inconsistencies; (3) to increase statistical power and (4) to close “interpretation possibilities.” This is the first, and in some areas the only, aquatic vertebrate test for sub-lethal endpoints, therefore it is important that these parameters can be assessed accurately. Hence the minimum hatching success and the minimum post-hatch success was increased and a recommended minimum length for control fish was introduced in combination with a recommended tank size. The most important clarifications and amendments are:

  • Increasing the quality of controls
  • Increasing the statistical power (four replicates per concentration compared with two)
  • Reducing the number of recommended species
  • Introduction of a detailed chapter on statistical interpretations

To reflect the aim of reducing the number of animals for testing, the number of individuals used per replicate was reduced from 30 to 20 fish per tank. However, it was recognized that with the higher replication, more animals would actually be needed per test, but with the increased confidence in the results the need for repeating studies should be minimized. After 26 January 2015 studies conducted according to the 1992 version can be rejected by regulatory authorities, which subsequently could also increase the number of organisms used in the future.

Alternative Testing Strategy for the Fish Early Life-stage Test for Predicting Chronic Toxicity
Dries Knapen (University of Antwerp, Belgium) presented the Cefic Long-range Research Initiative project related to developing an alternative test strategy for predicting chronic ecotoxicity. To ensure an ecologically relevant basis for environmental quality standards, toxicity information needs to be obtained for a large number of regulated chemicals using high quality testing strategies. Testing for chronic fish toxicity is one of the most animal demanding areas in environmental risk assessment. Industry and regulatory bodies have expressed the need to develop alternative testing strategies that focus on non-animal alternatives and mechanistic information. The main objective of this project is to develop a strategy that reduces the need for performing FELS tests. The project focuses on combining alternative test methods with mechanistic information on non-apical endpoints, which should result in a more focused assessment of chemical toxicity. The research approach is based on the adverse outcome pathway AOP) concept as a mechanistic framework. During the project a number of AOPs are being developed such as, narcosis leading to respiratory failure, thyroperoxidase or deiodinase inhibition leading to impaired swim bladder inflation, and acetylcholinesterase inhibition leading to motor activity impairment. A combination of a modified zebrafish embryo toxicity tests (ZFET, OECD TG 236) and in vitro tests are being used to study molecular initiating or key events along the selected AOPs, with the purpose of investigating the predictivity of these events for FELS toxicity.

Adverse Outcome Pathways
Knut Erik Tollefsen (NIVA, Norway) summarized the discussions of the Workshop “Advancing AOPs for Integrated Toxicology and Regulatory Applications” (Somma Lombardo, Italy, 2-7 March, 2014). This workshop focused on the use of the Adverse Outcome Pathway (AOP) concept as a framework to characterize, organize, and define predictive relationships between measurable key events that reflect the progression from a chemical-induced perturbation to an adverse outcome considered relevant to regulatory decision-making. Issues such as:

  • Research priorities for development of AOPs
  • Strategic approaches to AOP development
  • Weight of evidence evaluation to define uncertainties associated with predictive relationships represented in an AOP
  • Acceptance of AOPs for regulatory applications, and
  • Applying AOPs to support Informed Approaches to Testing and Assessment were covered in targeted work groups.

The results from the discussions are currently submitted as a number of papers to peer-review journals and should be expected to become available in the next 6 months.

Humane Anaesthesia for Fish
Stewart Owen (Astrazeneca, UK) highlighted work led by AZ Global SHE on the humane choice of anaesthetics for laboratory fish, which suggests we might be able to improve the welfare of zebrafish by choosing to use an anaesthetic agent that is not aversive (i.e. the fish do not sense and avoid the chemical agent). The work by Readman et al. (2013), and others since, has been highlighted by a lead editorial in Nature (Fish Have... 2014) that suggested it “should prompt reassessment of how much we know about some routine practices of animal research." There are at least two independent studies that use different methodologies to reach the same conclusion that zebrafish detect and actively avoid MS-222 in the water. Therefore, as a humane choice, the use of this agent for routine zebrafish anaesthesia should be reviewed and considered for replacement. From the work of Readman et al. (2013), it is recommended researchers investigate the use Etomidate as a non-aversive alternative for anaesthesia and the first step of euthanasia of zebrafish in the laboratory. Further discussion is available in Nature (Cressey 2014).

United Kingdom National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs)
Natalie Burden (NC3Rs, UK) presented an update summarizing the work of the NC3Rs over the past two years in the area of ecotoxicology. Natalie described the role of the NC3Rs, and the mechanisms that the organization uses to support the development and application of new technologies and approaches which advance the 3Rs. She then informed the group of recent publications which initiated from discussions within the NC3Rs Ecotoxicology Working Group. The advisory group was invited to contribute to the NC3Rs blog on pertinent issues surrounding the use of animals in ecotoxicology. Finally, details were given on the future focus areas for the NC3Rs.

For more information please contact the chairpersons from the AAAG (contact information is provided at the end of the article), Adam Lillicrap and Teresa Norberg-King.

The AAAG currently is comprised of 70+ members, 40+ of whom have participated in the most recent AAAG meetings in North America and Europe.

Current AAAG Steering Committee

  • Adam Lillicrap, Norway, non-profit sector
  • Teresa Norberg-King, US, government sector
  • Natalie Burden, UK, non-profit sector
  • Scott Belanger, US, industry sector
  • Michelle Embry, US, non-profit sector
  • Marlies Halder, Europe, government sector
  • Bob Hoke, US, industry sector
  • Lucy Lee, Canada, academic sector
  • Marc Léonard, Europe, industry sector
  • Barnett Rattner, US, government sector
  • Kristin Schirmer, Europe, academic sector

References
Cressey D. 2014. Fish-kill Method Questioned. Nature. 506: 419-420. doi: 10.1038/506419a Fish Have Feelings Too. 2014. Nature. 506: 407. doi:10.1038/506407a

Readman GD, Owen SF, Murrell JC, Knowles TG. 2013. Do Fish Perceive Anaesthetics as Aversive? PLoS ONE. 8(9): e73773. doi:10.1371/journal.pone.0073773

Authors' contact information: ali@niva.no; norberg-king.teresa@epa.gov

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