The Center for Alternatives to Animal Testing is an academic center affiliated with the Division of Toxicological Sciences in the Department of Environmental Health Sciences of the Johns Hopkins University Bloomberg School of Public Health.

 

Johns Hopkins School of Public Health

Abstract for TestSmart--A Humane and Efficient Approach to Screening Information Data Sets (SIDS) Data

Ecotoxicology Session

Phil Sayre
Environmental Protection Agency

The ecotoxicity endpoints which make up the SIDS data set needed in the HPV Challenge program are:

EPA noted two decisions under consideration which would decrease the amount of testing done. First, EPA is considering elimination of the requirement for terrestrial tests due to insufficient terrestrial exposure information under SIDS to trigger such tests: this would eliminate terrestrial organism tests which use avian species, earthworms, and plants. Second, EPA is also considering bypassing acute toxicity testing in fish, aquatic invertebrates, and aquatic plants for those chemicals with a high log Kow: such chemicals are sparingly soluble in water and thus would likely have no effect on test organisms in short-term tests. Instead, EPA advocates chronic toxicitity testing with aquatic invertebrates for such chemicals resulting in a reduction in the number of fish used for such insoluble chemicals.

The focus of the talks presented in the session was on alternatives to acute toxicity testing in fish, with the goals of further decreasing the number of fish to be used and replacement of the fish assay if appropriate alternatives exist. Alternative species considered during the session were the bacterium Vibrio fischeri, the protozoon Tetrahymena, the aquatic invertebrate Daphnia, and the African clawed frog Xenopus. The concept for each species is to draw a correlation between these species and existing acute data on fish such as the EPA Duluth data on the fathead minnow. Then, values from the alternative species could be converted into fathead minnow acute toxicity estimates.

The MICROTOX assay (as discussed by Dr. Mark Cronin) which uses the bioluminsecent bacterium Vibrio fischeri showed a good correlation for chemicals which act by a non-polar narcosis mechanism such as some neutral organics, and for esters. Although MICROTOX has some level of regulatory acceptance for the qualitative screening of wastewater effluents, it is not currently a good candidate as a complete replacement for fish acute tests. However, further testing with chemicals used to generate the fathead minnow database could expand its utility.

The TETRATOX system was reviewed by Dr. Terry Schultz and uses growth impairment of the ciliate Tetrahymena pyriformis to assess aquatic toxicity. There is a high correlation between TETRATOX responses and that of fish for neutral organics, but physiological differences between the protozoon and fish (along with differences in test protocols) make it difficult to currently draw good correlations for other classes of chemicals. Future work on these two areas and doing further protozoon testing to generate values that parallel those generated in the fathead minnow database could further the acceptance/use of TETRATOX. There is ongoing work sponsored by the German and Danish EPAs aimed at ring testing with standard compounds and determining interlab variability.

Dr. Kurt Enslein showed that TOPKAT can be used to predict toxicity of acyclic compounds in the fathead minnow from the limited reliable daphnid data in the literature. However, for other classes of compounds there is insufficient daphnid data to draw correlations to the fathead minnow database. The recommendation was to do more daphnid tests to obtain values to match that of the EPA Duluth fathead minnow database.

Dr. John Bantle examined the feasibility of drawing a correlation between the FETAX assay results (for frog embryo toxicity) and fish data, but concluded that most of the current chemicals tested with FETAX are pharmaceuticals and hence show a poor correlation to those industrial chemicals (tested to generate the fathead minnow database). It is possible that FETAX could be used in the near future in the SIDS program for teratogenicity endpoints, is currently in review for regulatory acceptance with ICCVAM, and hence would be valuable if its findings could be extended to the fish acute toxicity endpoint.

Finally, the EPA ECOSAR set of QSARs for ecotoxicity were noted (http://www.epa.gov/oppt/newchems/) as presenting an alternative approach to those advocated above: instead of drawing correlations between a toxicity value in another species and fish, the ECOSAR approach goes directly from a chemical structure trait (such as log Kow) to a fish acute toxicity value. ECOSAR has achieved regulatory acceptance for new chemicals under the Toxic Substances Control Act and has been validated to some degree in a joint US EPA / European Union study. However, critics of ECOSAR noted that it is not firmly tied to a mechanism of action for many of its correlations. The recommendation to move ECOSAR forward would be to test more chemicals for their acute toxicity value in order to build more correlations such that no testing would be needed for chemicals in the given ECOSAR class.

An approach to move alternative testing forward could consist of several steps. First, a review of the utility of the different approaches advocated above could be done in conjunction with such organizations as ASTM and SETAC. Second, a screening of the current high production volume chemicals could be done to see which chemical classes have the most missing data which could be most easily accomodated by one or two of the approaches above. Those systems selected could be augmented with further testing as recommended above (perhaps through industry collaborations, NIEHS, EPA efforts, through partnerships established by Johns Hopkins University, etc.) Finally, the outcomes of these analyses could be presented to an organization such as ICCVAM for validation.