Validating a sexual development test using the 3-spined stickleback for addressing the 3Rs in fish toxicity testing

Lead Research Organisation: Centre for Env Fisheries Aqua Sci CEFAS
Department Name: CEFAS Weymouth Laboratory

Abstract

A wide range of man-made chemicals has the ability to interfere with the endocrine system that controls amongst other functions, sexual development. These are called endocrine disruptors (EDs). Steroidal oestrogens are often implicated in the causation of the widely observed sexual disruption in fish and are of both natural and synthetic origin (e.g. ethinyl-oestradiol, which is the active ingredient of the human contraceptive pill). As well as posing potential risks to humans (there are several studies that attempt to link exposure to EDs with the decline of sperm counts, the increased incidence of testicular and prostate cancers, male congenital reproductive abnormalities and infertility rates), EDs are of particular concern for fish as the aquatic environment is often the most important sink for man-made chemicals and sewage waste. There is clearly a need to develop tools for the detection of such chemicals and fish have been recognised as providing the ideal biological system for this purpose, leading to the development of a number of tests by the OECD, including the fish sexual development test (FSDT)
The species that have entered a validation phase for the FSDT to date are the zebrafish and the fathead minnow, both of which are routinely used in aquatic ecotoxicological studies. However, we argue that they are not the ideal test subjects for the FSDT. The main disadvantage of these fish species is the lack of a genetic sex marker, which could unequivocally assign sex, leading to an enormous wastage of experimental fish. This is because the main endpoint employed by the FSDT is sex ratio, which in the case of the stickleback can be assigned genetically using a simple test. In the case of alternative models, sex is assigned by means of gonadal histology, a strategy that presents many drawbacks. Firstly, if the stickleback is used, an acceptable sex ratio for the control groups does not need to be defined waiving the risk of test failure. Secondly, the mode of action of a chemical can be better defined as an oestrogen or an androgen for example because any differences between genetic and histological sex can be attributed to the chemical exposure. Thirdly, but most importantly the ability to assign genetic sex increases the power of the test and can dramatically reduce the number of animals used in scientific procedures for both research and regulatory purposes.

Technical Summary

This proposal aims to decrease the numbers of fish used in research and regulatory testing and to refine the testing protocols so that they are more informative. We propose to achieve this aim via the adoption of the 3-spined stickleback as the preferred model species for the Fish Sexual Development Test (FSDT), which is designed to assess early life-stage effects and potential adverse consequences of putative endocrine disruptors (EDs).
EDs are defined as ?exogenous substances or mixtures that alter function(s) of the endocrine system and consequently cause adverse health effects in an intact organism, or its progeny, or (sub)populations?. The concept of environmental endocrine disruption has generated significant controversy, however, it is evident that the effects of endocrine disruption on sexual development and reproduction of birds, mammals, and other wildlife, including fish, are of great concern. The lability of sex-determination systems in fish makes them sensitive to EDs. This sensitivity of fish to environmental EDs can therefore be utilised to develop useful tools.
To adopt the stickleback as the preferred species for the FSDT we need to produce relevant validation data. This means independent in vivo testing and analysis of the effects of at least two chemicals by at least three laboratories under the same conditions. The main advantage of the stickleback over other proposed species (the fathead minnow, Pimephales promelas, and the zebrafish, Danio rerio) is the availability of a genetic sex marker; the lack of such a marker in the currently utilised fish models generates enormous wastage of experimental fish. The main endpoint employed by the FSDT is sex ratio, which in the case of the stickleback can be assigned genetically using a simple PCR test. In the case of alternative models, sex is assigned by means of gonadal histology, a strategy that presents many drawbacks. Firstly, if the stickleback is used, an acceptable sex ratio for the control groups does not need to be defined waiving the risk of test failure. Secondly, the mode of action of a chemical can be better defined as an oestrogen or an androgen for example because any differences between genetic and histological sex can be attributed to the chemical exposure. Thirdly, but most importantly the ability to assign genetic sex increases the power of the test and can dramatically reduce the number of animals used in scientific procedures for both research and regulatory purposes.

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