OECD test strategies and methods for endocrine disruptors
H.P. Gelbkea, , , M. Kaysera, and A. Pooleb,
aDepartment of Product Safety, GUP – Z 470, 67056 Ludwigshafen, Germany
bDOW Europe S.A., Bachtobelstr. 3, 8810 Horgen, Switzerland
Abstract
The question whether (man-made or natural) chemical substances may have an adverse effect on the endocrine system has gained high visibility in the public as well as in the scientific community. This relates to possible effects on the environment as well as on human health for chemicals with (anti)estrogenic, (anti)androgenic or (anti)thyroid activity. Taking into account the broad universe of chemicals to which humans or the environment may be exposed, a sound testing strategy and robust test methods are urgently needed. Both subjects have been addressed by a specific OECD working group (EDTA – Endocrine Disruptor Testing and Assessment Task Force) involving regulatory agencies, the scientific community, chemical industry and NGOs. Like other organizations the OECD has adopted a tiered-testing strategy with the first tier using screening assays as quick and inexpensive tools, providing a way of generating alerts to potential endocrine activity that can be used to prioritize substances for definitive tests that then can determine the toxicological consequences of endocrine toxicity. The efforts of the OECD have therefore concentrated on the validation of specific screening and testing guidelines, like the uterotrophic, the Hershberger, and the “enhanced TG 407” test. The experimental testing necessary for this validation procedure is completed for the uterotrophic and the “enhanced TG 407” tests and near completion for the Hershberger assay. The data obtained so far have been published (for the uterotrophic assay) or will be submitted to the EDTA working group for final evaluation. Overall, the validation program has been very successful and should be sufficient for setting up OECD test guidelines for these experimental procedures. This will add substantially to the “tool-box” of OECD test methods that is available internationally to regulatory agencies and chemical industry for the identification and assessment of possible endocrine disruptors. Despite this success it is well recognized that the methodological “tool-box” should be supplemented by further screening and testing procedures related to effects on human health and the environment.
Keywords: Endocrine disruptors; Validation of OECD test methods; Uterotrophic assay; Hershberger assay; “Enhanced" subacute test (OECD TG 407)
1. Introduction
Chemical substances that have the potential to interfere with the endocrine system are nowadays generally referred to as “endocrine disruptors”. While this issue has gained high visibility in the public as well as in the scientific community, the overall impact on human health and the environment is still unclear. The basic question is:
? Do (the generally small concentrations of) man-made chemicals in the environment interfere with human health and/or wildlife?
To come to a firm conclusion, chemicals with endocrine activity have to be identified with regard to (anti)estrogenic, (anti)androgenic and (anti)thyroid activity. This represents an enormous task as under the upcoming EU chemicals regulation (REACH) risk assessments for human health and the environment have to be carried out for approximately 30,000 man-made chemicals. But, in addition, natural substances with endocrine activity (e.g., phytoestrogens or natural hormones) have to be taken into account for a holistic evaluation.
The identification of endocrine disruptors by toxicological testing will only determine the hazard of the compounds. As importantly, sound exposure assessments for humans and wildlife are necessary to finally arrive at reliable risk assessments.
All this illustrates that the evaluation of possible endocrine disruptors will have a high impact on the chemical industry worldwide. Additionally, in the EU REACH is a specific aggravating factor, since endocrine disruptors will be subjected to authorization. Therefore, the European chemical industry has formed a task force as part of the “Long-range Research Initiative” (LRI) of CEFIC (The European chemical industry association), the “Endocrine Modulators Steering Group (EMSG)”, which inter alia closely cooperated with the OECD “Endocrine Disruptor Testing and Assessment Task Force (EDTA)” for test method development.
During recent years, various international bodies, national authorities, and scientific associations have reviewed the available data and provided concise evaluations, e.g., the US-EPA (1997), IUPAC (1998), CSTEE (1999), IPCS (2000), SCOPE/IUPAC (2003). Although differing in details, the major conclusions are very similar:
? an interaction with the endocrine system is a mechanism for possible effects but not an adverse effect per se,
? an interaction between endocrine disruptors at environmental concentrations and human health is not proven,
? human exposure to natural phytoestrogens is higher as compared to man-made endocrine disrupting chemical substances,
? there is an indication for an interaction between endocrine disruptors at environmental concentrations and effects on reproduction of animals in the environment,
? there is proof for an interaction between high local concentrations of endocrine disruptors and effects on reproduction of animals in the environment,
? and above all, further scientific work is necessary to address the issue.
To arrive at a scientifically sound evaluation of this issue, the following steps have to be done systematically:
? Identification of a general screening and testing strategy.
? Availability of specific validated methods for screening assays and definitive tests detecting biologically relevant toxicological effects caused by endocrine toxicity.
? Exposure assessments.
? Establishment of a risk assessment methodology.
Possible future regulations require an adequate definition of endocrine disruptors. This was already recognized at the first international workshop involving regulators and scientists from various organisations like OECD, WHO, European Commission, and the European Environment Agency. The basic question was whether any substance that may interfere with the endocrine system has to be regarded as an endocrine disruptor. At this Weybridge Work shop the term “endocrine disruptor” was clearly restricted to chemicals causing adverse health effects: “an endocrine disrupter is an exogenous substance that causes adverse health-effects in an intact organism, or its progeny, consequent to changes in endocrine function” (Weybridge Workshop, 1996).
The following paper deals with OECD activities to develop an experimental “tool-box” and specific test methods, which can be conducted in a cost-effective way for hazard identification of endocrine disruptors.
2. Test strategy
Taking into account the universe of natural and man-made chemical substances, which may/should be evaluated for their endocrine disrupting potential, the OECD EDTA task force developed a “conceptual framework”. Basically a “tool-box” of screening assays and definitive test procedures is divided into levels (tiers) with increasing methodological complexity and strength of evidence for effects. Tests for human health and environmental effects are assigned to these levels. These various levels of screening assays and definitive tests should not be sequentially run through in a “box-ticking” manner. Instead, screening assays and tests from this “tool-box” should be used as appropriate on a case-by-case basis.
The scheme of the OECD “conceptual framework” is given in Fig. 1 with examples of test methods for each level. In principle,
? level 1 is used to prioritise the large universe of man-made and natural chemicals by using existing information,
? level 2 comprises of in vitro screening assays, also including consideration of (quantitative) structure activity relationships, which may also yield mechanistic information,
? level 3 contains in vivo screening assays for detection of specific endocrine activities,
? in level 4 the in vivo assays listed will allow for detection of adverse effects in several endocrine systems and finally,
? level 5 contains the most comprehensive in vivo assays for adverse effects, which can be used for a definitive risk assessment.
Fig. 1. OECD conceptual framework.
This OECD framework is a tiered approach in which the in vitro assays and short-term in vivo screening tests (levels 1, 2, and 3) are used to prioritise substances for definitive longer term studies (levels 4 and 5) to detect adverse effects to be used as a basis for risk assessment. This should be viewed as a hierarchal approach in which the results of definitive tests “outweigh” or supercede the results of screening assays. While tiered approaches provide a workable framework for addressing endocrine toxicity, it is however important to guard against having a proliferation of endpoints and test methods, which are not adequately understood or validated and often cannot be conducted in a cost-effective manner.
It is generally accepted that in vitro assays not only provide the opportunity for generating alerts for chemicals, which interact with hormone receptors but are also valuable tools for mechanistic research and in some instances for establishing experimental designs for higher level investigations. It is also necessary to recognize that while in vitro tests have strengths they also have limitations including:
? in vitro assays are generally tailored to one specific mechanism which might neither mimic in vivo modes of action nor reflect the complex interactions taking place in the intact organism,
? in vitro assays generally lack appropriate metabolic activation,
? absorption, distribution, metabolism and excretion of a substance often play a critical role in determining biological activity, and then results of in vitro assays cannot be extrapolated to the whole organism.
For these reasons in vitro screening assays cannot be used as a surrogate for definitive long-term in vivo hazard-tests, which conclusively demonstrate adverse effects via an endocrine-mediated mechanism. As such longer term in vivo test procedures are most relevant for hazard identification and risk assessment.
When toxicological test methods are being developed for the assessment and regulation of chemicals, specific requirements must be met before they can be accepted as an official OECD test guideline.
First of all, taking into account the global distribution and use of chemicals as well as their regulation by different national authorities, the OECD test guidelines and testing strategy as in the above-mentioned “conceptional framework” must be accepted worldwide.
Secondly the purpose, including possible limitations, of the test methods must be clearly defined: Are they primarily meant to be used for prioritisation, effect screening, hazard identification, risk assessment or as a mechanistic research tool?
Today’s test method development should strictly follow a stepwise approach to finally arrive at a validated test procedure acceptable for hazard/risk assessment purposes. The first step, the prevalidation or standardisation, serves to define the protocol by using a few chemicals with clearly defined modes of action. In the second phase, a broad set of reference chemicals (including negative control substances) is tested in several laboratories to define sensitivity, specificity and intra- as well as interlaboratory reproducibility.
Finally, the day-to-day practicability of a test procedure must be taken into consideration. The test method should
? be robust and practicable for routine use also by less experienced researchers/laboratories,
? be slim and not contain unnecessary parameters,
? be cost-efficient, especially if used for screening purposes,
? use numbers of animals as low as possible for ethical reasons,and
? ideally cover a broad array of endpoints, not related to only one specific endocrine mechanism.
3. Existing OECD test guidelines
Notwithstanding the development of new specific test guidelines for endocrine effects, it should be recognized that the existing repertoire of OECD test guidelines comprises many methods, which will give more or less firm indications for endocrine effects. The most relevant examples for human health testing are:
? multiple dose tests involving the examination of endocrine-related tissues ranging from subacute to chronic exposure (e.g., OECD test guideline 407, 409, 451 and 452),
? prenatal toxicity tests in the rat or rabbit (OECD test guideline 414) that are able to show disturbances in the development of reproductive organs,
? the OECD–SIDS screening tests (e.g., OECD test guideline 421 and 422) that not only include investigation of endocrine-related tissues but were also specifically designed to detect reproductive effects after relatively short exposure times,
? and finally the one- and two-generation tests (OECD test guideline 415 and 416) which—especially the two-generation test—may be considered as the most comprehensive methods to investigate endocrine related tissues and the reproductive function.
4. Development of new test methods
Although the above-mentioned existing test guidelines may readily give an indication of endocrine effects, already in 1998, the need was well recognized to develop specific test methods for endocrine effects. Therefore, an international task force was established under the umbrella of the OECD, the “Endocrine Disruptor Testing and Assessment Task Force (EDTA)”. The test method development was intended to be a multi-stakeholder process, therefore, the EDTA includes scientists from authorities, universities, industry and environmental and animal welfare groups. The European chemical industry participated from the very beginning with representatives from the “Endocrine Modulators Steering Group (EMSG)” of CEFIC and offered appreciable experimental resources. In the area of human health, the OECD has focussed on the development of the following in vivo screening assays:
? Uterotrophic assay for (anti)estrogenicity.
? Hershberger assay for (anti)androgenicity and,
? the so-called “enhanced subacute test (TG 407)” for (anti)estrogenicity, (anti)androgenicity, and (anti)thyroid effects in context with general toxicity.
For environmental toxicity the OECD has similarly focussed on developing screening assays in fish and assessing protocols to detect the chronic effects of endocrine modulators on reproduction of fish. For convenience, the remainder of this paper will focus on OECD achievements in the area of mammalian endocrine screening assays and tests.
The methods being developed by OECD and finally accepted by regulatory authorities worldwide as well as by industry and the scientific community should offer a solid basis for identifying and assessing possible endocrine-disrupting chemicals. Therefore, such activities have to follow very stringent rules as were laid out by the OECD at the beginning of this program:
? first of all a lead laboratory with in-depth knowledge of the method under development was selected. The lead laboratory acted as the focal point, whenever specific issues had to be addressed within the overall process,
? the test method development followed a stepwise process starting with the prevalidation phase to define the protocol for the subsequent final validation step. The validation process itself could further be subdivided into a non-blinded and a blinded testing phase,
? especially in the validation phase—after the protocol had been specified—all participating laboratories had to strictly adhere to the test protocols and procedures laid down during prevalidation,
? for each test method a set of chemicals with well-defined mechanisms of action (including appropriate negative control substances) was carefully selected by a group of experts under guidance of the lead laboratory,
? all participating laboratories used the same lots of test chemicals to avoid any possible confounding by different impurities. To provide the necessary infra-structure and logistics, a chemical repository was set up and funded by the European and Japanese chemical industry (Owens and Koeter, 2003),
? and finally the participating laboratories had to be able to cope with this program, especially to handle large numbers of animals and data in parallel and apply a variety of different techniques, e.g., histopathology, precise necropsy and tissue dissection, clinical chemistry and clinical examination.
5. Uterotrophic assay
The uterotrophic assay is a short-term in vivo screening assay for estrogenicity or antiestrogenicity. Either immature intact or adult ovariectomised female rats or mice are used. Administration of a test chemical with estrogenic activity will lead primarily to an increase in the uterine weight besides effects on other estrogen-dependent parameters. Antiestrogenic compounds will diminish this estrogen-dependent weight increase in intact immature or ovarectomised adult females. Validation was primarily done with intact immature rats but, to a limited extent, also with mice as well as with adult ovarectomised animals. The following test compounds were used:
? Ethinylestradiol (strong estrogen)
? ZM 189, 154 (antiestrogen)
? 4-Nonylphenol (weak estrogen)
? Bisphenol A (weak estrogen)
? Methoxychlor (weak estrogen)
? Genistein (weak (phyto) estrogen)
? o,p’-DDT (weak estrogen)
? Dibutylphthalat (negative control)
After defining the protocol in the prevalidation phase (phase 1) (Kanno et al., 2001), the validation is now successfully completed. The large amount of data obtained has been analysed and published. The overall conclusion is that the uterotrophic assay is well suited for an in vivo screen for (anti)estrogenicity to be used in both versions, either with the intact immature or the adult ovariectomised female rat (Kanno et al., 2003a and Kanno et al., 2003b). As a final step, an OECD guideline has now to be drafted and finally accepted by OECD.
6. Hershberger assay
In the Hershberger assay, castrated immature male rats are used as a short-term in vivo screening assay for (anti)androgenicity. Androgenic compounds will increase the weight of androgen-dependent tissues, e.g., the male accessory organs like the prostate. Likewise, (anti)androgenic chemicals will diminish this weight increase in testosterone-treated immature castrated male rats. For validation the following test chemicals were used:
? Testosterone (strong androgen).
? Methyltestosterone (strong androgen).
? Trenbolone (weak androgen).
? Flutamide (strong antiandrogen).
? Vinclozoline (weak antiandrogen).
? Procimidone (weak antiandrogen).
? Linuron (weak antiandrogen).
? p,p′-DDE (weak antiandrogen).
? Finasteride (5 α-reductase inhibitor).
Prevalidation and the non-blinded validation phase are successfully completed. Completion of the validation still requires blinded testing, which has been initiated.
In analogy to the uterotrophic assay, a test procedure has been proposed using intact juvenile male rats. Such a test procedure would avoid castration for animal welfare reasons. An abbreviated validation program for this modification of the Hershberger assay is now initiated within the OECD program.
The so far available data clearly indicate that the Hershberger assay–at least in its original form–will be a valuable tool for screening of (anti)androgenic substances.
7. The “enhanced subacute test (TG 407)”
The 28 days (subacute) test in rats—as defined by the OECD test guideline 407—is a valuable method to identify the toxicological profile of chemicals after an administration period of 28 days. It is required by many regulatory agencies, especially in Europe and Japan for new chemicals. In addition, it is one of the cornerstones of the OECD–SIDS program for the evaluation of existing high production volume (HPV) chemicals and, in future, it may be required within the EU for existing chemicals under the new chemical regulation (REACH). Taking into account the wide regulatory application of this test method, the OECD EDTA working group gave from the very beginning of its considerations a high priority to enhance this test procedure in such a way that it would detect adverse effects via an endocrine mechanism, in addition to the general toxicological profile. Thereby, test requirements already in place especially in Europe and Japan would automatically fill data gaps for endocrine-active chemicals to a large extent.
Enhancements to the TG 407 considered in the prevalidation and in the validation phase comprised of:
? Organ weights/histopathology of additional endocrine-dependent tissues.
? Hormone measurements in clinical chemistry.
? Determination of the female cyclicity and synchronisation of the date of sacrifice for female animals.
? Sperm parameters.
? Possible increase of the number of rats from 5 to 10 per sex and dose.
For validation of this enhanced subacute test, the following chemicals were selected:
? Ethinylestradiol (strong estrogen).
? Genistein (weak (phyto)estrogen).
? 4-Nonylphenol (weak estrogen).
? Tamoxifen (partial estrogen agonist/antagonist).
? CGS 18320B (aromatase inhibitor).
? Methyltestosterone (strong androgen).
? Flutamide (strong antiandrogen).
? p,p′-DDE (weak antiandrogen).
? Thyroxine (thyroid activity).
? Propylthiouracile (antithyroid activity).
The prevalidation and the experimental validation phase are now completed. In the validation phase, each chemical was independently tested in two laboratories leading to a total of 20 tests. The large amount of data produced from these studies by all laboratories have now been tabulated in a harmonised form, condensed and interpreted. This resulted in a draft OECD report of several hundred pages. After acceptance by the EDTA working group, a test guideline can be developed.
While the uterotrophic and Hershberger assays are basic screening assays that should not be used directly for risk assessment, the “enhanced TG 407” has the potential to detect specific endocrine-related adverse effects as well as general toxicity. Thus, in certain circumstances it will be possible to determine if a substance represents a true endocrine disruptor with a primary mode of toxicological action via an effect on endocrine systems or if the endocrine effects are secondary to other toxicological endpoints. This type of insight into toxicological modes of action can never be derived from in vitro assays or short-term in vivo endocrine screening assays.
To further substantiate the validity of the “enhanced TG 407”, a comparison was carried out with results obtained by tests with longer and/or in utero exposure. For this purpose, the literature was searched for subchronic/chronic, prenatal, one- or multi-generation tests with the reference chemicals used in the validation phase of the “enhanced TG 407”.
Comparisons with test results for foetal development are especially important, as it is generally accepted that the developing foeus is particularly susceptible to endocrine toxicants. Thus, a major limitation of the “enhanced TG 407” may be the lack of in utero exposure.
Investigations that were used for this comparison are listed in Fig. 2. As can be seen, unfortunately, the database is very heterogeneous, tests strictly carried out according to the generally accepted OECD guidelines were rarely available, and for thyroxine, no appropriate investigations could be found. In spite of these limitations, the following preliminary conclusions may be drawn (of course, the final evaluation must wait for the discussion and assessment of the data set by the EDTA working group.):
? The “enhanced TG 407” is able to reliably detect endocrine disruptors with a strong/moderate potency.
? The possibility to detect weak actors may be limited depending on the maximum dose that can be administered due to systemic toxicity.
? Prolongation of exposure from 28 to 90 days does not add substantial sensitivity for the detection of endocrine-related effects.
? When endocrine disruptors are administered to the developing offspring, effects related to sexual development are generally observed at lower dose levels as compared to the treatment of young adult animals.
Fig. 2. Tests for comparison with the “enhanced TG 407” Biegel et al., 1998a and Biegel et al., 1998b, Chapin et al. (1999), Cook et al. (1998), Cunny et al. (1997), Declos et al., 2001 and Declos et al., 2003, De Jager et al., 1999a and De Jager et al., 1999b, Faqi et al. (2002), Gao and Short (1993), Gill-Sharma et al. (2001), Gopalkrishnan et al. (1998), Kornbrust et al. (1986), Lewis et al. (2003), Loeffler and Peterson (1999), McIntyre et al. (2001), Mylchreest et al. (1999), Nagao et al. (2001), Okahara et al. (2000), Shigeru et al. (1993), Walker and Nogues (1994), Wolfe, 1997 and Wolfe, 2002, You et al. (1998).
8. Conclusions and outlook
The OECD has successfully engaged in the development of screening and testing procedures for the identification of endocrine-active chemicals. The data obtained during the validation process of the uterotrophic, the Hershberger and the “enhanced TG 407” tests are promising and allow for establishing worldwide accepted OECD test guidelines. Nevertheless, more validated, internationally recognized test procedures are needed to cope with the problem of endocrine-active chemicals in a systematic manner, taking into account the broad universe of man-made and natural chemicals to which humans and the environment may be exposed. In addition to the activities described here, the OECD is also discussing:
? screening procedures (e.g., receptor binding, transcriptional activation, and steroid metabolism),
? a cost-efficient mammalian in utero exposure test guideline apart from the extremely laborious OECD multigeneration test, which requires a large number of test animals. This may, for example, be some kind of an “enhanced one-generation test”,
? wildlife test systems, primarily in fish (e.g., vitellogenin assay, gonadoal histopathology, partial- or even full-lifecycle tests) or for other target species if necessary (e.g., birds, amphibians, invertebrates).
As described earlier, there is a need to guard against proliferation of new and/or revised test methods, which often produce random or spurious endpoints making a clear interpretation impossible. For this and other reasons, development of specific testing or screening procedures must follow the same general principles as for any other toxicological endpoint. Risk assessments for endocrine disruptors must be based on definitive tests. Data for regulatory decisions should stem from robust, validated and internationally recognized screening assays and test methods that should ideally originate from the “tool-box” of OECD test guidelines. If the “tool-box” has to be complemented, such additional test methods should be subjected to a stringent validation process carried out in partnership between regulatory authorities, the scientific community and the chemical industry. The OECD program that is now coming close to its finalisation for the uterotrophic, the Hershberger, and the “enhanced TG 407” assays, these examples should serve as guidance for any such future activities.
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