Amphibian toxicity testing for identification of thyroid disrupting chemicals.

Both amphibian metamorphosis assay (AMA) and larval amphibian growth and development assay (LAGDA) can detect thyroid-mediated modality and adversity on the basis of morphological changes during the thyroid hormone-dependent metamorphosis. They are used for identification of thyroid hormone system disrupting chemicals (TDCs) for non-target organisms or the environment. The EU Guidance recommends that the AMA and the LAGDA should be used to address sufficient investigation of the thyroid-mediated modality and adversity, respectively. In the EU discussions over identification of TDCs, the necessity of using LAGDA as a follow-up of positive results of the AMA has been questioned because of the overlap between the endpoints and the exposure of both tests. This study analyzed similarities, differences, and sensitivity of these two assays in detection of TDCs. For agonists and most of antagonists of the hypothalamic-pituitary-thyroid (HPT) axis, both AMA and LAGDA can detect the thyroid-mediated modality and adversity. The LAGDA, as a follow-up of the positive results of the AMA, may not be needed because the results of AMA are considered enough for identification of TDCs. For chemicals like inhibitors of iodotyrosine deiodinase, the LAGDA is considered necessary for identification of TDCs because the thyroid-mediated adversity cannot be detected until Nieuwkoop and Faber (NF) stage 62. Incorporation of mechanistic endpoints into existing test guidelines and the use of Xenopus Eleutheroembryo Thyroid Assay (XETA), extended amphibian metamorphosis assay (EAMA) and adverse outcome pathways (AOPs) for testing and identification of TDCs are further discussed.

Fish toxicity testing for identification of thyroid disrupting chemicals.

Identification of thyroid disrupting chemicals (TDCs), one of the most studied types of endocrine disruptors (EDs), is required according to EU regulations on industrial chemicals, pesticides, and biocides. Following that requirement, the use of fish as a unique non-mammalian model species for identification of EDs may be warranted. This study summarized and evaluated effects of TDCs on fish thyroid sensitive endpoints including thyroid hormones, thyroid related gene expression, immunostaining for thyroid follicles, eye size and pigmentation, swim bladder inflation as well as effects of TDCs on secondary sex characteristics, sex ratio, growth and reproduction. Changes in thyroid sensitive endpoints may reflect the balanced outcome of different processes of the thyroid cascade. Thyroid sensitive endpoints may also be altered by non-thyroid molecular or endocrine pathways as well as non-specific factors such as general toxicity, development, stress, nutrient, and the environmental factors like temperature and pH. Defining chemical specific effects on thyroid sensitive endpoints is important for identification of TDCs. Application of the AOP (adverse outcome pathway) concept could be helpful for defining critical events needed for testing and identification of TDCs in fish.

The Role of Behavioral Ecotoxicology in Environmental Protection.

For decades, we have known that chemicals affect human and wildlife behavior. Moreover, due to recent technological and computational advances, scientists are now increasingly aware that a wide variety of contaminants and other environmental stressors adversely affect organismal behavior and subsequent ecological outcomes in terrestrial and aquatic ecosystems. There is also a groundswell of concern that regulatory ecotoxicology does not adequately consider behavior, primarily due to a lack of standardized toxicity methods. This has, in turn, led to the exclusion of many behavioral ecotoxicology studies from chemical risk assessments. To improve understanding of the challenges and opportunities for behavioral ecotoxicology within regulatory toxicology/risk assessment, a unique workshop with international representatives from the fields of behavioral ecology, ecotoxicology, regulatory (eco)toxicology, neurotoxicology, test standardization, and risk assessment resulted in the formation of consensus perspectives and recommendations, which promise to serve as a roadmap to advance interfaces among the basic and translational sciences, and regulatory practices.

Changes in fish sex ratio as a basis for regulating endocrine disruptors.

Fish sex ratio (SR) is an endpoint potentially indicating both endocrine activity and adversity, essential elements for identifying Endocrine Disrupting Chemicals (EDCs) as required by the EU regulations. Due to different protocols and methods in the literature studies, SR data vary greatly. This study analyses literature SR data and discusses important considerations for using SR data in the regulatory context for the hazard identification, classification, PBT (persistent, bioaccumulative and toxic) assessment, testing, and risk assessment. A total number of 106 studies were compiled for SR of zebrafish, medaka and fathead minnow exposed to 84 chemicals or mixtures. About 53% of literature studies determined SR by methods different from the standard histology method, leading to uncertainty of quantifying SR and differential sensitivity. SR was determined after depuration in 40 papers, which may lead to chemical-induced SR changes reversible to the control. SR was responsive to chemicals with EAS (estrogen, androgen, steoroidogenesis) activity and also to those with thyroid and progesterone activity. Besides, SR was influenced by non-chemical factors, e.g., inbreeding and temperature, leading to difficulty in data interpretation. The ECHA/EFSA/JRC Guidance suggests that SR and gonad histology data can be used for identifying EDCs. Due to reversibility, influence of confounding factors, and responsiveness to chemicals with endocrine activity other than EAS, this study suggests that SR/gonad histology should be combined with certain mode of action evidence for identifying EDCs. Important considerations for using SR data in the identification, classification, PBT assessment, testing, and risk assessment are discussed.

Endpoint sensitivity in Amphibian Metamorphosis Assay.

The Amphibian Metamorphosis Assay (AMA) is a screening test for detecting chemicals with thyroid activity. There is little experience in data interpretation and in using AMA data for screening, testing and identifying endocrine disruptors. To investigate the sensitivity of different endpoints of the AMA, the publically available data for 57 thyroid active and inactive chemicals were compiled and analyzed. Endpoints body weight and length appeared as sensitive as apical thyroid responsive endpoints hind limb length (HLL) and developmental stage (DS) for 12 thyroid active chemicals. The sensitivity of body weight, length and HLL was comparable, which is higher than that of DS for 45 thyroid inactive chemicals. The decision logic of the AMA suggests that an advanced development alone indicates thyroid activity. The analysis here showed that advanced development at day 7 could indicate thyroid activity of a chemical. However, advanced development at day 21 may be influenced by thyroid inactive chemicals. Among 39 thyroid inactive chemicals, which affected one or more endpoints, 33% and 77% induced changes in HLL and/or DS at day 7 and 21, respectively; only 10% influenced thyroid histology. These results showed that apical thyroid responsive endpoints HLL and DS are influenced by thyroid active chemicals as well as thyroid inactive chemical. Both endpoints should be combined with thyroid histology for the identification of thyroid active chemicals. The use of the AMA in a testing strategy to identify chemicals with thyroid activity is discussed.

Applicability of the fish embryo acute toxicity (FET) test (OECD 236) in the regulatory context of Registration, Evaluation, Authorisation, and Restriction of Chemicals (REACH).

In 2013 the Organisation for Economic Co-operation and Development (OECD) test guideline (236) for fish embryo acute toxicity (FET) was adopted. It determines the acute toxicity of chemicals to embryonic fish. Previous studies show a good correlation of FET with the standard acute fish toxicity (AFT) test; however, the potential of the FET test to predict AFT, which is required by the Registration, Evaluation, Authorisation, and Restriction of Chemicals (REACH) regulation (EC 1907/2006) and the Classification, Labelling and Packaging (CLP) Regulation (EC 1272/2008), has not yet been fully clarified. In 2015 the European Chemicals Agency (ECHA) requested that a consultant perform a scientific analysis of the applicability of FET to predict AFT. The purpose was to compare the toxicity of substances to fish embryos and to adult fish, and to investigate whether certain factors (e.g., physicochemical properties, modes of action, or chemical structures) could be used to define the applicability boundaries of the FET test. Given the limited data availability, the analysis focused on organic substances. The present critical review summarizes the main findings and discusses regulatory application of the FET test under REACH. Given some limitations (e.g., neurotoxic mode of action) and/or remaining uncertainties (e.g., deviation of some narcotic substances), it has been found that the FET test alone is currently not sufficient to meet the essential information on AFT as required by the REACH regulation. However, the test may be used within weight-of-evidence approaches together with other independent, relevant, and reliable sources of information. The present review also discusses further research needs that may overcome the remaining uncertainties and help to increase acceptance of FET as a replacement for AFT in the future. For example, an increase in the availability of data generated according to OECD test guideline 236 may provide evidence of a higher predictive power of the test. Environ Toxicol Chem 2018;37:657-670. © 2017 SETAC.

Fish embryo toxicity test, threshold approach, and moribund as approaches to implement 3R principles to the acute fish toxicity test.

The acute fish toxicity test (AFT) is requested by EU legal frameworks for hazard classification and risk assessment. AFT is one of the few regulatory required tests using death as an endpoint. This paper reviews efforts made to reduce, refine and replace (3Rs) AFT. We make an inventory of information requirements for AFT, summarize studies on 3Rs of AFT and give recommendations. The fish embryo toxicity test (FET) is proposed as a replacement of AFT and analyses have focused on two aspects: assessing the capacity of FET in predicting AFT and defining the applicability domain of FET. Six comparison studies have consistently shown a strong correlation of FET and AFT. In contrast, the applicability domain of FET has not yet been fully defined. FET has not yet been accepted as a replacement of AFT by any EU legal frameworks to fulfill information requirements because FET is insensitive to some chemicals. It is recommended that the outlier chemicals that do not correlate between FET and AFT should be further investigated. When necessary, additional FET data should be generated. Another effort to reduce and refine AFT is incorporation of FET into the threshold approach. Furthermore, moribund as an endpoint of fish death has been introduced in revising AFT guideline to reduce the duration of suffering for refinement. This endpoint, however, needs further work on the link of moribund and death. Global regulatory acceptance of the moribund endpoint would be critical for this development.

Recommended approaches to the scientific evaluation of ecotoxicological hazards and risks of endocrine-active substances.

A SETAC Pellston Workshop® "Environmental Hazard and Risk Assessment Approaches for Endocrine-Active Substances (EHRA)" was held in February 2016 in Pensacola, Florida, USA. The primary objective of the workshop was to provide advice, based on current scientific understanding, to regulators and policy makers; the aim being to make considered, informed decisions on whether to select an ecotoxicological hazard- or a risk-based approach for regulating a given endocrine-disrupting substance (EDS) under review. The workshop additionally considered recent developments in the identification of EDS. Case studies were undertaken on 6 endocrine-active substances (EAS-not necessarily proven EDS, but substances known to interact directly with the endocrine system) that are representative of a range of perturbations of the endocrine system and considered to be data rich in relevant information at multiple biological levels of organization for 1 or more ecologically relevant taxa. The substances selected were 17α-ethinylestradiol, perchlorate, propiconazole, 17ß-trenbolone, tributyltin, and vinclozolin. The 6 case studies were not comprehensive safety evaluations but provided foundations for clarifying key issues and procedures that should be considered when assessing the ecotoxicological hazards and risks of EAS and EDS. The workshop also highlighted areas of scientific uncertainty, and made specific recommendations for research and methods-development to resolve some of the identified issues. The present paper provides broad guidance for scientists in regulatory authorities, industry, and academia on issues likely to arise during the ecotoxicological hazard and risk assessment of EAS and EDS. The primary conclusion of this paper, and of the SETAC Pellston Workshop on which it is based, is that if data on environmental exposure, effects on sensitive species and life-stages, delayed effects, and effects at low concentrations are robust, initiating environmental risk assessment of EDS is scientifically sound and sufficiently reliable and protective of the environment. In the absence of such data, assessment on the basis of hazard is scientifically justified until such time as relevant new information is available. Integr Environ Assess Manag 2017;13:267-279. © 2017 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals, Inc. on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Challenges in assigning endocrine-specific modes of action: Recommendations for researchers and regulators.

As regulatory programs evaluate substances for their endocrine-disrupting properties, careful study design and data interpretation are needed to distinguish between responses that are truly endocrine specific and those that are not. This is particularly important in regulatory environments where criteria are under development to identify endocrine-disrupting properties to enable hazard-based regulation. Irrespective of these processes, most jurisdictions use the World Health Organization/International Programme on Chemical Safety definition of an endocrine disruptor, requiring that a substance is demonstrated to cause a change in endocrine function that consequently leads to an adverse effect in an intact organism. Such a definition is broad, and at its most cautious might capture many general mechanisms that would not specifically denote an endocrine disruptor. In addition, endocrine responses may be adaptive in nature, designed to maintain homeostasis rather than induce an irreversible adverse effect. The likelihood of indirect effects is increased in (eco)toxicological studies that require the use of maximum tolerated concentrations or doses, which must produce some adverse effect. The misidentification of indirect effects as truly endocrine mediated has serious consequences for prompting animal- and resource-intensive testing and regulatory consequences. To minimize the risk for misidentification, an objective and transparent weight-of-evidence procedure based on biological plausibility, essentiality, and empirical evidence of key events in an adverse outcome pathway is recommended to describe the modes of action that may be involved in toxic responses in nontarget organisms. Confounding factors such as systemic toxicity, general stress, and infection can add complexity to such an evaluation and should be considered in the weight of evidence. A recommended set of questions is proffered to help guide researchers and regulators in discerning endocrine and nonendocrine responses. Although many examples provided in this study are based on ecotoxicology, the majority of the concepts and processes are applicable to both environmental and human health assessments. Integr Environ Assess Manag 2017;13:280-292. © 2016 SETAC.

Interpretation of fish biomarker data for identification, classification, risk assessment and testing of endocrine disrupting chemicals.

Chemical induced changes in fish biomarkers vitellogenin (VTG), secondary sex characteristics (SSC), and sex ratio indicate modes/mechanisms of action (MOAs) of EAS (estrogen, androgen and steroidogenesis) pathways. These biomarkers could be used for defining MOAs and the causal link between MOAs and adverse effects in fish for the identification of endocrine disrupting chemicals (EDCs). This paper compiled data sets of 150 chemicals for VTG, 57 chemicals for SSC and 38 chemicals for sex ratio in fathead minnow, medaka and zebrafish. It showed 1) changes in fish biomarkers can indicate the MOAs as anticipated; 2) in addition to EAS pathways, chemicals with non-EAS pathways induced changes in fish biomarkers; 3) responses of fish biomarkers did not always follow the anticipated patterns of EAS pathways. These responses may result from the interaction of chemical-induced multiple MOAs and confounding factors like fish diet, infection, culture conditions, general toxicity and stress response. The complex response of fish biomarkers to a chemical of interest requires EDC testing at multiple biological levels. Interpretation of fish biomarker data should be combined with relevant information at different biological levels, which is critical for defining chemical specific MOAs. The utility of fish biomarker data for identification, classification, PBT assessment, risk assessment, and testing of EDCs in the regulatory context was discussed. This paper emphasizes the importance of fish biomarker data in the regulatory context, a weight of evidence approach for the interpretation of fish biomarker data and the need for defining levels of evidence for the identification of EDCs.

Fish biomarkers for regulatory identification of endocrine disrupting chemicals.

Demonstrating chemical-induced adverse effects, endocrine mechanisms/modes of action (MOAs) and their causal link is needed for regulatory identification of endocrine disrupting chemicals (EDCs). This paper addresses critical issues over MOAs and their causal link to changes in endpoints. Vitellogenin (VTG), secondary sex characteristics (SSC), and sex ratio (also an apical endpoint) are indicative of chemicals interfering with EAS (estrogen, androgen and steroidogenesis) pathways. These biomarkers, however, can be changed by non-EAS chemicals, systemic toxicity and the stress response. Examples are shown that proving causal link between MOAs and changes in endpoints may be difficult for regulatory identification of EDCs. The paper concludes that both in vitro and in vivo data are needed to define MOAs for regulatory identification of EDCs. Further development of guidance documents for data interpretation and for defining the level of evidence is needed for regulatory identification of EDCs.

[Effects of perfluorooctane sulfonate (PFOS) exposure on vitellogenin mRNA level in zebrafish (Brachydanio rerio)].

To study the endocrine disrupting effects and action mechanism of environmental levels of perfluorooctane sulfonate (PFOS) on the aquatic species, the research for the effects of PFOS exposure on vitellogenin (VTG) mRNA level in livers of zebrafish (Brachydanio rerio) was conducted. Zebrafish were exposed to PFOS at four environmental low concentrations (0.1, 1, 10, 100 microg x L(-1)) for 21 days. Livers from male and female zebrafish were collected for RNA extraction, VTG1 and VTG3 mRNA levels were measured respectively using real-time polymerase chain reaction (RT-PCR). The results show that: 1) The VTG1 and VTG3 mRNA level in the livers of male zebrafish increased after PFOS exposure. The VTG1 mRNA level increased with a positive dose response pattern, with the maximum response at 100 microg x L(-1) PFOS exposure where a significant difference compared with the control was observed. The VTG3 mRNA level increased as an inverted U-shaped dose response pattern, indicated as hormesis effects, where significant differences compared with the control were observed at 10 and 100 microg x L(-1) PFOS exposure. 2) The VTG1 mRNA level in the livers of female zebrafish increased where a significant difference compared with the control was observed at 10 microg x L(-1) PFOS exposure, but the standard errors for mRNA level at 10 and 100 microg x L(-1) PFOS exposure were distinct. The VTG3 mRNA level in the livers of female zebrafish increased at 10 microg L(-1) PFOS exposure but had no significant difference compared with the control. Thus, it deduced that PFOS exposure could be active on the endocrine system of zebrafish with the oestrogenic simulation action mechanism, and the VTG1 and VTG3 mRNA level in the livers of zebrafish might be sensitive biomarkers for the endocrine disrupting effects evaluation after PFOS exposure, with different responding patterns related to the gene subtypes and sex.

Evaluation of the Daphnia magna reproduction test for detecting endocrine disruptors.

The Daphnia 21 d reproduction test is considered as a comprehensive and decisive test in the OECD Conceptual Framework for testing and assessment of endocrine disrupting chemicals (EDCs). However, how to interpret results of the Daphnia 21 d reproduction test for identification, risk assessment and testing strategy of EDCs remains an unsolved issue. This study analysed a total number of 135 published studies encompassing 86 known EDCs and non-EDCs with different modes of action. Our results show that the majority of effects on apical endpoints (survival, molting, growth, time to reproductive maturity, brood size, the number of broods, and the total number of offspring) do not seem to be EDC-specific. In contrast, the endpoint sex ratio is likely specific to juvenile hormones and their mimics. Variability is quantified for three most reported endpoints survival, the total number of offspring and sex ratio. Quantification of the endpoint sensitivity shows that the sensitivity of the sex ratio is lower than that of the total number of offspring. The Daphnia 21 d reproduction test gives insufficient information to conclude if a substance is an EDC or not. EDCs that are potent in assays in vitro may not be potent in the Daphnia 21 d reproduction test. We conclude that the Daphnia 21 d reproduction test is important for deriving No Observed Effect Concentrations for risk assessment but may produce false negatives in identification of EDCs when used on its own. A targeted testing strategy for selection of species, tests, and endpoints is suggested for identifying EDCs.

Evaluation of the fish short term reproduction assay for detecting endocrine disrupters.

In a fish testing strategy, positive results of the fish short term reproduction assay (FSTR), often trigger a definitive test like the fish sexual development test (FSDT) or the fish full life cycle test (FFLC), entailing ethical and economic problems. This study analysed 137 studies encompassing 35 chemicals with different modes of actions (MOAs). Variability is quantified for MOA endpoints vitellogenin (VTG) and secondary sex characteristics (SSCs) as well as for apical endpoints. Two MOA endpoints could indicate estrogenic, anti-estrogenic, androgenic, anti-androgenic and steroidogenesis activities. Great variability, however, has been observed for chemicals with anti-androgenic and steroidogenesis activities, suggesting that TG229/230 may not be sensitive enough to detect these types of chemicals and may produce false negatives. Changes in apical endpoints like fecundity are not limited to endocrine disrupting chemicals (EDCs). Non-EDCs could induce the similar effects on these apical endpoints. If elucidating MOA is needed, targeted in vitro MOA tests are suggested. Positive in vitro MOA results trigger a definitive test, which could be used for confirmation of the MOA in vivo and for deriving a no observed effect concentration (NOEC). Based on positive MOA results of TG229, a definitive test such as the FSDT or the FFLC is still needed, because the current TG229 has limitation on the derivation of a NOEC. An extended TG229 with more power to detect reproduction effects, as recently proposed in the OECD test guideline program, would improve the possibility to derive a NOEC and increase its usefulness in risk assessment.

Endpoint sensitivity in fish endocrine disruption assays: regulatory implications.

Identifying potential endocrine disrupting chemicals (EDCs) needs screening and testing for mode of action (MOA) and intrinsic toxicological properties. MOA is often indicated by biomarker endpoints, whereas toxicity by apical endpoints. Risk assessment is mainly based on apical but not on biomarker endpoints. The 21-day fish assay (OECD TG229) is considered a screening test. But it includes both biomarker and apical endpoints. This study explores the utility of results of the 21-day fish assay for risk assessment purposes. Endpoint sensitivity was analysed by compiling 142 data sets for 21-day fish assays and 38 data sets for the fish sexual development test (FSDT), encompassing 62 chemicals with different MOAs. Conclusions from this analysis include: (1) vitellogenin (VTG), fecundity and gonad histology are the most sensitive endpoints for fathead minnow, medaka and zebrafish in 21-day fish assays; secondary sex characteristics (SSC) are a less sensitive endpoint and is likely inadequate to detect all known MOAs. (2) Biomarker endpoints like VTG and apical endpoints like fecundity from the 21-day fish assay can be used for risk assessment. (3) Lowest observed effect concentrations (LOECs) of the most chemicals are comparable for the 21-day fish assay and for the FSDT, further supporting that results of 21-day fish assays can be used for risk assessment. However, a significant difference in LOECs was observed for some chemicals, suggesting that chemical specific effects should be taken into account. This paper emphasizes that a weight of evidence approach is important for interpretation of results of the 21-day fish assay.

Comparison of chemical-induced transcriptional activation of fish and human estrogen receptors: regulatory implications.

Under the current EU chemical regulation REACH (Registration, Evaluation, Authorization and Restriction of Chemicals), revised plant protection products and biocides directives, evaluation of endocrine disrupting properties of chemicals becomes a regulatory need. Transcriptional activation (TA) testing of estrogen receptors (ERs) could be one important first step in the screening and testing of endocrine disrupting chemicals (EDCs) for regulatory purposes. However up to now there is no consensus on which species or subtype of ERs should be used for TA testing. This study collected data from publications on TA testing with fish and human ERs for 90 chemicals, covering strong, moderate, and weak or non-ER binders. Each chemical has been reported at least twice, with differential ER TA values that result from different cellular contexts, from intra-/inter-species and subtypes of ERs and from intra-/inter-laboratory differences. All assays could distinguish the differential transcriptional activity induced by chemicals of strong, moderate, and weak or non-ER binders. It is concluded that transactivation of ERs in one vertebrate species or one subtype of ERs could be extrapolated to other species or subtypes of ERs for the purpose of chemical screening. It is emphasized that results from ER TA assays can only be used in a weight-of-evidence approach for further testing in regulatory programs. These results are of importance for regulatory testing strategies and decision making for EDCs.

An Integrated Assessment Scheme for assessing the adequacy of (eco)toxicological data under REACH.

REACH requires all available (eco)toxicological information, whether protocol studies, other experiments, or non-testing approaches such as read-across or (Q)SAR, to be collected and evaluated. However, guidance documents only limitedly address how adequacy of (eco)toxicological information can be assessed consistently and transparently. We propose an Integrated Assessment Scheme (IAS) for the evaluation of (eco)toxicological data. The IAS consists of three modules: (i) the reliability of the data; (ii) the validity of the methods the data are generated from and; (iii) the regulatory need of the data. Each module is assessed and documented using adjusted OECD principles for the validation of (Q)SARs. These adjusted principles provide a harmonised set of criteria for the evaluation of all types of (eco)toxicological data. Assessment codes, similar to Klimisch codes, are assigned to the evaluated information in each module. The coherent combination of the assessment codes of all three modules determines the overall adequacy of information for fulfilling the information requirement in REACH, and can serve as a weight in a Weight of Evidence procedure as mentioned in REACH Annex XI.

Comparison of relative binding affinities to fish and mammalian estrogen receptors: the regulatory implications.

Screening and testing of chemicals binding to estrogen receptors (ERs) emerge as an important issue in several regulatory programs or frameworks. Discrepancies exist, however, whether fish ERs should be included in the regulatory programs. In view of the differences in binding affinities to ERalpha and ERbeta and the significant contribution of ERbeta to biological effects of chemicals, it remains unknown whether both types of ERs are needed for the regulatory purposes. This study collected publications on binding affinities to both mammalian and fish ERs for 65 chemicals, covering a wide range of strong, moderate, weak and non-ER binders. Systematic evaluation of the data was performed in order to compare the difference in binding affinity of chemicals to fish and mammalian ERs and to subtypes of ERs. Except the reference estrogen 17beta-estradiol, all 64 chemicals have differential values of relative binding affinity (RBA), which result mostly from the inter-laboratory tests other than interspecies differences. It is concluded that ER binding in one vertebrate species or one subtype of ERs could be extrapolated to other species or subtypes of ERs for most of chemicals for the regulatory purpose. Fish ERs are likely more sensitive to some chemicals of weak binders than mammalian ERs, suggesting the importance of including fish ERs in the regulatory programs. Issues on data interpretation and testing strategy for the regulatory purpose have been discussed.

Retrospective analysis of relative parameter sensitivity in multi-generation reproductive toxicity studies.

Current suggestions towards amending the OECD two-generation protocol include omission of the second generation and inclusion of additional parameters. This study analysed the relative parameter sensitivity in 18 individually published multi-generation studies with substances toxic to fertility. Among parameters that most often determined the reproductive LOAEL were weight of testis, dam and pup as well as litter size. Several other parameters were found to be unaffected in all studies evaluated. Some substances affected a specific set of parameters, indicating that rarely affected parameters may prove crucial in individual situations. This argues for the inclusion of a wide spectrum of parameters to cover all possible effects. Less sensitive parameters, mechanistically related to more sensitive ones, may be omitted as they will unlikely contribute to the overall LOAEL. This study gives first insights and needs follow-up by more extensive analyses before firm conclusions on the design of the two-generation study protocol can be drawn.

Sixteen hours of fasting differentially affects hepatic and muscle insulin sensitivity in mice.

Fasting readily induces hepatic steatosis. Hepatic steatosis is associated with hepatic insulin resistance. The purpose of the present study was to document the effects of 16 h of fasting in wild-type mice on insulin sensitivity in liver and skeletal muscle in relation to 1) tissue accumulation of triglycerides (TGs) and 2) changes in mRNA expression of metabolically relevant genes. Sixteen hours of fasting did not show an effect on hepatic insulin sensitivity in terms of glucose production in the presence of increased hepatic TG content. In muscle, however, fasting resulted in increased insulin sensitivity, with increased muscle glucose uptake without changes in muscle TG content. In liver, fasting resulted in increased mRNA expression of genes promoting gluconeogenesis and TG synthesis but in decreased mRNA expression of genes involved in glycogenolysis and fatty acid synthesis. In muscle, increased mRNA expression of genes promoting glucose uptake, as well as lipogenesis and beta-oxidation, was found. In conclusion, 16 h of fasting does not induce hepatic insulin resistance, although it causes liver steatosis, whereas muscle insulin sensitivity increases without changes in muscle TG content. Therefore, fasting induces differential changes in tissue-specific insulin sensitivity, and liver and muscle TG contents are unlikely to be involved in these changes.