The benefits of validation of methods for toxicity testing outweigh its costs

The 4th Annual Forum on Endocrine Disrupters organized by the European Commission brought together authors of this article around the topic: "From bench to validated test guidelines: (pre)validation of test methods". Validation activities are meant to demonstrate the relevance and reliability of methods and approaches used in regulatory safety testing. These activities are essential to facilitate regulatory use, still they are largely underfunded and unattractive to the scientific community. In the last decade, there has been large amounts of funding invested in European research towards the development of approaches that can be used in regulatory decision-making, including for the identification of endocrine disrupters. There is a vast pool of candidate test methods for potential regulatory applications, but most of them will not be used due to the absence of consideration of their relevance and reliability outside the method developer's laboratory. The article explains the reasons why such a gap exists between the outputs of research projects and the uptake in a regulatory context. In parallel, there are also increasing expectations from the regulatory science community that validation becomes more efficient with respect to time and resources. This article shares some of the lessons learned and proposes paths forward for validation of new methods that are not intended as one-to-one replacements of animal studies. This includes submitting only mature methods for validation that were developed following good practices and good documentation, proposing a greater emphasis on well-documented transferability studies, and adopting a cost-sharing model between those who benefit from validated methods.

Validation activities for methods intended to be used to assess chemical safety have a cost but also bring substantial benefits when the validated methods are established as OECD Test Guidelines which results in mutual acceptance of data generated by the methods. The article discusses some of the challenges faced when method validation is underfunded and unattractive for researchers. Proposals are made to improve the current situation, gain efficiency and make validation a shared responsibility.

ReadEDTest: A tool to assess the readiness of in vitro test methods under development for identifying endocrine disruptors.

Growing evidence shows that endocrine disruptors (EDs), known to affect the reproductive system, may also disturb other hormone-regulated functions leading to cancers, neurodevelopmental defects, metabolic and immune diseases. To reduce exposure to EDs and limit their health effects, development of screening and mechanism-based assays to identify EDs is encouraged. Nevertheless, the crucial validation step of test methods by regulatory bodies is a time- and resource-consuming process. One of the main raisons of this long duration process is that method developers, mainly researchers, are not fully aware of the regulatory needs to validate a test. We propose an online self-assessment questionnaire (SAQ) called ReadEDTest easy to be used by all researchers. The aim of ReadEDTest is to speed up the validation process by assessing readiness criteria of in vitro and fish embryo ED test methods under development. The SAQ is divided into 7 sections and 13 sub-sections containing essential information requested by the validating bodies. The readiness of the tests can be assessed by specific score limits for each sub-section. Results are displayed via a graphical representation to help identification of the sub-sections having sufficient or insufficient information. The relevance of the proposed innovative tool was supported using two test methods already validated by the OECD and four under development test methods.

Current EU regulatory requirements for the assessment of chemicals and cosmetic products: challenges and opportunities for introducing new approach methodologies.

The EU Directive 2010/63/EU   on the protection of animals used for scientific purposes and other EU regulations, such as REACH and the Cosmetic Products Regulation advocate for a change in the way toxicity testing is conducted. Whilst the Cosmetic Products Regulation bans animal testing altogether, REACH aims for a progressive shift from in vivo testing towards quantitative in vitro and computational approaches. Several endpoints can already be addressed using non-animal approaches including skin corrosion and irritation, serious eye damage and irritation, skin sensitisation, and mutagenicity and genotoxicity. However, for systemic effects such as acute toxicity, repeated dose toxicity and reproductive and developmental toxicity, evaluation of chemicals under REACH still heavily relies on animal tests. Here we summarise current EU regulatory requirements for the human health assessment of chemicals under REACH and the Cosmetic Products Regulation, considering the more critical endpoints and identifying the main challenges in introducing alternative methods into regulatory testing practice. This supports a recent initiative taken by the International Cooperation on Alternative Test Methods (ICATM) to summarise current regulatory requirements specific for the assessment of chemicals and cosmetic products for several human health-related endpoints, with the aim of comparing different jurisdictions and coordinating the promotion and ultimately the implementation of non-animal approaches worldwide. Recent initiatives undertaken at European level to promote the 3Rs and the use of alternative methods in current regulatory practice are also discussed.

Identification of non-validated endocrine disrupting chemical characterization methods by screening of the literature using artificial intelligence and by database exploration.

BACKGROUND: Exposure to endocrine disrupting chemicals (EDCs) represents a critical public health threat. Several adverse health outcomes (e.g., cancers, metabolic and neurocognitive/neurodevelopmental disorders, infertility, immune diseases and allergies) are associated with exposure to EDCs. However, the regulatory tests that are currently employed in the EU to identify EDCs do not assess all of the endocrine pathways. OBJECTIVE: Our objective was to explore the literature, guidelines and databases to identify relevant and reliable test methods which could be used for prioritization and regulatory pre-validation of EDCs in missing and urgent key areas. METHODS: Abstracts of articles referenced in PubMed were automatically screened using an updated version of the AOP-helpFinder text mining approach. Other available sources were manually explored. Exclusion criteria (computational methods, specific tests for estrogen receptors, tests under validation or already validated, methods accepted by regulatory bodies) were applied according to the priorities of the French Public-privatE Platform for the Pre-validation of Endocrine disRuptors (PEPPER) characterisation methods. RESULTS: 226 unique non-validated methods were identified. These experimental methods (in vitro and in vivo) were developed for 30 species using diverse techniques (e.g., reporter gene assays and radioimmunoassays). We retrieved bioassays mainly for the reproductive system, growth/developmental systems, lipogenesis/adipogenicity, thyroid, steroidogenesis, liver metabolism-mediated toxicity, and more specifically for the androgen-, thyroid hormone-, glucocorticoid- and aryl hydrocarbon receptors. CONCLUSION: We identified methods to characterize EDCs which could be relevant for regulatory pre-validation and, ultimately for the efficient prevention of EDC-related severe health outcomes. This integrative approach highlights a successful and complementary strategy which combines computational and manual curation approaches.

Regulatory needs and activities to address the retinoid system in the context of endocrine disruption: The European viewpoint.

Endocrine disruption continues to be a matter of high concern, and a subject of intensive activities at the public, political, regulatory and academic levels. Currently, available regulatory test guidelines (TGs) relevant to the identification of endocrine disrupters are largely limited to estrogen, androgen, thyroid and steroidogenesis (EATS) pathways. Thus, there is an increasing interest and need to develop test methods, biomarkers, and Adverse Outcome Pathways (AOPs), for identification and evaluation of endocrine disrupters in addition to the EATS pathways. An activity focusing on the retinoid system has been jointly initiated by the Swedish Chemicals Agency and the European Commission. The retinoid system is involved in fundamental life processes and has been described, in previous work at the OECD, as a system susceptible to environmental endocrine disruption, the disruption of which could contribute to the increasing incidence of certain disorders in humans and wildlife populations.

Guidance for the identification of endocrine disruptors in the context of Regulations (EU) No 528/2012 and (EC) No 1107/2009.

This Guidance describes how to perform hazard identification for endocrine-disrupting properties by following the scientific criteria which are outlined in Commission Delegated Regulation (EU) 2017/2100 and Commission Regulation (EU) 2018/605 for biocidal products and plant protection products, respectively.

Considerations for the development of in vitro dissolution tests to reduce or replace preclinical oral absorption studies.

The pharmaceutical development of new chemical entities can be hampered by their solubility and/or dissolution limitations. Currently, these properties are characterised mostly during in vivo pre-clinical studies. The development of appropriate in vitro methods to study the solubility and dissolution properties in preclinical species would lead to a significant reduction or replacement of the animal experiments at this stage of development. During clinical development, media simulating the human gastrointestinal tract fluids are commonly used and a similar approach mimicking laboratory animals' gastrointestinal tract fluids would impact on the preclinical stage of development. This review summarises the current knowledge regarding the gastrointestinal physiology of the most common laboratory animals, and animal simulated gastric and intestinal media are proposed.

Weak estrogenic transcriptional activities of Bisphenol A and Bisphenol S.

In 2011, the European Commission has restricted the use of Bisphenol A in plastic infant feeding bottles. In a response to this restriction, Bisphenol S is now often used as a component of plastic substitutes for the production of babybottles. One of the major concerns leading to the restriction of Bisphenol A was its weak estrogenic activity. By using two highly standardised transactivation assays, we could demonstrate that the estrogenic activity of Bisphenol A and Bisphenol S is of a comparable potency. Furthermore, some insights about the structure-activity relationships of these two chemicals and their metabolites could be gained from in silico predictions of their relative estrogen receptor-binding affinities and their liver phase-I biotransformation.

Alternative (non-animal) methods for cosmetics testing: current status and future prospects-2010.

The 7th amendment to the EU Cosmetics Directive prohibits to put animal-tested cosmetics on the market in Europe after 2013. In that context, the European Commission invited stakeholder bodies (industry, non-governmental organisations, EU Member States, and the Commission's Scientific Committee on Consumer Safety) to identify scientific experts in five toxicological areas, i.e. toxicokinetics, repeated dose toxicity, carcinogenicity, skin sensitisation, and reproductive toxicity for which the Directive foresees that the 2013 deadline could be further extended in case alternative and validated methods would not be available in time. The selected experts were asked to analyse the status and prospects of alternative methods and to provide a scientifically sound estimate of the time necessary to achieve full replacement of animal testing. In summary, the experts confirmed that it will take at least another 7-9 years for the replacement of the current in vivo animal tests used for the safety assessment of cosmetic ingredients for skin sensitisation. However, the experts were also of the opinion that alternative methods may be able to give hazard information, i.e. to differentiate between sensitisers and non-sensitisers, ahead of 2017. This would, however, not provide the complete picture of what is a safe exposure because the relative potency of a sensitiser would not be known. For toxicokinetics, the timeframe was 5-7 years to develop the models still lacking to predict lung absorption and renal/biliary excretion, and even longer to integrate the methods to fully replace the animal toxicokinetic models. For the systemic toxicological endpoints of repeated dose toxicity, carcinogenicity and reproductive toxicity, the time horizon for full replacement could not be estimated.

Testicular steroidogenesis is not altered by 137 cesium Chernobyl fallout, following in utero or post-natal chronic exposure.

The testis is especially sensitive to pollutants, including radionuclides. Following the Chernobyl nuclear power plant accident, several of these radionuclides were emitted and spread in the environment. Subsequently, children presented some disruptions of the endocrine system. To determine whether these disruptions were due to 137 cesium ((137)Cs) exposure, the effects of chronic contamination with low doses of (137)Cs in utero or from birth on testicular steroidogenesis in rats were studied. Contamination was continued for 9 months. No modification was observed in circulating level of hormones (17beta-estradiol, testosterone, follicle-stimulating hormone, luteinizing hormone) following in utero or post-natal contamination. Expression of several genes involved in testicular steroidogenesis was affected (cyp19a1, fxr, sf-1), without modification of protein expression or activity. Our results suggest that growing organisms may be affected at the molecular level by (137)Cs contamination at this post-accidental dose.

Contamination with depleted or enriched uranium differently affects steroidogenesis metabolism in rat.

Uranium is a naturally occurring heavy metal found in the Earth's crust. It is an alpha-emitter radioactive element from the actinide group that presents both radiotoxicant and chemotoxicant properties. Some studies revealed that uranium could affect the reproductive system. To distinguish chemical versus radiological effects of uranium on the metabolism of the steroids in the testis, rats were contaminated via their drinking water with depleted or enriched uranium. Animals were exposed to radionuclides for 9 months at a dose of 40 mg/L (560 Bq/L for depleted uranium, 1680 Bq/L for enriched uranium). Whereas depleted uranium did not seem to significantly affect the production of testicular steroid hormones in rats, enriched uranium significantly increased the level of circulating testosterone by 2.5-fold. Enriched uranium contamination led to significant increases in the mRNA levels of StAR (Steroidogenic Acute Regulatory protein; 3-fold, p = .001), cyp11a1 (cytochrome P45011a1; 2.2-fold, p < .001), cyp17a1 (cytochrome P45017a1; 2.5-fold, p = .014), cyp19a1 (cytochrome P45019a1; 2.3-fold, p = .021), and 5alpha -R1 (5alpha reductase type 1; 2.0-fold, p = .02), whereas depleted uranium contamination induces no changes in the expression of these genes. Moreover, expression levels of the nuclear receptors LXR (Liver X Receptor) and SF-1 (Steroidogenic Factor 1), as well as the transcription factor GATA-4, were modified following enriched uranium contamination. Altogether, these results show for the first time a differential effect among depleted or enriched uranium contamination on testicular steroidogenesis. It appears that the deleterious effects of uranium are mainly due to the radiological activity of the compound.

GPX5, the selenium-independent glutathione peroxidase-encoding single copy gene is differentially expressed in mouse epididymis.

Using various molecular approaches, including reverse transcription-polymerase chain reaction (RT-PCR), rapid amplification of cDNA ends-PCR, sequencing, northern and western blotting, we found that the mouse GPX5 gene gives rise to at least three different transcripts that are not expressed at the same levels in the mouse epididymis. In addition to the major GPX5 transcript, we show that minor GPX5 transcripts exist, arising either from precocious termination of transcription or an alternative splicing event within intron 4 of the 5 exon-encoding GPX5 single copy gene. Furthermore, we demonstrate that variants of the GPX5 protein that are correlated with the shorter GPX5 transcripts can be detected in caput epididymidis protein extracts and that the various GPX5 isoforms are subject to differential post-transcriptional maturation processes in the mouse epididymis that essentially involve the addition of O-glycosyl extensions. Using a sensitive poly-A+ mRNA tissue blot, as well as RT-PCR and northern assays, we further show that in addition to being expressed in the epididymis, the GPX5 gene is also expressed, albeit at lower levels, in other tissues of the male genital tract, including the testis and prostate. Finally, we present evidence suggesting that the GPX5 gene is expressed in a temporally regulated manner during mouse embryonic development.

In vivo effects of chronic contamination with 137 cesium on testicular and adrenal steroidogenesis.

More than 20 years after Chernobyl nuclear power plant explosion, radionuclides are still mainly bound to the organic soil layers. The radiation exposure is dominated by the external exposure to gamma-radiation following the decay of (137)Cs and by soil-to-plant-to-human transfer of (137)Cs into the food chain. Because of this persistence of contamination with (137)Cs, questions regarding public health for people living in contaminated areas were raised. We investigated the biological effects of chronic exposure to (137)Cs on testicular and adrenal steroidogenesis metabolisms in rat. Animals were exposed to radionuclide in their drinking water for 9 months at a dose of 6,500 Bq/l (610 Bq/kg/day). Cesium contamination decreases the level of circulating 17beta-estradiol, and increases corticosterone level. In testis, several nuclear receptors messenger expression is disrupted; levels of mRNA encoding Liver X receptor alpha (LXRalpha) and LXRbeta are increased, whereas farnesoid X receptor mRNA presents a lower level. Adrenal metabolism presents a paradoxical decrease in cyp11a1 gene expression. In conclusion, our results show for the first time molecular and hormonal modifications in testicular and adrenal steroidogenic metabolism, induced by chronic contamination with low doses of (137)Cs.

GPX5 orthologs of the mouse epididymis-restricted and sperm-bound selenium-independent glutathione peroxidase are not expressed with the same quantitative and spatial characteristics in large domestic animals.

We report here on the cloning of cDNAs coding bovine and equine orthologs of mouse epididymis-restricted and sperm-bound glutathione peroxidase 5 (GPX5), a selenium-independent member of the multigenic GPX family in mammals. The complete sequence of bovine GPX5 as well as a partial sequence of the equine GPX5 were characterized, conceptually translated and aligned with other known mammalian GPX5 proteins. Using Northern blotting assays, we show that the level of expression of GPX5 is high in bovine but low in equine and that in both species the regionalization of GPX5 expression in epididymis is not totally identical to what was reported for rodent mouse GPX5. An antibody was produced against GPX5 and used in Western blot assays as well as in immunohistochemistry assays on bovine epididymis sections. It shows that the protein is essentially present in the cytoplasmic compartment of the caput segment 2 epithelium of the bovine epididymis. Unlike in the mouse model, bovine GPX5 seems to be poorly secreted and does not seem to be present on cauda epididymal spermatozoa.