Is normalized hindlimb length measurement in assessment of thyroid disruption in the amphibian metamorphosis assay relevant?

The amphibian metamorphosis assay represents an OECD Level 3 and EDSP Tier 1 ecotoxicity test assessing thyroid activity of chemicals in African clawed frog (Xenopus laevis). To evaluate the effectiveness of snout-vent length (SVL) normalization of hindlimb length (HLL), correlation between the HLL and SVL or body weight was evaluated in the control groups of 10 individual studies from three laboratories. Two studies required separate analysis of the Nieuwkoop-Faber (NF) stage ≤60 and >60 animals creating a total of 12 data sets. On study day 7, significant positive correlation between HLL and SVL or body weight was observed in eight and seven of the 10 data sets, respectively (r = 0.608-0.843 and 0.583-0.876). On study day 21, significant positive correlation between HLL and SVL or body weight was found in three and four of the 12 data sets, respectively (r = 0.452, 0.480 and 0.553 and r = 0.621, 0.546, 0.564 and 0.378). Significant positive correlation between HLL and SVL was found in three of five studies, including ≤NF stage 60 data (r = 0.564, 0.546 and 0.621). In one of eight studies, including >NF stage 60 data, the positive correlation between HLL and body weight was determined (r = 0.378). Negative or no correlation between HLL and SVL or body weight was found in the other late stage data sets. Therefore, use of SVL-normalized HLL to assess thyroid-mediated effects in X. laevis tadpoles is not warranted. HL stage relative to body stage should be considered.

Endocrine Disruption: Current approaches for regulatory testing and assessment of plant protection products are fit for purpose.

The ongoing debate concerning the regulation of endocrine disruptors, has increasingly led to questions concerning the current testing of chemicals and whether this is adequate for the assessment of potential endocrine disrupting effects. This paper describes the current testing approaches for plant protection product (PPP) active substances in the European Union and the United States and how they relate to the assessment of endocrine disrupting properties for human and environmental health. This includes a discussion of whether the current testing approaches cover modalities other than the estrogen, androgen, thyroid and steroidogenesis (EATS) pathways, sensitive windows of exposure, adequate assessment of human endocrine disorders and wildlife species, and the determination of thresholds for endocrine disruption. It is concluded, that the scope and nature of the core and triggered data requirements for PPP active substances are scientifically robust to address adverse effects mediated through endocrine mode(s) of action and to characterise these effects in terms of dose response.

Risk assessment considerations for plant protection products and terrestrial life-stages of amphibians.

Some amphibians occur in agricultural landscapes during certain periods of their life cycle and consequently might be exposed to plant protection products (PPPs). While the sensitivity of aquatic life-stages is considered to be covered by the standard assessment for aquatic organisms (especially fish), the situation is less clear for terrestrial amphibian life-stages. In this paper, considerations are presented on how a risk assessment for PPPs and terrestrial life-stages of amphibians could be conducted. It discusses available information concerning the toxicity of PPPs to terrestrial amphibians, and their potential exposure to PPPs in consideration of aspects of amphibian biology. The emphasis is on avoiding additional vertebrate testing as much as possible by using exposure-driven approaches and by making use of existing vertebrate toxicity data, where appropriate. Options for toxicity testing and risk assessment are presented in a flowchart as a tiered approach, progressing from a non-testing approach, to simple worst-case laboratory testing, to extended laboratory testing, to semi-field enclosure tests and ultimately to full-scale field testing and monitoring. Suggestions are made for triggers to progress to higher tiers. Also, mitigation options to reduce the potential for exposure of terrestrial life-stages of amphibians to PPPs, if a risk were identified, are discussed. Finally, remaining uncertainties and research needs are considered by proposing a way forward (road map) for generating additional information to inform terrestrial amphibian risk assessment.

Prospective aquatic risk assessment for chemical mixtures in agricultural landscapes.

Environmental risk assessment of chemical mixtures is challenging because of the multitude of possible combinations that may occur. Aquatic risk from chemical mixtures in an agricultural landscape was evaluated prospectively in 2 exposure scenario case studies: at field scale for a program of 13 plant-protection products applied annually for 20 yr and at a watershed scale for a mixed land-use scenario over 30 yr with 12 plant-protection products and 2 veterinary pharmaceuticals used for beef cattle. Risk quotients were calculated from regulatory exposure models with typical real-world use patterns and regulatory acceptable concentrations for individual chemicals. The results could differentiate situations when there was concern associated with single chemicals from those when concern was associated with a mixture (based on concentration addition) with no single chemical triggering concern. Potential mixture risk was identified on 0.02 to 7.07% of the total days modeled, depending on the scenario, the taxa, and whether considering acute or chronic risk. Taxa at risk were influenced by receiving water body characteristics along with chemical use profiles and associated properties. The present study demonstrates that a scenario-based approach can be used to determine whether mixtures of chemicals pose risks over and above any identified using existing approaches for single chemicals, how often and to what magnitude, and ultimately which mixtures (and dominant chemicals) cause greatest concern. Environ Toxicol Chem 2018;37:674-689. © 2017 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Development and validation of an OECD reproductive toxicity test guideline with the mudsnail Potamopyrgus antipodarum (Mollusca, Gastropoda).

Mollusks are known to be uniquely sensitive to a number of reproductive toxicants including some vertebrate endocrine disrupting chemicals. However, they have widely been ignored in environmental risk assessment procedures for chemicals. This study describes the validation of the Potamopyrgus antipodarum reproduction test within the OECD Conceptual Framework for Endocrine Disrupters Testing and Assessment. The number of embryos in the brood pouch and adult mortality serve as main endpoints. The experiments are conducted as static systems in beakers filled with artificial medium, which is aerated trough glass pipettes. The test chemical is dispersed into the medium, and adult snails are subsequently introduced into the beakers. After 28 days the reproductive success is determined by opening the brood pouch and embryo counting. This study presents the results of two validation studies of the reproduction test with eleven laboratories and the chemicals tributyltin (TBT) with nominal concentrations ranging from 10 to 1000 ng TBT-Sn/L and cadmium with concentrations from 1.56 to 25 µg/L. The test design could be implemented by all laboratories resulting in comparable effect concentrations for the endpoint number of embryos in the brood pouch. After TBT exposure mean EC10, EC50, NOEC and LOEC were 35.6, 127, 39.2 and 75.7 ng Sn/L, respectively. Mean effect concentrations in cadmium exposed snails were, respectively, 6.53, 14.2, 6.45 and 12.6 µg/L. The effect concentrations are in good accordance with already published data. Both validation studies show that the reproduction test with P. antipodarum is a well-suited tool to assess reproductive effects of chemicals.

Validation of the OECD reproduction test guideline with the New Zealand mudsnail Potamopyrgus antipodarum using trenbolone and prochloraz.

The Organisation for Economic Cooperation and Development (OECD) provides several standard test methods for the environmental hazard assessment of chemicals, mainly based on primary producers, arthropods, and fish. In April 2016, two new test guidelines with two mollusc species representing different reproductive strategies were approved by OECD member countries. One test guideline describes a 28-day reproduction test with the parthenogenetic New Zealand mudsnail Potamopyrgus antipodarum. The main endpoint of the test is reproduction, reflected by the embryo number in the brood pouch per female. The development of a new OECD test guideline involves several phases including inter-laboratory validation studies to demonstrate the robustness of the proposed test design and the reproducibility of the test results. Therefore, a ring test of the reproduction test with P. antipodarum was conducted including eight laboratories with the test substances trenbolone and prochloraz and results are presented here. Most laboratories could meet test validity criteria, thus demonstrating the robustness of the proposed test protocol. Trenbolone did not have an effect on the reproduction of the snails at the tested concentration range (nominal: 10-1000 ng/L). For prochloraz, laboratories produced similar EC10 and NOEC values, showing the inter-laboratory reproducibility of results. The average EC10 and NOEC values for reproduction (with coefficient of variation) were 26.2 µg/L (61.7%) and 29.7 µg/L (32.9%), respectively. This ring test shows that the mudsnail reproduction test is a well-suited tool for use in the chronic aquatic hazard and risk assessment of chemicals.

Development and validation of an OECD reproductive toxicity test guideline with the pond snail Lymnaea stagnalis (Mollusca, Gastropoda).

The OECD test guideline development program has been extended in 2011 to establish a partial life-cycle protocol for assessing the reproductive toxicity of chemicals to several mollusk species, including the great pond snail Lymnaea stagnalis. In this paper, we summarize the standard draft protocol for a reproduction test with this species, and present inter-comparison results obtained in a 56-day prevalidation ring-test using this protocol. Seven European laboratories performed semi-static tests with cultured snails of the strain Renilys® exposed to nominal concentrations of cadmium chloride (from 53 to 608µgCdL(-1)). Cd concentrations in test solutions were analytically determined to confirm accuracy in the metal exposure concentrations in all laboratories. Physico-chemical and biological validity criteria (namely dissolved oxygen content >60% ASV, water temperature 20±1°C, control snail survival >80% and control snail fecundity >8 egg-masses per snail over the test period) were met in all laboratories which consistently demonstrated the reproductive toxicity of Cd in snails using the proposed draft protocol. Effect concentrations for fecundity after 56days were reproducible between laboratories (68

Mind the gap: Concerns using endpoints from endocrine screening assays in risk assessment.

Endocrine screening assays not only provide mechanistic information on the potential of a substance to interact with the endocrine system, but also data potentially relevant for risk assessment. However, these screening assays have a number of limitations that should be considered before the direct use of such data for risk assessment purposes. This paper discusses the limitations that should be considered for both human and environmental risk assessment. A proposal is made to provide an objective and transparent process in order to consider which endpoint(s) might be incorporated into a risk assessment, and when more definitive studies may be of value. The proposal is complemented with an easy-to-follow flowchart to aid industry scientists and regulators when evaluating the relevance of these data. Such an approach is necessary to ensure the appropriate use of screening data to further our understanding of the eco/toxicological profile of substances undergoing screening.

Risk assessment of endocrine active chemicals: identifying chemicals of regulatory concern.

The European regulation on plant protection products (1107/2009) (EC, 2009a), the revisions to the biocides Directive (COM[2009]267) (EC, 2009b), and the regulation concerning chemicals (Regulation (EC) No. 1907/2006 'REACH') (EC.2006) only support the marketing and use of chemical products on the basis that they do not induce endocrine disruption in humans or wildlife species. In the absence of agreed guidance on how to identify and evaluate endocrine activity and disruption within these pieces of legislation a European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) task force was formed to provide scientific criteria that may be used within the context of these three legislative documents. The resulting ECETOC technical report (ECETOC, 2009a) and the associated workshop (ECETOC, 2009b) presented a science-based concept on how to identify endocrine activity and disrupting properties of chemicals for both human health and the environment. The synthesis of the technical report and the workshop report was published by the ECETOC task force (Bars et al., 2011a,b). Specific scientific criteria for the determination of endocrine activity and disrupting properties that integrate information from both regulatory (eco)toxicity studies and mechanistic/screening studies were proposed. These criteria combined the nature of the adverse effects detected in studies which give concern for endocrine toxicity with an understanding of the mode of action of toxicity so that adverse effects can be explained scientifically. A key element in the data evaluation is the consideration of all available information in a weight-of-evidence approach. However, to be able to discriminate chemicals with endocrine properties of low concern from those of higher concern (for regulatory purposes), the task force recognised that the concept needed further refinement. Following a discussion of the key factors at a second workshop of invited regulatory, academic and industry scientists (ECETOC, 2011), the task force developed further guidance, which is presented in this paper. For human health assessments these factors include the relevance to humans of the endocrine mechanism of toxicity, the specificity of the endocrine effects with respect to other potential toxic effects, the potency of the chemical to induce endocrine toxicity and consideration of exposure levels. For ecotoxicological assessments the key considerations include specificity and potency, but also extend to the consideration of population relevance and negligible exposure. It is intended that these complement and reinforce the approach originally described and previously published in this journal (Bars et al., 2011a,b).

Aquatic toxicity tests with substances that are poorly soluble in water and consequences for environmental risk assessment.

Aquatic toxicity tests with substances that are poorly soluble in water have been conducted using different methods, and estimates of toxicity have varied accordingly. The present study illustrates differences in toxicity values resulting from variation in test designs and solution preparation methods, and offers guidance on the best way to conduct these tests. Consequences for environmental risk assessment and classification are also discussed. The present study mainly considers active ingredients of plant protection products, but is also considered relevant to other chemicals. It is recommended that toxicity tests be conducted only up to the saturation limit, dispersants avoided, and solvents used only if necessary to support handling and speed of dissolution. Analytical measurements of exposure concentrations should reflect what organisms are exposed to. If acute toxicity testing at the saturation limit yields no adverse effects, further testing should not normally be required; the toxicity value of the endpoints should be considered as the saturation limit and adverse classification should not be required. Chronic testing, if required, should then be conducted at the practical saturation limit as this is the most realistic worst-case exposure scenario. If no adverse effects occur, the risk should be acceptable because higher aqueous exposure cannot occur. This could be substantiated by testing additional species. Assessment factors on no observed effect concentration (NOEC) values at the saturation limit require careful consideration in the risk assessment to avoid unnecessarily low regulatory acceptable concentrations.

Science based guidance for the assessment of endocrine disrupting properties of chemicals.

The European legislation on plant protection products (Regulation (EC) No. 1107/2009) and biocides (Directive 98/8/EC), as well as the regulation concerning chemicals (Regulation (EC) No. 1907/2006 'REACH') only support the marketing and use of chemical products on the basis that they do not induce endocrine disruption in humans or non-target species. However, there is currently no agreed guidance on how to identify and evaluate endocrine activity and disruption. Consequently, an ECETOC task force was formed to provide scientific criteria that may be used within the context of these three legislative documents. Specific scientific criteria for the determination of endocrine disrupting properties that integrate information from both regulatory (eco)toxicity studies and mechanistic/screening studies are proposed. These criteria combine the nature of the adverse effects detected in studies which give concern for endocrine toxicity with an understanding of the mode of action of toxicity so that adverse effects can be explained scientifically. The criteria developed are presented in the form of flow charts for assessing relevant effects for both humans and wildlife species. In addition, since not all chemicals with endocrine disrupting properties are of equal hazard, assessment of potency is also proposed to discriminate chemicals of high concern from those of lower concern. The guidance presented in this paper includes refinements made to an initial proposal following discussion of the criteria at a workshop of invited regulatory, academic and industry scientists.

Chironomids: suitable test organisms for risk assessment investigations on the potential endocrine disrupting properties of pesticides.

Selecting an appropriate invertebrate assay has been a primary goal of national and international testing programs for endocrine disrupting chemicals. The available information on the endocrine system, its hormones and their modes of action in controlling physiological processes in invertebrates is limited and the selection of appropriate test species still presents a challenge. This paper outlines the development of a higher-tier full life cycle (FLC) test for pesticides with the non-biting midge Chironomus riparius (Insecta, Diptera, Chironomidae). As an insect, C. riparius represents the species' richest and ecologically one of the most important groups of invertebrates. In addition, the endocrine system of insects is one of the best studied among the invertebrates. Acute and chronic tests with Chironomus spp. are commonly used for testing and risk assessment of agrochemicals. A chironomid FLC test protocol has been developed and its suitability investigated in an inter-laboratory comparison. The protocol used is based on existing OECD and US-EPA test methods. To verify the suitability of the test to generate endpoints that encompass adverse effects on the arthropod endocrine system, a juvenile hormone analog was selected as positive control substance. Results have demonstrated that the proposed chironomid FLC can be performed in separate laboratories and that the selected arthropod juvenile hormone mimic causes effects. However, the observed toxicity is not proof of an endocrine disruptive mechanism and could equally be evoked by other compounds. Contrary to a screening assay, which aims at revealing a substance's mode-of-action, the FLC test generates robust, population-relevant endpoints that can be used in the risk assessment of agrochemicals. Since the initial results presented in this paper are encouraging we propose to complete the validation of this assay under OECD with high priority.