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.     

Assessment of reproductive toxicity under REACH

The European REACH regulation requires the evaluation of reproductive toxicity in screening tests according to OECD TG 421 and 422 for substances above the tonnage level of 10 tons/year. The overall aim of this paper is to increase flexibility in combination with a reduced number of experimental animals. Therefore, in contrast to the existing approach the registrant should have the possibility to file a dossier for a substance at the level of 10 tons/year and above also on the basis of data from a developmental toxicity study (OECD TG 414) plus a full-scale subacute toxicity study (OECD TG 407 according to the 1995 protocol). The proposed new test strategy takes into account overall considerations of duty of care and animal welfare. It enables an assessment of developmental toxicity on a definitive instead of a screening level. Registrants should be allowed to select between these two options, either the existing approach (OECD TG 421/407 and alternatively TG 422) or the approach proposed in this paper (OECD TG 407 plus TG 414).     

A regulatory approach to assess the potency of substances toxic to the reproduction

In order to develop a method for setting specific concentration limits (SCLs) for substances toxic to the reproduction within the European classification and labelling system, this study investigated possible parameters for reproductive toxicity potency and the quantitative distribution of those parameters. For that purpose, two databases were created comprising substances classified in the European Union for developmental toxicity or for effects on sexual function and fertility. For these substances six parameters including NOAEL, LOAEL and ED₁₀ were determined for effects on reproduction based on existing data summaries. The potency was defined independent of the type of reproductive effect as generally severe effects on reproduction warranting classification were already observed at the lowest dose showing reproductive effects. The reproductive toxicity potency range of substances in the databases was a factor of approximately one million. This shows that SCL setting is needed to adjust the classification of mixtures. The average potency distribution of substances classified according to the hazard classification as required by the European CLP regulation in category 1 versus category 2 was similar. The ED₁₀ for effects warranting classification is proposed as the best parameter for the potency based on its independence of administered dose levels.     

Hazard classification of chemicals inducing haemolytic anaemia: An EU regulatory perspective

Haemolytic anaemia is often induced following prolonged exposure to chemical substances. Currently, under EU Council Directive 67/548/EEC, substances which induce such effects are classified as dangerous and assigned the risk phrase R48 'Danger of serious damage to health by prolonged exposure.' Whilst the general classification criteria for this endpoint are outlined in Annex VI of this Directive, they do not provide specific information to assess haemolytic anaemia. This review produced by the EU Working Group on Haemolytic Anaemia provides a toxicological assessment of haemolytic anaemia and proposes criteria that can be used in the assessment for classification of substances which induce such effects. An overview of the primary and secondary effects of haemolytic anaemia which can occur in rodent repeated dose toxicity studies is given. A detailed analysis of the toxicological significance of such effects is then performed and correlated with the general classification criteria used for this endpoint. This review intends to give guidance when carrying out an assessment for classification for this endpoint and to allow for better transparency in the decision-making process on when to classify based on the presence of haemolytic anaemia in repeated dose toxicity studies. The extended classification criteria for haemolytic anaemia outlined in this review were accepted by the EU Commission Working Group on the Classification and Labelling of Dangerous Substances in September 2004.     

About hazard and risk assessment: Regulatory approaches in assessing safety in the European Union chemicals legislation

Hazard classification and labelling is the main and basic requirement for all industrial and consumer chemicals in the European Union, if they are not regulated under more specific legislation such as drugs, food ingredients or cosmetics. The first approach in hazard classification is hazard identification describing the hazardous properties of chemicals. Refinements in the classification criteria include the assessment of toxic potency (hazard characterisation) where feasible and the possibility to set higher or lower specific concentration limits for classification. In the past only a minor portion of the classified chemicals underwent a risk assessment including exposure assessment and risk characterisation. Risk assessment will become more frequent with the implementation of REACH. However, as risk assessment is rather labour-intensive even under REACH risk assessment will be performed in a targeted approach for a selected number of chemical substances while hazard classification and labelling will remain the basic approach for all chemicals.     

Efficacy of predictive modeling as a scientific criterion in dermal hazard identification for assignment of skin notations

Skin notations (SNs) represent a hazard characterization tool for alerting workers of health hazards associated with dermal contact with chemicals. This study evaluated the efficacy of a predictive model utilized by the National Institute for Occupational Safety and Health to identify dermal hazards based on potential of systemic absorption compared to hazard assignments based on dermal lethal dose 50% or logarithm of octanol-water partition coefficient. A total of 480 chemicals assigned an SN from at least one of seven institutes were selected and partitioned into seven hazard categories by frequency of SN assignment to provide a basis of evaluation for the predictivity of the examined criteria. We find that all three properties serve as a qualitative indicator in support of a dichotomous decision on dermal hazard; the predictive modeling was identified from a multiple regression analysis as the most significant indicator. The model generated estimates that corresponded to anticipated hazard potentials, suggesting a role of the model to further serve as a hazard-ranking tool. The hazard-ranking capability of the model was consistent with the scheme of acute toxicity classification in the Globally Harmonized System of Classification and Labeling of Chemicals.     

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 and Bars et al., 2011b). 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 and Bars et al., 2011b).     

A proposal to improve clarity and communication in the EU Classification process for chemicals for carcinogenicity and reproductive and developmental toxicity

There is an issue in the EU classification of substances for carcinogenicity and for reproductive or developmental toxicity which has brought difficulties to those involved in the process. The issue lies in the inability of the classification system to distinguish between carcinogens and reproductive toxicants with different levels of concern. This has its origins in the early years of toxicology when it was thought that a relatively small number of chemicals would be either carcinogens or reproductive toxicants, but this has turned out not to be the case. This can cause problems in communicating to the users of chemicals, including the public, the nature of the hazard presented by chemicals. Processes have been developed within the classification system for setting specific concentration limits which assess the degree of hazard for carcinogens and reproductive toxicants as high, medium or low. However these categories are not otherwise used in classification. It is proposed that their wider use would bring the advantages of transparency, clarity of communication, certainty of the process and would allow chemicals with a high degree of hazard to be identified and managed in an appropriate way. Copyright © 2014. The Authors. Journal of Applied Toxicology Published by John Wiley & Sons Ltd.     

MeClas: An online tool for hazard identification and classification of complex inorganic metal-containing materials

MeClas is a web-based tool to generate (eco)toxicity hazard categories and corresponding classification & labelling information of inorganic metal-containing complex materials such as ores, concentrates, intermediates or alloys for which the manual application of the GHS/CLP rules is very complex and requires a high level of consistency. The tool comprises several tiers, aimed at the progressive refinement of classification through recognition of specific mineral content, speciation/mineralogy up to bio-availability corrections. Where relevant in a regional jurisdiction (EU and US), mandatory classification references are used complementary to high quality (eco)toxicity reference values (ERV/TRV) and self-classifications. MeClas addresses the GHS human health and environmental hazard endpoints, is based on an unambiguous algorithm defined under GHS/CLP, has a well defined domain of applicability and robust predictability. MeClas allows a consistent approach across companies in line with GHS ruling (and regional implementations), considering the metal specificities and related classification GHS/CLP Guidance, and the most up to date (eco)-toxicological hazard information on self-classifications and ERV/TRV.     

A consistent and transparent approach for calculation of Derived No-Effect Levels (DNELs) for petroleum substances

The REACH legislation introduced Derived No-Effect Levels (DNELs) which are defined as 'the levels of exposure above which humans should not be exposed'. DNELs were required for several categories of petroleum substances and CONCAWE developed a consistent approach for their derivation. First, the No-Observed Effect Level from a relevant study was corrected for pattern and route of exposure to obtain a modified Point-of-Departure (POD(modified)). Subsequently, the DNEL was calculated by dividing the POD(modified) by Assessment Factors (AFs) to adjust for inter- and intraspecies differences. If substance-specific information allowed, Informed Assessment Factors (IAFs), developed by CONCAWE were utilised. When little or no substance-specific information on those differences was known, default AFs from the guidance provided by ECHA were used. Some hazard endpoints did not lend themselves to calculation of DNELs (e.g. aspiration, dermal irritation, mutagenicity). DNEL calculation was considered not appropriate if adverse effects were not observed in tests conducted at a limit dose or if meaningful dose-response curves could not be developed. However, DNELs were calculated when hazards were identified, regardless of whether or not risk characterisation was required under REACH. Examples for gasoline, Lubricating Base Oils, gas oils and bitumen are provided to illustrate CONCAWE's approach.     

An enhanced tiered toxicity testing framework with triggers for assessing hazards and risks of commodity chemicals

This paper presents an enhanced integrated testing framework based on tiered testing and endpoint-specific decision triggers envisioned for application to commodity chemical safety assessments. The framework has two tiers in which exposure information can be integrated with hazard data at each Tier. Tier 1 tests are used to screen chemicals for major toxic effects (i.e., acute toxicity potential, target organs of repeat dose toxicity, genotoxicity potential, neurotoxicity potential, reproductive toxicity potential, immunotoxicity potential, and developmental toxicity potential), and to direct planning for more complex and targeted testing in Tier 2. The proposed decision triggers coupled with information on use and potential for exposure allow for scientifically-based decisions to be made about further testing in Tier 2, indicating which specific endpoints and tests warrant further evaluation, and which do not. The testing framework addresses risks to humans during all stages of development and provides data relevant to assessing hazards to sensitive subpopulations, such as infants and children. The REACH program in Europe and TSCA in the United States have led to an increased focus on development of hazard and risk information for chemicals used in industrial processes and consumer products. This framework and its toxicity decision triggers will allow for scientifically justified evaluation of chemicals that is comprehensive in terms of hazard screening, focuses resources on the specific complex tests that are most important for hazard characterization, and minimizes the use of animals.     

Potency values from the local lymph node assay: Application to classification,labelling and risk assessment

Hundreds of chemicals are contact allergens but there remains a need to identify and characterise accurately skin sensitising hazards. The purpose of this review was fourfold. First, when using the local lymph node assay (LLNA), consider whether an exposure concentration (EC3 value) lower than 100% can be defined and used as a threshold criterion for classification and labelling. Second, is there any reason to revise the recommendation of a previous ECETOC Task Force regarding specific EC3 values used for sub-categorisation of substances based upon potency? Third, what recommendations can be made regarding classification and labelling of preparations under GHS? Finally, consider how to integrate LLNA data into risk assessment and provide a rationale for using concentration responses and corresponding no-effect concentrations. Although skin sensitising chemicals having high EC3 values may represent only relatively low risks to humans, it is not possible currently to define an EC3 value below 100% that would serve as an appropriate threshold for classification and labelling. The conclusion drawn from reviewing the use of distinct categories for characterising contact allergens was that the most appropriate, science-based classification of contact allergens according to potency is one in which four sub-categories are identified: ‘extreme’, ‘strong’, ‘moderate’ and ‘weak’. Since draining lymph node cell proliferation is related causally and quantitatively to potency, LLNA EC3 values are recommended for determination of a no expected sensitisation induction level that represents the first step in quantitative risk assessment.     

Decision trees for evaluating skin and respiratory sensitizing potential of chemicals in accordance with European regulations

Guidance for determining the sensitizing potential of chemicals is available in EC Regulation No. 1272/2008 Classification, Labeling, and Packaging of Substances; REACH guidance from the European Chemicals Agency; and the United Nations Globally Harmonized System (GHS). We created decision trees for evaluating potential skin and respiratory sensitizers. Our approach (1) brings all the regulatory information into one brief document, providing a step-by-step method to evaluate evidence that individual chemicals or mixtures have sensitizing potential; (2) provides an efficient, uniform approach that promotes consistency when evaluations are done by different reviewers; (3) provides a standard way to convey the rationale and information used to classify chemicals. We applied this approach to more than 50 chemicals distributed among 11 evaluators with varying expertise. Evaluators found the decision trees easy to use and recipients (product stewards) of the analyses found that the resulting documentation was consistent across users and met their regulatory needs. Our approach allows for transparency, process management (e.g., documentation, change management, version control), as well as consistency in chemical hazard assessment for REACH, EC Regulation No. 1272/2008 Classification, Labeling, and Packaging of Substances and the GHS.     

Effect of maternal feed restriction on prenatal development in rats and rabbits – A review of published data

With respect to hazard classification for developmental toxicity under the CLP Regulation it is important to consider the possible influence of maternal toxicity. The aim of the present review was to characterize to which extent developmental effects could be caused by non-specific maternal toxicity. Such effects would not be relevant for classification. In prenatal developmental toxicity studies, the administration of high doses is given in the guideline. The associated non-specific systemic toxicity often affects the maternal body weight. Therefore, published results of studies in rats and rabbits, where maternal weight gain during gestation was inhibited by restricted feeding, were examined regarding developmental effects. In summary, maternal feed restriction resulted in a reduction of fetal body weight that was sometimes accompanied by delayed ossification in both species. Considering their magnitude these effects could be interpreted as secondary non-specific (i.e. not caused by a developmental toxicant) effects. Based on the limited number of available publications in total no further consequences on prenatal development by maternal feed restriction were observed.     

Classification schemes for carcinogenicity based on hazard-identification have become outmoded and serve neither science nor society

Classification schemes for carcinogenicity based solely on hazard-identification such as the IARC monograph process and the UN system adopted in the EU have become outmoded. They are based on a concept developed in the 1970s that chemicals could be divided into two classes: carcinogens and non-carcinogens. Categorization in this way places into the same category chemicals and agents with widely differing potencies and modes of action. This is how eating processed meat can fall into the same category as sulfur mustard gas. Approaches based on hazard and risk characterization present an integrated and balanced picture of hazard, dose response and exposure and allow informed risk management decisions to be taken. Because a risk-based decision framework fully considers hazard in the context of dose, potency, and exposure the unintended downsides of a hazard only approach are avoided, e.g., health scares, unnecessary economic costs, loss of beneficial products, adoption of strategies with greater health costs, and the diversion of public funds into unnecessary research. An initiative to agree upon a standardized, internationally acceptable methodology for carcinogen assessment is needed now. The approach should incorporate principles and concepts of existing international consensus-based frameworks including the WHO IPCS mode of action framework.     

The impact of REACH on classification for human health hazards

The REACH Regulation represents a major piece of chemical legislation in the EU and requires manufacturers and importers of chemicals to assess the safety of their substances. The classification of substances for their hazards is one of the crucial elements in this process. We analysed the effect of REACH on classification for human health endpoints by comparing information from REACH registration dossiers with legally binding, harmonised classifications. The analysis included 142 chemicals produced at very high tonnages in the EU, the majority of which have already been assessed in the past. Of 20 substances lacking a harmonised classification, 12 chemicals were classified in REACH registration dossiers. More importantly, 37 substances with harmonised classifications for human health endpoints had stricter classifications in registration dossiers and 29 of these were classified for at least one additional endpoint not covered by the harmonised classification. Substance-specific analyses suggest that one third of these additional endpoints emerged from experimental studies performed to fulfil information requirements under REACH, while two thirds resulted from a new assessment of pre-REACH studies. We conclude that REACH leads to an improved hazard characterisation even for substances with a potentially good data basis.     

The multi-dimensional embryonic zebrafish platform predicts flame retardant bioactivity

Flame retardant chemicals (FRCs) commonly added to many consumer products present a human exposure burden associated with adverse health effects. Under pressure from consumers, FRC manufacturers have adopted some purportedly safer replacements for first-generation brominated diphenyl ethers (BDEs). In contrast, second and third-generation organophosphates and other alternative chemistries have limited bioactivity data available to estimate their hazard potential. In order to evaluate the toxicity of existing and potential replacement FRCs, we need efficient screening methods. We built a 61-FRC library in which we systemically assessed developmental toxicity and potential neurotoxicity effects in the embryonic zebrafish model. Data were compared to publicly available data generated in a battery of cell-based in vitro assays from ToxCast, Tox21, and other alternative models. Of the 61 FRCs, 19 of 45 that were tested in the ToxCast assays were bioactive in our zebrafish model. The zebrafish assays detected bioactivity for 10 of the 12 previously classified developmental neurotoxic FRCs. Developmental zebrafish were sufficiently sensitive at detecting FRC structure-bioactivity impacts that we were able to build a classification model using 13 physicochemical properties and 3 embryonic zebrafish assays that achieved a balanced accuracy of 91.7%. This work illustrates the power of a multi-dimensional in vivo platform to expand our ability to predict the hazard potential of new compounds based on structural relatedness, ultimately leading to reliable toxicity predictions based on chemical structure.     

The Threshold of Toxicological Concern for prenatal developmental toxicity in rats and rabbits

The Threshold Toxicological Concern (TTC) is based on the concept that in absence of experimental data reasonable assurance of safety can be given if exposure is sufficiently low.Using the REACH database the low 5th percentile of the NO(A)EL distribution, for prenatal developmental toxicity (OECD guideline 414) was determined. For rats, (434 NO(A)ELs values) for maternal toxicity, this value was 10 mg/kg-bw/day. For developmental toxicity (469 NO(A)ELs): 13 mg/kg-bw/day. For rabbits, (100 NO(A)ELs), the value for maternal toxicity was 4 mg/kg-bw/day, for developmental toxicity, (112 NO(A)EL values): 10 mg/kg-bw/day. The maternal organism may thus be slightly more sensitive than the fetus. Combining REACH- (industrial chemicals) and published BASF-data (mostly agrochemicals), 537 unique compounds with NO(A)EL values for developmental toxicity in rats and 150 in rabbits were evaluated. The low 5th percentile NO(A)EL for developmental toxicity in rats was 10 mg/kg-bw/day and 9.5 mg/kg-bw/day for rabbits. Using an assessment factor of 100, a TTC value for developmental toxicity of 100 μg/kg-bw/day for rats and 95 μg/kg-bw/day for rabbits is calculated. These values could serve as guidance whether or not to perform an animal experiment, if exposure is sufficiently low. In emergency situations this value may be useful for a first tier risk assessment.     

A retrospective analysis of developmental toxicity studies in rat and rabbit: What is the added value of the rabbit as an additional test species?

In contrast to most toxicological tests, developmental studies are usually required in both a rodent and a non-rodent species. This study retrospectively assessed the added value of the rabbit developmental test when a rat developmental test is available. In contrast with previous reviews, we looked at developmental toxicity instead of teratogenicity, and took into account maternal toxicity in the evaluation of developmental toxicity. We analyzed data for 54 substances classified for developmental toxicity and 73 substances considered to be teratogenic in the rabbit and not in the rat in two previous reviews. On average, the rat and the rabbit developmental toxicity studies were similarly sensitive: the average ratio of the NOAELs between the two species was about one, and for most compounds there were no differences between rat and rabbit studies in terms of classification for developmental toxicity. For certain substances the developmental study in either one of the two species appeared to be more sensitive than in the other species. However, these differences are partly due to differences between studies other than the test species used. Overall, our analysis does not clearly indicate that the evaluation of developmental toxicity, as opposed to other types of toxicity, would specifically require the rabbit as an additional test species. The discrimination between direct and indirect (i.e., as a consequence of maternal toxicity) developmental effects was often doubtful, and is one of the factors that could explain the apparent differences between the two species. A more accurate assessment of maternal toxicity might improve the reliability of the results from a single developmental toxicity study. More knowledge about the interaction between maternal and developmental effects is required before decisions on omitting the requirement for the developmental toxicity testing in a second species can be considered.