Risk assessment of local dermal effects and skin sensitisation under the EU Chemicals Regulation REACH: a proposal for a qualitative, exposure scenario specific, approach

Within the framework of REACH, an assessment regarding local dermal effects and skin sensitisation should be performed for substances. Quantitative hazard information for these effects is often not available. Furthermore, it is difficult to relate the way in which animals are exposed in dermal toxicity studies directly to dermal exposure in practice. In the absence of quantitative information, a qualitative assessment for dermal effects is the most reasonable option. The qualitative approach as proposed in the REACH guidance recommends only general risk management measures (RMM) for three categories with a low, moderate and high identified hazard, without specifying which RMM are needed for a specific exposure scenario. We propose to differentiate frequency of exposure based on differences in activities and to compare measured and estimated local skin exposure levels with rules of thumb for evaluation of control of risks per hazard category. For workers, specific RMM regimes are assigned to each combination of hazard category and process category (PROC). For consumers, a strategy in which RMM are arranged from product-integrated measures to the use of personal protective equipment (PPE) is presented. Our approach may be transferred into automated assessment tools like Chesar and CEFIC GES.     

An assessment of the skin sensitisation hazard of a group of polyfunctional silicones using a weight of evidence approach

Discordant results were observed when testing five prototype polyfunctional silicone materials for skin sensitization potential in the murine local lymph node assay (LLNA) and in the guinea pig maximization test (GPMT). While all five silicone materials were consistently negative in the GPMT, the testing in the LLNA revealed weak to moderate skin sensitisation potential for four of the five test materials. Neither study quality nor other known chemical factors could explain these findings. Further analysis did not provide sufficient evidence for a link between the LLNA responses and the irritancy of the test substances. Only in the case of one of the test materials, the occurrence of an excessive level of irritation could be linked to the positive LLNA result. Considering all existing information including physico–chemical and structure activity and animal data as well as existing human experience from silicone exposures at the workplace or their use in cosmetic products, the weight of evidence suggests that none of the examined silicone materials represents a significant skin sensitization hazard to humans. The suitability of the LLNA appears questionable for this class of materials. In case of any additional data needs for other or new silicone materials, the skin sensitization testing strategy will require careful evaluation and will need to be set up on a case by case basis.     

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.     

Classification of contact allergens according to potency: proposals

It is clear that contact allergens vary substantially with regard to the relative potency with which they are able to induce skin sensitisation. Considerations of potency will in the future become a significant factor in the classification of skin sensitising chemicals. It is therefore appropriate to establish what is known of potency and thresholds in the induction of skin sensitisation and the elicitation of allergic contact dermatitis, and to identify approaches that might be available for assessment of relative potency for the purposes of categorising chemical allergens. This paper was prepared by an ECETOC (European Centre for Ecotoxicology and Toxicology) Task Force that had the objective of recommending approaches for the measurement of potency and definition of thresholds for both the induction and elicitation of contact sensitisation. The deliberations recorded here build upon recommendations made previously by an ECETOC Task Force that considered the conduct of standard skin sensitisation test methods for the purposes of hazard identification and risk assessment (ECETOC, Monograph No. 29, Brussels, 2000). The emphasis in this present paper is also on standard and accepted methods for the assessment of skin sensitisation, and for which OECD guidelines are available: the local lymph node assay (LLNA), the guinea pig maximisation test and the occluded patch test of Buehler. For various reasons, discussed in detail herein, attention focused primarily upon consideration of categorisation of chemical allergens and the identification of thresholds with respect to the induction of skin sensitisation, rather than the elicitation of allergic contact dermatitis. It is concluded that although the LLNA is the method of choice for the determination of skin sensitisation potency for the purposes of categorisation, if data are already available from appropriate guinea pig tests then their judicious interpretation may provide information of value in determinations of potency and categorisation. Included here are detailed and specific recommendations for how best the results of the three test methods considered can be used for the categorisation of chemical allergens as a function of skin sensitisation potency.     

Behaviour of chemical respiratory allergens in novel predictive methods for skin sensitisation

Asthma resulting from sensitisation of the respiratory tract to chemicals is an important occupational health issue, presenting many toxicological challenges. Most importantly there are no recognised predictive methods for respiratory allergens. Nevertheless, it has been found that all known chemical respiratory allergens elicit positive responses in assays for skin sensitising chemicals. Thus, chemicals failing to induce a positive response in skin sensitisation assays such as the local lymph node assay (LLNA) lack not only skin sensitising activity, but also the potential to cause respiratory sensitisation. However, it is unclear whether it will be possible to regard chemicals that are negative in in vitro skin sensitisation tests also as lacking respiratory sensitising activity. To address this, the behaviour of chemical respiratory allergens in the LLNA and in recently validated non-animal tests for skin sensitisation have been examined. Most chemical respiratory allergens are positive in one or more newly validated non-animal test methods, although the situation varies between individual assays. The use of an integrated testing strategy could provide a basis for recognition of most respiratory sensitising chemicals. However, a more complete picture of the performance characteristics of such tests is required before specific recommendations can be made.     

The OSIRIS Weight of Evidence approach: ITS for skin sensitisation

Within the EU FP6 project OSIRIS approaches to Integrated Testing Strategies (ITSs) were developed, with the aim to facilitate the use of non-test and non-animal testing information in regulatory risk assessment of chemicals. This paper describes an analytical Weight-of-Evidence (WoE) approach to an ITS for the endpoint of skin sensitisation. It specifically addresses the European chemicals legislation REACH, but the concept is readily applicable to ITS and WoE procedures in other regulatory frameworks, and for other toxicological endpoints. Bayesian statistics are applied to estimate the reliability of a conclusion on the sensitisation potential of a chemical, combining evidence from different information sources such as QSAR model predictions, in vitro and in vivo test results. The methodology allows for adaptation of the weight of individual information sources to account for the different levels of reliability of the individual ITS components. The calculated reliability of the WoE conclusion gives an objective, transparent and reproducible measure to decide if the information requirements for data evaluation are satisfied. Furthermore, in case the WoE is not sufficient, it gives the possibility to evaluate a priori if and how it will be possible to fulfil the information requirements with additional tests and/or model predictions.     

Extension of the Dermal Sensitisation Threshold (DST) approach to incorporate chemicals classified as reactive

The evaluation of chemicals for their skin sensitising potential is an essential step in ensuring the safety of ingredients in consumer products. Similar to the Threshold of Toxicological Concern, the Dermal Sensitisation Threshold (DST) has been demonstrated to provide effective risk assessments for skin sensitisation in cases where human exposure is low. The DST was originally developed based on a Local Lymph Node Assay (LLNA) dataset and applied to chemicals that were not considered to be directly reactive to skin proteins, and unlikely to initiate the first mechanistic steps leading to the induction of sensitisation. Here we have extended the DST concept to protein reactive chemicals. A probabilistic assessment of the original DST dataset was conducted and a threshold of 64 μg/cm² was derived. In our accompanying publication, a set of structural chemistry based rules was developed to proactively identify highly reactive and potentially highly potent materials which should be excluded from the DST approach. The DST and rule set were benchmarked against a test set of chemicals with LLNA/human data. It is concluded that by combining the reactive DST with knowledge of chemistry a threshold can be established below which there is no appreciable risk of sensitisation for protein-reactive chemicals.     

An approach for the delineation of a generic cut-off value for local respiratory tract irritation by irritating or corrosive substances as a pragmatic tool to fulfill REACH requirements

Under the current European legislation for the Registration, Evaluation, Authorisation and restriction of Chemicals (REACHs) a Derived No Effect Level (DNEL) has to be delineated for acute and chronic inhalation effects. The majority of available experimental studies are performed by the oral route of exposure. Route to route extrapolation poses particular problems for irritating or corrosive substances but the necessity for additional animal studies with inhalation exposure needs to be balanced with the regulatory information requirements. Existing occupational exposure limits (OEL) as surrogate for cut-off limits representing safe exposure under working conditions were grouped under certain criteria for substances that are legally classified in Europe as irritating or corrosive. As a result, it was shown that the OEL for irritating substances in this dataset is not lower than 10 mg/m³ and for corrosives not lower than 1 mg/m³. Under certain conditions these generic limits could be applied as a pragmatic, but still sufficiently reliable and protective upper cut-off limit approach to avoid additional animal tests with irritating or corrosive chemicals. The respective systemic toxicity profiles and physical–chemical properties need to be considered. Specific exclusion criteria for the discussed concept apply.     

Refinement of the Dermal Sensitisation Threshold (DST) approach using a larger dataset and incorporating mechanistic chemistry domains

An essential step in ensuring the toxicological safety of ingredients in consumer products is the evaluation of their skin sensitising potential. Where skin exposure is low, it is possible to conduct a risk assessment using the Dermal Sensitisation Threshold (DST), a process similar to that of the Threshold of Toxicological Concern. This paper describes work building on that previously published, whose aim was to produce a more robust tool for assessing the safety of consumer products. This consisted of expanding the Local Lymph Node Assay dataset used to define the original DST and classifying chemicals in the dataset according to their mechanistic chemistry domains. A DST of 900μg/cm(2) was derived for chemicals classified as non-reactive and non-proreactive. This value was benchmarked against human potency data for 58 fragrance allergens and was lower than the measured No Expected Sensitisation Levels for those classified as non-reactive. Use of this DST will help to eliminate the need for animal testing of non-reactive and non-proreactive chemicals where skin exposure is sufficiently low. For chemicals where a Quantitative Risk Assessment based on the DST does not give an adequate margin of safety, and those classified as reactive, a case-by-case risk assessment will be required.     

Azodicarbonamide (ADCA): A reconsideration of classification as a respiratory sensitiser

Azodicarbonamide (ADCA) is widely used by industry in the manufacture of a variety of products. ADCA has been classified as a respiratory allergen, and the purpose of this article was to consider whether this classification is appropriate based upon the available data. Here both clinical experience and relevant experimental data have been reviewed. Although there have been reports of an association between workplace exposure to ADCA and symptoms of respiratory allergy and occupational asthma, the evidence is less than persuasive, with in many instances a lack of properly controlled and executed diagnostic procedures. In addition, ADCA fails to elicit positive responses in mouse and guinea pig predictive tests for skin sensitisation; a lack of activity that is regarded as being inconsistent with respect to respiratory sensitising potential. Collectively, the data reviewed here do not provide an adequate basis for the classification of ADCA as a respiratory allergen.     

A comparative survey of chemistry-driven in silico methods to identify hazardous substances under REACH

This paper presents an inventory of in silico screening tools to identify substance properties of concern under the European chemicals’ legislation REACH. The objective is to support the selection and implementation of appropriate tools as building blocks within integrated testing strategies (ITS). The relevant concerns addressed are persistence, bioaccumulation potential, acute and long-term aquatic toxicity, PBT/vPvB properties ((very) persistent, (very) bioaccumulative, toxic), CMR (carcinogenicity, mutagenicity, reproductive toxicity), endocrine disruption and skin sensitisation. The inventory offers a comparative evaluation of methods with respect to the underlying algorithms (how does the method work?) and the applicability domains (when does the method work?) as well as their limitations (when does the method not work?). The inventory explicitly addresses the reliability of predictions of different in silico models for diverse chemicals by applicability domain considerations. The confidence in predictions can be greatly improved by consensus modelling that allows for taking conflicting results into account. The inventory is complemented by a brief discussion of socio-economic tools for assessing the potential efficiency gains of using in silico methods compared to traditional in vivo testing of chemical hazards.     

Quantitative risk assessment for skin sensitisation: Consideration of a simplified approach for hair dye ingredients

With the availability of the local lymph node assay, and the ability to evaluate effectively the relative skin sensitizing potency of contact allergens, a model for quantitative-risk-assessment (QRA) has been developed. This QRA process comprises: (a) determination of a no-expected-sensitisation-induction-level (NESIL), (b) incorporation of sensitization-assessment-factors (SAFs) reflecting variations between subjects, product use patterns and matrices, and (c) estimation of consumer-exposure-level (CEL). Based on these elements an acceptable-exposure-level (AEL) can be calculated by dividing the NESIL of the product by individual SAFs. Finally, the AEL is compared with the CEL to judge about risks to human health.     

Principles for identification of High Potency Category Chemicals for which the Dermal Sensitisation Threshold (DST) approach should not be applied

An essential step in ensuring the toxicological safety of chemicals used in consumer products is the evaluation of their skin sensitising potential. The sensitising potency, coupled with information on exposure levels, can be used in a Quantitative Risk Assessment (QRA) to determine an acceptable level of a given chemical in a given product. Where consumer skin exposure is low, a risk assessment can be conducted using the Dermal Sensitisation Threshold (DST) approach, avoiding the need to determine potency experimentally. Since skin sensitisation involves chemical reaction with skin proteins, the first step in the DST approach is to assess, on the basis of the chemical structure, whether the chemical is expected to be reactive or not. Our accompanying publication describes the probabilistic derivation of a DST of 64 μg/cm² for chemicals assessed as reactive. This would protect against 95% of chemicals assessed as reactive, but the remaining 5% would include chemicals with very high potency. Here we discuss the chemical properties and structural features of high potency sensitisers, and derive an approach whereby they can be identified and consequently excluded from application of the DST.     

Exposure-triggered reproductive toxicity testing under the REACH legislation: a proposal to define significant/relevant exposure

Under the new REACH legislation, toxicological testing is required in relation to annual tonnages produced or imported. Requirements for toxicological information increase when production volume increases. The respective information requirements are laid down in the REACH Annexes VII-X. Concerning human toxicology, certain toxicological tests may be waived under specific conditions. Aside from waiving criteria such as technical feasibility, exposure plays a decisive role in the waiving process with the consequence that toxicological testing will not be required in case of "no relevant exposure", "limited exposure", "no exposure" or "no significant exposure" (as expressed in the documents). However, up to now criteria are lacking which precisely define these terms. Attempts have been made to establish cut-off criteria between "non-relevant" and "relevant" (detrimental) exposure based on external exposure concentrations and the threshold of toxicological concern (TTC) principle. In this paper we make a proposal and describe a strategy how to define the currently insufficiently described terms "relevant/significant" exposure. We propose to define relevant/significant exposure based on an endpoint-specific TTC approach, starting from a comparison of the tentative external exposure to the specific TTC. This can be followed by a refinement of exposure estimates and may culminate in the experimental determination of internal and target tissue exposure. This strategy enables a well-founded assessment of what "no relevant exposure" is and safeguards an appropriate level of protection of the general population. The feasibility of the approach is demonstrated for reproductive toxicity endpoints.     

A future approach to measuring relative skin sensitising potency: a proposal

Current approaches to skin sensitisation risk assessment are dependent upon the availability of information regarding two fundamental parameters. Firstly, data relating to the relative skin sensitising potency of the chemical, and secondly, information regarding likely conditions of human exposure. During the past two decades, much has been achieved in terms of refining methods capable of informing these parameters. For example, the development of the local lymph node assay (LLNA) has made it possible to predict skin sensitising hazard, and to determine relative skin sensitising potency, in a way that was not possible previously. Taken together with accurate information about predicted exposure, such potency data can be used to facilitate the derivation of effective risk assessments. However, although the LLNA provides an integrated assessment of skin sensitising activity, it does require the use of experimental animals and there is growing enthusiasm for designing robust alternative approaches that will reduce or obviate that need. Progress is being made in defining alternative experimental strategies that avoid animal use, but it is clear that accurate characterisation of skin sensitisation hazards will require the effective integration of various sources of information. For this reason, we exemplify here one possible approach that, in theory, provides a framework for not only the identification of skin sensitising chemicals, but also the estimation of relative sensitising potency. This paradigm depends upon development of an understanding of the various biological, biochemical and chemical factors that impact on the allergenic properties of chemicals and the acquisition of skin sensitisation, and an ability to measure these in vitro.     

Human biomonitoring as a pragmatic tool to support health risk management of chemicals--examples under the EU REACH programme

REACH requires health risk management for workers and the general population and introduced the concept of Derived No-Effect Level (DNEL). DNELs must be derived for all substances that are classified as health hazards. As with analogues to other health-risk based guidance values, such as reference doses (RfDs) and tolerable daily intakes (TDIs), risk to health is considered negligible if the actual exposure is less than the DNEL. Exposure assessment is relatively simple for occupational situations but more complex for the general public, in which exposure may occur via multiple pathways, routes, and media. For such complex or partially defined exposure scenarios, human biomonitoring gives a snapshot of internal or absorbed dose of a chemical and is often the most reliable exposure assessment methodology as it integrates exposures from all routes. For human risk management human biomonitoring data can be interpreted using the recently developed concept of Biomonitoring Equivalents (BE). Basically, a BE translates an established reference value into a biomarker concentration using toxicokinetic data. If the results of an exposure assessment using human biomonitoring indicate that the levels measured are below the DNEL-based BE (BE(DNEL)), it would indicate that the combined exposure via all potential exposure routes is unlikely to pose a risk to human health and that health risk management measures might not be needed. Hence, BEs do not challenge existing risk assessments but rather build upon them to help risk management, the ultimate goal of any risk assessment. A challenge in implementing this approach forms the limited availability of toxicokinetic information for many substances. However, methodologies such as generic physiologically-based toxicokinetic models, which allow estimation of biomarker concentrations based on physicochemical properties, are being developed for less data-rich chemicals. Use of BE by regulatory authorities will allow initial screening of population exposure to chemicals to identify those chemicals requiring more detailed risk and exposure assessment, assisting in priority setting and ultimately leading to improved product stewardship and risk management.     

Statistical Evaluation of the Orthogonal Pooling Strategy for High-Throughput Screening

In high-throughput screening (HTS), compounds can be tested in mixtures using the orthogonal pooling strategy. However, several experimental studies that used two-way orthogonal pooling have shown that the rate of false negative testing errors is still not negligible. Because replicate screening is prohibited due to the large number of compounds that need to be tested, the false negative results eventually lead to loss of active compounds. This article generalizes the two-way orthogonal pooling strategy to a three-way pooling and develops statistical methods to evaluate the false negative rate (FNR) and the false positive rate (FPR) for the two-way and three-way pooling strategies. Calculations show that the three-way pooling strategy decreases the FNR but increases the FPR to approximately three times that of the two-way pooling strategy. In terms of strategy selection, three-way pooling requires 50% more tests than two-way pooling but gains benefit when the testing technique has low sensitivity and high specificity, while two-way pooling may be preferred when the technique has high sensitivity and low specificity.