Grouping approaches based on structure alone are insufficient to conclude about toxicological properties-the example of monoamine-based chelates.

Aminocarboxylic acid (monoamine-based) chelating agents such as GLDA, MGDA, NTA, and EDG are widely used in a variety of products and processes. In the European Union, based on the Green Deal and the Chemicals Strategy for Sustainability (CSS), there is an increasing tendency to speed up chemical hazard evaluation and to regulate chemicals by grouping substances based on molecular structure similarity. Recently, it was proposed to group polycarboxylic acid monoamines, hydroxy derivatives and their salts with monovalent cations, and to consider all group members as potential carcinogens based on the official CLP classification of one group member, viz. NTA, which is classified as suspected carcinogen Cat. 2. In this review, we show that a grouping approach for harmonized classification and labeling based on molecular structure alone, disregarding existing animal test data as well as current scientific and regulatory knowledge, would result in incorrect classification. Using such a simplistic, although considered pragmatic approach, classification of all group members upfront would not improve protection of human health. Instead, it could not only lead to unnecessary additional vertebrate animal testing but also to onerous and disproportionate restrictions being placed on the use of these valuable substances; some of these even being considered as green chemicals.

Identification of true chemical respiratory allergens: Current status, limitations and recommendations.

Asthma in the workplace is an important occupational health issue. It comprises various subtypes: occupational asthma (OA; both allergic asthma and irritant-induced asthma) and work-exacerbated asthma (WEA). Current regulatory paradigms for the management of OA are not fit for purpose. There is therefore an important unmet need, for the purposes of both effective human health protection and appropriate and proportionate regulation, that sub-types of work-related asthma can be accurately identified and classified, and that chemical respiratory allergens that drive allergic asthma can be differentiated according to potency. In this article presently available strategies for the diagnosis and characterisation of asthma in the workplace are described and critically evaluated. These include human health studies, clinical investigations and experimental approaches (structure-activity relationships, assessments of chemical reactivity, experimental animal studies and in vitro methods). Each of these approaches has limitations with respect to providing a clear discrimination between OA and WEA, and between allergen-induced and irritant-induced asthma. Against this background the needs for improved characterisation of work-related asthma, in the context of more appropriate regulation is discussed.

Zinc deficiency induced by the chelating agent DTPA and its regulatory interpretation for developmental toxicity classification.

Aminocarboxylic acid (ethylenediamine-based) chelating agents such as DTPA are widely used in a variety of products and processes. Recently, DTPA was classified in the European Union as a developmental toxicant CLP Category 1B. However, according to the CLP regulation (CLP, 2008) classification as a developmental toxicant requires a chemical to possess an intrinsic, specific property to do so. This paper provides overwhelming evidence that shows the developmental toxicity only seen at a sustained high dose of 1000 mg DTPA/kg bw/day in rats during pregnancy is mediated by zinc depletion which leads to non-specific secondary effects associated with zinc deficiency. Therefore, based on the CLP regulation itself, viz. the lack of a specific, intrinsic property, supported by significant differences in zinc kinetics and physiology between pregnant rats and pregnant women, DTPA should not be classified as a developmental toxicant. Moreover, classification for developmental toxicity resulting from zinc deficiency, and only observed at high doses, would not increase protection of human health; instead, it will only lead to onerous and disproportionate restrictions being placed on the use of this substance.

Investigations on learning and memory function in extended one-generation reproductive toxicity studies - when considered needed and based on what?

To justify investigations on learning and memory (L&M) function in extended one-generation reproductive toxicity studies (EOGRTS; Organization for Economic Co-operation and Development (OECD) test guideline (TG) 443) for registration under Registration, Evaluation, Authorization, and Restriction of Chemical (REACH), the European Chemicals Agency has referred to three publications based on which the Agency concluded that "perturbation of thyroid hormone signaling in offspring affects spatial cognitive abilities (learning and memory)" and "Therefore, it is necessary to conduct spatial learning and memory tests for F1 animals". In this paper, the inclusion of the requested L&M tests in an EOGRTS is challenged. In addition, next to the question on the validity of rodent models in general for testing thyroid hormone-dependent perturbations in brain development, the reliability of the publications specifically relied upon by the agency is questioned as these contain numerous fundamental errors in study methodology, design, and data reporting, provide contradicting results, lack crucial information to validate the results and exclude confounding factors, and finally show no causal relationship. Therefore, in our opinion, these publications cannot be used to substantiate, support, or conclude that decreases in blood thyroid (T4) hormone level on their own would result in impaired L&M in rats and are thus not adequate to use as fundament to ask for L&M testing as part of an EOGRTS.

Grouping of orally ingested silica nanomaterials via use of an integrated approach to testing and assessment to streamline risk assessment.

BACKGROUND: Nanomaterials can exist in different nanoforms (NFs). Their grouping may be supported by the formulation of hypotheses which can be interrogated via integrated approaches to testing and assessment (IATA). IATAs are decision trees that guide the user through tiered testing strategies (TTS) to collect the required evidence needed to accept or reject a grouping hypothesis. In the present paper, we investigated the applicability of IATAs for ingested NFs using a case study that includes different silicon dioxide, SiO2 NFs. Two oral grouping hypotheses addressing local and systemic toxicity were identified relevant for the grouping of these NFs and verified through the application of oral IATAs. Following different Tier 1 and/or Tier 2 in vitro methods of the TTS (i.e., in vitro dissolution, barrier integrity and inflammation assays), we generated the NF datasets. Furthermore, similarity algorithms (e.g., Bayesian method and Cluster analysis) were utilized to identify similarities among the NFs and establish a provisional group(s). The grouping based on Tier 1 and/or Tier 2 testing was analyzed in relation to available Tier 3 in vivo data in order to verify if the read-across was possible and therefore support a grouping decision. RESULTS: The measurement of the dissolution rate of the silica NFs in the oro-gastrointestinal tract and in the lysosome identified them as gradually dissolving and biopersistent NFs. For the local toxicity to intestinal epithelium (e.g. cytotoxicity, membrane integrity and inflammation), the biological results of the gastrointestinal tract models indicate that all of the silica NFs were similar with respect to the lack of local toxicity and, therefore, belong to the same group; in vivo data (although limited) confirmed the lack of local toxicity of NFs. For systemic toxicity, Tier 1 data did not identify similarity across the NFs, with results across different decision nodes being inconsistent in providing homogeneous group(s). Moreover, the available Tier 3 in vivo data were also insufficient to support decisions based upon the obtained in vitro results and relating to the toxicity of the tested NFs. CONCLUSIONS: The information generated by the tested oral IATAs can be effectively used for similarity assessment to support a grouping decision upon the application of a hypothesis related to toxicity in the gastrointestinal tract. The IATAs facilitated a structured data analysis and, by means of the expert's interpretation, supported read-across with the available in vivo data. The IATAs also supported the users in decision making, for example, reducing the testing when the grouping was well supported by the evidence and/or moving forward to advanced testing (e.g., the use of more suitable cellular models or chronic exposure) to improve the confidence level of the data and obtain more focused information.

Refinement of the selection of physicochemical properties for grouping and read-across of nanoforms.

Before placing a new nanoform (NF) on the market, its potential adverse effects must be evaluated. This may e.g. be done via hazard and risk assessment. Grouping and read-across of NFs is a possible strategy to reduce resource consumption, maximising the use of existing data for assessment of NFs. The GRACIOUS project provides a framework in which possible grouping and read-across for NFs is mainly based on an evaluation of their similarity. The impact of NFs on human health and the environment depends strongly on the concentration of the NF and its physicochemical properties, such as chemical composition, size distribution, shape, etc. Hence, knowledge of the most relevant physicochemical properties is essential information for comparing similarity. The presented work aims to refine existing proposals for sets of descriptors (descriptor array) that are needed to describe distinct NFs of a material to identify the most relevant ones for grouping and read-across. The selection criteria for refining this descriptor array are explained and demonstrated. Relevant protocols and methods are proposed for each physicochemical property. The required and achievable measurement accuracies of the refined descriptor array are reviewed, as this information is necessary for similarity assessment of NFs based on individual physicochemical properties.

Evaluation of the carcinogenicity of dichloromethane in rats, mice, hamsters and humans.

Dichloromethane (DCM) is a high production volume chemical (>1000 t/a) mainly used as an industrial solvent. Carcinogenicity studies in rats, mice and hamsters have demonstrated a malignant tumor inducing potential of DCM only in the mouse (lung and liver) at 1000-4000 ppm whereas human data do not support a conclusion of cancer risk. Based on this, DCM has been classified as a cat. 2 carcinogen. Dose-dependent toxicokinetics of DCM suggest that DCM is a threshold carcinogen in mice, initiating carcinogenicity via the low affinity/high capacity GSTT1 pathway; a biotransformation pathway that becomes relevant only at high exposure concentrations. Rats and hamsters have very low activities of this DCM-metabolizing GST and humans have even lower activities of this enzyme. Based on the induction of specific tumors selectively in the mouse, the dose- and species-specific toxicokinetics in this species, and the absence of a malignant tumor response by DCM in rats and hamsters having a closer relationship to DCM toxicokinetics in humans and thus being a more relevant animal model, the current classification of DCM as human carcinogen cat. 2 remains appropriate.

Variation in dissolution behavior among different nanoforms and its implication for grouping approaches in inhalation toxicity.

Different nanoforms (NF) of the same substance each need to be registered under REACH, but similarities in physiological interaction -among them biodissolution- can justify read-across within a group of NFs, thereby reducing the need to perform animal studies. Here we focused on the endpoint of inhalation toxicity and explored how differences in physical parameters of 17 NFs of silica, and organic and inorganic pigments impact dissolution rates, half-times, and transformation under both pH 7.4 lung lining conditions and pH 4.5 lysosomal conditions. We benchmarked our observations against well-known TiO2, BaSO4 and ZnO nanomaterials, representing very slow, partial and quick dissolution respectively. By automated image evaluation, structural transformations were observed for dissolution rates in the order of 0.1 to 10 ng/cm2/h, but did not provide additional decision criteria on the similarity of NFs. Dissolution half-times spanned nearly five orders of magnitude, mostly dictated by the substance and simulant fluid, but modulated up to ten-fold by the subtle differences between NFs. Physiological time scales and benchmark materials help to frame the biologically relevant range, proposed as 1 h to 1 y. NFs of ZnO, Ag, SiO2, BaSO4 were in this range. We proposed numerical rules of pairwise similarity within a group, of which the worst case NF would be further assessed by in vivo inhalation studies. These rules divided the colloidal silica NFs into two separate candidate groups, one with Al-doping, one without. Shape or silane surface treatment were less important. The dissolution halftimes of many organic and inorganic pigment NFs were longer than the biologically relevant range, such that dissolution behavior is not an obstacle for their groupings.

Is there a rationale for direct dosing of chemicals to nursing pups in the EOGRTS (OECD 443)?

In OECD guideline 443 - Extended One Generation Reproductive Toxicity Study (EOGRTS) - to be used for testing industrial and agrochemicals, it has been indicated that careful consideration of benefits and disadvantages should be made prior to conducting direct-dosing studies in nursing pups. Nursing pups will not be directly dosed in dietary and drinking water studies whereas in oral gavage studies this possibility exists. Besides the risk of intubation trauma and overdosing due to direct exposure and exposure via the mother's milk, direct dosing could lead to a different hazard assessment of chemicals depending on the choice of the route of administration. In addition, in case of industrial and agrochemicals used in industrial or professional settings only, there will never be direct exposure of newborns. Moreover, direct dosing of nursing pups is an artificial, non-physiological, route of exposure and as such it would hamper risk assessment. It should therefore only be considered in exceptional cases and justified on a case-by-case approach.

How to assess respiratory sensitization of low molecular weight chemicals?

There are no validated and regulatory accepted (animal) models to test for respiratory sensitization of low molecular weight (LMW) chemicals. Since several decades such chemicals are classified as respiratory sensitizers almost exclusively based on observations in workers. However, both respiratory allergens (in which process the immune system is involved) as well as asthmagens (no involvement of the immune system) may induce the same type of respiratory symptoms. Correct classification is very important from a health's perspective point of view. On the other hand, over-classification is not preferable in view of high costs to overdue workplace engineering controls or the chemical ultimately being banned due to Authorities' decisions. It would therefore be very beneficial if respiratory sensitizers can be correctly identified and distinguished from skin sensitizers and non-sensitizers/respiratory irritants. The purpose of this paper is to consider whether LMW chemicals can be correctly identified based on the currently available screening methods in workers, and/or via in silico, in vitro and/or in vivo testing. Collectively, based on the available information further effort is still needed to be able to correctly identify respiratory sensitizers and to distinguish these from skin sensitizers and irritants, not at least because of the far-reaching consequences once a chemical is classified as a respiratory sensitizer.

Evaluation of developmental toxicity of Methyl Chloride (Chloromethane) in rats, mice, and rabbits.

Methyl Chloride (MeCl; Chloromethane) is a high production volume chemical (>1000 t/a) and is used as an industrial solvent. Based on cardiac lesions reported in developmental toxicity studies in mice, but not in rats, manufacturers decided to classify MeCl as a developmental toxicant, cat. 2. Recently, the European Chemical Agency required a developmental toxicity study in a non-rodent species. No developmental toxicity was observed in rabbits in the recently completed, GLP, OECD 414 guideline study. In view of the absence of cardiac effects in rats and rabbits, the purpose of this review is to consider whether the cardiac effects reported in mice should be considered real effects and, if so, their potential for relevance to humans. This paper provides substantive new evidence with data from a third species and shows that an evaluation of the integrated scientific evidence indicates the reported developmental cardiac effects in mice, if not an artifact, are unlikely to be relevant to humans. As such the classification of MeCl for developmental toxicity was reconsidered.

Should DTPA, an Aminocarboxylic acid (ethylenediamine-based) chelating agent, be considered a developmental toxicant?

Aminocarboxylic acid (ethylenediamine-based) chelating agents, such as DTPA and EDTA, are widely used in a variety of products and processes. Recently the European RAC proposed to classify DTPA as a developmental toxicant Category 1B according to CLP. This paper provides unequivocal and significant evidence that developmental effects cannot be considered an intrinsic property of the chelating substances themselves since: (1) animals fed a zinc deficient diet during gestation exhibit developmental toxicity of a similar nature and severity to that observed in studies involving such chelates, (2) sufficient supplementation of zinc in the diet, or administration of zinc bound chelates, completely negates the developmental effects. Moreover, the bioavailability of DTPA is very low with >95% of oral doses excreted unchanged via the feces within 24 h. If DTPA would possess the intrinsic property to be developmentally toxic, simple zinc supplementation should not be sufficient to negate these effects. Furthermore, the relevance of classification is highly questionable since worker or consumer exposure could not lead to a scenario whereby sufficient zinc deficiency would manifest itself. Therefore classification of DTPA for such effects is not protective of human health; instead it leads to onerous and disproportionate restrictions being placed on this substance.

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.

Case studies putting the decision-making framework for the grouping and testing of nanomaterials (DF4nanoGrouping) into practice.

Case studies covering carbonaceous nanomaterials, metal oxide and metal sulphate nanomaterials, amorphous silica and organic pigments were performed to assess the Decision-making framework for the grouping and testing of nanomaterials (DF4nanoGrouping). The usefulness of the DF4nanoGrouping for nanomaterial hazard assessment was confirmed. In two tiers that rely exclusively on non-animal test methods followed by a third tier, if necessary, in which data from rat short-term inhalation studies are evaluated, nanomaterials are assigned to one of four main groups (MGs). The DF4nanoGrouping proved efficient in sorting out nanomaterials that could undergo hazard assessment without further testing. These are soluble nanomaterials (MG1) whose further hazard assessment should rely on read-across to the dissolved materials, high aspect-ratio nanomaterials (MG2) which could be assessed according to their potential fibre toxicity and passive nanomaterials (MG3) that only elicit effects under pulmonary overload conditions. Thereby, the DF4nanoGrouping allows identifying active nanomaterials (MG4) that merit in-depth investigations, and it provides a solid rationale for their sub-grouping to specify the further information needs. Finally, the evaluated case study materials may be used as source nanomaterials in future read-across applications. Overall, the DF4nanoGrouping is a hazard assessment strategy that strictly uses animals as a last resort.

Thresholds in chemical respiratory sensitisation.

There is a continuing interest in determining whether it is possible to identify thresholds for chemical allergy. Here allergic sensitisation of the respiratory tract by chemicals is considered in this context. This is an important occupational health problem, being associated with rhinitis and asthma, and in addition provides toxicologists and risk assessors with a number of challenges. In common with all forms of allergic disease chemical respiratory allergy develops in two phases. In the first (induction) phase exposure to a chemical allergen (by an appropriate route of exposure) causes immunological priming and sensitisation of the respiratory tract. The second (elicitation) phase is triggered if a sensitised subject is exposed subsequently to the same chemical allergen via inhalation. A secondary immune response will be provoked in the respiratory tract resulting in inflammation and the signs and symptoms of a respiratory hypersensitivity reaction. In this article attention has focused on the identification of threshold values during the acquisition of sensitisation. Current mechanistic understanding of allergy is such that it can be assumed that the development of sensitisation (and also the elicitation of an allergic reaction) is a threshold phenomenon; there will be levels of exposure below which sensitisation will not be acquired. That is, all immune responses, including allergic sensitisation, have threshold requirement for the availability of antigen/allergen, below which a response will fail to develop. The issue addressed here is whether there are methods available or clinical/epidemiological data that permit the identification of such thresholds. This document reviews briefly relevant human studies of occupational asthma, and experimental models that have been developed (or are being developed) for the identification and characterisation of chemical respiratory allergens. The main conclusion drawn is that although there is evidence that the acquisition of sensitisation to chemical respiratory allergens is a dose-related phenomenon, and that thresholds exist, it is frequently difficult to define accurate numerical values for threshold exposure levels. Nevertheless, based on occupational exposure data it may sometimes be possible to derive levels of exposure in the workplace, which are safe. An additional observation is the lack currently of suitable experimental methods for both routine hazard characterisation and the measurement of thresholds, and that such methods are still some way off. Given the current trajectory of toxicology, and the move towards the use of non-animal in vitro and/or in silico) methods, there is a need to consider the development of alternative approaches for the identification and characterisation of respiratory sensitisation hazards, and for risk assessment.

A decision-making framework for the grouping and testing of nanomaterials (DF4nanoGrouping).

The European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) 'Nano Task Force' proposes a Decision-making framework for the grouping and testing of nanomaterials (DF4nanoGrouping) that consists of 3 tiers to assign nanomaterials to 4 main groups, to perform sub-grouping within the main groups and to determine and refine specific information needs. The DF4nanoGrouping covers all relevant aspects of a nanomaterial's life cycle and biological pathways, i.e. intrinsic material and system-dependent properties, biopersistence, uptake and biodistribution, cellular and apical toxic effects. Use (including manufacture), release and route of exposure are applied as 'qualifiers' within the DF4nanoGrouping to determine if, e.g. nanomaterials cannot be released from a product matrix, which may justify the waiving of testing. The four main groups encompass (1) soluble nanomaterials, (2) biopersistent high aspect ratio nanomaterials, (3) passive nanomaterials, and (4) active nanomaterials. The DF4nanoGrouping aims to group nanomaterials by their specific mode-of-action that results in an apical toxic effect. This is eventually directed by a nanomaterial's intrinsic properties. However, since the exact correlation of intrinsic material properties and apical toxic effect is not yet established, the DF4nanoGrouping uses the 'functionality' of nanomaterials for grouping rather than relying on intrinsic material properties alone. Such functionalities include system-dependent material properties (such as dissolution rate in biologically relevant media), bio-physical interactions, in vitro effects and release and exposure. The DF4nanoGrouping is a hazard and risk assessment tool that applies modern toxicology and contributes to the sustainable development of nanotechnological products. It ensures that no studies are performed that do not provide crucial data and therefore saves animals and resources.