Agnes Schulte

Animal testing and alternative approaches for the human health risk assessment under the proposed new European chemicals regulation

During the past 20xa0years the EU legislation for the notification of chemicals has focussed on new chemicals and at the same time failed to cover the evaluation of existing chemicals in Europe. Therefore, in a new EU chemicals policy (REACH, Registration, Evaluation and Authorisation of Chemicals) the European Commission proposes to evaluate 30,000 chemicals within a period of 15xa0years. We are providing estimates of the testing requirements based on our personal experiences during the past 20xa0years. A realistic scenario based on an in-depth discussion of potential toxicological developments and an optimised “tailor-made” testing strategy shows that to meet the goals of the REACH policy, animal numbers may be significantly reduced below 10xa0million if industry would use in-house data from toxicity testing, which are confidential, if non-animal tests would be used, and if information from quantitative structure activity relationships (QSARs) would be applied in substance-tailored testing schemes. The procedures for evaluating the reproductive toxicity of chemicals have the strongest impact on the total number of animals bred for testing under REACH. We are assuming both an active collaboration with our colleagues in industry and substantial funding of the development and validation of advanced non-animal methods by the EU Commission, specifically in reproductive and developmental toxicity.

The carcinogenic potential of nanomaterials, their release from products and options for regulating them

A summary of a critical review by a working group of the German Federal Environment Agency and the German Federal Institute for Risk Assessment on the carcinogenic potential of nanomaterials is presented. After a critical review of the available data, we conclude that the potential carcinogenic risk of nanomaterials can currently be assessed only on a case-by-case basis. There is certain evidence that different forms of CNTs (carbon nanotubes) and nanoscale TiO(2) particles may induce tumours in sensitive animal models. It is assumed that the mode of action of the inhalation toxicity of asbestos-like fibres and of inhalable fractions of biopersistent fine dusts of low toxicity (nano-TiO(2)) is linked to chronic inflammatory processes. Existing epidemiological studies on carcinogenicity for these manufactured nanomaterials are not sufficiently conclusive. Generally speaking, the database is not adequate for an assessment of the carcinogenic potential of nanomaterials. Whereas a number of studies provide evidence of a nano-specific potential to induce tumours, other studies did not. This is possibly due to insufficient characterisation of the test material, difference in the experimental design, the use of different animal models and species and/or differences in dosimetry (both with regard to the appropriate dose metric and the estimated effective dose quantities). An assessment of the carcinogenic potential and its relevance for humans are currently fraught with uncertainty. Furthermore, the nano-specificity of the carcinogenic effects observed cannot be conclusively evaluated. Specific carcinogenic effects of nanomaterials may be both quantitative and qualitative. In quantitative terms, the carcinogenic effects of nanoparticles are thought to be simply more pronounced compared to the corresponding bulk material (due, for example, to the considerably larger surface area and higher number of particles relative to the mass concentration). On the other hand, certain nano-properties such as small size, shape and reactivity, retention time and distribution in the body after overcoming biological barriers, as well as subcellular and molecular interactions may play a role in determining the toxicity in qualitative terms, i.e. the carcinogenic potential of the nanomaterial and the non-nanoscale comparison substance may be fundamentally different. All of these factors leave no doubt about the fact that there is a great need for research in this area and that new standardised test methods need to be developed or existing ones adapted at the very least to achieve valid answers regarding the carcinogenic potential of nanomaterials. Global production of nanomaterials is set to increase in the years to come, and new materials with new properties will be developed, so that greater human exposure to them must be anticipated. No reliable conclusions can currently be drawn about exposure to nanoparticles and their release from products. Firstly, there are substantial deficits in information about the processing of nanomaterials in products and preparations. Secondly, there are only a small number of studies on nanoparticle release, and reliable techniques for measuring and monitoring nanomaterials in different environmental media are still being developed which is both complex and costly. Despite the uncertainties, the findings to date on the carcinogenic potential of nanomaterials must be taken seriously, and precautionary measures to minimise exposure should go hand in hand with the development of a comprehensive and conclusive toxicological methodology and testing procedure for nanostructured materials that includes all possible exposure routes. With regard to possible legal classification of nanomaterials and the transferability of classifications of their non-nanomaterial counterparts, we believe it is necessary to have separate procedures for nano and non-nano forms. Furthermore, criteria for evaluating nano-specific carcinogenic properties should be constantly updated and adapted to the state of knowledge. There is a need here for amendments to be made to EU legislation, as currently nanoforms do not represent a separate category of substance in their own right.

1st international ESTP expert workshop: "Larynx squamous metaplasia". A re-consideration of morphology and diagnostic approaches in rodent studies and its relevance for human risk assessment.

Invited international experts participated in a 2-day workshop organized by the European Society of Toxicologic Pathology (ESTP) to evaluate and discuss spontaneous and induced laryngeal lesions in rodents. The main purpose of the workshop was to agree upon the terminology and relevance of a range of laryngeal changes that varied from very subtle epithelial alterations up to severe metaplastic or neoplastic lesions. The workshop experts concluded that minimal, focal epithelial changes of the laryngeal epithelium, predominantly occurring at the base of the epiglottis, should be given the descriptive term of epithelial alteration and assessed as non-adverse. Although observed as induced effects they may also occur in non-treated animals and were not considered to have a potential for a laryngeal dysfunction. Also, cases of minimal to slight laryngeal squamous metaplasia that are not observed diffusely could occur spontaneously or as treatment-induced lesions and should be assessed as non-adverse. Cases of moderate to severe laryngeal squamous metaplasia observed diffusely in multiple levels should be regarded as adverse, as there is a potential for dysfunction of the larynx. The occurrence of dysplasia or cellular atypia linked to laryngeal squamous metaplasia should always be reported separately and described in detail. In the evaluation of treatment-related effects of the larynx in studies utilizing aged animals, it has to be considered that moderate or even severe cases of focal laryngeal squamous metaplasia may occasionally be found as age-related, spontaneous lesions. Although inhalation exposure of rodents to non-genotoxic compounds may cause laryngeal squamous metaplasia, none of the workshop experts were aware of any reported cases of tumor induction in the larynx with a non-genotoxic compound. Therefore, for non-genotoxic compounds, the workshop experts did not regard laryngeal squamous metaplasia by itself as a precancerous lesion.

Workshop report. Children as a special subpopulation: focus on immunotoxicity. Federal Institute for Health Protection of Consumers and Veterinary Medicine (BgVV), 15-16 November 2001, Berlin, Germany

Abstract. An international symposium on the impact of environmental hazards, chemicals and drugs on the developing immune system of children was held in Berlin (Germany) organized by the BgVV. Epidemiological evidence indicates that an immature immune system challenged early in life by bacterial antigens may prevent, to some extent, allergic reactions including asthma bronchiale triggered by environmental pollutants. However, the prevalence for infectious disease is increased in childhood, especially when exposure to contaminants takes place in the period of pregnancy and breast-feeding. The effects of chlorinated biphenyls, dioxin, endotoxins, hexachlorobenzene, and direct and indirect in utero tobacco smoke exposure are examples. All participants recommend comparative and follow-up epidemiological studies and clinical examination of infants and children at risk during upbringing. There is ample evidence from experimental studies that indicates adverse effects on the developing immune system after in utero and postnatal exposure to chemicals and drugs. The adverse reactions of aciclovir, benzodiazepines, hexachlorobenzene, organotins (di-n-octyltin dichloride, tributyltin oxide), pesticides (methoxychlor, heptachlor) and polyhalogenated aromatic hydrocarbons (2,3,7,8-tetrachlorodibenzo-p-dioxin) are presented and reviewed. To determine the predictive value of test data in risk assessment for neonates and children, development, differentiation and maturation of the immune system in humans and laboratory rodents is compared in their pre- and postnatal stages. Considering some differences in immunocompetence at birth and after lactation, and differences in the time frame for maturation of the immune system, reaction types are thought to be common, comparable and similar in human childhood and early adolescence and the postnatal lifetime of laboratory rodents. The participants of the symposium felt strongly that regulatory steps urgently need to be initiated to incorporate some relevant aspects into existing test guidelines for testing developmental immunotoxicity. In this context, it is recommended that animals culled otherwise in one- and two-generation studies be examined for developmental immunotoxicity according to the valid methods and parameters discussed. The majority of participants agreed that a safety factor of 10 is too low in risk assessment and management to protect a sensitive subpopulation of children against man-made environmental pollutants.

Characterizing “Adversity” of Pathology Findings in Nonclinical Toxicity Studies Results from the 4th ESTP International Expert Workshop

The identification of adverse health effects has a central role in the development and risk/safety assessment of chemical entities and pharmaceuticals. There is currently a need for better alignment regarding how nonclinical adversity is determined and characterized. The European Society of Toxicologic Pathology (ESTP) therefore coordinated a workshop to review available definitions of adversity, weigh determining and qualifying factors of adversity based on case examples, and recommend a practical approach to define and characterize adversity in toxicology reports, to serve as a valuable prerequisite for future organ- or lesion-specific workshops planned by the ESTP.

Letter to the Editor: A regulatory view on the discussion on the role of alternative methods in the risk assessment of chemicals in the context of REACH

In a paper published recently in Archives of Toxicology, Lilienblum et al. present their view on available and ready to use alternative methods for the risk assessment of chemicals (Lilienblum et al. 2008). As they refer speciWcally to the new European legislation (REACH) which came into force mid 2007 we would like to comment some of the aspects of the paper from the perspective of regulators. First of all there is general agreement with the evaluation of the current status of alternative methods of the authors (Lilienblum et al. 2008). Considering complex endpoints, we consent that emphasis should be given to reWne tests with the aim to reduce the number of animals rather than to try to replace the tests by developing in vitro alternatives to the current testing methods. However, we would more clearly express the potential for the vitro methods for some of the endpoints which are discussed in the publication. For example, the in vitro dermal resorption test and the in vitro test on phototoxicity are both validated test methods and accepted at the regulatory level. Hence, in our view no in vivo tests are necessary to cover these endpoints. In addition, we see a high value in the available prediction tool for skin irritation and corrosion (ECB 2008) which is extremely helpful in a tiered approach. For classiWcation and labelling negative results can be used as they have clearly been identiWed in the validation study (Hulzebos et al. 2005). Hence, even under the GHS rules where two levels for positive results are required (which the prediction tool is not able to distinguish) the tool is nevertheless helpful considering that negative results for skin irritation and corrosion account for more than 70% of the cases in the EU New Chemicals Database. It can be assumed that using this alternative method would reduce the need for in vivo testing under REACH in 70% of 15,500 chemicals with a production volume below 10 tons per year. We also would put more emphasis in the potential the currently available in vitro test strategy can oVer for genotoxicity and mutagenicity testing. We agree with the assessment and the critique presented in the Lilienblum paper (Lilienblum et al. 2008). However, we would like to draw the attention to the fact that the currently used regulatory tiered testing approach reduces the necessity to perform in vivo testing to a small number of cases. Based on the numbers in the New Chemicals Data Base, the current approach to start with in vitro testing and to proceed with in vivo testing only in the positive in vitro cases was required in only around 25% of the cases which reduces the number of required in vivo tests under REACH to a quite remarkable low number of cases. Furthermore, we would like to give some considerations to the topic of toxicokinetic and metabolism which is extensively discussed in the Lilienblum paper. We agree with the assessment that toxicokinetic and metabolism is of importance and is a crucial issue and that metabolism often plays a key role in intraand inter-species diVerences (to document the regulatory need we refer to work of Bernauer et al. 2000, 2002, 2003, 2006; Abraham et al. 2005; Mielke et al. 2005; Bernauer and Gundert-Remy 2008). We agree with other scientists that the interpretation of testing results need additional data for extrapolation of the results to humans (Greim 2007) and toxicokinetic information is regarded as very helpful for the development of integrated testing strategies (ITS) which are propagated in the REACH guidance documents according to REACH legislation. In addition, toxicokinetic data can be used to support waiving arguments (Annex VIII, 8.6.1 90 days study; Annex IX, 8.7 studies on reprotoxiccity; Annex X, 8.4 germ cell mutageU. Gundert-Remy (&) · U. Bernauer · S. Madle · A. Oberemm · A. Schulte · H.-B. Richter-Reichhelm Berlin, Germany e-mail: ursula.gundert-remy@bfr.bund.de

Characterizing Adversity of Lysosomal Accumulation in Nonclinical Toxicity Studies: Results from the 5th ESTP International Expert Workshop:

Lysosomes have a central role in cellular catabolism, trafficking, and processing of foreign particles. Accumulation of endogenous and exogenous materials in lysosomes represents a common finding in nonclinical toxicity studies. Histologically, these accumulations often lack distinctive features indicative of lysosomal or cellular dysfunction, making it difficult to consistently interpret and assign adverse dose levels. To help address this issue, the European Society of Toxicologic Pathology organized a workshop where representative types of lysosomal accumulation induced by pharmaceuticals and environmental chemicals were presented and discussed. The expert working group agreed that the diversity of lysosomal accumulations requires a case-by-case weight-of-evidence approach and outlined several factors to consider in the adversity assessment, including location and type of cell affected, lysosomal contents, severity of the accumulation, and related pathological effects as evidence of cellular or organ dysfunction. Lysosomal accumulations associated with cytotoxicity, inflammation, or fibrosis were generally considered to be adverse, while those found in isolation (without morphologic or functional consequences) were not. Workshop examples highlighted the importance of thoroughly characterizing the biological context of lysosomal effects, including mechanistic data and functional in vitro readouts if available. The information provided here should facilitate greater consistency and transparency in the interpretation of lysosomal effects.

The German REACH Congress 2016: a workshop report

In October 2016, the German REACH Congress was held at the German Federal Institute for Risk Assessment (BfR) in Berlin. Here, the associated improvement made in the fields of consumer protection and the progress in and experiences gained from the implementation of the authorisation procedure were discussed. Several speakers from EU institutions, German authorities, industry, and civil society organisations were invited to present their views. There was a shared consensus that REACH contributes to the advancement of consumer protection against chemical risks, mainly because more and higher quality information on substance-related hazards and potential exposures becomes available. In addition, risk management measures, particularly regarding restrictions on uses, scale down consumer exposures to chemicals. Opportunities for improvements identified at the congress include the quality of registration dossiers and the management of and communication on substances of very high concern (SVHC) that may be present in consumer articles. Although regarded as being in an early implementation phase, the authorisation process was generally found to be operational and progressing well. Criticism was expressed with regard to the consistency of authorisation decisions and the costs and uncertainties related to authorisation applications. Consumer protection legislation consists of several legal provisions which are interlinked. The congress participants agreed that REACH is an important element of this legal framework as it enhances and complements other legal provisions.