Paving the way for application of next generation risk assessment to safety decision-making for cosmetic ingredients.

Next generation risk assessment (NGRA) is an exposure-led, hypothesis-driven approach that has the potential to support animal-free safety decision-making. However, significant effort is needed to develop and test the in vitro and in silico (computational) approaches that underpin NGRA to enable confident application in a regulatory context. A workshop was held in Montreal in 2019 to discuss where effort needs to be focussed and to agree on the steps needed to ensure safety decisions made on cosmetic ingredients are robust and protective. Workshop participants explored whether NGRA for cosmetic ingredients can be protective of human health, and reviewed examples of NGRA for cosmetic ingredients. From the limited examples available, it is clear that NGRA is still in its infancy, and further case studies are needed to determine whether safety decisions are sufficiently protective and not overly conservative. Seven areas were identified to help progress application of NGRA, including further investments in case studies that elaborate on scenarios frequently encountered by industry and regulators, including those where a 'high risk' conclusion would be expected. These will provide confidence that the tools and approaches can reliably discern differing levels of risk. Furthermore, frameworks to guide performance and reporting should be developed.

Workshop on the validation and regulatory acceptance of innovative 3R approaches in regulatory toxicology - Evolution versus revolution.

At a joint workshop organized by RIVM and BfR, international experts from governmental institutes, regulatory agencies, industry, academia and animal welfare organizations discussed and provided recommendations for the development, validation and implementation of innovative 3R approaches in regulatory toxicology. In particular, an evolutionary improvement of our current approach of test method validation in the context of defined approaches or integrated testing strategies was discussed together with a revolutionary approach based on a comprehensive description of the physiological responses of the human body to chemical exposure and the subsequent definition of relevant and predictive in vitro, in chemico or in silico methods. A more comprehensive evaluation of biological relevance, scientific validity and regulatory purpose of new test methods and assessment strategies together with case studies that provide practical experience with new approaches were discussed as essential steps to build up the necessary confidence to facilitate regulatory acceptance.

Workshop on acceleration of the validation and regulatory acceptance of alternative methods and implementation of testing strategies.

This report describes the proceedings of the BfR-RIVM workshop on validation of alternative methods which was held 23 and 24 March 2017 in Berlin, Germany. Stakeholders from governmental agencies, regulatory authorities, universities, industry and the OECD were invited to discuss current problems concerning the regulatory acceptance and implementation of alternative test methods and testing strategies, with the aim to develop feasible solutions. Classical validation of alternative methods usually involves one to one comparison with the gold standard animal study. This approach suffers from the reductionist nature of an alternative test as compared to the animal study as well as from the animal study being considered as the gold standard. Modern approaches combine individual alternatives into testing strategies, for which integrated and defined approaches are emerging at OECD. Furthermore, progress in mechanistic toxicology, e.g. through the adverse outcome pathway approach, and in computational systems toxicology allows integration of alternative test battery results into toxicity predictions that are more fine-tuned to the human situation. The road towards transition to a mechanistically-based human-focused hazard and risk assessment of chemicals requires an open mind towards stepping away from the animal study as the gold standard and defining human biologically based regulatory requirements for human hazard and risk assessment.

Skin sensitisation quantitative risk assessment (QRA) based on aggregate dermal exposure to methylisothiazolinone in personal care and household cleaning products.

Contact allergy to preservatives is an important public health problem. Ideally, new substances should be evaluated for the risk on skin sensitisation before market entry, for example by using a quantitative risk assessment (QRA) as developed for fragrances. As a proof-of-concept, this QRA was applied to the preservative methylisothiazolinone (MI), a common cause of contact allergy. MI is used in different consumer products, including personal care products (PCPs) and household cleaning products (HCPs). Aggregate exposure to MI in PCPs and HCPs was therefore assessed with the Probabilistic Aggregated Consumer Exposure Model (PACEM). Two exposure scenarios were evaluated: scenario 1 calculated aggregate exposure on actual MI product concentrations before the restricted use in PCPs and scenario 2 calculated aggregate exposure using the restrictions for MI in PCPs. The QRA for MI showed that in scenarios 1 and 2, the proportion of the population at risk for skin sensitisation is 0.7% and 0.5%, respectively. The restricted use of MI in PCPs does not seem very effective in lowering the risk on skin sensitization. To conclude, it is important to consider aggregate exposure from the most important consumer products into consideration in the risk assessment.

Probabilistic derivation of the interspecies assessment factor for skin sensitization.

An interspecies sensitization assessment factor (SAF) is used in the quantitative risk assessment (QRA) for skin sensitization when a murine-based NESIL (No Expected Sensitization Induction Level) is taken as point of departure. Several studies showed that, on average, the murine sensitization threshold is in good correspondence with that determined in humans. However, on an individual level, the murine and human sensitization thresholds may differ considerably. In this study, the interspecies SAF was quantified by using a probabilistic approach, to be able to take these cases into account. As expected, the geometric means of the probability distributions of murine and human sensitization threshold ratios were close to one, but taking the 95 th percentile of these distributions resulted in an interspecies SAF of 15. By using this value, one is sure that with 95% probability, the sensitization threshold determined in mice does not underestimate the human threshold. It can be concluded that a murine-based NESIL requires the use of an interspecies SAF (of 15) in the QRA for skin sensitization, to correct for the differences between mice and humans. This empirically derived interspecies SAF contributes to refinement of the risk assessment methodology.

Quantitative risk assessment of the aggregate dermal exposure to the sensitizing fragrance geraniol in personal care products and household cleaning agents.

A quantitative risk assessment was performed to establish if consumers are at risk for being dermally sensitized by the fragrance geraniol. Aggregate dermal exposure to geraniol was estimated using the Probabilistic Aggregate Consumer Exposure Model, containing data on the use of personal care products and household cleaning agents. Consumer exposure to geraniol via personal care products appeared to be higher than via household cleaning agents. The hands were the body parts receiving the highest exposure to geraniol. Dermal sensitization studies were assessed to derive the point of departure needed for the estimation of the Acceptable Exposure Level (AEL). Two concentrations were derived, one based on human studies and the other from dose-response analysis of the available murine local lymph node assay data. The aggregate dermal exposure assessment resulted in body part specific median exposures up to 0.041 µg/cm(2) (highest exposure 102 µg/cm(2)) for hands. Comparing the exposure to the lowest AEL (55 µg/cm(2)), shows that a range of 0.02-0.86% of the population may have an aggregated exposure which exceeds the AEL. Furthermore, it is demonstrated that personal care products contribute more to the consumer's geraniol exposure compared to household cleaning agents.

Effects of Bifidobacterium animalis administered during lactation on allergic and autoimmune responses in rodents.

Probiotics are promoted as being beneficial to health and positive effects on the immune system have been reported. Beneficial immune effects have been attributed to several mechanisms, including stimulating T helper 1 (Th1) immunity. To explore the effects of the probiotic Bifidobacterium animalis on Th1- and Th2-mediated immune responses, two different animal models representing either Th1- or Th2-mediated immune responses were used: a rat model for experimental autoimmune encephalomyelitis (EAE) (Th1) and a mouse model for respiratory allergy induced by ovalbumin (OVA) (Th2). B. animalis administration started when the mice or rats were 2 weeks old. Respiratory allergy or EAE were induced when the animals were 6-7 weeks old. In the allergy model, B. animalis modestly reduced the number of infiltrating eosinophils and lymphocytes in the lungs, but no effects on allergen-specific serum immunoglobulin E levels were found. Cytokine profiles assessed after culturing spleen cells with the mitogen concanvalin A (ConA) showed that B. animalis skewed the Th1/Th2 balance towards Th1 in females. However, allergen-induced cytokine production in females was not affected by B. animalis. In males, B. animalis significantly decreased ConA-induced interleukin-13 and a trend towards lower levels of OVA-induced Th2 cytokines. In the EAE model, B. animalis significantly reduced the duration of clinical symptoms by almost 2 days in males and improved the body weight gain during the experimental period compared with the control group. Our data show that B. animalis reduced several immune parameters in the allergy as well as in the autoimmunity model.

Chemical-specific properties co-determine the type of adverse immune response.

Many drugs but also environmental pollutants may cause adverse reactions in susceptible individuals that are reminiscent of autoimmune syndromes. Apart from a number of predisposing often inherent, idiosyncratic determinants, chemical-specific properties might be involved as well. Notably, reactive chemicals or metabolites may provoke formation or release of immunosensitizing neo-antigens (a.o. hapten-carrier complexes or cryptic epitopes). In addition reactive chemicals but also certain inert chemicals may trigger macrophages and other inflammatory cells to release proinflammatory products that, via elicitation of costimulatory help, support hapten- or neo-antigen-specific T cell activation. In addition, chemicals may influence immunoregulatory processes and modulate for instance the balance between type 1 and type 2 responses. Here, we review data showing that chemically induced upregulation of second or costimulatory signals co-determines not only whether, but also what type of an adverse immune response (type 1 or type 2) is triggered.

Predictive testing for autoimmunity.

Many chemicals, in particular drugs, cause systemic allergy or autoimmune-like disorders. Due to complex pathogenesis and strong dependence on genetic make-up, these immunotoxicological effects are usually missed in standard toxicity testing. Besides, animal studies that demonstrate chemically induced systemic allergy or autoimmune-like disorders are scarce. Here, animal models are presented that would fit into a predictive two-tiered strategy, designed to allow screening for immunostimulatory potential in the first tier, and more elaborate testing for allergenic or autoimmunogenic potential of selected chemicals in the second tier. The popliteal lymph node assay (PLNA), with or without reporter antigens, would fit in the first tier, and relevant route of exposure protocols with selected strains of mice or rats may be further developed to compose the second tier. To date, the relevant route of exposure models mentioned here (with 'normal' inbred mice and/or Brown Norway rats) has been tested with only a few chemicals, and the PLNA, although tested with over 100 chemicals, is not validated as yet. Conceivably, a major challenge in immunotoxicology is to incorporate the present knowledge on chemical-induced systemic allergy and autoimmunity in further development and validation of predictive models and strategies.

DR2 antigens are associated with severity of disease in toxic oil syndrome (TOS).

Toxic oil syndrome (TOS) was an epidemic which broke out in Spain in 1981, caused by the ingestion of rapeseed oil denatured with 2% aniline and sold illegally as edible oil. More than 20,000 people were affected and mortality rate was 8.4%. Genetic susceptibility appears to be involved in the pathology of this disease. Several reports have described association between the chronic stage of the disease and DR-DQ antigens (DR3, DR4, DR2 and DQ8). In the present work, we have reassessed the HLA class II antigens in a well-designed case-control study. Triplets of subjects (n=265) composed by chronic patients (n=117), non-affected family members (n=71) and non-related controls (n=77) were studied. Also, HLA class II antigens were analyzed in patients who had died from TOS (n= 34) and in TOS control patients who died from other non-TOS related causes (n=13). Regarding surviving patients no significant association was found between HLA and disease. In contrast, an increase in phenotypic frequency of DR2 antigen, was found in patients who had died from TOS (73.5%) compared with the whole study group: TOS-affected alive patients (25.6%, corrected P<0.001), non-affected family members (28.5%, corrected P<0.001), non-related controls (23.9%, corrected P<0.001) and dead controls (38.4%, P=0.03).

Epidermal cell kinetics by combining in situ hybridization and immunohistochemistry.

Double labelling can serve as a useful tool for providing information about cell kinetics in normal and hyperproliferative tissues in general, and skin in particular. We have developed a double-labelling method that combines immunohistochemistry using the monoclonal antibody MIB1 and non-isotopic in situ hybridization using either a digoxigenin-labelled RNA probe specific for histone 3 mRNA sequences or a Fluorescein-labelled oligonucleotide probe specific for histone 2b, 3, 4 mRNA sequences. Double labelling was performed on normal, tape-stripped normal skin and psoriatic skin. The three proliferation markers were also examined by single labelling. The ratio of cells in the S-phase (Ns) and the growth fraction (Ncy) was determined. In normal skin, psoriatic skin and tape-stripped normal skin after 24 h and after 48 h, we calculated that 15%, 16%, 3% and 12% of growth fraction consisted of cells in the S-phase respectively. The S-phase lasts approximately 10 h, so the cell cycle time in normal and psoriatic skin is approximately 62.5 h. At present, the MIB1/H3 digoxigenin or MIB1/H2b-H3-H4 Fluorescein double-labelling technique cannot be used routinely. Therefore, in order to understand the cell kinetic processes better, experiments are recommended to optimize these methods. From a practical point of view and for reasons of specificity and sensitivity, we prefer the Fluorescein-labelled oligonucleotide probe method.