A critical appraisal of the sensitivity of in vivo genotoxicity assays in detecting human carcinogens.

Genotoxicity testing is an important part of standard safety testing strategies. Animal studies have always been a key component, either as a mandatory part of the regulatory test battery, or to follow-up questionable in vitro findings. The strengths and weaknesses of in vivo assays is a continuous matter of debate, including their capacity to predict (human) carcinogenicity. We have therefore analysed the sensitivity of five routinely used in vivo tests to determine, in addition to other aspects, which tests or combination of tests best identify 73 chemicals classified as IARC Group 1 and 2A carcinogens. The in vivo tests included the micronucleus (MN), unscheduled DNA synthesis (UDS), comet, Pig-a and transgenic rodent assays (TGR). The individual assays detect 74.2% (49/66, MN), 64.3% (9/14, UDS), 92.1% (35/38, comet), 82.4% (14/17, Pig-a) and 90.3% (28/31, TGR) of the probable and confirmed human carcinogens that were tested in these assays. Combining assays that cover different genotoxicity endpoints and multiple tissues, e.g. the bone marrow MN and the liver comet assays, increases the sensitivity further (to 94%). Correlations in terms of organ-specificity for these assays with human cancer target organs revealed only a limited correlation for the hematopoietic system but not for other organs. The data supports the use of the comet and TGR assays for detection of 'site-of-first-contact' genotoxicants, but these chemicals were generally also detected in assays that measure genotoxicity in tissues not directly exposed, e.g. liver and the hematopoietic system. In conclusion, our evaluation confirmed a high sensitivity of the five in vivo genotoxicity assays for prediction of human carcinogens, which can be further increased by combining assays. Moreover, the addition of the comet to the in vivo MN test would identify all DNA reactive human carcinogens. Importantly, integration of some of the study readouts into one experiment is an animal-saving alternative to performing separate experiments.

Predictive performance and inter-laboratory reproducibility in assessing eye irritation potential of water- and oil-soluble mixtures using the Short Time Exposure test method.

The Short Time Exposure (STE) test method is an alternative method for assessing eye irritation potential using Statens Seruminstitut Rabbit Cornea cells and has been adopted as test guideline 491 by the Organisation for Economic Co-operation and Development. Its good predictive performance in identifying the Globally Harmonized System (GHS) No Category (NC) or Irritant Category has been demonstrated in evaluations of water-soluble substances, oil-soluble substances, and water-soluble mixtures. However, the predictive performance for oil-soluble mixtures was not evaluated. Twenty-four oil-soluble mixtures were evaluated using the STE test method. The GHS NC or Irritant Category of 22 oil-soluble mixtures were consistent with that of a Reconstructed human Cornea-like Epithelium (RhCE) test method. Inter-laboratory reproducibility was then confirmed using 20 water- and oil-soluble mixtures blind-coded. The concordance in GHS NC or Irritant Category among four laboratories was 90%-100%. In conclusion, the concordance in comparison with the results of RhCE test method using 24 oil-soluble mixtures and inter-laboratory reproducibility using 20 water- and oil-soluble mixtures blind-coded were good, indicating that the STE test method is a suitable alternative for predicting the eye irritation potential of both substances and mixtures.

Guiding principles for the implementation of non-animal safety assessment approaches for cosmetics: skin sensitisation.

Characterisation of skin sensitisation potential is a key endpoint for the safety assessment of cosmetic ingredients especially when significant dermal exposure to an ingredient is expected. At present the mouse local lymph node assay (LLNA) remains the 'gold standard' test method for this purpose however non-animal test methods are under development that aim to replace the need for new animal test data. COLIPA (the European Cosmetics Association) funds an extensive programme of skin sensitisation research, method development and method evaluation and helped coordinate the early evaluation of the three test methods currently undergoing pre-validation. In May 2010, a COLIPA scientific meeting was held to analyse to what extent skin sensitisation safety assessments for cosmetic ingredients can be made in the absence of animal data. In order to propose guiding principles for the application and further development of non-animal safety assessment strategies it was evaluated how and when non-animal test methods, predictions based on physico-chemical properties (including in silico tools), threshold concepts and weight-of-evidence based hazard characterisation could be used to enable safety decisions. Generation and assessment of potency information from alternative tools which at present is predominantly derived from the LLNA is considered the future key research area.

A tiered approach to the use of alternatives to animal testing for the safety assessment of cosmetics: genotoxicity. A COLIPA analysis.

For the assessment of genotoxic effects of cosmetic ingredients, a number of well-established and regulatory accepted in vitro assays are in place. A caveat to the use of these assays is their relatively low specificity and high rate of false or misleading positive results. Due to the 7th amendment to the EU Cosmetics Directive ban on in vivo genotoxicity testing for cosmetics that was enacted March 2009, it is no longer possible to conduct follow-up in vivo genotoxicity tests for cosmetic ingredients positive in in vitro genotoxicity tests to further assess the relevance of the in vitro findings. COLIPA, the European Cosmetics Association, has initiated a research programme to improve existing and develop new in vitro methods. A COLIPA workshop was held in Brussels in April 2008 to analyse the best possible use of available methods and approaches to enable a sound assessment of the genotoxic hazard of cosmetic ingredients. Common approaches of cosmetic companies are described, with recommendations for evaluating in vitro genotoxins using non-animal approaches. A weight of evidence approach was employed to set up a decision-tree for the integration of alternative methods into tiered testing strategies.

A tiered approach to the use of alternatives to animal testing for the safety assessment of cosmetics: skin irritation.

Evaluation of the skin irritancy and corrosivity potential of an ingredient is a necessity in the safety assessment of cosmetic ingredients. To date, there are two formally validated alternatives to the rabbit Draize test for skin corrosivity in place, namely the rat skin transcutaneous electrical resistance (TER) assay and the Human Skin Model Test using EpiSkin, EpiDerm and SkinEthic reconstructed human epidermal equivalents. For skin irritation, EpiSkin, EpiDerm and SkinEthic are validated as stand-alone test replacements for the rabbit Draize test. Data from these tests are rarely considered in isolation and are evaluated in combination with other factors to establish the overall irritating or corrosive potential of an ingredient. In light of the deadlines established in the Cosmetics Directive for cessation of animal testing for cosmetic ingredients, a COLIPA scientific meeting was held in Brussels on 30th January, 2008 to review the use of alternative approaches and to set up a decision tree approach for their integration into tiered testing strategies for hazard and safety assessment of cosmetic ingredients and their use in products. In conclusion, the safety assessments for skin irritation/corrosion of new chemicals for use in cosmetics can be confidently accomplished using exclusively alternative methods.

A tiered approach to the use of alternatives to animal testing for the safety assessment of cosmetics: eye irritation.

The need for alternative approaches to replace the in vivo rabbit Draize eye test for evaluation of eye irritation of cosmetic ingredients has been recognised by the cosmetics industry for many years. Extensive research has lead to the development of several assays, some of which have undergone formal validation. Even though, to date, no single in vitro assay has been validated as a full replacement for the rabbit Draize eye test, organotypic assays are accepted for specific and limited regulatory purposes. Although not formally validated, several other in vitro models have been used for over a decade by the cosmetics industry as valuable tools in a weight of evidence approach for the safety assessment of ingredients and finished products. In light of the deadlines established in the EU Cosmetics Directive for cessation of animal testing for cosmetic ingredients, a COLIPA scientific meeting was held in Brussels on 30th January, 2008 to review the use of alternative approaches and to set up a decision-tree approach for their integration into tiered testing strategies for hazard and safety assessment of cosmetic ingredients and their use in products. Furthermore, recommendations are given on how remaining data gaps and research needs can be addressed.

Chemoimmunotherapy for melanoma with dacarbazine and 2,4-dinitrochlorobenzene elicits a specific T cell-dependent immune response.

An empirically established chemoimmunotherapy for metastatic melanoma combines the systemic administration of the chemotherapeutic agent dacarbazine (DTIC) with the epifocal application of the contact sensitizer 2,4-dinitrochlorobenzene (DNCB) on cutaneous metastases. Although this therapy yields high response rates resulting in prolonged survival, the mechanisms involved remain unknown. Here, we investigated whether treatment of tumor-bearing mice with DTIC and DNCB resulted in a specific immune response against the tumor. Subcutaneous (s.c.) tumors and lung metastases were induced in C57BL/6 mice by injecting syngeneic B16-melanoma cells s.c. or into the lateral tail vein, respectively. Mice were treated with intraperitoneal injections of DTIC followed by epifocal application of DNCB. This therapeutic approach significantly reduced the growth of s.c. tumors as well as lung metastases. Our data showed that the effector mechanisms involved are dependent on T cells. No therapeutic effect was observed in immunodeficient RAG-1(-/-) mice, or when the contact sensitizer DNCB was replaced by skin irritants (croton oil or tributyltin). Splenic lymphocytes obtained from treated mice displayed a three-fold higher specific cytolytic activity against B16 cells than in tumor-bearing controls. Both CD8(+) and CD4(+) T cells were able to lyse B16 cells. No changes were observed in natural killer (NK) cell activity. Likewise, tumor-infiltrating lymphocytes (TIL) of treated mice showed higher cytolytic activity than that of controls. Analysis of cytokine expression in s.c. tumors revealed increased mRNA levels of interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6) in treated tumors. Together, these findings demonstrate the ability of DTIC/DNCB treatment to induce an effective T cell-dependent host immune response against a syngeneic tumor.

Expression of functional kappa-opioid receptors on murine dendritic cells.

Endogenous and exogenous opioids are known to exert direct effects on the immune system and the expression of functional opioid receptors has been reported for several immune cell types. Since dendritic cells are important inducers and regulators of immune responses, we investigated whether murine dendritic cells express functional kappa-opioid receptors. FACScan analysis and radioligand binding studies revealed the expression of kappa-opioid receptors by murine dendritic cells, which by RT-PCR were also shown to express kappa-opioid mRNA. In a primary allogenic mixed-lymphocyte reaction the kappa-agonists dynorphin A and U50488H suppressed the capacity of dendritic cells to induce T-cell proliferation in a concentration-dependent manner. Preincubation with the kappa-specific antagonist nor-binaltrophimine abolished the observed effect, indicating specificity. In contrast, antigen uptake by dendritic cells as well as phenotypic maturation of dendritic cells were not influenced by the kappa-agonists dynorphin A and U50488H. In summary our data demonstrate that dendritic cells express functional kappa-opioid receptors and that specific agonists exert a direct effect on these cells. Therefore, dendritic cells might be involved in the interaction of the neuroendocrine hormones and the immune system.