Frank Gerberick

A dataset on 145 chemicals tested in alternative assays for skin sensitization undergoing prevalidation.

Skin sensitization is a key endpoint for cosmetic ingredients, with a forthcoming ban for animal testing in Europe. Four alternative tests have so far been submitted to ECVAM prevalidation: (i) MUSST and (ii) h‐Clat assess surface markers on dendritic cell lines, (iii) the direct peptide reactivity assay (DPRA) measures reactivity with model peptides and (iv) the KeratinoSensTM assay which is based on detection of Nrf2‐induced luciferase. It is anticipated that only an integrated testing strategy (ITS) based on a battery of tests might give a full replacement providing also a sensitization potency assessment, but this concept should be tested with a data‐driven analysis. Here we report a database on 145 chemicals reporting the quantitative endpoints measured in a U937‐ test, the DPRA and KeratinoSensTM . It can serve to develop data‐driven ITS approaches as we show in a parallel paper and provides a view as to the current ability to predict with in vitro tests as we are entering 2013. It may also serve as reference database when benchmarking new molecules with in vitro based read‐across and find use as a reference database when evaluating new tests. The tests and combinations thereof were evaluated for predictivity, and overall a similar predictivity was found as before on three‐fold smaller datasets. Analysis of the dose–response parameters of the individual tests indicates a correlation to sensitization potency. Detailed analysis of chemicals false‐negative and false‐positive in two tests helped to define limitations in the tests but also in the database derived from animal studies. Copyright

Predictive identification of human skin sensitization thresholds.

For years, methods have been available for the predictive identification of chemicals that possess the intrinsic potential to cause skin sensitization. However, many have proven less suitable for the determination of relative sensitizing potency. In this respect, the local lymph node assay (LLNA) has been shown to have a number of important advantages. Through interpolation of LLNA dose–response data, the concentration of a chemical required to produce a threshold positive response (a 3‐fold increase in activity compared with concurrent vehicle controls, the EC3 value) can be measured. The robustness of this parameter has been demonstrated rigorously in terms of inter‐ and intralaboratory reproducibility. Additionally, the relationship between potency estimates from the LLNA and an appreciation of human potency based on clinical experience has been reported previously. In the present investigations, we have sought to consolidate further our understanding of the association between EC3 values and human skin‐sensitization potency by undertaking a thorough and extensive analysis of existing human predictive assays, particularly where dose–response information is available, from historical human repeated insult patch tests (HRIPTs). From these human data, information on the approximate threshold for the induction of skin sensitization in the HRIPT was determined for 26 skin‐sensitizing chemicals. These data were then compared with LLNA‐derived EC3 values. The results from each assay, expressed as dose per unit area (μg/cm2), revealed a clear linear relationship between the 2 values, thereby substantiating further the utility of LLNA EC3 values for prediction of the relative human sensitizing potency of newly identified skin sensitizers.

The local lymph node assay and the assessment of relative potency : status of validation

For the prediction of skin sensitization potential, the local lymph node assay (LLNA) is a fully validated alternative to guinea‐pig tests. More recently, information from LLNA dose–response analyses has been used to assess the relative potency of skin sensitizing chemicals. These data are then deployed for risk assessment and risk management. In this commentary, the utility and validity of these relative potency measurements are reviewed. It is concluded that the LLNA does provide a valuable assessment of relative sensitizing potency in the form of the estimated concentration of a chemical required to produce a threefold stimulation of draining lymph node cell proliferation compared with concurrent controls (EC3 value) and that all reasonable validation requirements have been addressed successfully. EC3 measurements are reproducible in both intra‐ and interlaboratory evaluations and are stable over time. It has been shown also, by several independent groups, that EC3 values correlate closely with data on relative human skin sensitization potency. Consequently, the recommendation made here is that LLNA EC3 measurements should now be regarded as a validated method for the determination of the relative potency of skin sensitizing chemicals, a conclusion that has already been reached by a number of independent expert groups.

Bayesian integrated testing strategy to assess skin sensitization potency: from theory to practice

Frameworks to predict in vivo effects by integration of in vitro, in silico and in chemico information using mechanistic insight are needed to meet the challenges of 21st century toxicology. Expert‐based approaches that qualitatively integrate multifaceted data are practiced under the term ’weight of evidence’, whereas quantitative approaches remain rare. To address this gap we previously developed a methodology to design an Integrated Testing Strategy (ITS) in the form of a Bayesian Network (BN). This study follows up on our proof of concept work and presents an updated ITS to assess skin sensitization potency expressed as local lymph node assay (LLNA) potency classes. Modifications to the ITS structure were introduced to include better mechanistic information. The parameters of the updated ITS were calculated from an extended data set of 124 chemicals. A detailed validation analysis and a case study were carried out to demonstrate the utility of the ITS for practical application. The improved BN ITS predicted correctly 95% and 86% of chemicals in a test set (n = 21) for hazard and LLNA potency classes, respectively. The practical value of using the BN ITS is far more than a prediction framework when all data are available. The BN ITS can develop a hypothesis using subsets of data as small as one data point and can be queried on the value of adding additional tests before testing is commenced. The ITS represents key steps of the skin sensitization process and a mechanistically interpretable testing strategy can be developed. These features are illustrated in the manuscript via practical examples. Copyright

Identifying and characterizing chemical skin sensitizers without animal testing: Colipa's research and method development program

The sensitizing potential of chemicals is usually identified and characterized using one of the available animal test methods, such as the mouse local lymph node assay. Due to the increasing public and political concerns regarding the use of animals for the screening of new chemicals, the Colipa Skin Tolerance Task Force collaborates with and/or funds research groups to increase and apply our understanding of the events occurring during the acquisition of skin sensitization. Knowledge gained from this research is used to support the development and evaluation of novel alternative approaches for the identification and characterization of skin sensitizing chemicals. At present one in chemico (direct peptide reactivity assay (DPRA)) and two in vitro test methods (cell based assays (MUSST and h-CLAT)) have been evaluated within Colipa inter-laboratory ring trials and accepted by the European Centre for the Validation of Alternative Methods (ECVAM) for pre-validation. Data from all three test methods will be used to support the development of testing strategy approaches for skin sensitizer potency prediction. The replacement of the need for animal testing for skin sensitization risk assessment is viewed as ultimately achievable and the next couple of years should set the timeline for this milestone.

Nothing is perfect, not even the local lymph node assay: a commentary and the implications for REACH

For many regulatory authorities, the local lymph node assay (LLNA) is the preferred assay for the predictive identification of skin‐sensitizing chemicals. It is the initial requirement for sensitization testing within the new REACH (Registration, Evaluation, Authorization and Restriction of Chemical substances) regulations in the European Union. The primary reasons for the preferment of the LLNA are the animal welfare benefits it provides compared with traditional guinea‐pig methods (refinement and reduction of animal usage) and the general performance characteristics of the assay with regard to overall reliability, accuracy, and interpretation. Moreover, a substantial published literature on the LLNA is available making it appropriate for use as a benchmark against which new approaches, including in vitro alternatives, can be evaluated and validated. There is, therefore, a view that the LLNA represents the ‘gold standard’ for skin sensitization testing. However, although this is probably correct, it is important to recognize and acknowledge that in common with all other predictive tests (whether they be validated or not), the LLNA has limitations, in addition to strengths, some of which were mentioned above. Arguably, it is the limitations (e.g., the occurrence of false positive and false negative results) of test methods that are most important to understand. With respect to the LLNA, these limitations are similar to those associated with guinea‐pig skin sensitization methods. Among these are the occurrence of false positive and false negative results, susceptibility of results to changes in vehicle, and the possibility that interspecies differences may confound interpretation. In this commentary, these issues are reviewed and their impact on the utility of the LLNA for identification, classification, and potency assessment of skin sensitizers are considered. In addition, their relevance for the future development and validation of novel in vitro and in silico alternatives is explored.

Inducible interleukin 4 (IL-4) production and mRNA expression following exposure of mice to chemical allergens.

Previous investigations have indicated that different classes of chemical allergen provoke discrete immune responses in mice characterized by the development of selective cytokine secretion patterns by draining lymph node cells. It was found that, in mice exposed chronically to chemical respiratory allergens, lymph node cells produced high levels of mitogen-inducible interleukin 4 (IL-4), a cytokine necessary for the development of IgE antibody responses. In contrast, exposure of mice to contact allergens resulted in only low levels of IL-4 production. The studies described here were conducted to determine whether differentially induced expression of IL-4 messenger RNA was independent of the need for mitogen stimulation, unlike secretion of IL-4 protein. Mice were exposed to concentrations of trimellitic anhydride (TMA), a respiratory allergen, or to 2,4-dinitrochlorobenzene (DNCB), a contact allergen, under conditions of equivalent immunogenicity and where the former, but not the latter, resulted in significant production of mitogen-inducible IL-4 protein. The steady state levels of IL-4 mRNA in draining lymph nodes were measured by reverse transcription-polymerase chain reaction after a single or repeated daily application of the test chemical to the ears of chronically sensitized mice. Expression of mRNA for IL-4 was evaluated relative to the expression of a housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase. Both chemicals elicited increased steady state levels of IL-4 mRNA by draining lymph node cells compared with vehicle-treated and naive controls. However, using two different treatment protocols, a chronic and an abbreviated chronic protocol and at all times examined, the elevation in IL-4 mRNA steady state levels induced by TMA was greater than that observed with DNCB. These data provide further support for the differential stimulation by contact and respiratory chemical allergens of cytokine secretion patterns in mice and demonstrate that the divergent production of IL-4 in response to these classes of allergen is at least partly transcriptionally regulated.

Evaluation of an optimized protocol using human peripheral blood monocyte derived dendritic cells for the in vitro detection of sensitizers: Results of a ring study in five laboratories.

Allergic contact dermatitis is a delayed T-cell mediated allergic response associated with relevant social and economic impacts. Animal experiments (e.g. the local lymph node assay) are still supplying most of the data used to assess the sensitization potential of new chemicals. However, the 7th amendment to the EU Cosmetic Directive have introduced a testing ban for cosmetic ingredients after March 2013. We have developed and optimized a stable and reproducible in vitro protocol based on human peripheral blood monocyte derived dendritic cells to assess the sensitization potential of chemicals. To evaluate the transferability and the predictivity of this PBMDCs based test protocol, a ring study was organized with five laboratories using seven chemicals with a known sensitization potential (one none-sensitizer and six sensitizers, including one pro-hapten). The results indicated that this optimized test protocol could be successfully transferred to all participating laboratories and allowed a correct assessment of the sensitization potential of the tested set of chemicals. This should allow a wider acceptance of PBMDCs as a reliable test system for the detection of human skin sensitizers and the inclusion of this protocol in the toolbox of in vitro methods for the evaluation of the skin sensitization potential of chemicals.

Joint meeting of the Experimental Contact Dermatitis Research Group and the American Contact Dermatitis Society in Baltimore, MD, September 28 to 30, 2006

T ONY GASPARI introduced the fifth meeting of the Experimental Contact Dermatitis Research Group (ECDRG), the fourth to be combined with a meeting of the American Contact Dermatitis Society (ACDS). The meeting theme is again ‘‘Blending Science with Best Practice.’’ This meeting brings together clinicians, academicians, government authorities, and scientists who are passionate about furthering the understanding and application of knowledge of contact dermatitis. For their excellent efforts, Dr. Gaspari thanked his fellow organizing committee members: Drs. Ian Kimber, Frank Gerberick, Boris Lushniak, and Denis Sasseville. Dr. Sasseville will also be organizing the next meeting, to be held in Montreal, Canada, from August 28 to 30, 2008, in conjunction with the International Contact Dermatitis Research Group and the Groupe d’Etudes et de Recherche en DermatoAllergologie.