David A. Basketter

A roadmap for the development of alternative (non-animal) methods for systemic toxicity testing - t4 report

Systemic toxicity testing forms the cornerstone for the safety evaluation of substances. Pressures to move from traditional animal models to novel technologies arise from various concerns, including: the need to evaluate large numbers of previously untested chemicals and new products (such as nanoparticles or cell therapies), the limited predictivity of traditional tests for human health effects, duration and costs of current approaches, and animal welfare considerations. The latter holds especially true in the context of the scheduled 2013 marketing ban on cosmetic ingredients tested for systemic toxicity. Based on a major analysis of the status of alternative methods (Adler et al., 2011) and its independent review (Hartung et al., 2011), the present report proposes a roadmap for how to overcome the acknowledged scientific gaps for the full replacement of systemic toxicity testing using animals. Five whitepapers were commissioned addressing toxicokinetics, skin sensitization, repeated-dose toxicity, carcinogenicity, and reproductive toxicity testing. An expert workshop of 35 participants from Europe and the US discussed and refined these whitepapers, which were subsequently compiled to form the present report. By prioritizing the many options to move the field forward, the expert group hopes to advance regulatory science.

Compilation of historical local lymph node data for evaluation of skin sensitization alternative methods.

Background: Within the toxicology community, considerable effort is directed toward the development of alternative methods for skin sensitization testing. The availability of high‐quality, relevant, and reliable in vivo data regarding skin sensitization is essential for the effective evaluation of alternative methodologies. Ideally, data derived from humans would be the most appropriate source because the test methods are attempting to predict a toxicologic effect in humans. Unfortunately, insufficient human data of the necessary quality are available, so it is necessary to rely on the best available animal data. In recent years, the local lymph node assay (LLNA) has emerged as a practical option for assessing the skin sensitization potential of chemicals. In addition to accurately identifying skin sensitizers, the LLNA can also provide a reliable measure of relative sensitization potency, information that is pivotal to the successful management of human health risks. Objective: To provide a database of robust in vivo data to calibrate, evaluate, and eventually validate new approaches for skin sensitization testing. Methods: LLNA data derived from previously conducted studies were compiled from the published literature and unpublished sources. Results: We provide a database that comprises LLNA data on 211 individual chemicals. This extensive chemical data set encompasses both the chemical and biologic diversity of known chemical allergens. To cover the range of relative allergenic potencies, the data set includes data on 13 extreme, 21 strong, 69 moderate, and 66 weak contact allergens, classified according to each allergens mathematically estimated concentration of chemical required to induce a threefold stimulation index. In addition, there are also 42 chemicals that are considered to be nonsensitizers. In terms of chemical diversity, the database contains data pertaining to the chemical classes represented by aldehydes, ketones, aromatic amines, quinones, and acrylates, as well as compounds that have different reactivity mechanisms. In addition to two‐dimensional chemical structures, the physicochemical parameters included are log Kp, log KO/W, and molecular weight. Conclusions: The list of chemicals contained in the data set represents both the chemical and biologic diversity that is known to exist for chemical allergens and non‐allergens. It is anticipated that this database will help accelerate the development, evaluation, and eventual validation of new approaches to skin sensitization assessment.

The murine local lymph node assay : a commentary on collaborative studies and new directions

The murine local lymph node assay is a predictive test for the identification of contact allergens. This paper provides a historical background to the development of the assay and describes the performance of a recently completed interlaboratory trial designed to evaluate further the utility of the method as an alternative or adjunct to guinea-pig predictive tests. On the basis of these and supplementary investigations, a number of recommendations can be made regarding the use and interpretation of the local lymph node assay. Finally, a number of issues arising from recent studies are discussed, including comparisons of the local lymph node assay with guinea-pig methods.

The local lymph node assay: developments and applications.

The murine local lymph node assay is a predictive test method for the identification of contact allergens in which sensitizing activity is measured as a function of induced proliferative responses in lymph nodes draining the site of application. In this article the development and validation of the assay are described and comparisons with guinea pig predictive test methods discussed. In addition we examine the advantages and limitations of the method and consider new opportunities and applications of the assay in the context of the toxicological evaluation of sensitizing potential.

Comparison of the local lymph node assay with the guinea-pig maximization test for the detection of a range of contact allergens

The guinea-pig maximization test (GMPT) has been in use as a method for the prediction of skin sensitization potential for over 20 years, and is widely accepted by regulatory authorities because of its reliable detection of a wide variety of potential human contact allergens. Nevertheless, the method has some limitations and drawbacks, including the use of an adjuvant, the injection of the test substance at induction thus bypassing the normal skin barrier and metabolic function, a subjective endpoint, interference by irritant and/or coloured chemicals, and a relatively long and complex protocol. To address these points, an alternative technique, the local lymph node assay (LLNA), has been proposed and has become the focus of much attention. Recent data from interlaboratory trials have shown a good level of agreement between test facilities and with existing guinea-pig data. The present work investigated the correlation between LLNA results and those derived from the GPMT for 40 chemicals covering a range of chemical types and levels of skin sensitization potential. The LLNA assay was capable of detecting chemicals that exhibit a strong sensitization potential in the GPMT. For chemicals classified as moderate sensitizers in the GPMT, the LLNA was usually positive or provided an indication of sensitizing activity (that was not sufficient to satisfy the current criteria for regarding the result as positive). Weaker sensitizers in the GPMT were usually not detected by the LLNA. With the single exception of copper chloride, non-sensitizers were not positive in the LLNA. The results support the view that the LLNA can provide a rapid and objective screening test for strong sensitizers.

A comparison of statistical approaches to the derivation of EC3 values from local lymph node assay dose responses.

Effective risk assessment and management of allergic contact dermatitis require three key factors: adequate hazard identification, measurement of the relative potency of identified hazards and an understanding of the nature, extent and duration of exposure. Suitable methods for hazard identification, such as the murine local lymph node assay (LLNA) and the guinea‐pig maximization test, are well established and conditions of human exposure normally can be well anticipated. Thus, the need is for a robust and quantitative method for the estimation of relative skin sensitizing potency. One possible approach is via the analysis of LLNA dose–response data, In the LLNA, contact allergens are defined currently as those chemicals that cause a threefold or greater increase in lymph node cell proliferative activity compared with concurrent vehicle‐treated controls. It is possible to estimate the concentration of a sensitizer required to generate a threefold stimulation of proliferation in draining lymph nodes; such a concentration is known as the EC3 value. Using a variety of statistical approaches to derive EC3 values from LLNA dose–response data for 10 chemicals, it has been demonstrated that simple linear interpolation between the values either side of the threefold stimulation index provides a robust assessment of the EC3 value without the need for recourse to more sophisticated statistical techniques. Provided that the appropriate concentrations of test chemical have been selected, EC3 values obtained in this way are reproducible both within and between laboratories and form the basis for examination of the utility of this approach for the estimation of relative skin sensitizing potency.

Further evaluation of the local lymph node assay in the final phase of an international collaborative trial.

The local lymph node assay (LLNA) is a method used for the prospective identification in mice of chemicals that have the potential to cause skin sensitization. We report here the results of the second and final phase of an international trial in which the performance of the assay has been evaluated using seven test materials in five independent laboratories. The additional chemicals examined here included compounds which are considered less potent allergens than some of those tested in the first phase of the investigation, and includes hexylcinnamic aldehyde (HCA), a chemical recommended by the Organization for Economic Cooperation and Development (OECD) as a positive control for skin sensitization studies. In each laboratory all skin sensitizing chemicals examined (2,4-dinitrochlorobenzene {DNCB}, HCA, oxazolone, isoeugenal and eugenol) elicited positive responses of comparable magnitude as judged by the derived lowest concentration of test chemical required to elicit a 3-fold or greater increase in the proliferative activity of draining lymph node cells compared with vehicle-treated controls. We observed that sodium lauryl sulphate, considered to be a non-sensitizing skin irritant, also induced a positive response in the assay. Para-aminobenzoic acid (pABA), a nonsensitizing chemical, was negative at all test concentrations in each laboratory. Some laboratories incorporated minor modifications into the standard assay procedure, including the evaluation of lymph nodes pooled from individual mice rather than treatment groups and the use of statistical analyses. The use of statistics did not markedly change the determination of the lowest concentration yielding a positive response. These data confirm that the local lymph node assay is robust and yields equivalent results when performed independently.

The local lymph node assay: A viable alternative to currently accepted skin sensitization tests

The prospective identification of skin sensitizing chemicals is a vital prerequisite for their proper risk management. Traditionally this has been achieved largely by the conduct of guinea pig assays such as the maximization and Buehler tests. These methods are recommended by the Organisation for Economic Cooperation and Development (OECD) and are required by the European Union (EU) for the evaluation of new substances. However, a novel mechanistically based method, the local lymph node assay (LLNA), has been the focus of substantial validation activity in recent years. This material is reviewed in this paper. It is shown that the LLNA has been validated successfully by five interlaboratory assessments as well as by comparisons with guinea pig tests and human data. The method also offers clear advantages to the user in terms of objectivity, time and cost, and delivers important animal welfare benefits. In consequence, it is recommended that the LLNA be formally adopted by the OECD in Guideline 406 and accepted by the EU and US EPA as a method suitable for the classification of the skin sensitizing potential of chemicals.

Dermal sensitization quantitative risk assessment (QRA) for fragrance ingredients.

Based on chemical, cellular, and molecular understanding of dermal sensitization, an exposure-based quantitative risk assessment (QRA) can be conducted to determine safe use levels of fragrance ingredients in different consumer product types. The key steps are: (1) determination of benchmarks (no expected sensitization induction level (NESIL)); (2) application of sensitization assessment factors (SAF); and (3) consumer exposure (CEL) calculation through product use. Using these parameters, an acceptable exposure level (AEL) can be calculated and compared with the CEL. The ratio of AEL to CEL must be favorable to support safe use of the potential skin sensitizer. This ratio must be calculated for the fragrance ingredient in each product type. Based on the Research Institute for Fragrance Materials, Inc. (RIFM) Expert Panels recommendation, RIFM and the International Fragrance Association (IFRA) have adopted the dermal sensitization QRA approach described in this review for fragrance ingredients identified as potential dermal sensitizers. This now forms the fragrance industrys core strategy for primary prevention of dermal sensitization to these materials in consumer products. This methodology is used to determine global fragrance industry product management practices (IFRA Standards) for fragrance ingredients that are potential dermal sensitizers. This paper describes the principles of the recommended approach, provides detailed review of all the information used in the dermal sensitization QRA approach for fragrance ingredients and presents key conclusions for its use now and refinement in the future.

The local lymph node assay: past, present and future

The local lymph node assay (LLNA) was developed originally as a method for the identification of chemicals that have the potential to cause skin sensitization and allergic contact dermatitis. The assay is based on an understanding that the acquisition of contact sensitization is associated with, and dependent upon, the stimulation by chemical allergens of lymphocyte proliferative responses in skin‐draining lymph nodes. Those chemicals that provoke a defined level of lymph node cell (LNC) proliferation (a 3‐fold or greater increase compared with concurrent vehicle controls) are classified as skin sensitizers. Following its original inception and development, the LLNA was the subject of both national and international interlaboratory collaborative trials, and of very detailed comparisons with other test methods and with human skin sensitization data. The assay has now been validated fully as a stand‐alone test for the purposes of hazard identification. In recent years, there has been a growing interest also in the use of the LLNA to assess the potency of contact allergens and in risk assessment. There is reason to believe that the extent of skin sensitization achieved is associated with the vigour of LNC proliferation induced in draining nodes. Given this relationship, the relative potency of skin sensitizing chemicals is measured in the LLNA by derivation of an EC3 value, this being the concentration of chemical required to provoke a 3‐fold increase in the proliferation of LNC compared with controls. Experience to date indicates that relative potency as determined using this approach correlates closely with what is known of the activity of skin sensitizing chemicals in humans. In this article, we review the development, evaluation and validation of the LLNA for the purposes of hazard identification, and the more recent application of the method for evaluation of potency in the context of risk assessment. In addition, we consider what new applications and modifications are currently being investigated.