Mouse Local Lymph Node Assay Research Articles

Quantum-Mechanics Calculations Elucidate Skin-Sensitizing Pharmaceutical Compounds.

Skin sensitization is a critical end point in occupational toxicology that necessitates the use of fast, accurate, and affordable models to aid in establishing handling guidance for worker protection. While many in silico models have been developed, the scarcity of reliable data for active pharmaceutical ingredients (APIs) and their intermediates (together regarded as pharmaceutical compounds) brings into question the reliability of these tools, which are largely constructed using publicly available nonspecialty chemicals. Here, we present the quantum-mechanical (QM) Computer-Aided Discovery and REdesign (CADRE) model, which was developed with the bioactive and structurally complex chemical space in mind by relying on the fundamentals of chemical interactions in key events (versus structural attributes of training-set data). Validated in this study on 345 APIs and intermediates, CADRE achieved 95% accuracy, sensitivity, and specificity and a combined 79% accuracy in assigning potency categories compared to the mouse local lymph node assay data. We show how historical outcomes from CADRE testing in the pharmaceutical space, generated over the past 10 years on ca. 2500 chemicals, can be used to probe the relationships between sensitization mechanisms (or the underlying chemical classes) and the probability of eliciting a sensitization response in mice of a given potency. We believe this information to be of value to both practitioners, who can use it to quickly screen and triage their data sets, as well as to model developers to fine-tune their structure-based tools. Lastly, we leverage our experimentally validated subset of APIs and intermediates to show the importance of dermal permeability on the sensitization potential and potency. We demonstrate that common physicochemical properties used to assess permeation, such as the octanol-water partition coefficient and molecular weight, are poor proxies for the more accurate energy-pair distributions that can be computed from mixed QM and classical simulations using model representations of the stratum corneum.

Reduced specificity for the local lymph node assay for lipophilic chemicals: Implications for the validation of new approach methods for skin sensitization

Meaningful and accurate reference data are crucial for the validation of New Approach Methodologies (NAMs) in toxicology. For skin sensitization, multiple reference datasets are available including human patch test data, guinea pig data and data from the mouse local lymph node assay (LLNA). When assessed against the LLNA, a reduced sensitivity has been reported for in vitro and in chemico assays for lipophilic chemicals with a LogP ≥3.5, resulting in reliability restrictions within the h-CLAT OECD test guideline.Here we address the question of whether LLNA data are an appropriate reference for chemicals in this physicochemical range. Analysis of LLNA vs human reference data indicates that the false-discovery rate of the LLNA is significantly higher for chemicals with LogP ≥3.5. We present a mechanistic hypothesis whereby irritation caused by testing lipophilic chemicals at high test doses leads to unspecific cell proliferation. The accompanying analysis indicates that for lipophilic chemicals with negative calls in in vitro and in chemico assays, resorting to the LLNA is not necessarily a better option. These results indicate that the validation of NAMs in this particular LogP range should be based on a more holistic evaluation of the reference data and not solely upon LLNA data.

Exploring structure/property relationships to health and environmental hazards of polymeric polyisocyanate prepolymer substances-2. Dermal sensitization potential in the mouse local lymph node assay.

The sensitization potencies of twenty custom-designed monomer-depleted polymeric polyisocyanate prepolymer substances and their associated toluene diisocyanate (TDI), methylene diphenyl diisocyanate (MDI), hexamethylene diisocyanate (HDI), and isophorone diisocyanate (IPDI) monomer precursors were investigated by means of the mouse Local Lymph Node Assay (LLNA). These polymeric prepolymers were designed to represent the structural features and physical-chemical properties exhibited by a broad range of commercial polymeric polyisocyanate prepolymers that are produced from the reaction of aromatic and aliphatic diisocyanate monomers with aliphatic polyether and polyester polyols. The normalization of LLNA responses to the applied (15-45-135mM) concentrations showed that the skin sensitization potency of polymeric polyisocyanate prepolymers is at least 300 times less than that of the diisocyanate monomers from which they are derived. The sensitization potency of the prepolymers was shown to be mainly governed by their hydrophobicity (as expressed by the calculated octanol-water partition coefficient, log Kow) and surfactant properties. Neither hydrophilic (log Kow <0) nor very hydrophobic (log Kow >25) prepolymers stimulated lymphocyte proliferation beyond that of the dosing vehicle control. The findings of this investigation challenge the generally held assumption that all isocyanate (-N=C=O) bearing substances are potential skin (and respiratory) sensitizers. Further, these findings can guide the future development of isocyanate chemistries and associated polyurethane applications toward reduced exposure and health hazard potentials.

Evaluation of in vitro assays for the assessment of the skin sensitization hazard of functional polysiloxanes and silanes

The skin sensitization potential of chemicals has traditionally been evaluated in vivo according to OECD testing guidelines in guinea pigs or the mouse local lymph node assay. There has lately been a great emphasis on establishing in vitro test methods reflecting the key biological events in the adverse outcome pathway (AOP) for skin sensitization as published by the OECD. Against this background, a group of 8 polysiloxanes and silanes, seven of them aminofunctionalised, for which in vivo data were already available, has been tested in vitro in the direct peptide reactivity assay (DPRA), the KeratinoSens™ and the human cell line activation test (h-CLAT) and in the modified myeloid U937 skin sensitization test (mMUSST) as far as technically feasible. The main objective of the programme was to determine the utility of these systems for this heterogeneous group of silicone-based substances, recognizing that some substances are outside the assays applicability domains. The presented data provided some interesting mechanistical insights into the performance of these assays for functionalised siloxanes and silanes. The data also allow for a preliminary evaluation of proposed integrated testing strategies (ITS) to determine the skin sensitization potential of chemicals which were not considered in the training sets of the respective ITS.

LB756 Determination of contact sensitization potential of chemicals using in vitro reconstructed normal human epidermal model EpiDerm: Impact of the modality of application

Assessment of skin sensitization potential has traditionally been conducted in animal models, such as the Mouse Local Lymph Node Assay (LLNA) and the Guinea Pig Maximisation Test (GPMT). However, a growing focus and consensus for minimizing animal use have stimulated the development of in vitro methods to assess skin sensitization. Interleukin-18 (IL-18) release in reconstructed human epidermal models has been identified as a potentially useful endpoint for the identification and classification of skin sensitizing chemicals, including chemicals of low water solubility or stability. The purpose of this study was to investigate the impact of the modality of chemical exposure on the predictive capacity of the assay. EpiDerm tissue viability assessed by MTT assay and IL-18 release assessed by ELISA were evaluated after 24 h topical exposure to test chemicals either impregnated in 8 mm diameter paper filters or directly applied to the surface of EpiDerm. Acetone: olive oil (4:1) was used as vehicle in all cases. A total of five chemicals from 3 different sources were tested. The testing set included 3 senzitizers, namely 2,4-dinitrochlorobenzene, cinnamaldehyde and isoeugenol/eugenol, and 2 non senzitizers, lactic acid and salicylic acid. Four independent dose – response experiments were conducted in 3 laboratories, resulting in correct prediction of the sensitizing potency of test chemicals. The assessment of IL-18 release using in vitro reconstructed normal human epidermal model EpiDerm appears to be a promising tool for in vitro determination of contact sensitization potential.

An in vitro human skin test for assessing sensitization potential.

Sensitization to chemicals resulting in an allergy is an important health issue. The current gold-standard method for identification and characterization of skin-sensitizing chemicals was the mouse local lymph node assay (LLNA). However, for a number of reasons there has been an increasing imperative to develop alternative approaches to hazard identification that do not require the use of animals. Here we describe a human in-vitro skin explant test for identification of sensitization hazards and the assessment of relative skin sensitizing potency. This method measures histological damage in human skin as a readout of the immune response induced by the test material. Using this approach we have measured responses to 44 chemicals including skin sensitizers, pre/pro-haptens, respiratory sensitizers, non-sensitizing chemicals (including skin-irritants) and previously misclassified compounds. Based on comparisons with the LLNA, the skin explant test gave 95% specificity, 95% sensitivity, 95% concordance with a correlation coefficient of 0.9. The same specificity and sensitivity were achieved for comparison of results with published human sensitization data with a correlation coefficient of 0.91. The test also successfully identified nickel sulphate as a human skin sensitizer, which was misclassified as negative in the LLNA. In addition, sensitizers and non-sensitizers identified as positive or negative by the skin explant test have induced high/low T cell proliferation and IFNγ production, respectively. Collectively, the data suggests the human in-vitro skin explant test could provide the basis for a novel approach for characterization of the sensitizing activity as a first step in the risk assessment process.

Appraisal of within- and between-laboratory reproducibility of non-radioisotopic local lymph node assay using flow cytometry, LLNA:BrdU-FCM: Comparison of OECD TG429 performance standard and statistical evaluation

Mouse local lymph node assay (LLNA, OECD TG429) is an alternative test replacing conventional guinea pig tests (OECD TG406) for the skin sensitization test but the use of a radioisotopic agent, 3H-thymidine, deters its active dissemination. New non-radioisotopic LLNA, LLNA:BrdU-FCM employs a non-radioisotopic analog, 5-bromo-2′-deoxyuridine (BrdU) and flow cytometry. For an analogous method, OECD TG429 performance standard (PS) advises that two reference compounds be tested repeatedly and ECt(threshold) values obtained must fall within acceptable ranges to prove within- and between-laboratory reproducibility. However, this criteria is somewhat arbitrary and sample size of ECt is less than 5, raising concerns about insufficient reliability. Here, we explored various statistical methods to evaluate the reproducibility of LLNA:BrdU-FCM with stimulation index (SI), the raw data for ECt calculation, produced from 3 laboratories. Descriptive statistics along with graphical representation of SI was presented. For inferential statistics, parametric and non-parametric methods were applied to test the reproducibility of SI of a concurrent positive control and the robustness of results were investigated. Descriptive statistics and graphical representation of SI alone could illustrate the within- and between-laboratory reproducibility. Inferential statistics employing parametric and nonparametric methods drew similar conclusion. While all labs passed within- and between-laboratory reproducibility criteria given by OECD TG429 PS based on ECt values, statistical evaluation based on SI values showed that only two labs succeeded in achieving within-laboratory reproducibility. For those two labs that satisfied the within-lab reproducibility, between-laboratory reproducibility could be also attained based on inferential as well as descriptive statistics.

Electrophilic Reactivity and Skin Sensitization Potency of SNAr Electrophiles

We published in 2011 a quantitative mechanistic model (QMM) for skin sensitization potency of SNAr electrophiles in the mouse local lymph node assay (LLNA). In this model, potency was correlated with a combination of σ* for the leaving group and the total σ(-) values of the other substituents in the aromatic ring. Shortly afterward Natsch et al. published a kinetic study in which rate constants were determined for reactions of SNAr electrophiles with the cysteine-based peptide Ac-RFAACAA (Cys-peptide) that is used in the direct peptide reactivity assay (DPRA), and correlations were sought between these rate constants and sensitization potency in the LLNA. These two publications together have enabled the present study, aiming to develop a linear free energy relationship (LFER) correlating Cys-peptide reactivity with a reactivity parameter (RP) based on a combination of σ* and σ(-) substituent constants and, by analyzing differences between the QMM based on RP and the QMM based on Cys-peptide rate constants, to gain further insights into the underlying chemistry of skin sensitization. For the 2,4-dinitro-X-subsituted benzenes (DNXB), the rate constants of Natsch et al. are well correlated with the reactivity parameter used in our earlier work, with two outliers. These are the compounds with X = F and X = SCN, which are both substantially more reactive toward Cys-peptide than predicted from comparison of their RP values with those of the other DNXB compounds. These two compounds are both negative outliers from a correlation of sensitization potency with experimental rate constants, but fit well to the correlation of sensitization potency with RP values. With these two compounds excluded, sensitization potency is well correlated with the experimental rate constants for the DNXB compounds (X = SO3(-), I, Br, Cl) together with 2,4-dichloro-1-nitrobenzene and 1,3,4,5-tetrachloro-2,6-dicyanobenzene. The regression equation is pEC3 = 0.88 log k + 4.03, R(2) = 0.966. The implication of DNFB being an outlier is that the model Cys-peptide nucleophile is substantially more sterically hindered than the cutaneous nucleophile(s) involved in the sensitization process. The pattern seen with 2,4-dinitrothiocyanatobenzene suggests that this compound reacts as an SNAr electrophile in the sensitization process, but by a different pathway, acting as a CN transfer agent, with the model Cys-peptide. For two further compounds, 2,4,6-trinitrochlorobenzene and 2,4,6-trinitrobenzenesulfonate, the Cys-peptide rate constants are well predicted by the reactivity parameter based on displacement of the Cl or SO3(-) substituent, but their sensitization potency is underestimated by both the Cys-peptide rate constant and this reactivity parameter. However, potency of these two compounds is well predicted by a reactivity parameter calculated on the basis of displacement of the 2-nitro group. This is interpreted as a case of sensitization being driven by the thermodynamically favored rather than the kinetically favored reaction product.

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.

Chemistry-Based Risk Assessment for Skin Sensitization: Quantitative Mechanistic Modeling for the SNAr Domain

There is a strong impetus to develop nonanimal based methods to predict skin sensitization potency. An approach based on physical organic chemistry, whereby chemicals are classified into reaction mechanistic domains and quantitative models or read-across methods are derived for each domain, has been the basis of several recent publications. This article is concerned with the S(N)Ar reaction mechanistic domain. Electrophiles able to react by the S(N)Ar mechanism have long been recognized as skin sensitizers and have been used extensively in research studies on the biology of skin sensitization. Although qualitative discriminant analysis approaches have been developed for estimating the sensitization potential for S(N)Ar electrophiles on a yes/no qualitative basis, no quantitative mechanistic model (QMM) has so far been developed for this domain. Here, we derive a QMM that correlates skin sensitization potency, quantified by murine local lymph node assay (LLNA) EC3 data on a range of S(N)Ar electrophiles. It is based on the Hammett σ(-) values for the activating groups and the Taft σ* value for the leaving group. The model takes the form pEC3=2.48 Σσ(-) + 0.60 σ* - 4.51. This QMM, generated from mouse LLNA data, provides a reactivity parameter 2.48 Σσ(-) + 0.60 σ*, which was applied to a set of 20 compounds for which guinea pig test results were available in the literature and was found to successfully discriminate the sensitizers from the nonsensitizers. The reactivity parameter correctly predicted a known human sensitizer 2,4-dichloropyrimidine. New LLNA data on two further S(N)Ar electrophiles are consistent with the QMM.

Encapsulating contact allergens in liposomes, ethosomes, and polycaprolactone may affect their sensitizing properties

Attempts to improve formulation of topical products are a continuing process and the development of micro- and nanovesicular systems as well as polymeric microparticles has led to marketing of topical drugs and cosmetics using these technologies. Encapsulation of some well-known contact allergens in ethanolic liposomes have been reported to enhance allergenicity compared with the allergens in similar vehicles without liposomes. The present report includes data on more sensitization studies using the mouse local lymph node assay with three contact allergens encapsulated in different dermal drug-delivery systems: liposomes, ethosomes, and polycaprolactone particles. The results show that the drug-delivery systems are not sensitizers in themselves. Encapsulating the hydrophilic contact allergen potassium dichromate in all three drug-delivery systems did not affect the sensitizing capacity of potassium dichromate compared with control solutions. However, encapsulating the lipophilic contact allergen dinitrochlorobenzene (DNCB) in polycaprolactone reduced the sensitizing capacity to 1211 ± 449 compared with liposomes (7602 ± 2658) and in acetone:olive oil (4:1) (5633 ± 666). The same trend was observed for encapsulating isoeugenol in polycaprolactone (1100 ± 406) compared with a formulation in acetone:olive oil (4491 ± 819) and in liposomes (3668 ± 950). Further, the size of DNCB-loaded liposomes did not affect the sensitizing properties. These results suggest that modern dermal drug-delivery systems may in some cases magnify or decrease the sensitizing capacity of the encapsulated contact allergen.

Recent Development in Animal Testing to Predict the Skin and Respiratory Sensitizing Potential of Chemicals

The identification of chemicals with skin and/or respiratory sensitizing potential is important for the prevention of allergic diseases in both living and work environments. Although a number of animal models for respiratory allergic diseases have been reported, none of these models meets the goals of broad assessments of chemical sensitizing potential. We are attempting to develop a test for predicting the respiratory sensitization of chemicals. In the evaluation of skin sensitization of chemicals, the mostly used predictive tests are the guinea pig maximization test, Buehler test, and mouse local lymph node assay (LLNA). However, only LLNA has been validated formally and independently. Recent studies have revealed that EC3 estimated by LLNA correlates well with human skin sensitizing potency and the threshold for the induction of skin sensitization in the human repeat patch test. Thus, LLNA can predict the potency of skin sensitizing potential of a chemical and its risk in humans.

A plasmacytoid dendritic cell (CD123+/CD11c−) based assay system to predict contact allergenicity of chemicals

A predictive allergenicity test system for assessing the contact allergenicity of chemicals is needed by the cosmetic and pharmaceutical industry to monitor product safety in the marketplace. Development of such non-animal alternative assay systems for skin sensitization and hazard identification has been pursued by policy makers and regulatory agencies. We investigated whether phenotypic and functional changes to a subset of dendritic cells (DC), plasmacytoid DC (pDC), could be used to identify contact allergens. To achieve this goal, normal human DC were generated from CD34+ progenitor cells and cryopreserved. Frozen DC were thawed and the pDC fraction (CD123+/CD11c−) was harvested using FACS sorting. The pDC were cultured, expanded, and exposed to chemical allergens ( N = 26) or non-allergens ( N = 22). Concentrations of each chemical that resulted in >50% viability was determined using FACS analysis of propidium iodide stained cells using pDC from 2 to 5 donors. Expression of the surface marker, CD86, which has been implicated in dendritic cell maturation, was used as a marker of allergenicity. CD86 expression increased (≥1.5-fold) for 25 of 26 allergens (sensitivity = 96%) but did not increase for 19 of 22 non-allergens (specificity = 86%). In a direct comparison to historical data for the regulatory approved, mouse local lymph node assay (LLNA) for 23 allergens and 22 non-allergens, the pDC method had sensitivity and specificity of 96% and 86%, respectively, while the sensitivity and specificity of the LLNA assay was 83% and 82%, respectively. In conclusion, CD86 expression in pDC appears to be a sensitive and specific indicator to identify contact allergenicity. Such an assay method utilizing normal human cells will be useful for high throughput screening of chemicals for allergenicity.

Is Toluene Diamine a Sensitizer and is there Cross-Reactivity between Toluene Diamine and Toluene Diisocyanate?

Toluene diamine (TDA) is formed when toluene diisocyanate (TDI), a potent sensitizer, comes in contact with an aqueous environment. The sensitizing capacity of TDA and the cross-reactivity between TDI and TDA are unknown. TDA (5-25%) and TDI (0.3%), dissolved in acetone/olive oil (AOO) (4:1) were tested in the mouse local lymph node assay (LLNA). To determine the capacity of TDA to elicit an asthmatic response and to determine the cross-reaction with TDI, a locally developed experimental mouse model of chemical-induced asthma was used. On days 1 and 8, BALB/c mice received 20 microl of TDI (0.3%), TDA (20%), or AOO (4:1) on each ear. On day 15, they received an intranasal instillation of TDI (0.1%), TDA (0.5%) or AOO (3:2). The EC(3) of TDA in the LLNA is 19%. In the model of chemical-induced asthma, TDI induced a ventilatory response [increased Penh after challenge; increased airway hyperreactivity (AHR)], inflammatory changes (bronchoalveolar lavage neutrophils), and immunological changes (increased CD19(+) lymphocytes, IL-4 and total serum IgE), whereas TDA did not show any of these responses. Mice sensitized with TDI and challenged with TDA also did not show any airway or inflammatory response, although they had increased levels of total serum IgE. Mice sensitized with TDA and challenged with TDI did not show any response. According to the classification of sensitizers in the LLNA, TDA is a weak dermal sensitizer. In the experimental mouse model of chemical-induced asthma, TDA does not act as a respiratory sensitizer, at the concentration used. No cross-reactivity between TDI and TDA was found.

Read‐across to rank skin sensitization potential: subcategories for the Michael acceptor domain

Eliminating animal testing for skin sensitization is a significant challenge in consumer safety risk assessment. To be able to perform resilient risk assessments in the future, one will need alternative approaches to fill the data gaps. To this end, we propose a subcategory-based read-across approach to estimate and rank skin sensitization potential of chemicals. The example described here is for the mechanism of Michael-type nucleophilic addition with subcategories being limited to carbonyl-containing compounds. In this approach, in silico tools based on structural alerts were used to determine both the mechanism of protein binding and the relative subcategories within that mechanism. Fifty compounds previously evaluated in the in vivo mouse local lymph node assay (LLNA) were placed in 10 subcategories defined by their polarized alpha,beta-unsaturated substructure. To offset the limitations and skewness of the published in vivo data, in chemico glutathione (GSH) depletion data also were included. It was shown that the read-across approach can be successfully used to rank qualitatively skin sensitization potential of an untested carbonyl-containing Michael acceptor chemical by using subcategories. Moreover, the use of the more resilient in chemico GSH depletion data added further support to the read-across result.

Application of a Systems Biology Approach to Skin Allergy Risk Assessment

We have developed an in silico model of the induction of skin sensitisation, in order to characterise and quantify the contribution of each pathway to the overall biological process. This analysis has been used to guide our research on skin sensitisation and in vitro test development programmes, and provides a theoretical rationale for the interpretation and integration of non-animal predictive data for risk assessment (RA) purposes. The in vivo mouse Local Lymph Node Assay (LLNA) is now in widespread use for the evaluation of skin sensitisation potential and potency. Recent changes in European Union (EU) legislation (i.e. the 7th Amendment to the EU Cosmetics Directive) have made the development of nonanimal approaches to provide the data for skin sensitisation RA a key business need. Several in vitro predictive assays have already been developed for the prediction of skin sensitisation. However, these are based on the determination of a small number of pathways within the overall biological process, and our understanding of the relative contribution of these individual pathways to skin sensitisation induction is limited. To address this knowledge gap, a "systems biology" approach has been used to construct a computer-based mathematical model of the induction of skin sensitisation, in collaboration with Entelos, Inc. The biological mechanisms underlying the induction phase of skin sensitisation are represented by nonlinear ordinary differential equations and defined by using information from over 500 published papers. By using the model, we have identified knowledge gaps for future investigative research, and key factors that have a major influence on the induction of skin sensitisation (e.g. TNF-alpha production in the epidermis). The relative contribution of each of these key pathways has been assessed by determining their contributions to the overall process (e.g. sensitiser-specific T-cell proliferation in the draining lymph node). This information provides a biologically-relevant rationale for the interpretation and potential integration of diverse types of non-animal predictive data. Consequently, the Skin Sensitisation Physiolab (SSP) platform represents one approach to integration that is likely to prove an invaluable tool for hazard evaluation in a new framework for consumer safety RA.

Assuring Consumer Safety without Animal Testing: A Feasibility Case Study for Skin Sensitisation

Allergic Contact Dermatitis (ACD; chemical-induced skin sensitisation) represents a key consumer safety endpoint for the cosmetics industry. At present, animal tests (predominantly the mouse Local Lymph Node Assay) are used to generate skin sensitisation hazard data for use in consumer safety risk assessments. An animal testing ban on chemicals to be used in cosmetics will come into effect in the European Union (EU) from March 2009. This animal testing ban is also linked to an EU marketing ban on products containing any ingredients that have been subsequently tested in animals, from March 2009 or March 2013, depending on the toxicological endpoint of concern. Consequently, the testing of cosmetic ingredients in animals for their potential to induce skin sensitisation will be subject to an EU marketing ban, from March 2013 onwards. Our conceptual framework and strategy to deliver a non-animal approach to consumer safety risk assessment can be summarised as an evaluation of new technologies (e.g. 'omics', informatics), leading to the development of new non-animal (in silico and in vitro) predictive models for the generation and interpretation of new forms of hazard characterisation data, followed by the development of new risk assessment approaches to integrate these new forms of data and information in the context of human exposure. Following the principles of the conceptual framework, we have been investigating existing and developing new technologies, models and approaches, in order to explore the feasibility of delivering consumer safety risk assessment decisions in the absence of new animal data. We present here our progress in implementing this conceptual framework, with the skin sensitisation endpoint used as a case study.

Proliferative responses in the local lymph node assay associated with concomitant exposure to 1,4‐phenylenediamine and methyldibromo glutaronitrile: evidence for synergy?

A key consideration when undertaking risk assessments should be the potential for synergy between contact allergens. Previously, this concept has only been investigated during elicitation in contact allergic individuals. To determine whether there exists evidence for synergy between contact allergens during the induction phase of skin sensitization using the mouse local lymph node assay (LLNA) as a model system. Proliferative responses in draining lymph nodes were assessed with increasing concentrations of 1,4-phenylenediamine (PPD), methyldibromo glutaronitrile (MDBGN), and a combination of PPD and MDBGN. Data from each of two independent experiments show that lymph node cell proliferation associated with combined exposure to PPD and MDBGN was, in general, only modestly increased relative to that predicted from a simple summation of their individual responses. Although the increase in response is very modest, it does imply a relationship between this combination of sensitizers that may not be simply additive in terms of their ability to stimulate proliferative responses in draining lymph nodes. The reproducibility of this observation should be confirmed in future studies with additional pairs of contact allergens to ascertain whether or not this represents evidence of synergy.

Interlaboratory Validation of 1% Pluronic L92 Surfactant as a Suitable, Aqueous Vehicle for Testing Pesticide Formulations Using the Murine Local Lymph Node Assay

The mouse local lymph node assay (LLNA) has become the preferred test for evaluating the dermal sensitization potential of chemicals and requirements are now emerging for its use in the evaluation of their formulated products, especially in the European Union. However, despite its widespread use and extensive validation, the use of this assay for directly testing mixtures and formulated products has been questioned, which could lead to repeat testing using multiple animal models. As pesticide formulations are typically a specific complex blend of chemicals for use as aqueous-based dilutions, traditional vehicles prescribed for the LLNA may change the properties of these formulations leading to inaccurate test results and hazard identification. The objective of this study was to evaluate the effectiveness of an aqueous solution of Pluronic L92 block copolymer surfactant (L92) as a vehicle in the mouse LLNA across five laboratories. Three chemicals with known sensitization potential and four pesticide formulations for which the sensitization potential in guinea pigs and/or humans had previously been assessed were used. Identical LLNA protocols and test materials were used in the evaluation. Assessment of the positive control chemicals, hexylcinnamaldehyde, formaldehyde, and potassium dichromate revealed positive results when using 1% aqueous L92 as the vehicle. Furthermore, results for these chemicals were reproducible among the five laboratories and demonstrated consistent relative potency determinations. The four pesticide formulations diluted in 1% aqueous L92 also demonstrated reproducible results in the LLNA among the five laboratories. Results for these test materials were also consistent with those generated previously using guinea pigs or from human experience. These data support testing aqueous compatible chemicals or pesticide formulations using the mouse LLNA, and provide additional support for the use of 1% aqueous L92 as a suitable, aqueous-based vehicle.

On the skin sensitisation potential of rosin and oxidised rosin

In the EU rosin is classified as a skin sensitiser, apparently on the basis of its oxidation to sensitising agents. Rosin (gum, tall oil or wood) is not a skin sensitiser when examined in the guinea pig maximisation test (GPMT). Oxidised rosins are sensitisers in the GPMT. Oxidised gum rosin was further tested in the mouse local lymph node assay (LLNA) and the Buehler test, but is not a sensitiser in either of these tests. Further, the outcome of the LLNA can be used to assess the potency of oxidised rosin as an inducing agent in humans, and oxidised rosin is, at most, a weak sensitiser in this test. Thus, oxidised rosin is not a potent inducing agent for skin sensitisation unless the dermal barrier is bypassed and/or there is deliberate use of Freund’s Complete Adjuvant to induce greater susceptibility. The material used for human patch testing (‘colophony’) is in oxidised form. A re-examination of epidemiological studies suggests that patients in dermatological clinics show higher response rates than do the general population or those occupationally exposed to presumably oxidised rosin. Thus, the differences seen in susceptibility in the regulatory tests may be reflected in the human population. These results are discussed in terms of possible testing and classification strategies for dealing with existing chemicals, with particular reference to the new European Union legislation.