Estimating uncertainty in LLNA EC3 data and its impact on regulatory classifications.

The murine Local Lymph Node Assay (LLNA) is a test that produces numerical results (EC3 values) quantifying the sensitization potency of chemicals. These results are broadly used in toxicology and serve as a basis for various classifications, which determine subsequent regulatory decisions. The continuing interest in LLNA data and the diminished likelihood of new experimental EC3 data being generated sparked this investigation of uncertainty. Instead of using the Gaussian distribution as a default choice for assessing variability in a data set, two strictly positive distributions were proposed and their performance over the available experimental EC3 values was tested. In the application stage, how the uncertainty in EC3 values affects the possible classifications was analyzed, and the percentage of the chemicals receiving ambiguous classification was determined. It was shown that this percentage is high, which increases the risk of improper classification. Two approaches were suggested in regulatory practice to address the uncertainty in the EC3 data: the approaches based on "grey zones" and the classification distribution. If a chemical cannot be classified unambiguously, the latter appears to be an acceptable means to assess the level of sensitization potency of chemicals and helps provide better regulatory decisions.

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 (15-45-135 mM) 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 the Skin-Sensitizing Potential of Brazilian Green Propolis.

Propolis is a resinous mixture produced by bees from their secretions and plant material, so its composition varies depending on its botanical origin. Propolis has several beneficial bioactivities, but its skin sensitization properties have long been suspected. Nevertheless, the skin sensitization potency of Brazilian green propolis (BGP) has not been scientifically evaluated. Here, we used scientifically reliable tests to evaluate it. In vitro antigenicity test based on the human cell line activation test (OECD TG 442E) was performed by measuring the expression of CD54 and CD86, which are indicators of the antigenicity of test substances, on THP-1 and DC2.4 cells. BGP did not affect the expression of either marker on THP-1 cells, but upregulated the expression of CD86 on DC2.4 cells, suggesting that BGP may be a skin sensitizer. Then, we performed local lymph node assay (LLNA, OECD TG 429) as a definitive in vivo test. LLNA showed that 1.70% BGP primed skin sensitization and is a "moderate sensitizer". Our results indicate scientific proof of the validity of arbitrary concentrations (1-2%), which have been used empirically, and provide the first scientific information on the safe use of BGP.

Skin Doctor: Machine Learning Models for Skin Sensitization Prediction that Provide Estimates and Indicators of Prediction Reliability.

The ability to predict the skin sensitization potential of small organic molecules is of high importance to the development and safe application of cosmetics, drugs and pesticides. One of the most widely accepted methods for predicting this hazard is the local lymph node assay (LLNA). The goal of this work was to develop in silico models for the prediction of the skin sensitization potential of small molecules that go beyond the state of the art, with larger LLNA data sets and, most importantly, a robust and intuitive definition of the applicability domain, paired with additional indicators of the reliability of predictions. We explored a large variety of molecular descriptors and fingerprints in combination with random forest and support vector machine classifiers. The most suitable models were tested on holdout data, on which they yielded competitive performance (Matthews correlation coefficients up to 0.52; accuracies up to 0.76; areas under the receiver operating characteristic curves up to 0.83). The most favorable models are available via a public web service that, in addition to predictions, provides assessments of the applicability domain and indicators of the reliability of the individual predictions.

Non-animal assessment of skin sensitization hazard: Is an integrated testing strategy needed, and if so what should be integrated?

There is an expectation that to meet regulatory requirements, and avoid or minimize animal testing, integrated approaches to testing and assessment will be needed that rely on assays representing key events (KEs) in the skin sensitization adverse outcome pathway. Three non-animal assays have been formally validated and regulatory adopted: the direct peptide reactivity assay (DPRA), the KeratinoSens™ assay and the human cell line activation test (h-CLAT). There have been many efforts to develop integrated approaches to testing and assessment with the "two out of three" approach attracting much attention. Here a set of 271 chemicals with mouse, human and non-animal sensitization test data was evaluated to compare the predictive performances of the three individual non-animal assays, their binary combinations and the "two out of three" approach in predicting skin sensitization potential. The most predictive approach was to use both the DPRA and h-CLAT as follows: (1) perform DPRA - if positive, classify as sensitizing, and (2) if negative, perform h-CLAT - a positive outcome denotes a sensitizer, a negative, a non-sensitizer. With this approach, 85% (local lymph node assay) and 93% (human) of non-sensitizer predictions were correct, whereas the "two out of three" approach had 69% (local lymph node assay) and 79% (human) of non-sensitizer predictions correct. The findings are consistent with the argument, supported by published quantitative mechanistic models that only the first KE needs to be modeled. All three assays model this KE to an extent. The value of using more than one assay depends on how the different assays compensate for each other's technical limitations. Copyright © 2017 John Wiley & Sons, Ltd.

What determines skin sensitization potency: Myths, maybes and realities. The 500 molecular weight cut-off: An updated analysis.

It is widely accepted that substances must have a molecular weight (MW) < 500 to penetrate effectively through the skin to induce sensitization. Roberts et al. (2012. Contact Dermatitis 68: 32-41) evaluated a data set of 699 substances taken from the TIMES-SS expert system and identified that of the 13 substances with a MW > 500, five were sensitizers. This provided good evidence to refute such a MW 500 threshold. While Roberts et al. (2012) made a convincing case that the MW > 500 cut-off was not a true requirement for sensitization, the number of counter examples identified were too few to draw any statistical conclusions. This updated analysis systematically interrogated a large repository of sensitization information collected under the EU REACH regulation. A data set of 2904 substances that had been tested for skin sensitization, using guinea pigs and/or mice were collected. The data set contained 197 substances with a MW > 500; 33 of these were skin sensitizers. Metal containing complexes, reaction products and mixtures were excluded from further consideration. The final set of 14 sensitizers substantiated the original findings. The study also assessed whether the same reaction chemistry principles established for low MW sensitizers applied to chemicals with a MW > 500. The existing reaction chemistry considerations were found appropriate to rationalize the sensitization behaviour of the 14 sensitizers with a MW > 500. The existence of the MW 500 threshold, based on the widespread misconception that the ability to penetrate efficiently the stratum corneum is a key determinant of skin sensitization potential and potency, was refuted. Copyright © 2016 John Wiley & Sons, Ltd.

Is skin penetration a determining factor in skin sensitization potential and potency? Refuting the notion of a LogKow threshold for skin sensitization.

It is widely accepted that substances that cannot penetrate through the skin will not be sensitizers. LogKow and molecular weight (MW) have been used to set thresholds for sensitization potential. Highly hydrophilic substances e.g. LogKow ≤ 1 are expected not to penetrate effectively to induce sensitization. To investigate whether LogKow >1 is a true requirement for sensitization, a large dataset of substances that had been evaluated for their skin sensitization potential under Registration, Evaluation, Authorisation and restriction of CHemicals (REACH), together with available measured LogKow values was compiled using the OECD eChemPortal. The incidence of sensitizers relative to non-sensitizers above and below a LogKow of 1 was explored. Reaction chemistry principles were used to explain the sensitization observed for the subset of substances with a LogKow ≤0. 1482 substances were identified with skin sensitization data and measured LogKow values. 525 substances had a measured LogKow ≤ 1, 100 of those were sensitizers. There was no significant difference in the incidence of sensitizers above and below a LogKow of 1. Reaction chemistry principles that had been established for lower MW and more hydrophobic substances were found to be still valid in rationalizing the skin sensitizers with a LogKow ≤ 0. The LogKow threshold arises from the widespread misconception that the ability to efficiently penetrate the stratum corneum is a key determinant of sensitization potential and potency. Copyright © 2016 John Wiley & Sons, Ltd.

Prediction of skin sensitization potency using machine learning approaches.

The replacement of animal use in testing for regulatory classification of skin sensitizers is a priority for US federal agencies that use data from such testing. Machine learning models that classify substances as sensitizers or non-sensitizers without using animal data have been developed and evaluated. Because some regulatory agencies require that sensitizers be further classified into potency categories, we developed statistical models to predict skin sensitization potency for murine local lymph node assay (LLNA) and human outcomes. Input variables for our models included six physicochemical properties and data from three non-animal test methods: direct peptide reactivity assay; human cell line activation test; and KeratinoSens™ assay. Models were built to predict three potency categories using four machine learning approaches and were validated using external test sets and leave-one-out cross-validation. A one-tiered strategy modeled all three categories of response together while a two-tiered strategy modeled sensitizer/non-sensitizer responses and then classified the sensitizers as strong or weak sensitizers. The two-tiered model using the support vector machine with all assay and physicochemical data inputs provided the best performance, yielding accuracy of 88% for prediction of LLNA outcomes (120 substances) and 81% for prediction of human test outcomes (87 substances). The best one-tiered model predicted LLNA outcomes with 78% accuracy and human outcomes with 75% accuracy. By comparison, the LLNA predicts human potency categories with 69% accuracy (60 of 87 substances correctly categorized). These results suggest that computational models using non-animal methods may provide valuable information for assessing skin sensitization potency. Copyright © 2017 John Wiley & Sons, Ltd.

Estimating skin sensitization potency from a single dose LLNA.

Skin sensitization is an important aspect of safety assessment. The mouse local lymph node assay (LLNA) developed in the 1990 s is an in vivo test used for skin sensitization hazard identification and characterization. More recently a reduced version of the LLNA (rLLNA) has been developed as a means of identifying, but not quantifying, sensitization hazard. The work presented here is aimed at enabling rLLNA data to be used to give quantitative potency information that can be used, inter alia, in modeling and read-across approaches to non-animal based potency estimation. A probit function has been derived enabling estimation of EC3 from a single dose. This has led to development of a modified version of the rLLNA, whereby as a general principle the SI value at 10%, or at a lower concentration if 10% is not testable, is used to calculate the EC3. This version of the rLLNA has been evaluated against a selection of chemicals for which full LLNA data are available, and has been shown to give EC3 values in good agreement with those derived from the full LLNA.

Extension of the Dermal Sensitisation Threshold (DST) approach to incorporate chemicals classified as reactive.

The evaluation of chemicals for their skin sensitising potential is an essential step in ensuring the safety of ingredients in consumer products. Similar to the Threshold of Toxicological Concern, the Dermal Sensitisation Threshold (DST) has been demonstrated to provide effective risk assessments for skin sensitisation in cases where human exposure is low. The DST was originally developed based on a Local Lymph Node Assay (LLNA) dataset and applied to chemicals that were not considered to be directly reactive to skin proteins, and unlikely to initiate the first mechanistic steps leading to the induction of sensitisation. Here we have extended the DST concept to protein reactive chemicals. A probabilistic assessment of the original DST dataset was conducted and a threshold of 64 µg/cm(2) was derived. In our accompanying publication, a set of structural chemistry based rules was developed to proactively identify highly reactive and potentially highly potent materials which should be excluded from the DST approach. The DST and rule set were benchmarked against a test set of chemicals with LLNA/human data. It is concluded that by combining the reactive DST with knowledge of chemistry a threshold can be established below which there is no appreciable risk of sensitisation for protein-reactive chemicals.

Principles for identification of High Potency Category Chemicals for which the Dermal Sensitisation Threshold (DST) approach should not be applied.

An essential step in ensuring the toxicological safety of chemicals used in consumer products is the evaluation of their skin sensitising potential. The sensitising potency, coupled with information on exposure levels, can be used in a Quantitative Risk Assessment (QRA) to determine an acceptable level of a given chemical in a given product. Where consumer skin exposure is low, a risk assessment can be conducted using the Dermal Sensitisation Threshold (DST) approach, avoiding the need to determine potency experimentally. Since skin sensitisation involves chemical reaction with skin proteins, the first step in the DST approach is to assess, on the basis of the chemical structure, whether the chemical is expected to be reactive or not. Our accompanying publication describes the probabilistic derivation of a DST of 64 µg/cm(2) for chemicals assessed as reactive. This would protect against 95% of chemicals assessed as reactive, but the remaining 5% would include chemicals with very high potency. Here we discuss the chemical properties and structural features of high potency sensitisers, and derive an approach whereby they can be identified and consequently excluded from application of the DST.

Skin Sensitization Evaluation of Carbon-Based Graphene Nanoplatelets.

Graphene nanoplatelets (GNPs) are one of the major types of carbon based nanomaterials that have different industrial and biomedical applications. There is a risk of exposure to GNP material in individuals involved in their large-scale production and in individuals who use products containing GNPs. Determining the exact toxicity of GNP nanomaterials is a very important agenda. This research aimed to evaluate the skin sensitization potentials induced by GNPs using two types of alternative to animal testing. We analyzed the physicochemical characteristics of the test material by selecting a graphene nanomaterial with a nano-size on one side. Thereafter, we evaluated the skin sensitization effect using an in vitro and an in vivo alternative test method, respectively. As a result, we found that GNPs do not induce skin sensitization. In addition, it was observed that the administration of GNPs did not induce cytotoxicity and skin toxicity. This is the first report of skin sensitization as a result of GNPs obtained using alternative test methods. These results suggest that GNP materials do not cause skin sensitization, and these assays may be useful in evaluating the skin sensitization of some nanomaterials.

Semi-correlations as a tool to model for skin sensitization.

Semi-correlation specifically assesses the correlation between a binary variable and a continuous variable. Semi-correlations were applied to develop binary models for various endpoints. We applied the semi-correlation to develop models of two kinds of skin sensitization one related to animals (local lymph node assay LLNA) and one to human beings (direct peptide reactivity assay DPRA and/or human cell line activation test h-CLAT). The models refer to binary classification for a two-level strategy: the first level (analysis of all compounds) is used in the format "sensitizer or non-sensitizer", and the second level (only sensitizers) is a further classification in the format "strong or weak sensitizer". The ranges of statistical characteristics of the models depend on the endpoint, LLNA or DPRA/h-CLAT: for the first level, sensitivity: 0.69-0.88, specificity: 0.75-0.89, accuracy: 0.77-0.87, Matthew's correlation coefficient (MCC): 0.54-0.57 and for the second level, sensitivity: 0.70-1.0, specificity: 0.78-0.83, accuracy: 0.77-0.87, MCC: 0.54-0.76. Thus, the described approach can be applied to building up models of the skin sensitization potency.

Evaluation of 4-methylcyclohexanemethanol (MCHM) in a combined irritancy and Local Lymph Node Assay (LLNA) in mice.

4-Methylcyclohexanemethanol (MCHM) is a flotation reagent used in fine coal beneficiation. On January 9, 2014, crude MCHM, a mixture containing predominantly MCHM, was inadvertently released into the Elk River, a municipal water source that serves about 300,000 people in the Charleston, WV area, resulting in temporary contamination of 15 percent of the state's tap water and causing significant dermal exposure. The current studies were undertaken to determine whether crude MCHM or MCHM has the potential to produce dermal irritancy and/or sensitization. BALB/c female mice were treated daily for 3 consecutive days by direct epicutaneous application of 25 µL of various concentrations of crude MCHM or MCHM to the dorsum of each ear. A mouse ear-swelling test was used to determine irritancy potential and was undertaken in combination with the standardized Local Lymph Node Assay (LLNA) to determine skin sensitizing potential. MCHM was found to produce skin irritation at concentrations above 20% and did not produce sensitization. Crude MCHM also produced irritation, although weaker, and in addition was found to be a weak to moderate skin sensitizer. The results are discussed in terms of potential human health hazard.