Direct Peptide Reactivity Assay Research Articles

The correlation of haptenation of gold nanoparticles and cysteine modified screen printed carbon electrode by impedance technique with local lymph node assay data

Skin sensitization occurs when a skin sensitizer binds covalently to skin proteins through the haptenation process. The objective of this study was to correlate the electrochemical impedance spectroscopy (EIS) data of a screen printed carbon electrode (SPCE) modified with cysteine and gold nanoparticles (AuNPs) with local lymph node assay (LLNA) data as a potential skin sensitizer biosensor. The EIS was used to quantify variations in charge transfer resistance of skin sensitizers (ΔRCTskin sensitizer) due to different binding rates of skin sensitizers to cysteine. SPCE was modified through electrodeposition of AuNPs/thiourea/self–assembly of AuNPs/cysteine (assigned as ETSC) for the detection of skin sensitizers. Surface analysis of modified SPCEs using FESEM and EDX revealed a smooth surface with an uneven distribution of cysteine with AuNPs molecules. The ETSC modified SPCE showed a significant skin sensitizer biosensor since the ΔRCTskin sensitizer readings were increased proportionally to the strength of the skin sensitizers, with strong/extreme skin sensitizers displaying higher ΔRCTskin sensitizer readings compared to moderate and weak/non–skin sensitizers. The skin sensitization analysis from this work was compared to LLNA (animal study), human cell line activation (h–CLAT), direct peptide reactivity assay (DPRA), and KeratinoSens™, surface plasmon resonance (SPR) matched the categorization of LLNA in the following descending order: 96%, 92%, 82%, 70%, 70%, and 12%. With just an 8% mismatch with LLNA data, the EIS approach used in this study could be used as a screening tool for skin sensitizers.

Pre-validation study of spectrophotometric direct peptide reactivity assay (Spectro-DPRA) as a modified in chemico skin sensitization test method.

Skin sensitization is induced when certain chemicals bind to skin proteins. Direct peptide reactivity assay (DPRA) has been adopted by the OECD as an alternative method to evaluate skin sensitization by assessing a substance's reaction to two model peptides. A modified spectrophotometric method, Spectro-DPRA, can evaluate skin sensitization, in a high throughput fashion, to obviate some limitations of DPRA. Pre-validation studies for Spectro-DPRA were conducted to determine transferability and proficiency, within- and between-laboratory reproducibility, and predictive ability based on GLP principles at three laboratories (AP, KTR, and KCL). All laboratories confirmed high (> 90%) concordance for evaluating the sensitivity induced by ten chemical substances. The concordance among the three tests performed by each laboratory was 90% for AP, 100% for KTR, and 100% for KCL. The mean accuracy of the laboratories was 93.3% [compared to the standard operating procedure (SOP)]. The reproducibility among the three laboratories was as high as 86.7%; the accuracy was 86.7% for AP, 100% for KTR, and 86.7% for KCL (compared to the SOP). An additional 54 substances were assessed in 3 separate labs to verify the prediction rate. Based on the result, 29 out of 33 substances were classified as sensitizers, and 19 out of 21 identified as non-sensitizers; the corresponding sensitivity, specificity, and accuracy values were 87.9%, 90.5%, and 88.9%, respectively. These findings indicate that the Spectro-DPRA can address the molecular initiating event with improved predictability and reproducibility, while saving time and cost compared to DPRA or ADRA.

Development of a 96-Well Electrophilic Allergen Screening Assay for Skin Sensitization Using a Measurement Science Approach.

The Electrophilic Allergen Screening Assay (EASA) has emerged as a promising in chemico method to detect the first key event in the adverse outcome pathway (AOP) for skin sensitization. This assay functions by assessing the depletion of one of two probe molecules (4-nitrobenzenethiol (NBT) and pyridoxylamine (PDA)) in the presence of a test compound (TC). The initial development of EASA utilized a cuvette format resulting in multiple measurement challenges such as low throughput and the inability to include adequate control measurements. In this study, we describe the redesign of EASA into a 96-well plate format that incorporates in-process control measurements to quantify key sources of variability each time the assay is run. The data from the analysis of 67 TCs using the 96-well format had 77% concordance with animal data from the local lymph node assay (LLNA), a result consistent with that for the direct peptide reactivity assay (DPRA), an OECD test guideline (442C) protein binding assay. Overall, the measurement science approach described here provides steps during assay development that can be taken to increase confidence of in chemico assays by attempting to fully characterize the sources of variability and potential biases and incorporate in-process control measurements into the assay.

Application of direct peptide reactivity assay for assessing the skin sensitization potential of essential oils.

Plant-derived products are frequently found as ingredients in cosmetics. However, the current data show non-neglectable skin sensitizing potential of these preparations suggesting an urgent need for data regarding their health safety profile. The aim of this study was to assess the skin sensitization potential of commercial essential oils by selected Lamiaceae species (Lavandula angustifolia, Melissa officinalis, Mentha longifolia, Thymus vulgaris, Salvia officinalis, and Rosmarinus officinalis) using a chemistry-based Direct Peptide Reactivity Assay (DPRA) in order to predict their potential allergic properties. In the DPRA assay, nucleophile-containing synthetic peptides (cysteine peptide and lysine peptide) were incubated with the test substance for 24 h. Depletion of the peptide in the reaction mixture was measured by high-pressure liquid chromatography (HPLC) using UV detection and the average peptide depletion data for cysteine and lysine was then calculated. Menthae longifoliae aetheroleum showed no or minimal reactivity with 4.48% cysteine depletion, Rosmarini aetheroleum and Salviae aetheroleum showed low reactivity with the 12.79% and 15.34% of cysteine depletion, respectively, while the other analyzed essential oils showed moderate reactivity with the cysteine depletion between 23.21 and 48.43%. According to DPRA predictive analysis, only Menthae longifoliae aetheroleum can be classified as negative, while all other essential oils may be classified as positive, thus having the potential to cause skin sensitization.

Application of Defined Approaches for Skin Sensitization to Agrochemical Products

Skin sensitization testing is a regulatory requirement for safety evaluations of pesticides in multiple countries. Globally harmonized test guidelines that include in chemico and in vitro methods reduce animal use, but no single assay is recommended as a complete replacement for animal tests. Defined approaches (DAs) that integrate data from multiple non-animal methods are accepted; however, the methods that comprise them have been evaluated using monoconstituent substances rather than mixtures or formulations. To address this data gap, we tested 27 agrochemical formulations in the direct peptide reactivity assay (DPRA), the KeratinoSens™ assay, and the human cell line activation test (h-CLAT). These data were used as inputs to evaluate three DAs for hazard classification of skin sensitization potential and two DAs for potency categorization. When compared to historical animal results, balanced accuracy for the DAs for predicting in vivo skin sensitization hazard (i.e., sensitizer vs. nonsensitizer) ranged from 56 to 78%. The best performing DA was the “2 out of 3 (2o3)” DA, in which the hazard classification was based on two concordant results from the DPRA, KeratinoSens, or h-CLAT. The KE 3/1 sequential testing strategy (STS), which uses h-CLAT and DPRA results, and the integrated testing strategy (ITSv2), which uses h-CLAT, DPRA, and an in silico hazard prediction from OECD QSAR Toolbox, had balanced accuracies of 56–57% for hazard classification. Of the individual test methods, KeratinoSens had the best performance for predicting in vivo hazard outcomes. Its balanced accuracy of 81% was similar to that of the 2o3 DA (78%). For predicting potency categories defined by the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (GHS), the correct classification rate of the STS was 52% and that of the ITSv2 was 43%. These results demonstrate that non-animal test methods have utility for evaluating the skin sensitization potential of agrochemical formulations as compared to animal reference data. While additional data generation is needed, testing strategies such as DAs anchored to human biology and mechanistic information provide a promising approach for agrochemical formulation testing.

Expansion of the Cosmetics Europe skin sensitisation database with new substances and PPRA data

The assessment of skin sensitisation is a key requirement in all regulated sectors, with the European Union's regulation of cosmetic ingredients being most challenging, since it requires quantitative skin sensitisation assessment based on new approach methodologies (NAMs).To address this challenge, an in-depth and harmonised understanding of NAMs is fundamental to inform the assessment. Therefore, we compiled a database of NAMs, and in vivo (human and local lymph node assay) reference data. Here, we expanded this database with 41 substances highly relevant for cosmetic industry. These structurally different substances were tested in six NAMs (Direct Peptide Reactivity Assay, KeratinoSens™, human Cell Line Activation Test, U-SENS™, SENS-IS, Peroxidase Peptide Reactivity Assay).Our analysis revealed that the substances could be tested without technical limitations, but were generally overpredicted when compared to reference results. Reasons for this reduced predictivity were explored through pairwise NAM comparisons and association of overprediction with hydrophobicity.We conclude that more detailed understanding of how NAMs apply to a wider range of substances is needed. This would support a flexible and informed choice of NAMs to be optimally applied in the context of a next generation risk assessment framework, ultimately contributing to the characterisation and reduction of uncertainty.

Next generation risk assessment for skin allergy: Decision making using new approach methodologies

Our aim is to develop and apply next generation approaches to skin allergy risk assessment (SARA) that do not require new animal test data and better quantify uncertainties. Significant progress has been made in the development of New Approach Methodologies (NAMs), non-animal test methods, for assessment of skin sensitisation and there is now focus on their application to derive potency information for use in Next Generation Risk Assessment (NGRA). The SARA model utilises a Bayesian statistical approach to infer a human-relevant metric of sensitiser potency and a measure of risk associated with a given consumer exposure based upon any combination of human repeat insult patch test, local lymph node, direct peptide reactivity assay, KeratinoSens™, h-CLAT or U-SENS™ data. Here we have applied the SARA model within our weight of evidence NGRA framework for skin allergy to three case study materials in four consumer products. Highlighting how to structure the risk assessment, apply NAMs to derive a point of departure and conclude on consumer safety risk. NGRA based upon NAMs were, for these exposures, at least as protective as the historical risk assessment approaches. Through such case studies we are building our confidence in using NAMs for skin allergy risk assessment.

Applicability of amino acid derivative reactivity assay (4mM) for the prediction of skin sensitization by combining multiple alternative methods to evaluate key events.

The amino acid derivative reactivity assay (ADRA) is an alternative method for evaluating key event 1 (KE-1) in the skin sensitization mechanism included in OECD TG442C (OECD, 2021). Recently, we found that ADRA with a 4-mM test chemical solution had a higher accuracy than the original ADRA (1mM). However, ADRA (4mM) has yet to be evaluated using integrated approaches to testing and assessment (IATA), a combination of alternative methods for evaluating KE. In this study, the sensitization potency of three defined approaches (DAs) using ADRA (4mM) as KE-1 was predicted and compared with those of two additional ADRAs or direct peptide reactivity assay (DPRA): (i) "2 out of 3" approach, (ii) "3 out of 3" approach, and (iii) integrated testing strategy (ITS). In the hazard identification of chemical sensitizers, the accuracy of human data and local lymph node assay (LLNA) remained almost unchanged among the three approaches evaluated. Potency classifications for sensitization were predicted with the LLNA and human data sets using ITS. The potency classifications for the sensitization potency prediction accuracy of LLNA data using any alternative method were almost unchanged, at approximately 70%, and those with ITS were not significantly different. When ITS was performed using DPRA, the prediction accuracy was approximately 73% for human data, which was similar to that of the LLNA data; however, the accuracy tended to increase for all ADRA methods. In particular, when ITS was performed using ADRA (4mM), the prediction accuracy was approximately 78%, which proved to be a practical level.

In chemico skin sensitization alternative method: development of ADRA and listing to OECD test guideline

Amino acid Derivative Reactivity Assay (ADRA) is an alternative method developed based on the principle of covalent bonding between sensitizer and proteins in the early stage of the mechanism of skin sensitization. The Direct Peptide Reactivity Assay (DPRA) with same principle previously listed in the OECD test guidelines (TG) have some problems such as precipitation of the test chemical in the reaction solution and co-elution of the peptide with the test chemical. While, instead of DPRA, the ADRA was developed using two chemically synthesized nucleophilic reagents-namely, NAC and NAL in which naphthalene rings with a high molar absorbance coefficient (MAC) in the ultraviolet range have been introduced to N-termini of the cysteine and lysine that can react with the test chemical. Therefore, in March 2016, we set up a validation team with the aim for adoption in the OECD TG, ADRA's validation tests were conducted. After reporting the results of validation study, holding a third-party evaluation meeting and two commenting rounds, ADRA was able to be adopted in the OECD TG in June 2019. In addition, since the introduction of naphthalene with a high MAC has made it possible to reduce the concentration, enabling the following items. 1) Decrease in the frequency of precipitation of the test chemicals in the reaction solution. 2) Decrease in the frequency of co-eluting of the nucleating reagent and the chemical. 3) Evaluation of chemicals with unknown molecular weight using the gravimetric approach. 4) High-sensitivity detection of nucleophilic reagents by the fluorescence method. 5) Evaluation of the mixture by a combination of the gravimetric approach and fluorescence detection.

Integrated skin sensitization assessment based on OECD methods (I): Deriving a point of departure for risk assessment_suppl2

Three guidelines covering key events in the skin sensitization adverse outcome pathway are endorsed by the Organisation for Economic Co-operation and Development (OECD). A recent guideline covers defined approaches (DA) to combine data from these tests for regulatory (sub)classification. The guideline methods provide continuous data that could characterize the sensitization potency on a more granular scale beyond (sub)classifications. We assembled a comprehensive database of in vitro and in vivo results from OECD guideline tests. Building on a previous approach using regression models, we provide quantitative models using input data from the kinetic direct peptide reactivity assay (kDPRA), the KeratinoSens™ (KS) assay, and the human cell line activation test (h-CLAT) to calculate a point of departure (PoD) in the form of a predicted local lymph node assay (LLNA) EC3 value for use in risk assessment. Predictive models include results from either two or all three assays. Detailed analysis versus in vivo data estimates redundancy between different tests and helps guide model selection. All models were tested on a set of case studies selected for their availability of multiple LLNA reference data in the OECD database. The predicted PoDs were within or close to the range of the variation of the historical LLNA data for most of these cases studies, and overall, the models predicted the in vivo value with a median fold-misprediction factor of around 2.5. The robustness of the models was characterized by comparing a comprehensive historical database versus the curated dataset provided by the OECD working group on DA.

Integrated skin sensitization assessment based on OECD methods (II): Hazard and potency by combining kinetic peptide reactivity and the “2 out of 3” Defined Approach_suppl1

Depending on regulatory requirements, the skin sensitization risk for new chemicals with potential consumer skin contact must be assessed by experimental testing by (i) binary hazard assessment to identify sensitizers, (ii) subclassification of sensitizers according to the Global Harmonized System (GHS), and (iii) derivation of a point of departure (PoD) for risk assessment. The Organisation for Economic Co-operation and Development (OECD) recently published a test guideline incorporating the “2 out of 3” defined approach (2o3 DA) for skin sensitization hazard assessment and added the kinetic direct peptide reactivity assay (kDPRA) as a stand-alone test guideline method for GHS subclassification. The 2o3 DA requires that at least two in vitro tests are conducted. The cell-based tests and the kDPRA generate, next to a binary outcome with a fixed threshold, continuous concentration-response data, which can be used in quantitative regression models to derive a PoD. The sequence of testing for the 2o3 DA is flexible. Here we compare different testing sequences and how they can be combined with kDPRA data to provide a PoD in parallel to hazard identification (hazard ID) and GHS subclassification. A set of 188 chemicals with available in vitro data was evaluated for the final PoD using these different testing sequences. The results indicate that testing can start with DPRA / kDPRA and either of the cell-based assays, and that testing can stop after two congruent tests without major impact on the final PoD for chemicals within the applicability domain of the kDPRA.

Integrated skin sensitization assessment based on OECD methods (I): Deriving a point of departure for risk assessment_suppl4

Three guidelines covering key events in the skin sensitization adverse outcome pathway are endorsed by the Organisation for Economic Co-operation and Development (OECD). A recent guideline covers defined approaches (DA) to combine data from these tests for regulatory (sub)classification. The guideline methods provide continuous data that could characterize the sensitization potency on a more granular scale beyond (sub)classifications. We assembled a comprehensive database of in vitro and in vivo results from OECD guideline tests. Building on a previous approach using regression models, we provide quantitative models using input data from the kinetic direct peptide reactivity assay (kDPRA), the KeratinoSens™ (KS) assay, and the human cell line activation test (h-CLAT) to calculate a point of departure (PoD) in the form of a predicted local lymph node assay (LLNA) EC3 value for use in risk assessment. Predictive models include results from either two or all three assays. Detailed analysis versus in vivo data estimates redundancy between different tests and helps guide model selection. All models were tested on a set of case studies selected for their availability of multiple LLNA reference data in the OECD database. The predicted PoDs were within or close to the range of the variation of the historical LLNA data for most of these cases studies, and overall, the models predicted the in vivo value with a median fold-misprediction factor of around 2.5. The robustness of the models was characterized by comparing a comprehensive historical database versus the curated dataset provided by the OECD working group on DA.

Integrated skin sensitization assessment based on OECD methods (II): Hazard and potency by combining kinetic peptide reactivity and the “2 out of 3” Defined Approach_suppl4

Depending on regulatory requirements, the skin sensitization risk for new chemicals with potential consumer skin contact must be assessed by experimental testing by (i) binary hazard assessment to identify sensitizers, (ii) subclassification of sensitizers according to the Global Harmonized System (GHS), and (iii) derivation of a point of departure (PoD) for risk assessment. The Organisation for Economic Co-operation and Development (OECD) recently published a test guideline incorporating the “2 out of 3” defined approach (2o3 DA) for skin sensitization hazard assessment and added the kinetic direct peptide reactivity assay (kDPRA) as a stand-alone test guideline method for GHS subclassification. The 2o3 DA requires that at least two in vitro tests are conducted. The cell-based tests and the kDPRA generate, next to a binary outcome with a fixed threshold, continuous concentration-response data, which can be used in quantitative regression models to derive a PoD. The sequence of testing for the 2o3 DA is flexible. Here we compare different testing sequences and how they can be combined with kDPRA data to provide a PoD in parallel to hazard identification (hazard ID) and GHS subclassification. A set of 188 chemicals with available in vitro data was evaluated for the final PoD using these different testing sequences. The results indicate that testing can start with DPRA / kDPRA and either of the cell-based assays, and that testing can stop after two congruent tests without major impact on the final PoD for chemicals within the applicability domain of the kDPRA.

Integrated skin sensitization assessment based on OECD methods (II): Hazard and potency by combining kinetic peptide reactivity and the "2 out of 3" Defined Approach.

Depending on regulatory requirements, the skin sensitization risk for new chemicals with potential consumer skin contact must be assessed by experimental testing by (i) binary hazard assessment to identify sensitizers, (ii) subclassification of sensitizers according to the Global Harmonized System (GHS), and (iii) derivation of a point of departure (PoD) for risk assessment. The Organisation for Economic Co-operation and Development (OECD) recently published a test guideline incorporating the “2 out of 3” defined approach (2o3 DA) for skin sensitization hazard assessment and added the kinetic direct peptide reactivity assay (kDPRA) as a stand-alone test guideline method for GHS subclassification. The 2o3 DA requires that at least two in vitro tests are conducted. The cell-based tests and the kDPRA generate, next to a binary outcome with a fixed threshold, continuous concentration-response data, which can be used in quantitative regression models to derive a PoD. The sequence of testing for the 2o3 DA is flexible. Here we compare different testing sequences and how they can be combined with kDPRA data to provide a PoD in parallel to hazard identification (hazard ID) and GHS subclassification. A set of 188 chemicals with available in vitro data was evaluated for the final PoD using these dif-ferent testing sequences. The results indicate that testing can start with DPRA / kDPRA and either of the cell-based assays, and that testing can stop after two congruent tests without major impact on the final PoD for chemicals within the applica-bility domain of the kDPRA.

Integrated skin sensitization assessment based on OECD methods (II): Hazard and potency by combining kinetic peptide reactivity and the “2 out of 3” Defined Approach_suppl2

Depending on regulatory requirements, the skin sensitization risk for new chemicals with potential consumer skin contact must be assessed by experimental testing by (i) binary hazard assessment to identify sensitizers, (ii) subclassification of sensitizers according to the Global Harmonized System (GHS), and (iii) derivation of a point of departure (PoD) for risk assessment. The Organisation for Economic Co-operation and Development (OECD) recently published a test guideline incorporating the “2 out of 3” defined approach (2o3 DA) for skin sensitization hazard assessment and added the kinetic direct peptide reactivity assay (kDPRA) as a stand-alone test guideline method for GHS subclassification. The 2o3 DA requires that at least two in vitro tests are conducted. The cell-based tests and the kDPRA generate, next to a binary outcome with a fixed threshold, continuous concentration-response data, which can be used in quantitative regression models to derive a PoD. The sequence of testing for the 2o3 DA is flexible. Here we compare different testing sequences and how they can be combined with kDPRA data to provide a PoD in parallel to hazard identification (hazard ID) and GHS subclassification. A set of 188 chemicals with available in vitro data was evaluated for the final PoD using these different testing sequences. The results indicate that testing can start with DPRA / kDPRA and either of the cell-based assays, and that testing can stop after two congruent tests without major impact on the final PoD for chemicals within the applicability domain of the kDPRA.

Integrated skin sensitization assessment based on OECD methods (I): Deriving a point of departure for risk assessment.

Three guidelines covering key events in the skin sensitization adverse outcome pathway are endorsed by the Organisation for Economic Co-operation and Development (OECD). A recent guideline covers defined approaches (DA) to combine data from these tests for regulatory (sub)classification. The guideline methods provide continuous data that could charac-terize the sensitization potency on a more granular scale beyond (sub)classifications. We assembled a comprehensive database of in vitro and in vivo results from OECD guideline tests. Building on a previous approach using regression models, we provide quantitative models using input data from the kinetic direct peptide reactivity assay (kDPRA), the KeratinoSens™ (KS) assay, and the human cell line activation test (h-CLAT) to calculate a point of departure (PoD) in the form of a predicted local lymph node assay (LLNA) EC3 value for use in risk assessment. Predictive models include results from either two or all three assays. Detailed analysis versus in vivo data estimates redundancy between different tests and helps guide model selection. All models were tested on a set of case studies selected for their availability of multiple LLNA reference data in the OECD database. The predicted PoDs were within or close to the range of the variation of the historical LLNA data for most of these cases studies, and overall, the models predicted the in vivo value with a median fold-misprediction factor of around 2.5. The robustness of the models was characterized by comparing a comprehensive historical database versus the curated dataset provided by the OECD working group on DA.

Integrated skin sensitization assessment based on OECD methods (II): Hazard and potency by combining kinetic peptide reactivity and the “2 out of 3” Defined Approach_suppl3

Depending on regulatory requirements, the skin sensitization risk for new chemicals with potential consumer skin contact must be assessed by experimental testing by (i) binary hazard assessment to identify sensitizers, (ii) subclassification of sensitizers according to the Global Harmonized System (GHS), and (iii) derivation of a point of departure (PoD) for risk assessment. The Organisation for Economic Co-operation and Development (OECD) recently published a test guideline incorporating the “2 out of 3” defined approach (2o3 DA) for skin sensitization hazard assessment and added the kinetic direct peptide reactivity assay (kDPRA) as a stand-alone test guideline method for GHS subclassification. The 2o3 DA requires that at least two in vitro tests are conducted. The cell-based tests and the kDPRA generate, next to a binary outcome with a fixed threshold, continuous concentration-response data, which can be used in quantitative regression models to derive a PoD. The sequence of testing for the 2o3 DA is flexible. Here we compare different testing sequences and how they can be combined with kDPRA data to provide a PoD in parallel to hazard identification (hazard ID) and GHS subclassification. A set of 188 chemicals with available in vitro data was evaluated for the final PoD using these different testing sequences. The results indicate that testing can start with DPRA / kDPRA and either of the cell-based assays, and that testing can stop after two congruent tests without major impact on the final PoD for chemicals within the applicability domain of the kDPRA.

Integrated skin sensitization assessment based on OECD methods (I): Deriving a point of departure for risk assessment_suppl1

Three guidelines covering key events in the skin sensitization adverse outcome pathway are endorsed by the Organisation for Economic Co-operation and Development (OECD). A recent guideline covers defined approaches (DA) to combine data from these tests for regulatory (sub)classification. The guideline methods provide continuous data that could characterize the sensitization potency on a more granular scale beyond (sub)classifications. We assembled a comprehensive database of in vitro and in vivo results from OECD guideline tests. Building on a previous approach using regression models, we provide quantitative models using input data from the kinetic direct peptide reactivity assay (kDPRA), the KeratinoSens™ (KS) assay, and the human cell line activation test (h-CLAT) to calculate a point of departure (PoD) in the form of a predicted local lymph node assay (LLNA) EC3 value for use in risk assessment. Predictive models include results from either two or all three assays. Detailed analysis versus in vivo data estimates redundancy between different tests and helps guide model selection. All models were tested on a set of case studies selected for their availability of multiple LLNA reference data in the OECD database. The predicted PoDs were within or close to the range of the variation of the historical LLNA data for most of these cases studies, and overall, the models predicted the in vivo value with a median fold-misprediction factor of around 2.5. The robustness of the models was characterized by comparing a comprehensive historical database versus the curated dataset provided by the OECD working group on DA.

Integrated skin sensitization assessment based on OECD methods (I): Deriving a point of departure for risk assessment_suppl3

Three guidelines covering key events in the skin sensitization adverse outcome pathway are endorsed by the Organisation for Economic Co-operation and Development (OECD). A recent guideline covers defined approaches (DA) to combine data from these tests for regulatory (sub)classification. The guideline methods provide continuous data that could characterize the sensitization potency on a more granular scale beyond (sub)classifications. We assembled a comprehensive database of in vitro and in vivo results from OECD guideline tests. Building on a previous approach using regression models, we provide quantitative models using input data from the kinetic direct peptide reactivity assay (kDPRA), the KeratinoSens™ (KS) assay, and the human cell line activation test (h-CLAT) to calculate a point of departure (PoD) in the form of a predicted local lymph node assay (LLNA) EC3 value for use in risk assessment. Predictive models include results from either two or all three assays. Detailed analysis versus in vivo data estimates redundancy between different tests and helps guide model selection. All models were tested on a set of case studies selected for their availability of multiple LLNA reference data in the OECD database. The predicted PoDs were within or close to the range of the variation of the historical LLNA data for most of these cases studies, and overall, the models predicted the in vivo value with a median fold-misprediction factor of around 2.5. The robustness of the models was characterized by comparing a comprehensive historical database versus the curated dataset provided by the OECD working group on DA.

Assessment of the skin sensitization potential of fragrance ingredients using the U-SENS™ assay

The U-SENS™ assay was developed to address the third key event of the skin sensitization adverse outcome pathway (AOP) and is described in OECD test guideline 442E, Annex II. A dataset of 68 fragrance ingredients comprised of 7 non-sensitizers and 61 sensitizers was tested in the U-SENS™ assay. The potential for fragrance ingredients to activate dendritic cells, measured by U-SENS™, was compared to the sensitization potential determined by weight of evidence (WoE) from historical data. Of the non-sensitizers, 4 induced CD86 cell surface marker ≥1.5-fold while 3 did not. Of the sensitizers, 50 were predicted to be positive in U-SENS™, while the remaining 11 were negative. Positive and negative predictive values (PPV and NPV) of U-SENS™ were 93% and 21%, respectively. No specific chemical property evaluated could account for misclassified ingredients. Assessment of parent and metabolite protein binding alerts in silico suggests that parent chemical metabolism may play a role in CD86 activation in U-SENS™. Combining the U-SENS™ assay in a “2 out of 3” defined approach with the direct peptide reactivity assay (DPRA) and KeratinoSens™ predicted sensitization hazard with PPV and NPV of 97% and 24%, respectively. Combining complementary in silico and in vitro methods to the U-SENS™ assay should be integrated to define the hazard classification of fragrance ingredients, since a single NAM cannot replace animal-based methods.