Assessment of commercial polymers with and without reactive groups using amino acid derivative reactivity assay based on both molar concentration approach and gravimetric approach.

The amino acid derivative reactivity assay (ADRA), an alternative method for testing skin sensitization, has been established based on the molar concentration approach. However, the additional development of gravimetric concentration and fluorescence detection methods has expanded its range of application to mixtures, which cannot be evaluated using the conventional testing method, the direct peptide reactivity assay (DPRA). Although polymers are generally treated as mixtures, there have been no reports of actual polymer evaluations using alternative methods owing to their insolubility. Therefore, in this study, we evaluated skin sensitization potential of polymers, which is difficult to predict, using ADRA. As polymers have molecular weights ranging from several thousand to more than several tens of thousand Daltons, they are unlikely to cause skin sensitization due to their extremely low penetration into the skin, according to the 500-Da rule. However, if highly reactive functional groups remain at the ends or side chains of polymers, relatively low-molecular-weight polymer components may penetrate the skin to cause sensitization. Polymers can be roughly classified into three major types based on the features of their constituent monomers; we investigated the sensitization capacity of each type of polymer. Polymers with alert sensitization structures at their ends were classified as skin sensitizers, whereas those with no residual reactive groups were classified as nonsensitizers. Although polymers with a glycidyl group need to be evaluated carefully, we concluded that ADRA (0.5 mg/ml) is generally sufficient for polymer hazard assessment.

Applicability of amino acid derivative reactivity assay (4 mM) 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 (1 mM). However, ADRA (4 mM) 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 (4 mM) 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 (4 mM), the prediction accuracy was approximately 78%, which proved to be a practical level.

The within- and between-laboratories reproducibility and predictive capacity of Amino acid Derivative Reactivity Assay using 4 mM test chemical solution: Results of ring study implemented at five participating laboratories.

Amino acid derivative reactivity assay (ADRA) for skin sensitization was adopted as an alternative method in the 2019 OECD Guideline for the Testing of Chemicals (OECD TG 442C). The molar ratio of the nucleophilic reagent to the test chemicals in the reaction solution was set to 1:50. Imamura et al. reported that changing this molar ratio from 1:50 to 1:200 reduced in false negatives and improved prediction accuracy. Hence, a ring study using ADRA with 4 mM of a test chemical solution (ADRA, 4 mM) was conducted at five different laboratories to verify within- and between-laboratory reproducibilities (WLR and BLR, respectively). In this study, we investigated the WLR and BLR using 14 test chemicals grouped into three classes: (1) eight proficiency substances, (2) four test chemicals that showed false negatives in the ADRA with 1 mM test chemical solution (ADRA, 1 mM), but correctly positive in ADRA (4 mM), and (3) current positive control (phenylacetaldehyde) and a new additional positive control (squaric acid diethyl ester). The results showed 100% reproducibility and 100% accuracy for skin sensitization. Hence, it is clear that the ADRA (4 mM) is an excellent test method in contrast to the currently used ADRA (1 mM). We plan to resubmit the ADRA (4 mM) test method to the OECD Test Guideline Group in the near future so that OECD TG 442C could be revised for the convenience and benefit of many ADRA users.

Improving predictive capacity of the Amino acid Derivative Reactivity Assay test method for skin sensitization potential with an optimal molar concentration of test chemical solution.

The Amino acid Derivative Reactivity Assay (ADRA) is a convenient and effective in chemico test method for assessing covalent binding of test chemicals with protein-derived nucleophilic reagents as a means of predicting skin sensitization potential. Although the original molar-concentration approach to ADRA testing was not suitable for testing multiconstituent substances of an unknown composition, a weight-concentration approach that is suitable for such substances was developed, which also led to the realization that test chemical solutions prepared to molar concentrations higher than the original 1 mM would reduce false negative results as well as enhance predictive capacity. The present study determined an optimal molar-concentration that achieves even higher predictive capacity than the original ADRA. Eight chemicals that were false negatives when tested with 1 mM test chemical solutions were retested with test chemical solutions between 2 and 5 mM, which showed 4 mM to be the optimal molar-concentration for ADRA testing. When 82 chemicals used in the original development were retested with 4 mM test chemical solutions, false negative results were reduced by four. When an additional 85 chemicals used to evaluate the weight-concentration approach to ADRA were retested, the results essentially replicated those obtained with 0.5 mg/ml test chemical solutions and gave 10 fewer false negatives than original ADRA with 1 mM solutions. A comparison of these results for 136 chemicals showed that ADRA testing with 4 mM solutions achieved a four percentage point improvement in accuracy over original ADRA and a two percentage point improvement over DPRA testing.

Development of photo-amino acid derivative reactivity assay: a novel in chemico alternative method for predicting photoallergy.

Photoallergy test of cosmetics and several types of pharmaceutical substances is often necessary for obtaining approval from authorities. However, there are no official test guidelines for photoallergy evaluation. Therefore, we tried to establish a photoallergy test by utilizing an in chemico alternative sensitization method, amino acid derivative reactivity assay (ADRA). To determine the criteria for judging the photoallergy potential, photo-ADRA with or without photoirradiation was performed using 60 photoallergenic chemicals, and cysteine and lysine derivatives were detected using high-performance liquid chromatography either by absorbance or fluorescence measurement. The accuracy of prediction was 81.4% (48 of 59) and 80.0% (48 of 60) using the absorbance and fluorescence methods, respectively. However, as chemicals can breakdown into multiple chemicals during photoirradiation, the absorbance method often cannot perform accurate detection due to co-elution, whereas the fluorescence method can do this due to lack of co-elution. Moreover, all eight chemicals that were found to be negative or false-positive for photoirritation in the 3T3 neutral red uptake phototoxicity test were confirmed as positive for photoallergy using this method. Furthermore, we prepared three types of pseudo-mixtures where we added one photoallergen along with five nonphotoallergens and performed the photo-ADRA by the ultraviolet and fluorescence methods. The result of the fluorescence method was almost the same as that obtained with the use of a single photoallergen and hence the outcome was not affected by the mixture. Thus, this study not only showed a method of evaluating the photoallergy potential of a single chemical but also a mixture, making it useful as an in chemico photoallergy alternative test.

The amino acid derivative reactivity assay with fluorescence detection and its application to multi-constituent substances.

The Amino acid Derivative Reactivity Assay (ADRA) is an in chemico alternative to animal testing for the prediction of skin sensitization potential. Although co-elution of test chemicals and nucleophilic reagents during HPLC analysis is sometimes problematic when using the Direct Peptide Reactivity Assay (DPRA), it rarely occurs when using ADRA. Nevertheless, the application of either of these tests to multi-constituent substances requires nucleophilic reagents capable of selective detection. With this issue in mind, the authors developed an ADRA fluorescence detection method (ADRA-FL), which utilizes the natural fluorescence of ADRA nucleophilic reagents. In this study, we demonstrate the efficacy of ADRA-FL by testing 82 test chemicals used in the development of both DPRA and the conventional ADRA (ADRA-UV) as well as establish a threshold value for distinguishing sensitizers and non-sensitizers. Our results show that not only are depletion values obtained using ADRA-FL virtually identical to those obtained using ADRA-UV, the threshold value for either test is 4.9%. Additionally, in order to demonstrate the applicability of ADRA-FL to multi-constituent substances, we prepared test samples that consisted of a set of 10 non-sensitizers combined with one of 10 different sensitizers and tested each using ADRA-FL. The test results were concordant with those obtained using ADRA-UV. Also, because ADRA-FL chromatograms showed a significant decrease in multiple peaks as well as extremely stable baselines, we conclude that ADRA-FL is a highly selective and highly accurate mans of quantifying nucleophilic reagents that is applicable to a wide variety of chemical substances.

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

Amino acid derivative reactivity assay (ADRA) has previously been developed as an alternative method to direct peptide reactivity assay (DPRA) to evaluate key event 1 in skin sensitization mechanisms. However, when using alternative methods for skin sensitization, integrated approaches to testing and assessment (IATA) that combine the results of multiple tests evaluating different key events are generally required. To verify whether ADRA can be used in IATA, we replaced DPRA with ADRA in five IATA methods combining DPRA, KeratinoSens, and h-CLAT: (i) the "2 out of 3" approach, (ii) the "3 out of 3" approach, (iii) sequential testing strategy (STS), (iv) integrated testing strategy by scoring approach (ITS-SA), and (v) the "ITS by two methods approach" (ITS-2MA). The prediction accuracy of the "2 out of 3" approach using ADRA (1 mM) and ADRA (0.5 mg/mL) was 90.0% and 91.1%, respectively, for human data, and was very similar to that obtained using DPRA (91.1%). The "3 out of 3" approach also showed good predictability (83.2%) using either ADRA (1 mM) or ADRA (0.5 mg/mL) compared to DPRA. Regarding the accuracy of the prediction of sensitization intensity for the human data by the third classification, prediction accuracy using ADRA was almost the same as STS, ITS-SA, or ITS-2MA using DPRA. As a result, this study showed that ADRA can be used as a test method for key event 1 in the evaluation of skin sensitization by combining multiple alternative methods.

Precipitation of test chemicals in reaction solutions used in the amino acid derivative reactivity assay and the direct peptide reactivity assay.

The Amino acid Derivative Reactivity Assay (ADRA) was developed by the authors as an in chemico alternative to animal testing for skin sensitization potential. Although ADRA is based on the same scientific principles as the Direct Peptide Reactivity Assay (DPRA), a comparison of the results from these two test methods shows a far lower incidence of precipitation of test chemicals in reaction solutions for ADRA than for DPRA. Specifically, a comparison of the results for 82 test chemicals that were tested using both DPRA and ADRA showed that while there were 30 chemicals tested using DPRA for which precipitation was found in the reaction solution, there were just three chemicals tested using ADRA for which even slight turbidity was found in the reaction solution. In contrast to the fact that many DPRA test chemicals with a n-Octanol/Water Partition Coefficient (LogKow) of 2.0 or higher exhibited precipitation, there were only three ADRA test chemicals that exhibited turbidity, and these were all highly hydrophobic with a LogKow of greater than 6.0. Moreover, one of the DPRA test chemicals that exhibited precipitation also gave a false negative result, suggesting that anytime a test chemical exhibits precipitation in the reaction solution during DPRA testing the results must be interpreted with the greatest care, although all false positives are not caused by precipitation of test chemicals. Therefore, since relatively few ADRA test chemicals exhibited precipitation relative to DPRA, we consider ADRA to be an extremely useful means of testing a wide variety of chemical substances.

A newly developed means of HPLC-fluorescence analysis for predicting the skin sensitization potential of multi-constituent substances using ADRA.

The Amino acid Derivative Reactivity Assay (ADRA) is an in chemico alternative to animal testing for skin sensitization potential, in which measurements of multi-constituent solutions were sometimes affected by co-elution with nucleophilic reagents. So, we established a means of using fluorescence detection and verified the utility of a newly developed ADRA-fluorescence detection (ADRA-FL) test method. We tested three types of plant extracts-aloe, green tea, and licorice-and although unable to quantify nucleophilic reagents using ultraviolet detection due to co-elution of multiple components, the use of fluorescence detection enabled us to detect nucleophilic reagents selectively and predict each of the extract solutions to be sensitizers. Given that plant extracts contain immunosuppressants, there is no reason to expect that positive results in ADRA-FL testing will always be concordant with in vivo results. But given its ability to predict the sensitization potential of cosmetics and other widely used multi-constituent substances that had previously been difficult to test, the newly developed ADRA-FL is expected to contribute to future assessments of sensitization risks.

The underlying factors that explain why nucleophilic reagents rarely co-elute with test chemicals in the ADRA.

The Amino acid Derivative Reactivity Assay (ADRA) is an in chemico alternative to animal testing for skin sensitization potential that uses two different nucleophilic reagents and it is known that ADRA hardly exhibts co-elution compared with the Direct Peptide Reactivity Assay (DPRA) based on the same scientific principles. In this study, we have analyzed the factors underlying why co-elution, which is sometimes an issue during DPRA testing, virtually never occurs during ADRA testing. Chloramine T and dimethyl isophthalate both exhibited co-elution during DPRA testing, but when quantified at both DPRA's 220 nm and ADRA's 281 nm, we found that when the later detection wavelength was used, these test chemicals produced extremely small peaks that did not interfere with quantification of the peptides. And although both salicylic acid and penicillin G exhibited co-elution during DPRA testing, when tested at a concentration just 1% of that used in DPRA, the very broad peak produced at the higher concentration was reduced significantly. However, both these test chemicals exhibited very sharp peaks when the pH of the injection sample was adjusted to be acidic. Based on these results, we were able to clarify that the reasons why nucleophlic reagents hardly co-elute with test chemicals during ADRA testing are depend on the following three major reasons: (1)differences in the detection wavelength, (2)differences in test chemical concentrations in the injection sample, (3)differences in composition of the injection solvent.

Expanding the applicability of the amino acid derivative reactivity assay: Determining a weight for preparation of test chemical solutions that yield a predictive capacity identical to the conventional method using molar concentration and demonstrating the capacity to detect sensitizers in liquid mixtures.

INTRODUCTION: The amino acid derivative reactivity assay (ADRA) is a novel in chemico alternative to animal testing for assessment of skin sensitization potential. The conventional ADRA protocol stipulates that test chemical solutions should be prepared to a specific molar concentration, allowing only for use of test chemicals with known molecular weights. Since many potential test substances are prepared by weight concentration or contain multiple unknown chemicals, this study was conducted to verify if it is possible to accurately assess the sensitization potential of test chemical solutions prepared at a specific weight concentration. METHODS: (1) Test chemical solutions for 82 chemicals were prepared at four different weight concentrations. Results were evaluated for agreement with in vivo results. (2) A liquid mixture comprising ten different non-sensitizers was prepared at 1 mg/mL. Ten different sensitizers of varying sensitization potencies were added individually to this mixture. The resulting pseudobinary mixtures were tested to confirm that the sensitizers could be detected. RESULTS: (1) The accuracies for test chemical solutions prepared at 0.5 and 0.2 mg/mL were 87.8% and 86.6%, respectively, which were roughly equivalent to the accuracy of 86.6% achieved with a solution prepared at the conventional molar concentration of 1 mM. In contrast, the accuracies for solutions prepared at 0.1 and 0.05 mg/mL were 82.9% and 74.4%, respectively, both of which were lower than that obtained with the conventional method. (2) Sensitizers added to the liquid mixture at 0.5 mg/mL were all correctly detected. DISCUSSION: Preparing test chemical solutions at a weight concentration of 0.5 mg/mL decreased false negatives and increased false positives while improving prediction accuracy, which suggests that the sensitization potential of mixtures can also be assessed with this method.

Quantitative analysis of γH2AX reveals distinct responses in multiple mouse organs after administration of mitomycin C or ethyl methanesulfonate.

Ser139-phosphorylated H2AX (γH2AX) is a functional biomarker of DNA double-strand breaks. However, its conventional detection for in vivo samples relies on immunological methods using anti-γH2AX antibodies, making quantitative analysis difficult. Here, we established an absolute γH2AX quantification in vivo method for multiple organs in mice using liquid chromatography-triple quadrupole tandem mass spectrometry. When applying the method to male Institute of Cancer Research (ICR) mice (8 weeks old), the testes showed the highest γH2AX level (2.3% of total H2AX), followed by the bone marrow (0.51%), stomach (0.28%), kidney (0.20%), spleen (0.20%), liver (0.15%) and lung, which had the lowest overall level (0.10%). After intraperitoneal administration of 2 mg/kg mitomycin C in mice, γH2AX levels increased until 2-4 h, followed by a monotonical decrease to the control level in the bone marrow and spleen, and increased moderately until 24 h, followed by a slight decrease by 48 h in the liver, stomach, lung and kidney. After oral administration of 400 mg/kg ethyl methanesulphonate, γH2AX levels increased until 8 h and then decreased to the control level by 24-48 h in the spleen and kidney, increased until 24 h and then slightly decreased until 48 h in the bone marrow and lung, increased until 8 h and plateaued by 48 h in the liver, and decreased until 8 h and then increased to the control level in the stomach. Both the genotoxic chemicals did not alter γH2AX levels in the testes. These results indicate that our novel method could reveal variation in the γH2AX state in mouse organs and allows monitoring of the in vivo dynamics induced by genotoxic chemicals.