National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, School of Pharmacy, The University of Mississippi, University, MS, 38677, USA.
Office of Cosmetics and Colors, Center for Food Safety and Applied Nutrition, Food and Drug Administration, College Park, MD, 20740, USA.
J Appl Toxicol. 2019 Feb;39(2):398-408. doi: 10.1002/jat.3732. Epub 2018 Oct 16.
Twenty-four pure fragrance ingredients of concern as potential skin sensitizers were previously subjected to degradation studies and evaluated using the high throughput with dansyl cysteamine (HTS-DCYA) method. The experimental results showed that two-thirds of the 24 fragrance ingredients underwent chemical degradation. In some cases, such degradation was accompanied by an increase in thio-reactivity. These results prompted us to investigate the reactivity of the same ingredients using the direct peptide reactivity assay (DPRA). In the present work, the 24 chemicals were subjected to forced degradation for 150 days, and evaluated with both DPRA and HTS-DCYA methods. At the end of the study, four and eight compounds remained non-reactive in the DPRA and DCYA assay, respectively. Coumarin, benzyl salicylate, benzyl cinnamate and hexyl cinnamal were found unreactive in both assays, while cinnamal, cinnamyl alcohol, hydroxycitronellal and lilial were found negative in the DCYA but positive in the DPRA method. The incongruity in reactivity of these four compounds was attributed to a possible role of pro-oxidants formed upon degradation, resulting in depletion of peptide without formation of apparent covalent adducts with the test chemical. To validate this hypothesis, the effect of hydrogen peroxide as model pro-oxidant on both lysine- and cysteine-heptapeptide depletion in the DPRA method was thus investigated. The obtained results showed little effect of oxidative conditions on lysine depletion, while cysteine depletion was significantly affected by concentrations above 1.1 mg/L of hydrogen peroxide. Overall, both in chemico methods confirmed chemical instability should be considered when assessing the skin sensitization potential of (un)known chemicals with alternative methods.
二十四种纯香精成分被认为是潜在的皮肤致敏物，此前曾进行过降解研究，并使用Dansyl Cysteamine（HTS-DCYA）方法进行了评估。实验结果表明，24 种香精成分中有三分之二发生了化学降解。在某些情况下，这种降解伴随着硫反应性的增加。这些结果促使我们使用直接肽反应性测定（DPRA）来研究相同成分的反应性。在本工作中，将 24 种化学品进行了 150 天的强制降解，并分别用 DPRA 和 HTS-DCYA 方法进行了评估。研究结束时，在 DPRA 和 DCYA 测定中，分别有四种和八种化合物保持非反应性。香豆素、水杨酸苄酯、肉桂酸苄酯和己基肉桂醛在两种测定中均未显示反应性，而肉桂醛、肉桂醇、羟基香茅醛和铃兰醛在 DCYA 中呈阴性，但在 DPRA 中呈阳性。这些四种化合物反应性的不一致归因于降解过程中形成的可能的促氧化剂的作用，导致肽的消耗而没有与测试化学物质形成明显的共价加合物。为了验证这一假设，因此研究了模型促氧化剂过氧化氢对 DPRA 方法中赖氨酸-和半胱氨酸七肽消耗的影响。结果表明，氧化条件对赖氨酸消耗的影响很小，而半胱氨酸消耗则受到浓度高于 1.1mg/L 过氧化氢的显著影响。总之，两种体外方法均证实，在使用替代方法评估（未知）化学物质的皮肤致敏潜能时，应考虑化学不稳定性。
