日本香粧品学会誌

Vol. 45, No. 4, pp. 331–335（2021）〈Regular Article〉

Establishment of a Threshold of Toxicological Concern Conceptfor Skin Sensitization by in Vitro/in Silico Approaches
Takao ASHIKAGA1, *, Kaori AMBE2, Masaharu SUZUKI2,
Masayuki KURIMOTO1, Takashi YAMADA1, Masahiro TOHKIN2(Accepted: October 25, 2021)

Abstract
Recently, multiple in vitro skin sensitization tests have been listed in the Organisation for Economic Co-operation andDevelopment (OECD) guidelines. The threshold of toxicological concern (TTC) is a threshold for human exposure whenchemicals do not show any obvious adverse eﬀects at lower doses. In this study, we aimed to develop a quantitative prediction
model using an in vitro/in silico dataset and to establish a TTC concept for skin sensitization. The EC3 value, the endpoint of thelocal lymph node assay (LLNA), was used as the objective variable and data on 120 substances were extracted from the dataset
published by Cosmetics Europe. In vitro tests (DPRA, KeratinoSens™ and h-CLAT) data and physico-chemical properties wereused as explanatory variables. A quantitative prediction model for EC3 was developed using support vector regression (SVR), amachine learning approach. Predicted EC3 values were used to establish no expected sensitization induction level (NESIL), andthe acceptable exposure level (AEL) for each chemical was calculated by dividing NESIL by the sensitization assessment factor(SAF). Then, by ﬁtting the gamma distribution of AELs using a negative log (10) scale, the 95th and 99th percentile probability
were calculated as the dermal sensitization threshold (DST) value. Finally, the conversion of the DST to the threshold concentration of a women s face cream was performed as an example of the application of this concept.
This prediction model was validated by a three-fold cross-validation, and the accuracy of prediction of potency class inﬁve categories was 45.8%. Assuming 20% of all chemicals are skin sensitizers, the DST (μg/cm2) for women s face creamwas 0.129 (99th percentile) and 3.99 (95th percentile). Furthermore, the threshold concentration of this type of products was
0.008% (for DST 99th percentile) and 0.26% (for DST 95th percentile). The TTC concept for skin sensitization can be applied as a non-animal approach in evaluating the safety proﬁle of cosmetic ingredients.
Key words: TTC, DST, skin sensitization, machine learning.

1.

Introduction

A threshold of toxicological concern (TTC) is a thresholdvalue for human exposure to a given chemical below which
no apparent adverse eﬀects are expected. TTC has been developed based on a statistical analysis of historical toxicity
testing data. Using TTC is one approach to assess the risk ofa chemical of unknown toxicity without conducting toxicity
testing because it requires only information on the chemical
structure of the target substance and the amount of human exposure. Thus, the use of TTC is eﬀective in comprehensively
evaluating groups of chemicals for which human exposureis expected to be extremely low and there is scarce toxicologic information available. The concept of TTC is primarily
applied to evaluate the systemic toxicity of orally exposedchemicals. This concept has been accepted by public regula-1

National Institute of Health Sciences, Division of Risk Assessment(3–25–26 Tonomachi, Kawasaki-ku, Kawasaki 210–9501, Japan)
2Department of Regulatory Science, Graduate School of Pharmaceutical Sciences, Nagoya City University* Corresponding author: National Institute of Health Sciences, Divi-sion of Risk Assessment, 3–25–26 Tonomachi, Kawasaki-ku, Kawasaki 210–9510, Japan (E-mail: takao.ashikaga@nihs.go.jp)

tions for speciﬁc applications such as impurities in fragrances1) and pharmaceuticals2). Furthermore, Saﬀord s research
introduced the concept of TTC to the evaluation of skinsensitizing potential, which is a major toxicity through skin
exposure. This concept of TTC is called dermal sensitizationthreshold or DST3, 4). The current circumstances surrounding
the development of cosmetics, such as the abolition of animal testing, diversiﬁcation of raw material procurement, andstricter safety assurance regulations, have made the concept ofTTC for skin sensitization attractive for cosmetics manufacturers because this evaluation approach does not require any
toxicity testing for substances such as impurities in raw ingredients and trace components in natural products.
Recently, in the OECD guidelines, several in vitro skin sensitization tests have been included. The OECD guidance on combined test methods, including combinations with in silico tests,