In Vitro Phototoxicity of Fragrance Materials: Evaluation in the 3T3 NRU Phototoxicity Test

Yuuko Okamoto, Makoto Mizuno, Hajime Asano, Takuji Masunaga
KOSE Corporation

Correspondence:
Yuuko Okamoto
Fundamental Research Laboratory, KOSE Corporation
1-18-4, Azusawa, Itabashi-ku, Tokyo 174-0051, Japan
Tel: 03-3967-6441, Fax: 03-3967-6649, E-mail: yu-okamoto@kose.co.jp

Original paper :AATEX 8(3-4):69-77
Abstract
The aim of the present study was to evaluate the suitability of Balb/c3T3 mouse fibroblast neutral red uptake phototoxicity test (3T3 NRU PT) for prediction of phototoxicity in fragrance materials. Twenty fragrance materials were assessed by the 3T3 NRU phototoxicity test. Of the fourteen test substances estimated to be phototoxic or photosensitive under International Fragrance Association (IFRA) guidelines, five test substances were classified as phototoxic in 3T3 NRU PT. Six test substances regarded as non-phototoxic fragrances under IFRA guidelines were predicted to be non-phototoxic by the 3T3 NRU PT. Nine false negative results, with respect to the classification set forth in IFRA guidelines, occurred in this in vitro assay. Of false negative results, seven were for natural essential oils. The amounts of four psoralen derivatives in each natural fragrance material used were analyzed using GC-MS. The contents of psoralens in each natural fragrance material were comparatively low, although the highest amount of psoralens found in this study was 33.5ppm of bergamot oil cold pressed. The correlation with the level of bergapten in the essential oil and the photo irritation factor (PIF) value was evaluated using a calibration curve. The concentration of bergapten (100 ppm), which caused a phototoxicity reaction in vivo, indicated a PIF value at 3.13. Furthermore, the total amount of bergapten required to obtain a PIF value of 5 (the value selected as a cut-off point) was more than 1000ppm. These results suggest that the essential oils, even those less than PIF value 5, may cause the phototoxicity in vivo.
Given these results, we conclude that it is necessary to pay attention to results of phototoxicity assessment based on PIF values when using 3T3 NRU PT for natural essential oil containing bergapten, in order to avoid an increased number of false negative results.

Keyword: in vitro phototoxicity, essential oil, bergapten, psoralen, 3T3NRU phototoxicity test

Validation Study in Korea for HET-CAM Test and NRU Assay as Alternatives to the Draize Eye Irritation Test

Bae-Hwan Kim¹, Seong-Joon Moon¹, Ih-Seop Chang¹, Young-Chul Sim¹, Jin-Cheon Hong², Ki-Moon Kim³, Moon-Uk Park⁴, Chang-Seok Rhoo⁵ and Min-Seok Jeong⁶
1 Skin Research Institute, Pacific Co. R&D Center, Kyonggi-do 449-729, Korea
2 Nadri Cosmetic Co., Kyonggi-do 451-860, Korea
3 Boryung Pharm. Co., Kyonggi-do 435-050, Korea
4 LG Chem. Research Park, Taejon 305-343, Korea
5 Coreana Cosmetic Co. Ltd., Chungchung 333-830 Nam-do, Korea
6 Hankuk Cosmetic Co. Ltd., Kyonggi-do 421-150, Korea
Correspondence: Bae-Hwan Kim, DVM
Tel.: +82-31-280-5904, Fax: +82-31-284-8478, E-mail: bhkim@pacific.co.kr

Original paper :AATEX 8(3-4):78-90
Abstract
The objective of this study is the validation of two alternative methods to rabbit eye irritation test (Draize test). The henfs egg chorioallantoic membrane (HET-CAM) test is actively studied by several countries as an alternative to the rabbit eye irritation test. It employs the vascular membrane of a fertile henfs egg to assess eye irritation potential. We assembled a co-operating organization including 6 cosmetic research centers and carried out validation studies to assure the HET-CAM test as an acceptable alternative to the Draize test. The NRU assay, a cytotoxicity test was also conducted in this study to complement the HET-CAM method.
We selected 25 substances for pre-validation study, 20 substances for 1st- validation study, and 12 substances for 2nd-validation study from common cosmetic ingredients. The correlation study to compare HET-CAM test, NRU assay and Draize test was performed with Pearson and Spearman correlation coefficient values. We also analyzed the inter-laboratory correlation based on the results of the HET-CAM obtained by 6 cosmetic research centers.
We conclude that the HET-CAM test and the NRU assay are good methods to replace the Draize test because of the high correlations observed among HET-CAM test, NRU assay and Draize test. However, additional validation studies are needed to accept these methods as integral components of the guideline due to the big inter-laboratory validation. Keywords: Validation study, in vitro alternatives, Draize test, HET-CAM, NRU assay, cytotoxicity

A Prevalidation Study for Three-dimensional Cultured Human Skin Models as Slternatives to Skin Irritation testing

Iwao Sonoda¹1, Hajime Kojima², Atsushi Sato², Matsumi Terasawa³, Maki Goda⁴, Michimasa Hori⁵, Hiroko Okamoto⁶, Makoto Mizuno⁶, Noriyasu Imai⁶, Mai Takei⁶, Nobuyuki Uetake⁷, Misako Goto⁸, Rumi Kawabata⁹, Yoshifumi Sasaki¹⁰, Kaoru Ukawa¹¹, Noriyasu Ozawa¹², Tamie Suzuki¹³, Masahito Usami¹⁴, Tomoko Kasahara¹⁵, Kyoko Goto¹⁶, Hisashi Torishima¹⁷, Hidekazu akahashi¹⁸, Takuya Ishibashi¹⁸, Noriyuki Morikawa¹⁹, and Isao Yoshimura¹
1 Tokyo Univ. of Science, 2 Nippon Menard Cosmetic, Co., Ltd., 3 IVY Cosmetic Co., Ltd.,
4 AJINOMOTO Co., Inc., 5 ICHIMARU PHARCOS Co., Ltd., 6 KOSE Corporation,
7 KOWA Co., Ltd., 8 KONICA Corporation, 9 TAIHO Pharmaceutical Co., Ltd.,
10 TANABE Seiyaku Co. Ltd., 11 Japan Bayer Agrochem K.K., 12 PIAS Corporation,
13 FANCL Corporation, 14 HOYU Co. Ltd., 15 POLA Substance Industries Inc.,
16 HOKKO Chemical Industry Co., Ltd., 17 KURABO Industries Ltd., 18 TOYOBO Co., Ltd.,
19 GUNZE Ltd.
Correspondence: Iwao Sonoda, Dept. of Management Science, Faculty of Engineering, Tokyo University of Science, 1-3 Kagurazaka, Shinjuku-ku, Tokyo 162-8601, Japan
Fax: 03-5228-8350, Email: sonoda@ms.kagu.tus.ac.jp

Original paper :AATEX 8(3-4):91-106
Abstract
Various cytotoxicity assays using cultured cells or physico-chemical tests are available as an alternative to testing skin irritation potential of substances on human subjects. Most, however, are unsatisfactory, having led to the development of three-dimensional cultured human skin models (3D skin models) that are closer to human skin. This paper presents results of a prevalidation study carried out to obtain data for designing future validation studies, being conducted in Japan using three 3D skin models (EPI-100, TESTSKIN, and Vitrolife-Skin).
Five substances including two vehicles are processed using these 3D skin models under masking the name of substances. Skin irritation potential is evaluated by the scores of the time (ET50) with 50% damage on the 3D-skin model used. The observed result was compared with scores on human bodies and animals obtained in the Research Labs., Nippon Menard Cosmetic Co., Ltd. The feasibility of the tested 3D-skin models as alternatives was suggested through the experiment, although inter-laboratory variation was not neglected. The study protocol should be improved for future studies concerning, for example, the number of time points to be measured and the method to get reliable ET50 scores.
Five masked substances including two vehicles were considered, with skin irritation potential being evaluated using time-dependent ET50 scores compared to rabbit and human scores obtained by a commercial laboratory. Experimental results suggest that the proposed 3D skin models are feasible alternatives, although inter-laboratory variation was clearly apparent. In addition, future validation studies should incorporate improved protocols, e.g., stating specific times and methods such that reliable ET50 scores can be obtained.

Keywords: skin irritation potential, human skin model, cytotoxicity assay alternative method, cosmetic testing, validation study

生体内遺伝子内組換え検出系としてのPunマウス澁谷　徹
(財)食品薬品安全センター秦野研究所
　 (pp. 107 -112)
苦痛による生命科学実験分類黒澤努、大谷若菜
大阪大学医学部附属動物実験施設
(pp. 113 -121)