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

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

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:

Original paper :AATEX 8(3-4):69-77
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