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Protocol no. 108
CAM-TBS TEST

In this modification of the HET-CAM test, trypan blue staining provides an objective and quantitative assessment of the degree of damage to the chicken chorioallantoic membrane (CAM).

CONTACT

Dr. Hiroshi Itagaki, Dr. Shinegobu Hagino, Dr. Shinobu Kato Shiseido Research and Analytical Center 1050 Nippa-cho Kohoku-ku Yokohama 233 Japan Fax: 045 546 4456

RATIONALE

The use of trypan blue staining, determined spectrophotometrically, to assess the amount of tissue damage occurring in the CAM test provides a more objective and quantitative measure of irritancy than obtained with the usual scoring system of the HET-CAM test, which is based on a subjective estimation of hyperaemia, haemorrhage and coagulation.

BASIC PROCEDURE

Fertile hen's eggs are rotated in an incubator for 10 days. The CAM is exposed by removing the shell above the air space and then the outer membrane. Test chemicals are applied to the CAM either undiluted or in aqueous solution for a set exposure period after which they are washed off with water. A 0.1% solution of trypan blue in PBS is applied to the CAM. After 1 minute, the CAM is extracted in formamide and the amount of dye absorbed is quantified spectrophotometrically.

TEST STATUS

Included in the Japanese inter-laboratory validation study "Study on test methods to evaluate the safety of cosmetics containing new ingredients" sponsored by the Japanese Ministry of Health and Welfare.

CHEMICALS TESTED

Surfactants
Cationic
Hexadecylpyridinium chloride
Octadecyltrimethylammonium chloride
Dodecyltrimethylammonium chloride
Anionic
Alkyloyltaurate
Sodium dodecyl sulphate
Acyl glutamate
POE dodecylether sulphate
Sodium caseinate Amphoteric
Alkylamidobetaine
Alkylbetaine
Nonionic
POE(10) octylphenylether
POE(20) octylphenylether
Other Chemicals
Cyclopentanone o-Diethoxybenzene
Ethanol Sodium lactate (60%)
p-Phenetidine Propylene glycol
Toluene p-Aminophenol
o-Aminophenol 1,6-Dibromohexane
Dipotassium EDTA Lanolin

REFERENCES

  1. Hagino S., Itagaki H., Kato S., Kobayashi T., and Tanaka M. (1991) Quantitative evaluation to predict the eye irritancy of chemicals: modification of chorioallantoic membrane test by using Trypan Blue. Toxicol. in Vitro 5: 301-304. Hagino S., Itagaki H., Kato S., and Kobayashi T. (1993) Further evaluation of the quantitative chorioallantoic membrane test using Trypan Blue stain to predict the eye irritancy of chemicals. Toxicol. in Vitro 7: 35-39. Kalweit S., Gerner I., and Spielmann H. (1987) Validation project of alternatives for the Draize eye test. Molecular Toxicology 1: 597-603.

IP-108 February/95