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Protocol no. 109
HAEMOGLOBIN DENATURATION AS A MODEL FOR PREDICTING IRRITANCY

The degree to which a surfactant causes denaturation of haemoglobin can be used to predict its potential to cause ocular irritation.

CONTACT

Dr Hiroshi Itagaki, Mr Toshikatsu Hayashi or Dr Shinobu Kato Shiseido Safety and Analytical Research Center 1050 Nippa-cho Kohoku-ku Yokohama 233 Japan Fax: 045 546 4456

RATIONALE

The percentage of haemoglobin denaturation caused by a surfactant has been shown to have a fairly high correlation with Draize rabbit eye test scores. Factor analysis has identified that two factors are involved, namely the low concentration factor and the high concentration factor. The haemoglobin denaturation ratio at defined surfactant concentrations above and below the critical micelle concentration can be used to obtain an estimate of the Draize corneal and total eye irritation scores.

BASIC PROCEDURE

Two-fold serial dilutions of the surfactant in deionised water (2-0.002% w/w) are pipetted in eight replicates into a 96-well plate. An equal volume of haemoglobin solution is added to four dilution series, and an equal volume of PBS to the remaining 4 series. Following a 5-minute incubation at 25%C, the absorbance of the wells at 418nm is determined using a plate reader. The haemoglobin denaturation ratio (HDR%) is determined for each dilution, and the HDR% at defined concentrations is used in calculationf to obtain estimates of Draize scores. TEST STATUS The haemoglobin denaturation method has been included in an inter-laboratory validation study sponsored by the Japanese Ministry of Health and Welfare. It is used routinely at Shiseido for preliminary screening of shampoos and rinses.

CHEMICALS TESTED

Nonionic Surfactants
POE(10) octylphenylether
POE(20) octlyphenylether
Cationic Surfactants
Hexadecylpyhridinium chloride
Octadecyltrimethylammonium chloride
Dodecyltrimethylammonium chloride
Anionic Surfactants
Alkyloyltaurate
Sodium dodecylsulphate
Acylglutamate
POE dodecylethersulphate
Sodium caseinate
Amphoteric Surfactants
Alkylbetaine
Alkylamidobetaine

REFERENCES

  1. Shopsis C., and Eng B. (1985) Rapid cytotoxicity testing using a semi-autimated protein denaturation on cultured cells. Toxicology Lett. 26: 1-8
  2. York M., Lawrence R.S., and Gibson G.B. (1982) An in vitro test for assessment of eye irritancy in consumer products - preliminary findings. Int. J. Cosmetic Sci. 4: 223-234.

IP-109 February/95