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The current OECD guideline for the assessment of eye irritation recommends, within its initial considerations, the use of data from skin irritation tests as a pre-screen to detect the most severely irritating materials, its being assumed that materials that are severely irritating to the skin are also significantly irritating to the eyes. However, analysis of data for 223 materials, tested in this laboratory for both dermal and ocular irritancy, revealed that only 23% of severe eye irritants were also severe skin irritants. This resulted in a significant number of rabbits developing severe ocular effects that had not been predicted from the dermal responses. This study reports the results of an alternative approach for predicting severe eye irritants. The approach was a two-stage in vitro test battery; the first stage was a cytoxicity assay using the K562 cell line; the second was the isolated rabbit eye test. In contrast to the use of skin irritation tests, the in vitro battery was significantly more predictive (83% of severe eye irritants were detected). Although the indicence of false positive responses in the assay precludes its routine use as a replacement for the in vivo rabbit eye test, the test battery provides a powerful aid to reducing animal use and guiding in vivo studies to minimize the severity of effects.

In vivo rabbit eye tests have attracted criticism on both scientific and ethical grounds. Consequently, there is a need to develop new approaches that still provide the necessary information on eye irritation hazard but that minimize or even avoid the use of whole laboratory animals. Cytotoxicity models have been used to predict the ocular irritancy of surfactants, since this class of chemicals has an essentially common action on cell membranes which involves membrane disruption. The aim of the present studies was to compare the predictive ability of two in vitro cytotoxicity tests, the K562 and the red blood cell lysis tests, in the assessment of the in vivo eye irritancy of surfactants. The results of these studies on 14 selected surfactant materials showed that the K562 assay was only modestly predictive of the in vivo response, with a specificity of 86% but a sensitivity of only 57%. In contrast, the red blood cell lysis test was more predictive, correctly identifying all irritants tested. In addition, all non-irritant surfactants examined were predicted and a high (89%) ability to rank irritant effect was demonstrated. The red blood cell lysis test could be a powerful addition to a testing strategy or pre-screen for the evaluation of surfactant chemicals.

As a follow-up to a prevalidation study on in vitro tests for replacing the in vivo rabbit test for skin corrosivity, an international validation study was conducted during 1996 and 1997 under the auspices of ECVAM. The main objectives of the study were to: (a) identify tests capable of discriminating corrosives from non-corrosives for selected types of chemicals and/or all chemicals; and (b) determine whether these tests could identify correctly known R35 (UN packing group I) and R34 (UN packing groups II & III) chemicals. The tests evaluated were the rat skin transcutaneous electrical resistance (TER) assay, CORROSITEX(TM), the Skin(2TM) ZK1350 corrosivity test and EPISKIN(TM). Each test was conducted in three independent laboratories. 60 coded chemicals were tested. All of the tests evaluated showed acceptable intralaboratory and interlaboratory reproducibilities, and the TER, Skin(2) and EPISKIN tests proved applicable to testing a diverse group of chemicals of different physical forms, including organic acids, organic bases, neutral organics, inorganic acids, inorganic bases, inorganic salts, electrophiles, phenols and soaps/surfactants. Two of the four tests evaluated, the TER assay and EPISKIN, met the criteria agreed by the Management Team concerning acceptable underprediction and overprediction rates for them to be considered scientifically validated for use as replacements for the animal test for distinguishing between corrosive and non-corrosive chemicals for all of the chemical types studied [objective (a)]. EPISKIN was the only test able to distinguish between known R35 (UN packing group I) and R34 (UN packing groups II & III) chemicals, for all of the chemical types included, on an acceptable number of occasions [objective (b)]. The corrosive potentials of about 40% of the test chemicals could not be assessed with CORROSITEX, and the assay did not meet all of the criteria for it to be considered acceptable as a replacement test. However, CORROSITEX may be valid for testing specific classes of chemicals, such as organic bases and inorganic acids. The Skin(2) assay did not meet the criteria for it to be considered scientifically validated. Thus, the validities of (i) the TER and EPISKIN assays for discriminating corrosives from non-corrosives, and (ii) the EPISKIN assay for identifying correctly known R35/I and R34/II & III chemicals, have been demonstrated in this study. CORROSITEX appears to be valid when used only with certain types of chemicals.

A list of 176 chemicals, all of high or consistent purity and stable on storage, has been developed using available comprehensive in vivo rabbit skin irritation data. No new in vivo testing was conducted to qualify a chemical for inclusion in the list. The chemicals were tested undiluted in in vivo studies, apart from those chemicals where high concentrations could be expected to cause severe effects. The in vivo data were generated in studies carried out since 1981 according to OECD Test Guideline 404 and following the principles of Good Laboratory Practice. The data were obtained from tests normally using at least three rabbits evaluated at the same time, involving application of 0.5 g or 0.5ml to the flank under semi-occlusive patches for 4 hr, and in which observations were made at least 24, 48 and 72 hr after removal of the patch. The chemicals represent a range of chemical classes [acids, acrylates/methacrylates, alcohols, aldehydes, alkalis, amines, brominated derivatives, chlorinated solvents, esters, ethers, fatty acids and mixtures, fragrance oils, halogenated aromatics, hydrocarbons (unsaturated), inorganics, ketones, nitrites, phenolic derivatives, S-containing compounds, soaps/surfactants, triglycerides] and different degrees of irritancy. They are ranked for skin irritation potential on the basis of a 'primary irritation index'. These chemicals could be used in validation tests of promising alternatives to the in vivo rabbit skin irritation/corrosion test. This is an essential step in the progression to regulatory acceptance of alternative procedures.

In vivo rabbit eye tests have attracted criticism on both scientific and ethical grounds. Consequently there is a need to introduce methods that provide relevant information whilst avoiding the use of whole laboratory animals. Cytotoxicity models have been used to predict the ocular irritancy of surfactants since the action of this class of chemicals on cell membranes is essentially common and involves membrane disruption. The aim of the present studies was to compare the predictive ability of two in vitro cytotoxicity tests (the K562 and the red blood cell lysis tests) in the assessment of the in vivo eye irritancy of surfactants. The results of these studies on 14 selected surfactant materials showed that the K562 assay was less likely to over-estimate effect (when assessed over a wide range of surfactant concentration) than had previously been observed. This increased specificity (86%) was accompanied by a decrease in sensitivity (57%), the ability to correctly identify 'irritant' surfactants. In contrast, the red blood cell lysis test was more predictive, correctly identifying all irritants tested. In addition, all non-irritant surfactants examined were predicted by this test and a high (89%) ability to rank irritant effect was demonstrated. The red blood cell lysis test could be a powerful addition to a testing strategy or pre-screen for the evaluation of surfactant chemicals.

The collaborative study reported here was performed to evaluate the reliability of the skin corrosivity test in vitro when performed in independent laboratories. Twenty substances were examined in each of three participating laboratories and the results were compared with existing data from standard assays in vivo. The skin corrosivity test is based on the assumption that corrosive substances destroy the skin's natural outer protective barrier, the stratum corneum. Corrosive action in vitro is measured by a fall in the transcutaneous electrical resistance (TER) below a predetermined threshold. A refined test using a MgSO(4) electrolyte solution for TER measurements has recently been shown to reduce the number of false positive results in the test, while maintaining excellent predictive value for skin corrosive substances. Although in the present study there was some variation between laboratories in terms of the absolute mean TER values obtained, all 6 substances corrosive in vivo were correctly predicted by the three laboratories. The other 14 substances ranged from being non-irritant to severe skin irritants in vivo, but the test was unable to discriminate between these different categories in any of the three laboratories. However, these inter-laboratory comparisons demonstrate that the refined skin corrosivity test is a robust and reliable method in vitro for identifying potential skin corrosive substances.