O. de Silva

A summary report of the COLIPA international validation study on alternatives to the draize rabbit eye irritation test.

The principal goal of this study was to determine whether the results from a set of selected currently available alternative methods as used by cosmetics companies are valid for predicting the eye irritation potential of cosmetics formulations and ingredients and, as a consequence, could be valid replacements for the Draize eye irritation test. For the first time in a validation study, prediction models (PMs) that convert the in vitro data from an assay to a prediction of eye irritation were developed for each alternative method before the study began. The PM is an unequivocal description of the relationship between the in vitro and the in vivo data and allows an objective assessment of the reliability and relevance of the alternative methods. In this study, 10 alternative methods were evaluated using 55 test substances selected as representative of substances commonly used in the cosmetics industry (23 ingredients and 32 formulations). Twenty of the single ingredients were common to the European Commission/British Home Office (EC/HO) eye irritation validation study (Balls et al., 1995b). The test substances were coded and supplied to the participating laboratories. The results were collected centrally and analysed independently, using statistical methods that had been agreed before the testing phase began. Each alternative method was then evaluated for reliability and relevance in assessing eye irritation potential. Using the criteria of both reliability and relevance as defined in the study, the preliminary results indicate that none of the alternative methods evaluated could be confirmed as a valid replacement for the Draize eye irritation test across the full irritation scale. However, three alternative methods-the fluorescein leakage test, the red blood cell assay (classification model) and the tissue equivalent assay-each satisfied one criterion of reliability or relevance. Further investigation of the decoded data from this study to explore more fully the relationship between the in vitro data and the in vivo data is recommended. Such a review may allow the development of new prediction models to be tested in a subsequent validation study.

EEC/COLIPA project on in vitro phototoxicity testing: First results obtained with a Balb/c 3T3 cell phototoxicity assay

In a joint validation project eight laboratories from the European Cosmetic Industry Association (COLIPA) as well as FRAME (England) and ZEBET (Germany) are trying to develop validated in vitro methods to be incorporated into new international guidelines for acute phototoxicity testing. The first stage of the study involved selection of the most promising in vitro phototoxicity tests for further validation. 20 chemicals with known phototoxic properties (12 phototoxins, four UV-absorbing non-phototoxins and four non-UV absorbing non-phototoxins) were tested under identical conditions of UV exposure conditions (sun simulator, UVA 5 J/cm(2)) in a standardized cytotoxicity assay with Balb/c 3T3 fibroblasts (endpoint: neutral red uptake, NRU). 19 of the 20 chemicals were correctly classified by the 3T3 NRU phototoxicity test, and therefore, this simple assay for phototoxicity seems very promising and should be validated further.

An evaluation of five potential alternatives in vitro to the rabbit eye irritation test in vivo

Five alternative techniques, each of which had been successfully used by one of the participating companies, were evaluated in the assessment of the eye-irritation potential of 32 samples. The 32 samples included chemical ingredients and preparations from household cleaning product, personal care, and cosmetic categories. Historical data from rabbit eye irritation tests in vivo existed for each sample; it was therefore not necessary to carry out any tests in vivo as part of this evaluation exercise. The five alternative methods used were the silicon microphysiometer test, the Microtox test, the neutral red uptake assay, the chorioallantoic membrane vascular assay (CAMVA) and the hen egg test-chorioallantoic membrane assay (HETCAM). Three of the assays were conducted in two laboratories, allowing an interlaboratory comparison of performance to be made. The CAMVA assay was carried out on 10-day-old as well as on 14-day-old fertile eggs. Correlations between the data sets in vivo and in vitro were determined for the five assays. The results demonstrated that for the materials tested, all of the assays show some promise as alternative methods to the rabbit eye test in vivo in the prediction of eye irritation, and that the reproducibility of results of those techniques carried out in two laboratories was very good. The results from 14-day and 10-day CAMVA assays were virtually identical. It is recommended that a larger-scale validation exercise be carried out to demonstrate the ultimate usefulness of these alternative procedures in the safety evaluation process.

IRAG working group 2: CAM-based assays

CAM-based assays, in which test material is applied to the chorion allantoic membrane (CAM) of embryonated chicken eggs, were assessed as alternatives to the Draize eye irritation test. Two general types of CAM-based assays are currently in use, the HET-CAM test and the CAMVA assay. Evaluations were made of five data sets produced with three different modifications of the HET-CAM test and two data sets obtained with the same CAMVA protocol. Data sets consisted of 9-133 test chemicals, usually from the sponsors product line, and also from a validation trial. Each data set and assay protocol were analysed for quality of data, purpose and proposed use of the assay, range of responses covered, range of test materials amenable, current use in safety and risk assessment both in-house and for regulatory purposes. Since the MMAS Draize score was not available for all in vivo data sets, the sigma MMMIS, which correlates well with the MMAS, was used instead. In vitro/in vivo correlations calculated with Pearsons linear coefficient ranged from r = 0.6 to r = 0.9 for six of seven data sets. Corneal opacity and inflammation of the iris showed the best correlation to in vitro data. Prediction rates were significantly improved when partial linear regression was used, and the predictivity of three different HET-CAM protocols was almost the same. HET-CAM assays showed the best prediction with surfactants and surfactant-based formulations, whereas the CAMVA assay provided the best performance with alcohols.

Local lymph node assay: study of the in vitro proliferation and control of the specificity of the response by FACScan analysis

The murine local lymph node assay (LLNA) has been proposed as a screening procedure to identify contact allergens (Kimber, Hilton and Weisenberger, Contact Dermatitis 1989, 21, 215; Kimber and Weisenberger, Archives of Toxicology 1989, 63, 274). In some cases irritants have given rise to proliferative responses and it is of interest to investigate whether these responses differ in the type of cells involved. We have studied the proliferative response in vitro to topically applied sodium lauryl sulphate (SLS) and 2,4-dinitrochlorobenzene (DNCB). The test chemical or vehicle alone was applied for 3 consecutive days to the dorsum of both ears of Balb/c strain mice at three different concentrations, the highest concentration being the maximum non-irritating concentration (MNIC). Cell cultures were made 72 hr after the final exposure: draining auricular lymph nodes were excised and a suspension of lymph node cells (LNC) was prepared. Cellularity (total number of LNC/animal) and proliferative activity were assessed; proliferation was measured by culture of LNC for 24 hr with [(3)H]thymidine. LNC were also studied by FACScan analysis: cells were incubated with fluorescein isothiocyanate (FITC)-labelled monoclonal antibodies against the T-cell marker CD3 and the activated T-cell marker CD25 (IL2 receptor). In the case of DNCB dose-response curves were obtained for both cellularity and proliferative response in comparison with the controls: there was a strong increase in both parameters for the MNIC.SLS, a non-sensitizing skin irritant, induced a much lower response, slightly increased at the MNIC in comparison with the controls. By FACScan analysis we measured the rates of CD3- and CD25-positive cells in the LNC. No significant difference was obtained for SLS in comparison with the controls. In the case of DNCB, there was a significant increase in CD3-positive cells and a large increase in CD25-positive cells in comparison with the controls and SLS. These parameters could be of great interest to help distinguish between contact sensitizers and irritants. We are presently investigating other irritants and sensitizers.

Evaluation of eye irritation potential: statistical analysis and tier testing strategies☆

Eye irritation testing, specifically the Draize test, has been the centre of controversy for many reasons. Several alternatives, based on the principles of reduction, refinement and replacement, have been proposed and are being used by the industry and government authorities. However, no universally applicable, validated non-animal alternative(s) is currently available. This report presents a statistical analysis and two testing approaches: the partial least squares multivariate statistical analysis of de Silva and colleagues from France, the tier-testing approach for regulatory purposes described by Gerner and colleagues from Germany, and the three-step tier-testing approach of the US Interagency Regulatory Alternatives Group described by Gupta and Hill. These approaches were presented as three separate papers at the November 1993 Interagency Regulatory Alternatives Group (IRAG) Workshop on Eye Irritation Testing; they have been summarized and combined into the following three-part report. The first part (de Silva et al.) presents statistical techniques for establishing test batteries of in vitro alternatives to the eye irritation test. The second (Gerner et al.) and third (Gupta and Hill) parts are similar in that they stage assessment of information by using a combination of screening information and animal testing to effect reductions in animal use and distress.

The use of in vitro methods in the ocular irritation assessment of cosmetic products

The ocular tissue is a complex system consisting of corneal and conjunctival epithelial cells, the underlying corneal stroma and associated endothelial cells. Exposure to chemicals may result in responses ranging from mild, slight redness and itching, to severe injury with loss of corneal epithelium, damage to stroma, inflammatory infiltration and loss of vision. This complexity hinders the development of in vitro methods able to replace animal testing. Various in vitro techniques have been proposed and subsequently developed as potential replacements for ocular toxicity screening on animals. Over the past 2 years, eight methods have been evaluated in these laboratories. The endpoint of these methods could be linked to one or to several clinical events occurring in the in vivo eye irritancy process described above. Using these systems, a battery of four complementary in vitro assays has been developed. For the categories of ingredients and cosmetic products investigated, the promising results obtained suggest that in vitro methods of ocular risk assessment may be used increasingly in the future.