Occurrence and distribution of polycyclic aromatic hydrocarbons (PAHs) in Bolgoda and Beira Lakes, Sri Lanka.

The pollution of polycyclic aromatic hydrocarbons (PAHs) has been widely used to assess the potential impact of anthropogenic activities on aquatic environments because their occurrence in water is closely tied to urban activities. Many PAHs possess mutagenic and carcinogenic properties (Menzie et al. 1992). PAH distribution and toxic potentials have therefore been the focus of numerous studies in waterways including the Great Lakes (USEPA Report 1994), Yanisei Bay (Dahle et al., 2003), and the Fraser River basin (Yunker et al., 2002). Sri Lanka, a small island nation with a dense population of about 20 million people, faces a multitude of environmental stresses ranging from deforestation to traffic congestion and the deterioration of water quality. This study was undertaken to understand the occurrence, sources, and potential impacts of PAHs in the waterways of Sri Lanka. Two lakes, Beira and Bolgoda, were selected for the study due to their economic value and high level of pollution. Beira Lake, situated in downtown Colombo, the capital city of Sri Lanka, is highly polluted. Sources of pollution are multifarious. For instance, clusters of communities have sprung up along the edges of the lake in recent times and many shacks have been built. These communities are generally not connected to municipal sewer systems and substantial quantities of domestic sewage and untreated wastewaters are discharged directly into the lake. Small industries have also grown rapidly around the lakes, most of which are not believed to have adequate facilities to treat industrial wastewater, especially organic wastes. In addition, Sri Lanka has experienced an upsurge of motor vehicles, including millions of three-wheelers and minivans that are powered by leaded gasoline and diesel fuels. Traffic congestion and severe air pollution due to vehicle emissions are now common daily occurrences and are considered a major potential source of PAHs in the lakes. Although Bolgoda Lake is situated some distance from Colombo, it is heavily polluted due to the growing number of towns with an attendant increase in small businesses and various industries along its shores. These new developments have undoubtedly impacted the lake through the discharge of PAHs and other anthropogenic chemicals present in industrial wastewater and from street runoffs. The lake, additionally, receives a large quantity of pollutants from the industrial zone in the north. The pollution caused by PAHs has led to various studies on the distribution and origin of PAHs in the environment (Yunker et al., 1996; Budzinski et al., 1997). Based on the proportions of different PAHs, most studies aim to distinguish PAHs of petrogenic sources from those of pyrolytic origins. The PAHs of petrogenic origin, prevalent in coals and fossil fuels, are formed from diagenesis of sedimentary organic material under low to moderate temperature and tend to consist of low-molecular-weight PAHs with two to three aromatic rings (Potter et al., 1998). The pyrolytic PAHs, on the other hand, are formed at much higher temperatures (greater than 500 degrees C for example) and consist mainly of four or more aromatic rings (Commins, 1969). Thus, an increase in the proportion of higher-molecular-weight PAHs is taken to be indicative of contaminations of mainly pyrolytic origin. The prevalence of high-molecular-weight PAHs in the urban dusts (Wise et al., 1988) and in atmospheric particles (Sicre et al., 1987) illustrates the chemistry of their formation at high temperature. The purpose of this study was to determine the PAH concentrations and distribution with respect to sampling location, origin and sources in two polluted lakes.

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Eye Irritation Testing: The Way Forward. The Report and Recommendations of ECVAM Workshop 34.

This is the report of the thirty-fourth of a series of workshops organised by the European Centre for the Validation of Alternative Methods (ECVAM). ECVAM's main goal, as defined in 1993 by its Scientific Advisory Committee, is to promote the scientific and regulatory acceptance of alternative methods which are of importance to the biosciences and which reduce, refine or replace the use of laboratory animals. One of the first priorities set by ECVAM was the implementation of procedures which would enable it to become well-informed about the state-of-the-art of non-animal test development and validation, and the potential for the possible incorporation of alternative tests into regulatory procedures. It was decided that this would be best achieved by the organisation of ECVAM workshops on specific topics, at which small groups of invited experts would review the current status of various types of in vitro tests and their potential uses, and make recommendations about the best ways forward (1). The workshop on Eye Irritation Testing: The Way Forward was held in Egham, UK, on 15-17 June 1998, under the chairmanship of Michael Balls (ECVAM, Italy). The workshop had two aims, the first of which was to review some of the previous multi-laboratory validation studies on alternatives to the Draize eye test and assess why many promising alternative methods were not successful in these studies. The second aim was to discuss strategies for making progress toward the short-term reduction, refinement, and eventual replacement, of the Draize test, including: a new approach to the validation of in vitro tests for eye irritancy, based on the use of reference standards, which promises to overcome some of the problems encountered in previous studies; the use of stepwise testing strategies which reduce and refine the use of animals in eye irritation testing; the use of multivariate and other statistical techniques for the further analysis of data generated in previous validation studies; and a programme of research aimed at understanding the underlying mechanisms of eye irritation.

The HET-CAM, a Useful In Vitro Assay for Assessing the Eye Irritation Properties of Cosmetic Formulations and Ingredients.

One of the most important biological properties of consumer products, and also of many raw materials, is the local compatibility to mucous membranes. Until now standardized in vivo tests are accepted by public health authorities as valid to estimate the irritation potential of chemicals and suitable for the risk assessment. Nevertheless, the controversial discussion on animal tests, and particularly on the Draize rabbit eye test, is increasing in the public and scientific domain. Efforts have been made to validate proper and suitable in vitro tests in international cosmetics industries during the last decade. One of the most important in vitro tests is the HET-CAM, the h en's e gg t est on the c horioa llantoic m embrane of fertilized chicken eggs. In this paper, the efforts to establish the HET-CAM protocol and the defined prediction model (PM) used in the COLIPA (The European Cosmetic, Toiletry and Perfumery Association) study on alternatives to the Draize rabbit eye test are described. Furthermore, the HET-CAM test results of the finalized phase I of the above-mentioned study are discussed in detail. Prior to the COLIPA validation study, the HET-CAM was prevalidated with about 100 test substances covering a broad spectrum of chemical structures and physical appearances and representing the range of chemicals in the cosmetics industry. This prevalidation was performed with a stringent in-house agreement in one company to test each chemical in the HET-CAM before any requested animal test was done. There was a high concordance of the HET-CAM results with in vivo data of the Draize test, especially for slightly irritating test articles. Based on these promising data, the HET-CAM protocol was taken as the final standard operating procedure (SOP) in the international COLIPA validation study, testing 55 coded chemicals in four different laboratories. The HET-CAM has been established and proven to be a robust test with a good prediction of irritation potential. According to strict associations of well-defined irritation categories (in vivo and in vitro), and with the concrete PM, the in vivo irritation potential of 29 out of 55 test articles (about 52%) were correctly predicted with the HET-CAM in at least three laboratories. This quality of prediction was of different success in the four categories of irritation severity. 90% of the slightly irritating chemicals but only 53% of the severely irritating articles were correctly predicted. The necessity to define a "gold standard" for validation purposes and the conflict with heterogeneous in vivo data were also pronounced this article. Here it is discussed, whether the evaluation of such heterogeneous responses and especially of persistent slight effects on the cornea can be done properly with additional data such as physicochemical data and biological information of the test substance.

Industry experience with alternative methods.

L'OREAL has been using alternative methods for almost 30 years and this has led to their current widespread in-house use for evaluation of local effects in safety and efficacy. Alternative methods are used daily to assess eye and skin tolerance, phototoxicity, photoprotection, skin sensitization, percutaneous absorption and skin and hair care. For eye irritation, many years of in-house studies have enabled us to develop and to select the most reliable tests. New in vitro methods have also been developed to help to understand the ocular irritation mechanisms which underlie the irritative properties of new chemicals. In the field of skin irritation, L'OREAL's work has focused mainly on the wide possibilities offered by reconstructed human skins to evaluate the skin tolerance of cosmetics. Today we have managed to introduce Langerhans cells in reconstructed epidermis to develop an alternative to skin sensitization. Besides these in-house investigations either in research or in evaluation, our laboratories have contributed actively to multicentric studies to help the prevalidation/validation process in various fields. The alternative approach is now totally integrated into the safety evaluation strategy, and this allowed L'OREAL to totally ban animal testing on cosmetic products several years ago. In vitro alternatives are very powerful tools: they allow the study of fine mechanisms and the use of human cells. This overall 'in vitro' approach is a scientific, ethical and industrial breakthrough.

The International EU/COLIPA In Vitro Phototoxicity Validation Study: Results of Phase II (Blind Trial). Part 1: The 3T3 NRU Phototoxicity Test.

To date, no standardized international guideline for the testing of chemicals for phototoxic potential has been accepted for regulatory purposes. In 1991, the European Commission (EC), represented initially by the Directorate General XI and later by ECVAM (the European Centre for the Validation of Alternative Methods) and COLIPA (the European Cosmetic, Toiletry and Perfumery Association), agreed to establish a joint EU/COLIPA programme on the development and validation of in vitro phototoxicity tests. The first phase (phase I, 1992-93) was designed as a prevalidation study, to identify in vitro test procedures and test protocols for a formal validation trial under blind conditions. In the second phase (phase II, 1994-95), the formal validation study, the most promising in vitro phototoxicity tests were validated with 30 carefully selected test chemicals in 11 laboratories in a blind trial. The 3T3 mouse fibroblast neutral red uptake phototoxicity test (3T3 NRU PT) was performed as a core test in nine laboratories, since it provided the best results in phase I of the study. The purpose of phase II was to confirm the reliability and relevance of the in vitro tests for predicting phototoxic effects and for identifying phototoxic chemicals. In phase II the phototoxic potential of test chemicals in the 3T3 NRU PT test was either assessed by determining the phototoxicity factor (PIF) by using a cut-off value of 5 as in phase I of the study, or by determining the mean photo effect (MPE) by using a cut-off value of 0.1, as recently proposed by Holzhütter (1997). Results obtained with both approaches in the 3T3 NRU PT test in phase II were reproducible in the nine laboratories, and the correlation between in vitro and in vivo data was very high. Therefore, ECVAM and COLIPA conclude from this formal validation trial under blind conditions that the 3T3 NRU PT test is a scientifically validated in vitro test which is ready to be considered for regulatory purposes for assessing the phototoxic potential of chemicals. A draft OECD Guideline for "In Vitro Phototoxicity Testing", incorporating the standard protocol of the 3T3 NRU PT test, will be submitted to the OECD test guidelines programme in due course.

A Study on UV Filter Chemicals from Annex VII of European Union Directive 76/768/EEC, in the In Vitro 3T3 NRU Phototoxicity Test.

In 1996, the Scientific Committee on Cosmetology of DGXXIV of the European Commission asked the European Centre for the Validation of Alternative Methods to test eight UV filter chemicals from the 1995 edition of Annex VII of Directive 76/768/EEC in a blind trial in the in vitro 3T3 cell neutral red uptake phototoxicity (3T3 NRU PT) test, which had been scientifically validated between 1992 and 1996. Since all the UV filter chemicals on the positive list of EU Directive 76/768/EEC have been shown not to be phototoxic in vivo in humans under use conditions, only negative effects would be expected in the 3T3 NRU PT test. To balance the number of positive and negative chemicals, ten phototoxic and ten non-phototoxic chemicals were tested under blind conditions in four laboratories. Moreover, to assess the optimum concentration range for testing, information was provided on appropriate solvents and on the solubility of the coded chemicals. In this study, the phototoxic potential of test chemicals was evaluated in a prediction model in which either the Photoirritation Factor (PIF) or the Mean Photo Effect (MPE) were determined. The results obtained with both PIF and MPE were highly reproducible in the four laboratories, and the correlation between in vitro and in vivo data was almost perfect. All the phototoxic test chemicals provided a positive result at concentrations of 1µ/ml, while nine of the ten non-phototoxic chemicals gave clear negative results, even at the highest test concentrations. One of the UV filter chemicals gave positive results in three of the four laboratories only at concentrations greater than 100µ/ml; the other laboratory correctly identified all 20 of the test chemicals. An analysis of the impact that exposure concentrations had on the performance of the test revealed that the optimum concentration range in the 3T3 NRU PT test for determining the phototoxic potential of chemicals is between 0.1µg/ml and 10µg/ml, and that false positive results can be obtained at concentrations greater than 100µg/ml. Therefore, the positive results obtained with some of the UV filter chemicals only at concentrations greater than 100µg/ml do not indicate a phototoxic potential in vivo. When this information was taken into account during calculation of the overall predictivity of the 3T3 NRU PT test in the present study, an almost perfect correlation of in vitro versus in vivo results was obtained (between 95% and 100%), when either PIF or MPE were used to predict the phototoxic potential. The management team and participants therefore conclude that the 3T3 NRU PT test is a valid test for correctly assessing the phototoxic potential of UV filter chemicals, if the defined concentration limits are taken into account.

Report on the COLIPA Workshop on Mechanisms of Eye Irritation.

This report summarises the discussions of a workshop sponsored by the European Cosmetics, Toiletries and Perfumery Association (COLIPA). The workshop discussed the state-of-the-art of eye irritancy testing, and made recommendations as to the best ways in which to validate alternatives to the Draize eye irritation test. The importance of understanding the mechanisms of eye irritation, particularly when attempting to improve in vitro prediction of in vivo eye irritancy, was also emphasised.

IRAG working group 2. CAM-based assays. Interagency Regulatory Alternatives Group.

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 sponsor's 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 Pearson's 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.

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.

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.

Modern biostatistical methods for assessing in vitro/in vivo correlation of severely eye irritating chemicals in a validation study of in vitro alternatives to the Draize eye test.

The HET-CAM test and 3T3 cell neutral red uptake (NRU) cytotoxicity assay were evaluated in a national German validation project to replace the Draize eye test for classifying severely eye irritating chemicals, which have to be labelled 'R-41' according to EU regulations. As testing of 200 chemicals in the two in vitro assays did not sufficiently allow severely eye irritating chemicals to be identified and since the scoring system of the HET-CAM assay has been derived empirically, it was investigated whether modern biostatistical methods, for example discriminant analysis, would improve the selection of predictive endpoints of the HET-CAM assay. Comparison of HET-CAM data with adverse reactions observed in different tissues of the rabbit's eye proved that complex regression models are better describing in vitro /in vivo correlations than simple linear models. Discriminant analysis revealed that among the nine endpoints routinely determined in the HET-CAM test, coagulation was the only acceptable endpoint to classify severely irritating chemicals 'R-41' according to EU regulations. To identify R-41 chemicals the reaction time of appearance of coagulation of a 10% solution was the best discriminating factor and coagulation of the undiluted chemical for the less water-soluble ones. The results suggest that only R-41 chemicals are inducing coagulation of the CAM within 50 sec, and can therefore be classified without further testing in vivo. Stepwise discriminant analysis allowed an in vitro testing strategy to be developed to identify R-41 chemicals by combining coagulation data of the HET-CAM assay with cytotoxicity data. Validity of the model for future data sets was assessed by cross-validation. The results obtained with 200 chemicals under blind conditions suggest that this approach will provide an acceptable sensitivity, predictivity and percentage of false positive data for severely eye irritating chemicals.

Alternatives in cosmetics testing.

This paper represents a summary of presentations made during a round-table discussion at the ECVAM Opening Symposium. After introductory comments on the cosmetic industry's use of alternative methods in the safety assessment process, the use of alternative methods by L'Oréal and by the Japanese cosmetic industry is outlined, current validation studies in Japan are noted, and the involvement of COLIPA, the European Cosmetic, Toiletry and Perfumery Association, in promoting the use of alternative methods is discussed. Two final sections deal with the effect of data variability on the performance of alternative methods in validation studies and on the integrated use of quantitative structure-activity relationship (QSAR) analysis with other approaches in the safety assessment process.

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.

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.

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.

In vitro methods: their relevance and complementarity in ocular safety assessment.

Ocular irritation includes a wide variety of mechanisms some of which can be explored by in vitro methods. For example, the effects on epithelial cells that constitute the outer layers of both the conjunctiva and the cornea may result in direct cytotoxicity or impairment of cellular functions -such as impermeability-, phenomena that can be explored in vitro. Irritancy may also involve inflammation of the conjunctival connective tissue and of the corneal stroma with its vascular and cellular features; effects on the stroma can lead to the opacification of the cornea; this last phenomenon may be the consequence of mechanisms such as modification of the structure of proteins or changes in stroma hydration which in particular is closely related to corneal endothelium metabolic activity. Recovery after eye injury depends partly on the extent of ocular damage and on the residual mitotic activity of the remaining cells. We have studied 41 surfactants, lotions and shampoos in 6 to 8 in vitro methods each one exploring one or two endpoints that could be linked to the ocular irritancy phenomena described above. In vivo ocular irritancy data for these materials from previous studies were compared to in vitro results. The results obtained show that -among the techniques that were investigated and for the categories of substances that were studied- the Het-CAM test and more particularly the endpoint that is related to vascular effects gives the best assessment of acute ocular irritancy (Spearman's rho coefficients between in vivo and in vitro data greater than 0.90); however, cell culture methods, especially one based on short contact time between cells and products and on evaluation of early toxic effects, also proved interesting (Spearman's rho coefficients between in vivo and in vitro data greater than 0.85). Moreover, the isolated cornea opacity and permeability test gave complementary information more related to recovery from surfactant-induced damage. These encouraging results lead us to consider in vitro ocular safety assessment with optimism for the categories of products investigated.

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.

Correlation and validation of alternative methods to the Draize eye irritation test (OPAL project).

A multicentre study of alternative methods to the Draize eye irritation test, involving six different laboratories was organized by OPAL (Oeuvre Pour l'Assistance aux Animaux de Laboratoire). Forty chemicals (including solvents, surfactants, acids, bases, and others) were selected for testing by three methods, namely Griffith's test (a low-volume eye irritation test on rabbits), the hen's egg chorioallantoic membrane (HET-CAM) assay for the evaluation of hyperaemia, haemorrhage and coagulation, and neutral red uptake by SIRC cells for the assessment of cytotoxicity. Each method was used in two or three laboratories. Intralaboratory reproducibility was good for each laboratory with values, for error, close to 10%. Interlaboratory agreement was also good, particularly for the cell culture method, a quantitative and objective technique. Griffith's test correlated well with the Draize test (r = 0.846; n = 37), while for the HET-CAM test (r = 0.670; n = 32) and the cell culture method (r = 0.579; n = 32) the correlation was satisfactory. A more complete statistical analysis is currently under way to confirm and extend these preliminary findings.