Low-volume Eye Test Research Articles

An Integrated Decision-tree Testing Strategy for Eye Irritation with Respect to the Requirements of the EU REACH Legislation

This paper presents some results of a joint research project, sponsored by Defra and conducted by FRAME and Liverpool John Moores University, on the status of alternatives to animal testing with regard to the European Union REACH (Registration, Evaluation and Authorisation of Chemicals) system for the safety testing and risk assessment of chemicals. The project covered all the main toxicity endpoints associated with REACH. This paper focuses on the use of alternative (non-animal) methods (both in vitro and in silico) for eye irritation testing. The manuscript reviews numerous in vitro tests and their possible collation into test batteries, in silico models and a refined in vivo method (the low volume eye test), before combining the use of all these methods into an integrated testing strategy. The aim of this strategy is a reduction in the number of animal tests which would need to be performed in the process of fulfilling the REACH system criteria; this would also lead to a lowering of the number of animals required in compliance with the REACH system requirements.

An Integrated Decision-tree Testing Strategy for Eye Irritation with Respect to the Requirements of the EU REACH Legislation

This paper presents some results of a joint research project, sponsored by Defra and conducted by FRAME and Liverpool John Moores University, on the status of alternatives to animal testing with regard to the European Union REACH (Registration, Evaluation and Authorisation of Chemicals) system for the safety testing and risk assessment of chemicals. The project covered all the main toxicity endpoints associated with REACH. This paper focuses on the use of alternative (non-animal) methods (both in vitro and in silico) for eye irritation testing. The manuscript reviews numerous in vitro tests and their possible collation into test batteries, in silico models and a refined in vivo method (the low volume eye test), before combining the use of all these methods into an integrated testing strategy. The aim of this strategy is a reduction in the number animal tests which would need to be performed in the process of fulfilling the REACH system criteria; this would also lead to a lowering of the number of animals required in compliance with the REACH system requirements.

Extent of Corneal Injury as a Biomarker for Hazard Assessment and the Development of Alternative Models to the Draize Rabbit Eye Test

ABSTRACTWe have characterized 22 ocular irritants differing in type (surfactants, acid, alkali, bleaches, alcohol, aldehyde, acetone) and severity (slight to severe) by using the low-volume rabbit eye test. Ocular irritation was evaluated by 1) light microscopy to assess pathological changes, 2) in vivo confocal microscopy (CM) to quantify 4-dimensionally (x, y, z, and t) initial corneal injury and later responses in the same eye, and 3) laser scanning CM to quantify initial cell death. These studies revealed that regardless of the processes leading to injury, slight irritants injure the corneal epithelium, mild irritants injure the corneal epithelium and the superficial stroma, and moderate/severe irritants injure the epithelium, deep stroma, and at times the corneal endothelium. Furthermore, extent of initial corneal injury was shown to predict subsequent responses and final outcomes. These findings suggest that extent of corneal injury may be used as a basis for the development of alternative ocular irritation tests. To test the validity of this approach, we have used an ex vivo, rabbit cornea culture model to measure extent of corneal injury following exposure to ocular irritants. Data indicate that the extent of ex vivo corneal injury significantly correlate with the extent of initial injury measured previously in live animals. Overall, these findings indicate that extent of initial corneal injury can be used as a new “gold standard” for the continued refinement and ultimate replacement of the Draize rabbit eye Ocular Irritation Test.

Pathology of ocular irritation with bleaching agents in the rabbit low-volume eye test.

Despite differences in the processes leading to tissue damage, the ocular irritation response to various surfactants, two concentrations of an acid and an alkali, and an acetone, alcohol, aromatic amine, and aldehyde has been shown to depend on the extent of initial injury. The purpose of this study was to assess the extent to which this fundamental relationship exists for bleaching agents in the rabbit low-volume eye test. Ten microl of sodium perborate monohydrate (NaBO3), sodium hypochlorite (NaOCl), 10% hydrogen peroxide (H2O2), and 15% H2O2 was applied directly to the cornea of the right eye of each rabbit. Macroscopic assessments for irritation were made 3 hours after dosing and periodically until 35 days. Light microscopic examinations were conducted on tissues obtained at 3 hr and on 1, 3, and 35 days. In vivo confocal microscopy (CM) and measurements of dead corneal epithelial cells and keratocytes at 3 hours and 1 day were used to characterize quantitatively initial corneal injury, while in vivo CM performed at 3 hours and 1, 3, 7, 14, and 35 days was used to characterize quantitatively the corneal changes over time. The changes with NaBO3 and NaOCl were consistent with mild irritancy. For both, corneal injury was limited to the epithelium and superficial stroma. The changes with 10% H202 and 15% H2O2 were consistent with severe irritation. Both concentrations affected the epithelium and deep stroma, with 15% H2O2 also at times affecting the endothelium. However, unlike other irritants previously studied, with 10% H2O2 and 15% H2O2 there was an incongruity between the extent of epithelial and stromal injury, with stromal injury being more extensive than epithelial injury. A similar, although less dramatic, effect was observed with NaBO3. Additionally, there was still significant keratocyte loss at 35 days with 10% H2O2 and 15% H2O2 even though the eyes at times were considered to be macroscopically normal. These observations highlight the need to include both epithelial and stromal components in an ex vivo or in vitro alternative assay. In conclusion, these results continue to support and extend our hypothesis that ocular irritation is principally defined by the extent of initial injury despite clear differences in the means by which irritants cause tissue damage. Importantly, we have identified unique differences in the ocular injury and responses occurring with bleaching agents that are important to consider in the development and validation of alternative ocular irritation tests to characterize a broad range of materials differing in type and irritancy.

Extent of initial corneal injury as a basis for alternative eye irritation tests

Based on studies that have characterized the extent of injury occurring with irritants of differing type and severity, we have proposed that extent of initial injury is the principal mechanism underlying ocular irritation. We report here our efforts to apply this hypothesis, as a mechanistic basis, to the development of an alternative eye irritation assay using an ex vivo rabbit corneal model. Rabbit eyes were obtained immediately after sacrifice or from an abattoir and 8.5-mm diameter corneal buttons were removed and cultured overnight at an air–liquid interface under serum-free conditions. Buttons were exposed to materials of differing type (surfactant, acid, base, alcohol and aldehyde) and irritancy (slight to severe) that had been previously characterized microscopically in the rabbit low-volume eye test. Exposure was accomplished by applying 1.5 μl of an irritant to a sterile, 3 mm diameter, filter paper disk and then placing the disk on the center of the corneal button for 10 s. After removal of the disk, buttons were washed and cultured for 3, 24 or 48 h. Buttons were then evaluated for extent of injury using a Live/Dead staining kit and fluorescent microscopy to measure cell size of live surface epithelial cells, area of epithelial denudation and depth of stromal injury. Ex vivo exposure to slight irritants generally reduced surface epithelial cell size (i.e. erosion) while exposure to mild irritants produced epithelial denudation with variable injury to the corneal stroma. Severe irritants generally produced extensive epithelial denudation and damaged the corneal stroma and endothelium. Overall, ex vivo extent of injury significantly correlated with in vivo extent of injury as measured in previous animal tests ( r=0.81, P<0.001). These findings indicate that extent of corneal injury, as shown to be associated with ocular irritation occurring in vivo, can be applied to the development of a mechanistically-based alternative eye irritation model. We believe that this approach may ultimately lead to an alternative assay to replace the use of animals in ocular irritation testing.

Pathology of ocular irritation with acetone, cyclohexanol, parafluoroaniline, and formaldehyde in the rabbit low-volume eye test.

The ocular irritation responses to 11 different surfactants and two concentrations of acetic acid and sodium hydroxide have been shown to depend on the extent of initial injury, despite marked differences in the processes leading to tissue damage. The purpose of these studies was to determine the extent to which this fundamental relationship applies to other nonsurfactants. Ten microl of acetone (ACT). cyclohexanol (CY), parafluoroaniline (PF), or 37% formaldehyde (FA) was directly applied to the cornea of the right eye of each rabbit. Eyes and eyelids were macroscopically scored for signs of irritation beginning 3 hours after dosing and periodically until recovery or 35 days. Tissues were obtained for light microscopic examination after 3 hours and on days 1, 3, and 35. Initial corneal injury was characterized quantitatively at 3 hours and I day using in vivo confocal microscopy (CM) and by postmortem quantitation of dead corneal epithelial cells and keratocytes using a Live Dead Assay (L/D, Molecular Probes) and scanning laser CM. Corneal changes over time were characterized quantitatively using in vivo CM performed at 3 hours and 1, 3, 7, 14, and 35 days. The changes with ACT were consistent with mild irritation. Corneal injury was limited to the epithelium and superficial stroma, with the mean normalized depth of injury (NDI) being less than 10% with the majority of regions showing no stromal injury. Changes with CY and PF were consistent with moderate to severe irritation, and FA caused severe irritation. Specifically, corneal injury by CY and PF tended to involve the epithelium and anterior stroma, with the mean NDI being 10.4% to 23.8%, while injury with FA involved the epithelium, deep stroma, and at times the endothelium. Interestingly, with FA significantly less injury was observed at 3 hours with a dramatic increase in injury observed at 1 day and thereafter. In conclusion, these results continue to support and extend our hypothesis that ocular irritation is principally defined by the extent of initial injury despite clear differences in the means by which irritants cause tissue damage. We believe this approach can be applied to developing alternative assays based on injury to ex vivo eyes or injury to an in vitro corneal equivalent system.

Quantitative characterization of acid- and alkali-induced corneal injury in the low-volume eye test.

Defining the extent of initial injury has proven to be a useful basis for differentiating the ocular irritation potential of surfactants; however, the applicability of this method to other types of irritants has not been demonstrated. In the following studies we characterized the extent of corneal injury following exposure to different concentrations of acetic acid and sodium hydroxide (NaOH) in the rabbit low-volume eye test. Groups of rabbits received 3% acetic acid, 10% acetic acid, 2% NaOH, or 8% NaOH and were evaluated in vivo by macroscopic and in vivo confocal microscopic examination and postmortem using a live/dead staining kit and scanning laser confocal microscopic examination. Quantitative assessment of macroscopic scores, corneal surface epithelial cell size, corneal epithelial thickness, corneal thickness, depth of stromal injury, corneal light scattering (confocal microscopy through focusing, CMTF), and number of dead cells was conducted at various times, including the following: at 3 hours and at 1, 3, 7, 14, and 35 days. Based on macroscopic scores, the order of ocular irritancy potential was 3% acetic acid < 2% NaOH < 10% acetic acid < 8% NaOH. Evaluation of the quantitative in vivo and postmortem microscopic live/dead data revealed a slight decrease in epithelial thickness and an increase in dead epithelial cell numbers with 3% acetic acid. With 2% NaOH, significant focal changes in epithelial cell size, epithelial thickness, corneal thickness, and number of dead surface epithelial cells occurred at 3 hours and at 1 day, with injury to only a very small number of corneal stromal keratocytes, despite the presence of epithelial denudation. Changes with 10% acetic acid were similar to those noted with 2% NaOH at 3 hours and 1 day, but these changes were more diffuse and included stromal injury to a depth of 7.2 +/- 9.3% of the corneal thickness, with significant numbers of dead keratocytes. Eight percent NaOH, on the other hand, caused focally extensive injury that averaged 26.3 +/- 18.4% of the corneal thickness at 1 day, with significant light scattering from the cornea, which did not return to normal by 35 days postinjury. Overall, these data indicate that ocular irritation as a result of acetic acid and NaOH was associated with changes similar to those observed with surfactants (ie, slight irritants damage the corneal epithelium, mild and moderate irritants damage the corneal epithelium and anterior stromal cells, and severe irritants damage the corneal epithelium and deep stroma). To our knowledge, this is the first time that the ocular irritation potential for different types of materials (acid/alkali, surfactants) has been shown to be primarily dependent on the initial area and depth of injury.

Microscopic changes with acetic acid and sodium hydroxide in the rabbit low-volume eye test.

Differences in ocular irritancy have been hypothesized to reflect differences in the extent of initial injury. Although differences in the processes leading to tissue damage may exist, extent of injury is believed to be the principal factor determining final outcome of ocular irritation. Previous studies characterizing the pathology of surfactant-induced ocular irritation support this premise. The purpose of this study was to begin to determine the applicability of this premise in terms of nonsurfactants; we planned to accomplish this by assessing the ocular irritancy of different concentrations of an acid and an alkali. Ten microliters of 3 or 10% acetic acid (C2H4O2) or 2 or 8% sodium hydroxide (NaOH) were directly applied to the cornea of the right eye of each test rabbit. Untreated left eyes served as the controls. Eyes and eyelids were macroscopically examined for signs of irritation beginning 3 hours after dosing and periodically until recovery or day 35. Eyes and eyelids from animals in each group were collected for microscopic examination after 3 hours and on days 1, 3, and 35. The macroscopic and microscopic changes were consistent with slight (3% C2H4O2), mild (2% NaOH, 10% C2H4O2), and severe (8% NaOH) irritancy. The spectra of changes were similar to those previously reported for surfactants of differing types and irritancies. As with surfactants, as the extent of initial injury increased, the intensity and duration of the subsequent responses increased. These results indicate that our hypothesis also applies to nonsurfactants. The results also support our belief that the initial extent of injury associated with ocular irritation may be used to predict the subsequent responses and final outcome. Finally, our results further indicate that such an approach may be applicable to the development of alternative assays that are based on either injury to ex vivo eyes or injury to an in vitro corneal equivalent system.

Eye irritation responses in rabbit and man after single applications of equal volumes of undiluted model liquid detergent products

A criticism of the use of the rabbit low-volume eye test to determine eye irritation hazard for man is the lack of comparative data in man and rabbit with undiluted products. To address this, such a study has been performed in man and rabbit using undiluted model liquid detergents. The hypothesis tested was that if, under identical test conditions, the effects in the rabbit were the same or greater than the response in man, then it is valid to use the low-volume eye test to assess eye irritation hazard for man. The studies were carried out using 29 human volunteers and 12 rabbits. The effects in the rabbit were unequivocally greater than the effects observed in man, but clearly less than the expected response from these types of product in the Draize test. The results from this study confirm the sensitivity of the rabbit as a test species, and support the use of the low-volume eye test to assess eye irritation hazard for man. Any in vitro/ ex vivo alternative to assess eye irritation should be developed against the rabbit low-volume eye test or human data where available.

Relative Eye Irritation Potential of Nonionic Surfactants: Correlation to Dynamic Surface Tension

AbstractTen nonionic surfactants, including five alcohol ethoxylates, were selected to test for correlations between measured eye irritation potential of surfactant solutions and a comprehensive set of surface tension measurements on the same solutions. The surfactants were evaluated in aqueous solutions at a concentration (0.2 M) chosen to represent the maximal use concentration in aqueous laundry and cleaning products. The low-volume eye test (LVET) was used to evaluate eye irritation potential of the surfactant solutions. Results were reported as maximum average scores (MAS) and medium days to clear (MDTC). The MAS values ranged from 0.0 to 13.3 (on a scale of 0-110) and the MDTC from 0 to 4 days. Based on these scores, the eye irritation potential of the surfactant solutions ranged from none to low. Analysis of variance revealed that the differences among the surfactant solutions in MAS scores were highly significant (p < 0.001). There were no significant regression correlations between MAS scores and...

A comparison of low volume, Draize and in vitro eye irritation test data. III. Surfactant-based formulations

The third phase in a series of investigations of the relationship between low volume eye test (LVET) data, Draize eye irritation test data, and comparable data from in vitro assay protocols is presented. These investigations utilize Draize eye test and in vitro endpoint data generated previously as part of the CTFA Evaluation of Alternatives Program. LVET data were generated de novo using the same 25 representative surfactant-based personal-care formulations. In general, these formulations were minimally to moderately irritating. The linear correlation between maximum average score as determined by the Draize test (MAS) and the LVET (LVET-MAS) was 0.87; LVET-MAS values were typically about 30% lower then corresponding MAS values. Comparison of in vitro assay performance with that of the LVET was determined by statistical analysis of the relationship between LVET-MAS and in vitro endpoint. Regression modelling was the primary means of enabling such a comparison, the objective being to predict LVET-MAS for a given test material (and to place upper and lower prediction bounds on the range in which the LVET-MAS is anticipated to fall with high probability) based on observation of an in vitro score for that material. The degree of 95% confidence in prediction is quantified in terms of the relative widths of prediction intervals constructed about the fitted regression curves. Twenty in vitro endpoints were shown to have the greatest agreement with the LVET (these endpoints included those with low discordance rates relative to the Draize test) and were therefore selected for regression modelling. Although prediction interval widths tended to be narrower when predicting LVET-MAS compared with predicting MAS, the confidence with which the selected in vitro endpoints predicted both LVET-MAS and MAS for surfactant-based formulations was greatest when values were close to the lower or upper limits of the observed irritation range (i.e. 95% prediction interval widths were most narrow in these areas). Overall precision of LVET-MAS prediction for surfactant-based formulations was similar to that previously reported for hydroalcoholic formulations and considerably better than was reported for oil/water emulsions.

A Comparison of Low Volume, Draize and in vitro eye irritation test data. II. Oil/water emulsions

The second phase in a series of investigations of the relationship between low volume eye test (LVET) data, Draize eye irritation test data, and comparable data from in vitro eye irritation test protocols is presented. These investigations utilize Draize eye test and in vitro endpoint data generated previously as part of the CTFA Evaluation of Alternatives Program. LVET data were generated de novo using the same 18 representative oil/water based personal-care formulations. In general, these formulations were minimally to mildly irritating; only three were classified as moderate eye irritants. The linear correlation between maximum average score as determined by the Draize test (MAS) and the LVET (LVET-MAS) was 0.85; LVET-MAS values were typically about half the corresponding MAS values. Comparison of in vitro assay performance with that of the LVET was determined by statistical analysis of the relationship between LVET-MAS and each in vitro endpoint. Regression modelling was the primary means of enabling such a comparison, the objective being to predict LVET-MAS for a given test material (and to place upper and lower 95% prediction bounds on the range in which the LVET-MAS is anticipated to fall with high probability) based on observation of an in vitro score for that material. The degree of confidence in prediction is quantified in terms of the relative widths of prediction intervals constructed about the fitted regression curves. Sixteen endpoints were shown to have the greatest agreement with the LVET (all but two were selected for modelling when compared with the Draize procedure). While the lower maximum average scores values (compared with the Draize test) in the LVET led to lower variability in LVET-MAS compared to MAS, the upper and lower bounds on predicted LVET-MAS values conditional on observed in vitro scores were still wide. Because there was overlap in the range of scores determined by the prediction bounds for many formulations, each of the selected endpoints was frequently unable to distinguish between test formulations in terms of statistically different predicted LVET-MAS values. In summary, none of the in vitro endpoints evaluated were able to reliably predict values of LVET-MAS among the set of oil/water emulsions considered here.

Comparison of low-volume, draize and in vitro eye irritation test data. I. Hydroalcoholic formulations

The first phase in a series of investigations of the relationship between low-volume eye test (LVET) data, Draize eye irritation test data, and comparable data from 25 in vitro assay protocols is presented. These investigations utilize Draize eye test and in vitro assay data generated previously as part of the Cosmetic, Toiletry and Fragrance Association (CTFA) Evaluation of Alternatives Program. LVET data were generated de novo using the same 10 representative hydroalcoholic personal-care formulations. The linear correlation between maximum average score (MAS) as determined by the Draize test and the LVET (LVET-MAS) was 0.93. Comparison of in vitro assay performance with that of the LVET was determined by statistical analysis of the relationship between LVET-MAS and in vitro endpoint. As in the CTFA program, regression modelling is the primary means of enabling such a comparison. The objective is to predict LVET-MAS for a given test material (and to place upper and lower prediction interval bounds in the range in which the LVET-MAS is anticipated to fall with high probability) conditional on observing an in vitro assay score for that material. The degree of confidence in prediction is quantified in terms of the relative widths of prediction intervals constructed about the fitted regression curves. Four assays [EYTEX™ MPA (membrane partition assay), HET-CAM (hen's egg test-chorioallantoic membrane HET-CAM) I, neutral red release and HET-CAM II] were shown to have the greatest agreement with the LVET. These assays were also among those with low discordance rates relative to the Draize test. Prediction of LVET-MAS values from experimentally determined in vitro scores was more accurate for hydroalcoholic formulations with lower rather than higher irritancy potential.

Determination of the Intra- and Interlaboratory Reproducibility of the Low Volume Eye Test and Its Statistical Relationship to the Draize Eye Test

The reproducibility of toxicologic test methods, including alternative tests, is a key scientific and regulatory concern. In the present work, historic rabbit eye irritation data were used to determine the intra- and interlaboratory reproducibility of the low volume eye test (LVET). The standard Draize eye irritation test was used as the basis for comparison. The LVET and Draize tests had similar degrees of intra- and interlaboratory reproducibility as determined by examination of their coefficients of variation, although the variability in LVET results was directionally lower. Results from 70 parallel Draize and LVET tests indicated a strong positive association between results from the two tests, for corneal, iridial, conjunctival, and maximum average scores (MAS). Correlation coefficients were 0.60, 0.73, 0.69, and 0.73, respectively (P≤ 0.0001). The linear relationship between LVET and Draize MAS values was examined by regression analysis and found to follow the relationship LVET MAS = 0.522 (Draize MAS). Thus, the LVET is at least as reproducible as the Draize test and gives responses that are (linearly) correlated to the Draize. The previous findings that the LVET is more predictive of human eye responses than the Draize test lends additional support for its use as a refined alternative to the Draize test.

Development and Intralaboratory Evaluation of an in Vitro Human Cell-Based Test to Aid Ocular Irritancy Assessments

Development and Intralaboratory Evaluation of an in Vitro Human Cell-Based Test to Aid Ocular Irritancy Assessments. Osborne, R., Perkins, M. A., and Roberts, D. A. (1995). Fundam. Appl. Toxicol. 28, 139-153. A human cell-based in vitro method was developed to screen for ocular irritancy potential of aqueous compatible and incompatible test agents, such as liquids, insoluble solids, powders, granulars, emulsions, and acids/alkalis. Methods were developed for topical application (an exposure that mimics in vivo testing) and wash-off of test substances on the epithelial surface of human skin derived epithelial-fibroblast cocultures (Skin 2 Model ZK1200 from Advanced Tissue Sciences). These cultures contain noncornified stratified squamous epithelium, providing a three-dimensional in vitro model that resembles noncornified mucosal epithelium, such as cornea and conjunctiva. The hypothesis tested and confirmed in this work was that the rate of cytotoxicity induced by topical application of test substances to the stratified epithelial cell cultures would correlate with ocular irritancy. Test substances were applied to the cell system for up to 30 min, and cytotoxicity was measured as decreased 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT) vital dye metabolism. The time (in minutes) of exposure to test agent that reduced MTT metabolism to 50% of control levels (the t50 value) was calculated for each test substance, and these values showed a good correlation ( r = 0.87) with historic rabbit low-volume eye test maximum average scores (MAS) for a range of liquid, solid, granular, powder, and other test materials, including consumer product formulations and ingredient chemicals. Additionally, t50 values confirmed the mild nature of selected cosmetic formulations. Taken together, these data indicate that this in vitro test, termed the tissue equivalent assay, is a valuable tool to screen for irritancy of test substances to mucosal tissues, such as cornea and conjunctiva.

Use of Clinical and Consumer Eye Irritation Data to Evaluate the Low-Volume Eye Test

AbstractThis paper evaluates further a previous finding that the low-volume eye test (LVET) is predictive of eye irritation potential in humans caused by exposure to specific consumer products. We reviewed consumer eye irritation comments from 1985 through 1992 on over 70 marketed beauty/personal care, laundry, and household cleaning products. Twenty-four products evaluated during this time were also tested in the LVET. A comparison was made of the time needed for consumer and rabbit eye irritation to clear after exposure to one of the 24 products. In addition, the results of clinical eye studies on two prototype shampoo formulations were evaluated against parallel LVET data. The clinical data and consumer experience showed less eye irritation in humans from exposure to these products than in the rabbit tests. The consistency of the data over a 13 year period presents strong evidence that human eye irritation is less severe than predicted by the LVET and that the LVET provides an adequate margin of safety...

Development and Intralaboratory Evaluation of an in Vitro Human Cell-Based Test to Aid Ocular Irritancy Assessments

A human cell-based in vitro method was developed to screen for ocular irritancy potential of aqueous compatible and incompatible test agents, such as liquids, insoluble solids, powders, granulars, emulsions, and acids/alkalis. Methods were developed for topical application (an exposure that mimics in vivo testing) and wash-off of test substances on the epithelial surface of human skin derived epithelial-fibroblast cocultures (Skin2 Model ZK1200 from Advanced Tissue Sciences). These cultures contain noncornified stratified squamous epithelium, providing a three-dimensional in vitro model that resembles noncornilled mucosal epithelium, such as cornea and conjunctiva. The hypothesis tested and confirmed in this work was that the rate of cytotoxicity induced by topical application of test substances to the stratified epithelial cell cultures would correlate with ocular irritancy. Test substances were applied to the cell system for up to 30 min, and cytotoxicity was measured as decreased 3-[4,5-dimethylthiazol-2-yl]2,5-diphe nyltetrazolium bromide (MTT) vital dye metabolism. The time (in minutes) of exposure to test agent that reduced MTT metabolism to 50% of control levels (the t50 value) was calculated for each test substance, and these values showed a good correlation (r = 0.87) with historic rabbit low-volume eye test maximum average scores (MAS) for a range of liquid, solid, granular, powder, and other test materials, including consumer product formulations and ingredient chemicals. Additionally, t50 values confirmed the mild nature of selected cosmetic formulations. Taken together, these data indicate that this in vitro test, termed the tissue equivalent assay, is a valuable tool to screen for irritancy of test substances to mucosal tissues, such as cornea and conjunctiva.

The use of low-volume dosing in the eye irritation test

The Draize rabbit eye test was developed to provide a method for assessing the irritation potential of materials that might come in contact with human eyes. The method involves the instillation of 0.1 ml of a test liquid (100 mg solid) into the conjunctival sac of an animal's eye. A refinement of the Draize test is the low-volume eye test in which 0.01 ml of a substance is placed directly on the cornea of the eye. Studies indicate that the low-volume method provides a better correlation to human eye irritation experience for some substances. The Interagency Regulatory Alternatives Group (IRAG) proposes that the low-volume eye test can be used to substantiate the irritancy of suspect severe ocular irritants that have not been eliminated by various pre-eye test ‘screens’. A substance testing positive by the low-volume method can be classified as an irritant; one that tests negative will require further testing by the use of the 0.1-ml volume procedure. For all other definitive testing, the Draize test (0.1 ml) should be used. Results from a questionnaire distributed at the IRAG workshop showed that many workshop participants thought that the low-volume test should be used as an eye irritation screening procedure.

Reducing the number of rabbits in the low-volume eye test

Although the Draize eye irritation test has provided important and useful information for eye safety assessments, considerable effort has been directed toward refining the assay procedure, reducing the number of animals used, and replacing this assay with alternative methods. The low-volume eye test (LVET) is a refinement of the Draize eye irritation test that uses 1 10 the volume of test substance placed directly on the cornea. The level and duration of eye irritation in the LVET are less than those in the Draize procedure, which means that it is a less stressful test. Furthermore, LVETs are more predictive of human response. Statistical studies have been conducted to determine the effects of reducing the number of animals used in the Draize test. These results suggested that a three-animal test would provide essentially the same information as the six-animal test. A similar analysis has not been performed on results from the LVET. Accordingly, the present study was undertaken to evaluate previously existing LVET data to determine if the number of animals used in a LVET can be decreased as has been shown for the Draize test. The results of the analysis are consistent with the findings of earlier evaluations of classical Draize data. Three-animal subsets from 119 six-animal LVETs provided the correct classification greater than 92% of the time for three different classification schemes. Furthermore, the discrepancies between the three-animal subsets and the six-animal maximum average score tended to be smaller than those observed for the Draize test. The analysis suggests that a three-animal LVET with provide an eye irritation classification similar to that obtained from a six-animal study.

Reducing the Number of Rabbits in the Low-Volume Eye Test

Reducing the Number of Rabbits in the Low-Volume Eye Test