Draize Rabbit Eye Irritation Test Research Articles

The EpiOcular™ Eye Irritation Test is the Method of Choice for the In Vitro Eye Irritation Testing of Agrochemical Formulations: Correlation Analysis of EpiOcular Eye Irritation Test and BCOP Test Data According to the UN GHS, US EPA and Brazil ANVISA Classification Schemes.

The Bovine Corneal Opacity and Permeability (BCOP) test is commonly used for the identification of severe ocular irritants (GHS Category 1), but it is not recommended for the identification of ocular irritants (GHS Category 2). The incorporation of human reconstructed tissue model-based tests into a tiered test strategy to identify ocular non-irritants and replace the Draize rabbit eye irritation test has been suggested (OECD TG 405). The value of the EpiOcular™ Eye Irritation Test (EIT) for the prediction of ocular non-irritants (GHS No Category) has been demonstrated, and an OECD Test Guideline (TG) was drafted in 2014. The purpose of this study was to evaluate whether the BCOP test, in conjunction with corneal histopathology (as suggested for the evaluation of the depth of the injury( and/or the EpiOcular-EIT, could be used to predict the eye irritation potential of agrochemical formulations according to the UN GHS, US EPA and Brazil ANVISA classification schemes. We have assessed opacity, permeability and histopathology in the BCOP assay, and relative tissue viability in the EpiOcular-EIT, for 97 agrochemical formulations with available in vivo eye irritation data. By using the OECD TG 437 protocol for liquids, the BCOP test did not result in sufficient correct predictions of severe ocular irritants for any of the three classification schemes. The lack of sensitivity could be improved somewhat by the inclusion of corneal histopathology, but the relative viability in the EpiOcular-EIT clearly outperformed the BCOP test for all three classification schemes. The predictive capacity of the EpiOcular-EIT for ocular non-irritants (UN GHS No Category) for the 97 agrochemical formulations tested (91% sensitivity, 72% specificity and 82% accuracy for UN GHS classification) was comparable to that obtained in the formal validation exercise underlying the OECD draft TG. We therefore conclude that the EpiOcular-EIT is currently the best in vitro method for the prediction of the eye irritation potential of liquid agrochemical formulations.

Identification of cornifelin and early growth response-1 gene as novel biomarkers for in vitro eye irritation using a 3D reconstructed human cornea model MCTT HCE™.

Evaluation of the eye irritation is essential in the development of new cosmetic products. Draize rabbit eye irritation test has been widely used in which chemicals are directly applied to rabbit eye, and the symptoms and signs of eyes are scored. However, due to the invasive procedure, it causes substantial pain and discomfort to animals. Recently, we reported in vitro eye irritation test method using a 3D human corneal epithelial model (MCTT HCE™) which is reconstructed from remaining human tissues after a corneal transplantation. This model exhibited an excellent predictive capacity for 25 reference chemicals (sensitivity 100%, specificity 77% and accuracy 88% vs. GHS). To improve the test performance, we explored new biomarkers for the eye irritation through transcriptomic approach. Three surfactants were selected as model eye irritants that include sodium lauryl sulfate, benzalkonium chloride and triton X-100. After test chemicals were treated, we investigated differentially expressed genes through a whole-gene microarray (Affymetrix GeneChip(®) Human Gene 2.0 ST Array, 48,000 probes). As a result, we identified that mRNAs of cornifelin (CNFN), a constituent of the insoluble cornified cell envelope of stratified squamous epithelia, and early growth response-1 (EGR1), a nuclear transcriptional regulator, were significantly up-regulated by all three irritants. Up-regulation of CNFN and EGR1 was further confirmed by Q-RT-PCR, and immunohistochemistry revealed increased level of CNFN in irritant-treated tissues, supporting the relevance of CNFN and EGR1 as new biomarkers for eye irritation.

Improvement of the Bovine Corneal Opacity and Permeability (BCOP) assay as an in vitro alternative to the Draize rabbit eye irritation test

Measurement of ocular irritancy is a necessary step in the safety evaluation of both industrial and consumer products. Assessment of the acute eye irritation potential is therefore part of the international regulatory requirements for testing of chemicals.The Bovine Corneal Opacity and Permeability (BCOP) assay is generally accepted as a valid in vitro alternative method to the Draize eye irritation test to detect corrosive and severe eye irritants (category 1), but has not proven sensitive enough to discriminate accurately moderate (category 2A/2B) to mild and non-irritating compounds. In the currently accepted BCOP assay, opacity is determined by the amount of light transmission through the cornea, and permeability is determined by the amount of sodium fluorescein dye that passes through all corneal cell layers. Both measurements are used to assign an In Vitro Irritancy Score (IVIS) for prediction of the in vivo ocular irritation potential of a test substance. Nowadays, opacity is measured by an OP-KIT opacitometer providing a center-weighted reading of light transmission by measuring changes in voltage when the transmission of white light passes through the cornea alters. As a consequence, this may underestimate opacity that develops as spots or heterogeneous opaque areas on the periphery of an isolated cornea.A prototype of a laser light-based opacitometer (PLLBO) allowing better measurement of opacities was developed by Van Goethem et al. (2010). This new device showed improved sensitivity to detect subtle changes in corneal transparency. Furthermore, the new opacitometer allowed the analysis of the complete corneal surface and was able to detect more efficiently opaque spots located along the sides of the excised corneas.A further improved prototype of the PLLBO was constructed in combination with a camera and a speckle noise reducer. Treatment conditions of the corneas in the cornea holders were optimized in order to mimic more the real in vivo situation. A set of test compounds with irritancy potencies especially in the mild and moderate range was tested. The improved LLBO showed some promising features which potentially could improve the usefulness of the BCOP test. Adaptation of cornea holders showed to be of limited value and only restricted to concentrations up to 15% which mimics more test conditions in industry.This 3-year research project was sponsored by the Stavros Niarchos Foundation (Greece).

In-house Validation of the EpiOcular™ Eye Irritation Test and its Combination with the Bovine Corneal Opacity and Permeability Test for the Assessment of Ocular Irritation

In 2009, the Bovine Corneal Opacity and Permeability (BCOP) test was accepted by the regulatory bodies for the identification of corrosive and severe ocular irritants (Global Harmonised System [GHS] Category 1). However, no in vitro test is currently accepted for the differentiation of ocular irritants (GHS Category 2) and non-irritants (GHS No Category). Human reconstructed tissue models have been suggested for incorporation into a tiered testing strategy to ultimately replace the Draize rabbit eye irritation test (OECD TG 405). The purpose of this study was to evaluate whether the EpiOcular(TM) reconstructed cornea-like tissue model and the COLIPA pre-validated EpiOcular Eye Irritation Test (EpiOcular-EIT) could be used as suitable components of this testing strategy. The in-house validation of the EpiOcular-EIT was performed by using 60 test substances, including a broad variety of chemicals and formulations for which in vivo data (from the Draize rabbit eye irritation test) were available. The test substances fell into the following categories: 18 severe irritants/corrosives (Category 1), 21 irritants (Category 2), and 21 non-irritants (No Category). Test substances that decreased tissue viability to ≤ 60% (compared to the negative control tissue) were considered to be eye irritants (Category 1/2). Test substances resulting in tissue viability of > 60% were considered to be non-irritants (No Category). For the assessed dataset and the classification cut-off of 60% viability, the EpiOcular-EIT provided 98% and 84% sensitivity, 64% and 90% specificity, and 85% and 86% overall accuracy for the literature reference and BASF proprietary substances, respectively. Applying a 50% tissue viability cut-off to distinguish between irritants and non-irritants resulted in 93% and 82% sensitivity, 68% and 100% specificity, and 84% and 88% accuracy for the literature reference and BASF proprietary substances, respectively. Further, in the EpiOcular-EIT (60% cut-off), 100% of severely irritating substances under-predicted by the BCOP assay were classified as Category 1/2. The results obtained in this study, based on 60 test substances, indicate that the EpiOcular-EIT and the BCOP assay can be combined in a testing strategy to identify strong/severe eye irritants (Category 1), moderate and mild eye irritants (Category 2), and non-irritants (No Category) in routine testing. In particular, when the bottom-up strategy with the 60% viability cut-off was employed, none of the severely irritating substances (Category 1) were under-predicted to be non-irritant. Sensitivity for Category 1/2 substances was 100% for literature reference substances and 89% for BASF SE proprietary substances.

Formulation of Indomethacin Eye Drops via Complexation with Cyclodextrins

Purpose: Topically administered indomethacin is used in the management and prevention of ocular inflammation and cystoid macular edema related to cataract surgery and in the maintenance of mydriasis during cataract surgery. Pharmaceutically, the main obstacle in formulating indomethacin as eye drops is its low solubility and aqueous instability. Inclusion complexation of indomethacin with cyclodextrins (CDs) was our interest in this research to obtain stable and effective aqueous indomethacin eye drops.Materials and Methods: The influence of β-CD, hydroxypropyl-β-CD (HP-β-CD), and sulfobutyl ether-β-CD (SBE-β-CD) on indomethacin solubility was investigated. Indomethacin-HP-β-CD complex was prepared and characterized by infrared spectroscopy, differential scanning calorimetry, and X-ray diffractometry and was subjected to in vitro release and stability studies. The anti-inflammatory effect of formulated indomethacin-HP-β-CD eye drops on chemically burned albino rabbit eyes was compared to those of Indocollyre® and Voltaren® eye drops.Results: HP-β-CD was found to have a high solubilizing effect towards indomethacin and was thus selected for its formulation. Formulated indomethacin-HP-β-CD eye drops exhibited delayed release and high drug stability compared to the drug solution. Draize rabbit eye irritation test and histological examination on albino rabbit eyes treated with indomethacin-HP-β-CD eye drops revealed that these eye drops were non-irritant. The anti-inflammatory studies indicated that formulating indomethacin eye drops via complexation with HP-β-CD significantly improves the therapeutic efficacy of the indomethacin compared to the investigated eye drops.Conclusion: An aqueous ocular delivery system for the poor water soluble anti-inflammatory indomethacin was prepared using HP-β-CD as a complexing agent. Besides a sufficient solubility for the drug, many factors were studied in the development of this system, such as stability and safety. In addition, indomethacin-HP-β-CD eye drops showed promising management to corneal inflammation.

Nanoemulsion as a Potential Ophthalmic Delivery System for Dorzolamide Hydrochloride

Dilutable nanoemulsions are potent drug delivery vehicles for ophthalmic use due to their numerous advantages as sustained effect and high ability of drug penetration into the deeper layers of the ocular structure and the aqueous humor. The aim of this article was to formulate the antiglaucoma drug dorzolamide hydrochloride as ocular nanoemulsion of high therapeutic efficacy and prolonged effect. Thirty-six systems consisting of different oils, surfactants, and cosurfactants were prepared and their pseudoternary-phase diagrams were constructed by water titration method. Seventeen dorzolamide hydrochloride nanoemulsions were prepared and evaluated for their physicochemical and drug release properties. These nanoemulsions showed acceptable physicochemical properties and exhibited slow drug release. Draize rabbit eye irritation test and histological examination were carried out for those preparations exhibiting superior properties and revealed that they were nonirritant. Biological evaluation of dorzolamide hydrochloride nanoemulsions on normotensive albino rabbits indicated that these products had higher therapeutic efficacy, faster onset of action, and prolonged effect relative to either drug solution or the market product. Formulation of dorzolamide hydrochloride in a nanoemulsion form offers, thus, a more intensive treatment of glaucoma, a decrease in the number of applications per day, and a better patient compliance compared to conventional eye drops.

The Immunohistochemical Characterisation of an SV40-immortalised Human Corneal Epithelial Cell Line

Alternatives to the Draize rabbit eye irritation test are currently being investigated. Because of morphological and biochemical differences between the rabbit and the human eye, continuous human cell lines have been proposed for use in ocular toxicology studies. Single cell-type monolayer cultures in culture medium have been used extensively in ocular toxicology. In the present study, an SV40-immortalised human corneal epithelial (HCE) cell line was characterised immunohistochemically, by using 13 different monoclonal antibodies to cytokeratins (CKs), ranging from CK3 to CK20. The results from the monolayer HCE cell cultures were compared with those from the corneal epithelium of human corneal cryostat sections. Previous studies have shown that the morphology of the HCE cell is similar to that of primary cultured human corneal epithelial cells, and that the cells express the cornea-specific CK3. In the study reported here, we show that the cell line also expresses CKs 7, 8, 18 and 19. These CKs are typically expressed by simple epithelial cells, and are not found in the human cornea in vivo. Therefore, the monolayer HCE cell line grown in culture medium does not express the CK pattern that is typical of human corneal epithelium. This should be taken into consideration when using HCE cell cultures in similar single cell-type experiments for ocular toxicology.

Using In Vitro Prediction Models Instead of the Rabbit Eye Irritation Test to Classify and Label New Chemicals: A Post Hoc Data Analysis of the International EC/HO Validation Study

The international validation study on alternative methods to replace the Draize rabbit eye irritation test, funded by the European Commission (EC) and the British Home Office (HO), took place during 1992-1994, and the results were published in 1995. The results of this EC/HO study are analysed by employing discriminant analysis, taking into account the classification of the in vivo data into eye irritation classes A (risk of serious damage to eyes), B (irritating to eyes) and NI (non-irritant). A data set for 59 test items was analysed, together with three subsets: surfactants, water-soluble chemicals, and water-insoluble chemicals. The new statistical methods of feature selection and estimation of the discriminant functions classification error were used. Normal distributed random numbers were added to the mean values of each in vitro endpoint, depending on the observed standard deviations. Thereafter, the reclassification error of the random observations was estimated by applying the fixed function of the mean values. Moreover, the leaving-one-out cross-classification method was applied to this random data set. Subsequently, random data were generated r times (for example, r = 1000) for a feature combination. Eighteen features were investigated in nine in vitro test systems to predict the effects of a chemical in the rabbit eye. 72.5% of the chemicals in the undivided sample were correctly classified when applying the in vitro endpoints lgNRU of the neutral red uptake test and lgBCOPo5 of the bovine opacity and permeability test. The accuracy increased to 80.9% when six in vitro features were used, and the sample was subdivided. The subset of surfactants was correctly classified in more than 90% of cases, which is an excellent performance.

Using In Vitro Prediction Models Instead of the Rabbit Eye Irritation Test to Classify and Label New Chemicals: A Post Hoc Data Analysis of the International EC/HO Validation Study

The international validation study on alternative methods to replace the Draize rabbit eye irritation test, funded by the European Commission (EC) and the British Home Office (HO), took place during 1992–1994, and the results were published in 1995. The results of this EC/HO study are analysed by employing discriminant analysis, taking into account the classification of the in vivo data into eye irritation classes A (risk of serious damage to eyes), B (irritating to eyes) and NI (non-irritant). A data set for 59 test items was analysed, together with three subsets: surfactants, water-soluble chemicals, and water-insoluble chemicals. The new statistical methods of feature selection and estimation of the discriminant function's classification error were used. Normal distributed random numbers were added to the mean values of each in vitro endpoint, depending on the observed standard deviations. Thereafter, the reclassification error of the random observations was estimated by applying the fixed function of the mean values. Moreover, the leaving-one-out cross-classification method was applied to this random data set. Subsequently, random data were generated r times (for example, r = 1000) for a feature combination. Eighteen features were investigated in nine in vitro test systems to predict the effects of a chemical in the rabbit eye. 72.5% of the chemicals in the undivided sample were correctly classified when applying the in vitro endpoints lgNRU of the neutral red uptake test and lgBCOPo5 of the bovine opacity and permeability test. The accuracy increased to 80.9% when six in vitro features were used, and the sample was subdivided. The subset of surfactants was correctly classified in more than 90% of cases, which is an excellent performance.

A compartment model to calculate time-dependent concentration profiles of topically applied chemical compounds in the anterior compartments of the rabbit eye.

Hitherto, none of the existing in vitro methods has been convincingly demonstrated to be suitable as a replacement for the Draize rabbit eye irritation test. We examine the hypothesis that one reason for this is that insufficient consideration has been given to the differences in the effective concentrations at which chemicals operate in vitro and in vivo. When a chemical is applied topically to the eye, the strength of the observed irritation that it elicits depends both on its toxic potential toward cells or tissues, and its effective concentration in the tissues of the eye. Most of the existing in vitro methods are based on isolated cells or tissues, and thus may be useful in assessing the cytotoxic potentials of chemicals. However, a reliable approach to assessing the effective concentrations of chemicals within the various tissues of the eye is lacking. A simplified compartment model is presented for calculating the time-dependent, intra-ocular concentration profiles of topically applied chemicals. The model encompasses the outer surface of the eye, three distinct segments of the cornea (subdivided into the epithelium, stroma and endothelium) and the conjunctiva. Transport through the membranes of these compartments is described as passive diffusion. For the transport coefficients, rate equations are established that contain, as free parameters, the molecular size and the partition coefficient of the chemical, as well as some intrinsic membrane parameters, such as thickness, viscosity and pore density. Numerical values for the unknown membrane parameters were estimated by fitting the theoretical rate equations to measured permeability coefficients. The compartment model was applied to an independent set of 52 test chemicals compiled from the European Commission/UK Home Office validation study. The calculated passage times (required to let 95% of the chemical reach the posterior eye tissues) varied between 0.33 minutes and 50.6 minutes, and are generally much shorter than the typical duration of observed impairments in the cornea or conjunctiva. This finding suggests that short-term contacts of the eye tissues with a chemical are sufficient to elicit long-term eye irritation. An example is given, showing how the conventional approach of using in vitro endpoints as predictors of eye irritation can be improved significantly by incorporating into the prediction the calculated intra-ocular concentration of a chemical.

The Performance of the Tissue Equivalent Assay using the Skin 2TM ZK1200 Model in the COLIPA International Validation Study on Alternatives to the Draize Eye Irritation Test

The tissue equivalent assay (TEA) ( Osborne et al., 1995) was used to evaluate 55 mixed ingredients and formulations in the COLIPA International Validation Study on Alternatives to the Draize Rabbit Eye Irritation Test ( Brantom et al., 1997). The TEA can be used to test all types of materials since it uses a topical application approach and is not limited to only testing liquid or soluble materials. A prediction model (PM) for the test was developed using historical eye irritation data from a total of 132 materials on which in vivo and in vitro data were available. A regression model was derived from these data and used to relate the in vitro endpoint (t 50) obtained in the study to a Draize MMAS (modified maximum average score). This provided a measure of the predicted in vivo eye irritation scores. In the current study, two separate laboratories used the same protocol to test the same set of coded materials and the results of both laboratories were compared to the initial PM. The TEA met the reliability criteria of the validation study in reproducing the predefined PM in both laboratories, and a good relationship between predicted and observed Draize MMAS values was obtained (r=0.906 and r=0.850). Good correlations were maintained when separate analyses were made of the formulations and ingredients included in the test set. Good relationships between the in vitro endpoint and individual Draize tissue scores (r>0.8) were also exhibited. Although insufficient data were available to make an assessment of interlaboratory variation, some difference in the reproducibility of the assay was noted between the two laboratories, particularly for the highly irritating materials. However, the consistency of data was encouraging and the discrepancies seen between the laboratories suggested a sensitivity of the model to subtle differences in application techniques, and in handling and timing. Taken together, these results indicate the utility of the TEA test for these types of substances and the need to more fully address the issue of interlaboratory reproducibility.

Interlaboratory validation of the in vitro eye irritation tests for cosmetic ingredients. (3) Evaluation of the haemolysis test

The haemolysis test using sheep red blood cells (RBC) was evaluated as an alternative method to the Draize rabbit eye irritation test (Draize test) by six to nine laboratories. The participating laboratories performed the test according to the standard operating procedure (SOP). Thirty-eight cosmetic ingredients and isotonic sodium chloride solution were used as test substances in this validation study. The concentrations of the test substances that induced 50% haemolysis (HC 50 value) was obtained to serve as a toxicological index and compared with in vivo Draize scores. HC 50 values were not obtained for coloured or water-insoluble (turbid) substances. Three acids caused denaturation of haemoglobin leaked from RBC and consequently interfered with the determination of the HC 50 value. Interlaboratory reproducibility was relatively good except in the case of water-insoluble substances. The average values of coefficient of variation (CV) was 37%. The correlation coefficient and Spearman's rank correlation between the HC 50 value and maximum average Draize total score (MAS) were −0.631 and 0.641, respectively. The equivalence ratio between the haemolysis test and MAS was 70.0% when MAS 15 was set as the in vivo cut-off point. On the other hand, strong irritants (MAS⩾50) could be correctly classified by this method. These results suggest that the haemolysis test might be applied to cosmetic ingredients as a screening method to distinguish strong irritants that directly affect the cell membrane permeability and do not disturb spectrophotometrical determination of haemoglobin. In order to evaluate the potential for eye irritation of cosmetic ingredients, a combination of haemolysis with other methods based on different mechanism should be employed to improve the predictability.

Interlaboratory validation of the In Vitro eye irritation tests for cosmetic ingredients (7) evaluation of cytotoxicity test by CornePack ®

The cytotoxicity test of neutral red (NR) uptake in normal rabbit corneal epithelial cells (CornePack ®) was validated as an alternative method to the Draize rabbit eye irritation test (Draize test). We tested 38 cosmetic ingredients as well as isotonic sodium chloride solution in three phases of the validation study. The test procedures were controlled among participating laboratories under a common standard operating procedure (SOP). The concentration of test substances that showed a 50% reduction in NR uptake relative compared with controls (median NR uptake concentration: NR 50, μg/ml) was determined and compared with in vivo Draize scores. Six laboratories participated in the first phase of the validation study, seven in the second, and five in the third. The average interlaboratory coefficient of variation (CV) was 32.9%. The correlation and rank correlation coefficients between the maximal average Draize total scores (MAS) and NR 50 were −0.583 and 0.587, respectively. When the anionic detergents were excluded from analysis, the correlation coefficient increased to −0.738. When the cut-off point for positive and negative irritation was set at MAS of 15 and the predictability of this method was assessed by liner regression line, six substances (two acids, two alkanolamines and two alcohols) were false negative. Through this project, it appeared that CornePack, supplied in kit form with frozen secondary cultured cell in serum-free medium, could provide an effective, highly sensitive and simple preliminary screen for determining the cytotoxicity of substances. These results suggested that CornePack might have the potential to predict the MAS if definite criteria can be established for the compounds to be applicable. However, it is important to understand the nature of CornePack responses since its NR 50 profile was quite different from other cytotoxicity assays.

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.

Development of a 10-day chorioallantoic membrane vascular assay as an alternative to the draize rabbit eye irritation test

The chorioallantoic membrane (CAM) of a fertilized hen's egg has been studied extensively as a promising alternative model for predicting eye irritation potential. The specific methodology used with this model has varied among investigators but the basic premise of applying test material to the membrane surface and evaluating changes in the vasculature is relatively consistent. The CAM vascular assay (CAMVA) has shown high correlation with in vivo rabbit eye irritation data. This method uses the CAM of a 14-day-old egg and the response at 30 min after treatment as the endpoint. The primary CAM methods being evaluated in Europe use 9–10-day-old eggs because older eggs are considered ‘live animals’; the possibility of using 10-day-old eggs to make the method more globally acceptable as a non-animal test was therefore investigated. By keeping the original CAMVA dosing and evaluation procedures the same, and only altering the age of the eggs from 14-day to 10-day, the results were found to be nearly identical for the two methods and both produce equivalent correlations to the in vivo eye irritation test results. Maintaining the original CAMVA methodology but using a younger egg, therefore, provides a good alternative method for predicting eye irritation potential that is more globally acceptable as a non-animal test.

Evaluation of the vascular components of the chorioallantoic membrane assay as a model for eye irritation potential: II

AbstractPrevious research has shown that the chorioallantoic membrane (CAM) of a fertile hen's egg can be a useful model for predicting eye irritation potential. However, the specific test procedure utilized will affect the degree of correlation between the CAM response and the results from the standard Draize rabbit eye irritation test. We studied how various components of a standard CAM assay, such as age of CAM and the endpoint evaluated, have an impact on the relationship of the CAM response to the in vivo eye response. These studies suggested the basis for a new CAM assay procedure that improves the correlation with the in vivo test. Herein is described an assay method that involves treating a 14-day-old CAM and evaluating the vascular changes that occur 30 min following treatment. This method is referred to as the CAM vascular assay (CAMVA). The CAMVA is shown to produce fewer false-positive responses than previously described CAM procedures and is especially effective at differentiating irritants f...

Toxicity of Detergent-based Commercial Products on Cells of a Mouse Line in Suspension Culture as a Possible Screen for the Draize Rabbit Eye Irritation Test

Summary The Draize rabbit eye irritation test has several disadvantages and inadequacies when used as an indicator for potential irritancy of detergent-based commercial products. As a possible in vitro screen, it was decided to use mouse LS cells cultured in suspension, taking 50% cell death (CD50) after exposure to the product for 4h as the endpoint. This figure for 11 formulations was compared with eye irritation data ranked using an arbitrary classification of mild, moderate and severe. All samples with a CD50 less than 0.5mg/ml were severe eye irritants, while most of those with a CD50 greater than 1.0mg/ml were mild irritants. Samples between these two cytotoxicity levels were, in general, moderately irritant to the rabbit eye. It would appear that this in vitro test is a possible screen for the irritancy of detergent-based products.

Rabbit Eye Irritation Test: Collaborative Study

Abstract The well known Draize rabbit eye irritation test was evaluated. Ten laboratories (3 government, 3 industrial, and 4 commercial) participated and 7 liquid materials were tested. Tests were conducted 2 at a time, using 1 of 6 different materials with the same control substance (70% isopropyl alcohol), for a total of 12 tests. Test materials were selected with stringency, by employing compounds which were difficult to evaluate as they were neither strongly irritant nor innocuous. By the Draize procedure, 5 of these materials were considered irritants, and 2 nonirritants. Results showed that if all 4 criteria for eye irritation (changes in cornea and iris and conjuctival redness and chemosis) were employed, collaborators were quite consistent in separating an irritant from a nonirritant substance. The method has been adopted as official first action.