Phillip L. Casterton

The CFTA Evaluation of Alternatives Program : an evaluation of in vitro alternatives to the draize primary eye irritation test. (Phase III) surfactant-based formulations

The CTFA Evaluation of Alternatives Program is an evaluation of the relationship between data from the Draize primary eye irritation test and comparable data from a selection of promising in vitro eye irritation tests. In Phase III, data from the Draize test and 41 in vitro endpoints on 25 representative surfactant-based personal care formulations were compared. As in Phase I and Phase II, regression modelling of the relationship between maximum average Draize score (MAS) and in vitro endpoint was the primary approach adopted for evaluating in vitro assay performance. The degree of confidence in prediction of MAS for a given in vitro endpoint is quantified in terms of the relative widths of prediction intervals constructed about the fitted regression curve. Prediction intervals reflect not only the error attributed to the model but also the material-specific components of variation in both the Draize and the in vitro assays. Among the in vitro assays selected for regression modeling in Phase III, the relationship between MAS and in vitro score was relatively well defined. The prediction bounds on MAS were most narrow for materials at the lower or upper end of the effective irritation range (MAS = 0-45), where variability in MAS was smallest. This, the confidence with which the MAS of surfactant-based formulations is predicted is greatest when MAS approaches zero or when MAS approaches 45 (no comment is made on prediction of MAS > 45 since extrapolation beyond the range of observed data is not possible). No single in vitro endpoint was found to exhibit relative superiority with regard to prediction of MAS. Variability associated with Draize test outcome (e.g. in MAS values) must be considered in any future comparisons of in vivo and in vitro test results if the purpose is to predict in vivo response using in vitro data.

The CTFA evaluation of alternatives program: An evaluation of in vitro alternatives to the draize primary eye irritation test. (Phase II) oil/water emulsions

The Cosmetic, Toiletry and Fragrance Association (CTFA) Evaluation of Alternatives Program is an evaluation of the relationship between Draize ocular safety test data and comparable data from a selection of in vitro tests. In Phase II, 18 representative oil/water-based personal-care formulations were subjected to the Draize primary eye safety test and 30 in vitro assay protocols (14 different types of in vitro endpoints were evaluated; the remainder were protocol variations). Correlation of in vitro with in vivo data was evaluated using analysis of sensitivity/specificity and statistical analysis of the relationship between maximum average Draize score (MAS) and in vitro endpoint. Regression modelling is the primary approach adopted in the CTFA Program for evaluating in vitro assay performance. The objective of regression analysis is to predict MAS for a given test material (and to place upper and lower prediction interval bounds on the range in which the 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: the narrower the prediction interval, the more predictive of the Draize score is the in vitro test result. 16 assays were shown to have the greatest agreement with the Draize procedure and were therefore selected for regression analysis. Based on the magnitude of the 95% prediction bounds of each of the 16 selected assays over the range of test data, it may be inferred that prediction of MAS values from experimentally determined in vitro scores is more accurate for oil/water-based formulations with lower rather than higher irritancy potential. The assays selected for modelling in Phase II generally exhibited weaker relationships with MAS than those selected in Phase I (evaluated using hydroalcoholic formulations), even though several assays were common to both Phases.

A Novel Approach to Assessing Eye Irritation Potential Using the Bovine Corneal Opacity and Permeability Assay

AbstractThe bovine corneal opacity and permeability assay (BCOP) as originally described by Gautheron is now utilized by several laboratories to assess the eye irritation potential of individual chemicals, chemical blends, and consumer formulations. We have applied the principles established by Gautheron to the equipment and data available in our laboratory to devise a variation of this assay. This variation differs in three ways: how we evaluate corneal opacity, how we apply opacity and permeability measurements to predict irritation potential, and how we evaluate solid materials. For opacity measurement, a UV-VIS spectrophotometer is used to measure light absorbance. This contrasts with Gautherons use of an opacitometer to measure changes in voltage as an indicator of opacity. Permeability is measured in the same manner but we interpret the results differently by applying them to a table of results relating to degrees of irritation potential. The corneal holding apparatus has also been modified to allo...

The SDA Alternatives Program Phase III: Comparison of in Vitro Data with Animal Eye Irritation Data on Solvents, Surfactants, Oxidizing Agents, and Prototype Cleaning Products

AbstractNine in vitro candidate tests for estimating eye irritation potential were evaluated as potential replacements for the Draize test. The tests examined were a cell protein assay, the chorioallantoic membrane vascularization assay, a cell protein assay, a fibroblast cytotoxicity assay, the Living Dermal Model and Living Skin Equivalent, two neutral red assays, an SIRC cytotoxicity assay, and a Tetrahymena thermophila motility assay. The results from these in vitro tests were compared to results from a modified Draize test with 22 test materials. The test materials were selected to represent various classes of cleaning products and ingredients. Ingredients were tested at concentrations representative of concentrations typically found in cleaning products. The correlation coefficients with all test materials considered ranged from 0.58 to 0.91. When only nonalkaline materials are considered, the correlation coefficients of all 10 tests were not significantly different from one another, ranging from 0....

A redesigned corneal holder for the bovine cornea opacity and permeability assay that maintains normal corneal morphology

The bovine cornea opacity and permeability assay (BCOP) has been in use for nearly 10 years but has not been submitted for regulatory approval. In previous reports we have presented corneal hydration and endothelial damage as additional endpoints in this assay and have suggested that the design of the BCOPs corneal holder should be modified. The standard holder used in the BCOP assay induces physical damage to the cornea because it contacts clear cornea causing edge damage to the epithelial, stromal and endothelial layers. Second, by forcing a curved, oval-shaped bovine cornea into a flat, circular opening, corneal wrinkling occurs which can alter the corneas optical characteristics and, most importantly, induces endothelial damage. We now report on a redesigned BCOP corneal holder that clamps onto the sclera, maintains normal corneal shape and does not cause damage to the endothelium. This ensures that irritancy tests are conducted using healthy, anatomically normal tissue. Tests of this holder using acetone, trichloroacetic acid, isopropanol and benzalkonium chloride show that it is now possible to evaluate effects of chemical substances on the endothelium. The effects of these compounds on corneal opacity and hydration in the new holder are similar to their effects on the cornea in the standard holder.

Effect of Hydration on Opacity in the Bovine Corneal Opacity and Permeability (BCOP) Assay

AbstractThe bovine corneal opacity and permeability (BCOP) assay has been proposed an an in vitro method for predicting the irritancy or toxicity of chemical substances. A loss of corneal transparency, which reduces visual acuity, can be the result of an increase in corneal hydration or direct damage to corneal tissues. The BCOP assay as currently practiced does not differentiate between these mechanisms. The purpose of this study was to determine the effect of increased hydration on transparency of the bovine cornea and to measure the hydration levels of corneas treated with compounds and substances known, from previous BCOP studies, to cause corneal opacity. Corneas were treated according to the BCOP protocol and corneal opacity was determined by measurement of light absorbance at 570 nm (A570). Corneal hydration was determined by measurement of wet and dry weights. Hydration of the intact cornea was 3.86 mg H2O/mg dry weight and A570 = 0.048. After removal of the epithelium and incubation in MEM, corne...

A dietary two-generation reproductive toxicity study of (2R,4R)-monatin salt in Crl:CD(SD) rats.

(2R,4R)-Monatin salt [sodium/potassium 2R,4R-2-amino-4-carboxy-4-hydroxy-5-(3-indolyl) pentanoate] was fed at 5000, 15,000, or 35,000 ppm to Crl:CD(SD) rats over two generations. Reduced body weights were observed at all dose levels. Sustained effect on body weight gain at 35,000 ppm in the F0 and F1 parental animals was associated with lower feed efficiency, soft stool, and slightly lower numbers of implantation sites. Lower numbers of pups born and live litter size at 35,000 ppm were considered secondary to slightly lower numbers of former implantation sites in the dams. Spermatogenic endpoints, estrous cyclicity, reproductive performance, mean gestation length, and parturition were unaffected in the F0 and F1 generations. There were no effects on F1 and F2 generation postnatal survival. Reduced pre-weaning pup body weights at 35,000 ppm resulted in lower F1 and F2 body weights at study termination. Slight delays in pubertal landmarks in the F1 offspring were considered secondary to the reduced pup body weights. The no-observed-adverse-effect level (NOAEL) was 15,000 ppm for systemic, reproductive, and neonatal effects based on test article-related effects on body weight and food efficiency, slight decrease in maternal implantation sites and corresponding reduction in live litter size, and reductions in pre-weaning pup body weights at 35,000 ppm.

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...