A dietary embryo/fetal developmental toxicity study of arruva, an R,R-monatin salt isomer, in Crl:CD(SD) rats.

R,R-Monatin is an intensely sweet substance originally identified in the root bark of Sclerochiton ilicifolius. R,R-Monatin salt, commonly known as "arruva", has potential for use as a high-potency sweetener food ingredient. Previously, arruva was concluded to present no toxicologically relevant effects to Crl:CD(SD) rats and Crl:CD-1(ICR) mice fed up to 35,000 ppm arruva in the diet for 90 days. In the present study, groups of mated Sprague-Dawley rats (25 Crl:CD(SD) females/group) were exposed continuously to 0 (control), 15,000, 30,000, or 50,000 ppm arruva in the diet during gestation days 6-21. There were no fetal malformations or developmental variations that were attributable to arruva at any exposure level, nor were there any test article-related effects on intrauterine survival. Maternal toxicity, evidenced by lower mean body weights, body weight gains and feed efficiency, was observed at 50,000 ppm. A developmental effect, in the form of lower mean fetal body weight, was noted in the 50,000 ppm group in the presence of maternal toxicity. Therefore, the dietary no-observed-adverse-effect level (NOAEL) for maternal and embryo/fetal developmental toxicity of arruva in pregnant rats during gestation days 6-21 was 30,000 ppm (equivalent to 2564 mg/kg bw/day) based on reductions in maternal and fetal body weights.

90-Day feeding and genotoxicity studies on a refined arachidonic acid-rich oil.

The safety of a refined arachidonic acid-rich oil (RAO) was evaluated for reverse mutation, chromosome aberration and gene mutation, and in a 90-day Wistar rat feeding study with in utero exposure. The results of the genotoxicity assays were all negative. The in utero phase of the 90-day study involved dietary exposure to 0.5%, 1.5% and 5% RAO and two controls diets, a standard feed low-fat diet and a high-fat diet supplemented with 5% corn oil. This exposure covered four-weeks prior to mating, through mating, gestation and lactation until offspring (F(1)) weaning. A subsequent 90-day feeding study in the F(1) rats evaluated the same test and control diets. Statistically significant effects were seen for selected histopathology, clinical chemistry and organ weight endpoints; however, other than increased absolute and relative monocytes seen in both sexes of high-dose rats, the observations were not attributed to treatment for one or more reasons. Based on these findings, no adverse treatment-related effects for RAO were seen at up to 5% in the diet, equivalent to an overall average RAO intake of 3170 mg/kg bwt/day. These and similar findings for other refined ARA-rich oils establish a strong body of evidence for the safety of this RAO.

Corneal permeability in a redesigned corneal holder for the bovine cornea opacity and permeability assay.

The bovine cornea opacity and permeability assay (BCOP) is a proposed alternative to the Draize rabbit test for potential eye irritants. In the standard BCOP, bovine corneas are mounted in a holder on a flat surface between two identical chambers. The flat configuration of the standard holder does not conform to the normal curved shape of the bovine cornea and it comes into direct contact with the cornea tissue. Mounting corneas in this holder causes extensive damage to both epithelial and endothelial corneal cell layers. Our laboratory has designed a new holder that allows the cornea to maintain its natural curvature and does not damage the cornea. Previous tests, using both the new and standard holders, and comparing corneal opacity, hydration and endothelial morphology, have shown that the new holder is a significant improvement over the standard holder. The present study extends the comparisons of the new and standard holders to measurement of corneal fluorescein permeability. The permeability (ng/cm(2)/min) of intact corneas, corneas with no epithelium, and corneas treated with 1% NaOH, isopropanol, acetone, 30% trichloroacetic acid or 30% sodium dodecysulfate for either 1 or 10 min was determined by measuring fluorescence of samples taken from the endothelial chamber after 90 min epithelial exposure to 0.04% sodium fluorescein. In all trials, the redesigned holders yielded not only lower permeability measurements but also decreased measurement variability. The data provide further evidence that the new holder is an improvement over the standard holder and should be incorporated into a new protocol for the BCOP.

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 BCOP's 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 cornea's 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.

Corneal opacity, hydration and endothelial morphology in the bovine cornea opacity and permeability assay using reduced treatment times.

The purpose of this study was to determine whether the standard bovine cornea opacity and permeability (BCOP) assay exposure time of 10 minutes overestimates the ocular irritancy of chemical substances. Corneas were subjected to BCOP protocol following 30-second and 1-minute exposures to irritants. Corneal opacity and hydration (mg H(2)O/mg cornea) were then measured and compared to data obtained after 10 minute irritant treatments. For most test substances corneal opacity and hydration were lower following reduced exposure times. It is suggested that using shorter exposure times in BCOP protocol may be more predictive of human response to ocular irritants, since irritants are usually in brief contact with the ocular surface during accidental exposure. A second purpose of this study was to examine effects of irritants on the corneal endothelium. Corneas were treated according to BCOP protocol following exposure to irritants for 1 or 10 minutes. The endothelium was stained with Alizarin Red and trypan blue, and examined using light microscopy. Severe irritants, such as NaOH and trichloroacetic acid, cause endothelial cell death. It was also determined that simply mounting the cornea in the BCOP assay holders caused damage to 20% of the endothelial cells. Because the endothelium is essential for normal corneal transparency and hydration, it is suggested that examination of the endothelium be added to the BCOP assay and that optimization of the assay will require modification of the cornea holders.

The CTFA 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.

Use of in vitro methods to rank surfactants for irritation potential in support of new product development.

11 surfactant raw materials with potential applications in light-duty liquid cleaning products were evaluated in vitro using a human skin analogue (ATS SKIN(2) Model ZK1100) for predicting cytotoxicity (MTT reduction) and inflammation [prostaglandin E(2) (PGE(2)) release]. Two of the 11 raw materials, both in the same compound family, were selected to be individually combined with each of the other nine in a 90:10 (raw:selected raw) mixture. Selection criteria were based on desired performance characteristics and low irritation potential as suggested from the individual surfactant assay data. To determine whether irritation potential was mitigated, MTT and PGE(2) scores were again determined for each of the 18 combinations with the resulting data being compared with the untreated raw material data. A plot of the data indicated that one of two selected materials may have an 'anti-irritant' effect. For raw materials with intrinsic MTT scores of less than 50 mug/ml and with the original data corrected for possible dilution effects, a statistical comparison between individual raw materials and the two sets of combinations was done using a one-sample analysis. Both cytotoxicity (MTT) and inflammation (PGE(2)) were significantly decreased by the milder of the two selected raw materials. By factoring the data into future new product decisions, this methodology has become a useful and practical tool for Amway product development.