M. Cottin

Interlaboratory assessment of the bovine corneal opacity and permeability (BCOP) assay

A multinational interlaboratory study to investigate the bovine corneal opacity and permeability (BCOP) assay is presented. The aim of this work was to determine the capability and possible limitations of this method to predict ocular irritancy of a large set of chemicals. The assays were carried out in 12 European laboratories with different types of activity. In each of these laboratories 52 substances, with a wide range of structure, physical form and irritant properties, were tested and in vitro scores were compared with those obtained from concurrent rabbit eye (Draize) tests. The technique was easily learned by workers in the participating laboratories, as shown by the fact that there were consistent responses between treated corneas within an individual laboratory. Interlaboratory variability was also very good. It was found that a given laboratory had a 96% chance of classifying irritants or non-irritants similarly to the other laboratories. In addition, it was observed that corneas preserved overnight responded similarly to freshly prepared tissues, thus allowing flexibility for those laboratories where the availability of corneas is limited. Comparisons between in vivo and in vitro data showed that the BCOP data correctly predicted whether a compound would be irritating or non-irritating for 44 of the 52 compounds (84.6%). Specificity and sensitivity were also greater than 84%, and the same number of substances were overestimated as were underestimated (four out of 52). All of the false negatives were solids whereas most of false positives were liquids, indicating that some adjustment in the protocol may be required depending on the physical state of the substance to be tested. All of the substances selected could be evaluated, with no limitation such as colour, insolubility, low or high pH. Given the number of products evaluated and the reproducibility within and among the laboratories involved, the overall results are quite satisfactory and therefore confirm the usefulness of the assay for screening chemicals for ocular irritation.

Experimental elicitation of contact allergy from a diazolidinyl urea‐preserved cream in relation to anatomical region, exposure time and concentration

The elicitation potential of the cosmetic preservative diazolidinyl urea was studied in formaldehyde‐ and diazolidinyl urea‐sensitized volunteer patients using a stepwise controlled exposure design. The test product was a facial moisturizer, preserved with varying concentrations of diazolidinyl urea, ranging from 0.05% to 0.6%. A repeated open application‐like exposure test was performed on volunteers and a control group with the test product containing increasing preservative concentrations, on arm, neck and face, sequentially, for 2 weeks or until dermatitis developed. The preservative action in the cream at different test concentrations was tested in microbial challenge tests and was found effective at all concentrations tested. The study established a non‐eliciting concentration of diazolidinyl urea of 0.05% in formaldehyde‐sensitive patients and showed that the skin reactivity depends on the anatomical region, increasing from the upper arm to neck and, possibly, to the face. The study design, beginning on the upper arm and moving on to the neck and face seems to be relevant for the study of reactions to cosmetic products. A clear dose–response relationship was seen regarding preservative concentration in the product.

The silicon microphysiometer for testing ocular toxicity in vitro

The silicon microphysiometer has been used for in vitro evaluation of the ocular irritancy potential of water soluble ingredients and formulations. This light-addressable potentiometric sensor detects changes in cell physiology by monitoring the rate at which cultured cells excrete their acidic products of metabolism. We have mainly determined the metabolic effect of 53 products (21 surfactants and 32 surfactant-based formulations). The related maximal average Draize score (MAS) were available from historical data and varied from 1.7 to 54. All of the Draize categories were represented. Murine fibroblastic cells (L929 clone) were exposed to increasing concentrations of the product for approximately 400 sec per dose. The MRD(50) (dose of product that decreased the metabolic rate of the cells by 50%) was determined by interpolation from a plot of metabolic rate versus test material concentration. Decreases in metabolic rate, as assessed by the MRD(50), occurred over a wide range of concentrations (40 mug/ml-200 mg/ml). The linear (Pearson) and rank (Spearman) correlation between in vivo (MAS) and in vitro (log MRD(50)) data were 0.91 and 0.89, respectively. This study indicates that the silicon microphysiometer method exhibits a high correlation with the Draize test for water-soluble raw materials and formulations and thus can be used as an in vitro screen for ocular irritation.

Fluorescein leakage test: a useful tool in ocular safety assessment.

The fluorescein leakage test (FLT) provides information on the effects of xenobiotics on the impermeability (gate function) of epithelial cell monolayers, and their recovery after exposure. The aim of this study was to assess the validity of this test in the ocular safety assessment of surfactant-based products with various irritant potencies. Madin-Darby canine kidney cells were grown to confluency on microporous membranes and exposed for 15 min to increasing concentrations of test substances. Damage was evaluated by measuring the amount of Na-fluorescein that passed through the monolayer in 30 min, starting just after exposure. Recovery was assessed 4, 24, 48 and 72 hr later. For each sample and each time point, the amounts of test substance that produced 10% and 20% leakage (FL(10) and FL(20)) compared with a cell-free control were calculated. For the 43 samples, FL(20) values ranged from 0.65 to 1000 mg/ml. These values increased or decreased with time according to the substance. In particular, cell monolayers showed very different recoveries after exposure to anionic and cationic substances with similar initial FL(20) values. These in vitro data correlated well with historical Draize in vivo test data (Spearmans varrho > 0.90). The FLT is therefore useful as a complement to other in vitro methods for the ocular safety evaluation of cosmetics.

Contact sensitizers specifically increase MHC class II expression on murine immature dendritic cells.

Contact sensitivity is a T-cell-mediated immune disease that can occur when low-molecular-weight chemicals penetrate the skin. In vivo topical application of chemical sensitizers results in morphological modification of Langerhans cells (LC). Moreover, within 18 h, LC increase their major histocompatibility complex (MHC) class II antigens expression and migrate to lymph nodes where they present the sensitizer to T lymphocytes. We wanted to determine if such an effect could also be observed in vitro. However, because of the high genetic diversity encountered in humans, assays were performed with dendritic cells (DC) obtained from a Balb/c mouse strain. The capacity of a strong sensitizer, DNBS (2,4-dinitrobenzene sulfonic acid), to modulate the phenotype of bone marrow-derived DC in vitro, was investigated. A specific and marked increase of MHC class II molecules expression was observed within 18 h. To eliminate the use of animals in sensitization studies, the XS52 DC line was tested at an immature stage. A 30-min contact with the strong sensitizers DNBS and oxazolone, or the moderate mercaptobenzothiazole, resulted in upregulation of MHC class II molecules expression, analyzed after 18-h incubation. This effect was not observed with irritants (dimethyl sulfoxide and sodium lauryl sulfate) nor with a neutral molecule (sodium chloride). These data suggested the possibility of developing an in vitro model for the identification of the sensitizing potential of chemicals, using a constant and non animal-consuming material.

Evaluation of eye irritation potential: statistical analysis and tier testing strategies☆

Eye irritation testing, specifically the Draize test, has been the centre of controversy for many reasons. Several alternatives, based on the principles of reduction, refinement and replacement, have been proposed and are being used by the industry and government authorities. However, no universally applicable, validated non-animal alternative(s) is currently available. This report presents a statistical analysis and two testing approaches: the partial least squares multivariate statistical analysis of de Silva and colleagues from France, the tier-testing approach for regulatory purposes described by Gerner and colleagues from Germany, and the three-step tier-testing approach of the US Interagency Regulatory Alternatives Group described by Gupta and Hill. These approaches were presented as three separate papers at the November 1993 Interagency Regulatory Alternatives Group (IRAG) Workshop on Eye Irritation Testing; they have been summarized and combined into the following three-part report. The first part (de Silva et al.) presents statistical techniques for establishing test batteries of in vitro alternatives to the eye irritation test. The second (Gerner et al.) and third (Gupta and Hill) parts are similar in that they stage assessment of information by using a combination of screening information and animal testing to effect reductions in animal use and distress.

IRAG Working Group 3: Cell function-based assays

Cell function-based tests measure responses of cells at sublytic concentrations of test agents. The fluorescein leakage assay measures effects of substances on the barrier function of epithelial monolayers or multilayers (MDCK or NHEK cells) as in vitro models of corneal epithelial function. Two IRAG data submissions suggest that the fluorescein leakage assay shows promise as a screening test for surfactants and alcohols. The test method requires further optimization, standardization and evaluation to fully determine its utility as an in vitro ocular irritancy test. The silicon microphysiometer test measures effects of test substances on the metabolic rate of cells; although a large number of cell types have been evaluated, L929 cells have been used for irritancy screening. Three IRAG data submissions on the silicon microphysiometer test showed strong in vivo/in vitro correlations for surfactants and surfactant-based personal care and household cleaning products in a range of mild to moderate ocular irritancy. This and published information support the reproducibility of the method and its use as an ocular irritancy screening test for aqueous-soluble liquid, surfactant-based formulations.

The use of in vitro methods in the ocular irritation assessment of cosmetic products

The ocular tissue is a complex system consisting of corneal and conjunctival epithelial cells, the underlying corneal stroma and associated endothelial cells. Exposure to chemicals may result in responses ranging from mild, slight redness and itching, to severe injury with loss of corneal epithelium, damage to stroma, inflammatory infiltration and loss of vision. This complexity hinders the development of in vitro methods able to replace animal testing. Various in vitro techniques have been proposed and subsequently developed as potential replacements for ocular toxicity screening on animals. Over the past 2 years, eight methods have been evaluated in these laboratories. The endpoint of these methods could be linked to one or to several clinical events occurring in the in vivo eye irritancy process described above. Using these systems, a battery of four complementary in vitro assays has been developed. For the categories of ingredients and cosmetic products investigated, the promising results obtained suggest that in vitro methods of ocular risk assessment may be used increasingly in the future.

In Vitro Methods in Ocular Toxicology: Introductory Remarks

Toxicologists should be aware of the rapid development of in vitro methods over the 10 last years, as they will have a major impact on this field (Frazier, 1993). The two main driving forces behind this development are a better understanding of cell biology, which has led to improvements in the use of cell culture models, and ethical pressures from animal rights movements (Balls, 1991; Goldberg and Frazier, 1989).

Biosensors in pharmacology and toxicology in vitro

The need of disposing of analytical information in real time in many fields of bioanalytical and bioindustries explains the great attention devoted to biosensor development. A biosensor is usually defined as resulting from the combination of a sensitive biological element capable of molecular recognition and a transductor (electrode, optical detector…) which gives a meaningful mostly electrical signal. During the last decade, major development has concerned electrode tipped with enzymes, antibodies or other reagents that interact chemically with analytes -the substances being analysed. Such biosensors are expected to have numerous applications in the field of medicine and biology (Brennan and Krull,1992).