Ph. Vanparys

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.

Toxicological Profile and Safety Evaluation of Antifungal Azole Derivatives

Summary: For the development of new sys‐temically acting, oral antifungal azoles, it is of key importance to compare them with ketoconazole, the first available drug in this therapeutic class.

Discrimination of aneuploidogens from clastogens by C-banding, DNA and area measurements of micronuclei from mouse bone marrow

Micronuclei (MN) obtained from mouse bone marrow cells, in vivo exposed to 3 typical clastogens (procarbazine, azathioprine, ethyl methanesulfonate) and 3 typical aneuploidogens (vinblastine, tubulazole, colchicine), were examined for C-band, area and DNA content. C-banding allows a clear discrimination between clastogens and aneuploidogens: the clastogens do not exceed 50% C-band-positive MN and the aneuploidogens all 3 produce 65-75% C-band-positive MN. Concerning the DNA content the percentages of MN containing more DNA than an average chromosome (chr) are lower than 12% for the clastogens and 38-60% for the aneuploidogens. As far as the area of the MN is concerned the percentages of MN which have a larger area than chr are lower than 23% for the clastogens and range from 47% to 71% for the aneuploidogens. Additionally 3 other mutagens were studied. Hydroquinone induces 43% C-band-positive MN with DNA content far below the content of chr; considering the area measurements, however, hydroquinone behaves as an aneuploidogen (65% of the MN are larger than chr). Mitomycin C lies between the clastogens and the aneuploidogens for all 3 criteria but 5-azacytidine is comparable to the model aneuploidogens.

Evaluation of a modified HET-CAM assay as a screening test for eye irritancy

The hens egg test-chorioallantoic membrane (HET-CAM) assay, an alternative to the Draize eye irritation test, was developed by Luepke and has been improved on by means of a microscopic examination and the use of a test substance applicator (TSA). The TSA is a double teflon ring in which a perlon mesh is locked, and has several advantages over conventional protocols, reducing subjectivity of the method and avoiding the need for rinsing after treatment. It was confirmed by statistical analysis that the HET-CAM-TSA method can reproduce potentialin vivo irritant effects on the conjunctiva. The classification based on thein vitro results was compared with fourin vivo classifications [MAS (maximal average score) with thresholds of 15.0 and 25.0; the Kay and Calandra method; and EC criteria]. Coopers parameters (specificity, sensitivity and concordance with the Draize test) were calculated according to these fourin vivo classifications. When the most rigorous classification (MAS threshold of 15.0) was taken into account, a sensitivity of 80%, a specificity of 81.3% and a concordance with the Draize test of 80.4% were obtained for this set of 46 compounds.

Evaluation of the bovine corneal opacity-permeability assay as an in vitro alternative to the Draize eye irritation test

The bovine corneal opacity-permeability assay (BCO-P) was evaluated as an in vitro alternative test model for the Draize eye irritancy test. Fifty pharmaceutical and commercially available compounds were tested in the BCO-P assay. The compounds were selected on the basis of their in vivo irritancy potential as determined in previous Draize tests. Liquids as well as solids were tested. Corneal opacity and permeability were measured to determine ocular irritation potential. When two irritancy classifications (non-irritant and irritant) were considered, 96% of the tested chemicals were classified correctly. A 72% concordance was obtained when four irritancy classifications (non-irritant, mild, moderate and severe irritant) were considered. Furthermore, all compounds that were severe eye irritants in vivo were equally scored in vitro. The results of this study show that the BCO-P assay is a competent in vitro test system for the prediction of ocular irritation of chemicals. This test model can be used as a first screen to avoid in vivo testing of severe ocular irritants.

Evaluation of the HET-CAM-TSA method as an alternative to the draize eye irritation test

The Hens Egg Test-Chorioallantoic Membrane (HET-CAM) method was modified in our laboratory by means of microscopic evaluation, a clear description of the three in vitro endpoints (haemorrhage, lysis and coagulation) and the use of a test substance applicator (TSA). A previous study on 46 chemicals demonstrated the usage of the HET-CAM-TSA assay as a screening test for eye irritancy. In order to extend our database and to come to a more reliable conclusion concerning the use of the HET-CAM-TSA method, a second set of 60 test substances was tested. The in vitro irritation scores (IS) were compared with the in vivo modified maximum average scores (MMAS) calculated 24 hr after instillation. The MMAS irritancy threshold was set at 15.0. The results were analysed according to the Coopers parameters (specificity, sensitivity and concordance with the Draize test) and the Pearsons correlation coefficient. It was concluded that the HET-CAM-TSA test was a valuable screening test. To compensate for the misclassifications generated, it was also concluded that the HET-CAM-TSA method should be considered as a part of a test battery, together with the Bovine Corneal Opacity-Permeability (BCOP) assay.

TOXICOLOGICAL PROPERTIES OF CLOSANTEL

The acute, subacute and chronic toxicity studies in laboratory animals showed that closantel is a well tolerated substance. At multiples of the clinical dose, overdosing might result in central nervous system effects and death. Repeated oral dosing was without effects up to 40 mg/kg in rats and dogs except for focal swelling of the epididymis in male rats at 40 mg/kg due to formation of spermatic granulomas. In sheep repeated dosing at 10 and 40 mg/kg orally and at 5 and 20 mg/kg intramuscularly every four weeks during 40 weeks demonstrated an acceptable safety margin in this target species. Reproduction studies including a three-generation study in rats showed that fertility was not affected except slightly in male rats at 40 mg/kg whereas an embryotoxic or teratogenic potential in rats and rabbits was absent. Peri- and postnatal parameters in rats were not affected. In target animals, reproduction was extensively studied in bulls, rams and ewes showing no risk of closantel for reproduction parameters. A mutagenic potential was found to be absent in a Salmonella Ames test, a sex-linked recessive lethal test in Drosophila melanogaster and a dominant lethal test in male and female mice. In 400 mice and 400 rats closantel was shown not to be carcinogenic. Tolerance studies in sheep and cattle demonstrated that oral and parenteral clinical doses were very well tolerated and devoid of serious side-effects.

Toxicity and Mutagenicity of the Molluscicidal Plant Ambrosia Maritima L.

The acute and subchronic toxicity of the molluscicidal plant, Ambrosia maritima L., has been tested on rats. No toxic signs could be detected neither after oral administration of 5 g/kg of dried leaves of the plant as a powder or as a methanolic extract, nor after the incorporation of 50,000 ppm powdered leaves in the feed during 4 weeks. Using an aqueous extract of the plant material of A. maritima or using ambrosin, one of the active molluscicidal components of the plant, no mutagenic activity could be detected in the S. typhimurium strains TA97, TA 98, TA1538, TA100 and TA1535.

Mutagenicity tests with astemizole in vitro and in vivo

Possible induction of chromosomal aberrations and/or sister chromatid exchanges by astemizole was studied in vitro on human lymphocytes. In vivo chromosomal damage was assessed by a micronucleus test on rats and a dominant lethal test on both male and female mice. All these tests yielded negative results for astemizole so that it can be concluded that astemizole has no potential to induce chromosomal aberrations.

Mutagenic and Leukemogenic Activity of Haloperidol: A Negative Study

The mutagenic and leukemogenic potential of haloperidol, a neuroleptic of the butyrophenone class, has been studied in an in vitro Ames Salmonella/microsome test and in an 18-month carcinogenicity study in mice. Three variants of the Salmonella mutation assay were included: the spot test, the standard plate incorporation test and the preincubation test. There was no evidence that haloperidol had any mutagenic activity in any of the Salmonella mutation tests with any of the Salmonella typhimurium tester strains in the presence or absence of Aroclor 1254-induced rat- or mouse-liver S9-mix. In the 18-month study, haloperidol was injected intraperitoneally as a solution (HaldolR) at a dosage of 5 mg/kg daily for 5, 10 and 20 consecutive days in 5-week-old mice. Leucocyte counts at several time points and histopathological tumor evaluation 18 months later did not reveal any leukemogenic or other carcinogenic effect. On the basis of these data, it may be concluded that haloperidol is not mutagenic in Salmonella nor leukemogenic in mice.