Nineteen scorers from seven Cuban laboratories participated in this slide exercise designed to test the influence of the scorer on the accuracy, sensitivity and variability of the comet assay when a visual method of DNA damage evaluation is used. The assay was performed using human lymphocytes from a single donor exposed in vitro for 5 min at 0 degrees C to doses of 0, 5, 10, 25, 50, 100 and 200 microM of hydrogen peroxide. Each participant scored the same set of 14 coded slides with silver stained comets. The comets were classified visually into five categories according to the appearance resulting from the relative proportion of DNA in the tail. The extent of DNA damage was expressed in arbitrary units. At zero dose the median values of 12 scorers out of 19 were included between the values of the overall 25 and 75 per thousand. This proportion remains practically the same as the dose increases. The lowest dose detected by this method for the majority of scorers (11) was 10 microM. The coefficient of variation at the control dose was the highest (median value 26%), progressively declined to 20%, and starting from 25 microM, values are around 10%. The results of the exercise show the reliability of the silver staining and visual scoring for the comet method.

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The determination of acute eye and skin irritation is included in international regulatory requirements for the testing of chemicals, because of the possibility of exposure during the production, transport, marketing, and disposal of products. Although there have been some advances in the areas of refinement and reduction, no single battery of tests has emerged as being acceptable as a complete replacement for the conventional Draize rabbit eye and skin irritation tests. Currently, dermal irritation and ocular irritation are generally evaluated in a sequential manner in the context of tiered assessment strategies. In this work, we show how 14 products, mostly designed to be used in agriculture, were evaluated in the Center of Experimental Toxicology of the Center for the Production of Laboratory Animals (Centro Nacional para la Producción de Animales de Laboratorio; CENPALAB) in order to assess their acute dermal and ocular effects. The performed studies include the acute dermal toxicity test, the acute dermal irritation/corrosion test, the hen's egg test-chorioallantoic membrane (HET-CAM) method, and the acute eye irritation/corrosion test. In general, it could be concluded that of the 14 products assessed, none of them showed systemic effects, but local reactions mainly to the eyes. The most significant effects were apparently related to the effects of azadirachtin, an active principle of 2 tested neem derivatives.

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