Alternatives to animal experiments for eye irritation

4. Test for recovery from ocular irritation

4.1 Method
4.2 Results
4.3 Discussion

Ocular irritancy is traditionally evaluated by use of the Draize test with albino rabbits receiving 0.1 mg or 0.1 ml of the test substance into the conjunctival sac of one of the eyes. The clinical responses of the cornea, conjunctiva and iris are observed until the initial lesions are cleared or up to 21 days. OECD guideline 405 on acute eye irritation/corrosion states that the eyes should be examined at 1, 24, 48 and 72 hours. If there is no evidence of irritation at 72 hours, the study may be ended. Extended observation may be needed if there is persistent corneal involvement or other ocular irritation in order to determine the progress of the lesions and their reversibility or irreversibility (OECD, 1987). The classification of ocular irritants by US-EPA/FDA is based on the use of maximum scores of corneal opacity, iritis and the conjunctival response, and the persistency of the lesions for more than 7 or 21 days (Gupta et al., 1993). The European Community is using the mean scores of the 24, 48 and 72 hour observation time for classification of R36 and R41 substances causing significant or severe ocular lesions, respectively. The persistency of the response is also evaluated, as the presence of ocular lesions at the 72 hours observation time or irreversible colouration of the eye are parameters releasing the R41 label (European Commission, 1996).

A wide range of in vitro assays for ocular irritation are being extensively used for screening purposes, but almost all of these tests have been designed to predict the severity of acute ocular effects of chemicals, and it has not been possible to demonstrate a correlation between the effects of chemicals over time in vitro and times to clear ocular damage in vivo. The Institute of Food Safety and Toxicology has as a part of the present project introduced a new testing procedure with SKIN² ZK1200 tissues to study the recovery from ocular irritation. Nine substances from the ECETOC eye irritation reference chemicals data bank were tested (see table 4.1).

4.1 Method

In the present exploratory study, the use of the three-dimensional ocular tissue model, SKIN^2TM ZK1200, was introduced for the study of tissue recovery after exposure to irritants. The study was designed to mimic the Draize test procedure with a topical exposure of the tissue specimens, and observation of the tissues at time points corresponding to observation times used in vivo. It was studied, whether the cellular viability of control tissue specimens could be maintained for periods of time comparable to the in vivo test procedure, and whether exposed tissues regained their viability in vitro in a manner comparable to days-to-clear the response in the Draize test.

The recovery model was developed before the identity of the COLIPA test substances was revealed to the participants. For this reason, the exposure times used in the recovery study were defined by a preliminary time range finding study, where exposure times reducing the MTT test response to 35-65% of the control level were established. The tissue specimens were exposed to the test substances according to the SKIN² ZK1200 dosing regime and the MTT test protocol used in the COLIPA study. The cellular viability of the tissue model was measured using the MTT assay immediately after chemical exposure and after incubation periods corresponding to observation times used in the Draize tests. The days-to-clear in vitro was defined as the number of days needed to obtain MTT activities in the exposed tissue specimens that were not significantly different from the matching control values.

Table 4.1
Days needed for exposed SKIN² ZK1200 tissues to regain the control MTT activity (days-to-clear in vitro) together with average and median values of days-to-clear in vivo and Draize test MMAS values.

*: Average of two Draize tests.

Control tissues and tissue specimens exposed to 1% benzalkonium chloride, 3% sodium lauryl sulphate, and 5% Triton X-100 were examined histologically. The tissues were fixed in 10% neutral buffered formalin, parafin embedded, sectioned, and stained with haematoxylin and eosin. The tissues were then subject to histomorphological examination of numbers of epithelial cell layers present and dermal/epidermal cell degeneration.

A one-way analysis of variance was used to analyse the results on MTT activities. Analyses of linear correlation were used for comparison of exposure times used in vitro and Draize test MMAS values and of in vitro and in vivo recovery data.

4.2 Results

The average MTT values for the unexposed control tissues remained fairly stable for up to 14 days of incubation, but after 21 days a considerable decrease in the MTT activity was observed. The mild ocular irritants (100% glycerol and 0.1% cetylpyrimidinium bromide) did not induce a significant depression of the cellular viability of the tissue specimens after 60 minutes of exposure, but significant decreases in MTT activities were observed after 2 to 3 days of culture. The MTT activity of the ZK1200 tissue specimens exposed to 3% sodium lauryl sulphate and 5% Triton X-100 returned to control levels within 1 day, and tissues exposed to 1% sodium hydroxide, 100% isopropanol and 100% methyl acetate returned to control MTT levels within 7 days. Tissue specimens exposed to various concentrations of benzalkonium chloride did not return to the control level of MTT activity in a stable manner. In tissue specimens exposed to 1% benzalkonium chloride for 5 seconds, the MTT activity was not affected after 1 day, but significantly lower MTT values than the matching controls were found on day 3, 7, 10, and 14. Tissues exposed to 1% benzalkonium chloride for 10 seconds did not return to control MTT levels during the 7 day observation period. Tissue specimens exposed to 10% benzalkonium chloride for 1 second did not regain control MTT values within 21 days.

Histological analysis of cellular degeneration and necrosis in SKIN² ZK1200 tissues.

Histomorphological analysis of control and exposed SKIN² ZK1200 tissues. Dermal/epidermal degeneration/necrosis: (+) traces, + mild, ++ moderate, +++ severe, ++++ very severe. n.d.: not determined.

A moderately good linear correlation (r = 0.73, p<0.05) was obtained between the exposure times used in vitro (log transformed) and Draize MMAS values. The number of days needed to regain tissue viability in vitro and Draize MMAS values is shown in table 4.1 together with the average and median days-to-clear the ocular responses in the Draize tests. Good linear correlations were found both between days-to-clear in vitro and days-to-clear in vivo using average (r = 0.92, p<0.001) and median (r = 0.91, p<0.001) values.

By histological examination, the number of epithelial cell layers were observed to vary between 1 and 3 in all tissue specimens analyzed (see table 4.2). No consistent cellular changes were seen on day 0, 1 or 3. On day 7 moderate degree of pyknosis and karyorrhexis of epithelial cells was observed in tissue specimens exposed to 3% sodium lauryl sulphate and 5% Triton X-100, while severe cellular changes including confluent areas of stromal cellular necrosis were present in tissue specimen exposed to 1% benzalkonium chloride. Only mild cellular changes were seen in the control tissue. No cellular regeneration was observed (Espersen et al., 1997).

4.3 Discussion

By histological examination no consistent cellular changes in the tissues were observed before day 7 after treatment, indicating that measurements of the MTT activity of the tissues appear to be a more sensitive marker of changes of the cellular viability. This is in line with results obtained by histopathological examinations and MTT conversion tests in skin organ cultures exposed to chemical irritants (van de Sandt & Rutten, 1995). The SKIN² ZK1200 model appeared to be adequate for determination of delayed cytotoxicity, as the viability of the system was stable for up to two weeks. The use of the model for more prolonged periods of time would be questionable due to the impaired tissue viability. A good overall correlation was found between the times for regain of a full MTT activity in the exposed tissue specimens and the in vivo recovery data. The results obtained suggest that the SKIN² ZK1200 model have shown promise for use in assessing chemicals and products for their ability to induce repairable or persistent cellular damage to the eyes.