Okra, ext.

Endpoint summary

Description of key information

skin irritation: not irritating

eye irritation: irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Additional information

The potential of the test substance to cause dermal irritation was assessed by a single topical application of ca. 25 μL bulk volume (about 10 mg) undiluted test substance to a reconstructed three-dimensional human epidermis model (EpiDerm™). Three EpiDerm™ tissues were incubated with the test substance for 1 hour followed by a 42-hour post-incubation period. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation by using a colorimetric test. The reduction of mitochondrial dehydrogenase activity measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the epidermal tissues treated with the test substance is compared to that of negative control tissues. The quotient of both values indicates the relative tissue viability. The following results were obtained in the EpiDerm skin irritation test:

The test substance is able to reduce MTT directly. Therefore, an additional MTT reduction control KC (freeze-killed control tissues) was introduced. The final relative mean viability of the tissues treated with the test substance determined after an exposure period of 1 hour with an about 42-hour post-incubation was 93.6%. Based on the results observed and by applying the evaluation criteria, it was concluded that the substance does not show a skin irritation potential in the EpiDerm™ in vitro skin irritation test under the test conditions chosen.

The potential of the substance to cause ocular irritation was assessed by a single topical application of ca. 50 μL bulk volume (about 14 mg) undiluted test substance to a reconstructed three-dimensional human cornea model (EpiOcular™). Three test runs were performed. Two EpiOcular™ tissues per test run were incubated with the test substance for 6 hours followed by a 18-hour post-incubation period.

Tissue destruction was determined by measuring the metabolic activity of the tissues after exposure/post-incubation by using a colorimetric test. The reduction of mitochondrial dehydrogenase activity measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the epidermal tissues treated with the test substance is compared to that of negative control tissues. The ratio of the values indicates the relative tissue viability. The following results were obtained in the EpiOcular™ eye irritation test:

The test substance is able to reduce MTT directly. Therefore, an additional MTT reduction control (freeze-killed control tissues (KC)) was introduced.

The following results were obtained within 3 test runs:

1st test run:

The final relative mean viability of the tissues treated with the test substance after the 1st test run was 36.6%. However, the acceptance criteria for the killed controls were not met: High values for direct MTT reduction (OD values > 0.35 and mean value of the killed control tissues 30.7% with individual values 30.7% and 30.6%) and high variability of the test-substance treated tissues (difference between the tissues 18.8% with inidividual values 57.9 and 76.6% close to the classification cut-off) a 2nd test run was performed to verify the result.

2nd test run:

The final relative mean viability of the tissues treated with the test substance after the 2nd test run was 20.6%. However, the acceptance criteria for the killed controls were not met, because exceptionally high values for direct MTT reduction and high variability between the single killed control tissues (OD values > 0.35 and mean value of the killed control tissues 30.7% with individual values 84.3% and 50.4%) were observed. Thus, a 3rd test run was performed.

3rd test run:

The final relative mean viability of the tissues treated with the test substance after the 3rd test run was 0.0%. However, the acceptance criteria for the killed controls were not met, because exceptionally high values for direct MTT reduction and high variability between the single killed control tissues (OD values > 0.35 and mean value of the killed control tissues 115.3% with single values 153.0% and 77.6%) were observed. In addition high variability of the testsubstance treated tissues (difference between the tissues 22%) was noted. Further, the final relative mean viability of the tissues treated with the test substance after the 3rd test run was set to zero after substracting the mean viabilaity of the killed controls form the man viability of the test-substance treated tisuues.

In summary the assessment of several test runs of the EpiOcular™ eye irritation assay showed that no valid results could be derived for the EpiOcular™ eye irritation assay. In summary, it must be concluded that the results of the test substance should be considered as “inconclusive” in the in vitro eye irritation test under the test conditions chosen.

Justification for classification or non-classification

As the EpiDerm test was clearly neagtive, the substance is not classified with regard to skin irriation. The EpiOcular test was considered to be inconclusive. Based on the result of the EpiDerm test and the characteristic of the substance, the substance is regarded to be not eye irritating. Based on the inconclusive result of the EpiOcular test, the substance is classified as "eye irritang".