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Key value for chemical safety assessment

Additional information

SKIN IRRITATION/ CORROSION

The skin irritation and corrosion potential of the main constituent 4,8,11-Dodecatrienal was investigated in 2 in vitro studies. Using the methods currently available, a single in vitro assay is not sufficient to cover the full range of skin irritating/corrosion potential. Therefore, two in vitro assays were part of this in vitro skin irritation and corrosion test strategy: The Skin Corrosion Test (SCT) and Skin Irritation Test (SIT), performed acc. to OECD 431 / OECD 439 and GLP.

In the SCT the potential of 4,8,11-Dodecatrienal to cause dermal corrosion was assessed by a single topical application of 50μL undiluted test substance to a reconstructed three-dimensional human epidermis model (EpiDerm™). Two EpiDerm™ tissues were incubated with 4,8,11-Dodecatrienal for 3 minutes and 1 hour, each. Due to the intense smell of the test substance, the plates were sealed with an adhesive protective foil immediately after application. 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 the values indicates the relative tissue viability. 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 4,8,11-Dodecatrienal determined after an exposure period of 3 minutes was 104.2%, and it was 101.3% after an exposure period of 1 hour. Based on the results of the skin corrosion test no skin corrosion potential of 4,8,11-Dodecatrienal can be concluded.

In the SIT the potential of 4,8,11-Dodecatrienal to cause dermal irritation was assessed by a single topical application of 30μL undiluted test substance to a reconstructed three-dimensional human epidermis model (EpiDerm™). The irritation test was performed with three EpiDerm™ tissues, which were incubated with 4,8,11-Dodecatrienal for 1 hour followed by a 42-hour post-incubation period. Due to the intense smell of the test substance, the plates were sealed with an adhesive protective foil immediately after application. 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 the values indicates the relative tissue viability. 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 9.3%.

Based on the results observed and by applying the evaluation criteria it was concluded that 4,8,11-Dodecatrienal shows a skin irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy under the test conditions chosen.

 

The skin irritation and corrosion potential of the constituent 4,8-Cyclododecadien-1-one was investigated in an analogous approach.

In the skin corrosion test (SCT), the mean viability of the test-substance treated tissues determined after an exposure period of 3 minutes was 118.0%, and 94.6% after an exposure period of 1 hour.

In the skin irritation test (SIT), the mean viability of the test-substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 3.3%.

It was concluded, that the constituent 4,8-Cyclododecadien-1-one shows a skin irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy under the test conditions chosen.

EYE IRRITATION

The eye irritation and damage potential of the main constituent 4,8,11-Dodecatrienal was investigated in 2 in vitro studies as part of an in vitro eye irritation test strategy: The Bovine Corneal Opacity and Permeability Test (BCOP Test) and EpiOcular Eye Irritation Test, performed acc. to OECD 437 / OECD 492 and GLP.

 

In the BCOP Test, Tthe potential of 4,8,11-Dodecatrienal to cause ocular irritation or serious damage to the eyes was assessed by a single topical application of 750μL undiluted test substance to the epithelial surface of isolated bovine corneas. Three corneas were treated with 4,8,11-Dodecatrienal for 10 minutes followed by a 2-hour postincubation period. In addition to the test substance, a negative control (NC; deionized water) and two positive controls (PC1 / PC2; 100% ethanol / 100% dimethylformamide) were applied to three corneas each. Corneal opacity was quantitatively measured as the amount of light transmitted through the cornea. Permeability was quantitatively measured as the amount of sodium fluorescein dye that passes across the full thickness of the cornea. Both measurements were used to calculate an In Vitro Irritancy Score of the test substance.

The mean IVIS (3.3) of the test substance treated corneas did not indicate a corrosive or severe eye irritation potential.

 

In the EpiOcular test, the potential of 4,8,11-Dodecatrienal to cause ocular irritation was assessed by a single topical application of 50μL undiluted test substance to a reconstructed three-dimensional, human cornea model (EpiOcular™). Two test runs were performed. Two EpiOcular™ tissues per test run were incubated with 4,8,11-Dodecatrienal for 30 minutes followed by a 2-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 ratio of the values indicates the relative tissue viability. The following results were obtained in the EpiOcular™ eye irritation assay: 4,8,11-Dodecatrienal is able to directly reduce MTT. Therefore, an additional MTT reduction control (freeze-killed control tissues (KC)) was introduced.

In the 1st test run the final relative mean viability of the tissues treated with 4,8,11-Dodecatrienal compared to the negative control was 67.9%. Another test run was performed to verify the result, because the OD570 value of the negative control tissues lied out of the acceptance range and the mean viability value for 4,8,11-Dodecatrienal lied slightly above the borderline range for evaluation. In the 2nd test run the final relative mean viability of the tissues treated with 4,8,11-Dodecatrienal compared to the negative control was 82.4% and therefore well above the value for eye irritation. The mean viability value of the negative control lied out of the acceptance range again. However, as meanwhile the acceptance range for the negative control was updated by the developer/supplier and the results of the 2nd test run verified the results of the 1st test run, the study is considered to be valid.

Based on the results of the BCOP and EpiOcular, it was concluded that 4,8,11-Dodecatrienal does not show an eye irritation potential in the in vitro eye irritation test strategy under the test conditions chosen.

 

The eye irritation and damage potential of the constituent 4,8-Cyclododecadien-1-one was investigated in an analogous approach.

In the BCOP test, the mean in vitro irritancy score (IVIS) of the test-substance treated tissues was 0.2 and 0.1 in the first and second test run. 

In the EpiOcular test, the mean viability of the test-substance treated tissues determined after an exposure period of 30 minutes with about 2 hours post-incubation was 45.5 %.

When applying the criteria of the OECD 437 for the assessment of the BCOP test results, the IVIS scores of 0.2 or 0.1 - obtained after incubation with 4,8-Cyclododecadien-1-one - would classify it as a substance that does not cause eye irritation or serious eye damage. However, the Guideline also states, that due to limited accuracy of the BCOP test to correctly identify substances that do not require classification for eye irritation or serious eye damage, this test method should not be the first choice to initiate a bottom-up approach. Based on this statement and the experience of the test facility, test substances leading to IVIS <= 3 are generally examined in the EpiOcular test as well. In the current case, the EpiOcular test was positive and the test substance was assessed to be irritating to the eyes.

Based on the results for BCOP and EpiOcular Test and applying the evaluation criteria described, 4,8-Cyclododecadien-1-one shows an eye irritation potential in the in vitro eye irritation test strategy under the test conditions chosen.

Justification for classification or non-classification

The present data on skin irritation and corrosion of the main constituent 4,8,11-Dodecatrienal do fulfill the criteria laid down in regulation (EU) 1272/2008 for a classification as "skin irritant" (category 2). Furthermore, skin irritation and corrosion data with the constituent 4,8-Cyclododecadien-1-one do fulfill the criteria laid down in regulation (EU) 1272/2008 for a classification as "skin irritant" (category 2). Thus, based on the current data for these two components (representing 55% to 95% of the reaction mass) a classification of the registered substance as "skin irritant" (category 2) is warranted.

 

The present data on eye irritation and damage of the main constituent 4,8,11-Dodecatrienal do not fulfill the criteria laid down in regulation (EU) 1272/2008. However, eye irritation and corrosion data with the constituent 4,8-Cyclododecadien-1-one do fulfill the criteria laid down in regulation (EU) 1272/2008 for a classification as "eye irritant" (category 2). Thus, based on the current data for the component 4,8-Cyclododecadien-1-one (representing 5% to 15% of the reaction mass) a classification of the registered substance as "eye irritant" (category 2) is warranted.