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Administrative data

Description of key information

The available information on 2-ethoxyethyl (2Z)-2-cyano-2-[3-(3-methoxypropylamino)cyclohex-2-en-1-ylidene]acetate (C-1701 B_C_3)

provides no indication for skin irritating properties in in vitro skin irritation studies (EpiDerm™ test). No eye irritating potential was demonstrated in in vitro eye irritation studies (EpiOcular™ and BCOP test) with 2-ethoxyethyl (2Z)-2-cyano-2-[3-(3-methoxypropylamino)cyclohex-2-en-1-ylidene]acetate (C-1701 B_C_3).

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:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation

In the chosen key study according to OECD TG 439 and GLP (BASF SE 2019; 68V0473/11A613), the potential of 2-ethoxyethyl (2Z)-2-cyano-2-[3-(3 methoxypropylamino)cyclohex-2-en-1-ylidene]acetate (C-1701 B_C_3) to cause dermal irritation was assessed by a single topical application of approx. 25 mg bulk volume of the undiluted test item (corresponding to about 39 mg/cm2) to a reconstructed three dimensional human epidermis model (EpiDerm™) for 1 hour followed by a 42-hour post-incubation period. Before application, the epidermis surface was moistened with 25 μL sterile PBS, then the test substance was distributed together with the fluid, so that the surface of the epidermis model was uniformly and completely covered. In addition, 30 μL / tissue of a negative control (PBS) and a positive control (5% SDS) were applied to three tissues each. Tissue destruction was determined by measuring the metabolic activity of the tissues after exposure/post-incubation by using a colorimetric test (formazan formation after MTT incubation).

The relative mean viability of the tissues treated with the test substance was 124.8% in relation to the negative control. The respective OD570 values determined for test substance treated tissues were higher than of the negative control. However, as all quality criteria of the test were met, the study is considered valid despite the higher viability values of the test substance. Application of the positive control 5% SDS showed a relative mean viability of the tissues of 3.6% and reflects the expected sensitivity of the tissues. The mean OD570 of the negative control (PBS) fulfill the acceptance criteria and demonstrate the validity of the assay. The test substance was not able to reduce MTT directly.

Based on the results observed it was concluded that 2-ethoxyethyl (2Z)-2-cyano-2-[3-(3 methoxypropylamino)cyclohex-2-en-1-ylidene]acetate (C-1701 B_C_3) does not show a skin irritation potential in the EpiDerm™ in vitro Skin Irritation Test under the test conditions chosen.

 

The supporting study with 2-ethoxyethyl (2Z)-2-cyano-2-[3-(3-methoxypropylamino)cyclohex-2-en-1-ylidene]acetate (C-1701 B_C_3) according to OECD TG 439 and GLP confirmed the absence of a skin irritating potential (BASF SE 2012; 61V0473/11A562). Three EpiDerm™ tissue samples were incubated with 25 μL (approx. 7 mg) bulk volume of the undiluted test item for 1 hour followed by a 42hours post-incubation period under similar test conditions.

The mean viability of the test item-treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 101 %. The test item was not able to reduce MTT directly. Yellow discoloration of the tissues was observed after washing. The PC item demonstrated appropriate sensitivity (relative viability 20 %) of the tissues. Under the conditions of this in vitro study, the test item did not show a skin irritation potential in the EpiDerm™ skin irritation test.

On the basis of these validated stand-alone in vitro tests, 2-ethoxyethyl (2Z)-2-cyano-2-[3-(3-methoxypropylamino)cyclohex-2-en-1-ylidene]acetate (C-1701 B_C_3) is not considered to be irritating to skin.

 

Eye irritation

The potential of 2-ethoxyethyl (2Z)-2-cyano-2-[3-(3-methoxypropylamino)cyclohex-2-en-1-ylidene]acetate (C-1701 B_C_3) to cause serious damage to the eyes or ocular irritation was assessed in different in vitro tests involving isolated bovine corneas and a reconstructed three dimensional human cornea model (EpiOcular™).

The serious damage of 2-ethoxyethyl (2Z)-2-cyano-2-[3-(3-methoxypropylamino)cyclohex-2-en-1-ylidene]acetate to the eyes was assessed in a BCOP test according to OECD TG 437 and GLP (BASF SE 2012; 63V0473/11A563). A single topical application of 750μL of a 20 % aqueous test item preparation was applied to the epithelial surface of 3 isolated bovine corneas for an exposure period of 4 hours. Deionised water served as negative control item (NC) and 20 % (w/v) Imidazole in deionised water served as positive control item (PC), applied to three corneas each. Corneal opacity was measured quantitatively as the amount of light transmission through the cornea using an opacitometer. Permeability was measured quantitatively as the amount of Sodium fluorescein dye that passed across the full thickness of the cornea by spectrophotometrical determination of the optical density at 490 nm. Both measurements were used to calculate an in vitro irritancy score (IVIS) of the test item relative to the control corneas. The mean IVIS of 2-ethoxyethyl (2Z)-2-cyano-2-[3-(3-methoxypropylamino)cyclohex-2-en-1-ylidene]acetate (5.4) was comparable to the NC value (4.5) and did not indicate a test itemrelated risk of serious damage to eyes. The PC item with a mean IVIS of 129.9 demonstrated appropriate sensitivity of the test system.

Based on the observed results it was concluded, that 2-ethoxyethyl (2Z)-2-cyano-2-[3-(3-methoxypropylamino)cyclohex-2-en-1-ylidene]acetate does not cause serious eye damage in the Bovine Corneal Opacity and Permeability Test (BCOP Test) under the test conditions chosen. The test method according to the regulatory accepted protocol currently does not allow for the final evaluation of an eye irritation potential. Thus, this result does not exclude an irritation potential and results from further studies would be needed for final assignment of a risk phrase.

 

In the chosen key study, occular irritation was investigated in an in vitro eye irritation test (EIT) according to OECD TG 492 and GLP (BASF SE 2019; 62V0473/11A614). A single topical application of approx. 50 mg/tissue undiluted (corresponding to 83 mg/cm²), approx. 50 μL/tissue undiluted (corresponding to 8 mg/tissue) or 50 μL/tissue of diluted (3% (w/w) in deionized water) 2-ethoxyethyl (2Z)-2-cyano-2-[3-(3-methoxypropylamino)cyclohex-2-en-1-ylidene]acetate was made on 2 EpiOcular™ tissues per dose group. The EpiOcular™ tissues were pretreated with 20 μL PBS in order to wet the tissue surface and then incubated with the registered substance for 6 hours followed by an 18-hour post-incubation period. In addition to the test substance, 50 μL per tissue of a negative control (deionized water) and a positive control (methyl acetate) were applied to two tissues each. Tissue destruction was determined by measuring the metabolic activity of the tissues after exposure/post-incubation by using a colorimetric test (formazan formation after MTT incubation). The relative mean viabilities observed in the tissues treated with the registered substance were 93.6% (application of approx. 50 mg/tissue), 106.5% (application of approx. 50 µl/tissue) and 103.6% (application of 3% aqueous preparation) in relation to the negative control. Slight or moderate compound residues (light yellowish colored) remained on the tissues treated with 50 mg or 50 µl test substance after the washing procedure. However, this did not interfere with the MTT assay as the test substance was not able to reduce MTT directly which was demonstrated in a pretest. The application of the positive control methyl acetate showed a relative mean viability of the tissues of 21.9% and reflects the expected sensitivity of the tissues. Furthermore, the mean OD570 of the negative control (deionized water) fulfilled the acceptance criteria and demonstrate the validity of the assay.

Based on the results observed, it was concluded that 2-ethoxyethyl (2Z)-2-cyano-2-[3-(3-methoxypropylamino)cyclohex-2-en-1-ylidene]acetate does not show an eye irritation potential in the EpiOcular™ in vitro Eye Irritation Test under the test conditions chosen.

The potential of 2-ethoxyethyl (2Z)-2-cyano-2-[3-(3-methoxypropylamino)cyclohex-2-en-1-ylidene]acetate to cause ocular irritation was further assessed in a second in vitro study by a single topical application of 50μL bulk volume (about 8 mg) of the undiluted test item on 2 EpiOcular™ tissues (BASF SE 2012; 62V0473/11A564). The EpiOcular™ tissues were incubated with the test item for 90 min followed by an 18-hour post-incubation period. Methyl acetate at a volume of 50 µL was used as positive control (PC) item. The mean viability of the test item treated tissues determined after an exposure period of 90 min with about 18 hours post-incubation was 104 %. The test item was not able to reduce MTT directly. Yellow discoloration of the tissues was observed after washing. The PC item demonstrated appropriate sensitivity of the tissues. Thus, 2-ethoxyethyl (2Z)-2-cyano-2-[3-(3-methoxypropylamino)cyclohex-2-en-1-ylidene]acetate was confirmed to show no eye irritation potential in the EpiOcular™ eye irritation test under the experimental conditions employed.

 

Overall, based on the key and the supporting studies for eye irritation and eye damage, 2-ethoxyethyl (2Z)-2-cyano-2-[3-(3-methoxypropylamino)-cyclohex-2-en-1-ylidene]acetate (C-1701 B_C_3) is not considered irritating to the eye.

Justification for classification or non-classification

The present data on skin and eye irritation do not fulfill the criteria laid down in 67/548/EEC and regulation (EU) 1272/2008, and therefore, a non-classification is warranted.