Registration Dossier

Administrative data

Description of key information

in vitro skin irritation/corrosion (OECD 431 and 439; EpiDerm test): irritating (BASF SE, 2012)
in vitro eye corrosion (OECD 437; BCOP): no risk of serious eye damage (BASF SE, 2012)
in vitro eye irritiation (EpiOcular): irritating (BASF SE, 2012)

Key value for chemical safety assessment

Skin irritation / corrosion

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In Vitro EpiDerm test (skin irritation and corrosion):

The potential of (Z)-4-[C11-13 (branched) alkylamino]-4-oxo-2-butenoic acid to cause dermal corrosion/irritation was assessed by a single topical application of 50 μL (corrosion test) or 30 μL (irritation test) of the test substance to a reconstructed three dimensional human epidermis model (EpiDerm™) according to OECD guideline 431 and 439 and GLP. For the corrosion test two EpiDerm™ tissue samples were incubated with the test substance for 3 minutes and 1 hour, respectively. The irritation test was performed with three EpiDerm™ tissue samples, which were incubated with the test substance for 1 hour followed by a 42-hours post-incubation period. Tissue viability was subsequently measured using a MTT assay comparing formazan production in test item-treated tissues as compared to negative controls. The quotient of the values indicated the relative tissue viability.

The mean viability of the test-substance treated tissues determined after an exposure period of 3 minutes was 98%, and it was 82% after an exposure period of 1 hour, indicating that the test item does not have a corrosive potential under the experimental conditions chosen. In contrast, the mean viability of the test-substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 5%, indicating that the test substance is irritating to the skin.

In Vitro BCOP test (eye corrosion):

The potential of the test item to cause serious damage to the eyes was assessed in an in vitro bovine cornea opacity and permeability (BCOP) test according to OECD guideline 437 and GLP. Three isolated cornea from freshly slaughtered cattle were prepared per treatment group (positive control (PC), negative control (NC) and test item); 750 μL of the undiluted liquid test substance was applied directly to the epithelial surface of the cornea using a pipette (open chamber method). Control tissues were concurrently applied into the anterior chamber with 750 μL of de-ionized water (NC) or with 750 μL of 1% (w/v) solution of sodium hydroxide in deionized water (PC) using a pipette. The corneas were incubated in a horizontal position at about 32 °C for approximately 10 minutes (liquids and surfactants) and the test item or controls removed thereafter. The corneas were incubated for further 2 hours at about 32 °C. After the incubation period the medium was removed and both chambers were then refilled with fresh Eagle’s MEM.

Corneal opacity was subsequently measured with an opacitometer; for determination of permeability the medium in the anterior chamber was replaced by 1 mL sodium fluorescein solution (4 mg/mL for liquid test substances and surfactants) and incubated for 90 ± 5 min in a horizontal position at about 32 °C. The amount of sodium fluorescein that permeated through the corneas into the posterior chamber was measured spectrophotometrically. Three aliquots per cornea were transferred to a 96-well microtiter plate and the optical density (OD490) was determined.

The mean corneal opacity and permeability values of each treatment group were used to calculate an In Vitro Irritancy Score (IVIS). The mean permeability score of the PC is out of the historical range. However, as all acceptance criteria were met in the test and due to the unambiguous results of the test substance, the evaluation of the study is not expected to be influenced by this deviation. Since the calculated IVIS value is 8.2 and therefore below the threshold value of 55, it can be concluded that the test item does not cause any serious damage to the eyes.

In vitro EpiOcular test (eye irritation):

The potential of (Z)-4-[C11-13 (branched) alkylamino]-4-oxo-2-butenoic acid to cause ocular irritation was assessed by a single topical application of 50 μL of the test substance to a reconstructed three dimensional human cornea model (EpiOcular™). Two EpiOcular™ tissue samples were incubated with the test substance for 30 minutes followed by a 2-hours post-incubation period. Water was used as negative control and methyl acetate as positive control.

Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation 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 testsubstance treated epidermal tissues is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability. Using this assay, the mean viability of the test-substance treated tissues was 2%. It can hence be concluded that the test item possesses an irritating potential to the eye.

Justification for classification or non-classification

Based on the available data and according to Directive 67/548/EEC (DSD) the test item is classified as irritating to the skin and labelled with Xi and the risk phrase R38 "Irritating to the skin". According to the Regulation (EC) No 1272/2008 (CLP), the substance is classified as category 2 and labelled with the signal word "Warning" and H315 "causes skin irritation"

According to the harmonised classification criteria laid down in Council Directive 67/548/EEC, Annex VI (and subsequent adaptations) the test substance should be classified as irritating to the eyes and assigned the symbol Xi, and the risk phrase R36: "Irritating to eyes" should be applied.

According to Annex I, Regulation (EC) No 1272/2008, the substance is classified as category 2 and labelled with "Warning" and H319 "causes serious eye irritation".