Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames-Test (BASF, 2012): negative
HPRT-Assay (Harlan, 2012): negative

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

in vivo Mirconucleus Assay (Harlan, 2013), negative

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

in vitro:

Ames-Test:

(Z)-4-[C11-13 (branched) alkylamino]-4-oxo-2-butenoic acidw as tested in a GLP compliant Ames reverse mutation assay according to OECD guideline 471 using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100 and Escherichia coli WP2 uvr A at 0.33 to 5000 µg/plate (three independent experiments (standard plate incorporation test and pre-incubation test) and each concentration was tested in triplicate) with and without metabolic activation (BASF SE, 2012). No precipitation of the test substance was found with and without S9 mix. A bacteriotoxic effect was observed depending on the strain and test conditions from about 33μg/plate onward. A relevant increase in the number of his+or trp+revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system. Thus, under the experimental conditions of this study, the test substance (Z)-4-[C11-13 (branched) alkylamino]-4-oxo-2-butenoic acid is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

HPRT-Test:

A GLP-compliant gene mutation assay, tested according to OECD guideline 476, was performed to investigate the potential of (Z)-4-[C11-13 (branched) alkylamino]-4-oxo-2-butenoic acid to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster (Harlan 2012). The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The concentration range of the main experiments was limited by cytotoxic effects.The test item was dissolved in ethanol.Relevant cytotoxic effects occurred in the first experiment with metabolic activation and in the second experiment without metabolic activation at 72μg/mL and above. In the second experiment with metabolic activation cytotoxic effects as described above were noted at 96μg/mL. Precipitation of the test item at the end of treatment was noted at 72.0μg/mL and above in both main experiments with metabolic activation. No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation. Therefore, 2-Butenoic acid, 4-oxo-4-(tridecylamino)-, (Z)-, branched is considered to be non-mutagenic in this HPRT assay.

Chromosome Aberration Test:

In a GLP-compliant chromosome aberration test, tested according to OECD guideline 473, the test item (Z)-4-[C11-13 (branched) alkylamino]-4-oxo-2-butenoic acid, dissolved in ethanol, was assessed for its potential to induce structural chromosome aberrations in V79cells of the Chinese hamster in vitroin the absence and presence of metabolic activation by S9 mix (Harlan 2012). Five independent experiments were performed. In Experiment IA and IB the exposure period was 4 hours with and without S9 mix. In Experiment IIA the exposure period was 4 hours with S9 mix and 28 hours without S9 mix. In Experiment IIB the exposure period was 4 hours with S9 mix and 18 hours without S9 mix. In Experiment IIC the exposure period was 4 hours with S9 mix. The chromosomes were prepared 18 and 28 hours after start of treatment with the test item. In each experimental group two parallel cultures were set up. At least 100 metaphases per culture were evaluated for structural chromosome aberrations, except for the positive control in Experiment IIA after 28 hours continuous treatment without metabolic activation, where only 50 metaphases were evaluated. The highest treatment concentration in this study, 3600.0 µg/mL was chosen with regard to the solubility properties of the test item and with respect to the OECD Guideline for in vitromammalian cytogenetic tests. Visible precipitation of the test item in the culture medium was observed at 450.0 µg/mL and above in Experiment IA in the absence and presence of S9 mix at the end of treatment. No relevant influence on osmolarity or pH value was observed. In Experiment IB in the absence of S9 mix and Experiment IIC in the presence of S9 mix concentrations showing clear cytotoxicity were not evaluable for cytogenetic damage. In Experiment IA in the presence of S9 mix clear cytotoxicity indicated as mitotic index (52.5 % of control) was observed at the highest evaluated concentration. In Experiment IIA after 28 hours continuous treatment in the absence of S9 mix and in Experiment IIB in the presence of S9 mix clear cytotoxicity indicated by reduced cell numbers was observed at the highest evaluated concentrations (41.7, 49.4 % of control, respectively). In Experiment IIB after 18 hours continuous treatment without S9 mix and in Experiment IIA with S9 mix concentrations showing clear cytotoxicity were not evaluable for cytogenetic damage. However, cell numbers were reduced below 60 % of control. In all experimental parts in the absence of S9 mix and in Experiment IA and IIB in the presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.5 - 3.8 % aberrant cells, excluding gaps) were slightly above the range of the solvent control values (1.0 - 3.0 % aberrant cells, excluding gaps) and within the range of the laboratory historical solvent control data. In Experiment IIA in the presence of S9 mix one single statistically significant increase in chromosomal aberrations (13.0 % aberrant cells, excluding gaps) was observed after treatment with 100.0 µg/mL. In Experiment IIB this finding could not be confirmed. However, concentrations higher than 50 µg/mL were not evaluable for cytogenetic damage. In the confirmatory experiment IIC with narrow concentration spacing a dose-dependent increase in cells carrying chromosomal aberrations was observed after treatment with 20 - 80 µg/mL (2.5, 3.5, 5.5, 9.0 % aberrant cells, excluding gaps) with the highest evaluated concentration being statistically significant and the two highest evaluated concentrations exceeding the laboratory historical control range (0.0 -4.0 % aberrant cells, excl. gaps). No biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (1.1 - 3.5 %) as compared to the rates of the solvent controls (1.2 - 3.3 %). No biologically relevant increase in the rate of endomitotic metaphases was found after treatment with the test item (0.0 - 0.1 %) as compared to the rates of the solvent controls (0.0 %). In both experiments, either EMS (1000.0, 600.0 or 500.0 µg/mL) or CPA (1.4, 2.0 or 2.4 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations. In conclusion, it can be stated that under the experimental conditions reported, the test item (Z)-4-[C11-13 (branched) alkylamino]-4-oxo-2-butenoic acid induced structural chromosomal aberrations in V79cells of the Chinese hamster in vitro in the presence of S9 mix, when tested up to cytotoxic or the highest evaluable concentrations.

in vivo:

Micronucleus Assay:

This study was performed to investigate the potential of (Z)-4-[C11-13 (branched) alkylamino]-4-oxo-2-butenoic acid to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The study was performed according to OECD 474 guideline and GLP (Harlan, 2013)

The test item was dissolved in corn oil, which was also used as vehicle control. The volume administered orally was 10 mL/kg b.w.. 24 h and 48 h after a single administration of the test item the bone marrow cells were collected for micronuclei analysis. A correction factor of 1.09 was applied.

Six males and six females per test group (except the vehicle and positive control groups with 5 males and 5 females each) were evaluated for the occurrence of micronuclei. Per animal 2000 polychromatic erythrocytes (PCEs) were scored for micronuclei.

To investigate a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes.

The following dose levels of the test item were investigated:

Males: 500, 1000 and 2000 mg/kg b.w. at the 24 h preparation interval
and 2000 mg/kg b.w. at the 48 h preparation interval,

Females: 375, 750 and 1500 mg/kg b.w. at the 24 h preparation interval
                and 1500 mg/kg b.w. at the 48 h preparation interval.

The highest dose levels were estimated by pre-experiments to be suitable.

The animals treated with the test item showed clinical signs such as reduced spontaneous activity, eyelid closure, tumbling, tremor, diarrhoea and/or ruffled fur in the high dose group (2000 and 1500 mg/kg b.w.) and few female mice showed eyelid closure at 375 mg/kg b.w. in the main experiment. One male of the high dose group (animal no. 67) died 24 hours after treatment.

After treatment with the test item the number of PCEs per 2000 erythrocytes was not substantially decreased as compared to the mean value of PCEs per 2000 erythrocytes of the vehicle control thus indicating that 2-Butenoic acid, 4-oxo-4-(tridecylamino)-, (Z)-, branched did not exert a cytotoxic effect in the bone marrow.

In comparison to the corresponding vehicle controls there was no biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used. All observed values were very well within the historical control data range.

40 mg/kg b.w. cyclophosphamide administered orally was used as positive control which showed a substantial increase of induced micronucleus frequenc

In conclusion, it can be stated that under the experimental conditions reported, the test item (Z)-4-[C11-13 (branched) alkylamino]-4-oxo-2-butenoic acid did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.

Therefore, (Z)-4-[C11-13 (branched) alkylamino]-4-oxo-2-butenoic acid is considered to be non-mutagenic in this micronucleus assay.


Justification for classification or non-classification

Negative results were obtained with an Ames-Test and with a HPRT-Test. The positive in vitro results in a chromosome aberration test could not be confirmed in an in vivo Micronucleus Assay. Therefore no classification for mutagenicity is required according to EU directive 67/548/EEC and EU classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.