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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In two Ames tests and an HPRT assay, 2-ethoxyethyl (2Z)-2-cyano-2-[3-(3-methoxypropylamino)cyclohex-2-en-1-ylidene]acetate was found to be non-mutagenic in bacteria and mammalian cells.

Clastogenicity by increases in micronuclei were observed in two in vitro micronucleus assays using primary human lymphocytes. The mode of action was defined to be predominantly clastogenic (chromosomal breakage). However, a clastogenic potential was not confirmed in a reconstructed skin micronucleus assay (RSMN) using the EpiDerm™ 3D skin model in vitro. Furthermore, an in vivo clastogenic potential of 2-ethoxyethyl (2Z)-2-cyano-2-[3-(3-methoxypropylamino)cyclohex-2-en-1-ylidene]acetate was not observed in a micronucleus assay in mice.

Additional information

Mutagenicity in bacteria

In the key study chosen, i.e. a bacterial reverse mutation assay (Ames test) according to OECD TG 471 and GLP, 2-ethoxyethyl (2Z)-2-cyano-2-[3-(3-methoxypropyl-amino)cyclohex-2-en-1-ylidene]acetate (C-1701 B_C_3) was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of the bacterial strains S. typhimurium TA1535, TA100, TA1537, TA98 and E. coli WP2 uvrA (BASF SE (2012), 40M0473/11M190). The standard plate test (SPT) and the preincubation test (PIT), both with and without a mammalian metabolic activation system (liver S9-mix from Phenobarbital and β-Naphthoflavone induced male Wistar rats), were performed using each a concentration range of 33 to 5250 µg/plate.

The test item did not induce a biologically relevant increase in the number of revertant colonies over background, either without S9-mix or with S9-mix in two independent experiments (SPT and PIT). The number of revertant colonies in the negative control plates was within the range of the historical data for each tester strain, with and without S9 mix. In addition, the positive controls (with and without S9-mix) induced a significant increase in the number of revertant colonies within the range of the historical control data or above. Bacteriotoxicity was observed in the SPT and PIT depending on the strain and test conditions from about 2625 μg/plate onward. No test item precipitation was found.

In conclusion, 2-ethoxyethyl (2Z)-2-cyano-2-[3-(3-methoxypropylamino)cyclohex-2-en-1-ylidene]acetate was not mutagenic in the bacterial reverse mutation test both in the absence and the presence of a mammalian metabolic activation system under the experimental conditions chosen.

In a supportive Ames test (BASF SE (2013), 40M473111M347) performed with the same study design, the same test item (batch C-1701/8) confirmed the data of the key study, i.e. showed no mutagenic potential in two independent experiments conducted each at concentrations ranging from 33 to > 5000 µg/plate.

 

Mutagenicity in mammalian cells

In the key study chosen for mutagenicity in mammalian cells (acc. to OECD 476 and GLP), 2-ethoxyethyl (2Z)-2-cyano-2-[3-(3-methoxypropylamino)cyclohex-2-en-1-ylidene]acetate (C-1701 B_C_3) was assessed for its potential to induce gene mutations at the Hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells (BASF SE (2012), 50M0473/11M219).

The test item was applied for 4 hours (with/without S9 mix) and 24 h (without S9 mix) at the following concentrations:

-      0;53.1*; 106.3*; 212.5*; 425*; 850*; 1700; 3400 μg/mL (without S9 mix; 4-hour exposure period)

-      0;53.1*; 106.3*; 212.5*; 425*; 850*;1700; 3400 μg/mL (with S9 mix; 4-hour exposure period)

-      0; 7.8; 15.6;31.3*; 62.5*; 125*; 250*;500; 1000 μg/mL (Follow up experiment: without S9 mix; 24-hour exposure period)

-      0; 31.3;62.5*; 125*; 250*; 500*;1000 μg/mL (Follow up experiment: with S9 mix; 4-hour exposure period)

*analysed for mutagenicity; other concentrations were not continued due to strong cytotoxicity or due to the fact, that a minimum of only four analysable concentrations are required.

After an attachment period of 20 - 24 hours and the respective treatment period, an expression phase of about 6 - 8 days and a selection period of about 1 week followed. Although performed according to the respective version of the OECD 476 guideline (21 Jul 1997) at the date of the study conduct, the number of cells incubated and plated for mutant selection were below numbers required according to the current OECD guideline (29 July 2016). The colonies of each test group were fixed with methanol, stained with Giemsa and counted.

No relevant increase in the number of mutant colonies was observed either without or with S9-mix. In the follow-up experiment with S9-mix, a statistically significant (p < 0.05, linear trend test) dose-related increase in the mutant frequency cells was found after 4 hours treatment (MFcorr.: 0.84-2.48 per 10^6 cells). However, the values obtained for the corrected mutation frequency of this experimental part were well within the respective historical NC data range (0.00–15.83 per 10^6 cells) and this dose response was not reproduced in the first experiment. Therefore, this finding was considered biologically irrelevant.

 

The negative controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, EMS and MCA, led to the expected increase in the frequencies of forward mutations, which clearly demonstrated the sensitivity of the test system used and of the S9-mix employed. At least the highest concentrations tested for gene mutations were clearly cytotoxic and revealed adversely influenced morphology and attachment of the cells. The cytotoxicity curve was very steep, and although valid mutation data could be obtained from 4 test substance concentrations in each part of the study, target range toxicity as required by the current OECD guideline (i.e. reduction in relative survival to 10-20% of concurrent solvent control) was not achieved. Osmolarity and pH values were not influenced by the test item treatment. At the end of the exposure period, test item precipitation was noted at 1700 and 3400 µg/mL, representing high concentrations, which induced strong cytotoxicity.

In conclusion, 2-ethoxyethyl (2Z)-2-cyano-2-[3-(3-methoxypropylamino)cyclohex-2-en-1-ylidene]acetate was determined to be non-mutagenic in the HPRT locus assay in the CHO cells under the experimental in vitro conditions used, in the absence and the presence of a mammalian metabolic activation system. Although, a potential lack in sensitivity is indicated due to a high fraction of plates with zero mutant colonies in the dataset and treatment concentrations achieving levels of cytotoxicity with a relative survival of 10-20% are lacking,the present study provides further evidence, that2-ethoxyethyl (2Z)-2-cyano-2-[3-(3-methoxypropylamino)cyclohex-2-en-1-ylidene]acetate is not mutagenic in mammalian cells.

 

Cytogenicity in mammalian cells/mammals

In an in vitro micronucleus assay acc. to OECD TG 487 and GLP, 2-ethoxyethyl (2Z)-2-cyano-2-[3-(3-methoxypropylamino)cyclohex-2-en-1-ylidene]acetate was tested for clastogenicity and aneugenicity using duplicate primary human lymphocyte cultures prepared from the pooled blood of two female donors in two independent experiments (Covance Laboratories Ltd 2013, 8256347). Cells were exposed to the test item in the vehicle DMSO for 3 hours (followed by 21 hours recovery) in the absence and the presence of a mammalian metabolic activation system or for 24 hours in the absence of S9-mix. Appropriate negative (vehicle; NC) and positive control cultures (PC) were included in the test system under each treatment condition. The following concentrations were tested for micronuclei:

-      750, 900, 1050 µg/mL (Experiment 1, 3-hour exposure, without S9 mix)

-      750, 900, 1000 µg/mL (Experiment 1, 3-hour exposure, with S9 mix)

-      80, 110, 155 µg/mL (Experiment 1, 24-hour exposure, without S9 mix)

-      400, 800, 1000 µg/mL (Experiment 2, 3-hour exposure, without S9 mix)

-      800, 950, 1000 µg/mL (Experiment 2, 3-hour exposure, with S9 mix)

In the first experiment, treatment of cells with the test item for 3 hours in the absence of S9 mix resulted in mean frequencies of micronucleated binucleate cells that were significantly higher than those observed in concurrent negative controls (NC) at the highest two concentrations analyzed. The MNBN cell frequencies exceeded the 95thpercentile and the upper limit of the observed historical NC range, and there was evidence of a concentration-related response. However, the only concentration at which the MNBN frequencies exceeded the upper limit of the historical NC range showed a cytotoxicity value above the target range of 50-60 %. The data therefore showed evidence of micronucleus induction under this treatment condition, but primarily at a cytotoxic concentration at which increased MNBN frequency might be a secondary effect of cytotoxicity.

Treatment of cells for 3 hours in the presence of S9-mix resulted in frequencies of MNBN cells that were significantly higher than those observed in concurrent NCs at the highest concentration analyzed. The MNBN cell frequencies exceeded the 95thpercentile and the upper limit of the historical NC range in both high concentration cultures.

Treatment of cells for 24 hours in the absence of S9-mix resulted in frequencies of MNBN cells that were similar to (and not significantly different from) those observed in concurrent NCs at all concentrations analyzed.

In the second experiment, treatment of cells for 3 hours in the absence of S9-mix resulted in frequencies of MNBN cells that were significantly higher than those observed in concurrent NCs at the highest two concentrations analyzed. The MNBN cell frequencies exceeded the 95thpercentile and the upper limit of the historical NC range with evidence of a concentration-related increase in MNBN cell frequency, thus fulfilling the criteria for a positive response. The data from Experiment 2 in the absence of S9-mix therefore confirmed the evidence of micronucleus induction seen in Experiment 1 at concentrations giving moderate levels of cytotoxicity, i.e. 31% and 39%.

Treatment of cells for 3 hours in the presence of S9-mix resulted in frequencies of MNBN cells that were significantly higher than those observed in concurrent NCs at all three concentrations analyzed. The MNBN cell frequencies exceeded the 95thpercentile and the upper limit of the historical NC range with evidence of a concentration-related increase in MNBN cell frequency. The data therefore showed evidence of micronucleus induction in the presence of S9-mix in Experiments 1 and 2.

In conclusion, 2-ethoxyethyl (2Z)-2-cyano-2-[3-(3-methoxypropylamino)cyclohex-2-en-1-ylidene]acetate induced micronuclei in cultured human peripheral blood lymphocytes when tested for 3 hours in the absence and presence of a mammalian metabolic activation system. In the same test system, the test item did not induce micronuclei when tested up to cytotoxic concentrations for 24 hours in the absence of metabolic activation. 

 

The potential of 2-ethoxyethyl (2Z)-2-cyano-2-[3-(3-methoxypropylamino)cyclohex-2-en-1-ylidene]acetate to induce micronuclei in cultured human peripheral blood lymphocytes was further investigated in a follow up study in the same testing laboratory using a comparable test protocol but applying fluorescence in situ hybridization (FISH) techniques with pan-centromeric DNA probes to discriminate between a chromosome breakage (clastogenicity) or a chromosome loss (aneuploidy) mechanism of action (Covance Laboratories Ltd 2014, 8303907). Lymphocyte cultures prepared from the pooled blood of two female donors and cells were treated with the test substance in the absence and presence of metabolic activation (S-9) from Aroclor 1254-induced rats. Three hour test substance treatments were conducted 48 hours following mitogen stimulation by phytohaemagglutinin (PHA). Appropriate negative (vehicle) control cultures (DMSO), clastogenic (Mitomycin C without S9; Cyclophosphamide with S9) and aneugenic (Noscapine) positive control chemicals were employed and provided evidence for the suitability of the test conditions chosen. Further, all acceptance criteria were considered met and the study was therefore accepted as valid. The following concentrations were tested for micronuclei:

-      400, 800,900*, 1000*µg/mL (3-hour exposure, without S9 mix)

-      800, 975,1025*, 1100*µg/mL (3-hour exposure, with S9 mix)

*analysed via fluorescence in situ hybridisation (FISH)

The treatment of cells with the test substance for 3+21 hours in the absence of S-9 resulted in frequencies of MNBN cells that were significantly higher than those observed in concurrent vehicle controls for the highest two concentrations analyzed (900 and 1000 µg/mL). The MNBN cell frequency of both treated cultures at 900 µg/mL and a single culture at 1000 µg/mL exceeded the normal range.

Treatment of cells with C-1701 B_C_3 for 3+21 hours in the presence of S-9 resulted in frequencies of MNBN cells that were significantly higher than those observed in concurrent vehicle controls for highest three concentrations analyzed (975, 1025 and 1100 µg/mL). The MNBN cell frequency of a single culture at 975 µg/mL and both replicate cultures at 1025 and 1100 µg/mL exceeded the normal range.

Incubation of the test substance at 900 and 1000 µg/mL following 3 hour treatments in the absence of S-9 resulted in 81 and 87% centromere-negative micronuclei, respectively.

Following 3 hour treatments in the presence of S-9 with 1025 and 1100 µg/mL, 81% and 94% centromere-negative micronuclei were recorded, respectively.

The aneugenic reference control gave 36% centromere-negative micronuclei and the clastogenic reference controls gave 69% and 91% respectively.

Overall, these data indicate that the mechanism of action of 2-ethoxyethyl (2Z)-2-cyano-2-[3-(3-methoxypropylamino)cyclohex-2-en-1-ylidene]acetate was predominantly clastogenic (chromosomal breakage) following treatments for 3 hours in the absence and presence of S-9 in this in vitro test system. As there are no noticeable differences between the responses in the absence and presence of S-9, there is no evidence that metabolism is involved in the clastogenic activity and a direct action of 2-ethoxyethyl (2Z)-2-cyano-2-[3-(3-methoxypropylamino)cyclohex-2-en-1-ylidene]acetate is indicated.

 

It needs to be noted, that test substance precipitations during treatment have been observed in all cultures, showing increased rates of micronuclei, whereas in cultures showing no precipitations, MN rates were comparable to respective controls (24 hour incubation without S9). Potential mechanic cell effects mediated by the test substance precipitate during the treatment of the cell suspension could not be excluded. Furthermore, full removal of the test substance precipitate after centrifugation of the cells was not ensured, leading to a potential exposure of cells beyond the duration intended. In the follow-up study, precipitation was also observed from 600 µg/ml onward during treatment. However, at the end of treatment, this precipitation was not considered suitable to limit concentrations for analysis in this study and was described as hazy media but not true test article precipitate in the assay system.

 

The induction of micronuclei by 2-ethoxyethyl (2Z)-2-cyano-2-[3-(3-methoxypropylamino)cyclohex-2-en-1-ylidene]acetate was further assessed in the reconstructed skin micronucleus assay (RSMN) using the EpiDerm™ 3D skin model (Bioreliance 2016; AE19SF358BTL). The test protocol chosen was derived from the OECD TG 487 and the study was conducted according to GLP. Tissues were treated by application of 10μL of the test article/vehicle mixture at the appropriate concentration on the top surface of the EpiDerm™ tissues. In the preliminary cytotoxicity and the definitive micronucleus assays, EpiDerm™ tissues were treated twice, 24 hours apart and tissues were processed at 48 hours (2-day dosing regimen). In the confirmatory micronucleus assay, the tissues were treated 3 times, 24 hours apart and tissues were processed at 72 hours (3-day dosing regimen). Acetone was used as the vehicle based on compatibility with the test system.

The preliminary cytotoxicity test was conducted by exposing a single tissue per concentration to vehicle alone and 15 concentrations of the test article ranging from 0.006 to 100 mg/mL. Cytotoxicity was assessed by the cytokinesis-block proliferation index (CBPI) and relative viable cell count (RVCC). In the preliminary assay,50% cytotoxicity by calculating CBPI relative to vehicle control was observed at concentrations50 mg/mL, while cytotoxicity by RVCC determination was not observed at any concentrations. Precipitate was observed on the tissue at concentrations50 mg/mL at the end of treatment. Based on these results, the definitive micronucleus assay was conducted using triplicate tissues at 9 concentrations of the test article ranging from 10 to 60 mg/mL. A 50 to 60% cytotoxicity (calculated by CBPI relative to vehicle control) was observed in the 3 replicates at the concentrations of 25 mg/mL and above, while cytotoxicity by RVCC determination was not observed at any concentrations. The concentrations selected for scoring micronuclei were 10, 20, 25, and 30 mg/mL. One thousand binucleated cells per tissue were scored for the presence of micronuclei.

The percentage of cells with micronucleated binucleated cells in the test article-treated tissues was not significantly increased relative to the vehicle control at any concentration tested.

Since the result of the micronucleus assay using a 2-day dosing regimen was negative, a confirmatory assay was conducted with a 3-day dosing regimen at concentrations ranging from 8 to 35 mg/mL using triplicate tissues. A 50 to 60% cytotoxicity (based on CBPI) was observed in the 3 replicates at the concentrations of 24 mg/mL and above, while cytotoxicity by RVCC determination was not observed at any concentrations. The concentrations selected for scoring micronuclei were 8, 20, and 26 mg/mL. The percentage of cells with micronucleated binucleated cells in the test article-treated tissues was not significantly increased relative to the vehicle control at any tested concentration.

In the definitive and confirmatory micronucleus assays, the percentage of micronucleated binucleated cells in the vehicle control was within the acceptable historical control range and the percentage of micronucleated binucleated cells in the positive control was statistically increased and within the historical positive range.

Based on the findings of this study 2-ethoxyethyl (2Z)-2-cyano-2-[3-(3-methoxypropylamino)cyclohex-2-en-1-ylidene]acetate was concluded to be negative for the induction of micronuclei in the reconstructed skin micronucleus assay (RSMN) in the EpiDerm™ model.

 

The in vivo relevance of the cytogenic potential (clastogenicity/aneugenicity) of 2-ethoxyethyl (2Z)-2-cyano-2-[3-(3-methoxypropylamino)cyclohex-2-en-1-ylidene]acetate was clarified in the chosen key study (acc. to OECD TG 474 and GLP), as measured by its ability to increase the incidence of micronucleated polychromatic erythrocytes (mnPCEs) in bone marrow of male and female Sprague-Dawley rats after repeated administration (BioReliance 2012, AD48SR.126.BTL). Groups of 5 Sprague-Dawley rats/sex/dose level received 2 applications of the test substance orally via gavage, suspended in Polyethylene glycol 300 (PEG 300) at dose levels of 500, 1000 or 2000 mg/kg bw (dose volume: 10 mL/kg bw). A concurrent control group of 5 rats/sex was treated in the same manner with the vehicle only. A positive control group of 5 rats/sex received a single oral gavage administration of Cyclophosphamide (CPA) in water at a dose level of 40 mg/kg bw. Bone marrow was harvested approximately 24 hours after the last (or only) treatment and evaluated microscopically for the presence of mnPCEs as well as for the proportion of PCEs (relative to total erythrocytes) as an indication of bone marrow cytotoxicity.

No mortality was observed in any of the treatment groups. Piloerection was noted after the second administration of 2000 mg/kg bw in all high dose group animals (day 1) and it persisted in the males until euthanasia (day 2). All other animals appeared normal during the study period. No appreciable changes in group mean body weights were observed in most groups, although a slight weight loss was observed in 3/5 top dose males between days 1 and 2. There were no statistically significant decreases in the proportion of PCEs to total erythrocytes at any dose level, indicating that the test item did not inhibit erythropoiesis. However, individual high dose males exhibited decreased PCE proportions (i.e ratios of 0.400 and 0.438) as compared to the concurrent control males (lowest ratio 0.514). Collectively, the clinical observations, the loss in body weight between days 1 and 2, and individually low PCE proportions are indicative of systemic exposure and systemic toxicity to the test substance in males at the highest dose evaluated, but do not provide a final proof as a definitive evidence of systemic (and therefore bone marrow) exposure. However, repeated oral dosing via gavage in rat reproductive/developmental and repeated dose toxicity studies showed systemic substance related effects at and above 300 mg/kg/day, which supports significant systemic exposure to 2-ethoxyethyl (2Z)-2-cyano-2-[3-(3-methoxypropylamino)cyclohex-2-en-1-ylidene]acetate.

No statistically significant increases in mnPCE frequencies were observed at any dose level of the test item as compared to the concurrent vehicle control. In contrast, the positive control item induced a statistically significant increase in mnPCE frequencies. All positive and vehicle control values were within acceptable ranges, and all criteria for a valid assay were met. Under the conditions of this in vivo study, 2-ethoxyethyl (2Z)-2-cyano-2-[3-(3-methoxypropylamino)cyclohex-2-en-1-ylidene]acetate was negative in the bone marrow micronucleus test in male and female rats after repeated administration of dose levels that produced effects indicative of systemic exposure.

 

Conclusion on genetic toxicity

The negative results obtained in bacterial reverse mutation assays (Ames tests; BASF SE (2012), 40M0473/11M190 and BASF SE (2013), 40M0473/11M347) and in an in vitro mammalian cell gene mutation test (HPRT; BASF SE (2012), 50M0473/11M219) provide a robust data base for the absence of any mutagenic potential of 2-ethoxyethyl (2Z)-2-cyano-2-[3-(3-methoxypropylamino)cyclohex-2-en-1-ylidene]acetate.

The potential of 2-ethoxyethyl (2Z)-2-cyano-2-[3-(3-methoxypropylamino)cyclohex-2-en-1-ylidene]acetate to induce clastogenic and/or aneugenic effects was assessed in in vitro and in vivo micronucleus tests. 

In the in vitro micronucleus tests (Covance Laboratories Ltd 2013 and 2014, 8256347 and 8303907), 2-ethoxyethyl (2Z)-2-cyano-2-[3-(3-methoxypropylamino)cyclohex-2-en-1-ylidene]acetate induced micronuclei in cultured human peripheral blood lymphocytes when tested for 3 hours in the absence and presence of a mammalian metabolic activation system. In the same test system, however, 2-ethoxyethyl (2Z)-2-cyano-2-[3-(3-methoxypropylamino)cyclohex-2-en-1-ylidene]acetate did not induce micronuclei when tested up to cytotoxic concentrations for 24 hours in the absence of metabolic activation. The data provide evidence, that a predominantly clastogenic (chromosomal breakage) mechanism of action was responsible for the effects observed in primary lymphocytes in vitro under the testing conditions chosen.

In contrast, no induction of micronuclei in vitro by 2-ethoxyethyl (2Z)-2-cyano-2-[3-(3-methoxypropylamino)cyclohex-2-en-1-ylidene]acetate was observed in a reconstructed skin micronucleus assay (RSMN) using the EpiDerm™ 3D skin model (Bioreliance 2016; AE19SF358BTL).

The in vivo micronucleus test (BioReliance (2012), AD48SR.126.BTL) assessed clastogenicity/ aneugenicity via the ability of 2-ethoxyethyl (2Z)-2-cyano-2-[3-(3-methoxypropylamino)cyclohex-2-en-1-ylidene]acetate to increase the incidence of micronucleated polychromatic erythrocytes (mnPCEs) in bone marrow of rats after repeated oral gavage treatment at dose levels above the limit dose. While effects indicative of systemic toxicity were noted at the high dose level in this study there was no relevant increase in mnPCEs at any dose level as compared with the concurrent controls. Although the decrease in proportions of PCE were relatively small and the systemic effects observed in the in vivo MN test do not provide a final proof of systemic (and therefore bone marrow) exposure, repeated oral dosing via gavage in rat reproductive/developmental and repeated dose toxicity studies showed systemic substance related effects at and above 300 mg/kg/day, which supports significant systemic exposure to 2-ethoxyethyl (2Z)-2-cyano-2-[3-(3-methoxypropylamino)cyclohex-2-en-1-ylidene]acetate. Since these were seen at daily doses lower than those used in the in vivo MN test, there must also have been systemic and therefore bone marrow exposure in the MN test. Therefore, the negative MN findings at doses up to 2000 mg/kg are considered robust, and demonstrate that the clastogenic activity of 2-ethoxyethyl (2Z)-2-cyano-2-[3-(3-methoxypropylamino)cyclohex-2-en-1-ylidene]acetate seen in human lymphocytes in vitro is not manifest at high doses in vivo.

The negative in vivo and in vitro test results in reconstituted skin do not confirm the in vitro findings after the 3-hour exposure period in primary lymphocytes. The EpiDerm™ 3D skin model contains cells of high relevance for human exposure and the exposure conditions chosen in the RSMN are of high comparability to exposure scenarios concerning the use of 2-ethoxyethyl (2Z)-2-cyano-2-[3-(3-methoxypropylamino)cyclohex-2-en-1-ylidene]acetate in cosmetic products. Furthermore, a higher degree of reliability and relevance is attributed to in vivo testing data (ECHA Guidance on information requirements and chemical safety assessment. Chapter R.7A: Endpoint specific guidance.) compared to the in vitro test results derived from human lymphocyte cultures. Thus, 2-ethoxyethyl (2Z)-2-cyano-2-[3-(3-methoxypropylamino)cyclohex-2-en-1-ylidene]acetate is considered to be non-clastogenic and non-aneugenic.

Taken together, the results obtained in the available test battery - addressing all relevant endpoints of genotoxicity - raise no concern, but indicate that 2-ethoxyethyl (2Z)-2-cyano-2-[3-(3-methoxypropylamino)cyclohex-2-en-1-ylidene]acetate is a non-genotoxic substance.

Justification for classification or non-classification

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