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Key value for chemical safety assessment

Additional information

Mutagenicity in bacteria

In the chosen key study, a bacterial reverse mutation assay (Ames test) according to OECD TG 471 and GLP,2-ethoxyethyl (2Z)-2-cyano-2-[3-(3-methoxypropylamino)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 NC plates was within the range of the historical NC data for each tester strain, without and with S9 mix. In addition, the PC items both without and with S9-mix induced a significant increase in the number of revertant colonies within the range of the historical PC data or above.

Bacteriotoxicity was observed in the SPT and PIT depending on the strain and test conditions from about 2625μg/plate onward. No test item precipitation was found without and with S9-mix. 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 chosen key study 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).

In this study, no relevant increase in the number of mutant colonies was observed either without or with S9-mix. 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. Osmolarity and pH values were not influenced by the test item treatment. At the end of the exposure period, test item precipitation was noted in Experiment II at 1700 and 3400 µg/mL only,i.e.at high concentrations which induced strong cytotoxicity. In conclusion, 2-ethoxyethyl (2Z)-2-cyano-2-[3-(3-methoxypropylamino)cyclohex-2-en-1-ylidene]acetate was not 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. 

 

 

Cytogenicity in mammalian cells/mammals

In the chosen key study in rats in vivo acc. to OECD TG 474 and GLP, 2-ethoxyethyl (2Z)-2-cyano-2-[3-(3-methoxypropylamino)cyclohex-2-en-1-ylidene]acetate (C-1701 B_C_3) was evaluated for its genotoxic potential (clastogenicity/aneugenicity) 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.

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 the high 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 as compared to the concurrent control males. Collectively, the clinical observations, the loss in body weight between days 1 and 2, and individually low PCE proportions are indicative of systemic exposure to the test substance in males, and systemic toxicity at the highest dose evaluated. 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.

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 using duplicate primary human lymphocyte cultures prepared from the pooled blood of two female donors in two independent experiments for clastogenicity and aneugenicity assessment (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.

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 NCs at the highest two concentrations analysed. The MNBN cell frequencies exceeded the 95th percentile 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 analysed. The MNBN cell frequencies exceeded the 95th percentile 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 analysed.

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 analysed. The MNBN cell frequencies exceeded the 95 % percentile 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 analysed. The MNBN cell frequencies exceeded the 95 % percentile 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. 

It needs to be noted, that test substance precipitations during treatment have been observed at 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 cannot be excluded. Furthermore, full removal of the test substance precipitate after centrifugation of the cells cannot be ensured, leading to a potential exposure of cells beyond the duration given in the study report. As given in the study report, no donor was suspected of any virus infection or exposed to high levels of radiation or hazardous chemicals. All donors are non-smokers and are not heavy drinkers of

alcohol. Donors were not taking any form of medication (contraceptive pill excluded). However, due to the use of primary cells in this study, a certain variability of the susceptibility for biological effects is to be expected, dependent on the hormone and/or health status of the respective donors. The historical control data used for comparison is based on 7 studies and54-70 single values respectively and might not adequately cover the whole effect range of untreated cells. 

 

 

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 databasis 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 an in vitro and in vivo micronucleus tests. 

In the in vitro micronucleus test (Covance Laboratories Ltd (2012), 8256347), 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 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, indicating that the bone marrow was exposed to the test item, there was no relevant increase in mnPCEs at any dose level as compared with the concurrent controls. Therefore, the negative in vivo test result does not confirm the questionable in vitro findings after the 3 -hour exposure period. Since 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.), 2-ethoxyethyl (2Z)-2-cyano-2-[3-(3-methoxypropylamino)cyclohex-2-en-1-ylidene]acetate is thus 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 non-genotoxic.


Short 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 not mutagenic. Increases in micronuclei observed in an in vitro micronucleus assay was not confirmed by an in vivo micronucleus assay in mice, prooving the absence of clastogenic and/or aneugenic potential of 2-ethoxyethyl (2Z)-2-cyano-2-[3-(3-methoxypropylamino)cyclohex-2-en-1-ylidene]acetate.

Endpoint Conclusion:

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.