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In vitro

Gene mutation in Bacteria

In a non-GLP, non-Guideline bacterial reverse mutation assay, strains TA1535 and TA100 of S. typhimurium and WP2 uvr A- of E. coli were exposed to the test item (purity unknown), using water as solvent, at concentrations of 1800 µM in the abscence of metabolic activation in the plate incorporation method (Thompson et al., 1981). The test substance was used as negative control in a test series were dialkylaminoalyl chlorides were evaluated for their mutagenicity. No data on determination of cytotoxicity and the use of a positive control substance was given. The study is classified as acceptable (reliability 2), but does not satisfy the requirement for OECD 471/472 for in vitro bacterial reverse gene mutagenicity.

In addition, there is no weight of evidence from a gene mutation assay in bacteria with the structural analogue 1-methylpiperidine (CAS No. 626 -67 -5). In this bacterial reverse gene mutation assay according to GLP and OECD 471 and 472 strains TA 98, TA 100, TA 1535 and TA 1537 of S. typhimurium and E.coli WP2 uvrA were exposed to the test item (analytical purity: 80.2 %), using water as solvent, at concentrations of 25, 125, 625, 3125, 6250 µg/plate (Standard plate incubation test) and 20, 100, 500, 2500, and 5000 µg/plate (Preincubation test, 20 minutes, 37 °C) in the presence and absence of mammalian metabolic activation (liver fraction (S-9) from Aroclor 1254 induced rat) (BASF, 40M0385/964256, 1997). The test item was tested up to cytotoxic concentrations. The positive control induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background. Cytotoxicity was observed at a concentration of > 2500 µg/plate with and without metabolic activation in the Preincubation test. The study is classified as acceptable (reliability 1), but does not fully satisfy the guideline requirements for a gene mutation assay (OECD 471/472) in bacteria. Despite the requirements of OECD Guideline 471 (adopted 1997), 2-Aminoanthracene was the sole positive control in the presence of S9-Mix.

The test item was negative in the Ames test using Salmonella typhimurium strains TA98, TA100, TA 1535 and TA1537 and E. coli WP2 uvrA with and without metabolic activation with S9 from Aroclor-induced rat liver. Therefore, due to the structural similarity, 1-ethylpiperidine is considered not to be mutagenic in bacterial cells with and without metabolic activation.

Gene mutation in mammalian cells

In a GLP conform in vitro mammalian cell gene mutation test (HPRT-Test) according to OECD Guideline 476 (Harlan 1464809 / BASF, 50M0721/11X361, 2012) the test substance was investigated for its potential to induce gene mutations at the HPRT locus in V79 cells. The assay was performed in two independent experiments, using two parallel cultures each. In the first main experiment cells were exposed for 4 hours to the test item at concentrations of 35.6; 71.3; 142.5; 285; 570; 1140 μg/mL with and without liver microsomal activation. The second experiment was performed with a treatment time of 4 hours with metabolic activation and a treatment time of 24 hours without metabolic activation, both at the same concentrations as in the first experiment.The maximum concentration of 1140μg/mL was equal to a molar concentration of about 10 mM. The test item was dissolved in deionised water.

Appropriate reference mutagens (EMS and DMBA), used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

No relevant toxic effects occurred up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment.

No substantial and reproducible dose dependent increase in mutant colony numbers/10E6 cells was observed up to the maximum concentration with and without metabolic activation. All mutant frequencies remained well within the historical range of solvent controls. The study is classified as acceptable (reliability 1) and satisfies the requirement for OECD 476 for in vitro gene mutation in mammalian cells.

According to the results of the study, the test substance did not induce gene mutations in the in vitro mammalian cell gene mutation test under the experimental conditions chosen.

Supportingly, in a non-GLP, non-Guideline mammalian cell gene mutation assay (thymidine kinase locus), mouse lymphoma L5178Y cells were exposed to the test item dissolved in water (Thompson et al., 1981). No data on test substance purty and concentrations were given. The test substance was used as negative control in a test series were dialkylaminoalyl chlorides were evaluated for their mutagenicity. No data on determination of cytotoxicity and the use of a positive control substance was given. The study is classified as acceptable (reliability 2), but does not satisfy the requirement for OECD 476 for in vitro gene mutation in mammalian cells.

DNA Damage and repair in rat hepatocytes

In a rat liver unscheduled DNA synthesis (UDS) indicator test (non-GLP, non-Guideline study), adult male rat hepatocytes were prepared by in situ perfusion of the livers of Fischer rats and treated with 1.999 nmol (226 µg/mL) of the test substance (Thompson et al., 1981). The autoradiographic assay for unscheduled DNA synthesis was conducted as described in Williams (1977) and modified by Probst et al. (1980). N-methyl-N-nitro-N-nitrosoguanidine (100 nmol/ml), 2-acetylaminofluorene (5 nmol/ml) and UV-light (300 ergs/mm²) served as positive control and yielded the expected results. The test substance was used as negative control in a test series were dialkylaminoalyl chlorides were evaluated for their mutagenicity. No data on determination of cytotoxicity or further details were given. The study is classified as acceptable (reliability 2), but does not satisfy the requirement for OECD 482/486 for genotoxic mutagenicity.

Cytogenicity in mammalian cells

The clastogenic potential of the test substance was evaluated in a chromosome aberration test in vitro according to GLP and OECD 473 (Bayer, T4076137, 2006). Chinese hamster V79 cells were exposed in the absence and in the presence of S9 mix for 4 hours to concentrations of 300, 600 and 1200 µg/ml of the test substance (puritiy was not given). Cultures of all concentrations were harvested 18 hours after the beginning of the treatment. In addition, cells treated with 1200 µg/ml were harvested 30 hours after the beginning of the treatment. All concentrations were used for reading. Without S9 mix an additional experiment was performed using continuous treatment for 18 hours and test substance-concentrations of 100, 200, 400, 600 and 800 µg/ml. Based on their cytotoxicity, which was determined 8 hours after the beginning of the treatment, three concentrations of this continuous treatment were selected for reading of metaphases.

Without S9 mix cytotoxic effects were observed at 1200 µg/ml after 4 hours treatment and at 400 µg/ml and above after 18 hours treatment. With S9 mix cytotoxic effects were observed at 1200 µg/ml. Precipitation of the test substance in the medium was not observed.

After 4 hours of treatment concentrations of 300, 600 and 1200 µg/ml test substance were used for reading in the absence as well as in the presence of S9 mix. In addition, 200, 400 and 600 µg/ml were chosen for reading after 18 hours of treatment without S9 mix. All of the cultures harvested 18 hours after the beginning of the treatment were included. In addition, cultures treated with 1200 µg/ml and harvested 30 hours after the beginning of the treatment were used.

None of the cultures treated with the test substance in the absence and in the presence of S9 mix showed biologically relevantly increased numbers of aberrant metaphases. The positive controls mitomycin C and cyclophosphamide induced clastogenic effects and demonstrated the sensitivity of the test system and the activity of the used S9 mix. The study is classified as acceptable (reliability 1), and does satisfy the guideline requirements for OECD 473 for in vitro mammalian cytogenicity.

Based on this test, the test item is considered not to be clastogenic for mammalian cells in vitro with and without metabolic activation with S9 from Aroclor-induced rat liver.


Short description of key information:
in vitro
Gene mutation in bacteria
(1) Ames test with S. typhimurium TA 1535 and TA 100 / E. coli WP2 uvr A- without metabolic activation: the test substance was used as negative control, non-GLP, non-Guideline, Val. 2 (Thompson et al., 1981)
(2) read across: 1-methylpiperidine (CAS No. 626 -67 -5) Ames test with S. typhimurium TA 98, TA 100, TA 1535 and TA 1537 and E. coli WP2 uvrA, with and without metabolic activation: the test substance was negative, GLP, OECD 471 and 472, Val. 1 (BASF, 40M0385/964256, 1997)

Gene mutation in mammalian cells
(1) Mammalian cell gene mutation assay with chinese hamster V79 cells: 35.6; 71.3; 142.5; 285; 570; 1140 μg/mL with (4 hours treatment) and without (4 and 24 hours treatment) metabolic activation: the test substance did not cause gene mutations, GLP, OECD 476, Val. 1 (Harlan, 1464809, 2012)
(2) Mammalian cell gene mutation assay with mouse lymphoma L5178Y cells; no details on metabolic activation: the test substance was used as negative control, non-GLP, non-Guideline, Val. 2 (Thompson et al., 1981).

DNA Damage and repair
Adult male rat hepatocytes were treated with 1.999 (226 µg/mL) of the test substance: the test substance was used as negative control, non-GLP, non-Guideline, Val. 2 (Thompson et al., 1981).

Cytogenicity in mammalian cells
Mammalian chromosome aberration test in V79 cells: 300, 600, 1200 µg/mL (4 hours treatment with and without metabolic activation) and 200, 400, 800 µg/mL (18 hours treatment without metabolic activation): the test substance did not cause clastogenic effects, GLP, OECD 473, Val. 1 (Bayer, T4076137, 2006).

in vivo
no data available

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available experimental test data for genetic mutagenicty are reliable and suitable for the purpose of classification under Directive 67/548/EEC. As a result the substance is not warranted to be classified for mutagenicity under Directive 67/548/EEC.

 

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data for genetic mutagenicity are reliable and suitable for the purpose of classification under Regulation (EC) No.1272/2008. As a result the substance is not warranted to be classified for mutagenicity, under Regulation (EC) No.1272/2008.