Reaction mass of (2S,6S)-2,6-Dimethylmorpholine and (2R,6R)-2,6-Dimethylmorpholine

Endpoint summary

Additional information

In vitro studies:

In an Ames test performed according to OECD Guideline 471 (Bacterial Reverse Mutation Assay) with Salmonella typhimurium TA 1535, TA 1537, TA 98 and TA 100, cis-2,6-dimethylmorpholine (CAS 6485-55-8) concentrations of 20, 100, 500, 2500 and 5000 µg/plate were employed in the presence and absence of Aroclor 1254-induced rat liver S9 mix. At non-toxic test substance concentrations, a significant increase in mutant frequency was not observed (BASF AG, 1995; Val. 2). In a similar AMES test according to OECD Guideline 471 and OECD 472 (Bacterial and E. coli, Reverse Mutation Assay) an isomer mixture of 2,6-dimethylmorpholine (CAS 141-91-3) showed also negative results in the absence and present of Aroclor 1254-induced rat liver S9 mix (BASF AG, 1998; Val. 2).

In two further Ames tests according a standard protocol approved by the National Toxicology Program, 2,6-dimethylmorpholine (CAS 141-91-3) was tested in four Salmonella typhimurium strains (TA98, TAl00, TAl535 and TAl537) in the presence and absence of Aroclor-induced rat or hamster liver S9 (NTP 1980, 1981; Val. 2). In one test a slight increase in revertants was noted in the Salmonella typhimurium strain TA 1535. No noteworthy increase in the number of revertants was observed for all other strains with and without metabolic activation and also in replicate no increase in revertants in the strain TA 1535 was observed.

 

In an in vitro cytogenetics assay using chinese hamster lung fibroblasts (V79), cis-2,6-dimethylmorpholine (CAS 6485-55-8) was tested both in the absence and presence of metabolic activation (S9 mix), according to the OECD Guideline 473 (BASF AG, 2007; Val.1). The test substance did not cause any biologically relevant increase in the number of structurally aberrant metaphases incl. and excl. gaps at both sampling times either without S9 mix and/or after adding a metabolizing system in two experiments performed independently of each other. The frequency of cells containing numerical chromosome aberrations was strongly increased in the 2nd Experiment after 18 hours exposure at 28 hours-sampling time in the absence of S9 mix. The rate of polyploid cells after treatment with 600 μg/mL (20.05%) clearly exceeded the value of the respective negative control group (3.0%). This observation was corroborated by an increased sample of evaluation of 1000 cells per culture scoring 2 cultures per test group. Cis-2,6-dimethylmorpholine is considered not to have a potent chromosome-damaging (clastogenic) effect under in vitro conditions in V79 cells in the absence and the presence of metabolic activation. However, based on the observation of a strongly increased frequency of polyploid cells after 18 hours treatment and 10 hours recovery in the absence of metabolic activation it has to be considered that the test substance has an aneugenic potential under the experimental conditions described under in vitro conditions in V79 cells.

 

In a in vitro gene mutation test in CHO cells according to OECD Guideline 476 (HPRT forward mutation assay), cis-2,6-dimethylmorpholine (CAS 6485-55-8) did not cause any increase in the mutant frequencies either without S9 mix or after adding a metabolizing system in two experiments performed independently of each other (BASF AG, 2007; Val. 1)

 

In vivo studies:

In a micronucleus assay with NMRI mice according to OECD TG 474, 5 male/female animals per group were orally administered single doses of 500, 1000, 2000 mg/kg bw cis-2,6-dimethylmorpholine (CAS 6485-55-8; BASF AG, 2007; Val.1). The single oral administration did not lead to any increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was always within the same range as that of the concurrent negative control in all dose groups and at all sacrifice intervals and within the range of the historical negative control data. An inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected in the dose of 2000 mg/kg body weight (48-hour sacrifice interval). The test substance did not have any chromosome-damaging (clastogenic) effect, and there were no indications of any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo.

 

Short description of key information:
In vitro studies:
Ames-Test: negative (OECD 471)
Chromosomal Aberration Test: negative (OECD 473)
HPRT-Test: negative (OECD 476)

In vivo studies:
Mouse Micronucleus Test: negative (OECD 474)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Several valid Ames-tests (OECD 471), with or without metabolic activation through S9-Mix, a valid HPRT forward mutation assay (OECD Guideline 476), a valid chromosomal aberration assay (OECD Guideline 473), and a valid in vivo Mammalian Bone Marrow Chromosome Aberration Test (OECD Guideline 474) failed to provide evidence for a clastogenic effect of cis-2,6-dimethylmorpholine. The observed increased frequency of polyploid cells (aneugenic potential) in an in vitro chromosomal aberration assay in V79 cells (OECD 473) couldn´t be confirmed in a in vivo Mammalian Bone Marrow Chromosome Aberration Test (OECD Guideline 474). Therefore cis-2,6-dimethylmorpholine is not considered to have a mutagenic potential.