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Genetic toxicity testing of Morpholine produced mostly negative findings as well as borderline positive findings.

Morpholine was tested for mutagenicity in vitro in the Ames test with and without metabolic activation. In the key reverse gene mutation assay (BASF AG, 1981), Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2 were exposed to 0, 15.8, 50, 158, 500, 1580, 5000, 15800, 31600 or 50000 µg/plate in the standard plate test (plate incorporation method). Higher doses were tested, but were too cytotoxic. Slight increases in the number of revertants were observed at 50000 µg/plate with TA100 in the presence and absence of S9 mix and with WP2 uvrA only with metabolic activation. Morpholine exposure at 0.5, 1.0, 1.5 or 2.5 mL/20 L by means of the desiccator method did not reveal mutagenicity of Morpholine at any dose. Morpholine (or possibly an impurity) was weakly mutagenic in the Ames test at a concentration of 50000 µg/plate. However, the observed increase in the number of mutants at this relatively high dose was not more than 2-fold. Therefore, this test result is considered as ambiguous. The positive controls induced the appropriate responses in the corresponding strains. An Ames test reported by Haworth et al. (1983) was considered as supporting study as well as a test by Huntsman (1979). Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 were exposed to Morpholine at concentrations of 0, 109, 363, 1090, 3633 or 10900 µg/plate in the preincubation assay. Morpholine was tested up to slightly cytotoxic concentrations (10900 µg/plate). The positive controls induced the appropriate responses in the corresponding strains. In this study (and in the study by Huntsman, 1979), there was no evidence of a concentration-related positive response of induced mutant colonies over background.

In a key mouse lymphoma mutation assay with L5176Y TK +/- cells (Huntsman, 1979), the mutagenic potential of Morpholine was assessed in vitro at concentrations up to 1.25 µL/mL in the presence and absence of metabolic activation (S9 mix). This assay was repeated and in an additional assay concentrations from 1.2 to 2 µL/mL were tested. The positive controls induced the appropriate responses. The test material induced small increases in the mutation frequency over the applied concentration range of 0.625 to 1.25 µL/mL under non-activation conditions. These treatments were highly toxic and the mutant frequency increases (approximately 2.5 -fold) were at the limit of detectability for this assay with microsomal activation; concentrations up to 1.0 - 1.25 µL/mL were not very toxic and not detectably mutagenic; concentrations from 1.2 - 1.5 µl/mL were excessively lethal. Under the conditions of this study, Morpholine was considered to be very weakly mutagenic in the assay without metabolic activation. In a supporting study (Huntsman, 1979), Morpholine is considered to be active in the BALB/3T3 in vitro transformation assay.

In a key in vitro sister chromatid exchange assay (Huntsman, 1980), Chinese hamster ovary cells were treated with Morpholine at concentrations of 3.13, 6.25, 12.50, 25.00, 50.00 or 100.00 nL/mL in the presence and absence of mammalian metabolic activation (S9 mix). Positive control items induced the appropriate responses. The maximum increases in sister chromatid exchange noted were only 25 % and 22 % with and without S9 mix, respectively. In the absence of a positive dose response these results indicated a very weak, or negative, response. Morpholine did not cause a meaningful increase in sister chromatid exchange under the conditions of this assay.

Morpholine was tested in vitro in a rat hepatocyte primary culture (HPC/DNA Repair Assay) at concentrations of 0.00001, 0.0001, 0.001, 0.01, 0.1 or 1% (Huntsman, 1982). Positive control items induced the appropriate responses in this key study. Under the conditions of this assay, Morpholine did not induce DNA repair over background at the highest non-toxic doses and lower doses.

In an in vivo transplacental mutagenesis study (Inui et al., 1979), sodium nitrite with Morpholine or Morpholine (500 mg/kg bw) alone were administered by single oral gavage to pregnant Syrian golden hamsters on day 11 or 12 of pregnancy. Twenty-four hours after treatment, the hamster embryos were excised and examined for chromosomal aberrations, micronucleus formation, morphological or malignant transformation and drug resistance mutation. Cells exposed in utero to Morpholine showed no increases in the numbers of chromosomal aberrations, micronuclei, 8-azaguanine- or ouabain-resistant mutants, or transformation rates. The number of resistant colonies was markedly increased after administration of sodium nitrite together with Morpholine only, showing that Morpholine alone displayed no mutagenic activity in vivo under the conditions of this study.

 


Short description of key information:
Morpholine was not mutagenic in several key or supporting in vitro mutagenicity studies (Hawoth et al., 1983; Huntsman, 1980; Huntsman, 1982) and in a key in vivo mutagenicity study (Inui et al., 1979). However, ambiguous results or borderline mutagenicity were noted in a key Ames test (BASF AG, 1981) and a key mouse lymphoma mutation assay (Huntsman, 1979), respectively, at high and/or cytotoxic doses. Taking the overall evidence into account and with special regard to the partially equivocal findings at high doses in vitro, Morpholine is not considered to be mutagenic.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

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

Considering the ambiguous or negative results observed in the Ames test, the borderline mutagenicity noted in a mouse lymphoma assay, and the negative findings in a sister chromatid exchange assay, a DNA repair assay and in a transplacental mutagenesis study including evaluation of micronuclei and chromosomal aberrations, morpholine is not subject to classification for mutagenicity according to Regulation 1272/2008/EC.