Morpholinium sulphamate

Endpoint summary

Additional information

For the assessment of genetic toxicity of morpholinium sulphamate several in vitro and in vivo gene mutation and in vitro cytogenetic studies conducted with morpholine, sulphamidic acid and morpholinium sulphamate were considered.

 

Morpholine (in vitro)

In an in vitro study (Huntsman, 1979), the analogous substance morpholine was evaluated for mutagenic potential in a mouse lymphoma forward mutation assay using L5178Y TK +/- cells at concentrations up to 1.25 µL/mL in the presence and absence of metabolic activation (S9 mix). In a third confirmatory assay concentrations from 1.2 to 2 µL/mL were also tested. The positive controls induced the appropriate responses. The test material induced an increase 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 cytotoxic and the mutant frequency increases (approximately 2.5 -fold) were at the limit of detectability for this assay. Concentrations up to 1.0 to 1.25 µL/mL were not very toxic and not detectably mutagenic in the presence of a metabolic activation system; concentrations from 1.25 to 1.5 µl/mL were highly cytotoxic. Under the conditions of this study, the analogous substance morpholine was considered to be very weakly mutagenic in the assay without metabolic activation. However, basic pH values of the culture medium at morpholine concentrations above 0.3 µg/mL followed by the induction of cell death suggested competition between toxic and mutagenic modes of action.

 

In a sister chromatid exchange assay (Huntsman, 1980), Chinese hamster ovary cells (CHO 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 25 % and 22 % over control with and without S9 mix, respectively. In the absence of a positive dose response these results indicated a negative response. The analogous substance morpholine was therefore considered to induce no increase in sister chromatid exchange under the conditions of this assay.

 

In another in vitro mutation study (Naylor Dana Institute, 1982), Morpholine was tested in a primary rat hepatocyte culture (HPC/DNA Repair Assay) at concentrations of 0.00001, 0.0001, 0.001, 0.01, 0.1 and 1%. As the two highest concentrations induced a high level of cytotoxicity these slides were not considered for mutation evaluation. Cytotoxicity was identified by a general absence of S-phase cells, and absence of grains in the few remaining hepatocytes and presence of hepatocytes with non-swollen nuclei. Counting of slides thus began with the slides exposed at 0.01 % and lower. The mean net nuclear grain counts of the slides exposed at 0.01 % and lower did not exceed 5. Therefore, it was concluded that the test substance was not genotoxic to the hepatocytes in this HPC/DNA repair assay. Parallel run DMSO and cell culture controls gave a mean net nuclear grain count of 0.3 ± 0.3 and 0.2 ± 0.4 respectively. The mean nuclear grain count of parallel run positive control was 20 ± 11.3 and negative control (pyrene) was 1.2 ± 1.1. Under the conditions of this assay, the analogous substance morpholine did not induce DNA repair over background at the highest non-toxic doses and lower doses.

 

Morpholine (in vivo)

In a transplacental mutagenesis study (Inui et al., 1979), sodium nitrite with Morpholine or Morpholine (500 mg/kg bw) alone were administered by a 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 has no mutagenic effect in vivo under the conditions of this study. Based on these results there was no evidence for a mutagenic effect of the analogous substance morpholine over background.

 

Sulphamidic acid (in vitro)

In an in vitro bacterial reverse mutation assay (De Flora, S. et al) the following tester strains were used to investigate the mutagenic potential of sulphamidic acid: Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli WP2 uvrA, WP67 uvrA-polA, CM871 uvrA- recA lexA. The test was performed using DMSO as vehicle. No data about the concentrations were specified in this publication. Instead, dose response relationship was evaluated. Sulphamidic acid was tested in each tester strain, both with and without S9 mix, at various dilutions (in duplicate or triplicate plates) performed by a geometric ratio of 2, starting from its solubility or toxicity limit for the Ames test. No substantial increases or decreases were observed in revertant colony numbers of any of the tester strains following treatment with sulphamidic acid at any concentration level, either in the presence or absence of metabolic activation (S9 mix). Therefore, the analogous substance sulphamidic acid is considered non-mutagenic in this bacterial reverse mutation assay.

 

In an in vitro mammalian cell gene mutation assay (CSERC Ltd, 2010) using chinese hamster ovary cells the mutagenic potential of sulphamidic acid was evaluated at concentrations up to 50 µg/mL in the presence and absence of metabolic activation (S9 mix).

In a preliminary cytotoxicity assay (by means of a growth inhibition test), the analogous substance was applied at a maximum concentration of 1200.0 mg/mL and 5 lower concentrations. Thereafter, five independent main assays were conducted. The treatment period was 4 h with and without S9 mix. Sulphamidic acid was dissolved in deionised water which was also used as solvent control, whereas the positive control substances ethylmethane sulfonate (EMS, not requiring activation) and 7,12-dimethylbenz(a)anthracene (DMBA, acting only after metabolic activation) were diluted in water and DMSO respectively. After expression, cells were dissociated and the total number of the passage cells was recorded, then cells were inoculated respectively into two groups. The first group was used for CFE expression (colony formation efficiency) determination (without 6-TG). Triplicate cultures were used for each group. The second group was used for determination of the mutagenicity factor. Triplicate cultures were used for each group. The sensitivity of the tests and the efficacy of the S9 mix were demonstrated by large increases in mutation frequency in the positive control cultures.

There were no biologically or statistically significant increases in mutation frequency at any concentration tested, either in the absence or in the presence of metabolic activation. There were no biologically differences between treatment and control groups and no dose-response relationships were noted. It was concluded that in this mutagenicity assay and under the experimental conditions reported, the analogous substance sulphamidic acid did not induce gene mutations at the HPRT-locus in chinese hamster ovary (CHO) cells.

 

In a further in vitro mammalian cell gene mutation assay (Bednarikova,M. et al., 2010) using V79 chinese hamster lung cells the analogous substance sulphamidic acid was tested at concentrations up to 3000 µg/mL in the presence and absence of metabolic activation (S9 mix).

Based on the results of the preliminary cytotoxicity study, the concentrations ranging from 31.25 µg/mL to 3000 µg/mL were chosen for in vitro mammalian cell gene mutation tests in the absence and presence of S9 mix. No marked changes of pH of treatment media were observed in the Range-Finder experiments at concentrations up to 3000 µg/mL tested as compared to the concurrent vehicle controls. In both experiments no cytotoxicity was observed at any concentration tested in the absence and presence of S9 mix.

In Experiment 1 no statistically significant increases in mean mutant frequency were observed following treatment with sulphamidic acid at any concentration tested either in the absence or in the presence of S9 mix.

In experiment 2 in the absence of S9 mix extended exposure of V79 cells to concentrations up to 2000 µg/mL for 24h resulted in a negative response. Statistically significant increase in mean mutant frequency was only observed at 31.25 µg/mL without S9 mix and at 125 µg/mL with S9 mix. In both cases fold increases greater than 3-fold over the vehicle control was not observed and mutant frequency fall within the range of historical control. Further this effect was not reproduced in independent experiment 1. Mutant frequencies in negative and positive control were consistent with the acceptable range. It was concluded that the analogous substance sulphamidic acid did not induce mutation at the hprt locus of V79 Chinese Hamster lung cells when tested under the conditions employed in this study.

 

The analogous substance sulphamidic acid was evaluated in an in vitro micronucleus assay (SLOVNAFT, 2010) according to Draft OECD 487 using human peripheral blood lymphocytes. The human peripheral blood lymphocytes were taken from two different healthy, non-smoking, young (between 31- 34 years of age) people (always man and women). The study was conducted at concentrations up to 860 µg/mL of blood cultivated in the presence and absence of metabolic activation (S9 mix). Due to the acidity of the test substance it was concluded that the highest test concentration in both test replicates was 344 µg/mL. The main test was performed with concentrations of 3.8, 68.8 and 344 µg/mL. 4000 to 6000 binucleate cells were analysed for one concentration of 2 - 3 experiments (test replicates). For measurement of cytotoxicity mitotic activity of cells was evaluated. The mitotic index was calculated as the percentage of mitotic cells and interphase cells. The results showed that the concentrations of the substance used in the test (2nd and 3rd test replicate) did not inhibit mitotic activity and did not raise the incidence of micronuclei in comparison with the intact control, in the variant without metabolic activation as well as with metabolic activation. Positive controls significantly increased the frequency of micronuclei compared with negative (intact) control. Under the conditions of the present study the test substance did not induce an increase of micronuclei and is therefore considered to be non-clastogenic and non-aneugenic.

 

Morpholinium sulphamate (in vitro)

In compliance with the OECD Guideline No. 471 and EU Method B.13/14, five bacterial strains, Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli WP2 uvrA were used to investigate the mutagenic potential of morpholinium sulphamate (Toxi Coop, 2012). Following concentrations were tested in two independent experiments composed of an initial mutation test (plate incorporation test) and a conformation mutation test (pre-incubation test): 5000, 1581, 500, 158, 50 and 15.8 μg test item/plate. Each assay was conducted with and without metabolic activation (S9 mix). The concentrations, including the respective positive and negative controls, were tested in triplicate.

No substantial increases or decreases were observed in revertant colony numbers of any of the five test strains following treatment with morpholinium sulphamate at any concentration level, either in the presence or absence of metabolic activation (S9 mix) in the performed experiments. Sporadic increases in revertant colony numbers compared to the vehicle control values were observed for certain strains in both independently performed main experiments. However, these increases remained in the corresponding historical control data ranges and there was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments.

It can be concluded that the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the strains used. Therefore, morpholinium sulphamate is considered non-mutagenic in this bacterial reverse mutation assay.

 

Conclusion

Due to the dissociation of the test item into the morpholinium cation and the sulphamidic anion in the culture medium as well as in the organism consideration of genetic toxicity data of only one part of the test item is justified.

Genetic toxicity testing of the analogous substances morpholine and sulphamidic acid revealed negative findings except one in vitro mouse lymphoma gene mutation assay with morpholine (Huntsman, 1979) in the absence of metabolic activity which produce an ambiguous result. As all further in vitro gene mutation assays as well as the available in vivo study with morpholine were clearly negative, morpholine is considered to be non-mutagenic.

It is concluded that testing of morpholinium sulphamate would not result in a positive result in any kind of genetic toxicity test both, in vitro and in vivo. This assumption is supported by the results of the bacterial reverse mutation assay with the test item (Toxi Coop, 2012) which did not revealed a biologically relevant induction of gene mutation in the tester strains used.

Justification for selection of genetic toxicity endpoint
For the assessment of genetic toxicity of morpholinium sulphamate several in vitro and in vivo gene mutation and in vitro cytogenetic studies conducted with morpholine, sulphamidic acid and morpholinium sulphamate were considered.

Short description of key information:
Genetic toxicity testing of the analogous substances morpholine and sulphamidic acid revealed negative findings except one in vitro mouse lymphoma gene mutation assay with morpholine (Huntsman, 1979) in the absence of metabolic activity which produce an ambiguous result. As all further in vitro gene mutation assays as well as the available in vivo study with morpholine were clearly negative, morpholine is considered to be non-mutagenic.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available in vitro and in vivo data the test item is not classified for genetic toxicity according to Regulation (EC) No 1272/2008 and Directive 67/548/EEC.