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Genetic toxicity (mutagenicity) in bacteria in vitro

A well conducted bacterial gene mutation assay (Ames test) was performed with aminoiminomethanesulphinic acid (CAS 1758 -73 -2) according to OECD guideline 471 and under GLP conditions (Shibuya, 1990).

The strains Salmonella typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E.coli WP2 uvr A were tested according to preincubation procedure in the absence and presence of a metabolic activation system (phenobarbital and 5,6-benzoflavone-induced rat liver S9-mix). The experiment was conducted in triplicates at concentrations from 313 to 5000 µg/plate (vehicle: water) and positive controls. No increase in the number of revertant colonies was noted in Salmonella typhimurium TA 1537, TA 98, TA 100 and E.coli WP2 uvr A bacterial strains, with and without metabolic activation system. The test item was found to be mutagenic in Salmonella typhimurium TA1535 with or without an exogenous metabolic activation system. Cytotoxicity was not observed up to the highest dose tested with and without metabolic activation. The included positive and negative controls showed the expected results. Under the conditions of the study, the test substance did not induce mutations in the bacterial mutation assay in the absence and presence of a metabolic activation system in all the strains tested, except for Salmonella typhimurium TA1535.

A second reliable study (of lower quality) conducted with the test material according to OECD guideline 471 (Robertson and McGregor, 1981).

The strains Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 1538 and E.coli WP2 uvr A were tested according to plate incorporation procedure in the absence and presence of a metabolic activation system (Aroclor 1254 rat liver S9-mix).

The first experiment was conducted in triplicates at concentrations from 33.3 to 10000 µg/plate (vehicle: water) and positive controls. A second experiment was conducted in triplicates at concentrations varying from 740 to 10000 µg/plate.

The results of the first test indicated that the substance was mutagenic to the base-pair substitution sensitive S. typhimurium strains TA 1535 and TA 100.

A doubling of the negative control values for TA 1535 was obtained at concentrations per plate 1000, 3300 and 10000 µg test substance (both in presence and absence of S9 mix). In presence of S9 mix, the test compound was mutagenic to TA 100 at 3300 µg per plate. In presence of S9 mix, the test compound was mutagenic at 3300 and 10000 µg per plate. Although a weak mutagenic response by TA 100 to test substance in the absence of S9 mix was apparent, the 1.5 fold increase of the negative control value was not achieved in this test.

The conclusion from this test were confirmed when the test substance retested over a narrower dose range at high concentrations of the test substance per plate. The minimum concentration of the test substance giving a doubling of the negative control values for TA 1535 was 750 µg/plate either in the presence or absence of S9 mix. The results obtained with strains TA 100 (in presence or in absence of S9 mix) suggested a weak mutagenic response but the number of revertant colonies obtained did not reach the 1.5-fold increase of the negative control which is the criterion for demonstration of mutagenicity by this strain.

The test substance was therefore shown to be weakly mutagenic to the strains sensitive to base-pair substitution mutations, TA 1535 and TA 100. Mutagenicity was detected both in presence and absence of S9 mix. The lowest concentration of test substance causing a mutagenic response was 750 µg/plate with TA 1535.

 

Genetic toxicity (cytogenicity) in mammalian cells in vitro

An in vitro mammalian chromosome aberration test was conducted with aminoiminomethanesulphinic acid (CAS 1758 -73 -2) in accordance with OECD guideline 473 under GLP conditions (Sasaki, 1990). The induction of structural chromosome aberrations was evaluated in vitro in Chinese hamster lung (CHL/IU) cultures, incubated for 6, 24 and 48h with and without a metabolic activation system (S9-mix from rats treated with phenobarbitone and beta-naphthoflavone).

Concentrations of 0.14-0.55 mg/mL (6 h incubation, + S9), 0.28-1.1 mg/mL (6 h incubation, -S9), 0.15-0.60 mg/mL (24 h incubation, -S9), and 0.038-0.15 mg/mL (48 h incubation, -S9) of the test substance in water were applied. The negative as well as the positive controls showed the expected results. Cytogenetic effects were observed from 0.60 mg/mL (-S9, 24 and 48 h exposure) and above 1.1 mg/mL (-S9, 6 h exposure) and 0.55 mg/mL (+S9, 6h exposure). Statistically significant increase (no dose-related effect) in the incidence of chromosome aberrations was observed at the highest dose (were in most of the cases cytotoxicity was detected) in each treatment system excluding the continuous treatment for 48 h. Furthermore polyploidy was not induced. Under this condition the test item is considered non-mutagenic in this system.

 

Genetic toxicity (mutagenicity) in mammalian cells in vitro

Aminoiminomethanesulphinic acid (CAS 1758 -73 -2) was tested according to OECD guideline 476 (Verhagen, 1991).

In the first HGPRT test, the cells were exposed to at least 6 concentrations (from 4.75-10000 µg/mL) of the test item for 4 h in presence and absence of S9 mix. In the second test in the absence of S9 mix cells were exposed to concentrations varying from 250 to 1200 µg/mL and in the presence of S9 mix to concentration range 250-2500 µg/mL. Ethylmethane-sulfonate (in absence of S9 mix) and dimethylnitrosamine and dimethylbenzanthracene (in presence of the S9 mix) were used and positive control substances, while culture medium alone served as negative control. Both in the presence and in the absence of S9, the test substance neither induced a concentration related increase in the mutant frequency nor a reproducible positive response at one of the test concentrations. The positive control substances induced the expected increase in mutant frequency.

Thus, the test item does not induce point mutation in cultured CHO cells, under the conditions used in the present study. 

 

Genetic toxicity in vivo

An in vivo micronucleus assay with aminoiminomethanesulphinic acid (CAS 1758 -73 -2) was conducted according to OECD guideline 474 and GLP (Völkner, 1995). The test item was administered orally to groups of 5 male and 5 female mice at a dose level of 600 mg/kg bw in 0.5% aqueous carboxymethylcellulose-suspension (CMC). Bone marrow samples were taken 24, 48 and 72 h after dosing.

The positive control (cyclophosphamide) induced statistically significant increase in induced micronucleus frequency. The mean number of normochromatic erythrocytes (NCEs) was not increased after treatment with test substance acid as compared to the mean values of NCEs of the negative control, indicating that the test item had no cytotoxic properties in the bone marrow. There was no enhancement in the frequency of the detected micronuclei in comparison to the negative control at any preparation interval after administration of the test item. 30 mg/kg bw cyclophosphamide administered orally was used as positive control which showed a statistically significant increase of induced micronucleus frequency. During Thus, under the experimental conditions, the test item did not induce micronuclei. Therefore, the test item is considered to be non-mutagenic in this micronucleus assay.

An additional micronucleus assay conducted with aminoiminomethanesulphinic acid (CAS 1758 -73 -2) according to the method of Schimd, 1975.

Five male and female NMRI mice were treated orally (gavage) on two consecutive days with 29.7, 148.6 and 730 mg/kg bw of the test substance. Eight male and female NMRI mice were treated on two consecutive days with vehicle (water) and served as negative control group. 24 h after the second treatment (48 h after the first treatment) mice were sacrificed and bone marrow was removed from the femora and prepared on slides for examination.

1000 polychromatic erythrocytes (PCE) were scored for the incidence of micronuclei. Additionally, 1000 normochromatic erythrocytes (NCE) were also scored for the presence of micronuclei. The ratio of polychromatic to normochromatic erythrocytes was determined based on 1500 cells (PCE + NCE) counted per animal. After treatment of the mice with the test compound no substance related increase of micronucleated erythrocytes was observed at any dose level test in comparison with the negative control group. The positive control group (cyclophosphamide) showed a significant increase in the number of micronucleated erythrocytes in comparison with the negative control group.

In conclusion the test item was not considered mutagenic in this micronucleus assay.

 

Conclusions

Studies on genetic toxicity showed a weakly mutagenic effect of aminoiminomethanesulphinic acid in Salmonella strains TA 1535 with and without metabolic activation. No gene mutation potential of the test substance was detected in a mammalian cell system (CHO cells with and without metabolic activation) in a study which was performed according to current OECD 476 guidelines under GLP condition. One chromosome aberration test was conducted with the test item in CHL cells according OECD guideline 473 and under GLP conditions. Statistically significant increase in the incidence of chromosome aberrations was observed at the highest dose. The effects observed were not dose-related and occurred at cytotoxic concentrations.  Furthermore polyploidy was not induced. Under this condition the test item is considered non-mutagenic in this system.

Two micronucleus tests performed with the test item in vivo in mouse bone marrow did not show mutagenic activity in micronucleus assay.

Thus, on the basis of the data available aminoiminomethanesulphinic acid is not considered to have mutagenic potential.

 

References

Schmid W., (1995).The micronucleus test, Mutation Res.31:9-15.

Short description of key information:

Negative results in Salmonella typhimurium TA 1537, TA 98 and TA 100and E.coli WP2 uvrA with and without metabolic activation. Positive results in Salmonella typhimurium TA 1535 with and without metabolic activation (OECD 471, GLP).

Negative results in mammalian cell gene mutation tests using Chinese Hamster ovary (CHO), with and without metabolic activation (OECD 476, GLP)

Negative results in micronucleus assay using for rat bone marrow cells (OECD 474, GLP)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The available data on the genetic toxicity of aminoiminomethanesulphinic acid (CAS 1758-73-2) do not meet the criteria for classification according to Regulation (EC) No 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but nor sufficient for classification.