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In vitro Gene Mutation in Bacteria Cells

In a GLP-compliant OECD guideline 471 study (BASF, 2014), ammonium carbamated was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strands, i.e. Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 98, and E. coli WP2 uvrA. No precipitation of the test substance was found with and without S9 mix. A weak bacteriotoxic effect was observed in the standard plate test only in tester strain TA 1535 without S9 mix at a concentration of 5 000 μg/plate. The test substance did not lead to a biologically relevant increase in the number of revertant colonies either without S9 mix or after adding a metabolizing system in two experiments carried out independently of each other (standard plate test and preincubation assay). Besides, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study. The number of revertant colonies in the negative and in the positive controls was within or only marginally above/below the range of the historical control data for each tester strain.

It was concluded that ammonium carbamate is not mutagenic in the bacterial reverse mutation test in the absence and the presence of metabolic activation.

This result was supported by an older OECD 471 study with acceptable restrictions, in which bacteria strains of S. typhimurium TA 98, TA 1535, TA 1537, TA 100 were exposed to ammonium carbamate (>99.8% pure) in distilled water up to the limit concentration (0, 20, 100, 500, 2500, 5000 µg/plate) in the presence and absence of mammalian metabolic activation using both the preincubation and the plate incorporation methods. No cytotoxity occurred. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background (BASF AG 1991).

In conclusion, ammonium carbamate is considered to not be mutagenic in the bacterial reverse mutation test.


In vitro Gene Mutation in Mammalian Cells

The ability of ammonium carbamate to induce gene mutations in mammalian cells was studied in a GLP-compliant HPRT locus assay (BASF SE, 2010d), performed according to OECD guideline 476, using Chinese hamster ovary (CHO) cell line. Two independent experiments were performed, both with and without metabolic activation. In the first one, using 4 hours exposure duration, cells were treated with the test substance in culture medium at test concentrations of 0, 100, 200, 400 and 800 μg/ml. In second experiment, 24-hour exposure period was used without S9-mix, using the same test substance concentrations. In the presence of S9 mix, exposure period was 4 hours and test concentrations were 0, 200, 400, 600 and 800 μg/ml.

No relevant increase in the number of mutant colonies was observed either without S9 mix or after the addition of a metabolising system. The positive control substances ethyl methanesulfonate (without S9 mix; 300 μg/ml) and methylcholanthrene (with S9 mix; 20 μg/ml) induced clearly increased mutant frequencies as expected. In both experiments, in the absence and presence of S9 mix, no cytotoxicity indicated by reduced relative cloning efficiency of below 20% relative survival was observed up to the highest required test substance concentration. However, the highest tested concentrations corresponded to the limit dose of 10 mM, recommended by the OECD guideline. Based on these results, ammonium carbamate is concluded not to induce gene mutations in mammalian cells in vitro.


In vitro Cytogenicity in Mammalian Cells

A broad base of literature data and the results of hydrolysis studies performed by the registrant indicate that ammonium carbamate rapidly decomposes into ammonium and carbonate/bicarbonate at physiological concentrations and pH (see rationale in chapter 5.1.3). Therefore, data from the surrogate substance ammonium hydrogen carbonate (CAS# 1066-33-7) was used for this endpoint. In a mammalian cell cytogenetics assay, Chinese hamster fibroblast cells were treated with 3 concentrations (maximal concentration of 250 µg/ml) in phosphate buffer for 24 and 48 hours without metabolic activation. Cells were treated with colcemid 2 hours prior harvest, fixed and stained with Giemsa. There was no evidence of chromosomal aberration induced over background (Ishidate et al, 1984).


In vivo Cytogenicity in Mammalian Cells

Due to the lack of data on in vivo genetic toxicity, a study with ammonium chloride (CAS# 12125-02-9) was taken into account for assessment. No data are available concerning the genetic toxicity of carbonate in vivo. However, carbonate is naturally present in cells, and its structure does not indicate a genotoxic potential. Therefore there is no reason to evaluate the potential genotoxicity of carbonate further, and no genotoxic effects are expected. In a ddY mouse bone marrow micronucleus assay (6 male/dose) were treated by intraperitoneal (ip) injection with ammonium chloride (99.7% pure). Ammonium chloride was tested at an adequate dose based on a preliminary dose finding experiment. Administration was performed as a single injection at doses of 0, 62.5, 125, 250, 500 mg/kg bw or as multiple injections (4 times) at 24 hour intervals at doses of 0, 31.3, 62.5, 125, 250 mg/kg bw. Bone marrow cells were harvested 24 hours after the last injection. Mytomycin C, administered at 2.0 mg/kg bw via ip injection served as the positive control. One thousand polychromatic erythrocytes (PCE) were scored for micronucleus. The vehicle was saline. No mortality occurred 24 hours after single dose treatment or the multiple dosing regimen. Percentage PCE/NCE (polychromatic/normochromatic erythrocytes)was comparable in the bone marrow of treated and control animals after single and multiple exposure. The positive control induced the appropriate response. There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time (Hayashi et al, 1988). This study is acceptable for assessment. The study is equivalent to OECD guideline 474 (Mammalian Erythrocyte Micronucleus Test) with accepatable restrictions (only 1000 PCE scored, incomprehensive reporting style).

Short description of key information:
Genetic toxicity:
- In vitro gene mutation in bacteria: negative (Ames);
- In vitro cytogenicity: negative (chromosome aberration test; read across ammonium bicarbonate);
- In vitro gene mutation in mammalian cells: negative (HPRT locus assay);
- In vivo cytogenicity: negative (MNT; read across ammonium chloride).

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available data, classification is not warranted according to EU Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.