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In vitro studies

Valid experimental data were available to assess the genetic toxicity in vitro.

 

Gene mutation in bacteria

Methylurea (CAS No. 598-50-5) with an analytical purity of ≥ 98.5 % was tested for its mutagenic potential at doses of 20, 100, 500, 1000, 2500, 3000, 4000 and 5000 µg/plate in Salmonella strains TA1535, TA100, TA1537 and TA98 with and without metabolic activation according to OECD guideline 471 and EU Method B.13/14 (Report 40M0085/914081, BASF 1992). The test was performed GLP conform, with the deviation, that analytical investigation of the stability of the test substance in water was not carried out. Testing with Salmonella typhimurium TA102 or Escherichia coli was not carried out. Therefore, the study does not completely fulfill the reliability criteria of today's guidelines. The study was performed as independent standard plate test and preincubation test. An S-9 mix of Aroclor 1254 -induced rat livers served as metabolic activation system. The substance was dissolved completely in water.

An increase in the number of his+ revertants was not observed and consistently a negative result was obtained. No cytotoxic effects were detected.

The positive control substances (2-aminoanthracene for all strains with metabolic activation; N-methyl-N´-nitro-N-nitrosoguanidine for the strains TA 100 and TA 1535, 4-nitro-o-phenylendiamine for the strain TA 98; 9-aminoacridine chloride monohydrate for the strain TA 1537 without metabolic activation; all substances dissolved in DMSO) produced valid responses.

In a second Ames test following no guideline (Ishidate 1981) mutagenicity was determined in Salmonella typhimurium TA98, TA100, TA1537 and assessed to be negative. The reliability of the study is not assignable due to insufficient documentation, methodological deficiencies and does not meet important criteria of today's standard methods (only 3 tester strains (Salmonella typhimurium TA 102 or Escherichia coli WP2 uvrA not included, no information about concentration levels, vehicle, solubility, cytotoxicity, and positive controls.

In an other bacterial gene mutation assay following no guideline (Tanooka 1977) Bacillus subtilis TKJ5211 with suppressible mutations in a hisB and a met gene were treated with 0.1 ml of the test chemical (500 μg) in DMSO without metabolic activation. After two days incubation at 37°C, colonies of His+ mutants were enhanced. No double mutations within the His+ colonies were detected. The authors assessed the test compound as positive.

 

Cytogenicity in mammalian cells

 

In mammalian chromosome aberration test following no guideline (Ishidate 1983, Ishidate and Kada 1980, Ishidate and Odashima 1977, Ishidate 1981), Chinese hamster fibroblast cell line (CHL cells) were exposed to Methylurea in physiological saline at concentrations of 1, 10 and 100 mM without metabolic activation for 24 and 48 hours. Chromatid or chromosomal breaks occurred at an incidence of 1% at the 24-hour harvest. No other types of chromosomal aberrations were observed after treatment for 24 h. No chromosomal aberrations were at the 48-hour harvest. The number of polyploid cells ranged from 0% to 2% at both 24 and 48 hours and was similar to negative controls. Vehicle control cells showed 1% polyploid cells and 0.8% chromosomal aberrations at 24 hours and 0.8% polyploid cells and 0.6% chromosomal aberrations at 48 hours. According to the authors, the final assessment for methylurea is negative.

The positive control substance (4-aminoquinoline 1-oxide dissolved in DMSO) produced a valid response.

 

In another mammalian chromosome aberration assay following no guideline (Abe 1977/ 1982, Ishidate 1980), pseudo diploid Chinese hamster cell line (Don) was exposed to Methylurea in Hanks' balanced salt solution at concentrations of 1, 10 and 100 mM without metabolic activation. No precipitation of the test substance, no toxic effects and no chromosomal aberrations were observed after treatment with the test substance.

 

In a mammalian cell micronucleus assay following no guideline (Von der Hude 2000), Chinese Hamster V79 cells were exposed to Methylurea (CAS No. 598-50-5) in water at concentrations up to 5000 µg/ml with (3 hours) and without S9-mix (24 hours). There was no genotoxicity. Marginal cytotoxicity effects were observed at 5000 µg/ml.

 

In a mammalian sister chromatid exchange assay following no guideline (Abe 1977/ 1982, Ishidate 1980), a pseudo diploid Chinese hamster cell line (Don) was exposed to Methylurea (CAS No. 598-50-5) in Hanks' balanced salt solution at concentrations of 1, 10 and 100 mM without metabolic activation. No precipitation of the test substance and no toxic effects were observed after treatment with the test substance.

No increase in the number of sister chromatid exchanges was detected after treatment with the test substance.

 

Cell transformation assay

In a mammalian cell transformation assay following no guideline (Ishidate and Kada 1980), Chinese hamster lung (CHL) cells were exposed to Methylurea at the test concentration 40 µg/ml. No cell transforming activity was observed in the colony assay.

 

 

In vivo studies

There are no studies available comparable to guideline. Other valid experimental data were available to assess the genetic toxicity in vivo.

 

Gene mutation 

In the Drosophila SLRL test following no guideline (Blijleven 1979), Methylurea was fed to Berlin K males (either singly or in combination with NaNO2and acetic acid) and sex-linked recessive lethals were determined. 

Methylurea, administered simultaneously with NaNO2and acetic acid produced a highly significant increase in the frequency of sex-linked recessive lethals, relative to treatment with methylurea alone, when both NaNO2and acetic acid were omitted. There is no further information about the results with methylurea alone.

 

In the Host-mediated Assay following no guideline (Braun 1977), the Mutagenicity of Methylurea in the presence of sodium nitrite (NaNO2) was screened in Salmonella typhimurium TA 1950 used as genetic indicator system after oral gavage of the test compound (ca. 107 - 215 mg/kg bw (1450 - 2900 µmole / kg bw) methylurea alone or simultaneously with equimolar amounts of NaNO2) in 0.9% NaCl solution to six male NMRI mice and simultaneous injection of bacteria. Three or five hours after bacterial injection, the animals were killed and the bacteria were recovered from the peritoneal cavity. An increased frequency of his+ revertants was only determined when methylurea was administered orally in combination with equimolar amounts of NaNO2. Methylurea alone was not mutagenic in the host-mediated assay.

 

In a second Host-mediated Assay following no guideline (Couch and Friedman 1975), 3 to 10 male Swiss mice were administered an aqueous solution of methylurea (2000 mg/kg bw methylurea alone or simultaneously with 150 mg/kg NaNO2) by gavage and inoculated with the S. typhimurium G46 strain LT2, a histidine-requiring auxotroph as indicator organism. Sodium nitrite solutions were administered by gavage at various times relative to treatment with methylurea. After killing the animals, peritoneal fluid was withdrawn, plated on minimal medium and Mutagenicity determined. Methylurea alone revealed no statistically significant change in mutant frequency. A highly significant increase in mutant frequency of Salmonella typhimurium was detected when methylurea was combined with NaNO2.

In an intragastric host-mediated assay (h.m.a.) following no guideline (Barale 1983), for analysis of the mutagenic activity of methylurea (0.135 and 6.750 mmoles / kg bw) in the gastrointestinal tract of mammals (4 – 6 adult male Swiss mice per dose group), the test substance (analytical purity of more than 99%) was injected as aqueous solution with the target cells of the yeast S. pombe, by single, oral gavage into the animals' stomachs. Target cells were recovered from the faeces for mutation-induction analysis. Additionally, yeast cells from parts of the gastrointestinal tract, the stomach and the small and large were recovered. Mutant and survivor colonies were scored 5 days after plating. Methylurea alone was shown to possess no mutagenic potential in the host mediated assay.

 

Cytogenicity in vivo

In a murine colonic nuclear aberration assay (Wargovich 1983), C57BL mice (male/female) were treated intrarectally with an aqueous solution of methylurea using a dose near the maximum tolerated dose as highest dose (250, 500, 750 mg/kg bw). After 24 hours animals were killed, longitudinal crypt columns of the colon were prepared and histologically examined. Nuclear aberrations were scored along longitudinal crypt columns. The test substance produced no response in the dose-response study and did not induce colonic nuclear aberrations.

The positive controls, a known colon carcinogen (Dimethylhydrazine) and a direct-acting intestinal carcinogen (N-nitroso-N-methylurea) produced valid responses. The study is of limited reliability due to very poor documentation, and the low number of animals used.

Conclusion: Methylurea alone is shown not to be mutagenic in different Mutagenicity assays. In combination with sodium nitrite it was clearly mutagenic, which is assumed to be based on the formation of Nitroso-methylurea.


Short description of key information:
in vitro
Gene mutation in bacteria
(1) Ames test with S. typhimurium TA1535, TA100, TA1537 and TA98 with and without metabolic activation: negative up to 5000 µg/plate (OECD 471; BASF AG 1992; 40M0085/914081).
(2) Ames test with S. typhimurium TA100, TA1537 and TA98, no data about metabolic activation: negative (no guideline followed; Ishidate 1981).

Cytogenicity in mammalian cells
(1) Sister chromatid exchange assay in mammalian cells with a Chinese hamster cell line (Don) without metabolic activation: negative up to 100 mM (no guideline followed; Abe and Sasaki 1977/1982).
(2) Mammalian chromosome aberration test with Chinese hamster cell line (Don) without metabolic activation: negative up to 100 mM (no guideline followed; Abe and Sasaki 1977/1982).
(3) Mammalian chromosome aberration test with Chinese hamster cell line (CHL): negative up to 1.4 mg/ml (no guideline followed; Ishidate 1983).
(4) Mammalian cell micronucleus test with Chinese hamster cell line (V79) without metabolic activation: negative up to 5000 µg/mL (no guideline followed; Von der Hude 2000)

Gene mutation in mammalian cells
no data

in vivo
Gene mutation
(1) Drosophila SLRL test: positive, when applied together with NaNO2 and acetic acid (no guideline followed; Blijleven WGH 1979).
(2) Host-mediated assay (Salmonella typhimurium), mouse: negative up to 107 to 215 mg/kg bw. (no guideline followed; Braun R 1977).
(3) Host-mediated assay (Salmonella typhimurium), mouse: negative up 2000 mg/kg bw. (no guideline followed; Couch and Friedman 1975).
(4) Intragastric host-mediated assay (yeast S. pombe), mouse: negative up to 6.750 mmoles / kg bw (mouse, no guideline followed, Barale 1983)

Cytogenicity
Murine colonic nuclear chromosome aberration assay: negative up to 750 mg/kg bw/d (no guideline followed; Wargovich MJ 1983)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the genetic toxicity data, 1-methylurea has not to be classified for genetic toxicity according to directive 67/548/EEC and regulation (EC) No. 1272/2008.