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Genotoxicity in vitro

Ames tests

The potential mutagenicity of urea was investigated in a screening assay performed in S. typhimurium TA98, TA100 and TA1537 according to the published method of Ames et al (1977), with a pre-incubation step and in the absence and presence of an exogenous source of metabolic activation. No evidence of mutagenicity was seen under the conditions of this assay (Ishidate et al, 1981).

The mutagenicity of urea was determined in the Ames test using Salmonella typhimurium and Escherischia coli, with and without S9 metabolic activation. The substance was not mutagenic at any of the 7 concentrations tested (Shimizu et al, 1985).

The potential mutagenicity of urea was investigated in a guideline-comparable Ames test (pre-incubation assay). Triplicate cultures of S. typhimurium TA97, TA98, TA100, TA1535 and TA1537 were exposed to urea (dissolved in water) at five concentrations between 10 -10000 ug/plate in the presence and absence of an exogenous metabolic activation system (Aroclor 1254 -induced male Sprague-Dawley rat and male Syrian hamster liver S9;). Exposure to urea caused cytotoxicity in some strains. The numbers of revertant colonies were not increased by exposure to urea. Appropriate positive controls confirmed the sensitivity of the assay (Mortelmans et al, 1986).

Clastogenicity

Ishidate et al (1981) report the results of a chromosomal aberration assay performed in CHL cells with a number of chemicals, including urea. Approximately 10e5 cells were plated and exposed to concentrations of urea up to the concentration causing 50% growth inhibition, in the absence and presence of PCB-induced Wistar rat liver S9-fraction. Cells were harvested at 24 and 48 hours (-S9) or at 24 hours following a 3-hour pre-incubation step (+S9). Chromosomal aberrations (including numerical aberrations) were scored from 100 well-spread metaphases per concentration. A positive result is reported in this assay, however the DT20 value (the concentration at which 20% of cells or approximately 4x background) of 13.0 mg/mL or 216 mM is well in excess of the limit concentration of 5 mg/ml or 10 mM recommended by OECD 473 (1997). The authors note a very low clastogenic potential. Considering the high concentrations of urea required to produce a response in this assay, which are well in excess of the limit concentration, it cannot be concluded that urea is clastogenic. The finding in this study is very likely to be a false positive due to osmotic effects.

The same group (Ishidate & Odashima, 1977) tested urea for the ability to cause chromosomal aberrations in a screening assay in CHL cells in vitro in the absence of metabolic activation and at concentrations up to those causing 50% growth inhibition. A positive result is reported at a concentration of 266.4 mMol/L, which is well in excess of the limit concentration of 10 mM. The result is considered to be a false positive and is attributable to the effects of osmolarity.

Mammalian cell mutation

The potential mutagenicity of urea was investigated in a mouse lymphoma assay in the absence of metabolic activation. A weak positive response was seen at concentrations of 265 -662 mMol/L, concentrations which also caused cytotoxicity and which are well in excess of the limit concentration of 10 mMol/L recommended in OECD 476 (1997). The result is considered to be a false positive. The authors conclude that the effect is due to the influence of high concentrations of urea on the osmolarity of the culture medium (Wangenheim & Bolcsfoldi, 1988).

Other studies

The ability of urea to cause DNA damage was assessed in two DNA-unwinding/alkaline elution assays. Garberg et al (1987), report positive effects in mouse lymphoma cells at high concentrations of 628 and 718 mMol/L, however negative results are reported by Sina et al (1983) at concentrations of up to 3 mMol/L in cultured rat hepatocytes.

Genotoxicity in vivo

Chaurasia & Sinha (1987) investigated the potential of urea to cause chromosomal aberrations in the bone marrow of Swiss mice. Mice (number unspecified) were administered urea in the diet at a dose level of 500 mg/day for 5 days. Animals were sacrificed after a recovery period of 7 days and the bone marrow harvested. A total 300 metaphases from treated animals and untreated controls were assessed for chromosomal aberration. A marked increase in the incidence of chromosomal aberrations was seen in the treated group (7x controls). However the dose level administered in this study is equivalent to 16 -17 g/kg bw/day and is thus far in excess of the limit dose of 1000 mg/kg bw. Signs of toxicity are not reported, but marked toxicity can be predicted at this dose level.

Conclusion

Positive results obtained in vitro are associated with concentrations well in excess of the recommended limit concentrations are not considered to be of biological relevance. A positive result in vivo is also associated with an excessive dose level. Considering the physiological role and presence of substantial quantities of urea in the human body, it is not considered likely that this substance is genotoxic. Further testing for genotoxicity is not proposed.


Short description of key information:
Negative results are reported in three Ames tests. Positive results are reported in assays for mutagenicity and clastogenicity in mammalian cells, however the value of these studies are limited by the extremely high test concentrations. A positive result is reported in a mouse bone marrow assay of unconventional design, however this study is not considered to be reliable. Based on its physiological role and presence in the body at high concentrations, urea is not considered to be genotoxic.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

No classification is proposed for genotoxicity. Urea is produced by the body in large quantities as a normal product of metabolism and is present in the bloodstream at high concentrations. Urea is therefore considered extremely unlikely to be genotoxic.