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In vitro

2 -methoxyethanol was consistently negative in a number of in vitro reverse mutation bacterial studies and also in a yeast mutation study both with and without metabolic activation. Across two Ames tests, all five strains of bacteria now required by modern protocols were assessed and found to be negative. A single reliable study reported no mutation in two mammalian cell systems (CHO K1BH4 and the HPRT locus and CHO AS52 at the GPT locus). This result was supported by other less robust studies which were either reported in less detail or were used without metabolic activation. All of the available in vitro data on cytogenics had shortcomings; none of the studies used metabolic activation but some did separate evaluations on known metabolites. From these studies it was possible to conclude that methoxyethanol does not increase SCE rates or the rates of chromosome abberations (in multiple cell lines). Weak positive results were obtained at high doses in two assays: a micronucleus assay where a weak increase in the percentage of micronuclei together with nuclear disorganisation was seen in V79 cells and at very high concentrations (131 -394mmol) aneuploidy was observed. This weakness in the cytogenotoxicity data is not critical as in vivo data is also available.

In vivo

A number of dominant lethal assays have been performed on 2 -methoxyethanol. The one study in mice was negative and one study in rats by the inhalation route was negative. Another study by the oral route reduced the total number of implants in a dose related manner and a simultaneous rise in pre-implantation loss, but did not produce clear evidence of a dominant lethal effect when administered at doses up to 1500 mg/kg. A further study reduced inconclusive results due to confounding caused by reproductive toxicity. It is probably that any adverse effects in this type of assay are due to testicular toxicity rather than genotoxicity, particularly in view of the negative results in other genotoxicity assays.

In vivo chromosome abberation studies have also been performed in both rats and mice. Rats subjected to an inhalation exposure of 500ppm showed no adverse effects and mice exposed to a single dose of 1900mg/l also produced similar results.

Fruit flies (Drosophila melanogaster) were briefly exposed by inhalation to 0, 25 or 500 ppm methoxyethanol ( the maximum tolerated concentration). Two independent tests were performed using different stocks of flies. After longer exposures for up to 7 days, sex-linked recessive lethals were found, but not after exposure for 10 days. The test data are difficult to interpret and the test considered invalid for this lack of repeatability.

A comet assay was employed for the evaluation of testicular cells and bone marrow cells after single gavage administrations at 0, 500, 1,000 or 1,500 mg/kg to rats. Two studies were conducted, one for effects 2 weeks after treatment and a second study for effects at 5 and 6 weeks after treatment. After 2 weeks, but not after 5 and 6 weeks, a dose-related increase in damage (as measured by the mean and median tail moment) was noted for haploid testicular cells; in diploid bone marrow cells there were also increases but without a clear dose relation (Anderson et al, 1996). Although the authors debate the possibility that methoxyethanol may be genotoxic, the results may also reflect cytotoxicity and apoptotic events which are known for methoxyethanol and its metabolite methoxyacetic acid, in these target cells.

The frequency of (sister chromatid exchange) SCE rates induced by methoxyethanol was evaluated in vitro in human peripheral blood and in bone marrow cells of mice. In human peripheral blood, SCE was observed after the addition of methoxyacetic acid to the culture medium but not of methoxyethanol. An increased SCE rate in mouse bone marrow cells was observed after i.p. injection of methoxyethanol (treatment in vivo, labelling in vitro)


Short description of key information:
In vitro gene mutation in bacteria and yeast: Negative with and without metabolic activation (4 studies)
In vitro cytogenicity: chromosome abberation: negative without metabolic activation (3 studies)
In vitro cytogenicity: sister chromatid exchange: negative without metabolic activation (2 studies)
In vitro cytogenicity: micronucleus: negative without metabolic activation (1 study)
In vitro mammalian cell mutation: negative (2 studies with and without metabolic activation), negative (2 studies without metabolic activation.)
In vivo dominant lethal assay, rat, oral dose of 1500mg/kg: no clear evidence of dominant lethality
In vivo dominant lethal assay, mouse, oral dose of 1500mg/kg: negative
In vivo dominant lethal assay, rat, inhalation of 300ppm: negative
In vivo dominant lethal assay, rat, inhalation of 500ppm: ambiguous due to confounding of fertility effects
In vivo chromosome abberation assay, rat, inhalation of 500ppm: negative
In vivo chromosome abberation assay, mouse, oral dose of 1900mg/kg: negative
In vivo sister chromatid exchange assay, mouse, intraperitoneal, 500mg/kg: positive but weak response
In vivo comet DNA damage assay, rat, oral 500-1500mg/kg: positive but reversible.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Weight of evidence suggests methoxyethanol is not mutagenic in vitro. Some positive results have been obtained with metabolites but the in vivo data is also negative indicating that the metabolite concentrations are not of concern in whole animal systems. In vivo chromosome abberation studies were consistently negative and the results from the dominant lethal assay studies, when interpreted in the context of methoxyethanol being a potent testicular toxin for which classification is required, can also be interpreted as negative.