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Litton Bionetics (1985) reported a bacterial reverse mutation assay (Ames test). Salmonella typhimurium strains used were TA 98, TA 100, TA 1535, TA 1537 and TA 1538. This test was done with and without metabolic activation. Test concentrations were 1.0, 10, 100, 500, 1000, 2500, 5000 and 10000 ug/plate. A negative result was obtained.

Another bacterial reverse mutation assay (Florin et al., 1980) using a concentration of 3 umol/plate was also negative for the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100. This test was done with and without metabolic activation.

Microbiological Associates (1991) reported also a negative result in a bacterial reverse mutation assay. The test concentrations used were 100, 300, 1000, 3333 and 5000 ug/plate (Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537, TA 1538).

An in vitro mammalian cell gene mutation assay was reported (Microbiological Associates, 1991). Mouse lymphoma L5178Y cells were used in this experiment. The test concentrations in this experiment were 100, 200, 400, 600, 800, 1000 and 1400 ug/ml (without metabolic activation) and 10, 20, 40, 60, 80, 100, 200, 400, 600 and 800 ug/ml (with metabolic activation). Results: With metabolic activation the mutant frequency was similar to controls for all dose levels except the low dose. Under non-activated conditions, the mutant frequency roughly increased at the highest concentration (which was highly toxic to the cells), indicating a positive mutagenic response. The data on colony size distributions showed an increase in the frequency of small colonies when the treated cultures were compared to the solvent control cultures. The molecular and cytogenetic nature of both large and small colony MLA mutants has been explored in detail [Honma et al. 2001; Applegate et al., 1990; Hozier et al., 1981+1985; Moore et al., 1985]. Slow growing mutants have suffered genetic damage that involves putative growth regulating gene(s) near the TK locus which results in prolonged doubling times and the formation of late appearing or small colonies [Amundson and Liber, 1992]. The induction of slow growing mutants has been associated with substances that induce gross structural changes at the chromosomal level. Thus, the observed increase in the frequency of small colonies is consistent with damage to multiple loci on chromosome 11 in addition to loss of the TK locus. Consequently, the test substance is considered to act by a clastogenic mode of action.

In another in vitro mammalian cell gene mutation assay (Litton Bionetics, 1985), also mouse lymphoma L5178Y cells were used. The test concentrations were 188, 375, 500, 750, 1000 and 1250 ug/ml (without metabolic activation) and 31.5, 125, 188, 250, 375 and 500 ug/ml (with metabolic activation). The test substance induced significant increases in the mutant frequency at the TK locus in l5178Y cells. Under non-activation conditions, the test material was assayed for mutant induction from 188 ug/ml to 1250 ug/ml and low to very high toxicities were induced. A significant increase in the mutant frequency was induced but only at very high toxicity (4.7% relative growth). The test material therefore has borderline activity without activation. In the presence of metabolic activation the test material was converted to a more active form or forms. Treatments from 31.5 ug/ml to 500 ug/ml were assayed and increases above the background mutant frequency (3.4-fold to 5.6-fold) were induced over a wide range of toxicities.

In an in vitro transformation test using BALB/C-3T3 cells a negative result was reported (Litton Bionetics, 1985). Test concentrations used were 62.5, 250, 400, 700 or 1125 mg/l.

Microbiological Associates Inc. (1991) reported an in vivo micronucleus test. Male and female mice were treated via the intraperitoneal route (one treatment) concentrations of 113, 225 or 450 mg/kg. Clinical signs which occurred on the days following treatment were lethargy, rough hair coat and hunched posture. One male of the high dose level was found dead on the 3rd day following treatment and was replaced at the time of bone marrow collection with an animal from the replacement group that was also doses with 450 mg/kg. With respect to the incidence of micronucleated polychromatic erythrocytes, no changes in the ratio of polychromatic erythrocytes to total erythrocytes was apparent in the substance-treatead mice when compared to vehicle control-treated animals. No significant increase of micronucleated polychromatic erythrocytes/1000 polychromatec erythrocytes was noted in male and female mice, regardless of dose level or bone marrow collection time.

Justification for classification or non-classification

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