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In the GLP compliant, key bacterial reverse mutation assay (according to OECD guideline 471), Butanediol diglycidyl ether was tested at doses of 0, 10, 100, 333.3, 1,000, or 5,000 µg/plate in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537, and TA 1538 both in the absence and presence of exogenous metabolic activation (Aroclor 1254-induced rat liver S9) (Timm, 1987).  The experiment was conducted in triplicate; however, an independent repeat experiment was not performed. Dimethyl sulfoxide (DMSO) was used as the vehicle and positive controls were included in all incubations. No cytotoxicity was observed at any Butanediol diglycidyl ether concentration either in the absence or presence of metabolic activation; however, increases in the reverse mutation rates were observed in strains TA 98, TA 100, and TA 1535 both in the absence and presence of metabolic activation and in strain TA 1538 in the absence of metabolic activation. Incubation with positive control substances in the presence or absence of metabolic activation resulted in anticipated increases in reverse mutation rates.

 

Similar mutagenic results were noted in a supportive, non-GLP bacterial reverse mutation assay (equivalent to OECD guideline 471) where Butanediol diglycidyl ether was tested at doses of 0, 25, 75, 225, 675, or 2,025 µg/0.1 mL in S. typhimurium strains TA 98, TA 100, TA 1535, and TA 1537 both in the absence and presence of exogenous metabolic activation (Aroclor 1254-induced rat liver S9) (Arni and Müller, 1978).  No cytotoxicity was noted; however, increases in the reverse mutation rates were noted in strain TA 98 in the absence of metabolic activation and in strains TA 100 and TA 1535 in the presence of metabolic activation. Incubation with positive control substances either in the presence or absence of metabolic activation resulted in anticipated increases in the reverse mutation rate. 

 

In a key, GLP compliant mammalian chromosome aberration test (equivalent to OECD guideline 473), Butanediol diglycidyl ether was tested at doses of 0, 1, 5, 10, 50, or 100 µg/mL in Chinese hamster lung fibroblast (V79) cells in the absence or presence of exogenous metabolic activation (Aroclor 1254-induced rat liver S9) (Stammberger, 1992).  Incubations at each concentration were done in duplicate; however, an independent repeat experiment was not performed.  Minimum essential medium was used as the vehicle control and ethylmethanesulfonate and cyclophosphamide were used as the positive control compounds in the absence and presence of metabolic activation, respectively. No cytotoxicity was observed; however, chromosome damage was noted at concentrations of 5 µg/mL and above. Incubations with the positive control compound resulted in anticipated increases in chromatid damage. The scoring of less than 200 metaphase cells per concentration rendered this study reliable with restrictions. 

 

In a key, GLP compliant mammalian gene mutation assay (equivalent to OECD guideline 476), Butanediol diglycidyl ether was tested at doses of 0, 5, 10, 20, 40, or 50 μg/mL in the absence of exogenous metabolic activation (Aroclor 1254-induced rat liver S9) and at doses of 0, 10, 50, 100, 175, or 200 μg/mL in the presence of exogenous metabolic activation in V79 cells (Stammberger, 1992).  The experiment was conducted in duplicate and an independent repeat experiment was performed. Minimum essential medium was used as the vehicle and ethylmethanesulfonate and 9,10-dimethyl-1,2-benzanthracene were used as the positive control compounds in the absence and presence of metabolic activation, respectively. Cytotoxicity was noted at 50 μg/mL in the absence of metabolic activation in the second experiment only. Increases in the mutant frequency were observed both in the absence and presence of metabolic activation. Incubation with positive control substances in the absence or presence of metabolic activation resulted in anticipated increases in the mutation frequencies.

 

Similar mutagenic results were noted in a non-GLP mammalian gene mutation assay (equivalent to OECD guideline 476) in which Butanediol diglycidyl ether was tested at doses of 0, 1.31, 2.63, 5.25, 10.5, or 21 nL/mL in the absence of exogenous metabolic activation (Aroclor 1254-induced rat liver S9)and at doses of 0, 49.4, 65.8, 87.8, 117, or 156 nL/mL in the presence of exogenous metabolic activation in mouse lymphoma L5178Y cells (Beilstein, 1983).  Cytotoxicity was noted at concentrations of 21 nL/mL and 156 nL/mL in the absence and presence of metabolic activation, respectively. Increases in the mutant frequency were observed both in the absence and presence of metabolic activation. Incubation with positive control substances ethylmethane sulfonate and dimethylnitrosamine in the absence and presence of metabolic activation, respectively, resulted in anticipated increases in the mutation frequencies.

 

In the key, GLP-compliant in vivo micronucleus assay (according to OECD guideline 474), Butanediol diglycidyl ether was administered via oral gavage to male ICR mice at doses of 0 (control), 187.5, 375, or 750 mg/kg body weight (Schisler, 2010).  0.5% Methocel was used as the vehicle and cyclophosphamide monohydrate was administered via gavage as the positive control compound. The test article was administered once/day for 2 consecutive days and animals were sacrificed 48 hours after the second administration (6 to 8 mice/dose). None of the mice died; however, 3 mice of the high-dose group had a decreased amount of feces (no other clinical signs of toxicity were noted). No statistically significant increases in the number of micronucleated polychromatic erythrocytes were noted following butanediol diglycidyl ether administration. The positive control compound caused anticipated increases in micronucleated polychromatic erythrocytes. 

 

In a non-GLP sister chromatid exchange study (no OECD guideline available with which to compare the study design), Butanediol diglycidyl ether was administered via oral (gavage) to male and female Chinese hamsters at doses of 600, 1,200, or 2,400 mg/kg body weight (Strasser, 1983).  A combination of sodium carboxymethylcellulose and Tween 80 was used as the vehicle and 7,12-dimethylbenzanthracene was administered via gavage as the positive control compound. The test article was administered once and animals were sacrificed 24 hours after exposure (4 hamsters/sex/dose). No clinical signs of toxicity were observed and no statistically significant increases in the number of sister chromatid exchanges were noted following epoxy resin administration. The positive control compound caused anticipated increases in the number of sister chromatid exchanges.

 

In an earlier, non-GLP in vivo micronucleus assay (equivalent to OECD guideline 474), Butanediol diglycidyl ether was administered via oral gavage to male and female Chinese hamsters at doses of 600, 1,000, 1,200, 1,500, 2,000, 2,400, 2,500, or 3,000 mg/kg body weight (Strasser, 1984).  Under the experimental conditions used, this report does not meet current guidelines for the in vivo micronucleus test and hence is not reliable for assessment of in vivo genetox potential. Although the current guideline standards (OECD 474, 1997) have changed from when this study was reported (1984), the study did not follow the guideline that was in effect at the time of the study conduct. Most notably, the protocol used for the study was a non-traditional and un-validated version of the micronucleus test and has a number of specific issues that preclude it from being suitable for inclusion. Most significantly, the methodological analysis of bone marrow cells was incompletely described in the report and used a flawed methodology that counted all bone marrow cells (both nucleated and non-nucleated). Specifically, the proportion of the 1000 cells counted for each individual cell type (i.e., single Jolly bodies, erythrocytes, erythroblasts, leucopoietic cells, and polyploid cells) may be highly variable between samples and makes a direct comparison impossible and, as a result, may inappropriately skew the counts due to the population of cells analyzed. For example, the small differences seen between the control and some of the treated groups could have been simply due to differences in cell populations among the 1000 analyzed cells. Furthermore, no assessment of bone marrow toxicity was undertaken, which is an essential to properly interpret the micronucleus data, especially when there were only marginal increases.

 

In addition, a number of other significant deviations from the current guideline raise questions as to the validity of the results, including: 1) the dose levels where positive effects are noted all exceed the current limit dose of 2000 mg/kg bw/day, 2) associated systemic toxicity including body temperature was not reported (except death), which is critical for assessment of a maximum tolerated dose for interpretation of the results, 3) the number of animals analyzed per group was less than the required 5/sex/dose, which may distort group means, 4) the samples do not appear to have been blinded to the scorer for analysis, 5) although mean body weights for each group are not presented, the range in body weights suggest potential for greater than ± 20% variability as stated in the guideline, 6) current guidelines require 2000 polychromatic erythrocytes/reticulocytes to be counted per animal (along with a PCE/NCE ratio), whereas only 1000 unspecified total cells were counted in this report, and 7) an inconclusive dose response was reported in the second experiment, where similar alterations were reported at 1500 and 2500 mg/kg, but no effect was seen at 2000 mg/kg.


Short description of key information:
The genetic toxicity of butanediol diglycidyl ether has been assessed in 5 in vitro studies (including 2 bacterial reverse mutation assays and a mammalian chromosome aberration test) as well as in 3 in vivo studies (two micronucleus assays and a sister chromatid exchange study). Positive results were reported in all studies in vitro studies, with the exception of the unreliable in vivo hamseter micronucleus study, all of the in vivo studies were negative.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The submission substance produced five positive genotoxic in vitro results, one unreliable positive in vivo somatic cell genotoxic result, and two negative in vivo somatic cell genotoxic results. According to the Guidance on the Application of the CLP Criteria (ECHA Reference ECHA-09-G-02-EN), if positive results in vitro are supported by at least one positive local in vivo, somatic cell test, such an effect should be considered as enough evidence to lead to classification in Category 2. If there is also negative or equivocal data, a weight of evidence approach using expert judgement has to be applied. The positive in vivo somatic cell study is not reliable for the evaluation of in vivo cytogenetic effects as a result of the significant deviations from both current and contemporary guideline requirements for an acceptable in vivo micronucleus test. The submission substance contains highly reactive oxirane functional groups, thus the positive in vitro results are not unexpected. As a result of expert judgement, the substance does not meet the criteria for classification according to Regulation (EC) No 1272/2008, Annex I section 3.5. (Additional details are reported in the field "Attached documents" and in section 13 "Assessment reports" see file "Assessment BDDGE Genotoxicity-Carcinogenicity").