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EC number: 231-640-0 | CAS number: 7665-72-7
The potential of the target substance t-butyl-glycidyl ether to induce genotoxic effects was assessed in a weight-of-evidence approach by using data from several in vitro and in vivo studies conducted with the target substance as well as with the source substance n-butyl glycidyl ether. The target substance was tested positive in several Ames tests as reported by Dabney, 1976 and Canter et al., 1986. Positive results were also obtained from an E. coli SOS Chromotest.
In one in vitro sister chromatid assay (von der Hude, 1991) and in two in vitro Unscheduled DNA Synthesis test the target substance was tested positive for genotoxic effects.
Data from the source substance n-butyl glycidyl ether were used to assess the mutagenicity. In the publication by Thompson et al., 1981 the results from several genotoxicity tests (Ames, Mouse Lymphoma Assay, UDS) conducted with n-butyl glycidyl ether were presented. The UDS was negative, but the Ames and the Mouse Lymphoma assay showed a positive potential to induce mutagenicity.
Furthermore, n-butyl glycidyl ether is classified as Muta. 2 (H314) in the harmonized classification according to Annex VI of Regulation (EC) No 1272/2008.
In this study, the mutagenicity of the test substance was tested in Salmonella strains TA98, TA100, TA1535, TA1537 and TA97 with and without S9 metabolic activation system. The test doses used were determined by the solubility or toxicity of the individual chemicals, but did not exceed 10 mg/plate.The test substance was found to be active in TA100 and TA1535 with and without activation and showed varying responses in other strains.
Table 1: DNA Repair Induction
(a): 4.6 µL tBGE added directly to culture (no DMSO)
(b): Dilution scheme employed in these assays was designed to minimise the DMSO concentrations to which cells are exposed. The maximum DMSO concentration of 0.357% corresponds to the DMSO control dose
(c): Meangrain counts calculated using only cells with at least one nuclear grain
In an in vitro DNA repair study, the DNA repair induction in human lymphocytes was tested with 1-tert-Butoxy-2-,3-expoxypropane at doses of 10.0, 100.0 and 1000.0 µg/mL (Donor 1) as well as 12.4, 37,0, 111.0, 333.0, 1000.0 and 3000.0 µg/mL (Donor 2). DNA repair induction occured optimally at 333 µg/mL with the test item in a dose-response relationship from 0 to 333 µg/mL. The viability assay employed may indicate little more than membrane integrity. Among the effects of these compounds on cellular metabolism at dose levels higher than 333 µg/mL appears to be an inhibitory effect on DNA repair synthesis. Any specificity for interference DNA repair function has not been examined, and may simply be a manifestation of overall cellular cytotoxicity.
Table 1: In vitro mutagenicity of test item using Salmonella typhimurium TA1535
µmoles / plate
100 µg / plate
5 µg / plate
In conclusion, in an bacterial reverse gene mutation assay, the target substance t-BGE reverted the base-pair substitution type strains (TA1535 and TA100), but produced no response in the frame-shift type strains (TA1537, TA1538 and TA98).
Table 1: Mutagenic assay of the urine of mice treated with t-BGE using Salmonella typhimurium TA1535 and TA98
In a body fluid analysis using a reverse bacterial mutation assay, Salmonella strains TA1535 and TA98 were tested directly with urine with and without the presence of beta-glucurondiase. The urine came from a group of ten mice dosed with 0, 100, 200, and 400 mg/kg bw/day t-BGE on four consecutive days. The mean colony forming units were counted. A somewhat dose-dependent mutagenicity was seen in strain TA1535 in the presence of beta-glucuronidase. No mutagenicity was seen in tester strain TA98 with or without beta-glucuronidase.
An unscheduled DNA synthesis assay was performed using normal human peripheral blood lymphocytes to determine DNA repair induction (via liquid scintillation counting (LSC), and slide autoradiography) and cell viability after a 5-hour exposure to different concentrations of (Tert-butoxymethyl) oxirane. Negative controls included saline and DMSO (vehicle), and MNNG was used a positive control. Exposure to increasing doses of (Tert-butoxymethyl) oxirane led to a reduction in cell viability in a dose-response manner. In cells from donor A, elevated repair activity occurred in (Tert-butoxymethyl) oxirane treated cultures at both 100 and 1000 µg/ml doses. In cells from donor B, DNA-repair induction occurs optimally at 333 µg/ml. A dose-related increase in percentage of nuclei with elevated gram counts was observed over the range of (Tert-butoxymethyl) oxirane concentration from 0 to 333 µg/ml. Under the described test conditions, (Tert-butoxymethyl) oxirane was seen to have genotoxic and cytotoxic effects.
RESULTS OF THE SOS-CHROMOTEST
Using the SOS -Chromotest, the test substance tert-butyl glycidyl ether showed a dose-related increase of the b-GAL activity and the induction factor. It also had a positive result during the Ames test. This indicates that the test substance has genotoxic effects.
Below is Table 1 showing the results of two independent experiments; each experimental point is the mean value of 25 metaphases±SD and the replication index (RI) determined from 100 cells.
Slope of dose-response curve calculated as the mean of 2 experiments.
Table 1. V79/SCE test with 2,3 -epoxypropyl-tert.-butylether *denotes statistically significant from solvent at p<0.0005
* SCE value different from the solvent, p< 0.005
The mutagenic action of t-BGE in mammalian cells was investigated using the Chinese hamster V79 cell line. After exposing the cells to DMSO as a control and test substances concentrations ranging from 0.31 to 5.0 mmol/L, the sister chromatid exchange (SCE)-inducing potency was calculated by determining the slopes of the dose-response relationships via computed linear regression analysis of the pooled results of 2 independent experiments. The SCE-inducing potency (SCEIP) was 1.9 for test substance 2,3-epoxypropyl-tert.-butylether according to experiments with V79 cells in vitro without an external metabolizing system.
A Salmonella/mammalian microsome plate assay was performed as described by Ames et al., 1975 with butyl glycidyl ether. The concentrations chosen for the main test were based on the results from a preliminary toxicity test. Concentrations of 2000, 666.7, 222.2, 74, 24.7, 8.2 and 0 µg/plate were tested in the presence and absence rat liver microsome activation system (S9) using Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538, and TA98. Both solvent (0.1 ml DMSO) and water negative controls were prepared. Three plates were prepared for each dose level and controls. In all cases the positive controls exhibited at least a 10-fold increase in the number of revertant colonies (mutation index >10). Butyl glycidyl ether was positive with and without metabolic activation in strains TA1535 and TA100, although enhancement of the response was seen (especially noticeable with TA1535) in the presence of the S9 fraction. The results of the assay with strains TA1537, TA1538 and TA98 were negative. Butyl glycidyl ether was considered mutagenetic in strains TA1535 and TA100 under the test conditions described.
The L5178Y mouse lymphoma assay was conducted as described by Clive and Spector, 1975 and later modified by Clive et al., 1979. Twelve concentrations of butyl glycidyl ether were tested in the presence and absence rat liver microsome activation system (S9) using L5178Y mouse lymphoma cells. In general, the highest mutagenic responses were obtained in the assays without metabolic activation. Responses obtained were slightly reduced in the assays that used the non-induced S9 preparations, whereas much lower responses were obtained with the Aroclor-induced S9 preparations. Cell toxicity was reduced proportionally, i.e. the same dose of chemical produced a more toxic response in the absence of any S9 and a less toxic response in the presence of the S9. Butyl glycidyl ether produced a positive response either with one of the S9 preparations or without activation. Under the described test conditions, butyl glycidyl ether was considered mutagenic.
An unscheduled DNA synthesis assay was performed using WI38 cells to determine DNA repair after exposure to five different concentrations of butyl glycidyl ether in the presence and absence rat liver microsome activation system (S9). The positive controls were 4-nitroquinoline-N-oxide (4NQO), a compound that induces UDS in the absence of metabolic activation, and dimethylnitrosamine (DMN), a compound that induces UDS in vitro only with metabolic activation. The negative control was DMSO diluted in culture medium. The response index was calculated by dividing the amount of thymidine incorporation in the test results by the thymidine incorporation in the solvent control. A compound was considered positive in this assay if a dose-related increased in the amount of [3H]thymidine incorporated into DNA over at least 3 concentrations with the highest response equal to at least twice the solvent control was attained. Demonstrable, although not considered positive, responses were obtained for butyl glycidyl ether in the presence of S9 and ethyl glycidyl ether in the non-activated assay.
In contrast to the in vitro data, the target substance was tested negative in several in vivo studies (in vivo chromosome aberration test, in vivo micronucleus test, dominant lethal test).
In an in-vivo cytogenicity toxicity test, t-BGE was orally exposed to ten female B6D2F1mice weighing 20 - 25 g at concentrations of 100 - 400 mg/kg once per day for five consecutive days. All animals were sacrificed 4 hours after the last treatment. Extraction of bone marrow, preparation of smears, staining and analysis was conducted and the metaphase cells from bone marrow preparations were scored for structural chromosomal aberrations. A total of 100 metaphases from each animal were scored. The frequency of gaps was essentially the same for both treated and control groups. The known positive controls Cytoxan and hycanthone demonstrated frequencies of damaged cells in the same range as prvious studies conducted in this laboratory. This test showed no differences between the control and the t-BGE dosage group oin the percentage of metaphases with chromosomal damage.
In an in vivo genetic toxicity test, conducted similar to guideline OECD 478, t-BGE was dermally exposed to 10 B6D2F1 male mice (per dosage group) at the concentrations 0, 0.375, 0.75, 1.5 g/kg bw. The male mice were exposed for 3 times per week for a total of 8 weeks and then mated with untreated females. The dominant lethal assay results indicated that increased dosage levels of t-BGE play no role in modifying either pregnancy rates, number of dead implants, number implants or the dead implant rates.
In an in-vivo genetic toxicity test, conducted similar to guideline OECD 474, t-BGE was orally exposed to ten female B6D2F1 mice weighing 20 - 25 g at concentrations 100 - 400 mg / kg once per day for five consecutive days. A total of 1000 polychromatic red cells from bone marrow preparations from each animal in control and t-BGE tested groups were scored for micronucleus. The test substance did not induce any increase in the frequency of the micronuclei in the polychromatic erythrocytes of the bone marrow. Thus, in this study, the test item is considered to be non-mutagenic.
In one in vitro sister chromatid assay (von der Hude, 1991) and in two in vitro unscheduled DNA synthesis tests the target substance was tested positive for genotoxic effects.
Data from the source substance n-butyl glycidyl ether were used to assess the in vitro mutagenicity. In the publication by Thompson et al., 1981 the results from several genotoxicity tests (Ames, Mouse Lymphoma Assay, UDS) conducted with n-butyl glycidyl ether were presented. The UDS was negative, but the Ames and the Mouse Lymphoma assay showed a positive potential to induce mutagenicity.
Furthermore, the structural analogue n-butyl glycidyl ether (source substance) is harmonized classified as Muta. 2 (H341) according to Annex VI of Regulation (EC) No 1272/2008.
Based on the results of the available data, and the fact that n-butyl glycidyl ether is harmonized classified as Muta. 2, H341 the target substance is considered as a Category 2 mutagen (Muta 2, H341).
Based on the results of the available data, and the fact that the structural analogue n-butyl glycidyl ether (source substance) is harmonized classified as Muta. 2, H341 the target substance is considered as a Category 2 mutagen (Muta 2, H341).
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