Registration Dossier
Registration Dossier
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EC number: 701-197-2 | CAS number: -
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Key, GE 100, Salmonella typhirium - Ames test - TA98, 100, 1535 - positive, 1537 and 1538 - unclear results (Banduhn, 1986)
Key, GE 100, Salmonella typhimurium and E. coli - positive (Ohtani and Nishioka, 1980)
Supp, ReA, Salmonella typhimurium - TA 98 and Ta 100 - positive, TA 1531 and 1533 - negative (Pattys, 1981)
Key, ReA, Mouse Lymphoma - positive (Thompson et al., 1981)
Key, ReA, UDS Assay -positive (with metabolic activation); negative (without metabolic activation) (Thompson et al., 1981)
Supp, ReA, Toolbox predictions - are all positive (in vitro and in vivo).
Link to relevant study records
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: DNA damage and/or repair
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: well-documented publication, which meets basic scientific principles, performed similar to OECD guideline 482 i.a. on the read-across substances glycidol und butylglycidyl ether
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)
- Deviations:
- not applicable
- GLP compliance:
- no
- Type of assay:
- DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro
- Target gene:
- not applicable
- Species / strain / cell type:
- mammalian cell line, other: WI38
- Details on mammalian cell type (if applicable):
- WI38 cells were obtained from the American Type Culture Collection.
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 of liver homogenate (250 mg of liver per ml) from adult male Swiss-Webster mice. The following cofactors were added to the S9: nicotinamide (3.05 mg/ml), glucose-6-phosphate (16.1 mg/ml), MgCI2 * 6 H20 (5.08 mg/ml), and NADP (0.765 mg/ml).
- Test concentrations with justification for top dose:
- The maximal testable level was set just below the level which produced cytotoxicity as demonstrated by a decrease in the amount of [3H]thymidine incorporated into the DNA. Test concentrations:
0, 0.375, 0.75, 1.5, 3.0, 6.0 µg/ml (glycidol without S9); 0, 0.037, 0.111, 0.333, 1.0, 3.0 µg/mL (glycidol with S9)
0, 0.24, 0.36, 0.53, 0.8, 1.2 µg/ml (butyl glycidyl ether without S9); 0, 0.5, 1.0, 2.0, 4.0, 8.0 µg/mL (butyl glycidyl ether with S9) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- N-dimethylnitrosamine
- Remarks:
- 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.
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: The cells were grown to confluency and maintained in medium containing 0.5% serum for 5-6 days preceding the UDS assays. The cultures were incubated for 1 h with 10 exp -2 M hydroxyurea (HU) before each assay.
- Exposure duration: For testing in the absence of metabolic activation, the cells were exposed simultaneously to the compounds and to 3H-TdR for 3 h. For testing with metabolic activation, the cells were incubated with the compound, 3H-TdR, and the metabolic activation preparation for 1 h and then with only 3H-TdR in culture medium for an additional 3 h.
NUMBER OF REPLICATIONS: 6
DETERMINATION OF CYTOTOXICITY
- Method: other: decrease in the amount of [3H]thymidine incorporated into the DNA - Statistics:
- Results are the average of 6 replicate samples at each dose level.
- Species / strain:
- mammalian cell line, other: WI38
- Metabolic activation:
- with
- Genotoxicity:
- positive
- Remarks:
- glycidol
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Species / strain:
- mammalian cell line, other: WI38
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Remarks:
- glycidol
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Species / strain:
- mammalian cell line, other: WI38
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- butyl glycidyl ether; demonstrable, although not considered positive responses were obtained for butyl glycidyl ether in the presence of S9.
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Conclusions:
- negative without metabolic activation both glycidol and butyl glycidyl ether
negative without metabolic activation butyl glycidyl ether
positive with metabolic activation glycidol
negative '1,2,3-propanetriol, glycidyl ethers' , derived from read-across
Unscheduled DNA synthesis was induced by glycidol in the presence of an uninduced mouse liver S9 fraction, whereas butyl glycidyl ether was clearly negative for UDS induction. The effect of S9 in this assay was just the opposite of that seen in the mouse lymphoma assay (Thompson, 1981), i.e. S9 appears to increase the amount of UDS whereas it reduced the mutagenic response in the mouse lymphoma assay: There, the mutagenic potential of the ethers increases in the presence of the S9 fraction, which reduces the mutagenic potential of glycidol. The epoxide hydrase present in induced S9 fractions has been reported to lower the mutagenic potential of an epoxide by converting it to the diol (Ortiz De Montellano, P.R., and A.S. Boparai (1978) Aliphatic 3,4-epoxyalcohols, Metabolism by epoxide hydrase and mutagenic activity, Biochim. Biophys. Acta 544, 504-513.).
The decrease in mutagenic potency in the presence of the metabolic activation system in the mouse lymphoma assay was probably due to inactivation of the epoxide group by epoxide hydrase in the induced rat liver S9 or by endogenous metabolism by the cells. It has been shown that rat liver S9 contains higher levels of epoxide hydrase than S9 from mice and that induction with Aroclor further increases the epoxide hydrase levels (Oesch, F. (1972) Mammalian epoxide hydrases; Inducible enzymes catalysing the inactivation of carcinogenic and cytotoxic metabolites derived from aromatic and olefinic compounds, Xenobiotica, 3,305-340.). However, these effects are consequently less relevant for humans and the results gained in the UDS assay by uninduced mouse S9 mix is more relevant for human risk assessment.
Ohtani et al. showed in a bacterial mutation assay that epoxide compounds which have higher molecular weight and lower solubility showed neither any killing effect nor mutagenic effect in the DNA repair test as well as the reversion test. The reason seems to be that their molecular sizes and solubilities are not small and high enough to pass through the cellular membrane and to reach DNA (Ohtani H., Nishioka H. (1981), Mutagenic activity of epoxide compounds as constituents of resins in bacterial test system, Science and engineering review of Doshisha University).
Similar conclusion can be drawn in this assay: Since '1,2,3-propanetriol, glycidyl ethers' has a higher molecular weight than the tested compounds, it can be reasonably concluded that '1,2,3-propanetriol, glycidyl ethers' will not induce any positive results in the UDS assay, and can be hence considered as negative with and without metabolic activation. - Executive summary:
In an unscheduled DNA synthesis assay similar to OECD guideline 482, WI38 cells were exposed to the read-across substances for '1,2,3-propanetriol, glycidyl ethers', glycidol and butyl glycidyl ether at concentrations of 0, 0.375, 0.75, 1.5, 3.0, 6.0 µg/ml (glycidol without S9); 0, 0.037, 0.111, 0.333, 1.0, 3.0 µg/ml (glycidol with S9); 0, 0.24, 0.36, 0.53, 0.8, 1.2 µg/ml (butyl glycidyl ether without S9); 0, 0.5, 1.0, 2.0, 4.0, 8.0 µg/ml (butyl glycidyl ether with S9).
Glycidol and butyl glycidyl ether were tested just below the level which produced cytotoxicity. The positive controls (4-nitroquinoline-N-oxide (4NQO), dimethylnitrosamine (DMN)), induced the appropriate response.
There was no evidence or a dose related positive response for both compounds without metabolic activation that unscheduled DNA synthesis, as determined by radioactive tracer procedures, was induced. Glycidol induced a dose-related positive response with metabolic activation, butyl glycidyl ether induced demonstrable, although not considered positive responses with metabolic activation. Based on the extrapolation from glycidol over butyl glycidyl ether to '1,2,3-propanetriol, glycidyl ethers', taking into account molecule size, side chain length(s) and lipophilicity, it can be concluded that '1,2,3-propanetriol, glycidyl ethers' would reveal negative results both with and without S9 mix in the UDS assay.
The study was classified as reliable with restrictions (Klimisch 2) and satisfies the requirements for OECD guideline 482 for other genotoxicity data.- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: well-documented publication, which meets basic scientific principles and was performed similar to OECD guideline 476, performed i.a. on the read-across substances glycidol und butylglycidyl ether
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- not applicable
- GLP compliance:
- no
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- tk+
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- The L5178Y mouse lymphoma cells were obtained from Dr. D. Clive, Burroughs Welcome Co.
- Metabolic activation:
- with and without
- Metabolic activation system:
- Liver homogenates prepared from Aroclor-1254-induced Sprague-Dawley rats; non-induced liver homogenate was prepared in the same manner from Sprague-Dawley rats injected with corn oil
- Test concentrations with justification for top dose:
- 5 or more concentrations were used for the mutation assay. The highest dose was set at twice the level that killed 50% of the organisms in the toxicity assay.
Glycidol: 8, 15, 23, 30, 45, 60, 75, 94, 125, 187, 250 µg/mL
Butyl glycidyl ester: 84, 100, 130, 164, 200, 256, 300, 320, 400, 500, 640, 800 µg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- 2-acetylaminofluorene
- N-dimethylnitrosamine
- ethylmethanesulphonate
- Remarks:
- Positive control without S9 fraction was ethyl methanesulfonate, 620 µg/ml; with induced S9 fraction, 2-acetylaminofluorene, 100 µg/ml; with uninduced S9 fraction, dimethylnitrosamine, 74 µg/ml.
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 48h
- Selection time (if incubation with a selection agent): 10-12 days
SELECTION AGENT (mutation assays): 1 µg/ml trifluorothymidine
NUMBER OF REPLICATIONS: A single tube was prepared for each dose level, and the treated cells were cloned in triplicate.
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; other: trypan blue exclusion - Evaluation criteria:
- A compound was considered positive if dose-related increases in the mutation index over at least 3 concentrations with the highest response at least equal to 3.0 were obtained.
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Remarks:
- both glycidol and butyl glycidyl ether
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Conclusions:
- positive with metabolic activation glycidol
positive without metabolic activation glycidol
positive with metabolic activation butyl glycidyl ester
positive without metabolic activation butyl glycidyl ester
The mutagenic potential of the the read-across substances glycidol and butyl glycidyl ester for '1,2,3-propanetriol, glycidyl ethers' was performed in a study similar to OECD 476, assessed with Klimisch 2. Hence, the results are considered as sufficiently reliable to cover this endpoint.
In this assay, a compound was considered positive if dose-related increases in the mutation index over at least 3 concentrations with the highest response at least equal to 3.0 were obtained. These criteria are met for both glycidol and butyl glycidyl ester, without metabolic activation as well as with the induced and non-induced S9 fraction. Hence, both substances and hence '1,2,3-propanetriol, glycidyl ethers' has to be considered as mutagenic in the mouse lymphoma assay. - Executive summary:
In a mammalian gene mutation assay (Mouse lymphoma assay, similar to OECD 476). L5178Y cell cultures were exposed to the read-across substances glycidol and butyl glycidyl ester for '1,2,3-propanetriol, glycidyl ethers' at concentrations of 8, 15, 23, 30, 45, 60, 75, 94, 125, 187, 250 µg/ml (glycidol) or 84, 100, 130, 164, 200, 256, 300, 320, 400, 500, 640, 800 µg/ml with and without metabolic activation. The metabolic activation system was either liver homogenates prepared from Aroclor-1254-induced Sprague-Dawley rats or rats injected with corn oil (non-induced).
Glycidol and butyl glycidyl ester were both tested up to cytotoxic concentrations, i.e. the highest dose was set at twice the level that killed 50% of the organisms in the toxicity assay.
Positive controls (ethyl methanesulfonate, 620 µg/ml, –S9; 2-acetylaminofluorene, 100 µg/ml, + induced S9; dimethylnitrosamine, 74 µg/ml, + uninduced S9) induced the appropriate responses. For both glycidol and butyl glycidyl ester there was a concentration-related positive response as well as the stipulated at least three-fold increase of the mutation frequency over background without or with both available metabolic activation systems. So it can be concluded that '1,2,3-propanetriol, glycidyl ethers' has to be considered as mutagenic in the mouse lymphoma assay, too.
This study is classified as acceptable, reliable with restrictions and satisfies the requirements for OECD guideline 476 for in vitro mammalian cytogenicity data.- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: well-documented publication, which meets basic scientific principles
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- The strains used were: TA98, TA100, E. coli WP 2, WP 2uvr A, CM 571, WP100. No metabolic activation system was used.
- GLP compliance:
- not specified
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his - for S. typhimurium and trp- for E. coli
- Species / strain / cell type:
- bacteria, other: E.coli strains: WP2, WP uvrA, CM 571 and WP 100; S.typhimurium: TA 98, TA 100
- Metabolic activation:
- without
- Metabolic activation system:
- not applicable
- Test concentrations with justification for top dose:
- 0.01, 0.1, 1.0, and 10%
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: not given - Untreated negative controls:
- yes
- Remarks:
- true negative control
- Negative solvent / vehicle controls:
- not specified
- True negative controls:
- yes
- Remarks:
- 10 mg/mL SM (streptomycin, non mutagenic antibiotics)
- Positive controls:
- yes
- Positive control substance:
- furylfuramide
- methylmethanesulfonate
- Remarks:
- Repair test (solid and liquid methods): Furylfuramide (10 µg/mL H2O), Methylmethanesulfonate (1% in H2O); Reversion test (spot and soft agar method): Furylfuramide (1 µg/mL H2O), Methylmethanesulfonate (10% in H2O).
- Details on test system and experimental conditions:
- For the DNA repair tests, solid method and liquid method, four strains of E. coli and two strains (WP2 and WP100) were used, respectively. For the reversion test, E. coli WP2uvrA and S. typhimurium TA98 and TA100 were used. It has been elucidated that TA98 is mutated by frameshift type mutagens and the other two strains by base-change type mutagens.
METHOD OF APPLICATION: in agar (plate incorporation)
Procedure of DNA repair test:
The strains of E. colt were grown in nutrient broth in an incubator shaker at 37°C to reach the early stationary phase (approximately 2-3x10E9cells/mL). Aftergrowth, bacterial cells were washed twice by centrifugations and resuspended in M 9 buffer giving the similar titers of cells at the early stationary phase. For solid method, four cell-suspensions of WP2, WP2uvrA, CM 157 and WP100 were streaked on YP agar from a central point to different directions by 0.1 mL pipetts. An aliquot (0.05 mL) of each sample solution which was dissolved in dimethylsulfoxide (DMSO) or acetone was dropped onto a filter paper disc (diameter 20 mm) which had been placed at the starting point of the streaks of four bacterial strains. After overnight incubation at 37°C, the distances from the end of each streak to the edge of filter paper disc, which shows inhibition for bacterial growth by the sample solutions, were measured. For liquid method, which has been developed for quantitative estimation of differential inhibiting effect of chemicals for bacterial growths at WP2 and WP100. M9GT (2.8 mL) and the sample solution (0.1 mL) at the different concentrations were put into several test tubes. Then, 0.1mL of the cellular suspensions of WP2 or WP100 in M9 buffer was added to each tube. After 18 hours incubation at 37°C without shaking, the absorbance of each test tube was measured at 660 nm by Shimadzu Spectronic 20 Colorimeter.
The differential inhibition for 50% growth (DIGso) in WP2 and WP100 by chemicals was calculated as follows, DIGso = log ((chemical concentration resulting 50% growth in WP2)/ (chemical concentration resulting 50 % growth in WP100))
Procedure of Reversion test:
For spot test, each 0.1 mL portion of the cell suspensions of E. coli WP2uvrA and S. typhimurium TA98 and TA100 were spread on SEM and SEM-B agar, respectively. An aliquot (0.05 mL) of each sample solution was dropped onto a filter paper disc (diameter 20 mm) which had been placed at the centre of the plate. After 48 hours incubation at 37°C, reversion colonies around the paper disc, that appeared against turbid back-ground resulting from residual growth of auxotrophic cells were counted.
Soft agar method was carried out with S. typhimurium TA98 and TA100. An aliquot (0.1 mL) of each sample solution, 0.5 mL of Na-K phosphate buffer and 0.1 mL of the cell suspensions of TA98 or TA100 in M9 buffer were put into several test tubes. After preincubation in an incubator shaker at 37°C for 20 minutes, 2.0 mL of soft agar dissolved previously were put into each tube. Then, the mixtures were immediately poured onto minimal glucose agar plates and spread evenly. After 48 hours incubation at 37°C, reversion colonies were scored as described above. - Species / strain:
- other: E. coli WP 2, E. coli WP 2uvr A, E. coli CM 571 and E. coli WP100
- Metabolic activation:
- without
- Genotoxicity:
- positive
- Remarks:
- DNA repair test of solid method.
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not applicable
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- other: E. coli WP2 100 and E.coli WP 2
- Metabolic activation:
- without
- Genotoxicity:
- positive
- Remarks:
- DNA repair test of liquid method
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not applicable
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- other: E. coli WP 2 uvrA and S. typhimurium TA 100
- Metabolic activation:
- without
- Genotoxicity:
- positive
- Remarks:
- Reversion Test (spot and agar soft methods)
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not applicable
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Remarks:
- Reversion Test (spot and agar soft methods)
- Vehicle controls validity:
- not applicable
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Results: positive without metabolic activation
This well-documented publication gives a detailed information about the experiments conducted and meets basic scientific principles. Therefore it is considered to be of the high quality (reliability Klimisch 2). The validity criteria of the test system are fulfilled. GE-100 was shown to cause gene mutation without metabolic activation in the bacterial strains investigated. GE-100 was negative in S.typhimurium TA98 in reversion tests (spot and soft agar method) without metabolic activation. - Executive summary:
Mutagenic activity of the target substance (1,2,3-propanetriol, glycidyl ethers) was examined with the DNA repair test (solid and liquid) and the reversion test (spot and soft agar methods) using the bacterial strains of E. coli (WP2, WP2uvrA, CM571 and WP100, these strains are nearly isogenic and have a tryptophan-deficiency that is suppressible by ochre suppressor mutation) and S. typhimurium (TA98 and TA100), (Ohtani and Nishioka, 1980). The study was performed similar to the OECD Guideline with deviations (different strains and no metabolic activation system was used) and considered to be of high quality (reliability Klimisch 2). For the DNA repair tests, solid method and liquid method, four strains of E. coli and two strains (WP2 and WP100) were used, respectively. For the reversion test, E. coli WP2uvrA and S. typhimurium TA98 and TA100 were used. It has been elucidated that TA98 is mutated by frameshift type mutagens and the other two strains by base-change type mutagens. The results of the DNA repair test of solid method showed that GE-100 (10 % in DMSO) were positive. The inhibiting zones in the DNA repair test of solid method for the positive samples were always greater in CM571 and WP100 which are deficient in recombination repair than in WP2 and WP2uvrA. This suggests that the epoxide resins cause DNA damage which can be repaired by the process of recombination. The results of the DNA repair test of liquid method (strains tested: E.coli WP100 and E.coli WP2) showed that GE-100 had a remarkable value of DIG50 (differential inhibition for 50% growth = log (chemical concentration resulting in 50% growth in WP2/chemical concentration resulting in 50% growth in WP100): 1.37 and it was almost comparable to positive controls, AF-2 and MMS.
From the results, it is possible to estimate that DNA damaging capacity of these epoxide resins could be similar to the positive controls 2-acetylaminofluorene (AF-2) and methylmethanesulfonate (MMS). The results of the reversion test with the strains of E. coli and S. typyimurium for the samples of epoxide compounds show that all those (data not shown here) which were positive in the DNA repair test induce revertants in E. coli WP2uvrA and in S. typhimurium TA100 but not in TA98. This suggests that these epoxide compounds induce the mutation of base-pair substitution type without metabolic activation. Those which showed no killing effect and no DNA damaging capacity were negative in the reversion test. In the experiment with the commercial adhesive agents, some of them (data not shown) were also mutagenic in TA100 with or without a hardening agent. These results obtained suggest that epoxide resins produce DNA damage which can be repaired by the process of recombination and induce mutation of base-pair substitution type without metabolic activation. Epoxide compounds which have a higher molecular weight and lower solubility showed neither any killing effect nor mutagenic effect in the DNA repair test as well as the reversion test. The reason seems to be that their molecular sizes and solubilities are not small and high enough to pass through the cellular membrane and to reach DNA.
In summary, the results indicate that some epoxide compounds which have relatively lower molecular weight induce mutation. It applies to the target substance GE-100. It is suggested that the mutagenicity of epoxide resins may be influenced by their solubilities and / or transportation through cellular membrane.
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- August 12, 1986 - September 22, 1986
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: GLP-guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- performed with the Salmonella typhimurium strains TA98, TA 100, TA 1535, TA 1537 and TA 1538
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Principles of method if other than guideline:
- Investigation of the potential gene mutagenic activity of GE 100 according to the plate incorporation test of Ames et al.
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his-, obtained from B.N. Ames, University of California, Berkeley CA, 94720, USA, May 1931.
Characterization of strains:
The strains used are mutants derived from Salmonella typhimurium 1_T2 and have the following genotypes:
S. typhimurium TA 1535 his G46 rfa- uvrB-
S. typhimurium TA 1537 his C3076 rfa- uvrB-
S. typhimuriumTA 1533 his D3052 rfa- uvrB-
S. typhimurium TA 98 his D3052 rfa- uvrB- R +
S. typhimurium TA 100 his G46 rfa- uvrB- R + - Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor-supplemented post-mitochondrial fraction prepared from the livers of rats treated with the enzyme-inducing agent Aroclor 1254.
- Test concentrations with justification for top dose:
- 1.58, 5, 15.8, 50, 158, 500, 1580 and 5000 micrograms per plate (mcg/pl.). Each concentration, including the controls, was tested in triplicate.
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: water (bidistilled water)
- Justification for choice of solvent/vehicle: On the day of the experiment, the test article was dissolved in bidistilled water at the highest investigated dose. The other doses were dilutions from this stock solution with the solvent in half-log intervals. All control data were established on the same day as the experiment. - Negative solvent / vehicle controls:
- yes
- Remarks:
- bidistilled water
- Positive controls:
- yes
- Positive control substance:
- other: MMS, 9AMA, 2 -N F and ENNG
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Preincubation period: none
- Exposure duration: the plates were incubated at 37 degrees centigrade in the dark for 3 days.
OTHER: To avoid any light effects on the test article, all experiments were performed under yellow light.
Storage:
The strain cultures are stored in sterile 0.5 ml ampoules (0.45 mL bacterial culture and 0.05 mL dimethylsuIfoxids) at -80 (+/- 5) degrees centigrade.
Pre-culture of strains:
The bacteria were grown in a shaking water bath for approximately 10 hours overnight at 37 °C in 2 .5% Nutrient Broth No. 2*. After centrifugation, the bacteria were resuspended to a concentration of approximately 1 x 10 exp. 8 to 2 x 10 exp.9 cells per milliliter in 0 .16 % Nutrient Broth and 0.5% sodium chloride. The concentration of germs was controlled photometrically and determined in an experimental test with histidine-rich potassium chloride solution on selective agar plates. - Evaluation criteria:
- A material is identified as a mutagen in this test system if there was a reproducible demonstration of a dose-effect relation with a 2-fold increase in the number of revertants compared with the concurrent negative controls in at least one strain. With the strain TA 100, a 1.5-fold increase was the criterion for a positive result. These criteria are generally in accordance with the internationally used standards for the evaluation of Ames Test results.
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Remarks:
- In the toxicity experiment with GE 100, neither quantitative nor qualitative evidence of a cytotoxic effect was observed
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- COMPARISON WITH HISTORICAL CONTROL DATA: Control plates with the solvent (negative control) showed numbers of spontaneous revertant colonies per plate which were within the normal range of those cited in the literature.
Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
No cytotoxic effect of the test article was observed. - Remarks on result:
- other: strain/cell type: TA 98, TA 100 and TA 1535 in the absence as well as in the presence of S-9 mix
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results: positive In TA 98, TA 100 and TA 1535 the test material (GE-100) induced genotoxicity in the absence as well as in the presence of S-9 mix.
The study was performed according to the OECD Guideline 471 and EU Method B13/14 with deviations (5 Salmonella strains tested) and still considered to be of highest quality quality (reliability Klimisch 1). The vehicle and the positive control substances fulfilled validity criteria of the test system. The Salmonella/microsome test, employing doses up to 5000 µg per plate, showed 1,2,3-propanetriol, glycidyl ethers not to produce cytotoxic effects. A dose-dependent elevation in the number of revertant colonies was observed with the strains TA 98, TA 100 and TA 1535 in the absence as well as in the presence of S-9 mix. The revertant rates were enhanced up to a 2-3-fold increase versus concurrent controls in TA 93, up to 14-fold in TA 100 and up to about 30-fold in TA 1535. A mutagenic activity of the test article was ascertained, starting at approx. 153 µg/µl in the experiments without metabolic activation and at approx. 50 µg/µl with activation. - Executive summary:
1,2,3-propanetriole, glycidyl ethers was investigated using the Salmonella/microsome test for point mutagenic effects in doses up to 5000 µg per plate on five Salmonella typhimurium LT2 mutants (TA98, TA 100, TA 1535, TA 1537 and TA 1538). The study was performed according to the OECD Guideline 471and EU Method B13/14 with deviations (five Salmonella strains tested) and considered to be of the highest quality (reliability Klimisch 1). The strains have the following genotypes: S. typhimurium TA 1535 his G46 rfa- uvrB-, S. typhimurium TA 1537 his C3076 rfa- uvrB-, S. typhimurium TA 1533 his D3052 rfa- uvrB-, S. typhimurium TA 98 his D3052 rfa- uvrB- R + and S. typhimurium TA 100 his G46 rfa- uvrB- R +. The bacteria were treated with the test material using the Ames plate incorporation method at up to eight dose levels (1.58, 5, 15.8, 50, 158, 500, 1580 and 5000 µg per plate), in triplicate, both with and without the addition of a rat liver homogenate metabolising system (cofactor-supplemented post-mitochondrial fraction prepared from the livers of rats treated with the enzyme-inducing agent Aroclor 1254.). Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. The vehicle (water) control plates gave counts of revertant colonies within the normal range. The positive controls methyl methanesulfonate (MMS), 9-Aminoacridine (9AMA), 2-nitrofluorene (2 -NF), N-ethyl-N'-nitro-N-nitrosoguanidine(ENNG), and benzo(a)pyrene (BaP) had a marked mutagenic effect, as was seen by a biologically relevant increase of induced revertant colonies compared to the corresponding negative controls. So all of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. No cytotoxic effect of the test article was observed. In the experiments, a dose-dependent elevation in the number of revertant colonies was observed with the strains TA 98, TA 100 and TA 1535 in the absence as well as in the presence of S-9 mix. The revertant rates were enhanced up to a 2-3-fold increase versus concurrent controls in TA 93, up to 14-fold in TA 100 and up to about 30-fold in TA 1535. No clear-cut enhancement of revertant rates was observed in TA 1537 and in TA 1533, except in the second experiment with TA 1538 in the absence of 3-9 mix, where a slight increase up to a doubling was observed at the highest concentration. A mutagenic activity of the test article was ascertained, starting at approx. 153 µg/µl. in the experiments without metabolic activation and at approx. 50 µg/µl with activation. These findings were confirmed in the second, independent experiment. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article, GE 100, showed a reproducible dose-dependent mutagenic activity. So during the described mutagenicity test and under the experimental conditions reported, the test article induced point mutations by base-pair changes and frameshifts in the genome of the strains used . Therefore, 1,2,3-propanetriol, glycidyl ethers is considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.
Referenceopen allclose all
The results of the unscheduled DNA synthesis assays are shown in Table 1. Preliminary tests were performed to define the maximal testable levels of the chemicals. 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. Positive responses were obtained for glycidol in the presence of the S9 activating system. Demonstrable, although not considered positive responses were obtained for butyl glycidyl ether in the presence of S9. The remainder of the assays were negative.Table 1: Levels of the UDS in WI38 cells following exposure to graded doses of glycidol and butyl glycidyl ether
Compound |
Without S9 |
With S9 |
||||
Concentration (µl/ml) |
Thymidine incorporationa |
Response indexb |
Concentration (µl/ml) |
Thymidine incorporationa |
Response indexb |
|
Glycidol |
6.0 |
94 |
<1 |
3.0 |
207 |
3.4 |
3.0 |
94 |
<1 |
1.0 |
171 |
2.8 |
|
1.5 |
89 |
<1 |
0.333 |
120 |
2.0 |
|
0.75 |
92 |
<1 |
0.111 |
86 |
1.4 |
|
0.375 |
82 |
<1 |
0.037 |
62 |
1 |
|
Solvent control |
111 |
1 |
Solvent control |
61 |
1 |
|
Positive controlc |
1087 |
9.8 |
Positive controlc |
346 |
5.7 |
|
Butyl glycidyl ether |
1.2 |
39 |
<1 |
8.0d |
68 |
1 |
0.8 |
46 |
<1 |
4.0d |
128 |
1.9 |
|
0.53 |
65 |
<1 |
2.0 |
75 |
1.1 |
|
0.36 |
75 |
<1 |
1.0 |
69 |
1 |
|
0.24 |
46 |
<1 |
0.5 |
67 |
1 |
|
Solvent control |
70 |
1 |
Solvent control |
67 |
1 |
|
Positive controlc |
1054 |
15.0 |
Positive controlc |
314 |
4.7 |
|
a DPM / µg DNA.
b Response index = ratio of test / control.
c With metabolic activation - dimethylnitrosamine (5 x 10 exp -2 M). Without metabolic activation - 4-nitroquinoline oxide (10 exp 5 M).
d Oil droplets formed on the surface of the medium.
The results of the mouse lymphoma assay are shown in Table 1. The assays were first performed over a wide range of concentrations, then rerun over the narrow ranges shown in the table to demonstrate dose response. The relative growth column is an index of the amount of toxicity to the cells. In most cases the range of concentrations was restricted to 5-fold or less due to toxicity. The mutation frequency was calculated by dividing the number of colonies appearing in the selective plates (TFT) by the number of surviving cells as determined by plating a sample of cells in the absence of the selective agent. The mutation index was calculated by dividing the mutation frequency of the test results by the mutation frequency of the solvent control. For this particular set of experiments, we considered a compound positive if dose-related increases in the mutation index over at least 3 concentrations with the highest response at least equal to 3.0 were obtained. The mouse lymphoma assays were performed without metabolic activation, with metabolic activation by non-induced rat liver microsome preparation, and with an Aroclor-1254-induced rat liver microsome preparation.
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.
Glycidol and the butyl glycidyl ether gave distinctly positive responses either with one of the S9 preparations or without activation. On a molar basis glycidol was considerably more potent than glycidyl ether.
Table 1: Results of the L5178Y TK+/- mouse lymphoma mutagenicity assay for glycidol and Butyl glycidyl ester ± S9 mix
Compound (µg/ml) | Relative growth (percent of control) | Mutation frequency per 10exp6 survivors | Mutation index (ratio of test/control) | ||||||
No S9a | I S9b | UI S9c | No S9a | I S9b | UI S9c | No S9a | I S9b | UI S9c | |
Glycidol | |||||||||
250 | - | 9.5 | - | - | 497 | - | - | 13.1 | - |
187 | - | 34.9 | - | - | 302 | - | - | 7.9 | - |
125 | - | 69.5 | - | - | 148 | - | - | 3.9 | - |
94 | - | 99.4 | - | - | 113 | - | - | 3.0 | - |
75 | - | - | 46.1 | - | - | 507 | - | - | 9.1 |
60 | - | - | 48.7 | - | - | 301 | - | - | 5.4 |
45 | - | - | 44.8 | - | - | 287 | - | - | 5.1 |
30 | 6.5 | - | 42.5 | 727 | - | 176 | 21.4 | - | 3.1 |
23 | 25.1 | - | - | 581 | - | - | 17.1 | - | - |
15 | 67.4 | - | 77.7 | 277 | - | 78 | 6.7 | - | 1.4 |
8 | 80.1 | - | - | 141 | - | - | 4.1 | - | - |
Solvent control | 100.0 | 100.0 | 100.0 | 34 | 38 | 56 | 1.0 | 1.0 | 1.0 |
Positive controle | 38.5 | 65.0 | 50.0 | 818 | 140 | 1280 | 24.5 | 3.7 | 22.9 |
Butyl glycidyl ester | |||||||||
800 | - | 5.3 | - | - | 751 | - | - | 26.7 | - |
640 | - | 34.1 | - | - | 236 | - | - | 8.4 | - |
500 | - | 53.6 | 7.7 | - | 193 | 353 | - | 6.9 | 17.6 |
400 | - | 59.4 | 22.3 | - | 164 | 235 | - | 5.8 | 11.7 |
320 | - | - | 39.9 | - | - | 125 | - | - | 6.2 |
300 | 4.2 | - | - | 805 | - | 64 | 17.9 | - | - |
256 | - | - | 61.7 | - | - | 59 | - | - | 3.2 |
200 | 16.7 | - | 54.2 | 681 | - | 63 | 15.1 | - | 2.9 |
164 | - | - | 46.3 | - | - | 35 | - | - | 3.1 |
130 | - | - | 81.8 | - | - | 37 | - | - | 1.7 |
100 | 58.1 | 116.6 | 79.6 | 133 | 4 | 37 | 3.0 | 0.3 | 1.8 |
84 | - | - | 80.9 | - | - | 38 | - | - | 1.9 |
Solvent control | 100.0 | 100.0 | 100.0 | 45 | 28 | 20 | 1.0 | 1.0 | 1.0 |
Positive controld | 8.4 | 48.1 | 47.5 | 1410 | 90 | 84 | 31.3 | 3.2 | 4.1 |
a Without exogenous metabolic activation.
b With Aroclor-induced S9 fraction.
c With uninduced S9 fraction.
d Positive control without S9 fraction was ethyl methanesulfonate, 620 µg/ml; with induced S9 fraction, 2-acetylaminofluorene, 100 µg/ml; with uninduced S9 fraction, dimethylnitrosamine, 74 µg/ml.
e Positive control with S9 fraction for this compound only was benzo[a]pyrene, 3 µg/ml.
DNA Repair Test
The results of DNA repair test of solid method were shown in Table 4.
GE-100 (10 % in DMSO) was positive in the DNA repair test of solid method.
The inhibiting zones in the DNA repair test of solid method for the positive samples were always greater in CM571 and WP100 which are deficient in recombination repair than in WP2 and WP2uvrA. This suggests that the epoxide resins cause DNA damage which can be repaired by the process of recombination.
In Table 5, the results of the DNA repair test of liquid method are shown. GE-100 showed remarkable values of DIG50 in the test. It was 1.37 and almost comparable to positive controls, AF-2 and MMS. From the results, it is possible to estimate that DNA damaging capacity of GE-100 is similar to AF-2 and MMS.
| Table 4. DNA repair test of solid method | |||||||
| Compounds | Concentration | Inhibition Zone (mm) | |||||
| WP2 | WP2uvrA | CM571 | WP100 | ||||
| control | |||||||
| AF-2 | 10 µg/mL | H20 | 2 | 8 | 10 | 16 | |
| MMS | 1.0 % | H20 | 0 | 1 | 1 | 3 | |
| SM | 10 mg/mL | H„0 | 8 | 8 | 8 | 8 | |
| epoxide compounds | |||||||
| GE-100 | 10% | DMSO | 1 | 2 | 5 | 9 | |
| Table 5. DNA repair test of liquid method | |
| Compounds | DIG60 |
| control | |
| AF-2 | 1.54 |
| MMS | 1.11 |
| SM | 0 |
| epoxide compound | |
| GE-100 | 1.37 |
The results of the reversion test with the strains of E. coli and S. typyimurium for GE-100 are shown in Table 6 and 7. It is obvious that all epoxides (data not shown here) which were positive in the DNA repair test induce revertants in E. coli WP2uvrA and in S. typhimurium TA100 but not in TA98. This suggests that these epoxide compounds induce the mutation of base-pair substitution type without metabolic activation. Those which showed no killing effect and no DNA damaging capacity were negative in the reversion test.
In the experiment with the commercial adhesive agents, AD and CH show also mutagenic in TA100 with or without a hardening agent. Mutagenic activity of AD seems more stronger than that of CH. These results obtained above suggest that epoxide resins produce DNA damage which can be repaired by the process of recombination and induce mutation of base-pair substitution type without metabolic activation. Epoxide compounds such as E-154, E-828, E-1004 and E-1007 which have higher molecular weight and lower solubility showed neither any killing effect nor mutagenic effect in the DNA repair test as well as the reversion test. The reason seems to be that their molecular sizes and solubilities are not small and high enough to pass through the cellular membrane "and to reach DNA.
Epoxide resins have been used for various purposes and their turnouts are increasing year by year. In order to avoid carcinogenic chemicals from our environment, it would be necessary to limit the unlimited use for these mutagenic epoxide resins because we have now sufficient evidence that carcinogenicity links mutagenicity closely.
| Table 6. Reversion test of spot test | |||||||
| Compounds | Concentration | Solvent | Number of revertant colonies/plate | ||||
| WP2uvrA | TA98 | TA100 | |||||
| control | 39 | 54 | 152 | ||||
| AF-2 | 1 µg/mL | H2O | 157 | 146 | 594 | ||
| MMS | 10% | H20 | 46 | 60 | 405 | ||
| epoxide compounds | |||||||
| PGE | 10% | DMSO | 83 | 69 | 317 | ||
| ECH | 1% | DMSO | 454 | 154 | > 10,000 | ||
| GE-100 | 10% | DMSO | 101 | 74 | 737 | ||
| E-191 | 10% | DMSO | 61 | 73 | 756 | ||
| adhesive agents | |||||||
| AD | main agent | undiluted | 102 | 171 | 1319 | ||
| hardening agent | undiluted | 41 | 59 | 167 | |||
| main + hardening | undiluted | 60 | 84 | 207 | |||
| CH | main agent | undiluted | 80 | 122 | 364 | ||
| hardening agent | undiluted | 45 | 62 | 176 | |||
| main+hardening | undiluted | 56 | 91 | 312 | |||
| Table7. Reversion test of soft agar method | ||||
| Compounds | Concentration | Solvent | Number of revertant colonies/plate | |
| TA98 | TA100 | |||
| control | 45 | 181 | ||
| AF-2 | 1 µg/mL | H20 | 216 | 554 |
| MMS | 10% | H20 | 61 | 385 |
| epoxide compounds | ||||
| PGE | 10% | DMSO | 86 | 397 |
| ECU | 1% | DMSO | 101 | > 10,000 |
| GE-100 | 10% | DMSO | 78 | > 10,000 |
| E-191 | 10% | DMSO | 72 | > 10,000 |
Control plates with the solvent (negative control) showed numbers of spontaneous revertant colonies per plate which were within the normal range of those cited in the literature.
Control plates with reference mutagens (positive controls) showed a distinct increase in revertant colonies with the tester strains. This confirms the reversion properties of each strain. The positive results of the mutagens 2 - aminoanthracene and benzo(a)pyrene indicate that the metabolizing system was functioning.
The aseptic control showed no contamination of the 3-9 mix.
Individual results:
| Table 4: | Salmonella test without S-9 mix (first experiment) | |||||||||
| Number of revertant colonies per plate with group means | ||||||||||
| Dose/plate | TA 98 | TA 100 | TA 1535 | TA 1537 | TA 1533 | |||||
| 0 mcg control | 33 | 28 | 72 | 88 | 10 | 11 | 7 | 7 | 19 | 17 |
| 28 | 95 | 13 | 5 | 21 | ||||||
| 24 | 97 | 11 | 10 | 11 | ||||||
| 1,58 mcg | 27 | 22 | 69 | 73 | 11 | 15 | 7 | 7 | 15 | 10 |
| 21 | 69 | 17 | 8 | 10 | ||||||
| 17 | 80 | 18 | 6 | 11 | ||||||
| 5 mcg | 23 | 27 | 81 | 83 | 20 | 16 | 7 | 6 | 17 | 12 |
| 29 | 88 | 14 | 7 | 10 | ||||||
| 29 | 79 | 14 | 5 | 10 | ||||||
| 15.8 mcg | 30 | 27 | 80 | 81 | 16 | 15 | 4 | 8 | 10 | 12 |
| 28 | 32 | 13 | C | 16 | ||||||
| 22 | --- | 16 | 12 | 11 | ||||||
| 50 mcg | 27 | 24 | 89 | 90 | 19 | 16 | 7 | 5 | 18 | 16 |
| 18 | 84 | 16 | 4 | 18 | ||||||
| 26 | 97 | 13 | 4 | 13 | ||||||
| 158 mcg | 31 | 33 | 109 | 112 | 31 | 23 | 14 | 9 | 13 | 15 |
| 32 | 111 | 19 | 7 | 17 | ||||||
| 35 | 116 | 20 | 5 | 16 | ||||||
| 500 mcg | 29 | 34 | 122 | 140 | 50 | 48 | 3 | 9 | 14 | 15 |
| 34 | 147 | 52 | 10 | 14 | ||||||
| 40 | 150 | 41 | 13 | 17 | ||||||
| 1580 mcg | 57 | 55 | 276 | 294 | 113 | 94 | 6 | 11 | 12 | 17 |
| 51 | 301 | 92 | 15 | 19 | ||||||
| 57 | 306 | 76 | 12 | 21 | ||||||
| 5000 mcg | 73 | 71 | 650 | 631 | 193 | 193 | 6 | 11 | 19 | 17 |
| 62 | 655 | 186 | 14 | 19 | ||||||
| 79 | 589 | 201 | 13 | 12 | ||||||
| MMS 500 mcg | --- | --- | 467 | 498 | --- | --- | --- | --- | --- | --- |
| --- | 525 | --- | --- | --- | ||||||
| --- | 502 | --- | --- | --- | ||||||
| 9-AMA 4 0 mcg | --- | --- | --- | --- | --- | --- | 550 | 515 | --- | --- |
| --- | --- | --- | 485 | --- | ||||||
| --- | --- | --- | 509 | --- | ||||||
| 2-NF 5 mcg | 321 | 380 | 498 | 503 | ___ | --- | 37 | 36 | 794 | 718 |
| 407 | 462 | --- | 32 | 717 | ||||||
| 413 | 550 | --- | 39 | 642 | ||||||
| ENNG 10 mcg | 137 | 160 | 1351 | 1178 | 842 | 977 | --- | --- | --- | --- |
| 179 | 1238 | 896 | --- | --- | ||||||
| 164 | 946 | 1194 | --- | --- | ||||||
| --- Not determined | ||||||||||
| C contaminated | ||||||||||
| Table 5: | Salmonella test with S-9 mix (first experiment) | |||||||||
| Number of revertant colonies per plate with group means | ||||||||||
| Dose/plate | TA 98 | TA 100 | TA 1535 | TA 1537 | TA 1533 | |||||
| 0 mcg control | 26 | 32 | 74 | 86 | 12 | 13 | 10 | 14 | 42 | 17 |
| 32 | 96 | 8 | 14 | 36 | ||||||
| 37 | 87 | 20 | 19 | C | ||||||
| 1,58 mcg | 34 | 34 | 115 | 111 | 18 | 13 | 16 | 15 | 39 | 10 |
| 31 | 92 | 8 | 18 | 40 | ||||||
| 37 | 127 | 13 | 12 | 35 | ||||||
| 5 mcg | 22 | 28 | 108 | 108 | 11 | 14 | 15 | 19 | 38 | 12 |
| 24 | --- | 21 | 21 | 45 | ||||||
| 38 | --- | 9 | 19 | 34 | ||||||
| 15.8 mcg | 43 | 38 | 70 | 68 | 22 | 22 | 16 | 16 | 43 | 12 |
| 36 | 71 | 25 | 17 | 48 | ||||||
| 34 | 64 | 20 | 14 | 48 | ||||||
| 50 mcg | 34 | 31 | 99 | 100 | 39 | 45 | 19 | 21 | 44 | 16 |
| 24 | 87 | 46 | 23 | 53 | ||||||
| 35 | 113 | 50 | 20 | 53 | ||||||
| 158 mcg | 38 | 34 | 162 | 171 | 159 | 161 | 14 | 15 | 46 | 15 |
| 34 | 168 | 143 | 20 | 48 | ||||||
| 31 | 182 | 181 | 11 | 37 | ||||||
| 500 mcg | 42 | 38 | 418 | 415 | 468 | 447 | 27 | 21 | 53 | 15 |
| 33 | 417 | 442 | 14 | 60 | ||||||
| 38 | 410 | 432 | 23 | 34 | ||||||
| 1580 mcg | 32 | 38 | 757 | 790 | 794 | 776 | 21 | 17 | 39 | 17 |
| 43 | 796 | 733 | 16 | 37 | ||||||
| 38 | 818 | 802 | 15 | 45 | ||||||
| 5000 mcg | 54 | 59 | 1256 | 1181 | 1093 | 1062 | 18 | 16 | 34 | 17 |
| 56 | 1188 | 1037 | 13 | 37 | ||||||
| 67 | 1099 | 1057 | 15 | 29 | ||||||
| 2-AA 0.5 mcg | 63 | 60 | 162 | 150 | --- | --- | --- | --- | 114 | 113 |
| 57 | 134 | --- | --- | 138 | ||||||
| 61 | 155 | --- | --- | 87 | ||||||
| 2-AA 1 mcg | --- | --- | --- | --- | 71 | 57 | --- | --- | --- | --- |
| --- | --- | 54 | --- | --- | ||||||
| --- | --- | 47 | --- | --- | ||||||
| 2-AA 10 mcg | --- | --- | --- | --- | --- | --- | 172 | 155 | --- | --- |
| --- | --- | --- | 135 | --- | ||||||
| --- | --- | --- | 159 | --- | ||||||
| BaP 5 mcg | 92 | 103 | 300 | 293 | --- | --- | 66 | 69 | 109 | 113 |
| 108 | 305 | --- | 71 | 114 | ||||||
| 109 | 274 | --- | 69 | 115 | ||||||
| --- Not determined | ||||||||||
| C contaminated | ||||||||||
| TABLE 6 : Toxicity test without S-9 mix (first experiment) | |||||
| DOSE | MEAN NUMBERS OF REVERTANT COLONIES | RELATIVE SURVIVAL RATE | |||
| TA 1537 | TA 1537 + RTA | ||||
| 0 mcg control | 7 | 236 | 1 | ||
| 1.58 mcg | 7 | 230 | 0,97 | ||
| 5 mcg | 6 | 226 | 0,96 | ||
| 15.8 mcg | 8 | 233 | 0,93 | ||
| 50 mcg | 5 | 243 | 1,04 | ||
| 158 mcg | 9 | 260 | 1,1 | ||
| 500 mcg | 9 | 243 | 1,02 | ||
| 1580 mcg | 11 | 254 | 1,06 | ||
| 5000 mcg | 11 | 253 | 1,06 | ||
| TABLE 7 : Toxicity test with S-9 mix (first experiment) | |||||
| DOSE | MEAN NUMBERS OF REVERTANT COLONIES | RELATIVE SURVIVAL RATE | |||
| TA 1537 | TA 1537 + RTA | ||||
| 0 mcg control | 14 | 172 | 1,00 | ||
| 1.58 mcg | 15 | 189 | 1,10 | ||
| 5 mcg | 18 | 202 | 1,16 | ||
| 15.8 mcg | 16 | 206 | 1,20 | ||
| 50 mcg | 21 | 223 | 1,28 | ||
| 158 mcg | 15 | 205 | 1,20 | ||
| 500 mcg | 21 | 210 | 1,20 | ||
| 1580 mcg | 17 | 199 | 1,15 | ||
| 5000 mcg | 16 | 204 | 1,19 | ||
| Table 8: | Salmonella test without S-9 mix (second experiment) | |||||||||
| Number of revertant colonies per plate with group means | ||||||||||
| Dose/plate | TA 98 | TA 100 | TA 1535 | TA 1537 | TA 1533 | |||||
| 0 mcg control | 33 | 26 | 116 | 104 | 8 | 18 | 14 | 11 | 15 | 14 |
| 29 | 76 | 19 | 12 | 6 | ||||||
| 16 | 120 | 27 | 8 | 22 | ||||||
| 1,58 mcg | 25 | 23 | 114 | 99 | 20 | 19 | 9 | 8 | 11 | 15 |
| 24 | 97 | 14 | 7 | 20 | ||||||
| 19 | 86 | 24 | 8 | 14 | ||||||
| 5 mcg | 28 | 25 | 107 | 107 | 17 | 16 | 9 | 9 | 11 | 11 |
| 27 | 114 | 14 | 7 | 12 | ||||||
| 21 | 99 | 17 | 10 | 10 | ||||||
| 15.8 mcg | 31 | 28 | 99 | 110 | 21 | 21 | 10 | 7 | 6 | 10 |
| 27 | 97 | 24 | 7 | 14 | ||||||
| 26 | 133 | 17 | 4 | 11 | ||||||
| 50 mcg | 27 | 27 | 113 | 116 | 21 | 21 | 10 | 10 | 16 | 17 |
| 28 | 125 | 23 | 13 | 17 | ||||||
| 26 | 111 | 19 | 8 | 19 | ||||||
| 158 mcg | 25 | 24 | 147 | 144 | 31 | 32 | 9 | 7 | 22 | 15 |
| 17 | 135 | 35 | 4 | 5 | ||||||
| 31 | 149 | 30 | 8 | 17 | ||||||
| 500 mcg | 23 | 24 | 195 | 200 | 60 | 57 | 10 | 11 | 22 | 20 |
| 23 | 202 | 61 | 12 | 14 | ||||||
| 25 | 204 | 50 | 10 | 25 | ||||||
| 1580 mcg | 38 | 42 | 345 | 359 | 128 | 135 | 13 | 11 | 20 | 16 |
| 40 | 355 | 127 | 9 | 18 | ||||||
| 49 | 376 | 150 | 11 | 11 | ||||||
| 5000 mcg | 60 | 73 | 666 | 701 | 240 | 241 | 10 | 14 | 26 | 29 |
| 82 | 726 | 237 | 12 | 31 | ||||||
| 78 | 710 | 246 | 19 | 29 | ||||||
| MMS 500 mcg | --- | --- | 526 | 537 | --- | --- | --- | --- | --- | --- |
| --- | 543 | --- | --- | --- | ||||||
| --- | 543 | --- | --- | --- | ||||||
| 9-AMA 4 0 mcg | --- | --- | --- | --- | --- | --- | 108 | 118 | --- | --- |
| --- | --- | --- | 129 | --- | ||||||
| --- | --- | --- | 116 | --- | ||||||
| 2-NF 5 mcg | 714 | 662 | 580 | 614 | ___ | --- | 95 | 80 | 945 | 940 |
| 657 | 581 | --- | 75 | 974 | ||||||
| 615 | 681 | --- | 69 | 901 | ||||||
| ENNG 10 mcg | 185 | 172 | 572 | 625 | 847 | 794 | --- | --- | --- | --- |
| 151 | 682 | 662 | --- | --- | ||||||
| 181 | 621 | 872 | --- | --- | ||||||
| --- Not determined | ||||||||||
| C contaminated | ||||||||||
| Table 9: | Salmonella test with S-9 mix (second experiment) | |||||||||
| Number of revertant colonies per plate with group means | ||||||||||
| Dose/plate | TA 98 | TA 100 | TA 1535 | TA 1537 | TA 1533 | |||||
| 0 mcg control | 46 | 56 | 97 | 100 | 11 | 15 | 32 | 30 | 23 | 29 |
| 57 | 91 | 13 | 34 | 26 | ||||||
| 64 | 113 | 21 | 25 | 37 | ||||||
| 1,58 mcg | 56 | 56 | 96 | 107 | 17 | 15 | 19 | 27 | 32 | 32 |
| 53 | 116 | 11 | 31 | 33 | ||||||
| 58 | 109 | 16 | 31 | 32 | ||||||
| 5 mcg | 43 | 38 | 109 | 112 | 19 | 18 | 22 | 28 | 34 | 29 |
| 37 | 129 | 19 | 29 | 30 | ||||||
| 33 | 99 | 17 | 34 | 24 | ||||||
| 15.8 mcg | 43 | 40 | 117 | 122 | 27 | 27 | 25 | 26 | 24 | 24 |
| 34 | 131 | 27 | 31 | 21 | ||||||
| 43 | 117 | 28 | 22 | 27 | ||||||
| 50 mcg | 46 | 44 | 152 | 147 | 68 | 65 | 26 | 27 | 24 | 29 |
| 35 | 159 | 67 | 29 | 22 | ||||||
| 51 | 129 | 60 | 26 | 41 | ||||||
| 158 mcg | 47 | 51 | 227 | 218 | 167 | 144 | 27 | 36 | 39 | 35 |
| 54 | 223 | 130 | 25 | 38 | ||||||
| 51 | 204 | 135 | 55 | 27 | ||||||
| 500 mcg | 53 | 55 | 435 | 328 | 307 | 308 | 19 | 21 | 28 | 33 |
| 69 | 252 | 292 | 21 | 37 | ||||||
| 44 | 297 | 324 | 22 | 33 | ||||||
| 1580 mcg | 63 | 57 | 585 | 587 | 610 | 614 | 19 | 25 | 26 | 29 |
| 65 | 604 | 657 | 25 | 28 | ||||||
| 44 | 573 | 576 | 31 | 32 | ||||||
| 5000 mcg | 67 | 73 | 1157 | 1175 | 980 | 975 | 26 | 23 | 31 | 30 |
| 78 | 1185 | 964 | 20 | 28 | ||||||
| 75 | 1182 | 981 | 23 | 31 | ||||||
| 2-AA 0.5 mcg | 122 | 182 | 200 | 185 | --- | --- | --- | --- | 77 | 73 |
| 236 | 172 | --- | --- | 65 | ||||||
| 187 | 183 | --- | --- | 76 | ||||||
| 2-AA 1 mcg | --- | --- | --- | --- | 112 | 107 | --- | --- | --- | --- |
| --- | --- | 115 | --- | --- | ||||||
| --- | --- | 93 | --- | --- | ||||||
| 2-AA 10 mcg | --- | --- | --- | --- | --- | --- | 160 | 150 | --- | --- |
| --- | --- | --- | 140 | --- | ||||||
| --- | --- | --- | 150 | --- | ||||||
| BaP 5 mcg | 128 | 156 | 341 | 332 | --- | --- | 90 | 83 | 67 | 71 |
| 133 | 357 | --- | 85 | 66 | ||||||
| 206 | 297 | --- | 74 | 80 | ||||||
| --- Not determined | ||||||||||
| TABLE 10 : Toxicity test without S-9 mix (second experiment) | |||||
| DOSE | MEAN NUMBERS OF REVERTANT COLONIES | RELATIVE SURVIVAL RATE | |||
| TA 1537 | TA 1537 + RTA | ||||
| 0 mcg control | 11 | 442 | 1,00 | ||
| 1.58 mcg | 8 | 466 | 1,06 | ||
| 5 mcg | 9 | 444 | 1,01 | ||
| 15.8 mcg | 7 | 437 | 1,00 | ||
| 50 mcg | 10 | 454 | 1,03 | ||
| 158 mcg | 7 | 440 | 1,00 | ||
| 500 mcg | 11 | 448 | 1,01 | ||
| 1580 mcg | 11 | 443 | 1,00 | ||
| 5000 mcg | 14 | 441 | 0,99 | ||
| TABLE 11 : Toxicity test with S-9 mix (second experiment) | |||||
| DOSE | MEAN NUMBERS OF REVERTANT COLONIES | RELATIVE SURVIVAL RATE | |||
| TA 1537 | TA 1537 + RTA | ||||
| 0 mcg control | 30 | 681 | 1,00 | ||
| 1.58 mcg | 27 | 649 | 0,96 | ||
| 5 mcg | 28 | 662 | 0,97 | ||
| 15.8 mcg | 26 | 622 | 0,92 | ||
| 50 mcg | 27 | 924 | 1,38 | ||
| 158 mcg | 36 | 941 | 1,39 | ||
| 500 mcg | 21 | 917 | 1,38 | ||
| 1580 mcg | 25 | 933 | 1,39 | ||
| 5000 mcg | 23 | 1015 | 1,52 | ||
In the described bacterial mutagenicity tests, a dose-dependent elevation in the number of revertant colonies was observed with the strains TA 93, TA 100 and TA 1535 in the absence as well as in the presence of S-9 mix. The revertant rates were enhanced up to a 2-3-fold increase versus concurrent controls in TA 93, up to 14-fold in TA 100 and up to about 30-fold in TA 1535.
A mutagenic activity of the test article was ascertained, starting at approx. 158 mcg/µl in the experiments without metabolic activation and at approx. 50 mcg/pl with metabolic activation. These findings were confirmed in the second, independent experiment
No clear-cut enhancement of revertant rates was observed in TA 1537 and in TA 1533, except in the second experiment with TA 1538 in the absence of 3-9 mix, where a slight increase up to a doubling was observed at the highest concentration.
In the experiments, a dose-dependent elevation in the number of revertant colonies was observed with the strains TA 98, TA 100 and TA 1535 in the absence as well as in the presence of S-9 mix. The revertant rates were enhanced up to a 2-3-fold increase versus concurrent controls in TA 93, up to 14-fold in TA 100 and up to about 30-fold in TA 1535. A mutagenic activity of the test article was ascertained, starting at approx. 153 mcg/µl in the experiments without metabolic activation and at approx. 50 mcg/µl with activation. These findings were confirmed in the second, independent experiment.
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article induced reproducible dose-dependent mutagenic activity (point mutations by base-pair changes and frameshifts in the genome of the strains used).
Therefore, GE 100 is considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed (positive)
Additional information
Mutagenicity in bacterial strains
Data on 1,2,3 - propanetriol, glycidyl ethers (GE-100):
1,2,3-propanetriole, glycidyl ethers was investigated using the Salmonella/microsome test for point mutagenic effects in doses up to 5000 µg per plate on five Salmonella typhimurium LT2 mutants (TA98, TA 100, TA 1535, TA 1537 and TA 1538; Banduhn, 1986). The study was performed according to the OECD Guideline 471and EU Method B13/14 with deviations (five Salmonella strains tested) and considered to be of the highest quality (reliability Klimisch 1). The strains have the following genotypes: S. typhimurium TA 1535 his G46 rfa- uvrB-, S. typhimurium TA 1537 his C3076 rfa- uvrB-, S. typhimurium TA 1533 his D3052 rfa- uvrB-, S. typhimurium TA 98 his D3052 rfa- uvrB- R + and S. typhimurium TA 100 his G46 rfa- uvrB- R +. The bacteria were treated with the test material using the Ames plate incorporation method at up to eight dose levels (1.58, 5, 15.8, 50, 158, 500, 1580 and 5000 µg per plate), in triplicate, both with and without the addition of a rat liver homogenate metabolising system (cofactor-supplemented post-mitochondrial fraction prepared from the livers of rats treated with the enzyme-inducing agent Aroclor 1254.). Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. The vehicle (water) control plates gave counts of revertant colonies within the normal range. The positive controls methyl methanesulfonate (MMS), 9-Aminoacridine (9AMA), 2-nitrofluorene (2 -NF), N-ethyl-N'-nitro-N-nitrosoguanidine(ENNG), and benzo(a)pyrene (BaP) had a marked mutagenic effect, as was seen by a biologically relevant increase of induced revertant colonies compared to the corresponding negative controls. So all of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. No cytotoxic effect of the test article was observed. In the experiments, a dose-dependent elevation in the number of revertant colonies was observed with the strains TA 98, TA 100 and TA 1535 in the absence as well as in the presence of S-9 mix. The revertant rates were enhanced up to a 2-3-fold increase versus concurrent controls in TA 93, up to 14-fold in TA 100 and up to about 30-fold in TA 1535. No clear-cut enhancement of revertant rates was observed in TA 1537 and in TA 1533, except in the second experiment with TA 1538 in the absence of S-9 mix, where a slight increase up to a doubling was observed at the highest concentration. A mutagenic activity of the test article was ascertained, starting at approx. 153 µg/µL in the experiments without metabolic activation and at approx. 50 µg/µL with activation. These findings were confirmed in the second, independent experiment. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article, GE 100, showed a reproducible dose-dependent mutagenic activity. So during the described mutagenicity test and under the experimental conditions reported, the test article induced point mutations by base-pair changes and frameshifts in the genome of the strains used. Therefore, 1,2,3-propanetriol, glycidyl ethers is considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.
Mutagenic activity of the target substance(1,2,3-propanetriol, glycidyl ethers) was examined with the DNA repair test (solid and liquid) and the reversion test (spot and soft agar methods) using the bacterial strains of E. coli (WP2, WP2uvrA, CM571 and WP100, these strains are nearly isogenic and have a tryptophan-deficiency that is suppressible by ochre suppressor mutation) and S. typhimurium (TA98 and TA100), (Ohtani and Nishioka, 1980). The study was performed similar to the OECD Guideline with deviations (different strains and no metabolic activation system was used) and considered to be of high quality (reliability Klimisch 2). For the DNA repair tests, solid method and liquid method, four strains of E. coli and two strains (WP2 and WP100) were used, respectively. For the reversion test, E. coli WP2uvrA and S. typhimurium TA98 and TA100 were used. It has been elucidated that TA98 is mutated by frameshift type mutagens and the other two strains by base-change type mutagens. The results of the DNA repair test of solid method showed that GE-100 (10 % in DMSO) caused a positive effect. The inhibiting zones in the DNA repair test of solid method for the positive samples were always greater in CM571 and WP100 which are deficient in recombination repair than in WP2 and WP2uvrA. This suggests that the epoxide resins caused DNA damage which can be repaired by the process of recombination. The results of the DNA repair test of liquid method (strains tested: E.coli WP100 and E.coli WP2) showed that GE-100 had a remarkable value of DIG50 (differential inhibition for 50% growth = log (chemical concentration resulting in 50% growth in WP2/chemical concentration resulting in 50% growth in WP100): 1.37 and it was almost comparable to positive controls, AF-2 and MMS.
From the results, it is possible to estimate that DNA damaging capacity of these epoxide resins could be similar to the positive controls 2-acetylaminofluorene (AF-2) and methylmethanesulfonate (MMS). The results of the reversion test with the strains of E. coli and S. typyimurium for the samples of epoxide compounds show that all those (data not shown here) which were positive in the DNA repair test induce revertants in E. coli WP2uvrA and in S. typhimurium TA100 but not in TA98. This suggests that these epoxide compounds induce the mutation of base-pair substitution type without metabolic activation. Those which showed no killing effect and no DNA damaging capacity were negative in the reversion test. In the experiment with the commercial adhesive agents, some of them (data not shown) were also mutagenic in TA100 with or without a hardening agent. These results obtained suggest that epoxide resins produce DNA damage which can be repaired by the process of recombination and induce mutation of base-pair substitution type without metabolic activation. Epoxide compounds which have a higher molecular weight and lower solubility showed neither any killing effect nor mutagenic effect in the DNA repair test as well as the reversion test. The reason seems to be that their molecular sizes and solubilities are not small and high enough to pass through the cellular membrane and to reach DNA.
In summary,the results indicate that some epoxide compounds which have relatively lower molecular weight induce mutation. It applies to the target substance GE-100. It is suggested that the mutagenicity of epoxide resins may be influenced by their solubilities and / or transportation through cellular membrane.
Data on Polyglycidyl Ether of Substituted Glycerin (EPON 562)
The "Patty´s Industial Hygiene and Toxicology" contains information about Polyglycidyl ether of substituted glycerine (EPON 562) (Hine et al., 1981). It is mentioned, that the substance, tested in S. typhimurium TA 98, Ta 100, TA 1531 and TA 1533, at concentrations of 50 to 1000 µg per plate, caused a positive results in TA 98 and 100 but negative at 50 µg; and negative results in TA 1531 and 1533.
Mutagenicity and Chromosome Aberration in mammalian cells
Data on read-across substances
The target chemical was profiled as "Epoxides" by the "US EPA New Chemical Categories" (OECD QSAR Toolbox, v3.1, 2013). Common properties of epoxides are high reactivity, cytotoxicity, and high probability of mutagenic potential and/or carcinogenicity. Therefore chemicals with the same profiling result have been retrieved from the database. The chemicals containing other chemical elements in their structure and/or other organic functional groups were considered dissimilar to the target chemical and have been removed from the domain. The target chemical is obtained by the reaction of epichlorohydrin with glycerol. Therefore, epichlorohydrin is considered to be a suitable candidate for read-across. Glycidol is the simplest representative of glycidyl ethers category (HPV, Epoxy Resin Systems Task Group (ERSTG), 2001). The other category members possess epoxy moieties in their structures and their profiling results regarding the ability to bind to proteins and to DNA (property which is likely responsible for genetic toxicity) are similar to those of the target chemical. Therefore, they considered to be suitable for read-across. Three chemicals have been removed from the domain as they are too lipophilic compared to the target chemical. The target chemical is predicted to be positive in Mouse lymphoma cells, in Chinese hamster Lung (CHL) cells as well as in in vivo Micronucleus Test.
In a mammalian gene mutation assay (Mouse Lymphoma Assay, similar to OECD 476), L5178Y cell cultures were exposed to the read-across substances glycidol and butyl glycidyl ether at concentrations of 8, 15, 23, 30, 45, 60, 75, 94, 125, 187, 250 µg/mL (glycidol) or 84, 100, 130, 164, 200, 256, 300, 320, 400, 500, 640, 800 µg/mL (butyl glycidyl ether) with and without metabolic activation (Thompson et al., 1981). The metabolic activation system was either liver homogenates prepared from Aroclor-1254-induced Sprague-Dawley rats or rats injected with corn oil (non-induced). Glycidol and butyl glycidyl ether were both tested up to cytotoxic concentrations, i.e. the highest dose was set at twice the level that killed 50% of the organisms in the toxicity assay. Positive controls (ethyl methanesulfonate, 620 µg/mL, –S9; 2-acetylaminofluorene, 100 µg/mL, + induced S9; dimethylnitrosamine, 74 µg/mL, + uninduced S9) induced the appropriate responses. For both glycidol and butyl glycidyl ether there was a concentration-related positive response as well as the stipulated at least three-fold increase of the mutation frequency over background without or with both available metabolic activation systems. Thus, it can be concluded that 1,2,3-propanetriol, glycidyl ethers possesses mutagenic activity in the Mouse Lymphoma Assay, too. This study is classified as acceptable, reliable with restrictions and satisfies the requirements for OECD Guideline 476 for in vitro mammalian cytogenicity data.
In an unscheduled DNA synthesis assay similar to OECD guideline 482, WI38 cells were exposed to the read-across substances glycidol and butyl glycidyl ether at concentrations of 0, 0.375, 0.75, 1.5, 3.0, 6.0 µg/mL (glycidol, without S9); 0, 0.037, 0.111, 0.333, 1.0, 3.0 µg/mL (glycidol, with S9); 0, 0.24, 0.36, 0.53, 0.8, 1.2 µg/mL (butyl glycidyl ether, without S9); 0, 0.5, 1.0, 2.0, 4.0, 8.0 µg/mL (butyl glycidyl ether, with S9) (Thompson et al., 1981). Glycidol and butyl glycidyl ether were tested just below the level which produced cytotoxicity. The positive controls (4-nitroquinoline-N-oxide (4NQO), dimethylnitrosamine (DMN)), induced the appropriate response.
There was no evidence or a dose related positive response for both compounds without metabolic activation that unscheduled DNA synthesis, as determined by radioactive tracer procedures, was induced. Glycidol induced a dose-related positive response with metabolic activation, butyl glycidyl ether induced demonstrable, although not considered positive responses with metabolic activation. The study was classified as reliable with restrictions (Klimisch 2) and satisfies the requirements for OECD guideline 482 for other genotoxicity data.
Justification for classification or non-classification
The target substance showed a reproducible dose-dependent mutagenic activity in Salmonella typhimurium reverse mutation assay (Banduhn, 1986). The chemical was predicted positive in Mouse lymphoma cells, in Chinese hamster Lung (CHL) cells as well as in in vivo Micronucleus Test (the OECD QSAR Toolbox v3.1). By this modelling tool, the epoxy ring was identified as a structural alert reacting with nucleophilic sites of DNA by SN2 mechanism and causing mutations.
There are numerous studies available publically for a variety of structurally similar epoxy compounds. The chemicals containing the same glycidyloxy moieties in their structures are almost all positive in a variety of genetic toxicity studies in vitro and in vivo. For instance, the read-across substances glycidol and butyl glycidyl ether showed a concentration-related positive response as well as the stipulated at least three-fold increase of the mutation frequency over background without or with metabolic activation in the Mouse Lymphoma Assay (Thompson et al., 1981). Ohtani et al. (1981) pointed to a trend in the genetic toxicity potency of epoxy compounds depending on their molecular weights and solubilities. The epoxy resins with large molecular size and low water solubility showed neither killing effect nor mutagenic effect in the DNA repair test as well as the reversion test in bacterial test system (Ohtani et al., 1981). Nevertheless, low molecular epoxides including GE-100 (the target substance) were positive (Ohtani et al., 1981). This assumption was confirmed in a lot of studies conducted with alkyl glycidyl ethers containing hydrocarbon chains of different lengths. The ethers with C-4 hydrocarbon side chain showed a definite response while the C-8 or higher ethers showed very weak or no responses in the Ames Test, Mouse Lymphoma Assay or UDS Test (Thompson et al., 1981).
Based on the extrapolation from glycidol, epichlorohydrin, allyl-, butyl-, isopropyl-, phenyl glycidyl ethers as well as diglycidyl ether and alkyl glycidyl ethers (see read-across statement in section 13) and taking into account molecule size, side chain length(s) and lipophilicity, it can be concluded that mutagenic activity of 1,2,3-propanetriol, glycidyl ethers cannot be ruled out. Therefore classification and labelling are warranted according to the criteria of the European regulation (EC) No. 1272/2008:
Mutagenicity, Cat 2, H 341 (suspected of causing genetic defects)
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