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EC number: 608-659-1 | CAS number: 31692-85-0
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 12 July - 21 Aug 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 017
- Report date:
- 2017
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- adopted in 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- adopted in 2008
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Version / remarks:
- adopted in Aug 1998
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- 2-[(oxolan-2-yl)methoxy]ethan-1-ol
- EC Number:
- 608-659-1
- Cas Number:
- 31692-85-0
- Molecular formula:
- C5H10O2[C2H4O]n, n = 0, 1, 2, 3, 4, ... (data given for max n of 4)
- IUPAC Name:
- 2-[(oxolan-2-yl)methoxy]ethan-1-ol
Constituent 1
Method
- Target gene:
- his operon
Species / strain
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor-supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male rats, treated with phenobarbital and β-naphthoflavone
- Test concentrations with justification for top dose:
- Prior to the main experiments, a range-finding study was performed using the TA 98 and TA 100 strains with following test concentrations: 0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5, 5.0 μL/plate with and without metabolic activation.
The following six concentrations were then used in the two main experiments: 0.0316, 0.100, 0.316, 1.0, 2.5, 5.0 μL/plate with and without metabolic activation. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent: good compatibility with the survival of the bacteria and the S9 activity
Controls
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- distilled water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- methylmethanesulfonate
- other: 4-nitro-o-phenylene-diamine (4-NOPD); 2-aminoanthracene (2AA)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation) (Range Finding Test and first main experiment); preincubation (second confirmatory experiment)
DURATION
- Preincubation period: 60 min
- Exposure duration: 48 h
NUMBER OF REPLICATIONS: triplicates each in two independent experiments
DETERMINATION OF CYTOTOXICITY
- Method: revertant colony number and inspection of the bacterial background lawn, toxicity is determined by a clearing or diminution of the background lawn or reduction of the number of revertants to a mutation factor of approximatively ≤ 0.5 in relation to the solvent control. - Evaluation criteria:
- Acceptance criteria
The study was considered valid if for each strain:
- the bacteria demonstrate their typical responses to ampicillin (TA 98, TA 100, TA 102)
- the negative/solvent control plates with and without S9 mix are within the historical control data ranges
- corresponding background growth on negative control, solvent control and test plates is observed
- the positive controls show a distinct enhancement of revertant rates over the control plate
- at least five different concentrations of each tester strain are analysable.
Evaluation criteria
The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.
According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system. - Statistics:
- According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
Results and discussion
Test resultsopen allclose all
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- RANGE-FINDING/SCREENING STUDIES: There was no reduced background lawn and barely any change in the mutation factor of the treated cells (TA98: -S9 Mix: 0.9 - 1.3, +S9 Mix: 0.9 - 1.2; TA100: -S9 Mix: 0.9 - 1.2, +S9 Mix: 1.0 - 1.2) compared to the solvent control. The positive controls induced a reversion rate more than two times higher than the reversion rate of the solvent control.
HISTORICAL CONTROL DATA (2014 - 2016)
- Positive historical control data:
Range values revertants, without S9:
TA 98 (4-NOPD): 141 - 1830
TA 100 (SA): 132 - 1423
TA 1535 (SA): 38 - 1854
TA 1537 (4-NOPD): 35 - 273
TA 102 (MMS): 272 - 3321
Range values revertants, with S9:
TA 98 (2AA): 70 - 3606
TA 100 (2AA): 169 - 3132
TA 1535 (2AA): 22 - 1954
TA 1537 (2AA): 26 - 682
TA 102 (2AA): 137 - 3588
- Negative (solvent/vehicle) historical control data:
Range values revertants, without S9:
TA 98: 11 - 58
TA 100: 49 - 155
TA 1535: 4 - 41
TA 1537: 3 - 35
TA 102: 141 - 472
Range values revertants, with S9:
TA 98: 15 - 59
TA 100: 62 - 160
TA 1535: 3 - 38
TA 1537: 3 - 36
TA 102: 157 - 586
Any other information on results incl. tables
Table 1:Summary of test results (experiment 1; Initial Mutation Test, Plate Incorporation Method)
With or without S9-Mix |
Test substance concentration (μL/plate) |
Mean number of revertant colonies per plate |
||||
Frameshift type |
Base-pair substitution type |
|||||
TA1537 |
TA98 |
TA100 |
TA1535 |
TA102 |
||
– |
Solvent control (distilled water) |
22 ± 2.1 |
29 ± 5.0 |
81 ± 13.6 |
21 ± 3.5 |
194 ± 29.4 |
0.0316 |
20 ± 1.5 |
26 ± 7.6 |
84 ± 3.1 |
25 ± 3.6 |
204 ± 12.2 |
|
0.100 |
21 ± 1.5 |
33 ± 4.2 |
76 ± 7.0 |
22 ± 3.0 |
170 ± 1.5 |
|
0.316 |
21 ± 1.5 |
31 ± 1.7 |
99 ± 11.7 |
25 ± 3.0 |
164 ± 13.3 |
|
1.0 |
21 ± 3.1 |
29 ± 3.0 |
78 ± 8.3 |
25 ± 5.5 |
199 ± 15.0 |
|
2.5 |
22 ± 3.2 |
32 ± 7.9 |
78 ± 6.1 |
22 ± 5.7 |
206 ± 19.7 |
|
5.0 |
20 ± 1.2 |
31 ± 10.6 |
98 ± 15.0 |
26 ± 5.0 |
158 ± 14.0 |
|
Positive controls (unit/plate) |
4-NOPD |
4-NOPD |
SA |
SA |
MMS |
|
Mean No. of colonies/plate (average of 3 plates) |
170 ± 13.3 |
480 ± 136.3 |
372 ± 43.1 |
751 ± 42.4 |
1042 ± 227.1 |
|
+ |
Solvent control (distilled water) |
21 ± 0.6 |
25 ± 7.5 |
67 ± 0.6 |
31 ± 4.6 |
281 ± 5.9 |
0.0316 |
19 ± 3.1 |
22 ± 0.0 |
73 ± 9.0 |
30 ± 5.0 |
279 ± 13.2 |
|
0.100 |
20 ± 2.5 |
27 ± 4.4 |
68 ± 12.9 |
24 ± 5.6 |
273 ± 8.5 |
|
0.316 |
18 ± 1.7 |
30 ± 0.6 |
78 ± 11.8 |
30 ± 5.3 |
281 ± 27.6 |
|
1.0 |
19 ± 3.2 |
23 ± 3.5 |
79 ± 12.5 |
26 ± 5.6 |
264 ± 12.4 |
|
2.5 |
18 ± 1.2 |
26 ± 9.5 |
66 ± 15.1 |
31 ± 2.1 |
275 ± 31.8 |
|
5.0 |
19 ± 3.2 |
28 ± 3.1 |
73 ± 4.6 |
25 ± 5.0 |
291 ± 12.1 |
|
Positive controls (µg/plate) |
2AA |
2AA |
2AA |
2AA |
2AA |
|
Mean No. of colonies/plate (average of 3 plates) |
413 ± 41.9 |
1797 ± 236.1 |
872 ± 113.3 |
175 ± 25.1 |
894 ± 58.4 |
|
SD = standard deviation
NPD = 4-nitro-1,2-phenylene-diamine
SA = sodium azide
MMS = methylmethanesulfonate
2AA = 2-aminoanthracene
Table 2: Summary of test results (experiment 2; Confirmatory Mutation Test, Pre-Incubation Method)
With or without S9-Mix |
Test substance (μg/plate) |
Mean number of revertant colonies per plate (average of 3 plates ± SD) |
||||
Frameshift type |
Base-pair substitution type |
|||||
TA1537 |
TA98 |
TA100 |
TA1535 |
TA102 |
||
– |
Solvent control (ultrapure water) |
8 ± 3.5 |
34 ± 13.1 |
92 ± 10.0 |
15 ± 2.0 |
209 ± 6.5 |
0.0316 |
9 ± 0.6 |
31 ± 2.6 |
86 ± 8.0 |
22 ± 4.0 |
222 ± 36.0 |
|
0.100 |
8 ± 3.1 |
25 ± 1.5 |
101 ± 10.4 |
17 ± 0.6 |
235 ± 13.8 |
|
0.316 |
12 ± 1.5 |
32 ± 4.2 |
89 ± 12.7 |
22 ± 5.8 |
211 ± 3.5 |
|
1.0 |
7 ± 3.2 |
33 ± 7.0 |
98 ± 6.1 |
19 ± 3.1 |
212 ± 11.1 |
|
2.5 |
9 ± 3.6 |
29 ± 6.4 |
88 ± 16.0 |
21 ± 7.6 |
193 ± 64.6 |
|
5.0 |
12 ± 1.7 |
29 ± 1.5 |
92 ± 8.4 |
18 ± 3.8 |
194 ± 13.8 |
|
Positive controls (unit/plate) |
4-NOPD |
4-NOPD |
SA |
SA |
MMS |
|
Mean No. of colonies/plate (average of 3 plates) |
89 ± 7.5 |
365 ± 25.7 |
368 ± 36.3 |
1146 ± 41.9 |
1462 ± 357.9 |
|
+ |
Solvent control (DMSO) |
14 ± 2.5 |
28 ± 9.0 |
72 ± 18.0 |
15 ± 5.0 |
234 ± 22.5 |
0.0316 |
15 ± 1.7 |
31 ± 3.8 |
74 ± 0.6 |
11 ± 2.5 |
245 ± 14.3 |
|
0.100 |
11 ± 2.1 |
34 ± 7.5 |
91 ± 2.9 |
17 ± 2.6 |
247 ± 23.4 |
|
0.316 |
15 ± 3.5 |
31 ± 1.2 |
71 ± 12.1 |
12 ± 1.5 |
251 ± 17.0 |
|
1.0 |
12 ± 3.0 |
33 ± 8.1 |
83 ± 18.9 |
13 ± 2.9 |
266 ± 10.5 |
|
2.5 |
13 ± 1.7 |
36 ± 4.0 |
77 ± 15.9 |
15 ± 3.8 |
274 ± 31.8 |
|
5.0 |
12 ± 3.2 |
36 ± 4.7 |
80 ± 7.0 |
14 ± 1.5 |
272 ± 44.2 |
|
Positive controls (µg/plate) |
2AA |
2AA |
2AA |
2AA |
2AA |
|
Mean No. of colonies/plate (average of 3 plates) |
128 ± 10.7 |
1470 ± 191.8 |
976 ± 229.7 |
134 ± 15.0 |
690 ± 38.2 |
|
SD = standard deviation
NPD = 4-nitro-1,2-phenylene-diamine
SA = sodium azide
MMS = methylmethanesulfonate
2AA = 2-aminoanthracene
Applicant's summary and conclusion
- Conclusions:
- Based on the results of the conducted study the test substance did not exhibit mutagenic properties in bacterial cells.
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