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EC number: 202-876-1 | CAS number: 100-66-3
Genetic toxicity: in vitro
Administrative 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
- Study period:
- From 23 April 2012 to 13 July 2012
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: OECD 471 guideline study in compliance with the GLP. No deviation from the protocol of the study.
Cross-referenceopen allclose all
- Reason / purpose for cross-reference:
- reference to same study
- Reason / purpose for cross-reference:
- reference to other study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 012
- Report date:
- 2012
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Deviations:
- not specified
- Qualifier:
- according to guideline
- Guideline:
- other: EPA, OPPTS 870.5100 (Public Draft, June 1996)
- Deviations:
- not specified
- Qualifier:
- according to guideline
- Guideline:
- other: ICH Harmonised Tripartite Guideline S2(R1), 09 November 2011
- Deviations:
- not specified
- Principles of method if other than guideline:
- Not applicable
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- Anisole
- EC Number:
- 202-876-1
- EC Name:
- Anisole
- Cas Number:
- 100-66-3
- Molecular formula:
- C7H8O
- IUPAC Name:
- anisole
- Test material form:
- other: liquid stored at controlled room temperature
- Details on test material:
- - Name of test material (as cited in study report): Anisole (methoxybenzene)
- Physical state: clear colourless liquid
- Stability under test conditions: the test is assumed to be stable by the sponsor
- Storage condition of test material: controlled room temperature (15-25°C, below 70 RH%)
Constituent 1
Method
- Target gene:
- - Histidine: TA 1535, TA 100, TA 1537 and TA 98
-Tryptophan: Escherichia coli WP2 uvr A
Species / strainopen allclose all
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Details on mammalian cell type (if applicable):
- Not applicable
- Additional strain / cell type characteristics:
- other: see Table 7.6.1/1
- Species / strain / cell type:
- E. coli WP2 uvr A
- Details on mammalian cell type (if applicable):
- Not applicable
- Additional strain / cell type characteristics:
- other: see Table 7.6.1/1
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 from induced phenobarbital/Beta-naphthoflavone rat liver.
- Test concentrations with justification for top dose:
- - Range finding test: 10; 31.6; 100; 316; 1000; 2500 and 5000 µg/plate
- Initial mutation test (plate incorporation method) and confirmatory mutation test (pre-incubation method): 5; 15.81; 50; 158.1; 500, 1581 and
5000 µg/plate
- Complementary confirmatory mutation test: 0.5; 15.81; 50; 158.1; 500 and 1581 µg/plate - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO (test item) and Water (positive control)
- Justification for choice of solvent/vehicle: in this study, two vehicle control groups were used depending on the solubility of the test item and the solubility of strain specific positive chemicals. The test item was partially insoluble in Distilled water. Therefore, the solubility of the test item was examined in Dimethyl sulfoxide (DMSO) and Acetone. The test item was soluble in the two examined solvents. Due to the better biocompatibility to the test system, DMSO was selected for solvent of the study.
Controls
- Untreated negative controls:
- yes
- Remarks:
- (untreated)
- Negative solvent / vehicle controls:
- yes
- Remarks:
- (two vehicle control groups: DMSO and water)
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- sodium azide
- methylmethanesulfonate
- other: 4-nitro-1,2-phenylene-diamine (NPD); 2-aminoanthracene (2AA)
- Remarks:
- see Table 7.6.1/2
- Details on test system and experimental conditions:
- METHOD OF APPLICATION:
- In agar (plate incorporation): In the Range Finding Test as well as in the Initial Mutation Test
- preincubation: in the Confirmatory Mutation Test and complementary mutation test
DURATION
- Incubation period: at 37°C for 48 hours.
- Preincubation period: 20 min at 37ºC
NUMBER OF REPLICATIONS: 3 plates/dose/strain. Two independent experiments were performed
DETERMINATION OF CYTOTOXICITY
- Method: the cytotoxicity of the test material was determined using TA100 and TA98 in the presence and absence of metabolic activation system (+/-S9 Mix) with appropriate untreated, negative (solvent) and positive controls. In the test each samples (including the controls) were tested in triplicate. The concentrations examined were 5000, 2500, 1000, 316, 100, 31.6 and 10 µg/plate. After 48 hours incubation at 37°C, the plates were scored for revertant colonies and examined for a thinning of the background lawn. - Evaluation criteria:
- The study was considered valid if:
- the number of revertant colonies of the negative (solvent) and positive controls were in the historical control range in all strains of the main tests;
- at least five analyzable concentrations were presented in all strains of the main tests.
- Criteria for a Positive Response:
A test item was considered mutagenic if:
- a dose–related increase in the number of revertants occurred and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.
Criteria for a Negative Response:
A test article was considered non-mutagenic if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation. - Statistics:
- No data
Results and discussion
Test resultsopen allclose all
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- See below for more details
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- See below for more details
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no data
RANGE-FINDING/SCREENING STUDIES: the range finding was performed using Salmonella typhimurium TA 98 and TA 100 strains. The concentrations examined were 5000, 2500, 1000, 316, 100, 31.6 and 10 µg/plate. Slightly higher numbers of revertant colonies compared to the solvent control plates were observed in TA 98 strain without S9 mix. However, they had no biological significance and were considered as reflecting the variability of the test system. Slightly lower numbers of revertant colonies compared to the DMSO solvent control plates were observed in TA 98 strain with S9 mix at some non-cytotoxic concentrations. However, the observed revertant counts were within the historical control range and without biological significance in all cases. Slight inhibitory, cytotoxic effect of the test item (slightly reduced background lawn) was observed in the preliminary experiment in both examined strains at 5000 µg/plate with S9 mix.
COMPARISON WITH HISTORICAL CONTROL DATA: the number of revertants colonies was within the historical control range (the initial mutation test, confirmatory mutation test, vehicle and positive controls)
ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the confirmatory mutation test, excessive cytotoxicity was observed in S.typhimurium TA100 and TA1535 strains without metabolic activation at the three highest concentrations. In these cases, the number of analyzable doses did not meet the recommandations of the test guidelines. Therefore, an additional experiment (complementary confirmatory mutation test) will be performed in these strains in an additional experimental period to complete the data. The experimental conditions were the same as in the confirmatory mutation test.
Slight inhibitory, cytotoxic effect of the test item was observed in the initial mutation test in S. typhimurium TA98 and TA100 strains at 5000 µg/plate with S9 mix. Similar, but stronger effect was observed in the confirmatory mutation test in S. typhimurium TA98 and TA1537 and Escherichia coli WP2 uvrA strains at 5000 and 1581 µg/plate without S9 mix, and in all the five tester strains at 5000 and 1581 µg/plate with S9 mix, and in the complementary confirmatory mutation test in S. typhimurium TA100 and TA1535 strains at 1581 and 500 µg/plate without S9 mix. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
Any other information on results incl. tables
Table 7.6.1/3: Summary Table of the Initial Mutation Test
Concentrations |
Mean |
Salmonella typhimurium tester strains |
Escherichia coli |
||||||||
TA98 |
TA100 |
TA1535 |
TA1537 |
WP2uvrA |
|||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
||
Untreated control |
Mean |
30.0 |
40.0 |
91.7 |
101.7 |
8.0 |
11.0 |
6.3 |
7.3 |
23.3 |
43.3 |
MF |
1.02 |
1.32 |
1.01 |
0.98 |
1.09 |
1.27 |
1.12 |
0.85 |
1.17 |
1.15 |
|
DMSO |
Mean |
29.3 |
30.3 |
90.7 |
104.0 |
7.3 |
8.7 |
5.7 |
8.7 |
20.0 |
37.7 |
MF |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
|
Distilled water control |
Mean |
-- |
-- |
94.7 |
-- |
8.0 |
-- |
-- |
-- |
29.0 |
-- |
MF |
-- |
-- |
1.04 |
-- |
1.09 |
-- |
-- |
-- |
1.45 |
-- |
|
5000 |
Mean |
31.3 |
30.3 |
67.7 |
92.0 |
6.3 |
6.7 |
4.7 |
6.7 |
26.7 |
27.3 |
MF |
1.07 |
1.00 |
0.75 |
0.88 |
0.86 |
0.77 |
0.82 |
0.77 |
1.33 |
0.73 |
|
1581 |
Mean |
30.0 |
36.0 |
77.7 |
96.7 |
10.0 |
10.0 |
5.0 |
6.7 |
26.0 |
34.3 |
MF |
1.02 |
1.19 |
0.86 |
0.93 |
1.36 |
1.15 |
0.88 |
0.77 |
1.30 |
0.91 |
|
500 |
Mean |
31.0 |
43.0 |
102.0 |
102.7 |
9.0 |
10.7 |
5.3 |
7.7 |
28.3 |
36.7 |
MF |
1.06 |
1.42 |
1.13 |
0.99 |
1.23 |
1.23 |
0.94 |
0.88 |
1.42 |
0.97 |
|
158.1 |
Mean |
34.7 |
40.0 |
94.0 |
120.0 |
10.7 |
12.0 |
3.0 |
7.0 |
35.3 |
32.7 |
MF |
1.18 |
1.32 |
1.04 |
1.15 |
1.45 |
1.38 |
0.53 |
0.81 |
1.77 |
0.87 |
|
50 |
Mean |
30.7 |
37.7 |
104.3 |
124.0 |
8.0 |
14.7 |
7.7 |
6.3 |
31.0 |
39.7 |
MF |
1.05 |
1.24 |
1.15 |
1.19 |
1.09 |
1.69 |
1.35 |
0.73 |
1.55 |
1.05 |
|
15.81 |
Mean |
28.7 |
38.7 |
99.0 |
111.7 |
10.3 |
12.3 |
3.7 |
8.3 |
32.3 |
37.0 |
MF |
0.98 |
1.27 |
1.09 |
1.07 |
1.41 |
1.42 |
0.65 |
0.96 |
1.62 |
0.98 |
|
5 |
Mean |
26.0 |
42.7 |
92.7 |
113.7 |
9.0 |
10.0 |
5.3 |
6.3 |
23.7 |
40.0 |
MF |
0.89 |
1.41 |
1.02 |
1.09 |
1.23 |
1.15 |
0.94 |
0.73 |
1.18 |
1.06 |
|
NPD (4µg) |
Mean |
353.3 |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
MF |
12.05 |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
|
2AA (2µg) |
Mean |
-- |
2370.7 |
-- |
2422.7 |
-- |
202.3 |
-- |
211.0 |
-- |
-- |
MF |
-- |
78.15 |
-- |
23.29 |
-- |
23.35 |
-- |
24.35 |
-- |
-- |
|
2AA (50µg) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
301.7 |
MF |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
8.01 |
|
SAZ (2µg) |
Mean |
-- |
-- |
1337.3 |
-- |
1172.0 |
-- |
-- |
-- |
-- |
-- |
MF |
-- |
-- |
14.13 |
-- |
146.50 |
-- |
-- |
-- |
-- |
-- |
|
9AA (50µg) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
521.3 |
-- |
-- |
-- |
MF |
-- |
-- |
-- |
-- |
-- |
-- |
92.00 |
-- |
-- |
-- |
|
MMS (2µL) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
1110.7 |
-- |
MF |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
38.30 |
-- |
|
Notes: NPD: 4-nitro-1,2-phenylene-diamine; 2AA: 2-aminoanthracene; SAZ: sodium azide; 9AA: 9-aminoacridine; MMS: Methyl-methanesulfonate.
Applicant's summary and conclusion
- Conclusions:
- Interpretation of results:
negative (with and without metabolic activation)
Under the test conditions of this study, Anisole had no mutagenic activity in the applied bacterium tester strains. - Executive summary:
In a reverse gene mutation assay in bacteria (Hargitai, 2012) performed according to the OECD N° 471 guideline and in compliance with GLP, Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were exposed to the test item Anisole diluted in DMSO at concentrations ranging from 0.5 to 5000 µg/plate in the presence and absence of metabolic activation system from liver fraction of phenobarbital/beta-naphthoflavone-induced rats (S9-mix).
The study included a Preliminary Solubility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method), a Confirmatory Mutation Test (Pre-Incubation Method) and a Complementary Confirmatory Mutation Test (Pre-Incubation Method) because of excessive cytotoxicity observed in the confirmatory mutation test for some conditions.
Based on the results of the range finding test, the test item concentrations in the initial mutation test and confirmatory mutation test were 5000; 1581; 500; 158.1; 50, 15.81 and 5µg/plate. Based on the results of the confirmatory mutation test, the test item concentrations in the complementary confirmatory mutation test were 1581; 500; 158.1; 50, 15.81; 5; 1.581 and 0.5 µg/plate.
In the initial mutation test, confirmatory mutation test and complementary confirmatory mutation test, none of the observed revertant colony numbers were above the respective biological threshold value. There were no consistent dose-related trends and no indication of any treatment effect. In all test item treated groups, the numbers of revertant colonies were below the biological relevance when compared with the solvent controls, within the historical control range and within the normal biological variability of the test system.
Slight inhibitory, cytotoxic effect of the test item was observed in the initial mutation test TA98 and TA100 strains at 5000 µg/plate with metabolic activation. Similar, but stronger effect was observed using the pre-incubation method, in the confirmatory mutation test in TA98, TA1537 andE.coli WP2uvrA strains at 5000 and 1581 µg/plate without metabolic activation, and in all the five tester strains at 5000 and 1581 µg/plate with metabolic activation, and in the complementary confirmatory mutation test in TA100 and TA1535 strains at 5000 and 1581 µg/plate without metabolic activation.
The positive controls induced the appropriate responses in the corresponding strains.
Under the test conditions, Anisole did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used with and without metabolic activation during the study.
In conclusion, Anisole had no mutagenic activity in the applied bacterium tester strains under the test conditions used in this study.
This study is considered as acceptable as it satisfied the criteria of the OECD guideline N° 471.
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