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EC number: 226-378-9 | CAS number: 5384-21-4
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- data from handbook or collection of data
- Justification for type of information:
- Data is from authoritative database
Data source
Reference
- Reference Type:
- other: J-check
- Title:
- Gene mutation toxicity study of the test chemical
- Author:
- NITE
- Year:
- 2 018
- Bibliographic source:
- J-check
Materials and methods
Test guideline
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Principles of method if other than guideline:
- Bacterial gene mutation assay was performed to determine the mutagenic nature of the test chemical
- GLP compliance:
- not specified
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- 6,6'-di-tert-butyl-2,2'-methylenedi-p-cresol
- EC Number:
- 204-327-1
- EC Name:
- 6,6'-di-tert-butyl-2,2'-methylenedi-p-cresol
- Cas Number:
- 119-47-1
- Molecular formula:
- C23H32O2
- IUPAC Name:
- 2,2'-methylenebis(6-tert-butyl-4-methylphenol)
- Test material form:
- solid
- Details on test material:
- - Name of test material: 2 ,2'-Methylenebis (6-tert-butyl-p-cresol)
- Molecular formula: C23H32O2
- Molecular weight: 340.5038 g/mol
- Substance type: Organic
- Physical state: white crystalline powder
- Purity: ≥98%
- Impurities (identity and concentrations): No data
Constituent 1
Method
- Target gene:
- Histidine
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:
- not specified
- Species / strain / cell type:
- E. coli WP2 uvr A
- Details on mammalian cell type (if applicable):
- Not applicable
- Additional strain / cell type characteristics:
- not specified
- Cytokinesis block (if used):
- No data
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 metabolic activation system isolated from 7 week old male Sprague-Dawley male rats administered concomitant administration of phenobarbital (PB) and 5,6-benzoflavone (BF).
- Test concentrations with justification for top dose:
- 0, 313, 625, 1250, 2500 or 5000 µg/plate
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: The test chemical was soluble in acetone
Controls
- Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Acetone
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- sodium azide
- other: 2- (2-furyl) -3- (5-nitro-2-furyl) acrylamide, and 2-aminoanthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Preincubation period: No data
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data
SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): No data
NUMBER OF REPLICATIONS: Triplicate
NUMBER OF CELLS EVALUATED: No data
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data
OTHER EXAMINATIONS:
- Determination of polyploidy:Yes
- Determination of endoreplication: No data
- Other: No data
OTHER: No data - Rationale for test conditions:
- No data
- Evaluation criteria:
- When the increase in the number of revertants/plate increased more than 2 times and the increase was found to be reproducible or dose dependent, it was decided that the test substance has mutagenicity (positive) in this test system.
- Statistics:
- No data
Results and discussion
Test results
- Species / strain:
- S. typhimurium, other: TA 100, TA 1535, TA 98, TA 1537,
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Additional information on results:
- Additional information on results
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Other confounding effects: No data
RANGE-FINDING/SCREENING STUDIES: When the test was carried out with a common ratio of about 3 in the range of 50 to 5000 μg / plate for the test chemical, it was confirmed that in all test bacteria, no antibacterial activity was observed in any of the addition tests.
COMPARISON WITH HISTORICAL CONTROL DATA: No data
ADDITIONAL INFORMATION ON CYTOTOXICITY: No data - Remarks on result:
- other: No muatgenic potential
Any other information on results incl. tables
Table: Mutation data for the test chemical (Test 1)
With or without S9 mix |
Dose (µg/plate) |
Number of revertants/plate |
||||
Base pair substitution type |
Frameshift type |
|||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||
- S9 mix |
0 |
125±10.5 |
13±3.6 |
23±4.7 |
23±5.7 |
7±1.0 |
313 |
137±6.0 |
16±2.0 |
24±5.9 |
24±5.0 |
8±2.6 |
|
625 |
145±6.5 |
15±3.0 |
18±3.8 |
32±1.7 |
11±0.0 |
|
1250 |
130±6.5 |
13±2.6 |
16±5.1 |
21±0.6 |
10±1.5 |
|
2500 |
129±4.7 |
14±3.0 |
22±2.6 |
16±2.6 |
6±1.2 |
|
5000 |
144±17.8 |
17±0.6 |
18±4.2 |
21±2.5 |
10±2.0 |
|
+ S9 mix |
0 |
148±2.3 |
14±4.9 |
26±12.1 |
39±7.6 |
12±4.5 |
313 |
141±2.6 |
15±4.0 |
29±6.1 |
38±3.5 |
19±3.1 |
|
625 |
136±8.5 |
19±4.5 |
16±1.2 |
28±4.0 |
16±5.5 |
|
1250 |
139±16.3 |
11±3.5 |
20±3.5 |
32±6.7 |
14±3.1 |
|
2500 |
147±14.6 |
13±5.6 |
24±2.1 |
32±1.5 |
17±4.5 |
|
5000 |
133±14.5 |
14±2.6 |
19±4.6 |
22±3.5 |
9±2.9 |
|
Positive control –S9 mix |
Chemical |
AF |
SA |
AG2 |
AF2 |
9AA |
Dose (µg/plate) |
0.01 |
0.5 |
0.01 |
0.1 |
80 |
|
No. Of colonies/plate |
833±41.4 |
188±17.2 |
184±22.5 |
915±22.6 |
1427±77.6 |
|
Positive control +S9 mix |
Chemical |
2AA |
2AA |
2AA |
2AA |
2AA |
Dose (µg/plate) |
1 |
2 |
10 |
0.5 |
2 |
|
No. Of colonies/plate |
1385±98.4 |
266±6.1 |
1231±60.5 |
401±20.0 |
309±32.0 |
|
Table: Mutation data for the test chemical (Test 2)
With or without S9 mix |
Dose (µg/plate) |
Number of revertants/plate |
||||
Base pair substitution type |
Frameshift type |
|||||
TA100 |
TA1535 |
WP2uvrA |
TA98 |
TA1537 |
||
- S9 mix |
0 |
124±8.5 |
15±4.2 |
22±2.6 |
26±2.5 |
9±1.5 |
313 |
114±3.1 |
15±1.0 |
26±5.9 |
19±6.2 |
8±4.2 |
|
625 |
128±13.3 |
18±0.6 |
25±4.2 |
25±1.5 |
9±1.2 |
|
1250 |
133±15.5 |
17±4.9 |
27±2.0 |
20±7.2 |
7±0.0 |
|
2500 |
129±8.6 |
15±5.5 |
22±1.0 |
18±1.2 |
6±1.2 |
|
5000 |
122±6.1 |
16±4.5 |
20±2.5 |
18±3.0 |
10±4.0 |
|
+ S9 mix |
0 |
138±6.8 |
15±2.0 |
27±1.5 |
29±6.0 |
14±5.7 |
313 |
118±17.0 |
16±3.5 |
29±12.5 |
33±5.0 |
17±1.2 |
|
625 |
133±14.9 |
13±2.9 |
25±6.1 |
28±4.9 |
10±3.1 |
|
1250 |
139±30.4 |
15±2.6 |
24±3.1 |
30±6.7 |
8±3.2 |
|
2500 |
157±8.7 |
15±4.0 |
18±3.1 |
25±3.8 |
9±4.2 |
|
5000 |
126±13.4 |
14±3.5 |
16±1.2 |
31±3.6 |
13±1.2 |
|
Positive control –S9 mix |
Chemical |
AF |
SA |
AG2 |
AF2 |
9AA |
Dose (µg/plate) |
0.01 |
0.5 |
0.01 |
0.1 |
80 |
|
No. Of colonies/plate |
721±110 |
151±8.5 |
172±16.1 |
884±21.7 |
1138±88.1 |
|
Positive control +S9 mix |
Chemical |
2AA |
2AA |
2AA |
2AA |
2AA |
Dose (µg/plate) |
1 |
2 |
10 |
0.5 |
2 |
|
No. Of colonies/plate |
1652±69.3 |
301±2.6 |
1558±43.3 |
479±33.3 |
381±30.6 |
|
Applicant's summary and conclusion
- Conclusions:
- The test chemical did not induce gene mutation in Salmonella typhimurium TA 100, TA 1535, TA 98, TA 1537, and Escherichia coli WP 2 uvrA in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.
- Executive summary:
Bacterial gene mutation assay was performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium TA 100, TA 1535, TA 98, TA 1537, and Escherichia coli WP 2 uvrA in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in acetone and used at dose level of 0, 313, 625, 1250, 2500 or 5000µg/plate. No increase in the number of mutant colonies that were more than twice the solvent control value in the S9 mix non-addition test and addition test of the five test bacteria used was observed. Based on the observations made, the test chemical did not induce gene mutation in Salmonella typhimurium TA 100, TA 1535, TA 98, TA 1537, and Escherichia coli WP 2 uvrA in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.
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