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Toxicological information

Genetic toxicity: in vitro

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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:
2018
Bibliographic source:
J-check

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
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
Name:
Unnamed
Type:
Constituent
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

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, thetest chemical did not induce gene mutation inSalmonella 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.