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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 peer reviewed publication

Data source

Reference
Reference Type:
publication
Title:
Gene mutation toxicity study for the test chemical
Author:
National Institute of Technology and Evaluation
Year:
2018
Bibliographic source:
Japan Chemicals Collaborative Knowledge Database, 2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: Refer below principle
Principles of method if other than guideline:
Ames 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: 4-methylbenzoic acid
- Molecular formula: C8H8O2
- Molecular weight: 136.1492 g/mol
- Substance type: Organic
- Physical state: No data
- Purity: 98.85%
- Impurities (identity and concentrations): 0.01% moisture as impurities and 0.01% ash content

Method

Target gene:
Histidne for Salmonella typhimurium strains and tryptophan for E.coli strains
Species / strain
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
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
S 9 is a 9000 × g centrifugal supernatant fraction of liver homogenate prepared by administering phenobarbital and 5,6-benzoflavone in combination with 7-week-old male SD rats
Test concentrations with justification for top dose:
-S9: 0, 313, 625, 1250, 2500 or 5000 µg/plate
+S9: 0, 156, 313, 625, 1250, 2500 or 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO
Controls
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
N-ethyl-N-nitro-N-nitrosoguanidine
other: 2-aminoanthracene and 2- (2-furyl) -3- (5-nitro-2-furyl) acrylamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: Preincubation

DURATION
- Preincubation period: 20 mins
- 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: Three plates were used for each dose in this test, and twice to confirm reproducibility

NUMBER OF CELLS EVALUATED: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

OTHER EXAMINATIONS: No data
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other: No data

OTHER: No data
Rationale for test conditions:
No data
Evaluation criteria:
Regardless of the presence or absence of S9 mix in any of the test strains, the number of reversed mutantcolonies (mean value) increased with the increase in the dose of the test substance to more than twice the negative (solvent) control value, When reproducibility was observed, the test substance was determined to be mutagenic (positive).
Statistics:
No statistical method was used for judging the test results

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium, other: TA98, TA100, TA1535, TA1537
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
Species / strain:
E. coli WP2 uvr A pKM 101
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:
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: Preliminary test was carried out at 7 doses of 5000, 1250, 313, 78.1, 19.5, 4.88 and 1.22 μg / plate using
Salmonella typhimurium TA 100, TA 1535, TA 98, TA 1537 and Escherichia coli WP 2 uvr A / pKM 101, no increase in the number of revertant colonies was observed in any of the strains. In all the strains coexisting withS9 mix, antibacterial activity was observed at 5000 μg / plate.

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data
Remarks on result:
other: No mutagenic potential

Any other information on results incl. tables

Table: Mutagenicity of the test chemical

Trial 1:

With and without

Test substance (µg/plate)

Number of revertants

Base pair change type

Frameshift type

TA100

TA1535

WP2uvrA/pKM101

TA98

TA1537

-S9

0

104±3

9±1

74±5

21±6

12±3

313

100±3

10±1

78±12

19±3

13±4

625

101±4

12±3

82±6

22±4

14±5

1250

97±4

10±1

71±4

24±1

15±3

2500

104±5

10±1

77±13

22±3

11±1

5000

59±10

10±1

57±5

16±1

11±5

+S9

0

118±13

10±1

98±7

24±1

23±3

156

113±6

11±2

98±11

27±5

22±1

313

102±6

11±4

97±4

27±3

29±1

625

102±2

11±2

93±10

28±5

29 v3

1250

104±3

11±1

84±9

26±4

27 v4

2500

99±4

10±1

86±13

25±3

24±3

5000

71±11

6±1

59±10

16±6

18±5

Positive control (-S9)

Name

AF2

NaN3

ENNG

AF2

9AA

Dose

0.01

0.5

2

0.1

80

Number of revertants

561±17

491±26

4071±180

655±19

253±36

Positive control (+S9)

Name

2AA

2AA

2AA

2AA

2AA

Dose

1

2

2

0.5

2

Number of revertants

1428±113

170±16

1176±145

470±29

214±21

 

Trial 2:

With and without

Test substance (µg/plate)

Number of revertants

Base pair change type

Frameshift type

TA100

TA1535

WP2uvrA/pKM101

TA98

TA1537

-S9

0

101±6

10±1

77±3

22±1

13±2

313

97±5

11±3

87±6

21±1

13±1

625

100±12

10±2

79±6

20±4

15±2

1250

101±6

10±3

76±4

19±2

12±1

2500

92±5

10±2

71±5

19±2

14±1

5000

73±8

8±1

56±3

16±2

12±1

+S9

0

108±12

10±1

98±6

28±5

22±1

156

104±11

11±1

104±7

26±3

21±3

313

102±8

12±3

98±5

24±1

26±3

625

99±1

12±0

92±10

29±5

24±2

1250

103±2

11±1

96±8

28±3

26±1

2500

100±7

11±2

81±8

29±2

26±3

5000

75±9

8±1

59±9

17±3

18±3

Positive control (-S9)

Name

AF2

NaN3

ENNG

AF2

9AA

Dose

0.01

0.5

2

0.1

80

Number of revertants

512±23

463±36

4127±321

675±7

253±44

Positive control (+S9)

Name

2AA

2AA

2AA

2AA

2AA

Dose

1

2

2

0.5

2

Number of revertants

1346±122

173±8

989±124

414±9

217±8

 

Applicant's summary and conclusion

Conclusions:
The test chemical did not induce gene mutation in Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and E. coli WP2 uvrA / pKM101 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:

Ames assay was performed to determine the mutagenic nature of the test chemical. The study was performed by the preincubation protocol using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and E. coli WP2 uvrA / pKM101 in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose level of 0, 313, 625, 1250, 2500 or 5000µg/plate in the absence of S9 and 0, 156, 313, 625, 1250, 2500 or 5000µg/plate in the presence of S9. Concurrent solvent and positive control plates were also included in the study. Based on the observations made, the test chemical did not induce gene mutation in Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and E. coli WP2 uvrA / pKM101 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.