[S(R*,R*)]-2,3-bis[(4-methylbenzoyl)oxy]succinic acid

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 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

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

Test material information

Constituent 1

Reference substance name:

p-toluic acid

EC Number:

202-803-3

EC Name:

p-toluic acid

Cas Number:

99-94-5

Molecular formula:

C8H8O2

IUPAC Name:

4-methylbenzoic acid

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 results

Test results 1

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

Test results 2

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.