3-nitrobenzoic acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27-02-2018 to 29-03-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from study report

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

This study was performed to investigate the potential of test item test substance to induce gene mutations in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3-nitrobenzoic acid

EC Number:

204-508-5

EC Name:

3-nitrobenzoic acid

Cas Number:

121-92-6

Molecular formula:

C7H5NO4

IUPAC Name:

3-nitrobenzoic acid

Test material form:

solid

Details on test material:

- Name (IUPAC): 3-Nitrobenzoic Acid
- Molecular formula: C7H5NO4
- Molecular weight: 167.1195 g/mol
- Smiles notation: c1(cc(ccc1)[N+](=O)[O-])C(=O)O
- InChl: 1S/C7H5NO4/c9-7(10)5-2-1-3-6(4-5)8(11)12/h1-4H,(H,9,10)
- Substance type: Organic
- Physical state: yellow crystalline powder

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other:

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced S9 metabolic activation system

Test concentrations with justification for top dose:

0 (NC), 0 (VC), 0.050, 0.158, 0.501, 1.582 and 5 mg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was solulble in DMSO

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-Nitro-o-phenylenediamine (TA 1537, TA 98, without S9); 2-Aminoanthracene (TA 1535, TA 1537, TA 98, TA 100 and TA 102, with S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation- Trial I); preincubation (Trial II)

DURATION
- Preincubation period: Trial I: Not applicable Trial II: 60 min
- 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: Each concentration, including the negative, vehicle and positive controls was tested in triplicate in two independent experiments performed

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Not applicable

NUMBER OF CELLS EVALUATED: No data

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): No data

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data
- Any supplementary information relevant to cytotoxicity: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): No data

- OTHER: No data

Rationale for test conditions:

No data

Evaluation criteria:

A test item is considered as a mutagen, if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100 and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding vehicle/solvent control is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative control and vehicle control such an increase is not considered biologically relevant.

Statistics:

No data

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102

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
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: To evaluate the toxicity of the test item, a pre-experiment was performed with strains TA 98 and TA 100. Eight concentrations (0.0 (NC), 0.0 (VC) 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate) were tested for toxicity and mutation induction with 3 plates each (triplicates). Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

In the pre-experiment, the concentration range of the test item was 0.002 – 5 mg/plate based on the solubility and precipitation test. There was no reduction in colony count as well as in background lawn in treated concentrations (5 (T8) mg/plate – 0.002 (T1) mg/plate) both in absence and in the presence of metabolic activation. Based on the results of pre-experiment following doses were selected for the main study trials: 0.0 (NC), 0.0 (VC), 0.050, 0.158, 0.501, 1.582 and 5 mg/plate both in the absence (-S9) as well as in the presence of metabolic activation (+S9).

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: No data

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: No data
- Indication whether binucleate or mononucleate where appropriate: No data

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: No data
- Negative (solvent/vehicle) historical control data: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No data
- Other observations when applicable: No data

Remarks on result:

other: No mutagenic potential

Any other information on results incl. tables

TABLE1- REVERTANT COUNT FOR PRE-EXPERIMENT

Dose (mg/plate)	R	Without metabolic activation (-S9)		With metabolic activation (+S9)
		TA100	TA 98	TA100	TA 98
NC (0.00)	R1	124	18	122	22
	R2	122	16	120	18
	R3	122	22	122	20
VC (0.00)	R1	130	26	132	27
	R2	132	24	130	25
	R3	136	24	134	22
T1 (0.002)	R1	124	19	122	22
	R2	124	21	120	18
	R3	126	20	120	20
T2 (0.005)	R1	130	22	122	22
	R2	126	20	124	22
	R3	124	22	122	22
T3 (0.016)	R1	126	22	124	18
	R2	130	24	126	24
	R3	126	16	120	22
T4 (0.050)	R1	132	22	124	24
	R2	126	24	120	22
	R3	126	20	122	22
T5 (0.158)	R1	124	22	126	22
	R2	130	24	124	24
	R3	124	22	124	24
T6 (0.501)	R1	126	24	126	25
	R2	132	22	124	22
	R3	128	24	126	24
T7 (1.582)	R1	130	22	124	22
	R2	126	24	128	20
	R3	128	22	126	22
T8 (5)	R1	134	24	126	26
	R2	132	26	132	22
	R3	134	22	126	24
PC	R1	1088	968	1376	1264
	R2	1112	944	1352	1280
	R3	1056	952	1328	1248

NC           =     Negative control

VC           =   Vehicle Control

PC            =     Positive control             

R              =     Replicate

T              =     Test concentration (T8: Highest, T1: Lowest)

4-Nitro-o-phenylenediamine [10μg/plate]: TA 98

Sodium azide [10μg/plate]: TA 100,

2-Aminoanthracene [2.5μg/plate]: TA98, TA100

 

TABLE 2 - REVERTANT COUNT IN PLATE INCORPORATION METHOD
(TRIAL I)

Dose (mg/plate)	R	In the Presence of Metabolic Activation (+S9)
		TA 1537	TA 1535	TA 98	TA 100	TA 102
NC (0.00)	R1	5	9	22	122	254
	R2	5	12	18	120	246
	R3	5	10	20	122	260
VC (0.00)	R1	8	13	27	132	280
	R2	7	15	25	130	290
	R3	7	15	22	134	272
T1 (0.050)	R1	5	12	24	124	248
	R2	6	10	22	120	266
	R3	5	10	22	122	254
T2 (0.158)	R1	5	12	22	126	272
	R2	6	12	24	124	262
	R3	6	10	24	124	254
T3 (0.501)	R1	7	12	25	126	266
	R2	6	12	22	124	254
	R3	6	12	24	126	260
T4 (1.582)	R1	6	12	22	124	274
	R2	5	11	20	128	280
	R3	7	12	22	126	266
T5 (5)	R1	7	12	26	126	272
	R2	6	14	22	132	282
	R3	7	12	24	126	268
PC	R1	180	596	1264	1376	1680
	R2	162	556	1280	1352	1712
	R3	174	576	1248	1328	1696

 

Dose (mg/plate)	R	In the Absence of Metabolic Activation (-S9)
		TA 1537	TA 1535	TA 98	TA 100	TA 102
NC (0.00)	R1	6	10	18	124	232
	R2	4	10	16	122	228
	R3	4	10	22	122	240
VC (0.00)	R1	6	16	26	130	264
	R2	8	14	24	132	288
	R3	6	14	24	136	276
T1 (0.050)	R1	5	10	22	132	238
	R2	6	10	24	126	246
	R3	5	12	20	126	240
T2 (0.158)	R1	5	10	22	124	258
	R2	5	12	24	130	262
	R3	5	12	22	124	244
T3 (0.501)	R1	6	12	24	126	238
	R2	5	10	22	132	246
	R3	6	13	24	128	252
T4 (1.582)	R1	6	14	22	130	266
	R2	6	10	24	126	258
	R3	6	12	22	128	260
T5 (5)	R1	6	12	24	134	272
	R2	6	14	26	132	260
	R3	7	14	22	134	272
PC	R1	172	1056	968	1088	1736
	R2	190	1096	944	1112	1704
	R3	188	1080	952	1056	1712

NC= Negative Control,VC= Vehicle Control,T =Test concentration (T5: Highest, T1: Lowest),R= Replicate

PC= Positive control                                                     2-Aminoanthracene [2.5μg/plate]: TA 1537, TA1535, TA 98, TA 100        
2- Aminoanthracene [10μg/plate]:TA 102                                           Sodium azide [10μg/plate]: TA 1535, TA 100                                              

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate], TA 98[10μg/plate]        Methyl methanesulfonate [4μl/plate]: TA 102

TABLE 3 - REVERTANT COUNT IN PRE-INCUBATION METHOD (TRIAL II)

Dose (mg/plate)	R	In the Presence of Metabolic Activation (+S9)
		TA 1537	TA 1535	TA 98	TA 100	TA 102
NC (0.00)	R1	6	10	15	86	230
	R2	4	11	19	96	238
	R3	4	10	21	88	244
VC (0.00)	R1	8	14	24	104	296
	R2	6	12	24	100	302
	R3	6	14	27	104	286
T1 (0.050)	R1	5	10	22	94	252
	R2	5	12	18	96	246
	R3	5	10	18	94	242
T2 (0.158)	R1	5	12	20	90	256
	R2	6	11	22	96	242
	R3	5	12	20	92	250
T3 (0.501)	R1	6	12	24	104	244
	R2	6	12	22	96	252
	R3	5	10	22	96	260
T4 (1.582)	R1	7	12	20	100	252
	R2	6	10	24	98	248
	R3	6	14	20	96	254
T5 (5)	R1	7	14	24	100	266
	R2	5	10	22	102	272
	R3	6	14	20	100	280
PC	R1	192	386	1124	1264	1344
	R2	186	402	1152	1288	1360
	R3	198	394	1136	1240	1336

 

 

Dose (mg/plate)	R	In the Absence of Metabolic Activation (-S9)
		TA 1537	TA 1535	TA 98	TA 100	TA 102
NC (0.00)	R1	5	10	20	104	224
	R2	4	10	18	98	238
	R3	6	10	15	100	250
VC (0.00)	R1	7	16	28	118	294
	R2	7	14	24	120	286
	R3	7	14	24	120	272
T1 (0.050)	R1	6	10	20	102	240
	R2	5	12	18	102	248
	R3	5	12	18	100	242
T2 (0.158)	R1	5	10	20	104	246
	R2	6	10	18	100	242
	R3	6	12	16	104	246
T3 (0.501)	R1	6	12	18	106	254
	R2	6	11	22	104	260
	R3	6	12	18	106	248
T4 (1.582)	R1	6	12	18	100	248
	R2	5	10	20	104	246
	R3	6	14	22	108	252
T5 (5)	R1	6	12	20	102	262
	R2	6	14	22	108	270
	R3	7	14	22	104	254
PC	R1	176	1206	912	1352	1576
	R2	162	1232	886	1376	1608
	R3	180	1218	924	1328	1584

NC= Negative Control,VC= Vehicle Control,T =Test concentration (T5: Highest, T1: Lowest), R= Replicate

PC= Positive control                                                                       2-Aminoanthracene [2.5μg/plate]: TA 1537, TA1535, TA98, TA100        
2-Aminoanthracene [10μg/plate]:TA 102                                              Sodium azide [10μg/plate]: TA 1535, TA 100,                                            

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate] TA 98[10μg/plate]        Methyl methanesulfonate [4μl/plate]: TA 10

TABLE 4 -    MEAN REVERTANT COUNT IN PLATE INCORPORATION METHOD (TRIALI)

Dose (mg/plate)	In the presence of Metabolic Activation (+S9)
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	5.00	0.00	10.33	1.53	20.00	2.00	121.33	1.15	253.33	7.02
VC (0.00)	7.33	0.58	14.33	1.15	24.67	2.52	132.00	2.00	280.67	9.02
T1 (0.050)	5.33	0.58	10.67	1.15	22.67	1.15	122.00	2.00	256.00	9.17
T2 (0.158)	5.67	0.58	11.33	1.15	23.33	1.15	124.67	1.15	262.67	9.02
T3 (0.501)	6.33	0.58	12.00	0.00	23.67	1.53	125.33	1.15	260.00	6.00
T4 (1.582)	6.00	1.00	11.67	0.58	21.33	1.15	126.00	2.00	273.33	7.02
T5 (5)	6.67	0.58	12.67	1.15	24.00	2.00	128.00	3.46	274.00	7.21
PC	172.00	9.17	576.00	20.00	1264.00	16.00	1352.00	24.00	1696.00	16.00

 

Dose (mg/plate)	In the Absence of Metabolic Activation (-S9)
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	4.67	1.15	10.00	0.00	18.67	3.06	122.67	1.15	233.33	6.11
VC (0.00)	6.67	1.15	14.67	1.15	24.67	1.15	132.67	3.06	276.00	12.00
T1 (0.050)	5.33	0.58	10.67	1.15	22.00	2.00	128.00	3.46	241.33	4.16
T2 (0.158)	5.00	0.00	11.33	1.15	22.67	1.15	126.00	3.46	254.67	9.45
T3 (0.501)	5.67	0.58	11.67	1.53	23.33	1.15	128.67	3.06	245.33	7.02
T4 (1.582)	6.00	0.00	12.00	2.00	22.67	1.15	128.00	2.00	261.33	4.16
T5 (5)	6.33	0.58	13.33	1.15	24.00	2.00	133.33	1.15	268.00	6.93
PC	183.33	9.87	1077.33	20.13	954.67	12.22	1085.33	28.10	1717.33	16.65

NC= Negative Control,VC= Vehicle Control,T =Test concentration (T5: Highest, T1: Lowest),SD= Standard Deviation

PC= Positive control

2-Aminoanthracene [2.5μg/plate]: TA 1537, TA 1535, TA 98, TA 100                  Methyl methanesulfonate [4μl/plate]: TA 102

2-Aminoanthracene [10μg/plate]:TA 102                                           

Sodium azide [10μg/plate]: TA 1535, TA 100

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate], TA 98 [10μg/plate]

TABLE 5 -    MEAN REVERTANT COUNT IN PRE-INCUBATIONMETHOD
(TRIAL II)

Dose (mg/plate)	In the presence of Metabolic Activation (+S9)
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	4.67	1.15	10.33	0.58	18.33	3.06	90.00	5.29	237.33	7.02
VC (0.00)	6.67	1.15	13.33	1.15	25.00	1.73	102.67	2.31	294.67	8.08
T1 (0.050)	5.00	0.00	10.67	1.15	19.33	2.31	94.67	1.15	246.67	5.03
T2 (0.158)	5.33	0.58	11.67	0.58	20.67	1.15	92.67	3.06	249.33	7.02
T3 (0.501)	5.67	0.58	11.33	1.15	22.67	1.15	98.67	4.62	252.00	8.00
T4 (1.582)	6.33	0.58	12.00	2.00	21.33	2.31	98.00	2.00	251.33	3.06
T5 (5)	6.00	1.00	12.67	2.31	22.00	2.00	100.67	1.15	272.67	7.02
PC	192.00	6.00	394.00	8.00	1137.33	14.05	1264.00	24.00	1346.67	12.22

 

Dose (mg/plate)	In the Absence of Metabolic Activation (-S9)
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	5.00	1.00	10.00	0.00	17.67	2.52	100.67	3.06	237.33	13.01
VC (0.00)	7.00	0.00	14.67	1.15	25.33	2.31	119.33	1.15	284.00	11.14
T1 (0.050)	5.33	0.58	11.33	1.15	18.67	1.15	101.33	1.15	243.33	4.16
T2 (0.158)	5.67	0.58	10.67	1.15	18.00	2.00	102.67	2.31	244.67	2.31
T3 (0.501)	6.00	0.00	11.67	0.58	19.33	2.31	105.33	1.15	254.00	6.00
T4 (1.582)	5.67	0.58	12.00	2.00	20.00	2.00	104.00	4.00	248.67	3.06
T5 (5)	6.33	0.58	13.33	1.15	21.33	1.15	104.67	3.06	262.00	8.00
PC	172.67	9.45	1218.67	13.01	907.33	19.43	1352.00	24.00	1589.33	16.65

NC= Negative Control,VC= Vehicle Control,T =Test concentration (T5: Highest, T1: Lowest),SD= Standard Deviation

PC= Positive control

2-Aminoanthracene [2.5μg/plate]: TA 1537, TA 1535, TA 98, TA 100

2-Aminoanthracene [10μg/plate]: TA 102

Sodium azide [10μg/plate]: TA 1535, TA 100

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate] TA 98[10μg/plate]

Methyl methanesulfonate: [4μl/plate]: TA 102

Applicant's summary and conclusion

Conclusions:

Test substance did not induce gene mutations by base pair changes or frame shifts in the genome of the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Executive summary:

Ames assay was performed to investigate the potential of test substance to induce gene mutations in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the negative, vehicle and positive controls were tested in triplicates. Based on the solubility and precipitation test results eight different concentrations viz., 0.0 (NC), 0.0 (VC) 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate were selected for pre-experiment. Based on the pre-experiment results, the test item was tested with the following concentrations:0.0 (NC), 0.0 (VC), 0.050, 0.158, 0.501, 1.582 and 5 mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9). No substantial increase in revertant colony numbers of any of the tester strains were observed following treatment with 3-nitrobenzoic acid (CAS no. 121-92-6) at any dose level in both the confirmatory trials, neither in the presence nor in the absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. The spontaneous reversion rates in the negative, vehicle and positive controls were within the range of in-house historical data. Whereas reference mutagens showed a distinct in­crease in induced revertant colonies in all the tester strains both in the presence as well as in the absence of metabolic activation without showing cytotoxicity. In conclusion, it is stated that during the described mutagenicity test and under the experimental conditions reported, the test substance did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.