3-nitrobenzoic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames assay

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.

In vitro chromosomal abbreviation study

Test chemical was evaluated for its mutagenic potential in Chinese hamster ovary cells by in vitro mammalian chromosome aberration test. The test result was considered to be negative both in the presence and absence of metabolic activation.

In vitro DNA damage and/or repair study

Test chemical was evaluated for its mutagenic potential in Chinese hamster ovary cells in vitro sister chromatid exchange assay. The test result was considered to be negative both in the presence and absence of metabolic activation.

Link to relevant study records

Reference

Reference 1

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

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

Target gene:

Histidine

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

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

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

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

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

data from handbook or collection of data

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Principles of method if other than guideline:

To evaluate the mutagenic potential of test chemical in Chinese hamster ovary cells by in vitro mammalian chromosome aberration test.

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not specified

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

not specified

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

not specified

Metabolic activation:

with and without

Metabolic activation system:

induced male Sprague Dawley rat liver S9

Test concentrations with justification for top dose:

0,1500,2500,3000 and 4000 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl Sulfoxide
- Justification for choice of solvent/vehicle: The test substance is soluble in DMSO.

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: S9 mix; Mitomycin-C +S9 mix; Cyclophosphamide

Details on test system and experimental conditions:

Details on test system and conditions
METHOD OF APPLICATION: In medium
DURATION
- Fixation time (start of exposure up to fixation or harvest of cells):
-S9;10.5hours
+S9: 12.5hours

NUMBER OF CELLS EVALUATED: 100 cells

Evaluation criteria:

The mammalian cells were observed for chromosome aberration, Chromosome gaps and breaks.

Statistics:

Yes, SD ± Mean was observed.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

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

Remarks on result:

other: No mutagenic effect were observed

Activation: No Activation    Date: 03/12/1985    Harvest Time: 10.5 hour(s)   Trial Call: Negative
		Dose µg/mL	Total Cells Examined	Total Aberrations			Complex Aberrations			Simple Aberrations			Other Abs
				No.of Abs	Abs Per Cell	% Cells With Abs	No.of Abs.	Abs Per Cell	% Cells With Abs	No.of Abs.	Abs Per Cell	% Cells With Abs	No.of Abs.	Abs Per Cell	% Cells With Abs
Vehicle Control:	Negative (Not Specified)	0	100	0	0.000	0.0	0	0.000	0.0	0	0.000	0.0	0	0.000	0.0
	Dimethyl Sulfoxide	0	100	1	0.010	1.0	0	0.000	0.0	1	0.010	1.0	0	0.000	0.0
Test Chemical:	m-Nitrobenzoic acid	1500	100	2	0.020	2.0	1	0.010	1.0	1	0.010	1.0	0	0.000	0.0
		2500	100	5	0.050	4.0	2	0.020	2.0	1	0.010	1.0	2	0.020	1.0
		3000	100	7	0.070	6.0	2	0.020	2.0	4	0.040	4.0	1	0.010	1.0
		4000	0	0	0.000	0.0	0	0.000	0.0	0	0.000	0.0	0	0.000	0.0
Positive Control:	Mitomycin-C	0.5	100	14	0.140	14.0	4	0.040	4.0	10	0.100	10.0	0	0.000	0.0
	Mitomycin-C	1	25	10	0.400	28.0	4	0.160	16.0	6	0.240	16.0	0	0.000	0.0
Trend:				2.106			1.458			1.339
Probability:				0.018			0.072			0.090

 

Activation: Induced Rat Liver S9   Date: 03/12/1985   Harvest Time: 12.5 hour(s)

Trial #:1_S9   Activation: Induced Rat Liver S9    Date: 03/12/1985    Harvest Time: 12.5 hour(s)   Trial Call: Negative
		Dose µg/mL	Total Cells Examined	Total Aberrations			Complex Aberrations			Simple Aberrations			Other Abs
				No.of Abs	Abs Per Cell	% Cells With Abs	No.of Abs.	Abs Per Cell	% Cells With Abs	No.of Abs.	Abs Per Cell	% Cells With Abs	No.of Abs.	Abs Per Cell	% Cells With Abs
Vehicle Control:	Negative (Not Specified)	0	100	2	0.020	2.0	1	0.010	1.0	1	0.010	1.0	0	0.000	0.0
	Dimethyl Sulfoxide	0	100	2	0.020	2.0	1	0.010	1.0	1	0.010	1.0	0	0.000	0.0
Test Chemical:	m-Nitrobenzoic acid	2000	100	2	0.020	2.0	1	0.010	1.0	1	0.010	1.0	0	0.000	0.0
		2500	100	1	0.010	1.0	1	0.010	1.0	0	0.000	0.0	0	0.000	0.0
		3000	100	2	0.020	2.0	0	0.000	0.0	2	0.020	2.0	0	0.000	0.0
		4000	0	0	0.000	0.0	0	0.000	0.0	0	0.000	0.0	0	0.000	0.0
Positive Control:	Cyclophosphamide	7.5	100	7	0.070	7.0	1	0.010	1.0	6	0.060	6.0	0	0.000	0.0
	Cyclophosphamide	37.5	25	20	0.800	48.0	10	0.400	32.0	9	0.360	28.0	1	0.040	4.0
Trend:				-0.198			-0.743			0.384
Probability:				0.578			0.771			0.351

Conclusions:

Test chemical was evaluated for its mutagenic potential in Chinese hamster ovary cells by in vitro mammalian chromosome aberration test. The test result was considered to be negative both in the presence and absence of metabolic activation.

Executive summary:

Genetic toxicity in vitro study was assessed for test chemical. For this purpose in vitro mammalian chromosome aberration test was performed .The test material was exposed to Chinese hamster ovary cells inthe presence and absence of metabolic activation S9. The concentration of test material used in the presence and absence of metabolic activation were 0,1500,2500,3000 and 4000 µg/mL. Chromosome aberration, Chromosome gaps and breaks were not observed in the presence or absence of metabolic activation. Therefore test chemical was considered to be non -mutagenic inChinese hamster ovary cells by in vitro mammalian chromosome aberration test. Hence the substance cannot be classified as mutagenic in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Test substance did not induce micronuclei in the polychromatic and normochromatic erythrocytes isolated from treated B6C3F1 mice during the 90 days study and hence the test chemical is likely to not classify as a gene mutant in vivo.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

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

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

Micronucleus study was performed in vivo to determine the mutagenic nature of m-nitrobenzoic acid

GLP compliance:

not specified

Type of assay:

not specified

Species:

mouse

Strain:

B6C3F1

Details on species / strain selection:

No data

Sex:

not specified

Details on test animals or test system and environmental conditions:

No data

Route of administration:

oral: feed

Vehicle:

No data

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test chemical was mixed with feed at dose level of 2% (2857.1 mg/Kg bw/day)

DIET PREPARATION
- Rate of preparation of diet (frequency): No data
- Mixing appropriate amounts with (Type of food): No data
- Storage temperature of food: No data

Duration of treatment / exposure:

90 days

Frequency of treatment:

No data

Post exposure period:

No data

Remarks:

2% (2857.1 mg/Kg bw/day)

No. of animals per sex per dose:

No data

Control animals:

not specified

Positive control(s):

Urethane
- Justification for choice of positive control(s): No data
- Route of administration: Drinking water
- Doses / concentrations: 0.2%

Tissues and cell types examined:

Polychromatic and normochromatic erythrocytes were screened for the presence of micronuclei.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: No data

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):

DETAILS OF SLIDE PREPARATION: VA. Blood was obtained immediately before or at the time of sacrifice by retro-orbital bleeding; less often, other methods were employed, including heart puncture, puncture of the ventral tail vessels, or tail clip. Drops of blood were spread on precleaned standard glass microscope slides, air dried, and were stained immediately before scoring with either Hoechst 33258/pyronin Y or acridine orange. All slides were coded prior to scoring by a person not involved in reading the slides. Slides stained with acridine orange or Hoechst 33258/pyronin Y were scored at 6303 or 10003 magnification by epifluorescence microscopy.

METHOD OF ANALYSIS: Criteria for identification of MN were that MN exhibit the fluorescence emission characteristic of the fluorescent stain used (blue with UV excitation and orange with green [540nm] excitation with Hoechst/pyronin stain, or yellow to greenish yellow with acridine orange stain). Polychromatic erythrocytes (PCE) were scored by direct manual counting. Normochromatic erythrocytes (NCE) were scored using a semiautomated method, in which cell counts were determined by counting a subfield of approximately 1/16th of the full microscope field. Routine micronucleus frequency scores were based on approximately 10,000 NCE or 1000 PCE per sample, and the percentage of PCE among the total erythrocyte population was based on the number of PCE among approximately 10,000 erythrocytes.

OTHER: No data

Evaluation criteria:

The erythrocytes were observed for micronuclei. The MN results were tabulated as the mean frequency of micronucleated erythrocytes per 1000 cells per animal, plus or minus the standard error of the mean among animals within a treatment group.

Generally, a test was considered positive if (1) the trend test P value was 0.025 or less or (2) the P value for any single exposure group was 0.025/N or less where N is the number of test chemical treatment groups. Trend test P values between 0.025 and 0.05 were considered to be equivocal if accompanied by a monotonic increase in the frequency of micronuclei over the dose range investigated. All other responses were considered to be negative.

Statistics:

The frequency of micronucleated cells among NCE or PCE was analyzed by a statistical software package that tested for increasing trend over exposure groups using a one-tailed Cochran-Armitage trend test, followed by pairwise comparisons between each exposure group and the control group. In the presence of excess binomial variation, as detected by a binomial dispersion test, the binomial variance of the Cochran-Armitage test was adjusted upward in proportion to the excess variation. Pairwise comparisons between each treatment group and the concurrent solvent control group were performed using an
unadjusted one-tailed Pearson x2 test that incorporated the calculated variance inflation factor for the study.

Although statistical analyses were used as an important aid in evaluating the test results, statistical significance was not the only determining factor in arriving at an overall call for a chemical. A decision to classify a test as negative, equivocal, or positive for induction of micronuclei in this in vivo assay was based on a broader evaluation of a number of factors that determined the biological relevance of the results, including the appropriateness of the concurrent control data, the magnitude of the observed response and the presence of a dose-dependent increase in the frequency of micronucleated cells.

The percentage of polychromatic erythrocytes (%PCE) data were analyzed by a standard ANOVA to determine if significant PCE suppression or stimulation occurred.

Sex:

not specified

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

other: No mutagenic potential

Additional information on results:

No data

Conclusions:

Test substance did not induce micronuclei in the polychromatic and normochromatic erythrocytes isolated from treated B6C3F1 mice during the 90 days study and hence the test chemical is likely to not classify as a gene mutant in vivo.

Executive summary:

Micronucleus study was performed in vivo to determine the mutagenic nature of test substance. The study was performed using B6C3F1 mice at dose level of 2% (2857.1 mg/Kg bw/day) for 90 days. Blood was obtained immediately before or at the time of sacrifice by retro-orbital bleeding; less often, other methods were employed, including heart puncture, puncture of the ventral tail vessels, or tail clip. Drops of blood were spread on precleaned standard glass microscope slides, air dried, and were stained immediately before scoring with either Hoechst 33258/pyronin Y or acridine orange. All slides were coded prior to scoring by a person not involved in reading the slides. Slides stained with acridine orange or Hoechst 33258/pyronin Y were scored at 6303 or 10003 magnification by epifluorescence microscopy. Generally, a test was considered positive if (1) the trend test P value was 0.025 or less or (2) the P value for any single exposure group was 0.025/N or less where N is the number of test chemical treatment groups. Trend test P values between 0.025 and 0.05 were considered to be equivocal if accompanied by a monotonic increase in the frequency of micronuclei over the dose range investigated. All other responses were considered to be negative. Based on the observations made, test substance did not induce micronuclei in the polychromatic and normochromatic erythrocytes isolated from treated B6C3F1 mice during the 90 days study and hence the test chemical is likely to not classify as a gene mutant in vivo.

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Data for the various publication was reviewed to determine the mutagenic nature of 3-nitrobenzoic acid (121-92-6). The studies are as mentioned below:

In Vitro studies

Ames assay

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.

Supported by other AMES study. Gene toxicity in vitro was performed for test substance in Salmonella typhimurium strains TA100 and TA98. The mutation test was performed by Ames's method with some modifications including a step of pre-incubation of the test chemicals (dimethyl sulfoxide solution) and norharman (200/µg per plate) with S9 mix and S. typhimurium for 20 min at 37°C. The S9 fraction was prepared from the liver of PCB-treated male rats. Chemical was assayed with 4 replicate plates at each dose level. No significant lethal effect on the tester strain was observed in the presence or absence of S9 mix at any of the dose levels tested. Test substance did not induce gene mutation in Salmonella typhimurium strains TA98 and TA100 without S9 metabolic activation system. It also did not induce gene mutation in strain TA98 in the presence of S9, with and without the presence of Norharman. Hence the test chemical is not likely to classify as a gene mutant in vitro.

Supported by another study. Salmonella/microsome test in the absence of exogenous metabolic activation and in the presence of liver S-9 from Aroclor-induced male Sprague-Dawley rats and Syrian hamsters was performed to evaluate the mutagenic nature of the test substance using S. typhimurium tester strains TA100, TA1535, TA1537 and TA98 in Lab 1 and TA1535, TA97, TA98 and TA100 in Lab 2. The study was performed as per the preincubation assay and the preincubation time was 20 mins and the plates were incubated for 48 hrs. The test substance was dissolved in DMSO and was used at a dosage level of 0, 100, 167, 333, 667, 1000, 1667, 3333 or 10000 µg/plate in Lab 1 and 0, 33, 100, 333, 1000, 3333 or 6666 µg/plate in Lab 2 respectively in the preincubation assay of 48 hrs. Concurrent solvent and positive control chemicals were included in the study. Test substance gives positive gene toxicity in vitro result in the presence and absence of metabolic activation system in the Salmonella typhimurium strains TA100 and TA1535 in Lab 1 study and strain TA100 and TA97 in Lab 2 study.

Supported by one other AMES study. Rec Assay was performed to evaluate the mutagenic role of test substance in Bacillus subtilis strains H17 and M45. Cultures were streaked from small pipettes onto the surface of a broth agar plate, without touching one another. Paper discs, 13 mm in diameter, were soaked in 0.05 ml of various concentrations (0.01, 0.05, 0.1, 0.5, 1 or 5 mg/plate) of the test compound and placed on the plate so as to cover the end of the bacterial streaks. After incubation for 24 h at 37°C, grown bacteria become visible except in the inhibition zone depending on the strain and on the compound used. A difference of more than 1 mm was a positive response. The genetic toxicity for Bacillus subtilis / strains H17 and M45 with test substance was considered to be positive.

Supported by another AMES study. Genetic toxicity test was performed on Salmonella typhimurium strains TA98, TA1538, TA1537, TA100, and TA1535 in the absence of S9 metabolic activation system. 3 strains, TA98, TA1538 and TA1537, were used for the detection of mutagens that cause frame shift mutations. TA100 and TA1535 were used for the detection of mutagens causing base-pair substitutions. The test compound was dissolved in sterilized dimethyl sulphoxide (DMSO) and used at dose levels of 0, 10, 50, 100, 500, 1000 and 5000 µg/Plate. A pour-plate/ preincubation method for the quantitative determination of mutagenic activity was carried out according to Ames' procedure. The plates were inverted and incubated at 37°C in the dark for 70 h. Colonies of his + revertants were counted after incubation, and chemicals inducing more than twice the number of revertant colonies on the control plate were considered as mutagenic. Test substance did not induce gene toxicity in vitro in the Salmonella typhimurium strains  TA 1538, TA1537, TA100 and TA1535 in the absence of metabolic activation system S9. It however showed a positive response in strain TA98 in the absence of S9 metabolic activation system.

 

In vitro chromosomal abbreviation study

Genetic toxicity in vitro study was assessed for test chemical. For this purpose in vitro mammalian chromosome aberration test was performed .The test material was exposed to Chinese hamster ovary cells in the presence and absence of metabolic activation S9. The concentration of test material used in the presence and absence of metabolic activation were 0,1500,2500,3000 and 4000 µg/mL. Chromosome aberration, Chromosome gaps and breaks were not observed in the presence or absence of metabolic activation. Therefore test chemical was considered to be non -mutagenic in Chinese hamster ovary cells by in vitro mammalian chromosome aberration test. Hence the substance cannot be classified as mutagenic in vitro.

In vitro DNA damage and/or repair study

Genetic toxicity in vitro study was assessed for test chemical. For this purpose in vitro sister chromatid exchange assay was performed .The test material was exposed to Chinese hamster ovary cells in the presence and absence of metabolic activation S9. The concentration of test material used in the presence and absence of metabolic activation were mention below

-S9;0,50,166.7,500and 1700 µg/mL 

+S9; 0,166.7,500,1666.7 and 5000 µg/mL

 

The mammalian cells were observed for number of scored exchanges correlates to the number of DNA breakage and reunion events in the presence or absence of metabolic activation. The test article did not induced sister chromatid exchange in the cultured cells in the presence and absence of metabolic activation. Therefore test chemical was considered to be non -mutagenic in Chinese hamster ovary cells by in vitro sister chromatid exchange assay. Hence the substance cannot be classified as mutagenic in vitro.

In Vivo studies

Micronucleus study was performed in vivo to determine the mutagenic nature of test substance. The study was performed using B6C3F1 mice at dose level of 2% (2857.1 mg/Kg bw/day) for 90 days. Blood was obtained immediately before or at the time of sacrifice by retro-orbital bleeding; less often, other methods were employed, including heart puncture, puncture of the ventral tail vessels, or tail clip. Drops of blood were spread on precleaned standard glass microscope slides, air dried, and were stained immediately before scoring with either Hoechst 33258/pyronin Y or acridine orange. All slides were coded prior to scoring by a person not involved in reading the slides. Slides stained with acridine orange or Hoechst 33258/pyronin Y were scored at 6303 or 10003 magnification by epifluorescence microscopy. Generally, a test was considered positive if (1) the trend test P value was 0.025 or less or (2) the P value for any single exposure group was 0.025/N or less where N is the number of test chemical treatment groups. Trend test P values between 0.025 and 0.05 were considered to be equivocal if accompanied by a monotonic increase in the frequency of micronuclei over the dose range investigated. All other responses were considered to be negative. Based on the observations made, test substance did not induce micronuclei in the polychromatic and normochromatic erythrocytes isolated from treated B6C3F1 mice during the 90 days study and hence the test chemical is likely to not classify as a gene mutant in vivo.

 

 Based on the data summarized, 3-nitrobenzoic acid (121-92-6) did not induce gene mutation .Hence it is not likely to be mutagenic in vitro.

Justification for classification or non-classification

Based on the data available, m-nitrobenzoic acid (CAS no 121 -92 -6) not likiy to exhibit gene mutation in vivo and vitro and hence is not likely to classify as a gene mutant in vitro and in vivo as per the criteria mentioned in CLP regulation.