Registration Dossier
Registration Dossier
Data platform availability banner - registered substances factsheets
Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.
The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.
Diss Factsheets
Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.
EC number: 204-508-5 | CAS number: 121-92-6
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Endpoint summary
Administrative data
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
- 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
- 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.
- 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
- 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.
Referenceopen allclose all
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
Activation: No Activation Date: 03/12/1985 Harvest Time: 10.5 hour(s) Trial Call: Negative |
||||||||||||||||
|
Dose |
Total |
Total Aberrations |
Complex Aberrations |
Simple Aberrations |
Other Abs |
|
|||||||||
No.of |
Abs |
% Cells |
No.of |
Abs |
% Cells |
No.of |
Abs |
% Cells |
No.of |
Abs |
% Cells |
|
||||
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 |
Total |
Total Aberrations |
Complex Aberrations |
Simple Aberrations |
Other Abs |
|
|||||||||
No.of |
Abs |
% Cells |
No.of |
Abs |
% Cells |
No.of |
Abs |
% Cells |
No.of |
Abs |
% Cells |
|
||||
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 |
|
|
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
- 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.
Reference
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.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.