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EC number: 202-723-9 | CAS number: 99-04-7
- 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
Genetic toxicity: in vivo
Administrative data
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2002-02-20 to 2002-03-27
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 002
- Report date:
- 2002
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Version / remarks:
- 1997
- Deviations:
- yes
- Remarks:
- source of test material not stated
- GLP compliance:
- yes
- Type of assay:
- other: Micronucleus Assay
Test material
- Reference substance name:
- m-toluic acid
- EC Number:
- 202-723-9
- EC Name:
- m-toluic acid
- Cas Number:
- 99-04-7
- Molecular formula:
- C8H8O2
- IUPAC Name:
- m-toluic acid
- Test material form:
- solid
- Details on test material:
- - Name as cited in the study report: 3-Methylbenzoic acid (MTA)
Constituent 1
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Lot No. 1015
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in a light-resistant container at room temperature
- Stability under test conditions:
- Solubility and stability of the test substance in the solvent/vehicle:
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
- pulverisation to a fine powder in an agate mortar
Test animals
- Species:
- rat
- Strain:
- Crj: CD(SD)
- Remarks:
- IGS
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS - Crj: CD(SD), IGS
- Source: Charles River Japan, Inc. (IGS refers to animals bred using the Charles River International Genetic Standardisation system)
- Age at study initiation (at administration): 7 weeks
- Weight at study initiation (at administration): 259 - 277 g (confirmation test: 266 - 289 g)
- Assigned to test groups randomly: yes
- Fasting period before study: not specified
- Housing:
- Diet (ad libitum): pellet diet for experimental animals (MF, Oriental Yeast Co., Ltd.)
- Water (ad libitum): tap water, filtered through 5-µm filter and irradiated with UV light
- Acclimation period: 5 days
- Levels of contaminants were below acceptable upper limits specified at the test facility
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 55 ± 15 %
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12
Administration / exposure
- Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle(s)/solvent(s) used: 1 w/v % MC (methylcellulose)
- Concentration of test material in vehicle: serial dilution: 200, 100, 50, 25 and 12.5 mg/mL (for usage in the main test and the confirmation test)
- Lot/batch no.: Metolose ® SM-100, Shin-Etsu Chemical, Inc., Lot no. 002658 - Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
- suspended by addition of 1 w/v % methylcellulose
- adjusting a concentration of 200 mg/mL
- in an stepwise dilution procedure by adding 1 w/v % methylcellulose, suspensions of 100 mg/mL and 50 mg/mL were prepared
- in the confirmation test the test material was prepared as described previously, but diluted to a concentration of 50 mg/mL, 25 mg/mL and 12.5 mg/mL
- the dose solutions were prepared under yellow light - Duration of treatment / exposure:
- 2 days
- Frequency of treatment:
- twice at 24 hour intervals
- Post exposure period:
- 24 hours (after the final administration)
Doses / concentrationsopen allclose all
- Dose / conc.:
- 125 mg/kg bw/day (actual dose received)
- Remarks:
- confirmation tes
- Dose / conc.:
- 250 mg/kg bw/day (actual dose received)
- Remarks:
- confirmation test
- Dose / conc.:
- 500 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 1 000 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 2 000 mg/kg bw/day (actual dose received)
- No. of animals per sex per dose:
- 5 animals (male) per dose
- Control animals:
- yes, concurrent vehicle
- yes, historical
- Positive control(s):
- cyclophosphamide (obtained from Sigma Chemical Company, Lot no. 86F-0101)
- Route of administration: intraperitoneally administered (once)
- Doses / concentrations: 10 mg/kg
Examinations
- Tissues and cell types examined:
- 1000 erythrocytes were scored from each slide
- Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION:
- doses were set according to a single dose toxicity study by the sponsor with LD50 >= 2000 mg/kg
- dose selection in the confirmation test: doses were set according to the main test which resulted in an unusual value in 1 of 5 animals of the low dose group (500 mg/kg)
TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
- the test substance and the negative control was administered by oral gavage twice at 24 hour intervals.
- the positive control was administered once intraperitoneally
- 24 hours after the last administration rats were sacrificed by exsanguination from the abdominal aorta under anesthesia (thiopental sodium) and femurs were dissected
DETAILS OF SLIDE PREPARATION:
- bone marrow cells were collected with phosphate buffered saline (PBS) and suspensions were centrifuged at 200 rpm for 5 min
- supernatant was transferred to 10 % neutral buffered formalin solution and centrifuged at 1000 rpm for 5 min
- cells were twice washed with 10 % neutral buffered formalin solution
- cells were resuspended in 10 % neutral buffered formalin solution
- suspension was stained with acridine orange and transferred to slides for observation
METHOD OF ANALYSIS:
- slides were observed and scored under blind condition with fluorescent microscope with B-2 excitation filter
- 1000 erythrocytes were scored from each slide in order to determine the ratio of polychromatic erythrocytes (PCEs) to the total erythrocytes (PCEs and normochromatic erythrocytes (NCEs))
- PCEs were further scored up to 2000 cells, the number of micronucleated PCEs (MNPCEs) in a slide were examined
- PCEs and NCEs were identified according to the method of Hayashi et al.*
*Reference:
Hayashi M, Sofuni T, Ishidate M Jr. (1983): An application of acridine orange fluorescent staining to the micronucleus test. Mutat. Res. 120, 241 - 247 - Evaluation criteria:
- When test substance induced a significant increase in the total number of micronucleated polychromatic erythrocytes with dose-dependency, the test is considered positive
- Statistics:
- - for the analysis of the percentage of polychromatic erythrocytes (PCEs): Student's t-test
- for the incidence of micronucleated PCEs (MNPCEs): tables of Kastenbaum and Bowman*
- statistics were conducted at the significance levels of 5 % and 1 %
*Reference:
Kastenbaum MA, Bowman KO (1970): Tables for determining the statistical significance of mutation frequencies. Mutat Res. 9, 527 - 549
Results and discussion
Test results
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay):
- significant induction of micronuclei in the 500 mg/kg dose group: mean incidence of MNPCE = 0.68 ± 1.19
- Ratio of PCE/NCE (for Micronucleus assay):
- 500 mg/kg dose group: mean ratio PCE/NCE: 54.0 ± 1.0
- 1000 mg/kg dose group: mean ratio PCE/NCE: 53.5 ± 2.3
- 2000 mg/kg dose group: mean ratio PCE/NCE: 52.2 ± 2.6
RESULTS OF CONFIRMATION TEST
- Induction of micronuclei (for Micronucleus assay): no significant induction observed
- Ratio of PCE/NCE (for Micronucleus assay):
- 125 mg/kg dose group: mean ratio PCE/NCE: 50.4 ± 3.3
- 250 mg/kg dose group: mean ratio PCE/NCE: 49.1 ± 5.1
- 500 mg/kg dose group: mean ratio PCE/NCE: 50.5 ± 2.8
Please refer to the field 'Any other information on results incl. tables'
Any other information on results incl. tables
Further observation:
body weight: no effects observed
clinical signs: no effects observed
Table 1: Results of micronucleus test
Treatment group |
Dosage (mg/kg) x times |
Animal no. |
Number of PCEs scored |
MNPCE |
PCE/(PCE+NCE)5 |
|
Number (total) |
Incidence Mean ± SD |
(%) Mean ± SD |
||||
Negative control 0 x 21 |
1 |
2000 |
2 |
0.10 |
53.9 |
|
2 |
2000 |
3 |
0.15 |
55.2 |
||
3 |
2000 |
1 |
0.05 |
54.9 |
||
4 |
2000 |
2 |
0.10 |
56.0 |
||
5 |
2000 |
1 |
0.05 |
54.0 |
||
|
|
(9) |
0.09 ± 0.04 |
54.8 ± 0.9 |
||
Test substance 500 x 21 |
1 |
2000 |
3 |
0.15 |
53.8 |
|
2 |
2000 |
3 |
0.15 |
55.5 |
||
3 |
2000 |
3 |
0.15 |
54.0 |
||
4 |
2000 |
3 |
0.15 |
52.7 |
||
5 |
2000 |
56 |
2.80 |
53.9 |
||
|
|
(68)3** |
0.68 ± 1.19 |
54.0 ± 1.0 |
||
Test substance 1000 x 21 |
1 |
2000 |
2 |
0.10 |
50.9 |
|
2 |
2000 |
4 |
0.20 |
55.7 |
||
3 |
2000 |
1 |
0.05 |
55.6 |
||
4 |
2000 |
4 |
0.20 |
54.1 |
||
5 |
2000 |
5 |
0.25 |
51.2 |
||
|
|
(16) |
0.16 ± 0.08 |
53.5 ± 2.3 |
||
Test substance 2000 x 21 |
1 |
2000 |
3 |
0.15 |
48.4 |
|
2 |
2000 |
1 |
0.05 |
55.0 |
||
3 |
2000 |
4 |
0.20 |
54.2 |
||
4 |
2000 |
4 |
0.20 |
52.0 |
||
5 |
2000 |
4 |
0.20 |
51.3 |
||
|
|
(16) |
0.16 ± 0.07 |
52.2 ± 2.6 |
||
Positive control 10 x 12 |
1 |
2000 |
40 |
2.00 |
50.4 |
|
2 |
2000 |
35 |
1.75 |
53.6 |
||
3 |
2000 |
44 |
2.20 |
50.4 |
||
4 |
2000 |
62 |
3.10 |
48.3 |
||
5 |
2000 |
48 |
2.40 |
50.9 |
||
|
|
(229)3** |
2.29 ± 0.51 |
50.7 ± 1.94## |
PCE: polychromatic erythrocytes, MNPCE: micronucleated PCE, NCE: normochromatic erythrocytes
1twice administered by oral gavage at 24 hour interval
2once administered by intraperitoneal injection
3significantly different from the negative control (**p<0.01) by Kastenbaum & Bowman’s method
4significantly different from the negative control (##p<0.01) by Student’s t-test
51000 erythrocytes were scored
Table 2: Results of confirmation test
Treatment group |
Dosage (mg/kg) x times |
Animal no. |
Number of PCEs scored |
MNPCE |
PCE/(PCE+NCE)5 |
|
Number (total) |
Incidence Mean ± SD |
(%) Mean ± SD |
||||
Negative control 0 x 21 |
1 |
2000 |
1 |
0.05 |
57.7 |
|
2 |
2000 |
3 |
0.15 |
50.8 |
||
3 |
2000 |
2 |
0.10 |
53.9 |
||
4 |
2000 |
1 |
0.05 |
58.2 |
||
5 |
2000 |
1 |
0.05 |
52.9 |
||
|
|
(8) |
0.08 ± 0.04 |
54.7 ± 3.2 |
||
Test substance 125 x 21 |
1 |
2000 |
2 |
0.10 |
46.5 |
|
2 |
2000 |
1 |
0.05 |
19.4 |
||
3 |
2000 |
1 |
0.05 |
52.3 |
||
4 |
2000 |
1 |
0.05 |
48.8 |
||
5 |
2000 |
3 |
0.15 |
55.0 |
||
|
|
(8) |
0.08 ± 0.04 |
50.4 ± 3.3 |
||
Test substance 250 x 21 |
1 |
2000 |
1 |
0.05 |
51.9 |
|
2 |
2000 |
1 |
0.05 |
52.3 |
||
3 |
2000 |
2 |
0.10 |
48.9 |
||
4 |
2000 |
2 |
0.10 |
40.3 |
||
5 |
2000 |
4 |
0.20 |
51.9 |
||
|
|
(10) |
0.10 ± 0.06 |
49.1 ± 5.1 |
||
Test substance 500 x 21 |
1 |
2000 |
3 |
0.15 |
47.8 |
|
2 |
2000 |
2 |
0.10 |
50.5 |
||
3 |
2000 |
2 |
0.10 |
54.9 |
||
4 |
2000 |
2 |
0.10 |
48.2 |
||
5 |
2000 |
1 |
0.05 |
51.1 |
||
|
|
(10) |
0.10 ± 0.04 |
50.5 ± 2.8 |
||
Positive control 10 x 12 |
1 |
2000 |
50 |
2.50 |
45.6 |
|
2 |
2000 |
28 |
1.40 |
43.1 |
||
3 |
2000 |
31 |
1.55 |
42.6 |
||
4 |
2000 |
44 |
2.20 |
37.3 |
||
5 |
2000 |
33 |
1.65 |
39.2 |
||
|
|
(186)3** |
1.86 ± 0.47 |
41.6 ± 3.3 |
PCE: polychromatic erythrocytes, MNPCE: micronucleated PCE, NCE: normochromatic erythrocytes
1twice administered by oral gavage at 24 hour interval
2once administered by intraperitoneal injection
3significantly different from the negative control (**p<0.01) by Kastenbaum & Bowman’s method
4significantly different from the negative control (##p<0.01) by Student’s t-test
51000 erythrocytes were scored
Applicant's summary and conclusion
- Conclusions:
- Negative
The test item did not induce biologically relevant increases in micronucleated polychromatic erythrocytes in the bone marrow of rats when administered up to the limit dose of 2000mg/kg bw via oral route.
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