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Genetic toxicity in vitro

Description of key information

In vitro bacterial gene mutation:

Study 1

The registered substance, N,N-Dimethyl-p-toluidine (CAS number: 99-97-8) was tested non-mutagenic (negative) up to the concentration of 333 µg/plate in Salmonella Typhimurium TA98, TA100, TA1525, TA1537 and TA1538 tester strains in the presence and absence of liver S9 microsomal activation.

Study 2

The registered substance, N,N-Dimethyl-p-toluidine (CAS number: 99-97-8) was tested non-mutagenic (negative) up to the concentration of 1500 µg/plate in Salmonella typhimurium TA98, TA100, and E.coli tester strains in the presence and absence of liver S9 microsomal activation.

In vitro cytogenicity assay:

The registered substance, N,N-Dimethyl-p-toluidine (CAS number: 99-97-8) showed both aneugenic and clastogenic activity in a concentration-related manner in the absence of metabolic activation in Chinese hamster V79 cells.

In vitro mammalian cell gene mutation study:

Gene mutation study in mammalian cells according to OECD TG 476 has been initiated with the registeed substance.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
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 journals
Qualifier:
according to guideline
Guideline:
other: As mentioned below
Principles of method if other than guideline:
A bacterial reverse mutagenicity study was performed to evaluate the mutagenic potential of the test chemical in Salmonella tryphimurium TA98, TA100, TA1535, TA1537 and TA1538.
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine
Species / strain / cell type:
S. typhimurium, other: Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535, TA1537, and TA1538
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system: Liver S9 homogenate was prepared from male Sprague-Dawley rats and Syrian golden hamsters that had been injected with Aroclor 1254 at 500 mg/kg body weight.
- source of S9
- method of preparation of S9 mix :The post-mitochondrial (microsomal) enzyme fractions were prepared as described by Ames et al.. The components of the S9 mix were 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 4 mM NADP, 100 mM sodium phosphate (pH 7.4), and the appropriate S9 homogenate at a concentration of 0.1 mL/mL of mix.
- concentration or volume of S9 mix and S9 in the final culture medium : For each plate receiving microsomal enzymes, 0.5 mL of S9 mix was added.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability)

Test concentrations with justification for top dose:
0, 3, 10, 33, 100, 333 microgram/ plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: [none; no data; acetone; arachis oil; beeswax; carbowaxe; castor oil; cetosteryl alcohol; cetyl alcohol; CMC (carboxymethyl cellulose); coconut oil; corn oil; cotton seed oil; DMSO; ethanol; glycerol ester; glycolester; hydrogenated vegetable oil; lecithin; macrogel ester; maize oil; olive oil; paraffin oil; peanut oil; petrolatum; physiol. saline; poloxamer; polyethylene glycol; propylene glycol; silicone oil; sorbitan derivative; soya oil; theobroma oil; vegetable oil; aqueous solvents (water or saline or culture medium)] : DMSO

- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO

- Justification for percentage of solvent in the final culture medium:
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 0.4 ug/Plate 2-Aminoanthracene; 0.75 ug/Plate 2-Aminoanthracene; 1.0 ug/Plate 2-Aminoanthracene; 2.0 ug/Plate 2-Aminoanthracene; 1.0 ug/Plate 4-Nitro-O-Phenylenediamine
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : triplicate
- Number of independent experiments: 3

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable):
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk : plate incorporation assay

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 48 hours
- Exposure duration/duration of treatment:
- Harvest time after the end of treatment (sampling/recovery times):

- OTHER: The doses that were tested in the mutagenicity assay were selected based on the levels of cytotoxicity observed in a preliminary dose range-finding study using strain TA100. Ten dose levels of the chemical, one plate per dose, were tested in both the presence and the absence of induced hamster S9. If no toxicity was observed, a total maximum dose of 10 mg of test chemical per plate was used.
Evaluation criteria:
The criteria used to evaluate a test were as follows: for a test chemical to be considered positive, it had to induce at least a doubling (TA98, TA100, and TA1535) in the mean number of
revertants per plate of at least one tester strain. This increase in the mean revertants per plate had to be accompanied by a dose response to increasing concentrations of the test chemical.
Statistics:
Values given as Mean or Mean ± Standard Error Mean
Key result
Species / strain:
S. typhimurium, other: Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535, TA1537, and TA1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH:
- Data on osmolality:
- Possibility of evaporation from medium:
- Water solubility:
- Precipitation and time of the determination:
- Definition of acceptable cells for analysis:
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES (if applicable): The doses that were tested in the mutagenicity assay were selected based on the levels of cytotoxicity observed in a preliminary
dose range-finding study using strain TA100. Ten dose levels of the chemical, one plate per dose, were tested in both the presence and the absence of induced hamster S9. If no toxicity was observed,
a total maximum dose of 10 mg of test chemical per plate was used. Initially the test chemical was tested at concentrations 3-1000 microgram/plate, but toxicity was observed in most of the tester strains at 500 and 1000 microgram/plate, hence the concentrations chosen for the final study were 3,10, 33, 100, 333 microgram/plate

STUDY RESULTS
- Concurrent vehicle negative and positive control data

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible
- Statistical analysis; p-value if any
- Any other criteria: e.g. GEF for MLA

Ames test:
- Signs of toxicity
- Individual plate counts :
- Mean number of revertant colonies per plate and standard deviation : The number of revertants/plate of all the doses tested both in the presence and absence of metabolic activation systems were significantly similar to negative controls in Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535, TA1537, and TA1538 tester strains. Hence, the test chemical can be considered to be non-mutagenic to Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535, TA1537, and TA1538.

Chromosome aberration test (CA) in mammalian cells:
- Results from cytotoxicity measurements:
o For lymphocytres in primary cultures: mitotic index (MI)
o For cell lines: relative population doubling (RPD), relative Increase in cell count (RICC), number of cells treated and cells harvested for each culture, information on cell cycle length, doubling time or proliferation index.
- Genotoxicity results (for both cell lines and lymphocytes)
o Definition for chromosome aberrations, including gaps
o Number of cells scored for each culture and concentration, number of cells with chromosomal aberrations and type given separately for each treated and control culture, including and excludling gaps
o Changes in ploidy (polyploidy cells and cells with endoreduplicated chromosomes) if seen

Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements:
o In the case of the cytokinesis-block method: CBPI or RI; distribution of mono-, bi- and multi-nucleated cells
o When cytokinesis block is not used: RICC, RPD or PD, as well as the number of cells treated and of cells harvested for each culture
o Other observations when applicable (complete, e.g. confluency, apoptosis, necrosis, metaphase counting, frequency of binucleated cells)

- Genotoxicity results
o Number of cells with micronuclei separately for each treated and control culture and defining whether from binucleated or mononucleated cells, where appropriate

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
o Relative total growth (RTG) or relative survival (RS) and cloning efficiency

- Genotoxicity results:
o Number of cells treated and sub-cultures for each cultures
o Number of cells plated in selective and non-selective medium
o Number of colonies in non-selective medium and number of resistant colonies in selective medium, and related mutant frequency
o When using the thymidine kinase gene on L5178Y cells: colony sizing for the negative and positive controls and if the test chemical is positive, and related mutant frequency. For the MLA, the GEF evaluation.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data:
- Negative (solvent/vehicle) historical control data:
Remarks on result:
other: not mutagenic

Results of the AMES assay

Result CAS # Dose TA98 TA100 TA102 TA1535 TA1537 TA1538 TA97
no S9 rat S9 Ham'r S9 no S9 rat S9 Ham'r S9 no S9 rat S9 Ham'r S9 no S9 rat S9 Ham'r S9 no S9 rat S9 Ham'r S9 no S9 rat S9 Ham'r S9 no S9 rat S9 Ham'r S9
Negative 99-97-8 Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative
DMSO 22 ± 6 24 ± 3 26 ± 3 160 ± 4 134 ± 12 189 ± 13 34 ± 9 28 ± 7 23 ± 6 7 ± 1 11 ± 7 7 ± 3 10 ± 3 15 ± 4 22 ± 5
3ug 21 ± 6 34 ±46 24 ± 4 113 ± 40 136 ± 3 186 ± 12 21 ± 2 31 ± 8 37 ± 10 9 ± 4 11 ± 1 18 ± 13 16 ± 2 21 ± 5 17 ± 2
10ug 15 ± 7 28 ± 7 26 ± 3 154 ± 6 157 ± 5 184 ± 5 22 ± 6 32 ± 2 31 ± 6 8 ± 1 12 ± 6 9 ± 2 9 ± 6 12 ± 3 23 ± 5
33ug 18 ± 3 27 ± 2 30 ± 6 133 ± 16 151 ± 6 146 ± 14 21 ± 6 38 ± 7 30 ± 7 8 ± 4 9 ± 2 8 ± 1 11 ± 2 19 ± 2 20 ± 3
100ug 15 ± 5 25 ± 2 27 ± 2 153 ± 23 185 ± 7 173 ± 21 30 ± 6 67 ± 13 59 ± 11 8 ± 1 8 ± 3 11 ± 5 11 ± 2 19 ± 4 25 ± 5
333ug 24 ± 4 23 ± 1 30 ± 2 136 ± 21 160 ± 6 159 ± 24 50 ± 4 21 ± 10 44 ± 6 6 ± 3 9 ±3 10 ± 4 12 ± 1 18 ± 3 18 ± 4
Positive 205 ± 67 722 ± 92 925 ± 68 667 ± 43 878 ± 47 1450 ± 114 483 ± 29 138 ± 21 162 ± 7 338 ± 16 81 ± 5 144 ± 22 253 ± 26 513 ± 34 938 ± 31
Positive 388 ± 33 1382 ± 123 1457 ± 29 764 ± 51 1642 ± 123 2603 ± 101 604 ± 28 216 ± 17 176 ± 8 1029 ± 150 159 ± 13 340 ± 41 421 ± 28 1428 ± 122 1732 ± 130
DMSO --- --- --- --- --- --- ---  21 ± 4 --- --- --- --- --- --- ---
33ug --- --- --- --- --- --- ---  39 ± 4 --- --- --- --- --- --- ---
100ug --- --- --- --- --- --- ---  52 ± 6 --- --- --- --- --- --- ---
333ug --- --- --- --- --- --- ---  23 ± 4 --- --- --- --- --- --- ---
Positive --- --- --- --- --- --- --- 185 ± 11 --- --- --- --- --- --- ---
Positive --- --- --- --- --- --- ---  237 ± 24 --- --- --- --- --- --- ---
DMSO --- --- --- --- --- --- --- 19 ± 5 20 ± 3 --- --- --- --- --- ---
10ug --- --- --- --- --- --- --- 18 ± 1 21 ± 5 --- --- --- --- --- ---
25ug --- --- --- --- --- --- --- 24 ± 6 19 ± 2 --- --- --- --- --- ---
50ug --- --- --- --- --- --- --- 20 ± 3 29 ± 3 --- --- --- --- --- ---
75ug --- --- --- --- --- --- --- 20 ± 5 26 ± 10 --- --- --- --- --- ---
100ug --- --- --- --- --- --- --- 22 ± 6 30 ± 2 --- --- --- --- --- ---
Positive 129 ± 7 131 ± 14
DMSO --- --- --- --- --- --- --- 15 ± 2 --- --- --- --- --- --- ---
10ug --- --- --- --- --- --- --- 19 ± 3 --- --- --- --- --- --- ---
33ug --- --- --- --- --- --- ---  18 ± 2 --- --- --- --- --- --- ---
100ug --- --- --- --- --- --- --- 10 ± 1 --- --- --- --- --- --- ---
333ug --- --- --- --- --- --- --- 9 ± 3 --- --- --- --- --- --- ---
Positive --- --- --- --- --- --- --- 161 ± 16 --- --- --- --- --- --- ---
Conclusions:
The Substance tested non-mutagenic (negative) up to the concentration of 333 µg/plate in Salmonella Typhimurium TA98, TA100, TA1525, TA1537 and TA1538 tester strains in the presence and absence of liver S9 microsomal activation.
Executive summary:

The mutagenic potential of the Substance, N, N,4-Trimethylbenzenamine (CAS number: 99-97-8) was assessed in a Salmonella/Mammalian-Microsome Mutagenicity Assay in the presence and absence of an exogenous metabolic activation system. The test was performed according to the plate incorporation method using Salmonella typhimurium TA98, TA100, TA1535, TA1537, and TA1538 tester strains. Cofactor-supplemented liver S9 microsomal fraction was used as an exogenous metabolic activation system. The liver S9 homogenate was prepared from male Aroclor 1254-injected (500 mg/kg body weight ) Sprague-Dawley rats and Syrian golden hamsters. Test concentrations were selected based on the levels of cytotoxicity observed in an initial dose range-finding study using strain TA100. In the main test, the following concentrations were tested: 0 (VC, DMSO) 3, 10, 33, 100, and 333 µg/plate. Five doses of test chemical, together with the appropriate concurrent solvent and positive controls (2-2-aminoanthracene and Sodium azide) were tested in triplicates on each tester strain without metabolic activation and also with activation by induced rat and hamster liver S9 preparations. The criteria used to evaluate a test were as follows: the test substance was considered positive (mutagenic) if induced at least a doubling (TA98, TA100, and TA1535) in the mean number of revertants per plate of at least one tester strain and this increase in the revertant counts was dose-dependent. If the results showed a dose-response with less than 3-fold increase with TA1537 or TA1538, the response had to be confirmed in a repeat experiment. Results: There was no significant or biologically relevant increase in the mean revertant counts at any concentrations tested up to 333 µg/plate either in the presence or absence of S9 metabolic activation in any tester strains when compared to the vehicle control. Conclusion: The registered substance, N,N,4-Trimethylbenzenamine (CAS number: 99-97-8) did not induce gene mutation by base-pair exchange or frameshifts in the histidine operon of Salmonella Typhimurium tester strains (TA 98, TA100, TA1535, TA1537 and TA1538) either with or without of liver S9 microsomal activation. 

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
The study contains experimental data on the registered sbstance.
Qualifier:
according to guideline
Guideline:
other: As mentioned below
Principles of method if other than guideline:
A bacterial reverse mutagenicity study was performed to evaluate the mutagenic potential of the registered substance in Salmonella tryphimurium TA98, TA100 and E.coli WP2.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine and tryptophan
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
Cofactor supplememnted liver S9 microsomal fraction (10%) was used as an exogenous metabolic activation system. S9 fraction was obtained from Aroclor 1254-induced male Sprague-Dawley rats.
Test concentrations with justification for top dose:
Concentrations:
0 (VC), 50, 100, 250, 500, 750, 1000 and 1500 ug/plate for TA98 and TA100
0 (VC), 50, 100, 250, 500, 750, 1000 and 1500 ug/plate for E.coli

The highest selected dose was limited by toxicity.
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylenediamine for TA98 without S9, 2-aminoanthracene for TA98, TA100, E.coli wih S9
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) :Triplicates
- Number of independent experiments : At least two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable):
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk : In agar according to preincubation method

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20 min at 37° C
- Exposure duration/duration of treatment: 48 hrs
- Harvest time after the end of treatment (sampling/recovery times): 48 hrs


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition; mitotic index (MI); relative population doubling (RPD); relative increase in cell count (RICC); replication index; cytokinesis-block proliferation index; cloning efficiency; relative total growth (RTG); relative survival (RS); other: Cytotoxicity was determined based on the reduction in the number of revertant counts and /or inhibition of the background lawn.


Evaluation criteria:
A positive response was defined as a reproducible, dose-related increase in histidine-independent (revertant) colonies in any one strain/activation combination. An equivocal response was defined as an increase in revertants that is not dose-related, was not reproducible, or was not of sufficient magnitude to support a determination of mutagenicity. A negative response was obtained when no increase in revertant colonies was observed following chemical treatment. There was no minimum percentage or fold increase required for a chemical to be judged positive or weakly positive, although positive calls were typically reserved for increases in mutant colonies that were at least twofold over the background.
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
A decrease in revertant counts was observed at ≥750 µg/plate and at 1500 µg/plate without and with S9 mix, respectively.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
A decrease in revertant counts was observed at ≥750 µg/plate without and with S9 mix.
Vehicle controls validity:
valid
Untreated negative controls validity:
not valid
True negative controls validity:
not valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
A decrease in revertant counts was observed at ≥750 µg/plate without and with S9 mix.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: Not mutagenic
Conclusions:
The registered substance, N,N-Dimethyl-p-toluidine (CAS number: 99-97-8) was tested non-mutagenic (negative) up to the concentration of 1500 µg/plate in Salmonella typhimurium TA98, TA100, and E.coli tester strains in the presence and absence of liver S9 microsomal activation.
Executive summary:

The mutagenic potential of N,N,4-Trimethylbenzenamine (CAS number: 99-97-8) was assessed in a bacterial mutagenicity assay in the presence and absence of an exogenous metabolic activation system. The test was performed according to the pre-incorporation method using Salmonella typhimurium TA98, TA100 and E. coliWP2 uvrA/pKM101tester strains. Cofactor-supplemented liver S9 microsomal fraction was used as an exogenous metabolic activation system. The liver S9 homogenate was prepared from male Aroclor 1254-injected Sprague-Dawley rats. Bacterial cells were exposed to 0 (VC), 50, 100, 250, 500, 750, 1000 and 1500 ug/plate test substance along with positive control substances (TA 100:sodium azidewithout S9 mix, TA 98:4-nitro-o-phenylenediaminewithout S9 mix,E. coli:Methyl methanesulfonatewithout S9 mix, and2-Aminoanthracenefor all strains with S9 mix) using triplicate plates. Thehistidine-independent mutant colonies arising on these plates were counted following incubation for 2 days at 37° C.Apositive response was defined as a reproducible, dose-related increase in histidine-independent (revertant) colonies in any strain/activation combination. An equivocal response was defined as an increase in revertants thatwasnotdose-related, wasnot reproducible, orwasnot of sufficient magnitude to support a determination of mutagenicity. A negative response was obtained when no increase in revertant colonies was observed following chemical treatment. There was no minimum percentage or fold increase required for a chemical to be judged positive or weakly positive, although positive calls were typically reserved for increases in mutant colonies that were at least twofold over the background.Results:There was no significant and dose-dependent increase in the number of revertant colonies up to 1500µg/plate in any tester strains used neither in the presence nor in the absence of S9 metabolic activationsystemwhen compared to the vehicle control.Conclusion:The registered substance did not induce gene mutation within the histidine and tryptophan operon of Salmonella typhimurium TA 98, TA 100 and E. coli tester strains, neither in the presence nor in the absence of S9 metabolic activation system.

Endpoint:
in vitro gene mutation study in mammalian cells
Data waiving:
other justification
Justification for data waiving:
other:
Justification for type of information:
The study is ongoing and this information will be submitted later based on ECHA communication/decision number CCH-D-2114539390-52-01/F
Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Data is from peer reviewed journal.
Qualifier:
according to guideline
Guideline:
other: As mentioned below
Principles of method if other than guideline:
CREST-Antibody Immunofluorescent Staining in V79 Cells was performed to evaluate the Micronuclei In Vitro Induction by the test chemical
GLP compliance:
no
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Metabolic activation system:
no data available
Test concentrations with justification for top dose:
0.3-1.2 millirnolar, Negative control
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: [none; no data; acetone; arachis oil; beeswax; carbowaxe; castor oil; cetosteryl alcohol; cetyl alcohol; CMC (carboxymethyl cellulose); coconut oil; corn oil; cotton seed oil; DMSO; ethanol; glycerol ester; glycolester; hydrogenated vegetable oil; lecithin; macrogel ester; maize oil; olive oil; paraffin oil; peanut oil; petrolatum; physiol. saline; poloxamer; polyethylene glycol; propylene glycol; silicone oil; sorbitan derivative; soya oil; theobroma oil; vegetable oil; aqueous solvents (water or saline or culture medium)] : DMSO

- Justification for choice of solvent/vehicle: The test chemical was dissolved in DMSO

- Justification for percentage of solvent in the final culture medium:
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Methylnitrosourea and Colchicine were used as positive controls
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : duplicate
- Number of independent experiments : 2 independent experiments

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): Cells were seeded onto 26 x 76 mm slides in Quadriperm plates (Haereus, Hanau, Germany), 1.2 X 105 cells per slide, and incubated at 37°C.
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable:
- Exposure duration/duration of treatment: 48 hours
- Harvest time after the end of treatment (sampling/recovery times):

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): indicate the identity of mitotic spindle inhibitor used (e.g., colchicine), its concentration and, duration and period of cell exposure.
- If cytokinesis blocked method was used for micronucleus assay: indicate the identity of cytokinesis blocking substance (e.g. cytoB), its concentration, and duration and period of cell exposure.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): In preliminary experiments micronuclei formation was evaluated by Giemsa staining, after both 24 and 48 hr of treatment, according to Countryman and Heddle [1976].
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored):
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification):
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): An immunological staining with antibodies against kinetochore proteins (CREST-antibodies) was used to discriminate between structural (CREST negative micronuclei) and numerical (CREST positive, kinetochore-micronuclei) chromosome aberrations [Nusse et al., 1989]. CREST-antibodies, obtained from a scleroderma patient, were a gift from Dr. Solberg (University Hospital of Frankfurt, Germany).
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): Each reported value was the mean of results obtained in at least two independent experiments in which at least 3,000 cells were scored.
- Determination of polyploidy:
- Determination of endoreplication:
Evaluation criteria:
Each reported value was the mean of results obtained in at least two independent experiments in which at least 3,000 cells were scored.
Statistics:
Statistical evaluation was done according to the x2 test or the Fisher Exact Test. The dose-dependence significance of CREST+ and CREST- micronuclei induction was evaluated using According to the Cochran-Armitage trend test.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Micronucleus test in mammalian cells: The test chemical was tested with a 0.3-1.2 millirnolar range of doses, the highest allowing > 10% survival (as estimated by colony formation). At lower survival rates, most of the nuclei are extensively fragmented and screening of micronuclei was not feasible. In terms of aneugenic effects,the test chemical gave the most significant positive response, with dose-dependcnt values for treated samples up to more than five times greater than concurrent controls. In terms of clastogenic effects, i.e., CREST negative micronuclei induction (CREST-), the test chemical gave a statistically significant result, at the highest testable dose. An overall trend of positivity was observable for the test chemical. According to the Cochran-Armitage trend test, the dose-dependence significance of CREST+ and CREST- micronuclei induction for the test chemical of P < 0.001 in both cases.
Remarks on result:
other: mutagenic potential observed
Conclusions:
The registered substance, N,N-Dimethyl-p-toluidine (CAS number: 99-97-8) showed both aneugenic and clastogenic activity in a concentration-related manner in the absence of metabolic activation in Chinese hamster V79 cells.
Executive summary:

CREST-Antibody Immunofluorescent Staining in V79 Cells was performed to evaluate the Micronuclei In Vitro Induction by the test chemical. Cells were seeded onto 26 x 76 mm slides in Quadriperm plates (Haereus, Hanau, Germany), 1.2 X 105 cells per slide, and incubated at 37°C.In preliminary experiments micronuclei formation was evaluated by Giemsa staining, after both 24 and 48 hr of treatment, according to Countryman and Heddle [1976]. Methylnitrosourea and Colchicine were used as positive controls.An immunological staining with antibodies against kinetochore proteins (CREST-antibodies) was used to discriminate between structural (CREST negative micronuclei) and numerical (CREST positive, kinetochore-micronuclei) chromosome aberrations [Nusse et al., 1989]. CREST-antibodies, obtained from a scleroderma patient, were a gift from Dr. Solberg (University Hospital of Frankfurt, Germany). Each reported value was the mean of results obtained in at least two independent experiments in which at least 3,000 cells were scored.Statistical evaluation was done according to the x2 test or the Fisher Exact Test. The dose-dependence significance of CREST+ and CREST- micronuclei induction was evaluated using According to the Cochran-Armitage trend test. The test chemical was  tested with a 0.3-1.2 millirnolar range of doses, the highest allowing > 10% survival (as estimated by colony formation). At lower survival rates, most of the nuclei are extensively fragmented and screening of micronuclei was not feasible. In terms of aneugenic effects,the test chemical gave the most significant positive response, with dose-dependcnt values for treated samples up to more than five times greater than concurrent controls. In terms ofclastogenic effects, i.e., CREST negative micronuclei induction (CREST-), the test chemical gave a statistically significant result, at the highest testable dose. An overall trend of positivity was observable for the test chemical. According to the Cochran-Armitage trend test, the dose-dependence significance of CREST+ and CREST- micronuclei induction for the test chemical of P < 0.001 in both cases. Hence, the test chemical can be considered to be mutagenic to V79 Cells when tested in vitro.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In vivo peripheral blood micronucleus test:

The registered substance, N, N-Dimethyl-p-toluidine (CAS number: 99-97-8), did not induce the formation of micronucleated erythrocytes, as an indicator of chromosomal damage, in peripheral blood erythrocytes of male or female B6C3F1/N mice treated with 15 to 125 mg/kg per day test substance by gavage for 3 months.

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:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
The study contains experimental data of the registered substance.
Qualifier:
according to guideline
Guideline:
other: As below explaned
Principles of method if other than guideline:
The ability of the registered substance to induce micronuclei in blood erythrocytes was tested in B6C3F1 mice after 14 weeks of oral administration.
GLP compliance:
not specified
Type of assay:
mammalian erythrocyte micronucleus test
Specific details on test material used for the study:
Purity: >99%
Species:
mouse
Strain:
B6C3F1
Details on species / strain selection:
Mice were obtained from Taconic Farms, Inc. (Germantown, NY). On receipt, the mice were 4 to 5 weeks old. Animals were quarantined for 11-14 days and were 5 to 7 weeks old on the first day of the study.
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
Corn oil
Details on exposure:
Groups of 10 male and 10 female mice received N,N-dimethyl-p-toluidine in corn oil by gavage, 5 days per week for 14 weeks.
Duration of treatment / exposure:
3 months (14 weeks)
Frequency of treatment:
Daily
Dose / conc.:
0
Remarks:
Vehicle control (corn oil)
Dose / conc.:
15 mg/kg bw/day
Dose / conc.:
30 mg/kg bw/day
Dose / conc.:
60 mg/kg bw/day
Dose / conc.:
125 mg/kg bw/day
No. of animals per sex per dose:
5 mice/sex/dose
Control animals:
yes, concurrent vehicle
Tissues and cell types examined:
Blood erythrocytes
Details of tissue and slide preparation:
Peripheral blood samples were obtained from male and female mice at the end of the 3-month gavage study. Smears were immediately prepared and fixed in absolute methanol. The methanol-fixed slides were stained with acridine orange and coded. Slides were scanned to determine the frequency of micronuclei in 2,000 normochromatic erythrocytes (NCEs, mature erythrocytes) per animal. In addition, the percentage of polychromatic erythrocytes (PCEs, reticulocytes) among a population of 1,000 erythrocytes in the peripheral blood was scored for each dose group as a measure of bone marrow toxicity.
Evaluation criteria:
In the slide-based micronucleus test, an individual trial is considered positive if the trend test P value is less than or equal to 0.025 or if the P value for any single dosed group is less than or equal to 0.025 divided by the number of dosed groups.
Statistics:

The results were tabulated as the mean of the pooled results from all animals within a treatment group plus or minus the standard error of the mean. The frequency of micronucleated cells among NCEs was analyzed by a statistical software package that tested for increasing trend over dose groups with a one-tailed Cochran-Armitage trend test, followed by pairwise comparisons between each dosed 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.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
not examined
Additional information on results:
No significant increases in the frequencies of micronucleated erythrocytes, an indicator of chromosomal damage, were observed in peripheral blood of male or female B6C3F1/N mice treated with 15 to 125 mg/kg per day test substance by gavage for 3 months. No significant alterations in the percentage of circulating reticulocytes were observed, suggesting that the test substance did not induce bone marrow toxicity over the dose range tested.
Conclusions:
The registered substance, N, N-Dimethyl-p-toluidine (CAS number: 99-97-8), did not induce the formation of micronucleated erythrocytes, as an indicator of chromosomal damage, in peripheral blood erythrocytes of male or female B6C3F1/N mice treated with 15 to 125 mg/kg per day test substance by gavage for 3 months.
Executive summary:

The ability of the registered substance, ,N, N-Dimethyl-p-toluidine (CAS number: 99-97-8), to induce micronuclei in blood erythrocytes was tested in B6C3F1 mice after 14 weeks of oral administration.Groups of 10 male and 10 female mice receivedthe test substancein corn oil by gavage at doses of 15, 30, 60, 125, or 250 mg/kg, 5 days per week for 14 weeks. Vehicle control animals received the corn oil vehicle alone. Feed and water were available ad libitum. The animals were weighed and clinical findings were recorded at study start, weekly thereafter, and at study termination.Mortality occurred at 125 mg/kg; three males and two females administered died before the end of the study. The final mean body weight of 125 mg/kg males and the mean body weight gains of 125 mg/kg males and females were significantly less than those of the vehicle controls.Clinical findings associated with administration ofthe test substanceincluded abnormal breathing, thinness, lethargy, cyanosis, and ruffled fur in 125 and 250 mg/kg males and females.At the end of theadministrating period (3months),peripheral blood samples were obtained from male and female mice. Smears were immediately prepared and fixed in absolute methanol. The methanol-fixed slides were stained with acridine orange.Slides were scanned to determine the frequency of micronuclei in 2000 normochromatic erythrocytes (NCEs, mature erythrocytes) per animal. In addition, the percentage of polychromatic erythrocytes (PCEs, reticulocytes) among a population of 1000 erythrocytes in the peripheral blood was scored for each dose group as a measure of bone marrow toxicity.The frequency of micronucleated cells among NCEs was analyzed by a statistical software package that tested for increasing trend over dose groups with a one-tailed Cochran-Armitage trend test, followed by pairwise comparisons between each dosed 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. In the slide-based micronucleus test, an individual trial is considered positive if the trend test P value is less than or equal to 0.025 or if the P value for any single dosed group is less than or equal to 0.025 divided by the number of dosed groups.Results:No significant increases in the frequencies of micronucleated erythrocytes were observed in peripheral blooderythrocytesof male or female B6C3F1/N mice treated with 15 to 125 mg/kg/day test substanceby gavage for 3 months.No significant alterations in the percentage of circulating reticulocytes were observed, suggesting thatthe test substancedid not induce bone marrow toxicity over the dose range tested.Conclusion:The test substance did not inducechromosomal damagein peripheral blooderythrocytesof male or female B6C3F1/N mice treatedup to125 mg/kg/dayby gavage for 3 months.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Genetic toxicity in vitro

In vitro bacterial gene mutation assays

Study 1:

The mutagenic potential of the Substance, N, N,4-Trimethylbenzenamine (CAS number: 99-97-8) was assessed in a Salmonella/Mammalian-Microsome Mutagenicity Assay in the presence and absence of an exogenous metabolic activation system. The test was performed according to the plate incorporation method using Salmonella typhimurium TA98, TA100, TA1535, TA1537, and TA1538 tester strains. Cofactor-supplemented liver S9 microsomal fraction was used as an exogenous metabolic activation system. The liver S9 homogenate was prepared from male Aroclor 1254-injected (500 mg/kg body weight ) Sprague-Dawley rats and Syrian golden hamsters. Test concentrations were selected based on the levels of cytotoxicity observed in an initial dose range-finding study using strain TA100. In the main test, the following concentrations were tested: 0 (VC, DMSO) 3, 10, 33, 100, and 333 µg/plate. Five doses of test chemical, together with the appropriate concurrent solvent and positive controls (2-2-aminoanthracene and Sodium azide) were tested in triplicates on each tester strain without metabolic activation and also with activation by induced rat and hamster liver S9 preparations. The criteria used to evaluate a test were as follows: the test substance was considered positive (mutagenic) if induced at least a doubling (TA98, TA100, and TA1535) in the mean number of revertants per plate of at least one tester strain and this increase in the revertant counts was dose-dependent. If the results showed a dose-response with less than a 3-fold increase with TA1537 or TA1538, the response had to be confirmed in a repeat experiment. Results: There was no significant or biologically relevant increase in the mean revertant counts at any concentrations tested up to 333 µg/plate either in the presence or absence of S9 metabolic activation in any tester strains when compared to the vehicle control. Conclusion: The registered substance, N,N,4-Trimethylbenzenamine (CAS number: 99-97-8) did not induce gene mutation by base-pair exchange or frameshifts in the histidine operon of Salmonella Typhimurium tester strains (TA 98, TA100, TA1535, TA1537 and TA1538) either with or without of liver S9 microsomal activation. 

Study 2:

The mutagenic potential of N,N,4-Trimethylbenzenamine (CAS number: 99-97-8) was assessed in a bacterial mutagenicity assay in the presence and absence of an exogenous metabolic activation system. The test was performed according to the pre-incorporation method using Salmonella typhimurium TA98, TA100 and E. coliWP2 uvrA/pKM101tester strains. Cofactor-supplemented liver S9 microsomal fraction was used as an exogenous metabolic activation system. The liver S9 homogenate was prepared from male Aroclor 1254-injected Sprague-Dawley rats. Bacterial cells were exposed to 0 (VC), 50, 100, 250, 500, 750, 1000 and 1500 ug/plate test substance along with positive control substances (TA 100:sodium azidewithout S9 mix, TA 98:4-nitro-o-phenylenediaminewithout S9 mix,E. coli:Methyl methanesulfonatewithout S9 mix, and2-Aminoanthracenefor all strains with S9 mix) using triplicate plates. Thehistidine-independent mutant colonies arising on these plates were counted following incubation for 2 days at 37° C.Apositive response was defined as a reproducible, dose-related increase in histidine-independent (revertant) colonies in any strain/activation combination. An equivocal response was defined as an increase in revertants that was not dose-related, wasnot reproducible, orwasnot of sufficient magnitude to support a determination of mutagenicity. A negative response was obtained when no increase in revertant colonies was observed following chemical treatment. There was no minimum percentage or fold increase required for a chemical to be judged positive or weakly positive, although positive calls were typically reserved for increases in mutant colonies that were at least twofold over the background. Results:There was no significant and dose-dependent increase in the number of revertant colonies up to 1500µg/plate in any tester strains used neither in the presence nor in the absence of S9 metabolic activation system when compared to the vehicle control.Conclusion: The registered substance did not induce gene mutation within the histidine and tryptophan operon of Salmonella typhimurium TA 98, TA 100 and E. coli tester strains, neither in the presence nor in the absence of S9 metabolic activation system.

Study 3:

The ability of the registered substance,N,N-dimethyl-p-toluidine (CAS number: 99-97-8) to induce gene mutation within the histidine operon was tested in Salmonella tryphimurium TA 97, TA98 and TA100 tester strains and in the presence and absence of an exogenous metabolic activation system. The test was performed according to the preincubation method described by Maron and Ames 1983.10% or 30% post-mitochondrial preparations (S9 fractions)were used as an exogenous metabolic activation system. S9 fractions were obtained from livers of Aroclor-induced male rats or male hamsters. For this purpose, the animals were injected i.p. with 500 mg/kg of Aroclor-1254 5 days before sacrifice. The substance was tested at concentrations of 0(vehicle control), 1, 2.5, 5, 10, 40, 70, 100µg/plate, and at 20, 30, 50, 60, and 80µg/plate in two independent experimentsusingduplicatecultures. Ethanol was selected as a vehicle for the test substance. Methyl methanesulfonate, 2-nitrofluorene, sodium azide (SA), were used in the absence of metabolic activation with TA 97, TA 98 and TA 100 strains, respectively; benzo(a)pyrene, which was used in all strains in the presence of S9served aspositive controls. The revertant colonies were counted 48 hrsafter plating of the treated bacteria on Vogel-Bonner medium.The substance was considered positive (mutagenic)if it induced a reproducible and dose-related increase in the number of revertant colonies over the background (according to Claxton et al.,1987). Results: There was no significant increase in the number of revertant colonies at any concentrations tested in the three tester strains when compared to the vehicle control neighter in the presence nor in the absence of S9 metabolic activation.Cytotoxicity was observed as the highest test concentration (at 100µg/plate) as a significant reduction in revertant counts in the three tester strains. Positive control substances induced reproducible,dose-dependentand significant increases in mutant colony counts with and without S9 metabolic activation. Conclusion: The Substance did not induce gene mutation within the histidine operon in Salmonella tryphimurium TA 97, TA98 and TA100 tester strains neither in the presence nor in the absence of S9 metabolic activation when tested up to a cytotoxic concentration.

Study 4:

The mutagenicity of the registered substance,N,N-Dimethyl-p-toluidine (CAS number: 99-98-7),was tested in a Spot test developed by Maron and Ames (1983). The Spot test was performed with Salmonella tryphimurium TA 97, TA 98, TA100 and TA 104 tester stains in the presence and absence of an exogenous metabolic activation system. Cofactor-supplemented liver S9 microsomal fraction was used as an exogenous metabolic activation system.For theSpot tests, the bacterial strain to be assayed was inoculated in sterile nutrient broth and grown overnight at 37°C. Bottom agar plates were prepared one day before testing by transferring 30 ml of Vogel-Bonner E medium to Falcon Petri plates. Top agar, 0.5% Difco agar and 0.5% NaCl was prepared in bulk. On the day of the experiment, 100 ml of top agar was heated in a microwave oven, and 10 ml of a solution of 0.5 mM histidine-biotin was added to the molten top agar. After mixing, 2 ml portions of the completed top agar were pipetted into sterile test tubes. The bacterial test strain and 0.5 ml of rat liver S9 mix, if appropriate, were added to the tubes. After mixing, the ingredients in the test tube were poured onto the bottom agar plate. As the top agar hardened, a 3µlspot of thetest substancewas applied directly to the top agar.Vehicle (DMSO) and positive control substances, i.e.,4-nitroquinoline-N-oxideand2-aminofluorenewere also included in the test.The plates were incubated for 48 h at 37°C. After incubation, plates were examined for spontaneous revertants, mutagenicity, and zones of inhibition. To evaluate the results, colonies were counted on each plate. The number of colonies on the DMSO plates(vehicle control)wassubtracted from the number of colonies on thetest substance-treatedplates.The scale developed by Ames (Maron and Ames, 1983) was used to score the experimental plates.Results:The average number of spontaneous reversions with DMSObutwithout S9 was 180 with TA97, 43 with TA98, 194 with TA100, and 510 with TA104. With S9 the average number of spontaneous reversions was 196 with TA97, 79 with TA98, 169 with TA100, and 544 with TA104.The test substance was found to be mutagenic (positive) in TA 100 both in the presence and absence of S9 metabolic activation and in TA 104 in the presence of metabolic activation. In TA98, the mean number of revertant counts was in the control range both with and without metabolic activation. Total growth inhibition was observed in TA97, and hence the mutagenicity was not possible to be assessed. The number of revertants/plate (spontaneous subtracted) was in the range of <20 (TA98), 20-100 (TA100) and <20 and 20-100 (TA104). Due to the partially positive results obtained in the Spot test, the Substance was further tested according to the plate incorporation method at concentrations of 0 (VC), 50, 250, 500, 2500 and 5000 µg/plate with and without S9 metabolic activation using TA100 and TA 104 tester strains. The registered substance tested non-mutagenic (negative) in TA100 and TA104 both in the presence and absence of S9 metabolic activation up to 5000µg/plate.Conclusion: N,N-Dimethyl-p-toluidine (CAS number: 99-98-7)did not induce gene mutation within the histidine operon in Salmonella tryphimurium TA 98, TA 100 and TA104 tester strains neither in the presence nor in the absence of S9 metabolic activation.

In vitro micronucleus test:

CREST-Antibody Immunofluorescent Staining in V79 Cells was performed to evaluate the Micronuclei In Vitro Induction by the test chemical. Cells were seeded onto 26 x 76 mm slides in Quadriperm plates (Haereus, Hanau, Germany), 1.2 X 105cells per slide, and incubated at 37°C.In preliminary experiments micronuclei formation was evaluated by Giemsa staining, after both 24 and 48 hr of treatment, according to Countryman and Heddle [1976]. Methylnitrosourea and Colchicine were used as positive controls.An immunological staining with antibodies against kinetochore proteins (CREST-antibodies) was used to discriminate between structural (CREST negative micronuclei) and numerical (CREST positive, kinetochore-micronuclei) chromosome aberrations [Nusse et al., 1989]. CREST-antibodies, obtained from a scleroderma patient, were a gift from Dr. Solberg (University Hospital of Frankfurt, Germany). Each reported value was the mean of results obtained in at least two independent experiments in which at least 3,000 cells were scored.Statistical evaluation was done according to the x2 test or the Fisher Exact Test. The dose-dependence significance of CREST+ and CREST- micronuclei induction was evaluated using According to the Cochran-Armitage trend test. The test chemical was  tested with a 0.3-1.2 millirnolar range of doses, the highest allowing > 10% survival (as estimated by colony formation). At lower survival rates, most of the nuclei are extensively fragmented and screening of micronuclei was not feasible. In terms of aneugenic effects,the test chemical gave the most significant positive response, with dose-dependcnt values for treated samples up to more than five times greater than concurrent controls. In terms ofclastogenic effects, i.e., CREST negative micronuclei induction (CREST-), the test chemical gave a statistically significant result, at the highest testable dose. An overall trend of positivity was observable for the test chemical. According to the Cochran-Armitage trend test, the dose-dependence significance of CREST+ and CREST- micronuclei induction for the test chemical of P < 0.001 in both cases. Hence, the test chemical can be considered to be mutagenic to V79 Cells when tested in vitro.

Gene mutation in mammalian cells:

Gene mutation study in mammalian cells according to OECD TG 476 has been initiated with the registered substance.

Genetic toxicity in vivo

Study 1:

The ability of the registered substance, N, N-dimethyl-p-toluidine (CAS number: 99-97-8), to induce DNA damage was tested in an In vivo Alkaline Comet assay using Sprague-Dawley rats. Six male rats per dose level received the test substance at 0 and 60 mg/kg bw/day in 1% acetone/corn oil for 4 consecutive days, at 24-hours intervals. Four hours after the fourth dose, tissue samples of the left liver lobe were collected to assess DNA damage. Ethyl methanesulfonate(200 mg/kg) was given by oral gavage to rats in the positive control group. The cell preparation and the single cell gel electrophoresis were performed according to Recio et al., 2010.For each cell, the extent of DNA migration was characterised using the percent tail DNA endpoint measurement (the intensity of all tail pixels divided by the total intensity of all pixels in the comet, expressed as a percentage). Pairwise comparison of the vehicle control and the dosed group was conducted using Student’s t-test (p˂0.05). There was a statistically significant increase in the percent tail DNA in liver cells at 60 mg/kg as compared with the vehicle control group (CTR, 10.5 ±1.23; 60 mg/kg, 14.6 ± 1.35; p=0.024). Hence, the test chemical caused DNA damage (DNA double-strand breaks) in liver cells of male SD rats administered with 60 mg/kg test substance for 4 days.

Study 2:

The potential of the test chemical to induce DNA strand breaks has been tested in an In vivo Alkaline Comet assay in mice. The test chemical was given by oral gavage to 5 male B6C3F1 mice per dose level at 0 (vehicle), 30, 60 or 75 mg/kg bw/day for 4 consecutive days at 24-hours intervals. Concurrent vehicle control (corn oil) and reference mutagen(ethyl methanesulfonate, 150 mg/kg) were also included in the assay. Four hours after the fourth dose, blood lymphocytes and tissue samples of the left liver lobe were collected to assess DNA damage. The cell preparation and the single cell gel electrophoresis were performed according to Recio et al., 2010.For each cell, the extent of DNA migration was characterised using the percent tail DNA endpoint measurement (the intensity of all tail pixels divided by the total intensity of all pixels in the comet, expressed as a percentage). The data was statistically analysed to determine if there was a significant increase in percent tail DNA in test chemical-exposed animals compared to the control (mice exposed to the vehicle). Additionally, the Kendall rank correlation test (Kendall, 1938) was applied to determine the presence of a dose-response; and the trend tests were considered statistically significant at p≤0.025. For a positive test, both a dose-response and at least one significant dose group were required. If only one of these measures was present, the test was considered to be equivocal. The absence of either condition resulted in a negative test. There was no dose-related and statistically significant increase in the percent tail DNA at doses tested either in the blood (30 mg/kg, 1.9 ± 0.23; 60 mg/kg, 1.5 ± 0.14; 75 mg/kg, 2.2 ± 0.3; p=0.943) and in the liver (30 mg/kg,5.7 ± 1.70; 60 mg/kg, 6.5 ± 0.42; 75 mg/kg, 6.3 ± 0.81; p=0.364) as compared to the control (blood 2.0 ± 0.24; liver 5.2 ± 0.59). The trend test was not significant (p= 0.5286). Based on the data of the present study, the repeated oral administration of the test chemical did not induce DNA damage (DNA double-strand breaks) in blood leukocytes and liver cells when it was given to male B6C3F1 for 4 days.

Study 3:

The potential of the test chemical to induce DNA damage has been tested by an alkaline DNA elution assay. The chemical was given by a single intraperitoneal injection to male BALB/c mice. The highest dose level used was based on the LD50values and was selected to allow at least 2/3 of the treated animals to survive up to the sacrifice time. The test was performed as previously described(Parodi et.al., 1978), with minor modifications. In short, aliquots of1-2 X 106liver nuclei were put on cellulose mixed ester Nuclepore filters and lysed for 30 min at room temperature with the lysing solution.After washing, single-stranded DNA was eluted in the dark, for 1hour at room temperature, with 10 ml of eluant containing 10mM Na2EDTA and tetraethylammonium hydroxide to give a pH value of 12.35. Three 20-min fractions were collected, and the DNA content in them and that remaining on the filter was then determined utilising a microfluorometric method using diaminobenzoic acid. The test chemical was given by intraperitoneal injection to rats at dose levels of 0, 4, and 8 mmol/kg bw/day and the DNA damage was evaluated at 2 and 24 hours after treatment. The positive control substance produced statistically significant increases in the elution rate over the control value in both mice and rats under all experimental conditions used. The elution rates were significantly increased both in the 4 and 8 mmol/kg groups at 2 hours (4 mmol/kg 3.14 ± 0.11, 8 mmol/kg 3.23 ± 0.25, p˂0.05) when compared to the control (CTR 1.63 ± 0.38). There was no difference in the elution rate between the control and treated groups at any doses tested at 24 hours (CTR 1.99 ± 0.05, 4 mmol/kg 2.06 ± 0.16). Under the experimental condition described, the test chemical had the potential to produce DNA damage/fragmentation in the liver of male Sprague Dawley rats following a single intraperitoneal administration.

 

Study 4:

The potential of the test chemical to induce DNA damage has been tested by an alkaline DNA elution assay. The chemical was given by a single intraperitoneal injection to male BALB/c mice. The highest dose level used was based on the LD50values and was selected to allow at least 2/3 of the treated animals to survive up to the sacrifice time. The test was performed as previously described(Parodi et.al., 1978), with minor modifications. In short, aliquots of1-2 X 106 liver nuclei were put on cellulose mixed ester. Nuclepore filters and lysed for 30 min at room temperature with the lysing solution. After washing, single-stranded DNA was eluted in the dark, for 1hour at room temperature, with 10 ml of eluant containing 10mM Na2EDTA and tetraethylammonium hydroxide to give a pH value of 12.35. Three 20-min fractions were collected, and the DNA content in them and that remaining on the filter was then determined utilising a microfluorometric method using diaminobenzoic acid. The chemical was given via intraperitoneal injection to male BALB/c mice at dose levels of 0 (control) 1 and 2 mmol/kg bw/day once, and the induced DNA damage demonstrated as changes in the elution rate was evaluated 2 and 24 hours after the administration. Mice injected with dimethylnitrosamine(0.05 and 0.15 mmol/kg) served as positive controls. Four to six experiments were performed at each sampling time, and in each experiment, the DNA determinations were done in duplicate. The positive control substance produced statistically significant increases in the elution rate over the control value in both mice and rats under all experimental conditions used. There were no positive results observed at the 2-hour time point, but a statistically significant increase in the elution rate was noted at 1 mmol/kg (CTR, 2.06 ±0.26; 1mmol/kg, 3.43 ± 0.28; p˂ 0.05) at 24 hours compared to the control in the liver cells of male BALB/c mice. Under the experimental condition described, the test chemical had the potential to produce DNA damage/fragmentation in the liver of male BALB/c mice following a single intraperitoneal administration.

Study 5:

The ability of the registered substance, , N, N-Dimethyl-p-toluidine (CAS number: 99-97-8), to induce micronuclei in blood erythrocytes was tested in B6C3F1 mice after 14 weeks of oral administration. Groups of 10 male and 10 female mice received the test substance in corn oil by gavage at doses of 15, 30, 60, 125, or 250 mg/kg, 5 days per week for 14 weeks. Vehicle control animals received the corn oil vehicle alone. Feed and water were available ad libitum. The animals were weighed, and clinical findings were recorded at study start, weekly thereafter, and at study termination. Mortality occurred at 125 mg/kg; three males and two females administered died before the end of the study. The final mean body weight of 125 mg/kg males and the mean body weight gains of 125 mg/kg males and females were significantly less than those of the vehicle controls. Clinical findings associated with administration of the test substance included abnormal breathing, thinness, lethargy, cyanosis, and ruffled fur in 125 and 250 mg/kg males and females. At the end of the administrating period (3months), peripheral blood samples were obtained from male and female mice. Smears were immediately prepared and fixed in absolute methanol. The methanol-fixed slides were stained with acridine orange.Slides were scanned to determine the frequency of micronuclei in 2000 normochromatic erythrocytes (NCEs, mature erythrocytes) per animal. In addition, the percentage of polychromatic erythrocytes (PCEs, reticulocytes) among a population of 1000 erythrocytes in the peripheral blood was scored for each dose group as a measure of bone marrow toxicity.The frequency of micronucleated cells among NCEs was analysed by a statistical software package that tested for increasing trend over the dose groups with a one-tailed Cochran-Armitage trend test, followed by pairwise comparisons between each dosed 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. In the slide-based micronucleus test, an individual trial is considered positive if the trend test P value is less than or equal to 0.025 or if the P value for any single dosed group is less than or equal to 0.025 divided by the number of dosed groups.Results: No significant increases in the frequencies of micronucleated erythrocytes were observed in peripheral blood erythrocytes of male or female B6C3F1/N mice treated with 15 to 125 mg/kg/day test substance by gavage for 3 months.No significant alterations in the percentage of circulating reticulocytes were observed, suggesting that the test substance did not induce bone marrow toxicity over the dose range tested. Conclusion: The test substance did not induce chromosomal damage in peripheral blood erythrocytes of male or female B6C3F1/N mice treated to125 mg/kg/day by gavage for 3 months

Study 6:.

The ability of the registered substance, , N, N-Dimethyl-p-toluidine (CAS number: 99-97-8) to induce micronuclei in blood erythrocytes was tested in B6C3F1 mice after 4 days of oral administration. Groups of 5 male mice received the test substance in corn oil by gavage at doses of  0 (VC), 30, 60, 75 mg/kg for 4 days. The highest dose was based on the toxicity information obtained in the 3-month mouse study. Ethyl methanesulfonate in 0.9% saline was used as the positive control. Four hours after the fourth dose, peripheral blood samples were collected and processed for flow cytometric evaluation of micronucleated erythrocytes as described by Witt et al. (2008). For each sample, 20,000 immature CD71+reticulocytes were analysed to determine the frequency of micronucleated reticulocytes. More than 106 mature erythrocytes were enumerated during the reticulocyte analysis, and the percentage of reticulocytes among total erythrocytes was calculated as a measure of bone marrow toxicity. Because measurements of micronucleus frequency using flow cytometry are obtained from a large number of cells, it was assumed that the proportion of micronucleated cells is approximately normally distributed within each sample. The authors used Levene’s test at α=0.05 to test for equal variances among the treatment groups. In the case of equal variances, linear regression was used to test for a dose-related trend, and Williams’ test (Williams, 1971, 1972) was used to test for pairwise differences between each treatment group and the vehicle control group. In the case of unequal variances, Jonckheere’s test (Jonckheere, 1954) was used to test for a linear trend, and pairwise differences with the control group were tested using Dunn’s test (Dunn, 1964). To correct for multiple pairwise comparisons, the P value for each comparison was multiplied by the number of comparisons made. Trend tests and pairwise comparisons with the controls were considered statistically significant at P≤0.025. A one-tailed independent t-test was used to verify a positive response (P≤0.05) to the control compound, ethyl methanesulfonate. Results: No significant increases in the frequencies of micronucleated erythrocytes were observed in peripheral blood erythrocytes of male B6C3F1/N mice treated with 30 to 75 mg/kg/day test substance by gavage for 4 days.No significant alterations in the percentage of circulating reticulocytes were observed, suggesting that the test substance did not induce bone marrow toxicity over the dose range tested. Conclusion: The test substance did not induce chromosomal damage in peripheral blood erythrocytes of male B6C3F1/N mice treated to75 mg/kg/day by gavage for 4 days.

Justification for classification or non-classification

The registered substance, N, N-Dimethyl-p-toluidine (CAS number: 99-97-8) does not induce gene mutationsin bacterial cell cultures or structural and numerical chromosome aberrations in somatic cells in vivo. The mutagenic potential of the N, N-Dimethyl-p-toluidine in mammalian cells is under testing according to OECD TG 476.