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Genetic toxicity: in vitro

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Administrative data

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

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Gene mutation toxicity study of the test chemical
Author:
Seifried et al
Year:
2006
Bibliographic source:
Chem. Res. Toxicol
Reference Type:
publication
Title:
Bacterial Gene Mutagenicity Results for the test chemical
Author:
NTP
Year:
2018
Bibliographic source:
Bacterial Gene Mutagenicity Results, NTP, 2018

Materials and methods

Test guideline
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

Test material

Constituent 1
Chemical structure
Reference substance name:
N,N-dimethyl-p-toluidine
EC Number:
202-805-4
EC Name:
N,N-dimethyl-p-toluidine
Cas Number:
99-97-8
Molecular formula:
C9H13N
IUPAC Name:
N,N,4-trimethylaniline
Test material form:
liquid
Details on test material:
- Name of test material: Benzenamine, N,N,N-trimethyl-
- Common Name: N,N-Dimethyl-p-toluidine
- Molecular formula: C9H13N
- Molecular weight: 135.209 g/mol
- Smiles notation: c1(N(C)C)ccc(C)cc1
- InChl: 1S/C9H13N/c1-8-4-6-9(7-5-8)10(2)3/h4-7H,1-3H3
- Substance type: Organic
- Physical state: liquid
- Purity: Greater than 99%.

Method

Target gene:
Histidine
Species / strain
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:
Controls
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

Results and discussion

Test results
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

Any other information on results incl. tables

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 --- --- --- --- --- --- ---

Applicant's summary and conclusion

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.