N,N-dimethyl-p-toluidine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The study study contains experimental data of the registered substance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Version / remarks:

Adopted: July 29 2016

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Test material information

Constituent 1

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

Specific details on test material used for the study:

Appearance: Clear light yellow colour oily liquid
Batch/ Lot Number: DMPT/22/10
Purity: 99.86%
Manufactured by: Industrial Solvents and Chemicals Pvt. Ltd.
Manufacturing date: February 2022
Expiry Date: January 2024
Storage condition: Room Temperature (20 to 30oC)

Method

Target gene:

Hprt gene

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

Cofactor-supplemented S9 liver microsomal fraction obtained from phenobarbital and β-naphthoflavone-injected rat.
The composition of S9 mix:
Glucose-6-phosphate (180 mg/ml): 1 ml
NADP (25 mg/ml): 1 ml
Potassium chloride (150 mM): 1 ml
S9 Fraction: 20ml
Final Volume: 5 ml
S9 Mix: 40 %
A volume of 2.5 ml S9 cofactor mix (40%) was added to 100 ml of culture medium to achieve 1 % v/v S9 in the culture medium.

Test concentrations with justification for top dose:

Test concentrations: 0 (NC), 0 (VC), 0.125, 0.25, 0.5, 1 mg/ml

Justification:
Test concentrations were selected based on the solubility, precipitation and pH checks and a preliminary cytotoxicity test. In the pre-test, CHO cells were exposed to the following test substance concentrations: 0 (NC), 0 (VC), 0.125, 0.25, 0.5, 1 and 2 mg/ml with and without S9 metabolic activation.Complete cytotoxicity was observed at the highest tested concentration, i.e. 2 mg/ml, both in the absence and presence of metabolic activation. At 1 mg/ml, the relative survival values were 15.49% and 13.79% in the absence and presence of S9 metabolic activation, respectively. Therefore, the main study was performed with the following test substance concentrations: 0 (NC), 0 (VC), 0.125, 0.25, 0.5 and 1 mg/ml and without metabolic activation.

Vehicle / solvent:

Dimethyl sulfoxide (DMSO)

Controls

Untreated negative controls:

yes

Remarks:

Distilled water

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): Triplicates were used.
- Number of independent experiments: One

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 10 × 106 cells /flask
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk: In medium; 5 ml of treatment media (RPMI 1640 media and 150 mM potassium chloride) and 50 µl of negative/vehicle/Test Item formulation/positive control were added to each respective flask.

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: NA
- Exposure duration/duration of treatment:4 hrs
- Harvest time after the end of treatment (sampling/recovery times): Expression period: 8 days, growing on selective medium: 8 days

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 8 days
- Selection time (if incubation with a selective agent): 8 hrs
- Fixation time (start of exposure up to fixation or harvest of cells): 16 days
- Method used: agar or microwell plates for the mouse lymphoma assay.
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure: 2x105cells / 10 ml of cloning media were seeded in the presence of 10 µg/ml of 6-thioguanine (6TG) in triplicate and incubated at 37±2 °C, 5 % CO2 in a CO2 incubator for 8 days for mutation frequency (MF) determination.

- Number of cells seeded and method to enumerate numbers of viable and mutants cells:
- Criteria for small (slow growing) and large (fast growing) colonies:

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 by the calculation of Relative survival (RS)
- Any supplementary information relevant to cytotoxicity:

METHODS FOR MEASUREMENTS OF GENOTOXICIY: Mutation Frequency (MF) was determined.

Evaluation criteria:

The test chemical was considered to be clearly positive if:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is concentration-related when evaluated with an appropriate trend test,
c) any of the results were outside the distribution of the laboratory negative control data.
When all of these criteria were met, the test chemical was then considered able to induce gene mutations in cultured mammalian cells in this test system.
The test chemical was considered clearly negative if:
a) none of the test concentrations exhibits a significant increase compared with the concurrent negative control,
b) all results are inside the distribution of the laboratory negative control data.
The test chemical was then considered unable to induce gene mutations in cultured mammalian cells in this test system.

Statistics:

Statistical analysis was performed to assess a possible dose-dependent increase of mutation frequency using Fisher’s Exact Test (NCSS statistics software). The mutation frequency of the Test Item-treated group was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Solubility and precipitation checks: The Substance was soluble up to 200 mg/ml in dimethyl sulfoxide. No precipitation was observed at the tested concentration of 200 mg/ml.
pH check: The pH values at the highest concentration of the Test Item in the medium were as follows:
Test Item
Concentration pH
0 hour 4th hour
R1 R2 Mean SD R1 R2 Mean SD
0 mg/ml 7.4 7.4 7.4 0.00 7.4 7.4 7.4 0.00
2 mg/ml 7.4 7.4 7.4 0.00 7.4 7.4 7.4 0.00

Preliminary cytotoxicity test: In the pre-test, CHO cells were exposed to the following test substance concentrations: 0 (NC), 0 (VC), 0.125, 0.25, 0.5, 1 and 2 mg/ml with and without S9 metabolic activation.Complete cytotoxicity was observed at the highest tested concentration, i.e. 2 mg/ml, both in the absence and presence of metabolic activation. At 1 mg/ml, the relative survival values were 15.49% and 13.79% in the absence and presence of S9 metabolic activation, respectively. Therefore, the main study was performed with the following test substance concentrations: 0 (NC), 0 (VC), 0.125, 0.25, 0.5 and 1 mg/ml and without metabolic activation.

Remarks on result:

other: Non-mutagenic

Any other information on results incl. tables

Appendix 1: Relative Survival – Preliminary Cytotoxicity Assay: Absence of metabolic activation

 

Dose  level	Concentration	No. of Cells		No. of colonies			Mean Colony count	No. of cells seeded	CE	Adjusted  CE	RS
		Before	After	R1	R2	R3
NC	Distilled water	20000000	23660000	242	228	265	245.00	100	2.450	2.898	100.00
VC	Dimethyl sulfoxide	20000000	23420000	232	232	240	234.67	100	2.347	2.748	94.81
T1	0.125 mg/ml	20000000	22400000	218	214	210	214.00	100	2.140	2.397	87.22
T2	0.25 mg/ml	20000000	21800000	205	211	201	205.67	100	2.057	2.242	81.58
T3	0.5 mg/ml	20000000	20400000	168	174	171	171.00	100	1.710	1.744	63.47
T4	1 mg/ml	20000000	18640000	51	41	45	45.67	100	0.457	0.426	15.49
T5	2 mg/ml	20000000	11200000	1	0	1	0.67	100	0.007	0.004	0.14

Key: NC = Negative Control, VC = Vehicle Control, T5 - T1= Test Item concentration from higher to lower, R = Replicate, CE = Cloning Efficiency, RS = Relative Survival, mg = milligram, ml = milliliter.

 

Appendix 2: Relative Survival – Preliminary Cytotoxicity Assay: Presence of metabolic activation

Dose  level	Concentration	No. of Cells		No. of colonies			Mean Colony count	No. of cells seeded	CE	Adjusted  CE	RS
		Before	After	R1	R2	R3
NC	Distilled water	20000000	23400000	235	242	230	235.67	100	2.357	2.757	100.00
VC	Distilled water	20000000	23160000	233	228	231	230.67	100	2.307	2.671	96.87
T1	0.125 mg/ml	20000000	22600000	204	196	208	202.67	100	2.027	2.290	85.74
T2	0.25 mg/ml	20000000	21460000	196	201	188	195.00	100	1.950	2.092	78.33
T3	0.5 mg/ml	20000000	19840000	155	161	168	161.33	100	1.613	1.600	59.92
T4	1 mg/ml	20000000	18120000	45	41	36	40.67	100	0.407	0.368	13.79
T5	2 mg/ml	20000000	9840000	0	0	1	0.33	100	0.003	0.002	0.06

Appendix 3: Relative Survival – Main Study: Absence of metabolic activation

Dose  level	Concentration	No. of Cells		No. of colonies			Mean Colony count	No. of cells seeded	CE	Adjusted  CE	RS
		Before	After	R1	R2	R3
NC	Distilled water	20000000	23600000	226	235	230	230.33	100	2.303	2.718	100.00
VC	Dimethyl sulfoxide	20000000	22800000	225	231	228	228.00	100	2.280	2.599	95.63
T1	0.125 mg/ml	20000000	21420000	214	224	214	217.33	100	2.173	2.328	89.55
T2	0.25 mg/ml	20000000	21420000	205	205	186	198.67	100	1.987	2.128	81.86
T3	0.5 mg/ml	20000000	20800000	165	162	155	160.67	100	1.607	1.671	64.29
T4	1 mg/ml	20000000	16620000	51	54	49	51.33	100	0.513	0.427	16.41
PC	400 µg/ml	20000000	19880000	225	236	231	230.67	100	2.307	2.293	88.21

 

Appendix 4: Relative Survival – Main Study: Presence of metabolic activation

Dose  level	Concentration	No. of Cells		No. of colonies			Mean Colony count	No. of cells seeded	CE	Adjusted  CE	RS
		Before	After	R1	R2	R3
NC	Distilled water	20000000	23820000	233	225	242	233.33	100	2.333	2.779	100.00
VC	Dimethyl sulfoxide	20000000	23280000	231	228	230	229.67	100	2.297	2.673	96.20
T1	0.125 mg/ml	20000000	21620000	211	215	209	211.67	100	2.117	2.288	85.59
T2	0.25 mg/ml	20000000	20880000	204	205	196	201.67	100	2.017	2.105	78.76
T3	0.5 mg/ml	20000000	18820000	170	158	162	163.33	100	1.633	1.537	57.49
T4	1 mg/ml	20000000	16280000	41	45	55	47.00	100	0.470	0.383	14.31
PC	30 µg/ml	20000000	19620000	228	217	226	223.67	100	2.237	2.194	82.08

Appendix 5: Cloning Efficiency (Non-selective medium) Main Study:  Absence of metabolic activation

Dose level	Non Selective medium
	Concentration	No. of cells  seeded	No. of colonies			Mean No. of colonies	CE
			R1	R2	R3
NC	Distilled water	100	211	208	199	206	2.06
VC	Dimethyl sulfoxide	100	202	189	194	195	1.95
T1	0.125 mg/ml	100	184	188	191	188	1.88
T2	0.25 mg/ml	100	174	169	184	176	1.76
T3	0.5 mg/ml	100	168	180	168	172	1.72
T4	1 mg/ml	100	166	168	165	166	1.66
PC	400 µg/ml	100	172	158	162	164	1.64

 

Appendix 6: Cloning Efficiency (Non-selective medium) Main Study:  Presence of metabolic activation

Dose level	Non Selective medium
	Concentration	No. of cells  seeded	No. of colonies			Mean No. of colonies	CE
			R1	R2	R3
NC	Distilled water	100	211	205	209	208	2.08
VC	Dimethyl sulfoxide	100	196	191	201	196	1.96
T1	0.125 mg/ml	100	196	211	210	206	2.06
T2	0.25 mg/ml	100	189	194	193	192	1.92
T3	0.5 mg/ml	100	178	168	178	175	1.75
T4	1 mg/ml	100	162	174	160	165	1.65
PC	30 µg/ml	100	180	175	193	183	1.83

 

Appendix 7: Cloning Efficiency (Selective medium): Absence of metabolic activation

Dose level	Selective medium
	Concentration	No. of cells  seeded	No. of colonies			Mean No. of colonies	CE
			R1	R2	R3
NC	Distilled water	200000	2	4	4	3.33	0.00001667
VC	Dimethyl sulfoxide	200000	4	3	3	3.33	0.00001667
T1	0.125 mg/ml	200000	4	2	4	3.33	0.00001667
T2	0.25 mg/ml	200000	3	3	4	3.33	0.00001667
T3	0.5 mg/ml	200000	4	4	5	4.33	0.00002167
T4	1 mg/ml	200000	4	5	2	3.67	0.00001833
PC	400 µg/ml	200000	86	80	74	80.00	0.00040000

Appendix 8: Cloning Efficiency (Selective medium) Phase I:  Presence of metabolic activation

 

Dose level	Selective medium
	Concentration	No. of cells  seeded	No. of colonies			Mean No. of colonies	CE
			R1	R2	R3
NC	Distilled water	200000	4	3	4	3.67	0.00001833
VC	Dimethyl sulfoxide	200000	2	5	2	3.00	0.00001500
T1	0.125 mg/ml	200000	2	4	4	3.33	0.00001667
T2	0.25 mg/ml	200000	3	3	3	3.00	0.00001500
T3	0.5 mg/ml	200000	4	4	4	4.00	0.00002000
T4	1 mg/ml	200000	4	4	5	4.33	0.00002167
PC	30 µg/ml	200000	92	84	96	90.67	0.00045333

Appendix 9: Mutation Frequency: Absence of metabolic activation

Dose level	Absence of metabolic activation
	Concentration	Mutation Frequency	MF x 10^-6
NC	Distilled water	0.00000809	8.09
VC	Dimethyl sulfoxide	0.00000855	8.55
T1	0.125 mg/ml	0.00000888	8.88
T2	0.25 mg/ml	0.00000949	9.49
T3	0.5 mg/ml	0.00001260	12.60
T4	1 mg/ml	0.00001102	11.02
PC	400 µg/ml*	0.00024390	243.90

Dose level	Presence of metabolic activation
	Concentration	Mutation Frequency	MF x 10^-6
NC	Distilled water	0.00000880	8.80
VC	Dimethyl sulfoxide	0.00000765	7.65
T1	0.125 mg/ml	0.00000810	8.10
T2	0.25 mg/ml	0.00000781	7.81
T3	0.5 mg/ml	0.00001145	11.45
T4	1 mg/ml	0.00001310	13.10
PC	30 µg/ml*	0.00024818	248.18

Key: NC = Negative Control, VC = Vehicle Control, PC = Positive Control (Absence of metabolic activation- Ethylmethanesulfonate, Presence of metabolic activation- Benzo[a]pyrene), T4-T1= Test item concentration from higher to lower, MF = Mutation Frequency, mg = milligram, µg =microgram, ml = milliliter, * = Stastically significant increse in mutation frequency.

 

Annexure 3: Historical Control Data

Mutation Frequency (10^-6)	NC		VC		PC
	-S9	+S9	-S9	+S9	-S9	+S9
Mean	7.08	6.97	7.71	7.92	222.00	229.09
SD	0.72	0.91	0.97	0.60	16.65	12.33
Min	6.09	6.02	6.35	6.51	197.57	209.32
Max	8.21	8.43	8.9	8.7	236.11	242.6

Key: - NC = Negative Control (Distilled water), VC = Vehicle Control (Dimethyl sulfoxide), PC = Positive Control (Absence of metabolic activation- Ethylmethanesulfonate, Presence of metabolic activation- Benzo[a]pyrene), SD = Standard Deviation,Min = Minimum, Max = Maximum, - S9 = Absence of metabolic activation, +S9 = Presence of metabolic activation. Number of studies = 09.

Applicant's summary and conclusion

Conclusions:

The registered substance, 4-Dimethylaminotoluene (CAS number: 99-97-8), tested non-mutagenic (negative) in cultured Chinese Hamster Ovary (CHO) cells both in the presence and absence of S9 metabolic activation.

Executive summary:

The potential of 4-Dimethylaminotoluene (CAS number: 99-97-8) to induce gene mutation at the hypoxanthine-guanine phosphoribosyltransferase (Hprt) locus in cultured Chinese Hamster Ovary (CHO) cells was tested in the presence and absence of S9 metabolic activation system and according to OECD TG 476. Cofactor-supplemented liver S9 microsomal fraction, derived from phenobarbital and β-naphthoflavone-injected rat, were used. Dimethyl sulfoxide was used as a vehicle for the test substance. The cytotoxicity of the test substance was assessed in a preliminary cytotoxicity assay. In this pre-test, CHO cells were exposed to the following test substance concentrations: 0 (NC), 0 (VC), 0.125, 0.25, 0.5, 1 and 2 mg/ml with and without S9 metabolic activation. Cytotoxicity was determined by the calculation of Relative survival (RS). Complete cytotoxicity was observed at 2 mg/ml in the absence and presence of metabolic activation. At 1 mg/ml, the RS values were 15.49% and 13.79% in the absence and presence of S9 metabolic activation, respectively. Therefore, the main study was performed with the following concentrations: 0 (NC), 0 (VC), 0.125, 0.25, 0.5 and 1 mg/ml. In the main study, cultures were exposed to the negative control (Distilled water), vehicle control (DMSO), different concentrations of the test item, and positive controls (Ethylmethanesulfonate and Beno[a]pyrene) for 4 hours in the absence and presence of metabolic activation. Result: In the absence of metabolic activation, the RS values were 100% (negative control), 95.63% (vehicle control), 89.55% (at 0.125 mg/ml), 81.86% (at 0.25 mg/ml), 64.29% (at 0.5 mg/ml), 16.41% (at 1 mg/ml) and 88.21% (at 400 µg/ml-positive control [Ehtylmethanesulfonate]). In the presence of metabolic activation, the RS values were 100% (negative control), 96.20% (vehicle control), 85.59% (at 0.125 mg/ml), 78.76% (at 0.25 mg/ml), 57.49% (at 0.5 mg/ml) 14.31% (at 1 mg/ml) and 82.08% (30 µg/ml-positive control[Benzo[a]pyrene]). No significant increase in the mutation frequency (MF) either in absence (8.88x10^-6, 9.49 x10^-6, 12.60 x10^-6 and 11.02 x10^-6 at 0.125 mg/ml, 0.25 mg/ml, 0.5 mg/ml and 1 mg/ml, respectively) or presence of metabolic activation  (8.10 x10^-6, 7.81x10^-6, 11.45x10^-6 and 13.10x10^-6 at 0.125 mg/ml, 0.25 mg/ml, 0.5 mg/ml and 1 mg/ml, respectively) was observed when compared to vehicle control (8.73 x10^-6, 8.00 x10^-6, absence and presence of S9, respectively). The positive controls (Ethylmethanesulfonate and Beno[a]pyrene in the absence and presence of metabolic activation, respectively) produced statistically significant increases in mutation frequency (243.90 x10^-6, p<0.0001 [Ethylmethanesulfonate], 248.18 x10^-6, p<0.0001 [Benzo(a)pyrene] in the absence and presence of metabolic activation, respectively). Conclusion: The registered substance (CAS number: 99-97-8) did not induce a statistically significant or biologically relevant increase in the mutation frequency at concentrations of 0.125, 0.25, 0.5 and 1 mg/ml when compared to the vehicle control either in the presence or in the absence of S9 metabolic activation.