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Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

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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

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
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.