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EC number: 209-218-2 | CAS number: 561-41-1
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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)
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 022
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
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Test material
- Reference substance name:
- 4,4'-bis(dimethylamino)-4''-(methylamino)trityl alcohol
- EC Number:
- 209-218-2
- EC Name:
- 4,4'-bis(dimethylamino)-4''-(methylamino)trityl alcohol
- Cas Number:
- 561-41-1
- Molecular formula:
- C24-H29-N3-O
- IUPAC Name:
- bis[4-(dimethylamino)phenyl][4-(methylamino)phenyl]methanol
Constituent 1
- Specific details on test material used for the study:
- Appearance: Violet Powder
Lot /Batch Number: KCP/FS/199/21
Purity/AI content :99.80%
Manufacturer Name: K.Patel Chemo Pharma Private Limited
Manufactured date: 26.05.2021
Expiry Date: 25.11.2022
Storage condition: Room Temperature (20 to 30oC)
Safety precautions: Standard safety precautions (gloves, mask, apron, head cap and goggles).
Method
- Target gene:
- hypoxanthine-guanine phosphoribosyltransferase (Hprt)
Species / strain
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- Source: NCCS, Pune, India
- Metabolic activation:
- with and without
- Metabolic activation system:
- Cofactor-supplemented liver S9 microsomal fraction was used as an exogenous metabolic activation system. The S9 microsomal (S9 homogenate) fraction derived from the liver of a phenobarbitone and β-naphthoflavone-injected rat.
S9 mixture:
Glucose-6-phosphate (180 mg/ml): 1 ml
NADP (25 mg/ml): 1ml
Potassium chloride (150 mM): 1ml
S9 Fraction: 2 ml
Final Volume (ml): 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:
0.0 (NC)
0.0(VC)
0.78 ug/ml
1.56 ug/ml
3.13 ug/ml
6.25 ug/ml
Justification:
The test concentrations were selected based on preliminary cytotoxicity tests I-II. Cytotoxicity was determined by calculating the Relative Survival (RS) values at each concentration. The test substance concentration which induced RS 10-20% was selected as the highest test concentration for this assay. In the preliminary cytotoxicity study, limiting cytotoxicity, i.e. >16% RS, was observed at 0.00625mg/ml both in the presence and absence of metabolic activation. Therefore, 0.00625 mg/ml was selected as the maximum test concentration used in the main study and three lower concentrations were included using spacing factor 2. - 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): Single culture per concnetration was used.
- Number of independent experiments: 1
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 10 x 106 cells/25 cm2
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk: in medium
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable:NA
- Exposure duration/duration of treatment: 4 hours
- Harvest time after the end of treatment (sampling/recovery times): Expression time: 8 days, plating for Mutation frequency: 10 days
FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 8 days
- Selection time (if incubation with a selective agent): 10 days
- Fixation time (start of exposure up to fixation or harvest of cells): ca 19 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: 10 µg/ml of 6-thioguanine (6TG) was added and incubated at 37±2 °C, 5 % CO2 in a CO2 incubator for 10 days.
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: Cloning efficiency (CE). At the end of the expression period, cells were trypsinised and counted. Cells were diluted to attain 100 cells /10 ml of cloning media and then plated in 60 mm culture plates in triplicate. The plates were incubated at 37±2 °C, 5 % CO2, in a CO2 incubator for 10 days.
- Criteria for small (slow growing) and large (fast growing) colonies: No
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 measured by calculating the Relative Survival (RS) at each concentration.
- Any supplementary information relevant to cytotoxicity:
METHODS FOR MEASUREMENTS OF GENOTOXICIY: Genotoxicity was demonstrated by calculating the Mutation frequency (MF) for each test concentration. - Evaluation criteria:
- The test chemical was considered to be clearly positive if in any of the experimental conditions examined:
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 are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.
The test chemical was considered clearly negative if, in all experimental conditions examined:
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 is 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 in 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 was judged as significant whenever the p-value (probability value) was 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
- Remarks:
- At 6.25 ug/ml, the RS values were 18.75% and 17.88% in the absence and presence of S9 metabolic activation system, respectively.
- 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 and pH checks:
The was found to be soluble in dimethyl sulfoxide up to 200 mg/ml. No precipitation was observed at the tested concentration of 200 mg/ml. The pH values at the highest concentration of the Test Item in the medium were not altered after 0 and 4 hours of incubation.
Preliminary cytotoxicity test:
The test concentrations were selected based on preliminary cytotoxicity tests I-II. In the preliminary cytotoxicity test I, CHO cells were exposed to 0.0 (Distilled water), 0.0 (DMSO), 0.125, 0.25, 0.5, 1 and 2 mg/ml of test substance in the culture medium, both in the presence and absence of S9 metabolic activation system (1 % v/v S9 mix). After the 4-hour treatment, complete toxicity was observed at all the tested concentrations in the absence and presence of metabolic activation; therefore, an additional cytotoxicity assay was performed. The preliminary cytotoxicity test-II was performed with the following test concentrations: 0.0 (NC), 0.0 (VC), 0.00625, 0.0125, 0.025, 0.05 and 0.1 mg/ml in the culture medium, both in the presence and absence of metabolic activation system (1 % v/v S9 mix). Complete cytotoxicity was observed within the concentration range of 0.0125 - 2 mg/ml, both in the absence and presence of metabolic activation. Whereas moderate cytotoxicity (>16% Relative Survival [RS is cloning efficiency (CE) of cells plated immediately after treatment adjusted by any loss of cells during treatment as compared with cloning efficiency in vehicle control]) was noted at 0.00625 mg/ml either in the absence (RS: 18.24%) or presence (RS: 16.25%) of metabolic activation. - Remarks on result:
- other: Non-mutagenic
Any other information on results incl. tables
Appendix 1: Relative Survival – Preliminary Cytotoxicity Assay-I: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 | 23400000 | - | - | - | - | - | - | - | - |
VC | Dimethyl sulfoxide | 20000000 | 23200000 | - | - | - | - | - | - | - | - |
T1 | 0.125 mg/ml | 20000000 | 0 | - | - | - | - | - | - | - | - |
T2 | 0.25 mg/ml | 20000000 | 0 | - | - | - | - | - | - | - | - |
T3 | 0.5 mg/ml | 20000000 | 0 | - | - | - | - | - | - | - | - |
T4 | 1 mg/ml | 20000000 | 0 | - | - | - | - | - | - | - | - |
T5 | 2 mg/ml | 20000000 | 0 | - | - | - | - | - | - | - | - |
Appendix 2: Relative Survival – Preliminary Cytotoxicity Assay-I: 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 | 23600000 | - | - | - | - | - | - | - | - |
VC | Distilled water | 20000000 | 23400000 | - | - | - | - | - | - | - | - |
T1 | 0.125 mg/ml | 20000000 | 0 | - | - | - | - | - | - | - | - |
T2 | 0.25 mg/ml | 20000000 | 0 | - | - | - | - | - | - | - | - |
T3 | 0.5 mg/ml | 20000000 | 0 | - | - | - | - | - | - | - | - |
T4 | 1 mg/ml | 20000000 | 0 | - | - | - | - | - | - | - | - |
T5 | 2 mg/ml | 20000000 | 0 | - | - | - | - | - | - | - | - |
Appendix 3: Preliminary Cytotoxicity Assay-II: 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 | 23700000 | 231 | 235 | 230 | 232.00 | 100 | 2.320 | 2.749 | 100.00 |
VC | Dimethyl sulfoxide | 20000000 | 23500000 | 236 | 228 | 229 | 231.00 | 100 | 2.310 | 2.714 | 98.73 |
T1 | 0.00625 mg/ml | 20000000 | 18800000 | 52 | 55 | 51 | 52.67 | 100 | 0.527 | 0.495 | 18.24 |
T2 | 0.0125 mg/ml | 20000000 | 6800000 | 4 | 10 | 5 | 6.33 | 100 | 0.063 | 0.022 | 0.79 |
T3 | 0.025 mg/ml | 20000000 | 0 | - | - | - | - | - | - | - | - |
T4 | 0.05 mg/ml | 20000000 | 0 | - | - | - | - | - | - | - | - |
T5 | 0.1 mg/ml | 20000000 | 0 | - | - | - | - | - | - | - | - |
Appendix 4: Relative Survival – Preliminary Cytotoxicity Assay -II: 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 | 23600000 | 233 | 230 | 226 | 229.67 | 100 | 2.297 | 2.710 | 100.00 |
VC | Dimethyl sulfoxide | 20000000 | 23400000 | 218 | 216 | 220 | 218.00 | 100 | 2.180 | 2.551 | 94.12 |
T1 | 0.00625 mg/ml | 20000000 | 16800000 | 46 | 48 | 54 | 49.33 | 100 | 0.493 | 0.414 | 16.25 |
T2 | 0.0125 mg/ml | 20000000 | 5400000 | 8 | 3 | 4 | 5.00 | 100 | 0.050 | 0.014 | 0.53 |
T3 | 0.025 mg/ml | 20000000 | 0 | - | - | - | - | - | - | - | - |
T4 | 0.05 mg/ml | 20000000 | 0 | - | - | - | - | - | - | - | - |
T5 | 0.1 mg/ml | 20000000 | 0 | - | - | - | - | - | - | - | - |
Appendix 5: 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 | 230 | 236 | 231 | 232.33 | 100 | 2.323 | 2.742 | 100.00 |
VC | Dimethyl sulfoxide | 20000000 | 23400000 | 228 | 218 | 225 | 223.67 | 100 | 2.237 | 2.617 | 95.45 |
T1 | 0.78 µg/ml | 20000000 | 22800000 | 179 | 182 | 186 | 182.33 | 100 | 1.823 | 2.079 | 79.43 |
T2 | 1.56 µg/ml | 20000000 | 21800000 | 158 | 168 | 155 | 160.33 | 100 | 1.603 | 1.748 | 66.78 |
T3 | 3.13 µg/ml | 20000000 | 19800000 | 115 | 126 | 128 | 123.00 | 100 | 1.230 | 1.218 | 46.53 |
T4 | 6.25 µg/ml | 20000000 | 18400000 | 58 | 50 | 52 | 53.33 | 100 | 0.533 | 0.491 | 18.75 |
PC | 400 µg/ml | 20000000 | 18600000 | 198 | 226 | 219 | 214.33 | 100 | 2.143 | 1.993 | 76.17 |
Appendix 6: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 | 23500000 | 226 | 235 | 232 | 231.00 | 100 | 2.310 | 2.714 | 100.00 |
VC | Dimethyl sulfoxide | 20000000 | 23300000 | 220 | 226 | 231 | 225.67 | 100 | 2.257 | 2.629 | 96.86 |
T1 | 0.78 µg/ml | 20000000 | 22600000 | 186 | 191 | 194 | 190.33 | 100 | 1.903 | 2.151 | 81.81 |
T2 | 1.56 µg/ml | 20000000 | 21600000 | 160 | 171 | 166 | 165.67 | 100 | 1.657 | 1.789 | 68.06 |
T3 | 3.13 µg/ml | 20000000 | 19800000 | 128 | 120 | 124 | 124.00 | 100 | 1.240 | 1.228 | 46.69 |
T4 | 6.25 µg/ml | 20000000 | 16300000 | 60 | 54 | 59 | 57.67 | 100 | 0.577 | 0.470 | 17.88 |
PC | 30 µg/ml | 20000000 | 18800000 | 218 | 214 | 225 | 219.00 | 100 | 2.190 | 2.059 | 78.30 |
Appendix 7: 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 | 219 | 226 | 224 | 223 | 2.23 |
VC | Dimethyl sulfoxide | 100 | 210 | 216 | 211 | 212 | 2.12 |
T1 | 0.78 µg/ml | 100 | 195 | 190 | 190 | 192 | 1.92 |
T2 | 1.56 µg/ml | 100 | 186 | 171 | 177 | 178 | 1.78 |
T3 | 3.13 µg/ml | 100 | 158 | 166 | 169 | 164 | 1.64 |
T4 | 6.25 µg/ml | 100 | 128 | 139 | 133 | 133 | 1.33 |
PC | 400 µg/ml | 100 | 192 | 190 | 186 | 189 | 1.89 |
Appendix 8: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 | 224 | 218 | 224 | 222 | 2.22 |
VC | Dimethyl sulfoxide | 100 | 220 | 218 | 216 | 218 | 2.18 |
T1 | 0.78 µg/ml | 100 | 193 | 191 | 186 | 190 | 1.90 |
T2 | 1.56 µg/ml | 100 | 178 | 177 | 170 | 175 | 1.75 |
T3 | 3.13 µg/ml | 100 | 160 | 166 | 164 | 163 | 1.63 |
T4 | 6.25 µg/ml | 100 | 128 | 130 | 121 | 126 | 1.26 |
PC | 30 µg/ml | 100 | 183 | 190 | 182 | 185 | 1.85 |
Appendix 9: 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 | 3 | 3 | 2.67 | 0.00001333 |
VC | Dimethyl sulfoxide | 200000 | 4 | 2 | 3 | 3.00 | 0.00001500 |
T1 | 0.78 µg/ml | 200000 | 4 | 4 | 3 | 3.67 | 0.00001833 |
T2 | 1.56 µg/ml | 200000 | 3 | 2 | 5 | 3.33 | 0.00001667 |
T3 | 3.13 µg/ml | 200000 | 4 | 4 | 5 | 4.33 | 0.00002167 |
T4 | 6.25 µg/ml | 200000 | 2 | 4 | 4 | 3.33 | 0.00001667 |
PC | 400 µg/ml | 200000 | 90 | 89 | 84 | 87.67 | 0.00043833 |
Appendix 10: 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 | 2 | 2 | 2.67 | 0.00001333 |
VC | Dimethyl sulfoxide | 200000 | 3 | 5 | 3 | 3.67 | 0.00001833 |
T1 | 0.78 µg/ml | 200000 | 2 | 4 | 4 | 3.33 | 0.00001667 |
T2 | 1.56 µg/ml | 200000 | 5 | 3 | 3 | 3.67 | 0.00001833 |
T3 | 3.13 µg/ml | 200000 | 4 | 3 | 3 | 3.33 | 0.00001667 |
T4 | 6.25 µg/ml | 200000 | 4 | 2 | 4 | 3.33 | 0.00001667 |
PC | 30 µg/ml | 200000 | 79 | 88 | 84 | 83.67 | 0.00041833 |
Appendix 11: Mutation Frequency: Absence of metabolic activation
Dose level | Absence of metabolic activation | ||
Concentration | Mutation Frequency | MF x 10-6 | |
NC | Distilled water | 0.00000598 | 5.98 |
VC | Dimethyl sulfoxide | 0.00000706 | 7.06 |
T1 | 0.78 µg/ml | 0.00000957 | 9.57 |
T2 | 1.56 µg/ml | 0.00000936 | 9.36 |
T3 | 3.13 µg/ml | 0.00001318 | 13.18 |
T4 | 6.25 µg/ml | 0.00001250 | 12.50 |
PC | 400 µg/ml* | 0.00023151 | 231.51 |
Dose level | Presence of metabolic activation | ||
Concentration | Mutation Frequency | MF x 10-6 | |
NC | Distilled water | 0.00000601 | 6.01 |
VC | Dimethyl sulfoxide | 0.00000841 | 8.41 |
T1 | 0.78 µg/ml | 0.00000877 | 8.77 |
T2 | 1.56 µg/ml | 0.00001048 | 10.48 |
T3 | 3.13 µg/ml | 0.00001020 | 10.20 |
T4 | 6.25 µg/ml | 0.00001319 | 13.19 |
PC | 30 µg/ml* | 0.00022613 | 226.13 |
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, µg =microgram, ml = millilitre, * = Statistically significant increase in mutation frequency.
Annexure 1: Historical control data
Mutation Frequency (10-6) | NC | VC | PC |
| ||||
-S9 | +S9 | -S9 | +S9 | -S9 | +S9 |
| ||
Mean | 7.45 | 7.33 | 7.78 | 7.92 | 229.63 | 236.21 |
| |
SD | 0.84 | 0.98 | 0.86 | 0.52 | 20.72 | 17.21 |
| |
Min | 6.09 | 6.02 | 6.35 | 6.51 | 197.57 | 209.32 |
| |
Max | 8.51 | 8.8 | 8.9 | 8.7 | 269 | 268.63 |
| |
|
|
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 = 12.
Applicant's summary and conclusion
- Conclusions:
- The registered substance, i.e., 4,4’-bis(dimethylamino)-4”-(methylamino)trityl alcohol [CAS No.: 561-41-1], did not induce gene mutation at the locus of hypoxanthine-guanine phosphoribosyltransferase (Hprt) in CHO cells up to the concentration of 6.25 µg/ml of culture medium, either in the presence or absence of S9 metabolic activation system. The test was performed according to the OECD TG 476 and in compliance with the OECD Principles of Good Laboratory Practice.
- Executive summary:
The potential of 4,4’-bis(dimethylamino)-4”-(methylamino)trityl alcohol [CAS No.: 561-41-1], 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 an exogenous metabolic activation system. The study was performed as per OECD Test Guideline No. 476 (Adopted: July 29 2016). Cofactor-supplemented liver S9 microsomal fraction was used as an exogenous metabolic activation system. The S9 microsomal (S9 homogenate) fraction derived from the liver of a phenobarbitone and β-naphthoflavone-injected rat. The test substance was soluble in dimethyl sulfoxide (DMSO) at 200 mg/ml; therefore, DMSO was selected as the vehicle of the substance in this assay. The test concentrations were selected based on preliminary cytotoxicity tests I-II. In the initial cytotoxicity test I, CHO cells were exposed to 0.0 (Distilled water), 0.0 (DMSO), 0.125, 0.25, 0.5, 1 and 2 mg/ml of test substance in the culture medium, both in the presence and absence of S9 metabolic activation system (1 % v/v S9 mix). After the 4-hour treatment, complete toxicity was observed at all the tested concentrations in the absence and presence of metabolic activation; therefore, an additional cytotoxicity assay was performed. The preliminary cytotoxicity test-II was performed with the following test concentrations: 0.0 (NC), 0.0 (VC), 0.00625, 0.0125, 0.025, 0.05 and 0.1 mg/ml in the culture medium, both in the presence and absence of metabolic activation system (1 % v/v S9 mix). Complete cytotoxicity was observed within the concentration range of 0.0125 - 2 mg/ml, both in the absence and presence of metabolic activation. Moderate cytotoxicity (>16% Relative Survival [RS is cloning efficiency (CE) of cells plated immediately after treatment adjusted by any loss of cells during treatment as compared with cloning efficiency in vehicle control]) was noted at 0.00625 mg/ml either in the absence (RS: 18.24%) or presence (RS: 16.25%) of metabolic activation. Hence, the gene mutation study was conducted with Test Item concentrations of 0.78, 1.56, 3.13 and 6.25 µg/ml, both in the presence (1 % v/v S9 mix) and absence of metabolic activation system along with negative (distilled water), vehicle (DMSO) and positive controls (Ethylmethanesulfonate: 400 µg/ml in the absence of S9 metabolic activation, Benzo (a) pyrene: 30 µg/ml in the presence of S9 metabolic activation). Results: In the main study, cultures were exposed to the negative control, vehicle control, different concentrations of the Test Item, and positive control for 4 hours (short-term exposure) in the absence and presence of metabolic activation. In the absence of metabolic activation, the Relative Survival values were 100% (negative control), 95.45% (vehicle control), 79.43% (at 0.78 µg/ml), 66.78% (at 1.56 µg/ml), 46.53% (at 3.13 µg/ml), 18.75% (at 6.25 µg/ml) and 76.17% (at 400 µg/ml-positive control [Ehtylmethanesulfonate]). In the presence of metabolic activation, the relative survival values were 100% (negative control), 96.86% (vehicle control), 81.81% (at 0.78 µg/ml), 68.06% (at 1.56 µg/ml), 46.69% (at 3.13 µg/ml) 17.88% (at 6.25 µg/ml) and 78.30% (30 µg/ml-positive control[Benzo[a]pyrene]). No significant increase in the mutation frequency (MF) either in the absence (9.57 x 10-6, 9.36 x 10-6, 13.18 x 10-6 and 12.50 x 10-6 at 0.78 µg/ml, 1.56 µg/ml, 3.13 µg/ml and 6.25 µg/ml, respectively) or presence of metabolic activation (8.77 x 10-6, 10.4 8 x 10-6, 10.20 x10-6 and 13.19 x10-6 at 0.78 µg/ml, 1.56 µg/ml, 3.13 µg/ml and 6.25 µg/ml, respectively) was observed when compared to vehicle control (5.98 x10-6, 6.01 x10-6, absence and presence of S9, respectively).
The dose concentrations of the Test Item in dose formulation were analysed by a validated test method for all concentrations, and the observed concentrations were within the acceptable limit of linearity (R) ≥0.99, % recovery 70-110% and % RSD ≤ 10%. The positive controls (Ethylmethanesulfonate and Beno[a]pyrene in the absence and presence of metabolic activation, respectively) used in the study produced statistically significant increases in mutation frequency (231.51 x10-6, p<0.0001 [Ethylmethanesulfonate], 226.13 x10-6, p<0.0001 [Benzo(a)pyrene] in the absence and presence of metabolic activation, respectively) indicating the sensitivity of the test system to specific mutagens, and confirmed that the test conditions were appropriate and that the metabolic activation system functioned properly. Conclusion: The registered substance, 4,4’-bis(dimethylamino)-4”-(methylamino)trityl alcohol (CAS No.: 561-41-1), did not induce a statistically significant or biologically relevant increase in the mutation frequency up to 6.26 µg/ml when compared to the vehicle control either in the presence or in the absence of S9 metabolic activation.
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