4,4'-bis(dimethylamino)-4''-(methylamino)trityl alcohol

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:

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

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:

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

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.