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REACH

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

EC number: 209-218-2 | CAS number: 561-41-1

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation test (OECD 471):

The registered substance, 4,4’-bis(dimethylamino)-4”-(methylamino) trityl alcohol (CAS 561-41-1), tested non-mutagenic (negative) in Salmonella Typhimurium TA98, TA100, TA1535, TA1537 and TA102 strains in the presence and absence of cofactor-supplemented liver S9 microsomal metabolic activation system. The test was performed according to OECD TG 471 and in compliance with the OECD Principles of Good Laboratory Practice.

In vitro mammalian chromosome aberration test (OECD 473):

The registered substance, 4,4’bis(dimethylamino)-4”-(methylamino)trityl alcohol [CAS No.: 561-41-1], did not induce chromosomal aberration up to the highest concentration of 0.00625 mg/ml of culture medium, either in the presence or absence of S9 metabolic activation system in CHO cells. The test was performed according to OECD TG 473 and in compliance with the OECD Principles of Good Laboratory Practice.

In vitro gene mutation test in mammalian cells:

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, 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.

Link to relevant study records

Referenceopen all close all

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

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

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).

Target gene:

hypoxanthine-guanine phosphoribosyltransferase (Hprt)

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)

Untreated negative controls:

yes

Remarks:

Distilled water

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

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.

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

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
Dose level	Concentration	Before	After	R1	R2	R3	Mean Colony count	No. of cells seeded	CE	Adjusted CE	RS
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
Dose level	Concentration	Before	After	R1	R2	R3	Mean Colony count	No. of cells seeded	CE	Adjusted CE	RS
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
Dose level	Concentration	Before	After	R1	R2	R3	Mean Colony count	No. of cells seeded	CE	Adjusted CE	RS
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
Dose level	Concentration	Before	After	R1	R2	R3	Mean Colony count	No. of cells seeded	CE	Adjusted CE	RS
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
Dose level	Concentration	Before	After	R1	R2	R3	Mean Colony count	No. of cells seeded	CE	Adjusted CE	RS
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
Dose level	Concentration	Before	After	R1	R2	R3	Mean Colony count	No. of cells seeded	CE	Adjusted CE	RS
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
	Concentration	No. of cells seeded	R1	R2	R3	Mean No. of colonies	CE
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
	Concentration	No. of cells seeded	R1	R2	R3	Mean No. of colonies	CE
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
	Concentration	No. of cells seeded	R1	R2	R3	Mean No. of colonies	CE
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
	Concentration	No. of cells seeded	R1	R2	R3	Mean No. of colonies	CE
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
Dose level	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
Dose level	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
Mutation Frequency (10^-6)	-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.

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^-6at 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.

Reference 2

Endpoint:: in vitro cytogenicity / chromosome aberration 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 contains experimental data of the registered substance.
Qualifier:: according to guideline
Guideline:: OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)
Version / remarks:: 29 July 2016
GLP compliance:: yes
Type of assay:: in vitro mammalian chromosome aberration test
Specific details on test material used for the study:: Appearance: Violet powder
Batch Number/Lot Number: KCP/FS/199/21
Purity / AI Content: 98.81%
Manufactured by: K. Patel Chemopharma Pvt. Ltd.
Manufacturing date: 26.05.2021
Expiry Date: 25.05.2022
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.
Test concentrations with justification for top dose:: Test concentrations:
0.0 (NC), 0.0 (VC), 0.0015625, 0.003125, 0.00625 mg/ml

Justification:
The cytotoxicity was evaluated based on the reduction in the RICC. A Test Item concentration causing approximately 45±5% reduction in RICC compared to that of the concurrent vehicle control (i.e. 55±5% cytotoxicity) was selected as the highest test concentration for the chromosome aberration assay. At 0.00625 mg/ml, RICC decreased to 55±5% compared to vehicle control both in the presence and absence of metabolic activation; RICC values were 46.86% (cytotoxicity: 53.14% and 42,69% (cytotoxicity: 57.31%) in the absence and presence of S9 metabolic activation system, respectively.
Vehicle / solvent:: dimethyl sulfoxide
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; methylmethanesulfonate
Details on test system and experimental conditions:: NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): Single cultures were used in Phase I- III.
- Number of independent experiments: 3 (Phase I, Phase II and Phase III)
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1 x 106 cells/25 cm2
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk: Test substance added in medium.
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable:
- Exposure duration/duration of treatment: 4 hours (Phase I-II), 24 hrs (Phase III)
- Harvest time after the end of treatment (sampling/recovery times): The cultures were harvested 24 hours after the beginning of treatment.
FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): indicate the identity of mitotic spindle inhibitor used (e.g., colchicine), its concentration and, duration and period of cell exposure: Two hours prior to harvesting, a volume of 50 µl from 0.1 mg/ml of colchicine stock was added to culture flask to achieve a final concentration of 1 µg/ml and incubated at 37 ±2 °C, 5 % CO2 in a CO2 incubator.
- If cytokinesis blocked method was used for micronucleus assay: indicate the identity of cytokinesis blocking substance (e.g. cytoB), its concentration, and duration and period of cell exposure: No
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): Slides were stained with freshly prepared 5 % Giemsa stain for 5 minutes and rinsed in distilled water for 2 minutes. After drying, slides were mounted using DPX mountant.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): At least 300 well-spread metaphases per concentration (single culture) were analysed using 100x magnification for the incidence of structural aberrations.
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification):
- Methods, such as kinetochore antibody binding, to characterise whether micronuclei contain whole or fragmented chromosomes (if applicable): No
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): As slide preparation procedures often result in the breakage of a proportion of metaphases with loss of chromosomes, cells that contain the number of centromeres equal to the number 2n ± 2 were scored. In CHO cells, the number of centromeres equal to the modal number is 22 ± 2.
- Determination of polyploidy:
- Determination of endoreplication:
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: Relative increase in cell count (RICC) was calculated.
- Any supplementary information relevant to cytotoxicity:
Evaluation criteria:: Test Item was considered to be clearly positive if, in any of the experimental conditions examined:
 At least one of the test concentrations exhibits a significant increase compared with the concurrent negative control,
 The increase is dose-related when evaluated with an appropriate trend test,
 Any of the results are outside of the distribution of the laboratory historical negative control database
Test Item was considered clearly negative if, in all experimental conditions examined:
 None of the test concentrations exhibits a significant increase compared with the concurrent negative control.
 There is no concentration-related increase when evaluated with an appropriate trend test.
 The results are inside the distribution of the laboratory historical negative control database.
Statistics:: Statistical analysis was performed to assess a possible dose-dependent increase of aberrant cell frequencies using Fisher’s Exact Test (NCSS statistics software). The percentage of aberrant cells from 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.
: Key result
Species / strain:: Chinese hamster Ovary (CHO)
Metabolic activation:: with and without
Genotoxicity:: negative
Cytotoxicity / choice of top concentrations:: cytotoxicity
Remarks:: At 0.00625 mg/ml, cytotoxicity was 54.15% (RICC: 45.85%), 58.62 (RICC: 41.38%) and 58.37% (RICC: 41.63) in Phase I, Phase II and Phase III experiments, respectively.
Vehicle controls validity:: valid
Untreated negative controls validity:: valid
True negative controls validity:: not specified
Positive controls validity:: valid
Additional information on results:: Solubility, precipitation, and pH checks:
The Test Item was found to be soluble in dimethyl sulfoxide at 200 mg/ml concentration. No precipitation was observed at the concentration of 2 mg/ml. The pH of the test concentrations was unaltered after 0 and 4 hours of incubation of the test item in the culture medium.
Preliminary cytotoxicity tests:
In the preliminary cytotoxicity test I, CHO cells were exposed to 0.0 (NC), 0.0 (VC), 0.125, 0.25, 0.5, 1 and 2 mg/ml of the test substance both in the presence and absence of S9 metabolic activation (1 % v/v S9 mix). Excessive cytotoxicity (defined by a Relative Increase in Cell Count [RICC] of <40% of the concurrent vehicle control data) was observed for the Test Item concentrations at ≥0.125 mg/ml, with and without S9 metabolic activation. In the preliminary cytotoxicity assay-II, CHO cells were treated with 0.0 (NC), 0.0 (VC), 0.0625, 0.0125, 0.025, 0.05 and 0.1 mg/ml of culture medium, both in the presence (1 % v/v S9 mix) and absence of metabolic activation system. In this second cytotoxicity assay, excessive cytotoxicity (defined by a Relative Increase in Cell Count [RICC] of <40% of the concurrent vehicle control data) was observed for the Test Item concentrations at ≥0.0125 mg/ml, both in the presence and absence of metabolic activation. At 0.00625 mg/ml, RICC decreased to 55±5% compared to vehicle control both in the presence and absence of metabolic activation; RICC were 46.86% (cytotoxicity: 53.14% and 42,69% (cytotoxicity: 57.31%) in the absence and presence of S9 metabolic activation system, respectively.
Remarks on result:: other: No clastogenic potential was observed
Conclusions:: The registered substance, 4,4’bis(dimethylamino)-4”-(methylamino)trityl alcohol [CAS No.: 561-41-1], did not induce chromosomal aberration up to the highest concentration of 0.00625 mg/ml of culture medium, either in the presence or absence of S9 metabolic activation system in CHO cells. The test was performed according to OECD TG 473 and in compliance with the OECD Principles of Good Laboratory Practice.
Executive summary:: This in vitro experiment was performed to evaluate the ability of4,4’bis(dimethylamino)-4”-(methylamino)trityl alcohol [CAS No.: 561-41-1] to cause structural chromosomal aberrations in cultured CHO cells in the presence and absence of an exogenous metabolic activation system (S9). The study was performed as per OECD Test Guidelines No.473 (Adopted: 29 July 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 substance was soluble in dimethyl sulfoxide (DMSO); therefore, DMSO was used as a vehicle of the test substance during the test. Test concentrations were selected based on preliminary cytotoxicity tests I-II. In the preliminary cytotoxicity test I, CHO cells were exposed to 0.0 (NC), 0.0 (VC), 0.125, 0.25, 0.5, 1 and 2 mg/ml of the test substance both in the presence and absence of S9 metabolic activation (1 % v/v S9 mix). Excessive cytotoxicity (defined by a Relative Increase in Cell Count [RICC] of <40% of the concurrent vehicle control data) was observed for the Test Item concentrations at ≥0.125 mg/ml, with and without S9 metabolic activation. In the preliminary cytotoxicity assay-II, CHO cells were treated with 0.0 (NC), 0.0 (VC), 0.0625, 0.0125, 0.025, 0.05 and 0.1 mg/ml of culture medium, both in the presence (1 % v/v S9 mix) and absence of metabolic activation system. In this second cytotoxicity assay, excessive cytotoxicity (defined by a Relative Increase in Cell Count [RICC] of <40% of the concurrent vehicle control data) was observed for the Test Item concentrations at ≥0.0125 mg/ml, both in the presence and absence of metabolic activation. At 0.00625 mg/ml, RICC decreased to 55±5% compared to vehicle control both in the presence and absence of metabolic activation; RICC were 46.86% (cytotoxicity: 53.14% and 42,69% (cytotoxicity: 57.31%) in the absence and presence of S9 metabolic activation system, respectively. Based on the cytotoxicity assay results, Phase I (short-term exposure in the absence of metabolic activation), Phase II (short-term exposure in the presence of metabolic activation) and Phase III (continuous exposure in the absence of metabolic activation) were conducted with the Test Item at the concentrations of 0.0015625, 0.003125 and 0.00625 mg/ml, along with vehicle (DMSO), negative (Distilled water) and concurrent positive controls (Methyl methanesulfonate: 20 µg/ml, without S9 mix; Benzo (a) pyrene: 30 µg/ml with S9 mix). Results: In Phase I, cultures were exposed to test substance, vehicle, and negative and positive control for 4 hours (short-term exposure) in the absence of metabolic activation. Average RICC values were 97.83 % (vehicle control), 68.85 % (at 0.0015625 mg/ml), 53.04 % (at 0.003125 mg/ml) and 45.85 % (at 0.00625 mg/ml). No significant increase in the mean percent aberrant cells at 0.0015625 mg/ml (the mean % aberrant cells: 0.67%, p=0.4992), 0.003125 mg/ml (the mean % aberrant cells: 0.33%, p=1.000), 0.00625 mg/ml (the mean % aberrant cells: 0.33%, p=1.000), was observed when compared to the vehicle control (the mean % aberrant cells 0.00 %). In Phase II, CHO cells were exposed to the test substance, vehicle, and negative and positive control for 4 hours (short-term exposure) in the presence of metabolic activation (1 % v/v S9 mix). Average RICC values were 96.47 % (vehicle control), 66.83 % (at 0.0015625 mg/ml), 56.34 % (at 0.003125 mg/ml) and 41.38 % (at 0.00625 mg/ml). No significant increase in the mean percent aberrant cells at 0.0015625 mg/ml (the mean % aberrant cells: 0.67%, p=0.4992), 0.003125 mg/ml (the mean % aberrant cells: 0.00%, p=1.0000), 0.00625 mg/ml (the mean % aberrant cells: 0.67%, p=0.4992), was observed when compared to the vehicle control (the mean % aberrant cells 0.00 %). In Phase III, cultures were exposed to the substance, vehicle, negative and positive control for 24 hours (continuous exposure) in the absence of metabolic activation. Average RICC values were 95.27 % (vehicle control), 64.31 % (at 0.0015625 mg/ml), 52.11 % (at 0.003125 mg/ml) and 41.63 % (at 0.00625 mg/ml). No significant increase in the mean percent aberrant cells at 0.0015625 mg/ml (the mean % aberrant cells: 0.33%, p=1.000), 0.003125 mg/ml (the mean % aberrant cells: 0.33%, p=1.0000), 0.00625 mg/ml (the mean % aberrant cells: 0.67%, p=0.4992), was observed when compared to the vehicle control (the mean % aberrant cells 0.00 %). In all phases of the study, no significant reduction in RICC (cytotoxicity) and no increase in percent aberrant cells were observed in vehicle control (DMSO) either in the presence or absence of metabolic activation when compared to the negative control (distilled water). Conclusion: Based on the results of this study, it is concluded that 4,4’bis(dimethylamino)-4”-(methylamino)trityl alcohol [CAS No.: 561-41-1]does not induce chromosomal aberration up to 0.00625 mg/ml of culture medium, either in the presence or absence of S9 metabolic activation system in CHO cells under the experimental conditions described.

Reference 3

Endpoint:: in vitro gene mutation study in bacteria
Type of information:: experimental study
Adequacy of study:: key study
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: guideline study
Qualifier:: according to guideline
Guideline:: OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:: Adopted on 21 July 1997 and corrected on 26 June 2020
GLP compliance:: yes
Type of assay:: bacterial reverse mutation assay
Specific details on test material used for the study:: Appearance : Violet Powder
Batch Number : KCP/FS/199/21
Purity : 99.80%
Manufacturer : K.Patel Chemo Pharma Private Limited
Manufacturing date: 26.05.2021
Expiry Date: 25.11.2022
Storage condition: Room Temperature (20 to 30o C).
Target gene:: Histidine operon
Species / strain / cell type:: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Remarks:: Supplied by Moltox, USA
Metabolic activation:: with and without
Metabolic activation system:: Cofactor-supplemented liver S9 microsomal fraction was used. The S9 microsomal fraction was obtained from the liver of phenobarbitone and β-naphthoflavone-injected rat.
Test concentrations with justification for top dose:: Test concentrations:
0.0, 0.0, 0.00244, 0.00488, 0.0097656, 0.01953125, 0.0390625 mg/plate

Justification:
Test concentrations were selected based on solubility and precipitation checks and a preliminary cytotoxicity test. The following concentrations of the Test Item were tested in triplicate plates: 0.0 (Distilled water), 0.0 (DMSO), 0.0390625, 0.078125, 0.15625, 0.3125, 0.625, 1.25, 2.5, and 5 mg/plate using tester strains TA98, and TA100. In tester strains TA98 and TA100, complete inhibition of background lawn was observed at ≥ 0.15625 mg/plate, both in the presence (10% v/v S9 mix) and absence of metabolic activation compared to the vehicle control. A substantial reduction in the revertant count and moderate inhibition of the background lawn was observed at 0.078125 mg/plate, and a slight reduction in the number of revertant colonies accompanied by a moderate inhibition of the background lawn growth was observed at 0.0390625 mg/plate, both in the presence (10% v/v S9 mix) and absence of metabolic activation compared to vehicle control.
Vehicle / solvent:: Dimethyl sulfoxide (DMSO)
Untreated negative controls:: yes
Remarks:: Distilled water
Negative solvent / vehicle controls:: yes
Remarks:: DMSO
True negative controls:: no
Positive controls:: yes
Positive control substance:: 2-nitrofluorene; sodium azide; benzo(a)pyrene; mitomycin C
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: Two. Trial I: Plate incorporation, Trial II: Pre-incubation

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1 to 2 ×109 bacteria/ml
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk: Trial I: in agar, Trial II: in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20 min at 37 ± 2 °C
- Exposure duration/duration of treatment: 48 hours
- Harvest time after the end of treatment (sampling/recovery times): 48 hours

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 detected as inhibition of the background lawn growth and/or reduction in revertant counts.
Evaluation criteria:: The substance was deemed mutagenic if a biologically relevant increase in the mean number of revertants exceeding the threshold of twice (strains TA98, TA100 and TA102) or thrice (strains TA1535 and TA1537) the colony count of the corresponding solvent control was observed.
A dose-dependent increase was considered biologically relevant if the threshold was exceeded at more than one concentration.
A dose-dependent increase exceeding the threshold at only one concentration was judged as biologically relevant if reproduced in an independent experiment.
A dose-dependent increase in the number of revertant colonies below the threshold was regarded as an indication of a mutagenic potential if reproduced in an independent experiment.
: Key result
Species / strain:: S. typhimurium TA 102
Metabolic activation:: with and without
Genotoxicity:: negative
Cytotoxicity / choice of top concentrations:: cytotoxicity
Remarks:: Moderate inhibition of the background lawn and slight reduction in the revertant count was observed at 0.0390625 mg/plate.
Vehicle controls validity:: valid
Untreated negative controls validity:: valid
True negative controls validity:: not examined
Positive controls validity:: valid
: Key result
Species / strain:: S. typhimurium TA 100
Metabolic activation:: with and without
Genotoxicity:: negative
Cytotoxicity / choice of top concentrations:: cytotoxicity
Remarks:: Moderate inhibition of the background lawn and slight reduction in the revertant count was observed at 0.0390625 mg/plate.
Vehicle controls validity:: valid
Untreated negative controls validity:: valid
True negative controls validity:: not examined
Positive controls validity:: valid
: Key result
Species / strain:: S. typhimurium TA 98
Metabolic activation:: with and without
Cytotoxicity / choice of top concentrations:: cytotoxicity
Remarks:: Moderate inhibition of the background lawn and slight reduction in the revertant count was observed at 0.0390625 mg/plate.
Vehicle controls validity:: valid
Untreated negative controls validity:: valid
True negative controls validity:: not examined
Positive controls validity:: valid
: Key result
Species / strain:: S. typhimurium TA 1537
Metabolic activation:: with and without
Genotoxicity:: negative
Cytotoxicity / choice of top concentrations:: cytotoxicity
Remarks:: Moderate inhibition of the background lawn and slight reduction in the revertant count was observed at 0.0390625 mg/plate.
Vehicle controls validity:: valid
Untreated negative controls validity:: valid
True negative controls validity:: not examined
Positive controls validity:: valid
: Key result
Species / strain:: S. typhimurium TA 1535
Metabolic activation:: with and without
Genotoxicity:: negative
Cytotoxicity / choice of top concentrations:: cytotoxicity
Remarks:: Moderate inhibition of the background lawn and slight reduction in the revertant count was observed at 0.0390625 mg/plate.
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 test substance was soluble in dimethyl sulfoxide. Slight precipitation was observed at 5 mg/plate, which was considered not to interfere with the scoring.

Preliminary cytotoxicity test:
Test concentrations were selected based on solubility and precipitation checks and a preliminary cytotoxicity test. The following concentrations of the Test Item were tested in triplicate plates: 0.0 (Distilled water), 0.0 (DMSO), 0.0390625, 0.078125, 0.15625, 0.3125, 0.625, 1.25, 2.5, and 5 mg/plate using tester strains TA98, and TA100. In tester strains TA98 and TA100, complete inhibition of background lawn was observed at ≥ 0.15625 mg/plate, both in the presence (10% v/v S9 mix) and absence of metabolic activation compared to the vehicle control. A substantial reduction in the revertant count and moderate inhibition of the background lawn was observed at 0.078125 mg/plate, and a slight reduction in the number of revertant colonies accompanied by a moderate inhibition of the background lawn growth was observed at 0.0390625 mg/plate, both in the presence (10% v/v S9 mix) and absence of metabolic activation compared to vehicle control.
Remarks on result:: other: Non-mutagenic
Conclusions:: The registered substance, 4,4’-bis(dimethylamino)-4”-(methylamino) trityl alcohol (CAS 561-41-1) tested non-mutagenic (negative) in Salmonella Typhimurium TA98, TA100, TA1535, TA1537 and TA102 strains in the presence and absence of cofactor-supplemented liver S9 microsomal metabolic activation system. The test was performed according to OECD TG 471 and in compliance with the OECD Principles of Good Laboratory Practice.
Executive summary:: The mutagenic potential of 4,4’-bis(dimethylamino)-4”-(methylamino) trityl alcohol (CAS 561-41-1) was tested according to OECD 471 in Salmonella Typhimurium TA98, TA100, TA1535, TA1537 and TA102 tester strains both in the presence and absence of an exogenous metabolic activation system. Cofactor-supplemented rat liver S9 microsomal fraction was applied as a metabolic activation system. The test substance was dissolved in dimethyl sulfoxide (DMSO). Slight precipitation was observed at a test substance concentration of 5 mg/plate, which was considered not to interfere with the scoring. Test concentrations were selected based on the solubility and precipitation checks and a preliminary cytotoxicity test. In this pre-test, the following concentrations were assessed using triplicate plates: 0.0 (NC), 0.0 (VC), 0.0390625, 0.078125, 0.15625, 0.3125, 0.625, 1.25, 2.5, and 5 mg/plate using tester strains TA98, and TA100. In tester strain TA98 and TA100, complete inhibition of background lawn was observed at ≥ 0.15625 mg/plate, both in the presence (10% v/v S9 mix) and absence of metabolic activation compared to the vehicle control. Furthermore, a substantial reduction in the revertant count and moderate inhibition of the background lawn was observed at 0.078125 mg/plate, and a slight reduction in the number of revertant colonies accompanied by a moderate inhibition of the background lawn growth was observed at 0.0390625 mg/plate, both in the presence (10% v/v S9 mix) and absence of metabolic activation compared to vehicle control. Hence, in the mutagenicity test, the following concentrations were applied: 0.0 (NC, distilled water), 0.0 (VC, DMSO), of 0.00244, 0.00488, 0.0097656, 0.01953125 and 0.0390625 mg/plate both in the presence (10 % v/v S9 mix) and absence of metabolic activation. Strain-specific positive control substances were also included in the test: Sodium azide (TA100, TA1535: 0.5 µg/plate, without S9 mix, 2-Nitrofluorene (TA 98: 10 µg/plate, without S9 mix), 9-Aminoacridine (TA1537: 50 µg/plate without S9 mix), Mitomycin-C (TA102: 0.4 µg/plate, without S9 mix), Benzo[a] pyrene (TA98, TA100, TA1535, TA1537: 10 µg/plate with S9 mix). Results: Trial I was performed according to the plate incorporation method in Salmonella typhimurium TA98, TA100, TA102, TA1535, and TA1537 strains using five concentrations of the test substance, negative, vehicle and positive controls both in the presence (10 % v/v S9 mix) and absence of metabolic activation. Cytotoxicity, demonstrated as moderate inhibition in the background lawn growth and a slight reduction in the revertant count, was observed at the concentration of 0.0390625 mg/plate both in the presence (10 % v/v S9 mix) and absence of metabolic activation when compared to the vehicle control data. No reduction in the revertant count was observed at the concentrations from 0.01953125 to 0.00244 mg/plate, neither in the presence (10% v/v S9 mix) nor the absence of metabolic activation when compared to the vehicle control data. Furthermore, no trend of an increased number of revertant colonies with increased dosing of the Test Item was observed. Trial II was conducted according to the preincubation method to confirm the negative results observed in Trial I. Trial II was performed in Salmonella typhimurium tester strains TA98, TA100, TA102, TA1535, and TA1537 employing test substance concentrations of 0.00244, 0.00488, 0.0097656, 0.01953125 and 0.0390625 mg/plate both in the presence (10 % v/v S9 mix) and absence of metabolic activation system with all the tester strains along with the negative, vehicle and positive controls. A moderate inhibition in the background lawn and a slight reduction in the revertant count (cytotoxicity) was observed at the concentration of 0.0390625 mg/plate both in the presence (10 % v/v S9 mix) and absence of metabolic activation compared to the vehicle control. No increase in the number of revertant colonies was observed up to the highest concentration of 0.0390625 mg/plate either in the presence (10 % v/v S9 mix) or absence of metabolic activation when compared to the vehicle control data. Furthermore, no trend of an increased number of revertant colonies with increased dosing of the test substance was observed. The dose concentration of the Test Item in dose formulation was analysed by a validated test method for all concentrations, and the observed concentration was within the acceptable limit of linearity (R) ≥0.99, % recovery 70-110 % and % RSD ≤ 10%. For each tester strain, the frequency of the spontaneous revertant colonies in the vehicle control was within the acceptable range of historical data of the lab. The positive controls used in the study produced significant increases in the mean number of revertant colonies in all of the tester strains compared to the control, thus confirming the validity of the test system to detect specific mutagens in the presence and absence of a metabolic activation system. Conclusion: The registered substance of 4,4’-bis(dimethylamino)-4”-(methylamino) trityl alcohol (CAS 561-41-1) did not induce gene mutation in the histidine operon of Salmonella Typhimurium tester strains (TA98, TA100, TA1535, TA1537 and TA102) either in the presence (10 % v/v S9 mix) or absence of S9 metabolic activation system when was tested up to a cytotoxic concentration.

Endpoint conclusion

Endpoint conclusion:: no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:: no study available

Additional information

Bacterial reverse mutation test (OECD 471):

The mutagenic potential of 4,4’-bis(dimethylamino)-4”-(methylamino) trityl alcohol (CAS 561-41-1) was tested according to OECD 471 in Salmonella Typhimurium TA98, TA100, TA1535, TA1537 and TA102 tester strains both in the presence and absence of an exogenous metabolic activation system. Cofactor-supplemented rat liver S9 microsomal fraction was applied as a metabolic activation system. The test substance was dissolved in dimethyl sulfoxide (DMSO). Slight precipitation was observed at a test substance concentration of 5 mg/plate, which was considered not to interfere with the scoring. Test concentrations were selected based on the solubility and precipitation checks and a preliminary cytotoxicity test. In this pre-test, the following concentrations were assessed using triplicate plates: 0.0 (NC), 0.0 (VC), 0.0390625, 0.078125, 0.15625, 0.3125, 0.625, 1.25, 2.5, and 5 mg/plate using tester strains TA98, and TA100. In tester strain TA98 and TA100, complete inhibition of background lawn was observed at ≥ 0.15625 mg/plate, both in the presence (10% v/v S9 mix) and absence of metabolic activation compared to the vehicle control. Furthermore, a substantial reduction in the revertant count and moderate inhibition of the background lawn was observed at 0.078125 mg/plate, and a slight reduction in the number of revertant colonies accompanied by a moderate inhibition of the background lawn growth was observed at 0.0390625 mg/plate, both in the presence (10% v/v S9 mix) and absence of metabolic activation compared to vehicle control. Hence, in the mutagenicity test, the following concentrations were applied: 0.0 (NC, distilled water), 0.0 (VC, DMSO), of 0.00244, 0.00488, 0.0097656, 0.01953125 and 0.0390625 mg/plate both in the presence (10 % v/v S9 mix) and absence of metabolic activation. Strain-specific positive control substances were also included in the test: Sodium azide (TA100, TA1535: 0.5 µg/plate, without S9 mix, 2-Nitrofluorene (TA 98: 10 µg/plate, without S9 mix), 9-Aminoacridine (TA1537: 50 µg/plate without S9 mix), Mitomycin-C (TA102: 0.4 µg/plate, without S9 mix), Benzo[a] pyrene (TA98, TA100, TA1535, TA1537: 10 µg/plate with S9 mix). Results: Trial I was performed according to the plate incorporation method in Salmonella typhimurium TA98, TA100, TA102, TA1535, and TA1537 strains using five concentrations of the test substance, negative, vehicle and positive controls both in the presence (10 % v/v S9 mix) and absence of metabolic activation. Cytotoxicity, demonstrated as moderate inhibition in the background lawn growth and a slight reduction in the revertant count, was observed at the concentration of 0.0390625 mg/plate both in the presence (10 % v/v S9 mix) and absence of metabolic activation when compared to the vehicle control data. No reduction in the revertant count was observed at the concentrations from 0.01953125 to 0.00244 mg/plate, neither in the presence (10% v/v S9 mix) nor the absence of metabolic activation when compared to the vehicle control data. Furthermore, no trend of an increased number of revertant colonies with increased dosing of the Test Item was observed. Trial II was conducted according to the preincubation method to confirm the negative results observed in Trial I. Trial II was performed in Salmonella typhimurium tester strains TA98, TA100, TA102, TA1535, and TA1537 employing test substance concentrations of 0.00244, 0.00488, 0.0097656, 0.01953125 and 0.0390625 mg/plate both in the presence (10 % v/v S9 mix) and absence of metabolic activation system with all the tester strains along with the negative, vehicle and positive controls. A moderate inhibition in the background lawn and a slight reduction in the revertant count (cytotoxicity) was observed at the concentration of 0.0390625 mg/plate both in the presence (10 % v/v S9 mix) and absence of metabolic activation compared to the vehicle control. No increase in the number of revertant colonies was observed up to the highest concentration of 0.0390625 mg/plate either in the presence (10 % v/v S9 mix) or absence of metabolic activation when compared to the vehicle control data. Furthermore, no trend of an increased number of revertant colonies with increased dosing of the test substance was observed. The dose concentration of the Test Item in dose formulation was analysed by a validated test method for all concentrations, and the observed concentration was within the acceptable limit of linearity (R) ≥0.99, % recovery 70-110 % and % RSD ≤ 10%. For each tester strain, the frequency of the spontaneous revertant colonies in the vehicle control was within the acceptable range of historical data of the lab. The positive controls used in the study produced significant increases in the mean number of revertant colonies in all of the tester strains compared to the control, thus confirming the validity of the test system to detect specific mutagens in the presence and absence of a metabolic activation system. Conclusion: The registered substance of 4,4’-bis(dimethylamino)-4”-(methylamino) trityl alcohol (CAS 561-41-1) did not induce gene mutation in the histidine operon of Salmonella Typhimurium tester strains (TA98, TA100, TA1535, TA1537 and TA102) either in the presence (10 % v/v S9 mix) or absence of S9 metabolic activation system when was tested up to a cytotoxic concentration.

In vitro mammalian chromosome aberration test (OECD 473):

This in vitro experiment was performed to evaluate the ability of4,4’bis(dimethylamino)-4”-(methylamino)trityl alcohol [CAS No.: 561-41-1] to cause structural chromosomal aberrations in cultured CHO cells in the presence and absence of an exogenous metabolic activation system (S9). The study was performed as per OECD Test Guidelines No.473 (Adopted: 29 July 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 substance was soluble in dimethyl sulfoxide (DMSO); therefore, DMSO was used as a vehicle of the test substance during the test. Test concentrations were selected based on preliminary cytotoxicity tests I-II. In the preliminary cytotoxicity test I, CHO cells were exposed to 0.0 (NC), 0.0 (VC), 0.125, 0.25, 0.5, 1 and 2 mg/ml of the test substance both in the presence and absence of S9 metabolic activation (1 % v/v S9 mix). Excessive cytotoxicity (defined by a Relative Increase in Cell Count [RICC] of <40% of the concurrent vehicle control data) was observed for the Test Item concentrations at ≥0.125 mg/ml, with and without S9 metabolic activation. In the preliminary cytotoxicity assay-II, CHO cells were treated with 0.0 (NC), 0.0 (VC), 0.0625, 0.0125, 0.025, 0.05 and 0.1 mg/ml of culture medium, both in the presence (1 % v/v S9 mix) and absence of metabolic activation system. In this second cytotoxicity assay, excessive cytotoxicity (defined by a Relative Increase in Cell Count [RICC] of <40% of the concurrent vehicle control data) was observed for the Test Item concentrations at ≥0.0125 mg/ml, both in the presence and absence of metabolic activation. At 0.00625 mg/ml, RICC decreased to 55±5% compared to vehicle control both in the presence and absence of metabolic activation; RICC were 46.86% (cytotoxicity: 53.14% and 42,69% (cytotoxicity: 57.31%) in the absence and presence of S9 metabolic activation system, respectively. Based on the cytotoxicity assay results, Phase I (short-term exposure in the absence of metabolic activation), Phase II (short-term exposure in the presence of metabolic activation) and Phase III (continuous exposure in the absence of metabolic activation) were conducted with the Test Item at the concentrations of 0.0015625, 0.003125 and 0.00625 mg/ml, along with vehicle (DMSO), negative (Distilled water) and concurrent positive controls (Methyl methanesulfonate: 20 µg/ml, without S9 mix; Benzo (a) pyrene: 30 µg/ml with S9 mix). Results: In Phase I, cultures were exposed to test substance, vehicle, and negative and positive control for 4 hours (short-term exposure) in the absence of metabolic activation. Average RICC values were 97.83 % (vehicle control), 68.85 % (at 0.0015625 mg/ml), 53.04 % (at 0.003125 mg/ml) and 45.85 % (at 0.00625 mg/ml). No significant increase in the mean percent aberrant cells at 0.0015625 mg/ml (the mean % aberrant cells: 0.67%, p=0.4992), 0.003125 mg/ml (the mean % aberrant cells: 0.33%, p=1.000), 0.00625 mg/ml (the mean % aberrant cells: 0.33%, p=1.000), was observed when compared to the vehicle control (the mean % aberrant cells 0.00 %). In Phase II, CHO cells were exposed to the test substance, vehicle, and negative and positive control for 4 hours (short-term exposure) in the presence of metabolic activation (1 % v/v S9 mix). Average RICC values were 96.47 % (vehicle control), 66.83 % (at 0.0015625 mg/ml), 56.34 % (at 0.003125 mg/ml) and 41.38 % (at 0.00625 mg/ml). No significant increase in the mean percent aberrant cells at 0.0015625 mg/ml (the mean % aberrant cells: 0.67%, p=0.4992), 0.003125 mg/ml (the mean % aberrant cells: 0.00%, p=1.0000), 0.00625 mg/ml (the mean % aberrant cells: 0.67%, p=0.4992), was observed when compared to the vehicle control (the mean % aberrant cells 0.00 %). In Phase III, cultures were exposed to the substance, vehicle, negative and positive control for 24 hours (continuous exposure) in the absence of metabolic activation. Average RICC values were 95.27 % (vehicle control), 64.31 % (at 0.0015625 mg/ml), 52.11 % (at 0.003125 mg/ml) and 41.63 % (at 0.00625 mg/ml). No significant increase in the mean percent aberrant cells at 0.0015625 mg/ml (the mean % aberrant cells: 0.33%, p=1.000), 0.003125 mg/ml (the mean % aberrant cells: 0.33%, p=1.0000), 0.00625 mg/ml (the mean % aberrant cells: 0.67%, p=0.4992), was observed when compared to the vehicle control (the mean % aberrant cells 0.00 %). In all phases of the study, no significant reduction in RICC (cytotoxicity) and no increase in percent aberrant cells were observed in vehicle control (DMSO) either in the presence or absence of metabolic activation when compared to the negative control (distilled water). Conclusion: Based on the results of this study, it is concluded that 4,4’bis(dimethylamino)-4”-(methylamino)trityl alcohol [CAS No.: 561-41-1]does not induce chromosomal aberration up to 0.00625 mg/ml of culture medium, either in the presence or absence of S9 metabolic activation system in CHO cells under the experimental conditions described.

In vitro gene mutation test (OECD 476):

Justification for classification or non-classification

The registered substance, 4,4’-bis(dimethylamino)-4”-(methylamino)trityl alcohol (CAS 561-41-1), has been tested non-mutagenic (negative) in five Salmonella Typhimurium tester strains according to OECD TG 471. The substance has been tested non-clastogenic (negative ) in cultured Chinese Hamster Ovary cells in the presence or absence of an exogenous metabolic activation system in an in vitro cytogenicity test performed according to OECD TG 473. In addition, the registered substance (CAS 561-41-1) did not induce gene mutation at the hypoxanthine-guanine phosphoribosyltransferase (Hprt) locus in cultured Chinese Hamster Ovary (CHO) cells in the presence and absence of S9 metabolic activation in an OECD 476 guideline study. Thus, adequate, relevant and reliable studies indicate that the registered substance, 4,4’-bis(dimethylamino)-4”-(methylamino)trityl alcohol (CAS No.: 561-41-1), is non-mutagenic in various in vitro test systems (both in bacterial and mammalian cell cultures). Hence it is concluded that 4,4’-bis(dimethylamino)-4”-(methylamino)trityl alcohol (CAS No.: 561-41-1) is classified as Non-Classified for Germ Cell Mutagenicity according to the Regulation (EC) No 1272/2008 on the classification, labelling and packaging of substances and mixtures (CLP Regulation).

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Dose Level	Conc. (mg/ml)	Absence of Metabolic activation				Presence of Metabolic activation
		Cell count		RICC	% Cytotoxicity	Cell count		RICC	% Cytotoxicity
		Starting	Final	RICC	% Cytotoxicity	Starting	Final	RICC	% Cytotoxicity
NC	Distilled water	1000000	3580000	100.00	0.00	1000000	3584000	100.00	0.00
VC	DMSO	1000000	3560000	99.22	0.78	1000000	3470000	95.59	4.41
T1	0.125	1000000	0	0.00	100.00	1000000	0	0.00	100.00
T2	0.25	1000000	0	0.00	100.00	1000000	0	0.00	100.00
T3	0.5	1000000	0	0.00	100.00	1000000	0	0.00	100.00
T4	1	1000000	0	0.00	100.00	1000000	0	0.00	100.00
T5	2	1000000	0	0.00	100.00	1000000	0	0.00	100.00

Dose Level	Conc.	Phase I -Absence of Metabolic activation
		Cell count		RICC	% Cytotoxicity
		Starting	Final	RICC	% Cytotoxicity
NC	Distilled water	1000000	3586000	100.00	0.00
VC	DMSO	1000000	3530000	97.83	2.17
T1	0.0015625 mg/ml	1000000	2742000	68.85	31.15
T2	0.003125 mg/ml	1000000	2342000	53.04	46.96
T3	0.00625 mg/ml	1000000	2160000	45.85	54.15
PC	20 µg/ml	1000000	2842000	72.81	27.19

Dose Level	Conc.	Phase II -Presence of Metabolic activation
		Cell count		RICC	% Cytotoxicity
		Starting	Final	RICC	% Cytotoxicity
NC	Distilled water	1000000	3550000	100.00	0.00
VC	DMSO	1000000	3460000	96.47	3.53
T1	0.0015625 mg/ml	1000000	2644000	66.83	33.17
T2	0.003125 mg/ml	1000000	2386000	56.34	43.66
T3	0.00625 mg/ml	1000000	2018000	41.38	58.62
PC	30 µg/ml	1000000	2642000	66.75	33.25

Dose Level	Conc.	Phase III -Absence of Metabolic activation
		Cell count		RICC	% Cytotoxicity
		Starting	Final	RICC	% Cytotoxicity
NC	Distilled water	1000000	3582000	100.00	0.00
VC	DMSO	1000000	3460000	95.27	4.73
T1	0.0015625 mg/ml	1000000	2582000	64.31	35.69
T2	0.003125 mg/ml	1000000	2282000	52.11	47.89
T3	0.00625 mg/ml	1000000	2024000	41.63	58.37
PC	20 µg/ml	1000000	2682000	68.37	31.63

Test Item Concentration (mg/plate)	TA 98				TA 100
	- S9		+ S9		- S9		+ S9
	Mean	SD	Mean	SD	Mean	SD	Mean	SD
NC (Distilled water)	21 (NI)	2.00	19 (NI)	1.15	104 (NI)	5.51	102 (NI)	5.51
VC (Dimethyl sulfoxide)	19 (NI)	2.31	19 (NI)	0.00	99 (NI)	2.52	99 (NI)	2.08
T1 (0.0390625)	16 (MI)	1.53	16 (MI)	1.15	76 (MI)	2.31	75 (MI)	3.06
T2 (0.078125)	11 (MI)	2.08	8 (MI)	1.15	30 (MI)	3.21	23 (MI)	1.15
T3 (0.15625)	0 (CI)	0.00	0 (CI)	0.00	0 (CI)	0.00	0 (CI)	0.00
T4 (0.3125)	0 (CI)	0.00	0 (CI)	0.00	0 (CI)	0.00	0 (CI)	0.00
T5 (0.625)	0 (CI)	0.00	0 (CI)	0.00	0 (CI)	0.00	0 (CI)	0.00
T6 (1.25)	0 (CI)	0.00	0 (CI)	0.00	0 (CI)	0.00	0 (CI)	0.00
T7 (2.5)	0 (CI)	0.00	0 (CI)	0.00	0 (CI)	0.00	0 (CI)	0.00
T8 (5.0)	0 (CI)	0.00	0 (CI)	0.00	0 (CI)	0.00	0 (CI)	0.00
PC	317 (NI)	8.50	339 (NI)	7.55	710 (NI)	14.00	726 (NI)	7.21

2-Nitrofluorene	:	TA98 (absence of metabolic activation)
Sodium azide	:	TA100 (absence of metabolic activation)
Benzo[a]pyrene	:	TA98 and TA100 (presence of metabolic activation)

	Absence of metabolic activation										Presence of metabolic activation (+S9 10% v/v S9 Mix)
Test Item Concentration (mg/plate)	TA 98		TA 100		TA 1535		TA 1537		TA 102		TA 98		TA 100		TA 1535		TA 1537		TA 102
Test Item Concentration (mg/plate)	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
NC (Distilled water)	20	2.89	93	6.43	13	1.15	7	1.15	230	6.66	19	0.58	98	4.36	14	2.65	6	0.58	224	7.81
VC (Dimethyl sulfoxide)	18	0.58	93	2.52	13	0.58	7	1.53	236	9.00	20	2.89	94	2.00	14	0.58	6	1.73	225	6.43
T1 (0.00244)	19	1.15	92	5.57	12	1.73	7	1.15	226	7.37	18	1.53	91	2.08	12	0.58	6	0.58	219	9.17
T2 (0.00488)	20	3.06	92	2.65	13	1.73	6	1.15	218	8.19	20	1.73	93	2.00	14	2.31	6	1.15	225	6.24
T3 (0.0097656)	20	2.89	88	2.08	14	0.58	7	1.15	210	4.93	20	2.89	86	3.51	13	0.58	6	0.00	218	8.08
T4 (0.01953125)	17	1.15	86	3.61	12	1.15	6	1.53	206	9.29	17	0.00	83	3.06	11	0.58	4	0.58	214	4.93
T5 (0. 0390625)	15	1.15	73	4.51	9	1.15	2	1.15	178	9.85	13	0.58	69	1.53	8	0.00	2	0.58	179	4.04
PC	321	4.16	673	14.84	317	10.02	188	6.24	1643	39.50	317	9.54	690	15.95	306	10.02	198	8.54	1627	7.51

	Trial I - Plate Incorporation Method										Trial II – Preincubation Method
Test Item Concentration (mg/plate)	TA 98		TA 100		TA 1535		TA 1537		TA 102		TA 98		TA 100		TA 1535		TA 1537		TA 102
Test Item Concentration (mg/plate)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
NC (Distilled water)	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
VC (Dimethyl sulfoxide)	0.90	1.02	0.99	0.96	0.95	0.98	0.91	0.95	1.02	1.01	0.90	1.03	0.98	0.96	0.91	0.93	0.89	0.90	0.97	1.01
T1 (0.00244)	1.09	0.93	0.99	0.96	0.95	0.90	1.00	1.06	0.96	0.97	0.95	1.03	1.00	0.98	0.95	0.92	1.18	1.11	0.99	0.96
T2 (0.00488)	1.15	1.02	0.99	0.99	1.03	1.00	0.95	0.94	0.92	1.00	1.04	0.87	1.03	0.99	1.08	1.00	1.00	1.22	1.03	0.92
T3 (0.0097656)	1.11	1.00	0.95	0.92	1.08	0.93	1.00	1.00	0.89	0.97	1.04	1.15	0.99	0.96	1.13	1.03	1.18	1.00	1.01	0.96
T4 (0.01953125)	0.98	0.86	0.93	0.88	0.97	0.78	0.95	0.72	0.87	0.95	0.93	0.87	0.95	0.95	0.87	0.92	0.76	0.78	0.93	0.89
T5 (0. 0390625)	0.83	0.68	0.79	0.73	0.68	0.59	0.35	0.39	0.75	0.80	0.73	0.63	0.72	0.70	0.59	0.56	0.29	0.33	0.77	0.70
PC	18.19	16.12	7.27	7.34	25.00	22.37	28.20	33.00	6.96	7.22	17.36	16.02	7.84	7.73	25.64	25.67	38.00	36.39	7.65	7.52

Summary of S9 efficacy check
	TA100		TA1535
	Mean	SD	Mean	SD
VC Dimethyl sulfoxide (-S9)	99	2.52	13	0.58
VC Dimethyl sulfoxide (+S9)	99	2.08	14	0.58
PC Benzo[a]pyrene (-S9)	100	3.06	15	2.31
PC Benzo[a]pyrene (+S9)	726	7.21	306	10.02

Test Item Concentration (mg/plate)	R	TA98				TA100
Test Item Concentration (mg/plate)	R	-S9	BL	+S9	BL	-S9	BL	+S9	BL
NC (Distilled water)	1	23	NI	20	NI	98	NI	102	NI
	2	21	NI	20	NI	109	NI	107	NI
	3	19	NI	18	NI	104	NI	96	NI
VC (Dimethyl sulfoxide)	1	18	NI	19	NI	96	NI	101	NI
	2	18	NI	19	NI	101	NI	98	NI
	3	22	NI	19	NI	99	NI	97	NI
T1 (0.0390625)	1	17	MI	15	MI	79	MI	78	MI
	2	16	MI	17	MI	75	MI	72	MI
	3	14	MI	17	MI	75	MI	76	MI
T2 (0.078125)	1	10	MI	9	MI	34	MI	24	MI
	2	13	MI	7	MI	28	MI	22	MI
	3	9	MI	7	MI	29	MI	22	MI
T3 (0.15625)	1	0	CI	0	CI	0	CI	0	CI
	2	0	CI	0	CI	0	CI	0	CI
	3	0	CI	0	CI	0	CI	0	CI
T4 (0.3125)	1	0	CI	0	CI	0	CI	0	CI
	2	0	CI	0	CI	0	CI	0	CI
	3	0	CI	0	CI	0	CI	0	CI
T5 (0.625)	1	0	CI	0	CI	0	CI	0	CI
	2	0	CI	0	CI	0	CI	0	CI
	3	0	CI	0	CI	0	CI	0	CI
T6 (1.25)	1	0	CI	0	CI	0	CI	0	CI
	2	0	CI	0	CI	0	CI	0	CI
	3	0	CI	0	CI	0	CI	0	CI
T7 (2.5)	1	0	CI	0	CI	0	CI	0	CI
	2	0	CI	0	CI	0	CI	0	CI
	3	0	CI	0	CI	0	CI	0	CI
T8 (5.0)	1	0	CI	0	CI	0	CI	0	CI
	2	0	CI	0	CI	0	CI	0	CI
	3	0	CI	0	CI	0	CI	0	CI
PC	1	309	NI	332	NI	716	NI	720	NI
	2	317	NI	347	NI	694	NI	734	NI
	3	326	NI	338	NI	720	NI	724	NI

Registration Dossier

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Diss Factsheets

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

Endpoint summary

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Link to relevant study records

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Reference 1

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Reference 3

Endpoint conclusion

Genetic toxicity in vivo

Endpoint conclusion

Additional information

Justification for classification or non-classification

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