α,α-bis[4-(dimethylamino)phenyl]-4-(phenylamino)naphthalene-1-methanol

Genetic toxicity: in vitro

Administrative data

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

Justification for type of information:

The study contains experimental data of the registered substance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted: 21st July 1997, Corrected: 26th June2020

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

α,α-bis[4-(dimethylamino)phenyl]-4-(phenylamino)naphthalene-1-methanol

EC Number:

229-851-8

EC Name:

α,α-bis[4-(dimethylamino)phenyl]-4-(phenylamino)naphthalene-1-methanol

Cas Number:

6786-83-0

Molecular formula:

C33H33N3O

IUPAC Name:

(4-anilino-1-naphthyl){bis[4-(dimethylamino)phenyl]}methanol

Test material form:

solid

Remarks:

Powder

Details on test material:

- Name of test material: (4-anilino-1-naphthyl){bis[4-(dimethylamino)phenyl]methanol
- Molecular formula: C33H33N3O
- Molecular weight: 487.6 g/mol
- Substance type: Organic
- Physical state: Blackish powder
- Purity: 99.62%
- Impurities (identity and concentrations): No data

Specific details on test material used for the study:

- Name of test material: (4-anilino-1-naphthyl){bis[4-(dimethylamino)phenyl]methanol
- Molecular formula: C33H33N3O
- SMILES: CN(C)C1=CC=C(C=C1)C(C2=CC=C(C=C2)N(C)C)(C3=CC=C(C4=CC=CC=C43)NC5=CC=CC=C5)O
- Physical state: Solid
- Purity: 99.62%
- Apperance: Blackish powder
- Batch Number: KCP/FS/742/20
- Expiry Date: 21.08.2021
- Storage condition: Room Temperature (20 to 30 C)

Method

Target gene:

Histidine operon

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9 mix [Cofactors (Cofactor ingredients: D-glucose-6-phosphate, β-NADP, magnesium chloride, potassium chloride, sodium phosphate and Liver homogenate] was prepared prior to use in the study. The post-mitochondrial fraction (liver homogenate) was used at the concentration of 10 % (v/v) in the S9 mix.

Test concentrations with justification for top dose:

0 (solvent control), 0.0625, 0.125, 0.25, 0.5 and 1 mg/plate.

Justification for the top dose: The highest test concentration was selected based on solubility, precipitation checks, and a preliminary cytotoxicity test. The test substance was soluble in DMSO up to 10 mg/ml, giving the final test concentration of 1 mg/ml. No precipitation that can interfere with scoring was observed at 1 mg/ml. A reduction in revertant counts and moderate inhibition of the background lawn was observed at 1 mg/m. Hence, 1 mg/ml was selected as the highest test dose for the reverse mutation test.

Vehicle / solvent:

dimethyl sulfoxide (DMSO)

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: Trial1 (plate incorporation) in agar, Trial 2 (preincubation) in medium

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 hours
- Expression time (cells in growth medium): 48 hours

SELECTION AGENT (mutation assays): Top agar was prepared with 0.6 % (w/v) bacto agar and 0.6 % (w/v) sodium chloride and was supplemented with 10 % (v/v) of 0.5 mM histidine/biotin solution for selection of histidine revertants

NUMBER OF REPLICATIONS: Triplicates

Evaluation criteria:

A Test Item is considered as a mutagen 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 is observed. A dose-dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration. A dose-dependent increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent experiment. A dose-dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent experiment.

Statistics:

The colonies were counted manually. Then, the mean number of revertant colonies and the standard deviation within the plates for each concentration were compared to the spontaneous reversion rates of the control. Microsoft Office Excel-based calculations were used for descriptive statistical analysis.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A decrease in mean revertant counts was observed at 1 mg/ml both with and without S9 mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A decrease in mean revertant counts was observed at 1 mg/ml both with and without S9 mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A decrease in mean revertant counts was observed at 1 mg/ml both with and without S9 mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A decrease in mean revertant counts was observed at 1 mg/ml both with and without S9 mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A decrease in mean revertant counts was observed at 1 mg/ml both with and without S9 mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

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. In addition, the positive controls used in the study produced significant increases in the mean number of revertant colonies in all of the tester strains when compared to the control data, thus confirming the validity of the test system to detect specific mutagens in the presence and absence of a metabolic activation system.

Remarks on result:

other: No mutagenic potential was observed

Any other information on results incl. tables

TABLES

Table1          Mean Revertant Colony Count – Preliminary Cytotoxicity Assay

Test Item Concentration (mg/plate)	TA 98				TA 100
	- S9		+ S9		- S9		+ S9
	Mean	SD	Mean	SD	Mean	SD	Mean	SD
NC (Distilled water)	22.00 (NI)	2.00	20.33 (NI)	0.58	101.33 (NI)	3.06	101.00 (NI)	6.24
VC (Dimethyl sulfoxide)	20.67 (NI)	2.08	21.00 (NI)	2.65	100.67 (NI)	6.11	99.00 (NI)	3.00
T1 (0.0000128)	22.00 (NI)	1.73	21.67 (NI)	1.15	95.67 (NI)	1.53	96.67 (NI)	3.06
T2 (0.000064)	20.00 (NI)	2.00	21.33 (NI)	2.31	100.67 (NI)	2.08	97.33 (NI)	1.53
T3 (0.00032)	20.33 (NI)	2.31	21.67 (NI)	1.15	95.67 (NI)	2.89	94.33 (NI)	1.53
T4 (0.0016)	20.33 (NI)	1.15	22.00 (NI)	2.00	95.00 (NI)	2.65	94.67 (NI)	2.08
T5 (0.008)	21.33 (NI)	1.53	19.67 (NI)	3.06	97.33 (NI)	4.93	93.00 (NI)	3.46
T6 (0.04)	19.67 (NI)	2.52	21.33 (NI)	0.58	95.67 (NI)	3.51	88.00 (NI)	5.29
T7 (0.2)	19.33 (NI)	1.53	18.67 (NI)	0.58	91.00 (NI)	5.00	88.00 (NI)	3.46
T8 (1.0)	15.00 (MI)	1.00	16.67 (MI)	1.15	86.00 (MI)	2.00	82.00 (MI)	1.73
PC	333.67 (NI)	13.58	323.33 (NI)	8.33	684.00 (NI)	5.00	673.67 (NI)	11.24

Key:   NC = Negative Control, VC = Vehicle control, PC = Positive Control, -S9 = Absence of metabolic activation, +S9 = Presence of metabolic activation, SD = Standard Deviation, T1-T8 = Test Item concentration from lower to higher, NI = No inhibition, MI = Moderate inhibition.

 

Positive Controls:

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

Table2          Mean Revertant Colony Count in Trial I(Plate Incorporation Method)

	Absence of metabolic activation										Presence of metabolic activation (+S9 10% v/v S9 Mix)
Test Item Concentration (mg/plate)	TA 1535		TA 1537		TA 102		TA 98		TA 100		TA 1535		TA 1537		TA 102		TA 98		TA 100
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
NC (Distilled water)	12.67	1.53	7.00	1.00	240.00	8.19	20.33	2.52	102.33	4.51	14.33	1.15	5.67	0.58	236.67	6.81	21.67	3.21	94.67	1.15
VC (Dimethyl sulfoxide)	13.00	1.00	5.33	0.58	241.67	4.04	21.00	2.65	97.67	4.73	12.33	0.58	6.33	1.15	233.00	6.24	20.67	2.08	102.67	4.04
T1(0.625)	12.67	1.15	6.67	1.15	237.67	8.62	21.33	2.89	104.33	2.52	12.33	1.15	5.67	0.58	230.67	6.11	20.67	2.31	97.00	5.57
T2 (0.125)	11.67	1.15	6.67	0.58	237.67	7.23	21.67	2.52	99.33	5.51	13.33	2.08	7.33	1.15	236.67	5.69	18.33	1.15	98.67	3.06
T3 (0.25)	13.33	1.15	6.00	2.00	231.67	4.93	19.33	1.15	98.33	4.51	12.67	1.53	6.33	1.15	232.33	7.37	20.67	1.15	96.00	2.65
T4 (0.5)	12.67	2.08	6.33	0.58	229.67	6.66	21.00	2.00	102.67	4.04	13.33	0.58	6.00	2.00	235.33	7.09	18.67	1.15	95.33	2.52
T5 (1.0)	10.33	0.58	5.67	1.15	198.33	12.66	16.67	1.15	82.00	2.00	10.00	1.00	5.00	1.73	193.67	11.93	16.00	1.00	84.67	4.73
PC	329.00	16.09	191.00	4.36	1606.00	27.84	329.00	15.87	682.33	9.71	315.00	12.29	196.00	10.44	1627.67	9.61	323.33	8.33	701.67	14.50

Key:   NC = Negative Control, VC = Vehicle control, PC = Positive Control, -S9 = Absence of metabolic activation, +S9 = Presence of metabolic activation, SD = Standard Deviation, T1-T5 = Test Item concentration from lower to higher.

 

Positive Controls:

2-Nitrofluorene		:		TA98 (absence of metabolic activation)
Sodium azide		:		TA100 and TA1535 (absence of metabolic activation)
9-Aminoacridine		:		TA1537 (absence of metabolic activation)
Mitomycin-C		:		TA102(absence of metabolic activation)
Benzo[a]pyrene		:		TA98, TA100, TA1535, TA1537 and TA102 (presence of metabolic activation)

 

Table3          Mean Revertant Colony Count in Trial II(Preincubation Method)

	Absence of metabolic activation										Presence of metabolic activation (+S9 10% v/v S9 Mix)
Test Item Concentration (mg/plate)	TA 1535		TA 1537		TA 102		TA 98		TA 100		TA 1535		TA 1537		TA 102		TA 98		TA 100
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
NC (Distilled water)	12.33	1.53	7.33	1.15	239.00	7.00	21.7	3.2	99.33	6.81	13.67	0.58	6.33	1.53	233.00	7.00	21.33	1.53	100.67	3.21
VC (Dimethyl sulfoxide)	12.00	1.73	6.33	1.15	239.67	9.45	20.0	2.0	101.33	7.02	12.67	1.15	5.33	0.58	236.67	7.09	21.33	0.58	97.33	2.89
T1(0.625)	12.33	1.53	6.67	1.15	238.00	8.72	20.0	3.0	95.33	2.08	13.33	0.58	6.67	0.58	235.00	10.15	21.67	3.21	91.67	2.65
T2 (0.125)	12.67	2.31	6.00	1.73	233.33	6.51	20.3	2.3	98.33	4.04	13.67	1.53	7.67	0.58	232.67	4.04	20.67	2.08	102.00	3.51
T3 (0.25)	12.67	0.58	5.33	0.58	234.67	2.31	22.0	2.0	94.33	2.52	12.33	1.15	5.67	1.53	236.00	7.00	22.00	1.73	96.00	4.36
T4 (0.5)	14.33	1.53	6.67	1.15	230.00	12.12	19.0	1.0	92.67	3.21	12.67	1.15	5.67	2.08	234.33	7.57	20.00	3.00	95.00	0.58
T5 (1.0)	10.67	1.15	5.00	1.44	194.33	8.50	15.7	1.5	81.67	3.79	9.33	1.15	3.33	1.15	185.67	6.66	14.67	0.58	82.00	3.21
PC	323.33	10.02	204.33	11.68	1590.33	15.63	325.7	10.6	700.67	14.57	313.00	6.56	201.00	13.45	1630.00	18.68	329.67	17.21	713.67	6.51

Key:     NC = Negative Control, VC = Vehicle control, PC = Positive Control, -S9 = Absence of metabolic activation, +S9 = Presence of metabolic activation, SD = Standard Deviation,T1-T5 = Test Item concentration from lower to higher.

 

Positive Controls:

2-Nitrofluorene	:	TA98 (absence of metabolic activation)
Sodium azide	:	TA100 and TA1535 (absence of metabolic activation)
9-Aminoacridine	:	TA1537 (absence of metabolic activation)
Mitomycin-C	:	TA102 (absence of metabolic activation)
Benzo[a]pyrene	:	TA98, TA100, TA1535, TA1537 and TA102 (presence of metabolic activation)

Table4          Fold Increase

	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
	-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)	1.03	0.95	0.95	1.08	1.03	0.86	0.76	1.12	1.01	0.98	0.92	1.00	1.02	0.97	0.97	0.93	0.86	0.84	1.00	1.02
T1(0.625)	1.02	1.00	1.07	0.94	0.97	1.00	1.25	0.89	0.98	0.99	1.00	1.02	0.94	0.94	1.03	1.05	1.05	1.25	0.99	0.99
T2 (0.125)	1.03	0.89	1.02	0.96	0.90	1.08	1.25	1.16	0.98	1.02	1.02	0.97	0.97	1.05	1.06	1.08	0.95	1.44	0.97	0.98
T3 (0.25)	0.92	1.00	1.01	0.94	1.03	1.03	1.13	1.00	0.96	1.00	1.10	1.03	0.93	0.99	1.06	0.97	0.84	1.06	0.98	1.00
T4 (0.5)	1.00	0.90	1.05	0.93	0.97	1.08	1.19	0.95	0.95	1.01	0.95	0.94	0.91	0.98	1.19	1.00	1.05	1.06	0.96	0.99
T5 (1.0)	0.79	0.77	0.84	0.82	0.79	0.81	1.06	0.79	0.82	0.83	0.78	0.69	0.81	0.84	0.89	0.74	0.79	0.63	0.81	0.78
PC	15.67	15.65	6.99	6.83	25.31	25.54	35.81	30.95	6.65	6.99	16.28	15.45	6.91	7.33	26.94	24.71	32.26	37.69	6.64	6.89

Key:   NC = Negative Control, VC = Vehicle control, PC = Positive Control, -S9 = Absence of metabolic activation, +S9 = Presence of metabolic activation, T1-T5 = Test Item concentration from lower to higher.

Table5          S9 Efficiency Check- Summary

Summary of S9 efficiency check
	TA100		TA1535
	Mean	SD	Mean	SD
VC Dimethyl Sulfoxide (-S9)	100.67	6.11	13.00	1.00
VC Dimethyl Sulfoxide (+S9)	99.00	3.00	12.33	0.58
PC Benzo[a]pyrene (-S9)	96.33	2.08	13.67	0.58
PC Benzo[a]pyrene (+S9)	673.67	11.24	315.00	12.29

Key:   VC = Vehicle control, PC = Positive control, -S9 = Absence of metabolic activation, +S9 = Presence of metabolic activation, SD = Standard Deviation.

Applicant's summary and conclusion

Conclusions:

The registered substance, α,α-bis[4-(dimethylamino)phenyl]-4-(phenylamino)naphthalene-1-methanol (CAS Number: 6786-83-0; EC Number: 229-851-8) tested non-mutagenic (negative) in Salmonella typhimurium tester strains TA1537, TA1535, TA98, TA100 and TA102 with and without S9 metabolic activation system. The test was performed according to OECD TG 471 and in compliance with OECD Principles of Good Laboratory Practice.

Executive summary:

The mutagenicity of the test substance, α,α-bis[4-(dimethylamino)phenyl]-4-(phenylamino)naphthalene-1-methanol (CAS Number: 6786-83-0; EC Number: 229-851-8) was tested according to OECD 471 using Salmonella typhimurium tester strains TA1537, TA1535, TA98, TA100 and TA102. The test was performed as two independent experiments according to the plate incorporation method (Trial I) and the preincubation method (Trial II), both in the presence and absence of rat liver microsomal activation (S9 mix). Concentrations used in the mutagenicity assay were selected based on a preliminary cytotoxicity assay. This preliminary cytotoxicity assay was performed using concentrations of 0.0000128, 0.000064, 0.00032, 0.0016, 0.008, 0.04, 0.2 and 1 mg/plate, with and without metabolic activation (10 % v/v S9 mix) using TA98 and TA100 strains. A slight reduction in the revertant counts and moderate inhibition of the background lawn was observed at 1mg/plate, and no reduction in the revertant count and/or inhibition in the background lawn was noted at concentrations from 0.0000128 to 0.2 mg/plate when compared to the vehicle control data. Based on the observations of the pre-test, the following doses were applied in the mutagenicity test: 0.0 (NC), 0.0 (VC), 0.0625, 0.125, 0.25, 0.5 and 1mg/plate, both in the presence and absence of metabolic activation, along with the negative, vehicle and positive controls. Trial I was performed according to the plate incorporation method. The test substance, together with the negative, vehicle and positive controls, were tested in triplicates. The Test Item doses were selected using a spacing factor of 2. There was slight precipitation, a slight reduction in revertant colony count and a moderate inhibition in the background lawn at 1 mg/plate. Furthermore, there was no increase in the number of revertant colonies and/or diminution of the background lawn at concentrations of 0.0625 - 0.5 mg/plate, neither in the presence nor the absence of metabolic activation compared to the vehicle control data. Trial II was performed to confirm the negative results observed in Trial I. Trial II was conducted according to the preincubation method; the substance was tested in all the tester strains in triplicates. There was slight precipitation, a slight reduction in revertant colony count and a moderate inhibition in the background lawn at 1 mg/plate. There was no increase in the number of revertant colonies and/or diminution of the background lawn at concentrations of 0.0625 - 0.5 mg/plate, neither in the presence nor in the absence of metabolic activation when compared to the vehicle control data. The numbers of revertant colonies of the vehicle (spontaneous revertant colonies) and positive controls (induced revertant colonies) of all the tester strains were within the range of historical data of the laboratory. The positive controls used in the study produced significant increases in the mean number of revertant colonies under both activated and non-activated conditions in all tester strains compared to the vehicle control data confirming the validity of the mutagenicity test. Based on the results of this study, it is concluded that(4-anilino-1-naphthyl){bis[4-(dimethylamino)phenyl]}methanol (CAS No. 6786-83-0) is non-mutagenic as it does not induce gene mutations (point and frameshift) in the histidine operon by base-pair changes or frameshifts, in the presence or the absence of metabolic activation system, in any of the five tester strains of Salmonella typhimurium (TA1537, TA1535, TA98, TA100 and TA102).