Leuco polysulfided 4-[(2,4-dinitrophenyl)amino]phenol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 March - 12 June, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July, 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

30 May, 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

August, 1998

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: – ICH Guidance S2(R1): Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use,

Version / remarks:

June, 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Leuco polysulfided 4-[(2,4-dinitrophenyl)amino]phenol

EC Number:

310-132-3

EC Name:

Leuco polysulfided 4-[(2,4-dinitrophenyl)amino]phenol

Cas Number:

61902-31-6

Molecular formula:

not applicable

IUPAC Name:

Reaction product of 4-[(2,4-dinitrophenyl)amino]phenol with sodium polysulfide, leuco derivatives

Test material form:

solid: particulate/powder

Details on test material:

Test item: Leuco Sulphur Blue 11 2P
Appearance: dark black powder
CAS No. 61902-31-6
EC No. 310-132-3
Storage: room temperature

Specific details on test material used for the study:

Date of production: 29.11.2016
Expiration date: 29.11.2021
Storage: Room temperature (15-25°C)

Method

Target gene:

his/trp
In addition to histidine (his) or tryptophan (trp) mutation, each strain has additional mutations which enhance its sensitivity to mutagens. The uvrB (uvrA) strains are defective in excision repair. It causes the strains to be more sensitive to the mutagenic and lethal effects of a wide variety of mutagens because they cannot repair DNA damages. rfa mutation increases the permeability of the bacterial lipopolysaccharide wall for larger molecules. The plasmid pKM101 (TA98, TA100) carries the muc+ gene which participates in the error-prone "SOS" DNA repair pathway induced by DNA damage. This plasmid also carries an ampicillin resistance transfer factor (R-factor) which is used to identify its presence in the cell. The Escherichia coli strain used in this test (WP2uvrA) is also defective in DNA excision repair.

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 mix of phenobarbital and β-naphthoflavone-induced rat liver

Test concentrations with justification for top dose:

5000, 1600, 500, 160, 50, 16 and 5 µg/plate.
Selection of the concentration range was done on the basis of a solubility test and a concentration range finding test (informatory toxicity test).

Vehicle / solvent:

Solvent used: dimethyl sulfoxide (DMSO)
- Justification for choice of solvent: Based on the results of a soliubility test, dimethyl sulfoxide (DMSO) was found to be the most appropriate solvent for preparing the test item stock formulations and dilutions. The solvent was chosen due to its non-toxicity to the bacteria and the compatibility to the S9 activity based on the available laboratory’s historical control database.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-Nitro-1,2-phenylenediamine

Remarks:

TA98, without S9 mix, 4 µg/plate

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA100 and TA1535, without S9 mix, 2 µg/plate

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA1537, without S9 mix, 50 µg/plate

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

E.coli WP2, without S9 mix, 2 µL/plate

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

All tested Salmonella strains, with S9 mix, 2 µg/plate; E.coli WP2, with S9 mix, 50 µg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: number of revertat colonies, background lawn growth

Rationale for test conditions:

Justification of concentrations:
Choice of the concentrations was done on the basis of a Solubility Test and a concentration Range Finding Test (Informatory Toxicity Test).
Based on the solubility test, the stock solution with a concentration of 50 mg/mL was prepared in the vehicle and diluted in at least 6 steps by factor of approximately √10.
The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98, TA100) were determined at the concentrations of 5000, 1600, 500, 160, 50, 16 and 5 µg/plate of the test item.
The revertant colony numbers of vehicle control plates in both strains with and without S9 Mix were in line with the corresponding historical control data ranges. The positive control treatments showed the expected, biological relevant increases in induced revertant colonies in both tester strains.

Evaluation criteria:

The colony numbers on the controls (untreated, vehicle, positive) and the test plates were determined (counted manually), the mean values and appropriate standard deviations and mutation rates were calculated.
A test item is considered mutagenic if:
- a dose–related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control,
- in strain Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.

According to the guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Criteria for a Negative Response:
A test item is considered non-mutagenic if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Statistics:

The mean values and appropriate standard deviations and mutation rates were calculated by EXCEL software.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without S9 mix, 500 µg/plate with S9 mix, 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Without S9 mix, 1600 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without S9 mix, 500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without S9 mix, 500 µg/plate; with S9 mix, 1600 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: non-interfering test item precipitate was noticed after about 48 hours incubation on the plates in the examined strains at the concentrations of 5000 and 1600 μg/plate in absence and in the presence of S9 following the plate incorporation and pre-incubation procedures.

Any other information on results incl. tables

Table 4: Summary Table of the Results of the Initial Mutation Test

Initial Mutation Test (Plate Incorporation Test)
Concentrations (mg/plate)	Salmonella typhimuriumtester strains																Escherichiacoli
	TA 98				TA 100				TA 1535				TA 1537				WP2uvrA
	-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9
Mean values of revertants per plate Mutation rate (MR)	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR
Untreated Control	17.7	1.02	23.0	0.92	100.0	1.04	109.0	1.18	10.0	0.97	14.3	1.02	7.3	1.22	7.3	0.96	32.0	0.99	39.3	1.18
DMSO Control	17.3	1.00	25.0	1.00	96.3	1.00	92.3	1.00	10.3	1.00	14.0	1.00	6.0	1.00	7.7	1.00	32.3	1.00	33.3	1.00
Ultrapure Water Control	–	–	–	–	90.0	1.00	–	–	11.3	1.00	–	–	–	–	–	–	36.3	1.00	–	–
5000	12.0	0.69	16.3	0.65	71.0	0.74	82.0	0.89	12.3	1.19	12.3	0.88	0.0	0.00	1.0	0.13	18.3	0.57	25.0	0.75
1600	23.0	1.33	19.7	0.79	89.7	0.93	100.3	1.09	9.3	0.90	13.0	0.93	1.7	0.28	6.0	0.78	28.3	0.88	31.3	0.94
500	21.7	1.25	35.7	1.43	96.0	1.00	113.3	1.23	14.3	1.39	12.0	0.86	5.3	0.89	6.3	0.83	29.0	0.90	29.7	0.89
160	20.3	1.17	57.3	2.29	90.0	0.93	117.3	1.27	8.7	0.84	11.3	0.81	5.3	0.89	8.3	1.09	32.7	1.01	31.3	0.94
50	19.3	1.12	50.0	2.00	87.0	0.90	121.3	1.31	9.3	0.90	11.7	0.83	4.7	0.78	6.3	0.83	31.3	0.97	24.3	0.73
16	21.0	1.21	42.7	1.71	88.3	0.92	110.7	1.20	15.7	1.52	12.0	0.86	6.7	1.11	6.3	0.83	32.7	1.01	29.7	0.89
5	23.7	1.37	27.7	1.11	86.0	0.89	109.0	1.18	12.7	1.23	12.7	0.90	7.0	1.17	5.3	0.70	28.0	0.87	37.7	1.13
NPD (4mg)	202.7	11.69	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
SAZ (2mg)	–	–	–	–	1234.7	13.72	–	–	765.3	67.53	–	–	–	–	–	–	–	–	–	–
9AA (50mg)	–	–	–	–	–	–	–	–	–	–	–	–	676.0	112.67	–	–	–	–	–	–
MMS (2mL)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	918.7	25.28	–	–
2AA (2mg)	–	–	2429.3	97.17	–	–	1632.0	17.68	–	–	228.3	16.31	–	–	239.3	31.22	–	–	–	–
2AA (50mg)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	197.3	5.92

MR: Mutation Rate;          NPD: 4-Nitro-1,2-phenylenediamine; SAZ: Sodium azide; 9AA: 9-Aminoacridine; MMS: Methyl methanesulfonate; 2AA: 2-aminoanthracene

Remarks:           DMSO was applied as solvent of the test item and positive control substances: NPD, 9AA and 2AA and the ultrapure water was applied as solvent for the SAZ and MMS. The mutation rate of the test item and the untreated control is given referring to the DMSO. The mutation rate of the NPD, 9AA and 2AA is given referring to the DMSO and the mutation rate of the SAZ and MMS positive control is given referring to the ultrapure water.

Table 5 :Summary Table of the Results of the Confirmatory Mutation Test

Confirmatory Mutation Test (Pre-Incubation Test)
Concentrations (mg/plate)	Salmonella typhimuriumtester strains																Escherichia coli
	TA 98				TA 100				TA 1535				TA 1537				WP2uvrA
	-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9
Mean values of revertants per plate Mutation rate (MR)	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR
Untreated Control	21.3	1.25	29.3	1.16	87.3	1.08	111.3	1.17	14.3	1.48	10.7	0.94	5.0	0.88	9.7	0.88	24.3	1.18	25.7	0.71
DMSO Control	17.0	1.00	25.3	1.00	81.0	1.00	95.3	1.00	9.7	1.00	11.3	1.00	5.7	1.00	11.0	1.00	20.7	1.00	36.0	1.00
Ultrapure Water Control	–	–	–	–	79.0	1.00	–	–	11.7	1.00	–	–	–	–	–	–	32.7	1.00	–	–
5000	8.0	0.47	14.3	0.57	45.0	0.56	89.0	0.93	5.7	0.59	7.7	0.68	0.0	0.00	2.7	0.24	16.7	0.81	24.3	0.68
1600	8.3	0.49	23.3	0.92	57.0	0.70	77.7	0.81	3.0	0.31	13.0	1.15	0.0	0.00	4.0	0.36	21.3	1.03	28.7	0.80
500	11.3	0.67	28.7	1.13	91.0	1.12	91.0	0.95	2.0	0.21	9.7	0.85	2.0	0.35	7.3	0.67	23.0	1.11	39.7	1.10
160	23.0	1.35	57.7	2.28	86.7	1.07	104.0	1.09	7.0	0.72	9.3	0.82	5.0	0.88	8.0	0.73	23.3	1.13	30.3	0.84
50	21.3	1.25	37.7	1.49	89.3	1.10	93.3	0.98	11.3	1.17	11.3	1.00	7.7	1.35	11.3	1.03	26.7	1.29	32.7	0.91
16	15.7	0.92	27.7	1.09	79.7	0.98	109.3	1.15	12.0	1.24	11.0	0.97	3.7	0.65	11.0	1.00	27.0	1.31	33.7	0.94
5	18.0	1.06	25.3	1.00	88.3	1.09	111.7	1.17	10.0	1.03	9.3	0.82	6.7	1.18	9.3	0.85	24.7	1.19	30.3	0.84
NPD (4mg)	241.3	14.20	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
SAZ (2mg)	–	–	–	–	1146.7	14.51	–	–	1314.7	112.69	–	–	–	–	–	–	–	–	–	–
9AA (50mg)	–	–	–	–	–	–	–	–	–	–	–	–	303.0	53.47	–	–	–	–	–	–
MMS (2mL)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	1058.7	32.41	–	–
2AA (2mg)	–	–	2122.7	83.79	–	–	2373.3	24.90	–	–	178.0	15.71	–	–	193.7	17.61	–	–	–	–
2AA (50mg)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	168.0	4.67

MR: Mutation Rate;          NPD: 4-Nitro-1,2-phenylenediamine; SAZ: Sodium azide; 9AA: 9-Aminoacridine; MMS: Methyl methanesulfonate; 2AA: 2-aminoanthracene

Remarks:           DMSO was applied as solvent of the test item and positive control substances: NPD, 9AA and 2AA and the ultrapure water was applied as solvent for the SAZ and MMS. The mutation rate of the test item and the untreated control is given referring to the DMSO. The mutation rate of the NPD, 9AA and 2AA is given referring to the DMSO and the mutation rate of the SAZ and MMS positive control is given referring to the ultrapure water.

 

Applicant's summary and conclusion

Conclusions:

In an in vitro bacterial reverse mutation assay (Ames) according to OECD guideline 471, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Executive summary:

The test item was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay (Ames) according to OECD guideline 471. The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coliWP2uvrA) in the presence and absence of a S9 mix prepared from livers of phenobarbital/beta-naphthoflavone-induced rats. The study included preliminary solubility test, preliminary concentration range finding test (informatory toxicity test), an initial mutation test (plate incorporation test), and a confirmatory mutation test (pre-incubation test). Based on the results of the solubility tests and the concentration range finding tests the test item was dissolved in dimethyl sulfoxide (DMSO). At the formulation of test item solutions correction of concentrations for active component content (97.4 % dyestuff) was made in the main experiments. Based on the results of the preliminary concentration range finding tests (informatory toxicity tests) the following concentrations of the test item (based on 97.4 % dyestuff) were prepared and investigated in the initial and confirmatory mutation tests: 5000, 1600, 500, 160, 50, 16, and 5 µg/plate. When evaluated by naked eye, non-interfering test item precipitate was noticed after about 48 hours incubation on the plates in the examined strains at concentrations of 5000 and 1600 µg/plate in absence and in the presence of S9 mix following the plate incorporation and pre-incubation procedures. Inhibitory effect of the test item was observed in the initial mutation test in the S. typhimurium TA1537 strain in the absence and also in the presence of S9 mix, in the confirmatory mutation test in all S. typhimurium strains in the absence of S9; in TA98 and TA1537 also in the presence of S9. The inhibitory effect was indicated by absent or decreased revertant colony counts (some of them below the corresponding historical control data ranges) and/or affected background lawn development (reduced or slightly reduced background lawn). In general, 500 µg/plate was considered as lowest concentration showing cytotoxicity. The revertant colony numbers of solvent control (dimethyl sulfoxide, DMSO) plates with and without S9 mix demonstrated the characteristic mean number of spontaneous revertants that was in line with the corresponding historical control data ranges.

The reference mutagen treatments (positive controls) showed the expected, biological relevant increases (more than 3-fold increase) in induced revertant colonies and the number of revertants mostly fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in all experimental phases, in all tester strains. No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment at any concentration level, either in the presence or absence of S9 mix.The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

In conclusion, the test item has no mutagenic activity on the applied bacterium tester strains, under the test conditions used in this study.