Phenol, 2,4-dinitro-, sulfurized, thiosulfonated

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:

21 June - 09 August, 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, June 2012

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:

Phenol, 2,4-dinitro-, sulfurized, thiosulfonated

EC Number:

215-445-8

EC Name:

Phenol, 2,4-dinitro-, sulfurized, thiosulfonated

Cas Number:

1326-83-6

Molecular formula:

not applicable

IUPAC Name:

Reaction product of 2,4-dinitrophenol with polysulfide, thiosulfonated

Test material form:

solid: particulate/powder

Details on test material:

Test item: Solubilised Sulphur Black 1
Appearance: black powder
CAS No: 1326-83-6

Specific details on test material used for the study:

Date of production: 30.11.2016
Expiration date: 30.11.2021

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. The 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 (WP2 uvrA) 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, and 16 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:

- Solvents used: ultrapure water (ASTM Type I), Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent: In the study two types of solvent control were used depending on the solubility of the test item and the solubility of positive control reference items.

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 revertant colonies, background lawn growth

Rationale for test conditions:

Based on the solubility test, a stock solution/suspension with a concentration of 50 mg/mL was prepared in ultrapure water (ASTM Type I) and diluted accordingly. In the informatory toxicity test a correction factor, based on the active component of the test item (80.2 %) was taken into consideration.
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.
In the informatory toxicity test the revertant colony numbers of solvent control plates 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 untreated, solvent control, positive control and the test item treated plates were determined visually by manual counting and the mean values, standard deviations, and the mutation rates were calculated:



* : untreated, solvent or positive control

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 solvent 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 solvent 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 1535

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

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

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

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

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

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

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

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: No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the study.

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				WP2 uvrA
	-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	21.0	1.03	26.0	1.18	93.7	1.05	96.7	1.13	8.3	0.89	11.3	0.87	7.0	0.81	8.3	1.09	26.7	1.00	25.7	0.99
DMSO Control	22.7	1.00	24.3	1.00	–	–	88.3	1.00	–	–	8.7	1.00	8.3	1.00	8.0	1.00	–	–	34.7	1.00
Ultrapure Water Control	20.3	1.00	22.0	1.00	89.0	1.00	85.7	1.00	9.3	1.00	13.0	1.00	8.7	1.00	7.7	1.00	26.7	1.00	26.0	1.00
5000	16.7	0.82	12.0	0.55	69.3	0.78	84.7	0.99	17.0	1.82	18.0	1.38	9.0	1.04	7.3	0.96	30.3	1.14	20.7	0.79
1600	15.3	0.75	14.0	0.64	77.7	0.87	82.0	0.96	8.0	0.86	9.7	0.74	11.0	1.27	10.3	1.35	25.0	0.94	20.3	0.78
500	17.0	0.84	15.3	0.70	80.0	0.90	89.7	1.05	8.7	0.93	10.3	0.79	7.7	0.88	9.3	1.22	29.3	1.10	21.0	0.81
160	16.3	0.80	23.3	1.06	72.3	0.81	90.0	1.05	7.3	0.79	11.0	0.85	9.3	1.08	6.3	0.83	30.7	1.15	26.3	1.01
50	17.3	0.85	29.0	1.32	72.3	0.81	83.3	0.97	10.7	1.14	14.0	1.08	11.7	1.35	6.7	0.87	28.0	1.05	28.3	1.09
16	18.0	0.89	25.7	1.17	80.3	0.90	94.0	1.10	8.3	0.89	11.7	0.90	6.7	0.77	6.0	0.78	27.0	1.01	32.0	1.23
NPD (4mg)	306.0	13.50	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
SAZ (2mg)	–	–	–	–	988.0	11.10	–	–	1428.0	153.00	–	–	–	–	–	–	–	–	–	–
9AA (50mg)	–	–	–	–	–	–	–	–	–	–	–	–	209.0	25.08	–	–	–	–	–	–
MMS (2mL)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	810.7	30.40	–	–
2AA (2mg)	–	–	1957.3	80.44	–	–	1001.3	11.34	–	–	222.3	25.65	–	–	191.3	23.92	–	–	–	–
2AA (50mg)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	202.0	5.83

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

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

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				WP2 uvrA
	-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	18.3	1.31	20.7	1.09	82.7	1.06	102.3	1.07	15.0	1.05	12.3	1.00	7.0	1.40	9.0	0.93	29.3	0.75	32.7	0.82
DMSO Control	14.7	1.00	20.0	1.00	–	–	92.0	1.00	–	–	14.7	1.00	6.0	1.00	8.7	1.00	–	–	37.3	1.00
Ultrapure Water Control	14.0	1.00	19.0	1.00	77.7	1.00	95.7	1.00	14.3	1.00	12.3	1.00	5.0	1.00	9.7	1.00	39.3	1.00	40.0	1.00
5000	13.0	0.93	14.7	0.77	55.0	0.71	88.3	0.92	17.0	1.19	24.3	1.97	4.7	0.93	7.0	0.72	33.3	0.85	32.0	0.80
1600	12.3	0.88	17.0	0.89	77.7	1.00	80.7	0.84	15.7	1.09	13.3	1.08	6.0	1.20	8.7	0.90	28.0	0.71	36.0	0.90
500	13.7	0.98	21.7	1.14	72.0	0.93	78.3	0.82	12.0	0.84	11.3	0.92	5.0	1.00	10.3	1.07	34.0	0.86	30.3	0.76
160	15.3	1.10	21.3	1.12	77.3	1.00	79.3	0.83	10.7	0.74	12.7	1.03	6.7	1.33	10.7	1.10	46.3	1.18	40.3	1.01
50	15.3	1.10	23.7	1.25	68.3	0.88	89.7	0.94	10.3	0.72	14.7	1.19	5.0	1.00	10.7	1.10	38.0	0.97	33.7	0.84
16	17.7	1.26	31.3	1.65	74.7	0.96	100.7	1.05	11.7	0.81	13.3	1.08	5.0	1.00	7.3	0.76	32.0	0.81	48.0	1.20
NPD (4mg)	304.0	20.73	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
SAZ (2mg)	–	–	–	–	981.3	12.64	–	–	978.7	68.28	–	–	–	–	–	–	–	–	–	–
9AA (50mg)	–	–	–	–	–	–	–	–	–	–	–	–	738.7	123.11	–	–	–	–	–	–
MMS (2mL)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	1544.0	39.25	–	–
2AA (2mg)	–	–	1411.3	70.57	–	–	2442.7	26.55	–	–	234.0	15.95	–	–	209.7	24.19	–	–	–	–
2AA (50mg)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	249.7	6.69

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

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

Table 6: Historical Control Values for Revertants/Plate (for the Period of 2008-2016)

			Bacterial strains
Historical control data of untreated control	‑S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	21.0	105.0	10.5	8.1	25.4
		SD	3.7	25.7	1.4	2.3	5.2
		Minimum	9	66	3	2	11
		Maximum	39	155	23	19	45
		n	226	236	216	214	215
	+S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	27.5	117.1	11.8	9.0	33.9
		SD	4.3	18.1	1.4	1.9	5.2
		Minimum	12	75	4	2	17
		Maximum	46	166	23	20	56
		n	226	236	216	214	215
			Bacterial strains
Historical control data of DMSO control	‑S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	20.4	100.1	10.3	7.9	24.7
		SD	3.6	24.8	1.3	2.4	4.6
		Minimum	10	64	3	2	11
		Maximum	38	147	23	20	45
		n	226	236	216	214	215
	+S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	26.5	113.8	11.8	8.8	33.7
		SD	4.1	18.3	1.5	1.9	5.0
		Minimum	15	71	3	3	16
		Maximum	47	162	25	20	57
		n	226	236	216	214	215
			Bacterial strains
Historical control data of Water control	‑S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	21.9	104.7	10.5	7.6	26.1
		SD	3.7	25.9	1.5	2.2	5.5
		Minimum	12	68	3	2	12
		Maximum	35	154	24	16	48
		n	89	236	216	89	215
	+S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	27.4	117.3	11.4	8.7	34.9
		SD	4.0	18.5	1.3	2.2	4.9
		Minimum	15	83	4	3	18
		Maximum	43	167	22	16	57
		n	89	152	149	89	148

			Bacterial strains
Historical control data of positive controls	‑S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	260.1	977.2	847.3	478.6	724.5
		SD	31.8	150.6	126.3	104.5	65.0
		Minimum	123	521	359	110	320
		Maximum	664	1970	1855	1601	1313
		n	226	236	216	214	215
	+S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	1222.7	1436.4	164.1	147.0	257.7
		SD	274.9	318.3	33.1	20.1	72.5
		Minimum	386	583	85	69	140
		Maximum	2676	2988	498	399	477
		n	226	236	216	214	215

Abbreviations:   TA98, TA100, TA1535, TA1537: Salmonella typhimuriumTA98, TA100, TA1535, TA1537; E. coli:Escherichia coliWP2uvrA

                                     SD: Standard deviation;   DMSO: Dimethyl sulfoxide;  n: number of studies

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 coli WP2uvrA) 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 in ultrapure water (ASTM Type I). At the formulation of test item solutions correction of concentrations for active component content (80.2 % 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, and 16 µg/plate. No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the study. The test item did not show unequivocal inhibitory, cytotoxic effects in the performed experiments. The colony and background lawn development was not affected in any case; the obtained revertant colony number decreases (compared to the revertant colony numbers of the vehicle control) were considered to be within the biological variability range of the applied test system. The revertant colony numbers of solvent control (ultrapure water (ASTM Type I)) plates with and without S9 mix demonstrated the characteristic mean number of spontaneous revertants that was solved in line with the corresponding historical control data ranges. The reference mutagen treatments (positive controls) showed the expected biologically 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 with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 mix) in the performed experiments.

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