Acetamide, N-(2,4-dinitrophenyl)-, reaction products with p-phenylenediamine, sodium sulfide (Na2(Sx)) and sulfur, leuco deriv.

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:

23 June 2017 - 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:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1998

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:

Acetamide, N-(2,4-dinitrophenyl)-, reaction products with p-phenylenediamine, sodium sulfide (Na2(Sx)) and sulfur, leuco deriv.

EC Number:

288-897-7

EC Name:

Acetamide, N-(2,4-dinitrophenyl)-, reaction products with p-phenylenediamine, sodium sulfide (Na2(Sx)) and sulfur, leuco deriv.

Cas Number:

85940-09-6

Molecular formula:

not applicable

IUPAC Name:

Reaction product of acetamide, N-(2,4-dinitrophenyl)- with p-phenylenediamine and sodium polysulfide, leuco derivatives

Test material form:

solid: particulate/powder

Details on test material:

Test item: Olive BW
Appearance: dark powder, near black
CAS No: 85940-09-6
EC No: 288-897-7
Storage: room temperature

Method

Target gene:

In addition to histidine and tryptophan 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:

The test bacteria were also exposed to the test item in the presence of an appropriate metabolic activation system, which is a cofactor-supplemented post-mitochondrial fraction (S9).

Test concentrations with justification for top dose:

Justification of concentrations:
Selection of the concentration range was done on the basis of solubility tests and concentration range finding test (informatory toxicity test).

Based on the results of the preliminary tests, the maximum test item concentration was 5000 µg/plate (±S9 mix). The test item concentrations investigated in the initial and confirmatory mutation tests were as follows:
5000, 1600, 500, 160, 50 and 16 µg/plate.

Vehicle / solvent:

Based on the results of these tests, 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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

methylmethanesulfonate

other: 4-Nitro-1,2-phenylenediamine; 2-aminoanthracene

Details on test system and experimental conditions:

Tester strains: Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA
Supplier: Trinova Biochem GmbH; Rathenau Str. 2; D-35394 Giessen, Germany;
Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA.
Frozen stock cultures were prepared from the disc cultures.

Genotypes of the Strains Used for Mutagenicity Testing

In addition to histidine and tryptophan 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.

Storage of Tester Strains

The strains are stored at -80 ± 10ºC in the form of lyophilized discs and in frozen permanent copies. Frozen permanent cultures of the tester strains are prepared from fresh, overnight cultures to which DMSO (8 % (v/v)) is added as a cryoprotective agent.

Confirmation of Phenotypes of Tester Strains

The phenotypes of the tester strains used in the bacterial reverse mutation assays with regard to membrane permeability (rfa), UV sensitivity (uvrA and uvrB), ampicillin resistance (amp), as well as spontaneous mutation frequencies are checked regularly according to Ames et al.

Spontaneous Reversion of Tester Strains

Each tester strain reverts spontaneously at a frequency that is characteristic for the strain. Spontaneous reversions of the test strains to histidine or tryptophan prototrophs are measured routinely in mutagenicity experiments and expressed as the number of spontaneous revertants per plate.

Procedure for Bacterial Cultures

The frozen bacterial cultures were thawed at room temperature and 200 µL inoculum was used to inoculate each 50 mL of Nutrient Broth No. 2 for the overnight cultures in the assay. The cultures were incubated for approximately 11-12 hours in a 37 oC Benchtop Incubator Shaker.

Viability and the Cell Count of the Testing Bacterial Cultures

The viability of each testing culture was determined by plating 0.1 mL of the 10-5, 10-6, 10-7 and 10-8 dilutions of cultures on nutrient agar plates. The viable cell number of the cultures was determined by manual colony counting.

Metabolic Activation System

The test bacteria were also exposed to the test item in the presence of an appropriate metabolic activation system, which is a cofactor-supplemented post-mitochondrial fraction (S9).

Rat Liver S9 Fraction

The S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF)-induced rat liver was provided by Trinova Biochem GmbH (Rathenau Str. 2; D-35394 Giessen, Germany; Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA).

Rationale for test conditions:

Selection of the concentration range was done on the basis of solubility tests and concentration range finding test (informatory toxicity test). Based on the solubility test, a stock suspension with a concentration of 50 mg/mL was prepared in dimethyl sulfoxide (DMSO) and further diluted accordingly. In the informatory toxicity test a correction factor, based on the active component of the test item (96.45 %) 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.
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:

No substantial increases were observed in revertant colony numbers of any of the five test strains following treatment with Olive BW at any concentration level, either in the presence or absence of metabolic activation (S9 mix) in the performed experiments.
In the performed experiments, sporadically increased revertant colony numbers were observed. These increases did not show a clear dose-response relationship, were mostly of minor intensity, and all of the increases remained far below the biologically relevant thresholds for being positive. The obtained increases were therefore considered as biologically not relevant, being in the range of the biological variability of the applied test system.
The highest revertant colony number increase was observed in the initial mutation test (plate incorporation test) in S. typhimurium TA98 strain, at 160 μg/plate, in the presence of metabolic activation (+S9). The revertant colony numbers at this treatment were above the corresponding solvent historical control data range; however the value was considered to be unique without clear concentration related increases. The mutation rate was 2.18, which was far below the genotoxicological threshold for being positive.
In the initial and confirmatory mutation tests, an unequivocal inhibitory effect of the test item on bacterial growth was not observed . All of the noticed lower revertant colony numbers (when compared to the revertant colony numbers of the corresponding vehicle control) remained in the range of the biological variability of the applied test system and the background lawn development was not affected in any case.
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 the highest examined concentration of 5000 µg/plate in the absence and also in the presence of exogenous metabolic activation following the plate incorporation and pre-incubation procedures.

Any other information on results incl. tables

Summary Table of the Results of the Concentration Range Finding Test

Concentration Range Finding Test (Informatory Toxicity Test)
Concentrations (mg/plate)	Salmonella typhimurium tester strains
	TA 98				TA 100
	-S9		+S9		-S9		+S9
Mean values of revertants per plate and Mutation rate (MR)	Mean	MR	Mean	MR	Mean	MR	Mean	MR
Untreated Control	16.3	0.92	18.3	0.82	85.7	0.94	120.0	1.10
DMSO Control	17.7	1.00	22.3	1.00	90.7	1.00	108.7	1.00
Ultrapure Water Control	–	–	–	–	86.0	1.00	–	–
5000	18.0	1.02	19.3	0.87	78.0	0.86	90.0	0.83
1600	13.3	0.75	16.7	0.75	94.7	1.04	89.3	0.82
500	22.0	1.25	21.3	0.96	83.7	0.92	90.3	0.83
160	14.3	0.81	31.3	1.40	86.0	0.95	100.3	0.92
50	16.7	0.94	25.0	1.12	82.0	0.90	110.0	1.01
16	21.3	1.21	20.0	0.90	87.3	0.96	103.0	0.95
5	17.7	1.00	20.3	0.91	93.0	1.03	99.3	0.91
NPD (4mg)	193.3	10.94	–	–	–	–	–	–
SAZ (2mg)	–	–	–	–	776.0	9.02	–	–
2AA (2mg)	–	–	1066.7	47.76	–	–	2017.3	18.56

MR:Mutation Rate

NPD:4-Nitro-1,2-phenylenediamine

SAZ:Sodium azide

2AA:2-aminoanthracene

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

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	0.93	26.0	1.07	93.7	1.30	96.7	1.09	8.3	0.78	11.3	1.31	7.0	0.84	8.3	1.04	26.7	1.25	25.7	0.74
DMSO Control	22.7	1.00	24.3	1.00	72.0	1.00	88.3	1.00	10.7	1.00	8.7	1.00	8.3	1.00	8.0	1.00	21.3	1.00	34.7	1.00
Ultrapure Water Control	–	–	–	–	89.0	1.00	–	–	9.3	1.00	–	–	–	–	–	–	26.7	1.00	–	–
5000	16.3	0.72	24.3	1.00	64.7	0.90	71.0	0.80	11.0	1.03	10.3	1.19	6.3	0.76	7.0	0.88	22.0	1.03	20.7	0.60
1600	17.7	0.78	30.0	1.23	64.3	0.89	71.3	0.81	8.0	0.75	11.3	1.31	10.3	1.24	6.3	0.79	19.7	0.92	29.0	0.84
500	15.0	0.66	46.7	1.92	67.7	0.94	72.0	0.82	10.7	1.00	12.0	1.38	6.7	0.80	9.7	1.21	26.3	1.23	28.7	0.83
160	18.7	0.82	53.0	2.18	72.7	1.01	72.3	0.82	11.3	1.06	8.7	1.00	11.7	1.40	7.7	0.96	23.0	1.08	31.7	0.91
50	17.0	0.75	36.7	1.51	65.7	0.91	79.7	0.90	8.3	0.78	10.0	1.15	6.3	0.76	9.0	1.13	20.7	0.97	31.7	0.91
16	20.3	0.90	28.3	1.16	79.3	1.10	96.7	1.09	11.3	1.06	11.0	1.27	9.0	1.08	7.7	0.96	18.3	0.86	27.3	0.79
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:           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.

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	1.25	20.7	1.03	82.7	1.11	102.3	1.11	15.0	0.82	12.3	0.84	7.0	1.17	9.0	1.04	29.3	1.00	32.7	0.88
DMSO Control	14.7	1.00	20.0	1.00	74.3	1.00	92.0	1.00	18.3	1.00	14.7	1.00	6.0	1.00	8.7	1.00	29.3	1.00	37.3	1.00
Ultrapure Water Control	–	–	–	–	77.7	1.00	–	–	14.3	1.00	–	–	–	–	–	–	39.3	1.00	–	–
5000	16.7	1.14	18.3	0.92	72.0	0.97	90.7	0.99	18.0	0.98	9.7	0.66	3.7	0.61	7.3	0.85	19.3	0.66	27.0	0.72
1600	15.0	1.02	22.0	1.10	70.3	0.95	93.3	1.01	13.0	0.71	12.0	0.82	6.3	1.06	9.0	1.04	22.0	0.75	34.0	0.91
500	14.0	0.95	36.3	1.82	72.0	0.97	104.3	1.13	16.0	0.87	11.7	0.80	7.3	1.22	10.3	1.19	22.3	0.76	32.3	0.87
160	15.0	1.02	42.0	2.10	63.0	0.85	102.0	1.11	13.0	0.71	13.3	0.91	5.7	0.94	10.0	1.15	28.0	0.95	35.0	0.94
50	15.7	1.07	28.3	1.42	71.3	0.96	95.7	1.04	15.0	0.82	12.7	0.86	7.0	1.17	8.0	0.92	23.7	0.81	39.7	1.06
16	16.0	1.09	25.0	1.25	84.7	1.14	101.3	1.10	14.3	0.78	14.0	0.95	5.7	0.94	7.0	0.81	27.3	0.93	41.7	1.12
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:           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:

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

Executive summary:

The test item was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay 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 post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/b-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 test and the concentration range finding test the test item was dissolved in dimethyl sulfoxide (DMSO). At the formulation of test item solutions correction of concentrations for active component content (96.45 %) was made in the experiments. Based on the results of the preliminary concentration range finding test (informatory toxicity test) the following concentrations of the test item were prepared and investigated in the initial and confirmatory mutation tests: 5000; 1600; 500; 160; 50 and 16 µg/plate. The selection of the concentration range was based on the recommendations in OECD 471 guideline. At the concentration choice the non-toxicity of the test item and the precipitation of the test item in the final treatment mixture were taken into consideration. The observations were made by naked eye. 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 the highest examined concentration of 5000 µg/plate in the absence and also in the presence of exogenous metabolic activation following the plate incorporation and pre-incubation procedures. 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 (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 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 item has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.