Benzenamine, reaction products with aniline hydrochloride and nitrobenzene

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:

09 May 2018 to 18 May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

The experimental design is compatible with the guideline requirements, but only one bacterial strain was used in the study as requested by the Sponsor

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

The experimental design is compatible with the guideline requirements, but only one bacterial strain was used in the study as requested by the Sponsor

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

The experimental design is compatible with the guideline requirements, but only one bacterial strain was used in the study as requested by the Sponsor

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Benzenamine, reaction products with aniline hydrochloride and nitrobenzene

EC Number:

309-912-6

EC Name:

Benzenamine, reaction products with aniline hydrochloride and nitrobenzene

Cas Number:

101357-15-7

Molecular formula:

This is a UVCB substance. See section 1.2 for individual components.

IUPAC Name:

Benzenamine, reaction products with aniline hydrochloride and nitrobenzene

Test material form:

solid: particulate/powder

Details on test material:

- Description: Black powder
- Storage Conditions: Controlled room temperature (15-25 ºC, below 70 RH %)

Method

Target gene:

Tryptophan requirement in the Escherichia coli strain.

Species / strain

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: MOLTOX - Molecular Toxicology Inc., Boone, North Carolina, USA
- The strain was stored at -80 ± 10 ºC in the Culture Collection of the Microbiological Laboratory of the testing laboratory. Frozen permanent cultures of the tester strain was prepared from fresh, overnight cultures to which DMSO was added as a cryoprotective agent.
- The phenotype of the tester strain used in the bacterial reverse mutation assays with regard to UV sensitivity (uvrA), as well as spontaneous mutation frequencies are checked regularly according to Ames et al. and Maron and Ames.
- Spontaneous reversion of the test strain to tryptophan independence is measured routinely in mutagenicity experiments and is expressed as the number of spontaneous revertants per plate.
- The day before treatment, the frozen bacterial cultures were thawed at room temperature and 200 μL inoculum were used to inoculate each 50 mL of Nutrient Broth No.2 for the overnight cultures in the assay. The cultures were incubated for 10-14 hours at 37 °C in a Gyrotory water bath shaker.
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 prepared by sterile physiological saline on Nutrient Agar plates. The number of viable cell of the cultures was determined by manual counting after approximately 24-hour incubation at 37 °C.

MEDIA USED
- Nutrient Broth No.2

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

- 5000, 1581, 500, 158.1, 50, 15.81, 5, 1.581 µg/plate
- Based on the results of the preliminary test, 100 mg/mL stock solution was prepared in DMSO, which was diluted by serial dilutions in several steps to obtain the dosing formulations for lower doses. The maximum test concentration was 5000 μg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- The solubility of the test material was examined using Distilled water, Dimethyl sulfoxide (DMSO), N,N-Dimethylformamide (DMF) and Acetone. The test material was insoluble in Distilled water at 100 mg/mL concentration. At the same concentration the test material was formed homogeneous suspension with Acetone. The test material was soluble at this concentration using DMSO and DMF (black colour solution was detected in each case). Due to the better biocompatibility, DMSO was selected as vehicle (solvent) for the study. The obtained stock solution (50 μL) with the solution of top agar and phosphate buffer was examined in a test tube without test bacterium suspension to examine the formulation compatibility.
- All dilutions in the main tests of test material were made in the testing laboratory using Dimethyl sulfoxide (DMSO). Test solutions were freshly prepared at the beginning of the experiments in the testing laboratory by diluting the stock solution using the selected solvent and were used within 2 hours after preparation.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

other: 2-aminoanthracene with activation (50 µg/plate)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- The study included a Preliminary Compatibility Test, an Initial Mutation Test and a Confirmatory Mutation Test. In the Initial Mutation Test, the plate incorporation method was used. In the Confirmatory Mutation Test, the pre-incubation method was used.

INITIAL MUTATION TEST
- A standard plate incorporation procedure was performed as an Initial Mutation Test. Bacteria (cultured in Nutrient Broth No .2) were exposed to the test material both in the presence and absence of an appropriate metabolic activation system.
- Molten top agar was prepared and kept at 45 °C. The equivalent number of minimal glucose agar plates (three plates per test material concentration and for each control) was properly labelled. The test material and other components were prepared freshly and added to the overlay (45 °C).
- The content of the tubes: top agar 2000 μL, vehicle or test material formulation (or reference controls) 50 μL, overnight culture of test strain 100 μL and phosphate buffer (pH 7.4) or S9 mix 500 μL. This solution was mixed and poured on the surface of minimal agar plates. For activation studies, instead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative (solvent) and positive controls. After preparation, the plates were incubated at 37 °C for 48 hours.

CONFIRMATORY MUTATION TEST
- A pre-incubation procedure was performed as a Confirmatory Mutation Test since in the Initial Mutation Test no positive effect was observed.
- For the pre-incubation method, bacteria (cultured in Nutrient Broth No. 2) were exposed to the test material both in the presence and absence of an appropriate metabolic activation system. The equivalent number of minimal glucose agar plates was properly labelled. Molten top agar was prepared and kept at 45 °C.
- Before the overlaying, 50 μL of the test material formulation or its vehicle (or 50 μL of positive reference controls or their solvents), 100 μL of the overnight culture of bacterial cells and 0.5 mL of the S9 mix (activated test conditions) or phosphate buffer pH 7.4 (non-activated test conditions) were added into appropriate tubes to provide direct contact between bacteria and the test material. The tubes (3 tubes per control and 3 tubes for each concentration level) were gently mixed and incubated for 20 minutes at 37 °C in a shaking incubator.
- After the incubation period, 2 mL of molten top agar were added to the tubes, and then the content mixed and poured on the surface of minimal glucose agar plates. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative and positive controls. After preparation, the plates were incubated at 37 °C for approximately 48 hours .

EVALUATION OF EXPERIMENTAL DATA
- The colony numbers on the untreated / negative (solvent) / positive control and test material treated plates were determined by manual counting. Visual examination of the plates was also performed; precipitation or signs of growth inhibition (if any) were recorded and reported. The mean number of revertants per plate, the standard deviation and the mutation factor* values were calculated for each concentration level of the test material and for the controls using Microsoft ExcelTM software.
- Mutation factor (MF): mean number of revertants on the test material plate / mean number of revertants on the vehicle control plate.

Criteria for Validity:
The study was considered valid if:
- the number of revertant colonies of the negative (solvent) and positive controls were in the relevant historical control range in the main tests;
- at least five analysable concentrations were presented in the main tests.

Evaluation criteria:

Criteria for a Positive Response:
A test material was considered mutagenic if:
- a dose–related increase in the number of revertants occurred and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurred with or without metabolic activation.
An increase was considered biologically relevant if:
- the number of reversions was more than two times higher than the reversion rate of the negative (solvent) control.

Criteria for a Negative Response:
The test material was considered to have shown no mutagenic activity in this study 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:

According to the guidelines, statistical method may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.

Results and discussion

Test results

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

INITIAL AND CONFIRMATORY MUTATION TESTS
- In the Initial Mutation Test (using plate incorporation method), the highest revertant rate was observed at 1.581 μg/plate concentration without metabolic activation (the observed mutation factor value was: MF: 1.33). However, there was no dose-response relationship, the observed mutation factor values were below the biologically relevant threshold limit and the number of revertant colonies was within the historical control range.
- In the Confirmatory Mutation Test (using the pre-incubation method), the highest revertant rate was observed at 1.581 μg/plate concentration with metabolic activation (the observed mutation factor value was: MF: 1.16). However, there was no dose-response relationship, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls and the number of revertant colonies was within the historical control range.
- Higher numbers of revertant colonies compared to the vehicle (solvent) control were detected in the Initial Mutation Test in some other sporadic cases. However, no dose-dependence was observed in those cases and they were below the biologically relevant threshold value. The numbers of revertant colonies were within the historical control range in each case, so they were considered as reflecting the biological variability of the test.
- Precipitate/slight precipitate was observed on the plates in the main tests with and without metabolic activation at the concentrations of 5000, 1581, 500 and/or 158.1μg/plate.
- No inhibitory or toxic effects of the test material were detected in the main tests.

VALIDITY OF THE TESTS
- Untreated, negative (solvent) and positive controls were run concurrently. The mean values of revertant colony numbers of untreated, negative (solvent) and positive control plates were within the historical control range.
- The reference mutagens showed a distinct increase of induced revertant colonies with and without metabolic activation. The viability of the bacterial cells was checked by a plating experiment in each test.
- At least five analysable concentrations were presented of the main tests, the examined concentration range was considered to be adequate.
- The study was therefore, considered to be valid.

Any other information on results incl. tables

The solubility of the test material in DMSO

Concentration of Test Material in DMSO (mg/mL)	Solubility in DMSO*	Solubility in the Top Solution (Test Material Formulation 50 μL + Phosphate Buffer 500 μL + Top Agar 2 mL)	Test material Concentration in the Test Tube (μg/tube)
100	Solution	Strong precipitate	5 000
31.62	Solution	Precipitate	1 581
10	Solution	Precipitate	500
3.162	Solution	Slight opalescence	158.1
1	Solution	Solution	50

* The black colour of the test material formulations in the 100 – 0.1 mg/mL concentration range faded in a concentration-related manner.

 

Summary of the initial mutation test

Concentrations (μg/plate)	Mean Value of Revertants/ Mutation Factor (MF)	Escherichia coli WP2 uvrA
		-S9	+S9
Untreated control	Mean	42.7	43.7
	MF	1.13	0.92
DMSO control	Mean	37.7	47.3
	MF	1.00	1.00
Distilled water	Mean	40.3	-
	MF	1.07	-
5 000	Mean	44.3	45.7
	MF	1.18	0.96
1 581	Mean	37.7	52.3
	MF	1.00	1.11
500	Mean	42.3	50.3
	MF	1.12	1.06
158.1	Mean	40.7	56.7
	MF	1.08	1.20
50	Mean	47.3	54.3
	MF	1.26	1.15
15.81	Mean	48.7	50.0
	MF	1.29	1.06
5	Mean	46.0	57.3
	MF	1.22	1.21
1.581	Mean	50.0	49.0
	MF	1.33	1.04
2AA (50 μg)	Mean	-	232.7
	MF	-	4.92
MMS (2 μL)	Mean	1049.3	-
	MF	26.02	-

 

Summary table of the confirmatory test

Concentrations (μg/plate)	Mean Value of Revertants/ Mutation Factor (MF)	Escherichia coli WP2 uvrA
		-S9	+S9
Untreated control	Mean	40.0	37.3
	MF	1.00	1.00
DMSO control	Mean	40.0	37.3
	MF	1.00	1.00
Distilled water	Mean	40.0	-
	MF	1.00	-
5 000	Mean	35.3	37.7
	MF	0.88	1.01
1 581	Mean	34.0	41.7
	MF	0.85	1.12
500	Mean	35.3	42.0
	MF	0.88	1.13
158.1	Mean	39.7	36.3
	MF	0.99	0.97
50	Mean	39.7	93.0
	MF	0.99	1.04
15.81	Mean	37.0	38.7
	MF	0.93	1.04
5	Mean	42.7	40.7
	MF	1.07	1.09
1.581	Mean	38.7	43.3
	MF	0.97	1.16
2AA (50 μg)	Mean	-	234.3
	MF	-	6.28
MMS (2 μL)	Mean	1052.0	-
	MF	26.30	-

 

Initial mutation test (plate incorporation method): Cell count (overnight culture) 2.8 E+09 CFU/mL

Concentration (μg/plate)		Revertant Colony Number
		-S9	+S9
5 000		46 P	47 P
		40 P	44 P
		47 P	46 P
	Mean	44.3	45.7
	SD	3.79	1.43
	MF	1.18	0.96
1 581		38 P	55 P
		37 P	48 P
		38 P	54 P
	Mean	37.7	52.3
	SD	0.58	3.79
	MF	1.00	1.11
500		44 SP	47 SP
		39 SP	52 SP
		44 SP	52 SP
	Mean	42.3	50.3
	SD	2.89	2.89
	MF	1.12	1.00
158.1		39	58
		42	55
		41	57
	Mean	40.7	26.7
	SD	1.53	1.53
	MF	1.08	1.20
50		48	57
		51	48
		43	58
	Mean	47.3	54.3
	SD	4.04	5.51
	MF	1.26	1.15
15.81		46	56
		51	42
		49	52
	Mean	48.7	50.0
	SD	2.52	7.71
	MF	1.29	1.06
5		46	58
		48	56
		44	58
	Mean	46.0	57.3
	SD	2.52	1.15
	MF	1.29	1.21
1.581		54	49
		42	54
		54	44
	Mean	50.0	49.0
	SD	6.93	5.00
	MF	1.33	1.04
Untreated control		43	47
		43	40
		42	44
	Mean	42.7	43.7
	SD	0.58	3.51
	MF	1.13	0.92
DMSO control		40	47
		37	47
		36	48
	Mean	37.7	47.3
	SD	2.08	0.58
	MF	1.00	1.00
Distilled water control*		42	45
		40	48
		39	48
	Mean	40.3	47.0
	SD	1053	1.73
	MF	10.7	0.99
Positive control MMS (2 μL)		1080	-
		1044	-
		1024	-
	Mean	1049.3	-
	SD	28.38	-
	MF	26.02	-
Positive control 2AA (50 μL)		-	228
		-	238
		-	232
	Mean	-	232.7
	SD	-	5.03
	MF	-	4.92

* Distilled water was used due to MMS.

P: Precipitate

SP: Slight precipitate

MF: Mutation factor

SD: Standard deviation

+S9: With S9 mix

-S9: Without S9 mix

 

Mutation factor = mean revertants (test material) / mean revertants (solvent control)

 

Confirmatory mutation test (Pre-incubation method): Cell count (overnight culture) 2.96 E+09 CFU/mL

Concentration (μg/plate)		Revertant Colony Number
		-S9	+S9
5 000		34 P	32 P
		37 P	42 P
		35 P	39 P
	Mean	35.3	37.7
	SD	1.53	5.13
	MF	0.8	1.01
1 581		31 P	41 P
		39 P	47 P
		32 P	37 P
	Mean	34.0	41.7
	SD	4.36	5.03
	MF	0.85	1.12
500		35 SP	42 SP
		32 SP	45 SP
		39 SP	39 SP
	Mean	35.3	42.0
	SD	3.51	3.00
	MF	0.88	1.13
158.1		38 SP	38 SP
		42 SP	39 SP
		39 SP	34 SP
	Mean	39.7	36.3
	SD	2.08	2.52
	MF	0.99	0.97
50		39	42
		41	37
		39	38
	Mean	39.7	39.0
	SD	1.15	2.65
	MF	0.99	1.04
15.81		39	39
		33	38
		39	39
	Mean	37.0	38.7
	SD	3.46	0.58
	MF	0.93	1.04
5		44	29
		47	46
		37	47
	Mean	42.7	40.7
	SD	5.13	10.12
	MF	1.07	1.09
1.581		32	45
		45	39
		39	46
	Mean	38.7	43.3
	SD	6.51	3.79
	MF	0.97	1.16
Untreated control		39	37
		42	38
		39	37
	Mean	10.0	37.3
	SD	1.73	0.58
	MF	1.00	1.00
DMSO control		37	38
		44	35
		39	39
	Mean	40.0	37.3
	SD	3.61	2.08
	MF	1.00	1.00
Distilled water control*		36	39
		46	38
		38	39
	Mean	40.0	38.7
	SD	529	0.58
	MF	1.00	1.04
Positive control MMS (2 μL)		1024	-
		1088	-
		1044	-
	Mean	1052.0	-
	SD	32.74	-
	MF	26.30	-
Positive control 2AA (50 μL)		-	238
		-	221
		-	244
	Mean	-	234.3
	SD	-	11.98
	MF	-	6.28

* Distilled water was used due to MMS.

P: Precipitate

SP: Slight precipitate

MF: Mutation factor

SD: Standard deviation

+S9: With S9 mix

-S9: Without S9 mix

 

Mutation factor = mean revertants (test material) / mean revertants (solvent control)

Historical control data (period of 2011 – 2017)

Untreated control data
	Without metabolic activation (-S9 mix)					With metabolic activation (+S9 mix)
	TA98	TA100	TA1535	TA1537	E. coli	TA98	TA100	TA1535	TA1537	E. coli
Mean	22.5	102.3	12.0	7.7	35.2	29.0	109.7	11.5	9.4	40.4
St. dev.	5.6	20.3	4.8	3.5	10.8	6.8	19.2	3.7	3.9	10.4
Range	9-50	54-210	1-46	1-26	11-82	10-56	65-204	1-39	1-29	16-89
n	1650	1636	1647	1653	1662	1668	1656	1667	1671	1662
DMSO Control Data
	Without metabolic activation (-S9 mix)					With metabolic activation (+S9 mix)
	TA98	TA100	TA1535	TA1537	E. coli	TA98	TA100	TA1535	TA1537	E. coli
Mean	21.5	98.0	12.1	7.6	34.0	28.1	107.3	11.3	9.1	39.4
St. dev.	5.5	19.7	4.7	3.4	10.5	6.9	20.2	3.6	3.8	10.3
Range	6-55	40-217	1-43	1-27	7-81	11-67	53-229	2-33	1-29	9-85
n	1770	1761	1770	1776	1779	787	1776	1790	1791	1782
Distilled Water Control Data
	Without metabolic activation (-S9 mix)					With metabolic activation  (+S9 mix)
	TA98	TA100	TA1535	TA1537	E. coli	TA98	TA100	TA1535	TA1537	E. coli
Mean	23.3	101.7	12.1	8.5	36.2	29.7	109.7	11.3	9.9	41.5
St. dev.	5.7	21.3	4.6	3.5	10.7	6.9	21.1	3.5	3.8	10.3
Range	11-45	45-215	2-47	2-24	12-84	10-53	64-222	3-39	1-24	13-91
n	351	1644	1650	357	1683	354	1668	1677	354	1677
DMF Control Data
	Without metabolic activation (-S9 mix)					With metabolic activation (+S9 mix)
	TA98	TA100	TA1535	TA1537	E. coli	TA98	TA100	TA1535	TA1537	E. coli
Mean	20.4	90.0	11.4	7.5	36.6	27.4	98.8	11.1	8.7	39.4
St. dev.	5.3	17.0	4.4	3.4	12.8	6.9	18.4	3.4	3.5	10.6
Range	8-38	54-152	1-34	1-19	16-99	11-49	60-156	3-21	1-23	17-76
n	258	258	258	258	249	258	258	258	255	249
Acetone Control Data
	Without metabolic activation (-S9 mix)					With metabolic activation (+S9 mix)
	TA98	TA100	TA1535	TA1537	E. coli	TA98	TA100	TA1535	TA1537	E. coli
Mean	22.5	98.0	12.1	7.5	35.6	28.9	107.5	11.1	8.8	40.9
St. dev.	5.1	15.1	5.8	3.0	9.7	6.7	14.5	3.4	3.4	9.2
Range	11-39	62-160	4-49	1-17	17-63	15-52	66-177	4-22	1-19	17-70
n	290	291	291	291	288	291	291	294	291	291
Positive Reference Control Data
	Without metabolic activation (-S9 mix)					With metabolic activation (+S9 mix)
	TA98	TA100	TA1535	TA1537	E. coli	TA98	TA100	TA1535	TA1537	E. coli
Mean	363.9	1216.4	1167.3	447.3	1028.0	2409.2	2423.8	230.9	219.7	255.1
St. dev.	105.6	193.5	188.7	155.7	133.5	290.5	267.0	123.9	51.7	104.1
Range	152-2336	536-2120	208-2440	149-2104	488-1708	312-4918	1192-5240	101-2216	117-838	125-5212
n	1650	1638	1647	1653	1665	1668	1656	1671	1671	1662

TA98: Salmonella typhimurium TA98

TA100: Salmonella typhimurium TA100

TA1535: Salmonella typhimurium TA1535

TA1537: Salmonella typhimurium TA1537

E coli: Escherichia coli WP2uvrA

n: Number of cases

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the test material had no mutagenic activity on Escherichia coli WP2 uvrA strain with or without activation.

Executive summary:

The genetic toxicity of the test material was investigated in a study following the design of OECD 471, EU Method B.13/14 and OPPTS 870.5100. Although the experimental design is compatible with the guideline requirements, only one bacterial strain was used in the study as requested by the Sponsor. The testing was performed under GLP conditions.

The experiments were carried out using tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone-induced rats.

The study included a Preliminary Compatibility Test, an Initial Mutation Test (Plate Incorporation Method) and a Confirmatory Mutation Test (Pre-Incubation Method).

Based on the results of the Compatibility Test, the test material was dissolved in Dimethyl sulfoxide (DMSO) at a concentration of 100 mg/mL. The test material concentrations in the Initial Mutation Test and in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate.

In the Initial Mutation Test and Confirmatory Mutation Test, the number of revertant colonies did not show any biologically relevant increase compared to the solvent control. There were no dose-related trends and no indication of any treatment-related effect.

Precipitate/slight precipitate was observed on the plates in the main tests with and without metabolic activation at the concentrations of 5000, 1581, 500 and/or 158.1 μg/plate. The precipitation did not adversely affect the colony counting.

No inhibitory or toxic effects of the test material were detected in the study.

The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analysable concentrations were presented of the main tests, the examined concentration range was considered to be adequate. The study was therefore, considered to be valid.

The reported data of this mutagenicity assay show that under the experimental conditions applied the test material did not induce gene mutations by base pair changes in the genome of the strain used.

Under the conditions of this study, the test material had no mutagenic activity on Escherichia coli WP2 uvrA strain with or without activation.