2-Propanone, reaction products with diphenylamine

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 to 24 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:

OECD Guidelines for testing of Chemicals, Section 4, No. 471 “Bacterial Reverse Mutation Test”, adopted 21st 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:

Commission Regulation (EC) No. 440/2008, B.13/14. “Mutagenicity: Reverse Mutation Test Using Bacteria”, 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:

EPA Health Effects Test Guidelines, OPPTS 870.5100 “Bacterial Reverse Mutation Test”, EPA 712-C-98-247, August 1998
EPA Health Effects Test Guidelines, OPPTS 870.5100 “Escherichia coli WP2 and WP2 uvrA Reverse Mutation Assays”, EPA 712-C-96-247, June 1996 (Public Draft)

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:

2-Propanone, reaction products with diphenylamine

EC Number:

270-192-0

EC Name:

2-Propanone, reaction products with diphenylamine

Cas Number:

68412-48-6

Molecular formula:

Variable.

IUPAC Name:

N-cyclohexylcyclohexanamine; propan-2-one

Test material form:

solid: particulate/powder

Details on test material:

Name: AMINOX®
Chemical name: 2-Propanone, reaction products with diphenylamine
Batch/Lot number: EL5D06K347
Appearance: Green to brown powder
CAS number: 68412-48-6
Purity: 100% (as a UVCB)
Retest date: 05 April 2018
Storage conditions: Room temperature
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.

Specific details on test material used for the study:

No further details specified in the study report.

Method

Target gene:

histidine and tryptophan

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix

Test concentrations with justification for top dose:

Based on the results of the Compatibility Test and available information, the test item formulated in Acetone. Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 μg/plate were examined in the Range Finding Test. Based on the results of the Range Finding Test, the test item concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50 and 15.81 g test item/plate and Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg test item/plate. Examined concentrations in the Complementary Confirmatory Mutation Test were 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg test item/plate in Salmonella typhimurium strains without metabolic activation and 500, 158.1, 50, 15.81, 5 and 1.581 μg test item/plate in Escherichia coli WP2 uvrA strain without metabolic activation.

Vehicle / solvent:

Acetone was used as vehicle to prepare the stock formulation of the test material.
Acetone:
Supplier: SIGMA-ALDRICH / VWR
Batch No.: 15J060514
Expiry date: 31 October 2020

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 4-nitro-1,2-phenylene-diamine (NPD); 2-aminoanthracene (2-AA)

Details on test system and experimental conditions:

The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test, a Confirmatory Mutation Test and a Complementary Confirmatory Mutation Test. In the Range Finding Test as well as in the Initial Mutation Test, the plate incorporation method was used. In the Confirmatory Mutation Test and Complementary Confirmatory Mutation Test, the pre-incubation method was used.

Concentrations
Concentrations were selected on the basis of the Preliminary Compatibility Test and Preliminary Concentration Range Finding Test (Informatory Toxicity Test). In the Initial Mutation Test, Confirmatory Mutation Test and Complementary Confirmatory Mutation Test different concentrations were used.

Preliminary Compatibility Test
The solubility of the test item was examined using Distilled water, Dimethyl sulfoxide (DMSO) and Acetone. Test item was insoluble at 100 mg/mL concentration using Distilled water. Partial dissolution was observed at the same concentration using DMSO. The test item was soluble at this concentration using Acetone, therefore Acetone was selected as vehicle (solvent) for the study.
The obtained stock formulation (50 μL) with the solution of top agar and phosphate buffer was examined in a test tube without test bacterium suspension.

Preliminary Concentration Range Finding Test (Informatory Toxicity Test)
Based on the solubility test, 100 mg/mL stock formulation was prepared in Acetone which was diluted in 6 steps by factors of 2, 2.5 and 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, 2500, 1000, 316, 100, 31.6 and 10 μg/plate of the test item. In the Preliminary Concentration Range Finding Test the plate incorporation method was used.

Test Item Concentrations in the Mutagenicity Tests (Initial Mutation Test, Confirmatory Mutation Test and Complementary Confirmatory Mutation Test)
Based on the results of the preliminary tests, 100 mg/mL stock formulation was prepared from the test item with Acetone, which was diluted by serial dilutions in at last five steps to obtain at last six dosing formulations. The maximum test concentration was 5000 μg test item/plate in the main tests.
Examined concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50 and 15.81 μg test item/plate.
Examined concentrations in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg test item/plate.
Examined concentrations in the Complementary Confirmatory Mutation Test were 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg test item/plate in Salmonella typhimurium strains without metabolic activation and 500, 158.1, 50, 15.81, 5 and 1.581 μg test item/plate in Escherichia coli WP2 uvrA strain without metabolic activation.

Control Groups Used in the Tests
Strain-specific positive and negative (vehicle/solvent) controls, both with and without metabolic activation were included in each test. In addition, untreated control was used demonstrating that the chosen vehicle (solvent) induced no deleterious or mutagenic effects.

Procedure for Exposure in the 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 item both in the presence and absence of an appropriate metabolic activation system.
Molten top agar was prepared and kept at 45 °C. 2 mL of top agar was aliquoted into individual test tubes (3 tubes per control or concentration level). The equivalent number of minimal glucose agar plates was properly labelled. The test item and other components were prepared freshly and added to the overlay (45 °C).
The content of the tubes:
top agar 2000 μL
vehicle (solvent) or test item formulation (or reference controls) 50 μL
overnight culture of test strain 100 μL
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 (vehicle/solvent) and positive controls. After preparation, the plates were incubated at 37 °C for 48 ± 1 hours.

Procedure for Exposure in the Confirmatory Mutation Test and Complementary Confirmatory Mutation Test
A pre-incubation procedure was performed as a Confirmatory Mutation Test and Complementary Confirmatory Mutation Test since in the Initial Mutation Test no positive effect was observed.
Before the overlaying, the test item formulation (or vehicle/solvent or reference control), the bacterial culture and the S9 mix or phosphate buffer was added into appropriate tubes to provide direct contact between bacteria and the test item (in its vehicle/solvent).
The tubes (3 tubes per control or concentration level) were gently mixed and incubated for 20 min at 37 °C in a shaking incubator. After the incubation period, 2 mL of molten top agar was added to the tubes; the content was mixed up and poured onto minimal glucose agar plates as described for the standard plate incorporation method. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative (vehicle/solvent) and positive controls. After preparation, the plates were incubated at 37 °C for 48 ± 1 hours.

Rationale for test conditions:

The experimental methods were conducted according to the methods described by Ames et al. and Maron and Ames, Kier et al., Venitt and Parry, OECD Guideline No. 471, 1997, Commission Regulation (EC) No. 440/2008, 2008, EPA Guidelines, OPPTS 870.5100, 1998, 1996, and according to the relevant SOPs of CiToxLAB Hungary Ltd.

Evaluation criteria:

The colony numbers on the untreated / negative (vehicle/solvent) / positive control and test item 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 item and for the controls using Microsoft ExcelTM software.
* Mutation factor (MF): mean number of revertants on the test item 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 (vehicle/solvent) and positive controls were in the historical control range in all strains of the main tests;
- at least five analyzable concentrations were presented in all strains of the main tests.

Criteria for a Positive Response:
A test item 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 in at least one strain with or without metabolic activation.

An increase was considered biologically relevant if:
- the number of reversions is more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains;
- the number of reversions is more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.

Criteria for a Negative Response:
A test article was considered non-mutagenic if it produced 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

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, but tested up to precipitating concentrations

Remarks:

Precipitate was observed in all tester strains at 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 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Precipitate was observed in all tester strains at 5000 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 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Precipitate was observed in all tester strains at 5000 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 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Precipitate was observed in all tester strains at 5000 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, but tested up to precipitating concentrations

Remarks:

Precipitate was observed in all tester strains at 5000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

PRELIMINARY RANGE FINDING TEST (INFORMATORY TOXICITY TEST)
In the Preliminary Range Finding Test, the plate incorporation method was used. The preliminary test was performed using Salmonella typhimurium TA98 and Salmonella typhimurium TA100 tester strains in the presence and absence of metabolic activation system (±S9 Mix) with appropriate untreated, negative (vehicle/solvent) and positive controls. In the test each sample (including the controls) was tested in triplicate.
In the Range Finding Test the concentrations examined were: 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate.
The observed number of revertant colonies was in the normal range. Minor differences compared to the solvent control numbers were observed. However, they had no biological relevance and were situated within the historical control range most probably reflecting the variability of the test system.
Precipitate / slight precipitate was observed in both tester strains with and without metabolic activation at the concentrations of 5000 and 2500 μg/plate. Precipitate did not interfere with the colony counting or the background lawn growth evaluation.
Inhibitory, cytotoxic effects of the test item (slightly reduced background lawn development) were observed in both tester strains with and without metabolic activation at the concentration of 5000 μg/plate in the Preliminary Range Finding Test.

INITIAL AND CONFIRMATORY MUTATION TESTS
In the Initial Mutation Test, the plate incorporation method; in the Confirmatory Mutation Test, the pre-incubation method was used. The Initial Mutation Test and Confirmatory Mutation Test were carried out using four Salmonella typhimurium strains (TA98, TA100, TA1535 and TA1537) and Escherichia coli WP2 uvrA strain.
The Initial Mutation Test and Confirmatory Mutation Test were performed in the presence and absence of metabolic activation system (±S9 mix). Each test was performed with appropriate untreated, negative (vehicle/solvent) and positive controls.
In the main tests each sample (including the controls) was tested in triplicate.
In the Confirmatory Mutation Test using the pre-incubation method, excessive cytotoxicity was observed in all bacterial strains without metabolic activation at several concentrations. In this case, the number of analyzable doses did not meet the recommendations of the test guidelines. Therefore, an additional experiment (Complementary Confirmatory Mutation Test) was performed in these strains in an additional experimental period (Experimental Period III) to complete the data. The experimental conditions were the same as in the Confirmatory Mutation Test.
Examined concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50 and 15.81 μg test item/plate.
Examined concentrations in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg test item/plate.
Examined concentrations in the Complementary Confirmatory Mutation Test were 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg test item/plate in Salmonella typhimurium strains without metabolic activation and 500, 158.1, 50, 15.81, 5 and 1.581 μg test item/plate in Escherichia coli WP2 uvrA strain without metabolic activation.
In the Initial Mutation Test and Confirmatory Mutation Test none of the observed revertant colony numbers were above the respective biological threshold value. There were no reproducible dose-related trends.
In the Initial Mutation Test (plate incorporation method), the highest revertant rate was observed in Salmonella typhimurium TA1535 bacterial strain without metabolic activation at the concentrations of 1581 μg/plate. The mutation factor value at each concentration was 1.65. However, there was no dose-response relationship, the observed mutation factor values were below the biologically relevant threshold limit and the numbers of revertant colonies were within the historical control range.
In the Confirmatory Mutation Test and Complementary Confirmatory Mutation Test (pre-incubation method), the highest revertant rate was observed in Salmonella typhimurium TA1535 bacterial strain at 5 μg/plate concentration with metabolic activation. The mutation factor value was 1.46. However, there was no dose-response relationship, the observed mutation factor values were below the biologically relevant threshold limit and the numbers of revertant colonies were within the historical control range.
Higher numbers of revertant colonies compared to the solvent control were detected in the Initial Mutation Test and Confirmatory Mutation Tests in some other cases as well.
However, no dose-dependence was observed and they were below the biologically relevant threshold value and were within the historical control range, they were considered as reflecting the biological variability of the test.
Slightly lower revertant counts compared to the solvent control were observed in the Initial Mutation Test and Confirmatory Mutation Tests at some non-cytotoxic concentrations. However, the mean numbers of revertant colonies were within the historical control range, thus they were considered as biological variability of the test system.
Inhibitory, cytotoxic effects of the test item (reduced / slightly reduced background lawn development) were observed in the Initial Mutation Test without metabolic activation at 5000 μg/plate concentration in Salmonella typhimurium TA100, in the Confirmatory Mutation Tests with metabolic activation at 5000 and 1581 μg/plate concentrations in all examined bacterial strains and in Complementary Confirmatory Mutation Test without metabolic activation at 50 μg/plate concentration in Salmonella typhimurium strains; at 500 μg/plate concentrations in Escherichia coli WP2 uvrA strain.
Precipitate was observed in the Initial Mutation Test in all tester strains at 5000 μg/plate concentrations with and without metabolic activation and Confirmatory Mutation Tests in all tester strains at 5000 μg/plate concentrations with metabolic activation. Precipitate did not interfere with the colony counting or the background lawn growth evaluation.

VALIDITY OF THE TESTS
Untreated, negative (vehicle/solvent) and positive controls were run concurrently. The mean values of revertant colony numbers of untreated, negative (vehicle/solvent) and positive control plates were within the historical control range. At least five analysable concentrations were presented in all strains of the main tests.
The reference mutagens showed a distinct increase of induced revertant colonies. The viability of the bacterial cells was checked by a plating experiment in each test. The tests were considered to be valid.

Any other information on results incl. tables

Summary Table of the Range Finding Test

Concentrations (μg/plate)	Mean values of revertants / Mutation factor (MF)	Salmonella typhimurium strains
		TA98		TA100
		-S9	+S9	-S9	+S9
Untreated control	Mean	22.0	23.3	110.0	114.3
	MF	0.99	1.00	1.01	1.01
Distilled water control	Mean	-	-	108.3	-
	MF	-	-	0.99	-
DMSO control	Mean	23.7	23.0	-	111.3
	MF	1.06	0.99	-	0.99
Acetone control	Mean	22.3	27.0	51.7	71.0
	MF	1.00	1.00	1.00	1.00
5000	Mean	24.3	27.0	51.7	71.0
	MF	1.09	1.16	0.47	0.63
2500	Mean	27.3	26.7	115.3	114.7
	MF	1.22	1.14	1.05	1.02
1000	Mean	24.3	26.0	119.3	112.0
	MF	1.09	1.11	1.09	0.99
316	Mean	23.3	28.0	114.3	124.0
	MF	1.04	1.20	1.05	1.10
100	Mean	21.0	26.7	122.7	133.0
	MF	0.94	1.14	1.12	1.18
31.6	Mean	23.3	26.0	125.0	121.0
	MF	1.04	1.11	1.14	1.07
10	Mean	22.3	23.0	117.7	120.7
	MF	1.00	0.99	1.08	1.07
NPD (4μg)	Mean	412.0	-	-	-
	MF	17.41	-	-	-
2AA (2μg)	Mean	-	2426.7	-	2373.3
	MF	-	105.51	-	21.32
SAZ (2μg)	Mean	-	-	1205.3	-
	MF	-	-	11.13	-

-: Not applicable

 

Summary Table of the Initial Mutation Test

Concentrations (μg/plate)	Mean values of revertants / Mutation factor (MF)	Salmonella typhimurium tester strains								Escherichia coli
		TA98		TA100		TA1535		TA1537		WP2 uvrA
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Untreated control	Mean	15.36	27.7	85.7	77.3	10.3	8.3	13.3	16.3	48.3	48.0
	MF	0.71	1.26	0.96	1.08	1.19	0.89	0.98	1.04	0.98	1.04
Distilled water control	Mean	-	-	81.0	-	10.7	-	-	-	46.0	-
	MF	-	-	0.91	-	1.23	-	-	-	0.93	-
DMSO control	Mean	14.7	20.3	-	83.3	-	11.3	14.0	14.7	-	53.0
	MF	0.68	0.92	-	1.16	-	1.21	1.02	0.94	-	1.15
Acetone control	Mean	21.7	22.0	89.0	71.7	8.7	9.3	13.7	15.7	49.3	46.0
	MF	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
5000	Mean	17.0	18.0	52.3	64.3	13.7	9.3	8.7	14.0	42.3	49.0
	MF	0.78	0.82	0.59	0.90	1.58	1.00	0.63	0.89	0.86	1.07
1581	Mean	21.0	20.0	95.3	69.3	14.3	13.0	13.7	10.3	44.0	52.0
	MF	0.97	0.91	1.07	0.97	1.65	1.39	1.00	0.66	0.89	1.13
500	Mean	18.7	20.7	85.0	60.7	12.3	12.3	15.0	14.7	46.0	44.7
	MF	0.86	0.94	0.96	0..85	1.42	1.32	1.10	0.94	0.93	0.97
158.1	Mean	22.7	21.7	83.0	68.0	11.3	9.0	14.7	16.0	36.7	47.7
	MF	1.05	0.98	0.93	0.95	1.31	0.96	1.07	1.02	0.74	1.04
50	Mean	21.3	21.3	83.7	73.3	11.7	10.0	17.3	13.7	40.3	46.7
	MF	0.98	0.97	0.94	1.02	1.35	1.07	1.27	0.87	0.82	1.01
15.81	Mean	21.0	24.0	84.0	79.3	13.7	9.7	10.7	13.0	45.3	42.3
	MF	0.97	1.09	0.94	1.11	1.58	1.04	0.78	0.83	0.92	0.92
NPD (4μg)	Mean	404.7	-	-	-	-	-	-	-	-	-
	MF	27.59	-	-	-	-	-	-	-	-	-
2AA (2μg)	Mean	-	2474.7	-	2405.3	-	206.7	-	207.3	-	-
	MF	-	121.70	-	28.86	-	18.24	-	14.14	-	-
2AA (50μg)	Mean	-	-	-	-	-	-	-	-	-	253.7
	MF	-	-	-	-	-	-	-	-	-	4.79
SAZ (2μg)	Mean	-	-	1205.3	-	1214.0	-	-	-	-	-
	MF	-	-	14.88	-	113.81	-	-	-	-	-
9AA (50μg)	Mean	-	-	-	-	-	-	418.7	-	-	-
	MF	-	-	-	-	-	-	29.90	-	-	-
MMS (2μL)	Mean	-	-	-	-	-	-	-	-	990.7	-
	MF	-	-	-	-	-	-	-	-	21.54	-

-: Not applicable

 

Summary Table of the Confirmatory Mutation Test and Complementary Confirmatory Mutation Test

Concentrations (μg/plate)	Mean values of revertants / Mutation factor (MF)	Salmonella typhimurium tester strains								Escherichia coli
		TA98		TA100		TA1535		TA1537		WP2 uvrA
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Untreated control	Mean	20.0	27.3	98.7	104.0	11.0	10.0	10.3	13.0	66.7	67.7
	MF	1.22	1.15	1.02	1.13	0.80	1.07	1.19	1.08	1.09	1.05
Distilled water control	Mean	-	-	94.3	-	12.3	-	-	-	62.7	-
	MF	-	-	0.97	-	0.90	-	-	-	1.03	-
DMSO control	Mean	17.3	27.0	-	87.3	-	11.0	8.3	15.3	-	71.0
	MF	0.96	1.14	-	0.95	-	1.18	0.96	1.28	-	1.10
Acetone control	Mean	18.0	23.7	97.0	92.3	13.7	9.3	8.7	12.0	61.0	64.7
	MF	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
5000	Mean	-	15.7	-	66.3	-	5.7	-	5.7	-	54.7
	MF	-	0.66	-	0.72	-	0.61	-	0.47	-	0.85
1581	Mean	-	15.3	-	54.3	-	9.3	-	6.3	-	56.0
	MF	-	0.65	-	0.59	-	1.00	-	0.53	-	0.87
500	Mean	-	27.3	-	77.3	-	11.7	-	11.3	29.3	54.7
	MF	-	1.15	-	0.84	-	1.25	-	0.94	0.48	0.85
158.1	Mean	-	29.7	-	96.3	-	11.3	-	16.0	50.7	56.7
	MF	-	1.25	-	1.04	-	1.21	-	1.33	0.83	0.88
50	Mean	14.0	28.7	60.3	94.7	5.3	8.0	2.7	13.7	59.3	59.7
	MF	0.78	1.21	0.63	1.03	0.39	0.86	0.31	1.14	0.97	0.92
15.81	Mean	16.7	28.3	82.0	93.7	11.7	12.7	6.3	13.7	65.7	68.3
	MF	0.93	1.20	0.85	1.01	0.85	1.36	0.73	1.14	1.08	1.06
5	Mean	21.7	24.3	94.0	102.3	11.3	13.7	7.3	13.7	68.3	67.7
	MF	1.20	1.03	0.97	1.11	0.83	1.46	0.85	1.14	1.12	1.05
1.581	Mean	19.7	-	96.7	-	9.3	-	9.7	-	68.0	-
	MF	1.09	-	1.00	-	0.68	-	1.12	-	1.11	-
0.5	Mean	21.3	-	94.7	-	9.7	-	8.3	-	-	-
	MF	1.19	-	0.98	-	0.71	-	0.96	-	-	-
0.1581	Mean	20.0	-	92.0	-	10.7	-	8.3	-	-	-
	MF	1.11	-	0.95	-	0.78	-	0.96	-	-	-
NPD (4μg)	Mean	338.7	-	-	-	-	-	-	-	-	-
	MF	19.54	-	-	-	-	-	-	-	-	-
2AA (2μg)	Mean	-	2325.3	-	2373.3	-	226.7	-	205.3	-	-
	MF	-	86.12	-	27.18	-	20.61	-	13.39	-	-
2AA (50μg)	Mean	-	-	-	-	-	-	-	-	-	286.0
	MF	-	-	-	-	-	-	-	-	-	4.03
SAZ (2μg)	Mean	-	-	1134.7	-	1068.0	-	-	-	-	-
	MF	-	-	12.03	-	86.59	-	-	-	-	-
9AA (50μg)	Mean	-	-	-	-	-	-	476.0	-	-	-
	MF	-	-	-	-	-	-	57.12	-	-	-
MMS (2μL)	Mean	-	-	-	-	-	-	-	-	1084.0	-
	MF	-	-	-	-	-	-	-	-	17.30	-

-: Not applicable

 

Historical Control Data

(Period of 2011 – 2016)

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	22.7	103.6	11.8	7.2	33.4	29.7	111.7	11.5	8.9	39.1
St. dev.	5.8	21.4	5.1	3.3	9.7	6.8	19.6	3.9	3.8	9.9
Range	9-50	54-210	1-46	1-24	11-82	10-56	65-204	1-39	1-29	19-89
n	1371	1357	1365	1371	1374	1377	1365	1373	1380	1371
DMSO control data
	Without metabolic activation (-S9 Mix)					With metabolic activation (+S9 Mix)
	TA98	TA100	TA1535	TA1537	E. coli	TA98	TA100	TA1535	TA1536	E. coli
Mean	21.7	98.9	12.0	7.1	32.3	28.7	109.5	11.3	8.7	38.1
St. dev.	5.7	20.7	5.0	3.3	9.6	7.0	20.7	3.8	3.7	9.7
Range	6-55	40-217	1-43	1-25	7-81	11-67	53-229	2-33	1-29	9-85
n	1482	1473	1479	1485	1482	1487	1476	1487	1491	1482
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	23.5	103.2	11.9	7.8	34.5	30.8	112.2	11.3	9.3	40.3
St. dev.	6.0	22.4	4.9	3.4	9.8	7.1	21.8	3.7	3.7	10.0
Range	11-45	45-215	2-47	2-24	12-84	10-53	64-222	3-39	1-24	13-91
n	267	1359	1365	270	1392	267	1371	1380	267	1383
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	89.9	11.2	6.9	34.7	28.1	100.3	11.0	8.0	38.0
St. dev.	5.6	17.8	4.7	3.1	12.3	7.0	19.2	3.6	3.1	10.2
Range	8-38	54-152	1-34	1-19	16-99	13-49	60-156	3-21	1-23	17-76
n	216	216	216	216	207	216	216	216	213	207
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	22.6	98.1	12.1	7.4	35.0	29.1	108.1	11.1	8.6	40.5
St. dev.	5.1	15.4	5.8	2.9	9.3	6.7	14.2	3.4	3.3	9.0
Range	11-39	62-160	4-49	1-17	17-62	15-52	66-177	4-22	1-19	17-69
n	278	279	279	279	276	279	279	282	279	279
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	357.2	1229.3	1169.8	454.1	1034.3	2410.2	2429.6	235.1	221.3	257.4
St. dev.	113.8	207.5	204.2	169.7	141.7	317.7	291.6	135.9	56.2	113.4
Range	152-2336	536-2120	208-2440	149-2104	488-1708	312-4918	1192-5240	101-2216	117-838	125-2512
n	1371	1359	1365	1371	1377	1378	1365	1377	1380	1371

TA98: Salmonella typhimurium TA98, TA100: Salmonella typhimurium TA100, TA1535: Salmonella typhimurium TA1535, TA1537: Salmonella typhimurium TA1537, E.coli: Escherichia coli WP2 uvrA, n: number of cases.

Applicant's summary and conclusion

Conclusions:

The test item AMINOX® was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.

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 WP2 uvrA) in the presence and absence of a metabolic activation system, which was a cofactor-supplemented post-mitochondrial S9 fraction prepared from the livers of phenobarbital/β-naphthoflavone-induced rats.

The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test, a Confirmatory Mutation Test and a Complementary Confirmatory Mutation Test. In the Range Finding Test as well as in the Initial Mutation Test, the plate incorporation method was used. In the Confirmatory Mutation Test and Complementary Confirmatory Mutation Test, the preincubation method was used.

The reported data of the 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 AMINOX® had no mutagenic activity in the applied bacterium tester strains under the test conditions used in this study.

Executive summary:

The test item AMINOX® was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.

 

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 WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavoneinduced rats.

 

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

 

Based on the results of the Compatibility Test and available information, the test item formulated in Acetone. Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 μg/plate were examined in the Range Finding Test. Based on the results of the Range Finding Test, the test item concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50 and 15.81 μg test item/plate and Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg test item/plate. Examined concentrations in the Complementary Confirmatory Mutation Test were 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg test item/plate in Salmonella typhimurium strains without metabolic activation and 500, 158.1, 50, 15.81, 5 and 1.581 μg test item/plate in Escherichia coli WP2 uvrA strain without metabolic activation.

 

In the Initial Mutation Test and Confirmatory Mutation Test none of the observed revertant colony numbers were above the respective biological threshold value when compared to the solvent control and were within the normal biological variability of the test system. There were no dose-related trends and no indication of any treatment effect.

 

Inhibitory, cytotoxic effects of the test item were observed in the Initial Mutation Test in Salmonella typhimurium TA100 strain without metabolic activation at 5000 μg test item/plate and in the Confirmatory Mutation Test in all examined strains with metabolic activation at 5000 and 1581 μg test item/plate and Complementary Confirmatory Mutation Test in all Salmonella typhimurium strains without metabolic activation at 50 μg/plate concentrations and in Escherichia coli WP2 uvrA strain without metabolic activation at 500 μg/plate.

 

Precipitate was detected on the plates in the Initial Mutation Test and Confirmatory Mutation Test in all examined strains with or without metabolic activation at 5000 μg concentration. Precipitate did not interfere with the colony counting or the background lawn growth evaluation.

 

The mean values of revertant colonies of the 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 analyzable concentrations were presented in all strains of the main tests. The tests were considered to be valid.

 

The reported data of the 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 AMINOX® had no mutagenic activity in the bacterium tester strains under the test conditions used in this study.