Reaction mass of 2-ethylhexyl (3-isocyanato-4-methylphenyl)carbamate and 2-ethylhexyl (5-isocyanato-2-methylphenyl)carbamate and 2-ethylhexyl (3-isocyanato-2-methylphenyl)carbamate

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:

12. Jun. 2018 to 21. Jun. 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)

Version / remarks:

OECD Guidelines for the Testing of Chemicals Part 471, adopted 21. Jul. 1997 “Bacterial Reverse Mutation Test“

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, EU-Method B.13/14 adopted 30. May 2008 “Mutagenicity –Reverse mutation test using bacteria”

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:

Reaction mass of 2-ethylhexyl (3-isocyanato-4-methylphenyl)carbamate and 2-ethylhexyl (5-isocyanato-2-methylphenyl)carbamate and 2-ethylhexyl (3-isocyanato-2-methylphenyl)carbamate

EC Number:

946-383-6

Molecular formula:

C17H24N2O3

IUPAC Name:

Reaction mass of 2-ethylhexyl (3-isocyanato-4-methylphenyl)carbamate and 2-ethylhexyl (5-isocyanato-2-methylphenyl)carbamate and 2-ethylhexyl (3-isocyanato-2-methylphenyl)carbamate

Test material form:

liquid: viscous

Details on test material:

Name: Trixene AS
Batch no.: OP544-153/1801239973
Appearance: clear, light yellow-green, viscous liquid
Composition: Reaction mass of 2-ethylhexyl (3-isocyanato-4-methylphenyl)carbamate and 2-ethylhexyl (5-isocyanato-2-methylphenyl)carbamate and 2-ethylhexyl (3-isocyanato-2-methylphenyl)carbamate
EINECS-No.: 946-383-6
Molecular formula: C17-H24-N2-O3
Purity: "multi-constituent substance made up of a mixture of isomers
Homogeneity: Homogeneous
Vapour pressure: not stated
Expiry date: 01. Sep. 2019
Storage Room: Temperature (20 ± 5°C)

Specific details on test material used for the study:

No further details specified in the study report.

Method

Target gene:

Histidene

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA97a, TA98, TA100, TA102 and TA1535

Details on mammalian cell type (if applicable):

Origin and Culture
All Salmonella typhimurium strains were obtained from TRINOVA BioChem GmbH (batch: TA97a: 4997D, TA98: 5011D, TA100: 4996D, TA102: 4982D, TA1535: 5012D) and were stored as lyophilizates in the refrigerator at 2-8 °C.
The lyophilizates were used to prepare permanent cultures which were filled into vials and stored at < - 75 °C.
Eight hours before the start of each experiment, an aliquot of a permanent culture per strain to be used was taken from the deep freezer to inoculate a culture vessel containing nutrient broth. After incubation overnight for eight hours at 37 ± 1 °C, the cultures were used in the experiment. During the test, the cultures were stored at room temperature as to prevent changes in the titre.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 was obtained by Trinova Biochem GmbH, Gießen.
Batch nos.: batch: 3850 for the first experiment and batch: 3913 and 3833 for the second experiment
Specification: produced from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254/kg body weight intraperitoneally.

Test concentrations with justification for top dose:

The stock solution was used to prepare the geometric series of the concentrations to be tested.
The following nominal concentrations were prepared for the first experiment:
5 μL/plate, 1.5 μL/plate, 0.5 μL/plate, 0.15 μL/plate and 0.05 μL/plate
Based on the non- toxic results of the first experiment, the test item was tested up to con-centrations of 5 μL/plate in the presence and absence of S9-mix in all bacteria strains using the pre-incubation method.
The following nominal concentrations were prepared for the second experiment:
5 μL/plate, 2.5 μL/plate, 1.25 μL/plate, 0.63 μL/plate, 0.31 μL/plate, 0.16 μL/plate, 0.08 μL/plate and 0.04 μL/plate

Vehicle / solvent:

In a non-GLP pre-test, the solubility of the test item was tested in DMSO.
The liquid test item is sufficiently soluble in DMSO.
Based on the non-GLP pre-test, DMSO was chosen as vehicle, because the test item was sufficiently soluble and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.
On the day of the start of the first and the second experiment, a stock solution containing 50 mL/L (nominal concentration) of the test item in DMSO was prepared. The test item solution was not sterile filtrated before use.

The following substances were used as solvent controls:
• DMSO, batch: 246245640 and batch: 187256959, for the positive controls nitro-phenylendiamine, benzo-a-pyrene and 2-amino-anthracene and the test item
• Demineralised water, batch: 20170309 and batch: 20180221 for the positive control sodium azide

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: 4-Nitro-1,2-phenylene Diamine; 2-Amino-Anthracene

Details on test system and experimental conditions:

Culture of Bacteria
Eight hours before the start of each experiment, one vial permanent culture of each strain was taken from the deep freezer and an aliquot was put into a culture flask containing nutri-ent broth. After incubation for eight hours at 37 ±1 °C, the cultures were used in the experi-ment. During the test, the cultures were stored at room temperature as to prevent changes in the titre.

Conduct of Experiment
Preparations
Different media and solutions were prepared preliminary (exact production dates are docu-mented in the raw data).
On the day of the test, the bacteria cultures were checked for growth visually. The incubation chambers were heated to 37 ±1 °C. The water bath was turned to 43 ±1 °C. The table sur-face was disinfected.
The S9 mix was freshly prepared and stored at 0 °C.

Experimental Parameters
First Experiment
Concentrations tested: 5 / 1.5 / 0.5 / 0.15 / 0.05 μL/plate
Incubation time: 48 h
Incubation temperature: 37 ±1 °C
Tested strains: TA97a, TA98, TA100, TA102, TA1535
Method: plate incorporation method

Second Experiment
Concentrations tested: 5 / 2.5 / 1.25 / 0.63 / 0.31 / 0.16 / 0.08 / 0.04 μL/plate
Incubation time: 48 h
Incubation temperature: 37 ±1 °C
Tested strains: TA97a, TA98, TA100, TA102, TA1535
Method: pre-incubation method

Description of the Method
General preparation
Per bacteria strain and concentration, three plates with and three plates without metabolic activation (-S9) were used.
For the top agar 100 mL agar basis was melted in a microwave oven, 10 mL of the histidine-biotin-solution 0.5 mM was added, then the mixture was placed in the water bath at 43 ±1 °C.

Plate incorporation method
The following materials were gently vortexed in a test tube and poured onto the selective agar plates:
100 μL test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control)
500 μL S9 mix or phosphate buffer (for test without metabolic activation).
100 μL bacteria suspension
2000 μL overlay agar (top agar)
The plates were closed and left to solidify for a few minutes, then inverted and placed in the dark incubator at 37 ±1 °C.

Pre-incubation method
The following materials were gently vortexed in a test tube and incubated at 37 ±1°C for 20 minutes:
100 μL test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control)
500 μL S9 mix or phosphate buffer (for test without metabolic activation).
100 μL bacteria suspension
After the pre-incubation for 20 minutes, 2000 μL top agar was added and the tube was gently vortexed. The mixture was poured onto the selective agar plate.
The plates were closed and left to solidify for a few minutes, then inverted and placed in the incubator at 37 ±1 °C.

References and Validity
Genotype Confirmation
Genotype confirmation is performed for each batch of lyophilized bacteria before stock cul-ture preparation.

Histidine requirement
Each strain was streaked on a biotin and a histidine-biotin-plate, using a sterilized wire loop. The plates were incubated for 24 hours at 37 ±1 °C.

Ampicillin/Tetracycline-Resistance (pKM 101, pAQ1)
Each strain was streaked on an ampicillin agar plate and on an ampicillin-tetracycline agar plate. TA1535 was used as control strain, since it is not ampicillin resistant. The plates were incubated for 24 hours at 37 ±1 °C.

UV-sensitivity (uvrB)
Each strain was streaked on a plate, and one half of the plate covered with aluminum foil so that one half of each streak was protected against light. The plates for the strain TA97a, TA100 and TA102 were irradiated for 8 seconds, the plates for the strain TA 98 were irradi-ated for 10 seconds and the plates for the strain TA1535 were irradiated for 6 seconds with a germicidal lamp (254 nm, 30W).
Keeping a distance of 33 cm for the strains TA97a, TA102 and TA1535.
Keeping a distance of 66 cm for the following strains: TA98, TA100.
Incubation for 24 hours at 37 ±1 °C followed.

Crystal violet sensitivity (deep rough/rfa)
For each strain, two plates were used. 0.1 mL of bacteria suspension were mixed with 2 mL Top-Agar and poured on nutrient agar. Sterile paper discs ( 9 mm), each soaked with 10 μL of crystal violet solution (0.1%) were placed into the middle of each plate, followed by incubation for 24 hours at 37 ±1°C.

Spontaneous Revertants
Three replicates, with/without S9, for each solvent which was used in the test, incubation for 48 hours at 37 ±1°C.

Determination of Titre
The titre was determined by dilution of the overnight culture using sodium chloride solution and placing 0.1 mL on maximal-soft agar. Incubation for 48 hours at 37 ±1 °C followed. It should give a density of 109 cells/mL (at the least), two replicates with and without metabolic activation.

Toxicity Control
Performed in experiment 1 only analogously to the titre control with the maximum dose of test item on maximal-soft agar, two replicates with and without metabolic activation, incuba-tion for 48 hours at 37 ±1°C.

Sterility Control
Performed analogously to the test with solvent only and S9 (without adding bacteria) on top agar, incubation for 48 hours at 37 ±1°C, four replicates.

Solubility
Plates were checked for precipitation of test item at the end of the incubation by visual in-spection.

Positive Controls
Using diagnostic mutagens, three replicates were prepared. The stock solutions of the substances were diluted to achieve an application volume of 0.1 mL/plate, incubation for 48 hours at 37 ±1°C.

Rationale for test conditions:

In accordance with test guidelines.

Evaluation criteria:

The colonies were counted visually and the numbers were recorded. A validated spread-sheet software (Microsoft Excel®) was used to calculate mean values and standard devia-tions of each treatment, solvent control and positive control.
The mean values and standard deviations of each threefold determination were calculated as well as the increase factor of revertant induction (mean revertants divided by mean spon-taneous revertants) of the test item solutions and the positive controls. Additionally, the ab-solute number of revertants (mean revertants minus less spontaneous revertants) was given.
A substance is considered to be mutagenic, if a reproducible increase with or without meta-bolic activation of revertant colonies per plate exceeding an increase factor of 2 for the bac-teria strains TA97a, TA98, TA100 and TA102 and an increase factor of 3 for the bacteria strain TA1535 compared to vehicle controls in at least one strain can be observed and there is a concentration-related increase.
A substance is not mutagenic if it does not meets these criteria.
If the criteria listed above are not clearly met, the results will be assessed as equivocal and will be discussed.

Statistics:

Not specified.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium, other: TA97a

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5, 2.5 and 1.25 μL/plate (decrease in the number of revertants)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5 and 2.5 μL/plate (decrease in the number of revertants)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5, 2.5 and 1.25 μL/plate (decrease in the number of revertants)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5 μL/plate (decrease in the number of revertants)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5 and 2.5 μL/plate (decrease in the number of revertants)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

First Experiment
Confirmation of the Criteria and Validity
All strains met the criterion of at least 109 bacteria/mL, and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory. All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and nearly all were within the historical control data ranges (one exception, only).

Solubility and Toxicity
In the first experiment, the test item showed no precipitates on the plates in all tested concentrations.
No signs of toxicity towards the bacteria strains could be observed. The bacterial back-ground lawn was visible and not affected. The number of revertant colonies was not reduced.

Mutagenicity
No increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found.
Therefore, the test item is stated as not mutagenic under the conditions of this experiment.
To verify this result, a further experiment was performed with the pre- incubation method.

Second Experiment
Confirmation of the Criteria and Validity
All strains met the criterion of at least 109 bacteria/mL, and no inconsistencies were found in the sterility control. Nearly all determined values for the spontaneous revertants of the neg-ative controls (one exception, only) were in the normal range of the test laboratory. All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and were within the historical control data ranges.

Solubility and Toxicity
In the second experiment, the test item showed no precipitates on the plates in all tested concentrations.
Signs of toxicity were observed in the following concentrations:
TA97a: 5, 2.5 and 1.25 μL/plate (decrease in the number of revertants)
TA98: 5 and 2.5 μL/plate (decrease in the number of revertants)
TA100: 5, 2.5 and 1.25 μL/plate (decrease in the number of revertants)
TA102: 5 μL/plate (decrease in the number of revertants)
TA1535: 5 and 2.5 μL/plate (decrease in the number of revertants)
In all concentrations the bacterial background lawn was visible and not affected.

Mutagenicity
No increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found.
Therefore, the test item is stated as not mutagenic under the conditions of this experiment.

Mutagenicity of Test Item
The test item Trixene AS showed no increase in the number of revertants in all bacteria strains in both experiments.
Nearly all negative (one exception, only) and nearly all strain-specific positive control (one exception, only) values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
Based on the results of this study it is concluded that Trixene AS is not mutagenic in the Salmonella typhimurium test strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in the present study.

Acceptability of Study, Discussion
In all experiments, no precipitation of the test item Trixene AS was observed at any of the tested concentrations up to 5 μL/plate.
In the first experiment, the test item caused no cytotoxicity towards all bacteria strains
In the second experiment, the test item caused cytotoxicity towards all bacteria strains in the different concentrations.
The confirmation tests of the genotype did not show any irregularities. The control of the titre was above the demanded value of 109 bacteria/mL.
Nearly all of the means of all replicates of the spontaneous revertants (in negative and sol-vent controls) were within the range of the historical data of the test facility. Nearly all num-bers of revertant colonies of the positive controls were within the range of the historical data of the laboratory, but all were increased in comparison with the negative controls, which demonstrated the mutagenic po-tential of the diagnostic mutagens.
Since all criteria for acceptability have been met, the study is considered valid.

Any other information on results incl. tables

Survey of the Findings

The mean revertant values of the three replicates are presented in the following table

Mean Revertants First Experiment

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. Water	Mean	79	90	43	49	78	77	324	339	13	16
	sd	13.3	16.4	5.5	9.0	3.5	3.2	45.4	19.7	1.0	4.2
DMSO	Mean	80	99	44	41	86	86	299	347	16	14
	sd	24.6	5.7	4.9	9.5	12.7	7.0	37.8	19.7	3.5	3.1
Positive Controls*	Mean	512	1125	392	227	1256	1312	1200	1163	251	224
	sd	72.8	50.8	28.0	40.9	133.1	181.7	104.0	46.9	26.0	90.9
	f(l)	6.40	11.36	8.91	5.54	16.10	15.26	4.01	3.35	19.31	16.00
5 µL/plate	Mean	77	79	42	40	75	77	315	264	11	11
	sd	6.7	11.2	2.1	8.1	10.5	2.6	39.5	36.7	3.5	1.2
	f(l)	0.96	0.80	0.95	0.98	0.87	0.90	1.05	0.76	0.69	0.79
1.5 µL/plate	Mean	81	87	38	40	86	89	251	256	13	15
	sd	11.0	7.0	2.6	9.9	5.9	1.2	31.1	6.9	3.2	3.0
	f(l)	1.01	0.88	0.86	0.98	1.00	1.03	0.84	0.74	0.81	1.07
0.5 µL/plate	Mean	81	91	38	39	73	93	276	323	13	15
	sd	15.5	18.7	2.6	5.3	11.0	16.0	62.5	45.0	1.0	3.1
	f(l)	1.01	0.92	0.86	0.95	0.85	1.08	0.92	0.93	0.81	1.07
0.15 µL/plate	Mean	73	91	40	42	85	74	252	272	17	12
	sd	2.3	15.3	3.1	12.5	5.0	1.0	34.9	58.9	3.0	2.1
	f(l)	0.91	0.92	0.91	1.02	0.99	0.86	0.84	0.78	1.06	0.86
0.05 µL/plate	Mean	80	79	43	38	83	79	284	297	13	18
	sd	8.5	6.1	7.9	8.0	3.1	10.1	46.1	28.4	2.1	3.6
	f(l)	1.00	0.80	0.98	0.93	0.97	0.92	0.95	0.86	0.81	1.29

f(l) = increase factor

*Different positive controls were used.

 

Survey of the Findings

The mean revertant values of the three replicates are presented in the following table

Mean Revertants First Experiment

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. Water	Mean	79	90	38	33	104	91	395	352	14	15
	sd	18.2	3.0	5.1	1.2	12.7	5.0	33.3	60.4	2.9	3.5
DMSO	Mean	71	81	40	36	87	86	419	384	13	15
	sd	2.5	12.3	4.9	1.7	19.3	5.9	59.0	113.4	2.1	4.6
Positive Controls*	Mean	465	701	311	135	543	1251	1253	1456	344	181
	sd	177.1	99.8	73.4	48.0	142.6	56.2	44.1	112.0	42.3	28.1
	f(l)	6.55	8.65	7.78	3.75	5.22	14.55	2.99	3.79	24.57	12.07
5 µL/plate	Mean	14	15	4	4	34	32	79	50	6	5
	sd	3.5	1.5	1.7	1.0	4.9	8.5	25.7	10.6	2.3	2.1
	f(l)	0.20	0.19	0.10	0.11	0.39	0.37	0.19	0.13	0.46	0.33
2.5 µL/plate	Mean	12	36	2	12	37	44	331	377	5	4
	sd	3.5	3.1	1.0	3.0	9.5	14.1	37.8	34.9	2.1	2.5
	f(l)	0.17	0.44	0.05	0.33	0.43	0.51	0.79	0.98	0.37	0.27
1.25 µL/plate	Mean	18	36	42	38	34	42	339	397	14	12
	sd	18.4	10.5	4.2	1.2	17.8	8.9	114.4	75.6	3.5	0.6
	f(l)	0.25	0.44	1.05	1.06	0.39	0.49	0.81	1.03	1.08	0.80
0.63 µL/plate	Mean	90	87	40	40	91	87	352	411	13	14
	sd	18.0	23.4	3.1	2.3	13.0	6.4	36.7	100.0	4.0	1.5
	f(l)	1.27	1.07	1.00	1.11	1.05	1.01	0.84	1.07	1.00	0.93
0.31 µL/plate	Mean	78	100	43	40	88	91	395	349	16	17
	sd	12.5	11.1	2.1	3.5	6.4	14.9	119.8	12.2	1.2	2.3
	f(l)	1.10	1.23	1.09	1.11	1.01	1.06	0.94	0.91	1.23	1.13
0.16 µL/plate	Mean	79	101	37	38	78	84	336	427	13	17
	sd	7.2	9.9	4.9	7.5	5.5	12.6	94.3	30.3	2.1	1.0
	f(l)	1.11	1.25	0.93	1.06	0.90	0.98	0.80	1.11	1.00	1.13
0.08 µL/plate	Mean	93	113	38	39	86	99	344	360	15	10
	sd	25.3	18.0	1.0	5.3	12.8	28.6	89.1	32.0	4.0	0.6
	f(l)	1.31	1.40	0.95	1.08	0.99	1.15	0.82	0.94	1.15	0.67
0.04 µL/plate	Mean	79	115	34	44	83	83	397	363	14	13
	sd	19.1	10.3	5.6	6.2	11.1	8.1	20.1	46.2	4.2	4.6
	f(l)	1.11	1.42	0.85	1.22	0.95	0.97	0.95	0.95	1.08	0.87

f(l) = increase factor

*Different positive controls were used.

 

DATA OF THE FIRST EXPERIMENT

 

Determination of Titre

Criterion: The determination of titre should give a number of at least 10⁹cells/mL, correlating to 100 colonies/plate after dilution.

The criterion was fulfilled, exact values are given in the table below.

Titre Values (colonies per plate)

Strain	TA97a		TA98		TA100		TA102		TA1535
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	1001	1001	1001	1001	1001	1001	1001	1001	1001	1001
Repl. 2	1001	1001	1001	1001	1001	1001	1001	1001	1001	1001
Mean	1001	1001	1001	1001	1001	1001	1001	1001	1001	1001
Stand. Dev.	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
Assessment	ok	ok	ok	ok	ok	ok	ok	ok	ok	ok

1001 colonies per plate means the bacteria growth was too strong for counting.

 

Sterility Control

Criterion: No colony per plate may grow.

Sterility (colonies per plate)

	Demin. water	DMSO
Repl. 1	0	0
Repl. 2	0	0
Repl. 3	0	0
Repl. 4	0	0
Assessment	ok	ok

 

Spontaneous Revertants

The determined values were within the normal range of the laboratory.

 

Demin. Water

Spontaneous Revertants demin. water (colonies per plate)

Strain	TA97a		TA98		TA100		TA102		TA1535
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	94	104	47	40	81	81	344	316	12	21
Repl. 2	70	94	46	48	74	75	356	348	14	13
Repl. 3	72	72	37	58	78	76	272	352	13	15
Mean	79	90	43	49	78	77	324	339	13	16
sd	13.3	16.4	5.5	9.0	3.5	3.2	45.4	19.7	1.0	4.2

 

DMSO

Spontaneous Revertants DMSO (colonies per plate)

Strain	TA97a		TA98		TA100		TA102		TA1535
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	61	93	42	40	84	83	256	356	16	17
Repl. 2	108	104	41	32	75	81	328	324	12	15
Repl. 3	72	101	50	51	100	94	312	360	19	11
Mean	80	99	44	41	86	86	299	347	16	14
sd	24.6	5.7	4.9	9.5	12.7	7.0	37.8	19.7	3.5	3.1

 

Positive Controls

Without metabolic activation:

4-Nitro-1,2-phenylene diamine (NPD) in DMSO, 20 µg/plate

Sodium azide (Na-azide) in demineralized water, 1 µg/plate

With metabolic activation

2-Amino anthracene (2-AA) in DMSO, 1 µg/plate

Benzo-a-pyrene (BaP) in DMSO, 20 µg/plate

 

Diagnostic Mutagens (colonies per plate)

Strain	TA97a		TA98		TA100		TA102		TA1535
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Substance	NPA	2-AA	NPD	BaP	Na-azide	2-AA	NPA	2-AA	Na-azide	2-AA
Repl. 1	596	1096	424	244	1160	1104	1304	1144	276	160
Repl. 2	468	1096	380	180	1408	1392	1096	1128	224	184
Repl. 3	472	1184	372	256	15200	1440	1200	1216	252	328
Mean	512	1125	392	227	1256	1312	1200	1163	251	224
sd	72.8	50.8	28.0	40.9	133.1	181.7	104.0	46.9	26.0	90.9
f(l)	6.40	11.36	8.91	5.54	16.10	15.26	4.01	3.35	19.31	16.00
Rev. abs.	432	1026	348	186	1178	1226	901	816	238	210

f(l) = increase factor.

Rev. abs. = absolute revertants.

 

Test Item Trixene AS

Mutagenicity Test

Concentration 5 µL/plate (colonies per plate)

Strain	TA97a		TA98		TA100		TA102		TA1535
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	84	76	44	44	85	75	288	296	13	10
Repl. 2	71	69	41	46	75	80	296	224	7	10
Repl. 3	75	91	40	31	64	76	360	272	13	12
Mean	77	79	42	40	75	77	315	264	11	11
sd	6.7	11.2	2.1	8.1	10.5	2.6	39.5	36.7	3.5	1.2
f(l)	0.96	0.80	0.95	0.98	0.87	0.90	1.05	0.76	0.69	0.79
Rev. abs.	-3	-20	-2	-1	-11	-9	16	-83	-5	-3

 

Concentration 1.5 µL/plate (colonies per plate)

Strain	TA97a		TA98		TA100		TA102		TA1535
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	70	80	37	45	93	90	216	260	9	15
Repl. 2	92	86	36	29	82	88	260	248	15	12
Repl. 3	81	94	41	47	84	90	276	260	14	18
Mean	81	87	38	40	86	89	251	256	13	15
sd	11.0	7.0	2.6	9.9	5.9	1.2	31.1	6.9	3.2	3.0
f(l)	1.01	0.88	0.86	0.98	1.00	1.03	0.84	0.74	0.81	1.07
Rev. abs.	1	-12	-6	-1	0	3	-48	-91	-3	1

 

Concentration 0.5 µL/plate (colonies per plate)

Strain	TA97a		TA98		TA100		TA102		TA1535
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	94	94	36	41	62	108	348	312	12	12
Repl. 2	86	71	41	43	74	76	244	372	14	18
Repl. 3	64	108	37	33	84	94	236	284	13	16
Mean	81	91	38	39	73	93	276	323	13	15
sd	15.5	18.7	2.6	5.3	11.0	16.0	62.5	45.0	1.0	3.1
f(l)	1.01	0.92	0.86	0.95	0.85	1.08	0.92	0.93	0.81	1.07
Rev. abs.	1	-8	-6	-2	-13	7	-23	-24	-3	1

 

Concentration 0.15 µL/plate (colonies per plate)

Strain	TA97a		TA98		TA100		TA102		TA1535
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	70	104	37	56	84	75	236	284	20	10
Repl. 2	74	94	43	38	90	74	292	208	17	11
Repl. 3	74	74	41	32	80	73	228	324	14	14
Mean	73	91	40	42	85	74	252	272	17	12
sd	2.3	15.3	3.1	12.5	5.0	1.0	34.9	58.9	3.0	2.1
f(l)	0.91	0.92	0.91	1.02	0.99	0.86	0.84	0.78	1.06	0.86
Rev. abs.	-7	-8	-4	1	-1	-12	-47	-75	1	-2

 

Concentration 0.05 µL/plate (colonies per plate)

Strain	TA97a		TA98		TA100		TA102		TA1535
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	83	78	49	38	86	78	336	292	11	21
Repl. 2	86	86	34	30	80	90	248	272	12	14
Repl. 3	70	74	46	46	84	70	268	328	15	19
Mean	80	79	43	38	83	79	284	297	13	18
sd	8.5	6.1	7.9	8.0	3.1	10.1	46.1	28.4	2.1	3.6
f(l)	1.00	0.80	0.98	0.93	0.97	0.92	0.95	0.86	0.81	1.29
Rev. abs.	0	-20	-1	-3	-3	-7	-15	-50	-3	4

 

 

DATA OF THE SECOND EXPERIMENT

 

Determination of Titre

Criterion: The determination of titre should give a number of at least 10⁹cells/mL, correlating to 100 colonies/plate after dilution.

The criterion was fulfilled, exact values are given in the table below.

Titre Values (colonies per plate)

Strain	TA97a		TA98		TA100		TA102		TA1535
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	1001	1001	1001	1001	1001	1001	1001	1001	1001	1001
Repl. 2	1001	1001	1001	1001	1001	1001	1001	1001	1001	1001
Mean	1001	1001	1001	1001	1001	1001	1001	1001	1001	1001
Stand. Dev.	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
Assessment	ok	ok	ok	ok	ok	ok	ok	ok	ok	ok

1001 colonies per plate means the bacteria growth was too strong for counting.

 

Sterility Control

Criterion: No colony per plate may grow.

Sterility (colonies per plate)

	Demin. water	DMSO
Repl. 1	0	0
Repl. 2	0	0
Repl. 3	0	0
Repl. 4	0	0
Assessment	ok	ok

 

Spontaneous Revertants

The determined values were within the normal range of the laboratory.

 

Demin. Water

Spontaneous Revertants demin. water (colonies per plate)

Strain	TA97a		TA98		TA100		TA102		TA1535
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	82	93	34	32	118	86	432	408	12	11
Repl. 2	59	87	37	32	93	90	368	288	12	18
Repl. 3	95	90	44	34	102	96	384	360	17	15
Mean	79	90	38	33	104	91	395	352	14	15
sd	18.2	3.0	5.1	1.2	12.7	5.0	33.3	60.4	2.9	3.5

 

DMSO

Spontaneous Revertants DMSO (colonies per plate)

Strain	TA97a		TA98		TA100		TA102		TA1535
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	71	95	46	37	83	93	440	256	15	19
Repl. 2	69	71	38	37	70	84	464	424	14	16
Repl. 3	74	78	37	34	108	82	352	472	11	10
Mean	71	81	40	36	87	86	419	384	13	15
sd	2.5	12.3	4.9	1.7	19.3	5.9	59.0	113.4	2.1	4.6

 

Positive Controls

Without metabolic activation:

4-Nitro-1,2-phenylene diamine (NPD) in DMSO, 20 µg/plate

Sodium azide (Na-azide) in demineralized water, 1 µg/plate

With metabolic activation

2-Amino anthracene (2-AA) in DMSO, 1 µg/plate

Benzo-a-pyrene (BaP) in DMSO, 20 µg/plate

 

Diagnostic Mutagens (colonies per plate)

Strain	TA97a		TA98		TA100		TA102		TA1535
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Substance	NPA	2-AA	NPD	BaP	Na-azide	2-AA	NPA	2-AA	Na-azide	2-AA
Repl. 1	664	740	336	184	528	1304	1256	1408	312	184
Repl. 2	324	776	228	134	692	1192	1208	1584	392	208
Repl. 3	408	588	368	88	408	1256	1296	1376	328	152
Mean	465	701	311	135	543	1251	1253	1456	344	181
sd	177.1	99.8	73.4	48.0	142.6	56.2	44.1	112.0	42.3	28.1
f(l)	6.55	8.65	7.78	3.75	5.22	14.55	2.99	3.79	24.57	12.07
Rev. abs.	394	620	271	99	439	1165	834	1072	330	166

f(l) = increase factor.

Rev. abs. = absolute revertants.

 

Test Item Trixene AS

Mutagenicity Test

Concentration 5 µL/plate (colonies per plate)

Strain	TA97a		TA98		TA100		TA102		TA1535
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	14	13	3	4	32	33	108	60	5	7
Repl. 2	17	16	6	3	40	23	70	52	9	3
Repl. 3	10	15	3	5	31	40	59	39	5	4
Mean	14	15	4	4	34	32	79	50	6	5
sd	3.5	1.5	1.7	1.0	4.9	8.5	25.7	10.6	2.3	2.1
f(l)	0.20	0.19	0.10	0.11	0.39	0.37	0.19	0.13	0.46	0.33
Rev. abs.	-57	-66	-36	-32	-53	-54	-340	-334	-7	-10

 

Concentration 2.5 µL/plate (colonies per plate)

Strain	TA97a		TA98		TA100		TA102		TA1535
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	8	33	3	12	26	59	360	348	7	4
Repl. 2	14	35	2	9	42	31	288	368	6	2
Repl. 3	14	39	1	15	43	42	344	416	3	7
Mean	12	36	2	12	37	44	331	337	5	4
sd	3.5	3.1	1.0	3.0	9.5	14.1	37.8	34.9	2.1	2.5
f(l)	0.17	0.44	0.05	0.33	0.43	0.51	0.79	0.98	0.38	0.27
Rev. abs.	-59	-45	-38	-24	-50	-42	-88	-7	-8	-11

 

Concentration 1.25 µL/plate (colonies per plate)

Strain	TA97a		TA98		TA100		TA102		TA1535
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	39	26	41	39	14	52	312	312	17	12
Repl. 2	5	47	39	37	48	39	464	424	14	12
Repl. 3	10	36	47	39	40	35	240	456	10	11
Mean	18	36	42	38	34	42	339	397	14	12
sd	18.4	10.5	4.2	1.2	17.8	8.9	114.4	75.6	3.5	0.6
f(l)	0.25	0.44	1.05	1.06	0.39	0.49	0.81	1.03	1.08	0.80
Rev. abs.	-53	-45	2	2	-53	-44	-80	13	1	-3

 

Concentration 0.63 µL/plate (colonies per plate)

Strain	TA97a		TA98		TA100		TA102		TA1535
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	110	108	43	37	84	84	320	512	9	12
Repl. 2	75	62	41	41	106	94	392	312	14	15
Repl. 3	85	92	37	41	83	82	344	408	17	14
Mean	90	87	40	40	91	87	352	411	13	14
sd	18.0	23.4	3.1	2.3	13.0	6.4	36.7	100.0	4.0	1.5
f(l)	1.27	1.07	1.00	1.11	1.05	1.01	0.84	1.07	1.00	0.93
Rev. abs.	19	6	0	4	4	1	-67	27	0	-1

 

Concentration 0.31 µL/plate (colonies per plate)

Strain	TA97a		TA98		TA100		TA102		TA1535
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	92	90	41	40	81	80	528	360	17	18
Repl. 2	69	98	44	36	93	108	360	336	17	18
Repl. 3	72	112	45	43	91	85	296	352	15	14
Mean	78	100	43	40	88	91	395	349	16	17
sd	12.5	11.1	2.1	3.5	6.4	14.9	119.8	12.2	1.2	2.3
f(l)	1.10	1.23	1.08	1.11	1.01	1.06	0.94	0.91	1.23	1.13
Rev. abs.	7	19	3	4	1	5	-24	-35	3	2

 

Concentration 0.16 µL/plate (colonies per plate)

Strain	TA97a		TA98		TA100		TA102		TA1535
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	84	106	40	45	81	96	416	392	12	18
Repl. 2	71	108	31	30	82	71	360	440	15	17
Repl. 3	83	90	39	38	72	86	232	448	11	16
Mean	79	101	37	38	78	84	336	427	13	17
sd	7.2	9.9	4.9	7.5	5.5	12.6	94.3	30.3	2.1	1.0
f(l)	1.11	1.25	0.93	1.06	0.90	0.98	0.80	1.11	1.00	1.13
Rev. abs.	8	20	-3	2	-9	-2	-83	43	0	2

 

Concentration 0.08 µL/plate (colonies per plate)

Strain	TA97a		TA98		TA100		TA102		TA1535
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	122	108	38	33	100	83	424	360	15	10
Repl. 2	84	98	39	43	83	132	248	328	11	10
Repl. 3	74	133	37	41	75	82	360	392	19	11
Mean	93	113	38	39	86	99	344	360	15	10
sd	25.3	18.0	1.0	5.3	12.8	28.6	89.1	32.0	4.0	0.6
f(l)	1.31	1.40	0.95	1.08	0.99	1.15	0.82	0.94	1.15	0.67
Rev. abs.	22	32	-2	3	-1	13	-75	-24	2	-5

 

Concentration 0.04 µL/plate (colonies per plate)

Strain	TA97a		TA98		TA100		TA102		TA1535
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	100	112	29	46	93	74	416	336	19	18
Repl. 2	73	106	33	49	84	90	376	336	13	10
Repl. 3	63	126	40	37	71	84	400	416	11	10
Mean	79	115	34	44	83	83	397	363	14	13
sd	19.1	10.3	5.6	6.2	11.1	8.1	20.1	46.2	4.2	4.6
f(l)	1.11	1.42	0.85	1.22	0.95	0.97	0.95	0.95	1.08	0.87
Rev. abs.	8	34	-6	8	-4	-3	-22	-21	1	-2

 

DATA OF THE CYTOTOXICITY TEST

The toxicity of the following concentration was tested: 5 µL/plate.

Per strain, 2 plates with and without metabolic activation were incubated with the corresponding dose of the test item on maximal soft agar.

 

Experiment Parameters

Concentration tested: 5 µL/plate

Incubation time: 48 h

Incubation temperature: 37 ± 1 °C

Tested strains: TA97a, TA98, TA100, TA102, TA1535

Method: Plate incorporation method

 

Determination of Titre

Criterion: The determination of titre should give a number of at least 10⁹cells/mL, correlating to 100 colonies/plate after dilution.

The criterion was fulfilled, exact values are given in the table below.

Titre Values (colonies per plate)

Strain	TA97a		TA98		TA100		TA102		TA1535
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	1001	1001	1001	1001	1001	1001	1001	1001	1001	1001
Repl. 2	1001	1001	1001	1001	1001	1001	1001	1001	1001	1001
Mean	1001	1001	1001	1001	1001	1001	1001	1001	1001	1001
Stand. Dev.	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
Assessment	ok	ok	ok	ok	ok	ok	ok	ok	ok	ok

1001 colonies per plate means the bacteria growth was too strong for counting.

 

Toxicity Control

The test item is considered non-toxic, if the quotient titre/toxicity is below 2.

5 µL/plate on maximal-soft-agar with culture diluted by 10⁶

Strain	TA97a		TA98		TA100		TA102		TA1535
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	1001	1001	1001	1001	1001	1001	1001	1001	1001	1001
Repl. 2	1001	1001	1001	1001	1001	1001	1001	1001	1001	1001
Mean	1001	1001	1001	1001	1001	1001	1001	1001	1001	1001
Stand. Dev.	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
Titre/Tox	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
Assessment	non-toxic	non-toxic	non-toxic	non-toxic	non-toxic	non-toxic	non-toxic	non-toxic	non-toxic	non-toxic

1001 colonies per plate means the bacteria growth was too strong for counting.

 

COMPAIRSON WITH HISTORICAL DATA

In the following table, the history of the spontaneous revertants and positive controls of the performed experiments with these strains up to 28. May 2018 is stated in comparison with the experiments performed within this study. Only experiments which were performed before the performance of the study were considered.

For the historical data, the plate incorporation method and the pre-incubation method were used.

Historical Data of Spontaneous Revertants

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. water	Mean	88	95	22	25	93	98	280	300	18	18
	Min	60	63	6	8	51	64	85	67	6	7
	Max	144	138	52	51	147	141	425	587	36	40
	SD	17	17	12	11	16	15	57	71	6	6
	Exp 1	79	90	43	49	78	77	324	339	13	16
	Exp 2	79	90	38	33	104	91	395	352	14	15
DMSO	Mean	88	97	22	24	90	93	280	293	18	17
	Min	58	67	7	8	44	62	79	80	8	6
	Max	135	144	47	50	138	199	413	459	35	37
	SD	17	16	12	12	16	17	55	60	6	6
	Exp 1	80	99	44	41	86	86	299	347	16	14
	Exp 2	71	81	40	36	87	86	419	384	13	15
Positive Controls*	Mean	534	526	413	129	487	781	1093	1197	263	134
	Min	264	228	77	39	220	273	491	408	55	45
	Max	1165	1181	1001	487	984	1912	2331	6083	515	712
	SD	169	168	169	101	155	284	409	558	84	79
	Exp 1	512	1125	392	227	1256	1312	1200	1163	251	224
	Exp 2	465	701	311	135	543	1251	1253	1456	344	181

*Different positive controls were used

 

The values which lie outside the range of the historical data are given in bold italics.

Bacteria strains are living biological systems, therefore variations in behavior are not unusual.

Each bacteria strain in this study has its own characteristic spontaneous revertant colony number. Day to day variations in the number of spontaneous revertant colonies are usual.

The variations of the values of all bacteria strains lie in an acceptable range.

Evaluation of the mutagenicity if the test item and validity of the study was not affected by the variations.

No critical impact on the outcome of the study is expected.

Applicant's summary and conclusion

Conclusions:

Based on the results of this study it is concluded that Trixene AS is not mutagenic in the Salmonella typhimurium test strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in the present study.

Executive summary:

Determination of the mutagenic potential of Trixene AS with the Bacterial Reverse Mutation Test following OECD 471 and EU B.13/14

 

Findings and Results:

Two valid experiments were performed.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

The test item Trixene AS was tested in the Salmonella typhimurium reverse mutation assay with five strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 and TA1535).

The test was performed in two experiments in the presence and absence of metabolic activation, with +S9 standing for presence of metabolic activation, and –S9 standing for absence of metabolic activation.

 

Experiment 1:

In the first experiment, the test item (dissolved in DMSO) was tested up to concentrations of 5 μL/plate in the presence and absence of S9-mix in the strains TA97a, TA98, TA100, TA102 and TA1535 using the plate incorporation method.

The test item showed no precipitates on the plates at any of the concentrations.

The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test item showed no signs of toxicity towards the bacteria strains in both the presence and absence of metabolic activation.

The results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

 

Experiment 2:

Based on the non- toxic results of the first experiment, the test item was tested up to concentrations of 5 μL/plate in the presence and absence of S9-mix in all bacteria strains using the pre-incubation method.

The test item showed no precipitates on the plates at any of the concentrations.

Signs of toxicity were observed in the following concentrations:

• TA97a: 5, 2.5 and 1.25 μL/plate (decrease in the number of revertants)

• TA98: 5 and 2.5 μL/plate (decrease in the number of revertants)

• TA100: 5, 2.5 and 1.25 μL/plate (decrease in the number of revertants)

• TA102: 5 μL/plate (decrease in the number of revertants)

• TA1535: 5 and 2.5 μL/plate (decrease in the number of revertants)

The bacterial background lawn was observed in all concentrations.

The results of this experiments showed that the test item caused no increase in the number of revertants in all bacteria strains compared to the solvent control, in both the presence and absence of metabolic activation. The test item did not induce a dose-related increase in the number of revertants colonies in all strains, in the presence and absence of metabolic activation.

 

Based on the results of this study it is concluded that Trixene AS is not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the presence and absence of metabolic activation under the experimental conditions in this study.