Quaternary ammonium compounds, C12-14 (even numbered) alkyl(hydroxyethyl)dimethyl, C12 alkylbis(hydroxyethyl)methyl, chlorides and amines, C12-14 (even numbered) alkyldimethyl, chlorides

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:

From October 9, 2018 to November 15, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted on 21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted 30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

liquid

Method

Species / strain

Species / strain / cell type:

other: Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535

Metabolic activation:

with and without

Metabolic activation system:

Rat liver homogenate metabolizing system (4% liver S9 in standard co-factors).

Test concentrations with justification for top dose:

Plate incorporation method
Experiment 1a: 0.05, 0.15, 0.5, 1.5 and 5 µL/plate, triplicate against each tester strain
Experiment 1b: 0.005, 0.015, 0.05, 0.15, 0.5, 1.5 µL/plate, triplicate against each tester strain

Pre-incubation method
Experiment 2: 0.006, 0.012, 0.023, 0.047, 0.094, 0.188, 0.375, 0.75 and 1.5 µL/plate

The maximum dose level of the test substance in the first experiment was selected as the maximum recommended dose level of 5 µL/plate. Both in test 1a and 1b no precipitation was observed in all the tested concentration. The test substance showed signs of toxicity towards all five bacterial strains in both absence and presence of metabolic activation in the highest concentration (5 µL/plate) and in the next lower concentration (1.5 µL/plate). The bacterial background lawn was not reduced at any of the concentrations in both the experiments. Based on the results of the experiment 1a and 1b, the test substance was tested up to concentrations of 1.5 µL/plate in the absence and presence of metabolic activation in all bacteria strain using the pre-incubation method. The test substance showed signs of toxicity (no bacteria growth and no bacterial background lawn) with 0.375, 0.75 and 1.5 µL/plate concentrations for all five tester strains, whereas TA100 and TA1535 showed signs of toxicity as decrease in the number of revertants with concentration of 0.188 µL/plate.

Vehicle / solvent:

In a non-GLP pre-test, the solubility of the test substance was tested in a concentration of 50 mL/L in de-mineralized water, dimethyl sulfoxide (DMSO) and acetone. The liquid test substance is sufficiently soluble in DMSO, only. Based on the non-GLP pre-test, DMSO was chosen as vehicle, because the test substance 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.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO and Demineralised water

True negative controls:

not specified

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:

Test system
All Salmonella typhimurium strains were obtained from TRINOVA BioChem GmbH (batch: TA97a: 5033D, TA98: 5136D, TA100: 5141D, TA102: 5145D, TA1535: 5138D) 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.

Preparation
In a non-GLP pre-test, the solubility of the test substance was tested in a concentration of 50 mL/L in de-mineralized water, dimethyl sulfoxide (DMSO) and acetone. The liquid test substance is sufficiently soluble in DMSO, only. Based on the non-GLP pre-test, DMSO was chosen as vehicle, because the test substance was sufficiently soluble, and this solvent does not have any effects on the viability of the bacteria or the number of sponta-neous revertants in the tested concentrations. On the day of the start of the experiment, a stock solution containing 50 mL/L of the test substance in DMSO was prepared. The test substance solution was not sterile filtrated before use. The stock solution was used to prepare the geometric series of the concentrations to be tested.

The following nominal concentrations were prepared for the experiment 1a for all bacteria strains: 5 µL/plate, 1.5 µL/plate, 0.5 µL/plate, 0.15 µL/plate and 0.05 µL/plate

On the day of the start of the experiment 1b and 2, a stock solution containing 15 µL/L of the test substance in DMSO was prepared. The following nominal concentrations were prepared for the experiment 1b for all bacteria strains: 5 µL/plate, 1.5 µL/plate, 0.5 µL/plate, 0.15 µL/plate, 0.05 µL/plate and 0.015 µL/plate

The following nominal concentrations were prepared for the experiment 2 for all bacteria strains: 1.5 µL/plate, 0.75 µL/plate, 0.38 µL/plate, 0.19 µL/plate, 0.09 µL/plate, 0.05 µL/plate, 0.03 µL/plate, 0.02 µL/plate and 0.01 µL/plate

Solvent Controls
The following substances were used as solvent controls:
Dimethylsulfoxide (DMSO), batch: 187256959, for the positive controls nitrophenylendiamine, benzo-a-pyrene and 2-amino-anthracene and batch: 218270919 for the test substance. Demineralised water, batch: 20180614 for the positive control sodium azide
 
Positive Controls
All mutagenic substances were stored as ready to use solution in the test facility in the deep freezer (-20 ± 5 °C). The respective positive control solution was thawed on the day of each experiment.

The following mutagenic substances were used as positive controls in all experiments:
A) 4-Nitro-1,2-phenylene diamine, C6H7N3O2; CAS-No.: 99-56-9
Concentration per plate: 20 µg
Solvent: DMSO
Strains: TA97a, TA98 and TA102
Metabolic activation: none

B) Sodium azide, NaN3; CAS-No.: 26628-22-8
Concentration per plate: 1 µg
Solvent: H2O
Strains: TA100 and TA1535
Metabolic activation: none

C) 2-Amino-anthracene, C14H11N; CAS-No.: 613-13-8
Concentration per plate: 1 µg
Solvent: DMSO
Strains: TA97a, TA100, TA102 and TA1535.
Metabolic activation: S9

D) Benzo-a-pyrene, C20H12; CAS-No.: 50-32-8
Concentration per plate: 20 µg
Solvent: DMSO
Strain: TA98
Metabolic activation: S9

Note: each batch of S9 is characterized with a mutagen that requires metabolic activation by microsomal enzymes (e.g., benzo(a)pyrene).

Evaluation criteria:

The colonies were counted visually and the numbers were recorded. A validated spreadsheet software (Microsoft Excel®) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control. The mean values and standard deviations of each threefold determination was calculated as well as the increase factor f(l) of revertant induction (mean revertants divided by mean spontaneous revertants) of the test substance solutions and the positive controls. Additionally, the absolute number of revertants (Rev. Abs.) (mean revertants less mean spontaneous revertants) was given. A substance is considered to have mutagenic potential, if a reproducible increase of revertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

other: Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535

Remarks:

Experiment 1a

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Highest concentration (5 µL/plate; no bacteria growth) and in the next lower concentration (1.5 µL/plate, decrease in the num-ber of revertants)

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

other: Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535

Remarks:

Experiment 1b

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Highest concentration (1.5 µL/plate; no bacteria growth)

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

other: Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535

Remarks:

Experiment 2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥0.19 µL/plate (decrease in the number of revertants, no bacteria growth and no bacterial background lawn)

Vehicle controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Results

Confirmation of genotype is performedfor each batch of lyophilized bacteria before stock culture preparation. The last performance showed no abnormalities.

 

A) Experiment 1a

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 were within the historical control data ranges.

 

Solubility and Toxicity

In this experiment, the test substance showed no precipitates on the plates in all tested concentrations. The test substance showed signs of toxicity towards all five bacteria strains in both the absence and presence of metabolic activation in the highest concentration (5 µL/plate; no bacteria growth) and in the next lower concentration (1.5 µL/plate, decrease in the number of revertants).The bacterial background lawn was not reduced at any of the concentrations.

 

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 substance is stated as not mutagenic under the conditions of this experiment. To verify this result, a further experiment was performed with lower concentrations.

 

Survey of the Findings

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

Table: Mean Revertants Experiment 1a

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. water	Mean	98	83	50	53	96	107	435	440	9	9
	sd	15.7	7.5	4.6	2.3	12.5	12.1	37.8	27.7	2.1	0.0
DMSO	Mean	91	96	48	45	90	95	445	437	10	9
	sd	13.3	6.0	2.1	1.2	8.7	12.2	25.7	24.1	3.0	1.7
Positive Controls*	Mean	456	484	349	417	1251	1261	1061	1064	349	109
	sd	79.7	144.4	75.6	6.1	48.2	72.6	115.5	92.3	46.2	23.4
	f(I)	5.01	5.04	7.27	9.27	13.03	13.27	2.38	2.43	38.78	12.11
5 µL/plate	Mean	0	0	0	0	0	0	0	0	0	0
	sd	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
	f(I)	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
1.5 µL/plate	Mean	2	2	9	10	1	4	3	6	1	3
	sd	1.2	1.2	1.0	0.6	0.6	4.2	1.7	3.8	0.0	0.6
	f(I)	0.02	0.02	0.19	0.22	0.01	0.04	0.01	0.01	0.10	0.33
0.5 µL/plate	Mean	85	87	36	39	31	15	63	241	10	12
	sd	7.2	6.0	4.7	4.5	7.0	6.0	11.1	22.7	0.0	1.2
	f(I)	0.93	0.91	0.75	0.87	0.34	0.16	0.14	0.55	1.00	1.33
0.15 µL/plate	Mean	91	84	42	43	75	92	387	433	11	11
	sd	10.0	11.5	1.5	1.5	4.9	5.3	67.1	24.4	0.6	1.5
	f(I)	1.00	0.88	0.88	0.96	0.83	0.97	0.87	0.99	1.10	1.22
0.05 µL/plate	Mean	98	102	39	42	101	103	352	456	11	8
	sd	11.0	4.0	0.6	1.2	17.2	24.3	32.7	78.7	2.6	0.6
	f(I)	1.08	1.06	0.81	0.93	1.12	1.08	0.79	1.04	1.10	0.89

f(I) = increase factor

* Different positive controls were used

B) Experiment 1b

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 were within the historical control data ranges.

 

Solubility and Toxicity

In this experiment, the test substance showed no precipitates on the plates in all tested concentrations. The test substance showed signs of toxicity towards all five bacteria strains in both the absence and presence of metabolic activation in the highest concentration (1.5 µL/plate; no bacteria growth).The bacterial background lawn was not reduced at any of the concentrations.

 

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 substance is stated as not mutagenic under the conditions of this experiment.

 

Survey of the Findings

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

Table: Mean Revertants Experiment 1b

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. water	Mean	77	117	45	44	90	93	239	245	12	13
	sd	4.0	7.6	9.0	2.6	11.0	9.0	20.5	52.8	3.5	3.6
DMSO	Mean	87	108	44	41	94	109	207	203	11	12
	sd	7.4	22.5	10.5	4.5	12.5	9.9	43.1	10.1	1.2	2.9
Positive Controls*	Mean	621	497	305	483	717	941	1291	1349	145	313
	sd	128.6	55.2	2.3	120.1	57.2	219.4	101.3	74.3	37.2	68.9
	f(I)	7.14	4.60	6.93	11.78	7.97	8.63	6.24	6.65	12.08	26.08
1.5 µL/plate	Mean	0	0	0	0	0	0	0	0	0	0
	sd	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
	f(I)	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
0.5 µL/plate	Mean	70	92	36	40	88	84	285	301	10	11
	sd	10.3	17.1	3.6	5.0	2.6	6.4	74.3	19.7	1.0	1.5
	f(I)	0.80	0.85	0.82	0.98	0.94	0.77	1.38	1.48	0.91	0.92
0.15 µL/plate	Mean	92	86	45	43	81	98	237	253	10	10
	sd	15.9	8.1	2.1	10.1	0.6	19.3	61.1	27.2	1.5	0.6
	f(I)	1.06	0.80	1.02	1.05	0.86	0.90	1.14	1.25	0.91	0.83
0.05 µL/plate	Mean	83	95	40	39	87	114	228	279	11	11
	sd	1.5	16.1	3.0	8.6	8.1	7.2	10.6	55.5	1.5	1.2
	f(I)	0.95	0.88	0.91	0.95	0.93	1.05	1.10	1.37	1.00	0.92
0.015 µL/plate	Mean	81	113	42	46	82	91	251	299	10	10
	sd	12.0	14.7	8.7	7.6	5.6	8.7	22.0	23.4	1.5	1.7
	f(I)	0.93	1.05	0.95	1.12	0.87	0.83	1.21	1.47	0.91	0.83
0.005 µL/plate	Mean	86	118	43	43	109	112	276	272	11	10
	sd	5.3	6.0	8.9	4.0	6.1	13.8	44.5	27.7	1.2	0.6
	f(I)	0.99	1.09	0.98	1.05	1.16	1.03	1.33	1.34	1.00	0.83

f(I) = increase factor

* Different positive controls were used

 

C) Experiment 2

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 were within the historical control data ranges.

 

Solubility and Toxicity

In this experiment, the test substanceshowed no precipitates on the plates in all tested concentrations.

The following signs of toxicity were observed towards the bacteria strains: 

- Strain TA97a: 1.5 µL/plate, 0.75 µL/plate, 0.38 µL/plate (no bacteria growth and no bacterial background lawn)

- Strain TA98: 1.5 µL/plate, 0.75 µL/plate, 0.38 µL/plate (no bacteria growth and no bacterial background lawn)

- Strain TA100: 1.5 µL/plate, 0.75 µL/plate, 0.38 µL/plate (no bacteria growth and no bacterial background lawn), 0.19 µL/plate (decreasein the number of revertants)

- Strain TA102: 1.5 µL/plate, 0.75 µL/plate (no bacteria growth and no bacterial background lawn), 0.38 µL/plate (decreasein the number of revertants)

- Strain TA1535: 1.5 µL/plate, 0.75 µL/plate, 0.38 µL/plate (no bacteria growth and no bacterial background lawn), 0.19 µL/plate (decreasein the number of revertants)

 

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 substance is stated as not mutagenic under the conditions of this experiment.

 

Survey of the Findings

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

Table: Mean Revertants Experiment 2

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. water	Mean	82	110	42	41	99	113	245	235	12	11
	sd	10.1	6.9	1.2	10.0	5.0	6.4	34.0	8.3	1.7	2.6
DMSO	Mean	75	105	43	45	91	91	229	235	12	12
	sd	8.7	12.9	9.7	2.1	14.2	8.1	66.6	53.7	1.5	3.1
Positive Controls*	Mean	513	456	379	456	552	1205	1277	1389	291	251
	sd	40.5	113.4	53.3	36.7	138.8	212.3	120.4	44.1	32.3	28.1
	f(I)	6.84	4.34	8.81	10.13	5.58	13.24	5.58	5.91	24.25	20.92
1.5 µL/plate	Mean	0	0	0	0	0	0	0	0	0	0
	sd	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
	f(I)	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
0.75 µL/plate	Mean	0	0	0	0	0	0	0	0	0	0
	sd	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
	f(I)	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
0.38 µL/plate	Mean	0	0	0	0	0	0	14	54	0	0
	sd	0.0	0.0	0.0	0.0	0.0	0.0	2.6	3.2	0.0	0.0
	f(I)	0.00	0.00	0.00	0.00	0.00	0.00	0.06	0.23	0.00	0.00
0.19 µL/plate	Mean	79	80	33	40	9	3	211	219	3	3
	sd	12.6	7.1	5.5	1.5	2.1	3.5	16.2	16.2	0.0	0.6
	f(I)	1.05	0.76	0.77	0.89	0.10	0.03	0.92	0.93	0.25	0.25
0.09 µL/plate	Mean	83	85	39	39	81	83	248	291	10	8
	sd	9.5	9.6	2.1	3.6	10.5	11.3	73.0	54.0	0.0	1.5
	f(I)	1.11	0.81	0.91	0.87	0.89	0.91	1.08	1.24	0.83	0.67
0.05 µL/plate	Mean	100	85	38	34	78	106	214	271	10	13
	sd	6.4	3.1	5.5	3.1	3.8	17.1	8.7	51.1	1.0	1.5
	f(I)	1.33	0.81	0.88	0.76	0.86	1.16	0.93	1.15	0.83	1.08
0.03 µL/plate	Mean	85	95	44	37	93	78	238	265	12	14
	sd	6.1	16.5	2.5	5.3	12.0	5.5	17.4	39.7	2.9	0.6
	f(I)	1.13	0.90	1.02	0.82	1.02	0.86	1.04	1.13	1.00	1.17
0.02 µL/plate	Mean	84	73	41	41	91	89	283	267	9	11
	sd	9.6	1.5	10.5	14.2	7.0	8.3	10.3	49.7	1.7	2.9
	f(I)	1.12	0.70	0.95	0.91	1.00	0.98	1.24	1.14	0.75	0.92
0.01 µL/plate	Mean	76	122	41	49	94	107	259	263	14	12
	sd	11.6	7.2	3.0	2.9	11.1	17.0	21.4	16.0	2.6	1.0
	f(I)	1.01	1.16	0.95	1.09	1.03	1.18	1.13	1.12	1.17	1.00

f(I) = increase factor

* Different positive controls were used

 

Mutagenicity of Test substance

The test substanceshowed no increase in the number of revertants in all bacteria strains in all experiments. All negative and all strain-specific positive control 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 test substance 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 substance was observed at any of the tested concentrations up to 5 µL/plate. 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. All of the means of all replicates of the spontaneous revertants (in negative and solvent controls) were within the range of the historical data of the test facility. All numbers of revertant colonies of the positive controls were within the range of the historical data of the laboratory and were increased in comparison with the negative controls, which demonstrated the mutagenic potential of the diagnostic mutagens. Since all criteria for acceptability have been met, the study is considered valid.

Applicant's summary and conclusion

Conclusions:

Under ths study conditions, the test substance was determined to be non-mutagenic with and without metabolic activation in the Ames test.

Executive summary:

An in vitro study was conducted to determine the mutagenicity potential of the test substance, C12 -14 HEDMAC (39.6% active) in a bacterial reverse mutation test, according to the OECD Guideline 471 and EU Method B13/B14, in compliance with GLP. Using five strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 and TA1535), the test was performed in three experiments in the presence and absence of metabolic activation. In the experiment 1a, the test substance (5, 1.5, 0.5, 0.15, 0.05 µL/plate dissolved in DMSO) was tested using the plate incorporation method. The test substance showed no precipitates on the plates at any of the concentrations. The test substance showed signs of toxicity towards all five bacteria strains in both the absence and presence of metabolic activation in the highest concentration (5 µL/plate; no bacteria growth) and in the next lower concentration (1.5 µL/plate, decrease in the number of revertants). The bacterial background lawn was not reduced at any of the concentrations. 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. Based on the toxicity results of the experiment 1a, the test substance (1.5, 0.5, 0.15, 0.05, 0.015, 0.005 µL/plate) was tested up to concentrations of 1.5 µL/plate using the plate incorporation method. This experiment was performed to verify the toxicity results of the experiment 1a with lower concentrations. The test substance showed no precipitates on the plates at any of the concentrations. The test substance showed signs of toxicity towards all five bacteria strains in both the absence and presence of metabolic activation in the highest concentration (1.5 µL/plate; no bacteria growth). The bacterial background lawn was not reduced at any of the concentrations. The results of this experiment showed that the test substance caused no increase in the number of revertants in all bacteria strains compared to the solvent control, in both the absence and presence of metabolic activation. The test substance 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 the experiment 1a and 1b, the test substance (1.5, 0.75, 0.38, 0.19, 0.09, 0.05, 0.02, 0.01, 0.006 µL/plate) was tested up to concentrations of 1.5 µL/plate using the pre-incubation method. The test substance showed no precipitates on the plates at any of the concentrations. The following signs of toxicity were observed towards the bacteria strains: Strain TA97a: 1.5 µL/plate, 0.75 µL/plate, 0.38 µL/plate (no bacteria growth and no bacterial background lawn); Strain TA98: 1.5 µL/plate, 0.75 µL/plate, 0.38 µL/plate (no bacteria growth and no bacterial background lawn); Strain TA100: 1.5 µL/plate, 0.75 µL/plate, 0.38 µL/plate (no bacteria growth and no bacterial background lawn), 0.19 µL/plate (decrease in the number of revertants); Strain TA102: 1.5 µL/plate, 0.75 µL/plate (no bacteria growth and no bacterial background lawn), 0.38 µL/plate (decrease in the number of revertants); Strain TA1535: 1.5 µL/plate, 0.75 µL/plate, 0.38 µL/plate (no bacteria growth and no bacterial background lawn), 0.19 µL/plate (decrease in the number of revertants). Vehicle and positive control studies were considered valid. The results of this experiment showed that the test substance caused no increase in the number of revertants in all bacteria strains compared to the solvent control, in both the absence and presence of metabolic activation. The test substance did not induce a dose-related increase in the number of revertants colonies in all strains, in the presence and absence of metabolic activation. Under the study conditions, the test substance was determined to be non-mutagenic with and without metabolic activation in the Ames test (Andres, 2019).