[No public or meaningful name is available]

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:

2019-04-04 to 2019-06-19

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:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1998

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: ICH Guideline S2 (R1): Genotoxicity testing and data interpretation for pharmaceuticals intended for human use, June 2012

Version / remarks:

2012

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 sodium bis(2-ethylhexyl) phosphate and sodium bis(2-ethylhexyl) phosphate

Molecular formula:

C8H19O4P.2Na - C16H35O4P.Na

IUPAC Name:

Reaction mass of sodium bis(2-ethylhexyl) phosphate and sodium bis(2-ethylhexyl) phosphate

Method

Target gene:

The Salmonella typhimurium histidine (his) reversion system measures his- → his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base pair (TA1535, TA100) and frameshift (TA1537, TA98) mutations. The Escherichia coli WP2 uvrA (trp) reversion system measures trp– → trp+ reversions. The Escherichia coli WP2 uvrA strain detects mutagens that cause other base-pair substitutions (AT to GC).

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

induced rat liver S9

Test concentrations with justification for top dose:

At the preparation of the test item stock solution a correction (multiplier) factor of 1.953 (1/0.512=1.953) based on the purity of 51.2% was taken into consideration.

Based on the cytotoxicity and solubility results of the preliminary concentration range finding test (informatory toxicity test) and based on the recommendations in OECD 471 guideline, the following concentrations of the test item were prepared and investigated in the initial and confirmatory mutation tests:

3200; 1600; 800; 320; 128; 51.2 and 20.5 μg/plate.

Vehicle / solvent:

- Vehicle/solvent used: ultrapure water

- Justification for choice of solvent/vehicle: suitable vehicle according to the guideline and sufficient solubility of the test item.

- Justification for percentage of solvent in the final culture medium: as recommended in the guideline

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 4-Nitro-1,2-phenylenediamine, NPD (TA 98; without S9; 4 µg/plate); 2-aminoanthracene, 2AA (all strains, with S9; 2 µg/plate (S. typhimurium), 50 µg/plate (E.coli)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation

DURATION
- Preincubation period: 20 min at 37 °C (bacterial culture and the S9 Mix or phosphate buffer)
- Exposure duration: 48 hours in the dark at 37 °C

NUMBER OF REPLICATIONS:
3

DETERMINATION OF CYTOTOXICITY
A dose level is considered toxic if:
- the reduced revertant colony numbers are observed as compared to the mean vehicle control value and the reduction shows a dose-dependent relationship, and / or
- the reduced revertant colony numbers are below the historical control data range and / or
- pinpoint colonies appear and / or
- reduced background lawn development occurs
- other: The toxicity of the test item was determined with strains Salmonella typhimurium TA98 and TA100 in a pre-experiment. 7 concentrations were tested for toxicity and mutation induction with 3 plates each. The experimental conditions in this pre-experiment were the same as for the main experiment I (plate incorporation test) and included non-activated and S9 activated test conditions with appropriate positive and negative controls. The test item concentrations, including the controls (untreated, vehicle and positive reference) were tested in triplicate. In the toxicity test the concentrations examined were: 5000, 1600, 500, 160, 50, 16 and 5 μg/plate.

Rationale for test conditions:

According to guidelines.

Evaluation criteria:

The colony numbers on the control, positive control and the test plates were determined, the mean values, standard deviations and the mutation rates were calculated.

Mutation Rate = Mean revertants at the test item (or control) treatments / Mean revertrants of vehicle control

A test item is considered mutagenic if:
- a dose-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- in strain TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control
- in strain TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.
According to the guidelines, the biological relevance of the results was the criterion for the interpretation of results, a statistical evaluation of the results was not regarded as necessary.
Criteria for a Negative Response:
A test article is considered non-mutagenic in this bacterial reverse mutation assay if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: yes
- Data on osmolality: yes
- Possibility of evaporation from medium: no
- Water solubility: sufficient soluble
- Precipitation and time of the determination: no

RANGE-FINDING/SCREENING STUDIES: yes

STUDY RESULTS
- Concurrent vehicle negative and positive control data

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible
- Statistical analysis; p-value

Ames test:
- Signs of toxicity : please refer to "any other information on results including tables"
- Individual plate counts: please refer to "any other information on results including tables"
- Mean number of revertant colonies per plate and standard deviation : please refer to "any other information on results including tables"

HISTORICAL CONTROL DATA
- Positive historical control data: please refer to "any other information on results including tables"
- Negative (solvent/vehicle) historical control data: please refer to "any other information on results including tables"

Any other information on results incl. tables

Table 1: Summary of Signs of Cytotoxicity in the Plate Incorporation and Pre-Incubation Tests

 

Plate Incorporation Test
Concentrations (µg/plate)	Salmonella typhimurium								Escherichia coliWP2uvrA
	TA98		TA100		TA1535		TA1537
	‑S9	+S9	‑S9	+S9	‑S9	+S9	‑S9	+S9	‑S9	+S9
3200	B<<	B<<	B<<	B<<	B<<	SB<	B0	B0	B<<	SB<<
1600	*	–	SB<<	<<	–	–	SB<	*	*	<<
800	–	*	<<	–	–	–	*	–	*	–
320	–	*	–	–	–	–	–	–	*	–
128	–	–	*	<<*	–	–	–	–	–	–
51.2	–	–	–	–	–	–	–	–	*	–
20.5	–	*	–	–	–	–	–	–	*	–
Pre-Incubation Test
Concentrations (µg/plate)	Salmonella typhimurium								Escherichia coliWP2uvrA
	TA98		TA100		TA1535		TA1537
	‑S9	+S9	‑S9	+S9	‑S9	+S9	‑S9	+S9	‑S9	+S9
3200	A	A	A	A	A	A	A	A	B<<	SB, PP<<
1600	B0	B0	B0	B0	B0	B0	B0	B<<	SB<<	<
800	B<<	SB<<	B0	B<<	SB<	SB<	B<	SB0	*	–
320	SB<<	–	SB#	–	SB<	–	B<	SB	–	–
128	–	–	–	–	–	–	–	–	–	–
51.2	–	–	–	–	–	*	–	–	–	–
20.5	–	–	–	–	–	–	–	–	–	–

A:          Absent background lawn development;

B0:       No revertant growth and reduced background lawn development;

B<<:     Reduced background lawn development and revertant colony numbers below the vehicle and historical control data ranges;

B<:        Reduced background lawn development and revertant colony numbers below the vehicle control data range;

SB0:     No revertant growth and slightly reduced background lawn development;

SB<<:  Slightly reduced background lawn development and revertant colony numbers below the vehicle and historical control data ranges;

SB<:     Slightly reduced background lawn development and revertant colony numbers below the vehicle control data range;

SB#:      Slightly reduced background lawn development and revertant colony numbers within the vehicle control data range; however, below the historical control data range;

SB:        Slightly reduced background lawn development and revertant colony numbers within the historical control data range and within the vehicle control data range;

PP:        Pinpoint colonies;

<<:         Revertant colony numbers below the vehicle and historical control data ranges;

<:           Revertant colony number significantly lightly below the corresponding vehicle control range; evaluated as inhibition;

‑:            No inhibition

*:           Revertant colony number slightly below the corresponding vehicle control range; however, within the biological variability range of the applied test system, evaluated as no inhibition

 

Table 2 Summary table of the results of the initial mutation test

Initial Mutation Test (Plate Incorporation Test)
Concentrations (mg/plate)	Salmonella typhimuriumtester strains																Escherichia coli
	TA 98				TA 100				TA 1535				TA 1537				WP2uvrA
	-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9
Mean values of revertants per plate Mutation rate (MR)	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR
Untreated Control	13.0	0.78	25.3	1.01	82.3	1.04	96.3	1.02	9.7	0.91	11.0	1.14	4.3	0.76	7.7	1.28	30.7	0.92	25.3	0.84
DMSO Control	22.0	1.00	23.7	1.00	–	–	97.7	1.00	–	–	11.0	1.00	4.3	1.00	6.0	1.00	–	–	39.0	1.00
Ultrapure Water Control	16.7	1.00	25.0	1.00	79.0	1.00	94.0	1.00	10.7	1.00	9.7	1.00	5.7	1.00	6.0	1.00	33.3	1.00	30.0	1.00
3200	1.0	0.06	7.3	0.29	10.0	0.13	4.0	0.04	1.7	0.16	5.3	0.55	0.0	0.00	0.0	0.00	9.7	0.29	13.3	0.44
1600	11.0	0.66	22.3	0.89	29.0	0.37	59.0	0.63	8.7	0.81	9.7	1.00	2.7	0.47	4.7	0.78	21.3	0.64	11.3	0.38
800	18.3	1.10	15.0	0.60	59.0	0.75	84.0	0.89	9.7	0.91	8.3	0.86	4.3	0.76	6.7	1.11	21.3	0.64	24.7	0.82
320	15.7	0.94	13.0	0.52	74.7	0.95	85.3	0.91	9.3	0.88	10.0	1.03	7.0	1.24	7.7	1.28	21.0	0.63	31.7	1.06
128	15.3	0.92	20.0	0.80	60.0	0.76	74.0	0.79	9.3	0.88	14.3	1.48	4.7	0.82	5.7	0.94	27.0	0.81	28.3	0.94
51.2	14.3	0.86	20.0	0.80	65.3	0.83	78.3	0.83	9.0	0.84	12.7	1.31	4.7	0.82	6.3	1.06	24.0	0.72	32.0	1.07
20.5	14.7	0.88	19.0	0.76	69.3	0.88	90.7	0.96	10.0	0.94	10.3	1.07	5.3	0.94	7.0	1.17	21.7	0.65	31.7	1.06
NPD (4mg/plate)	404.0	18.36	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
SAZ (2mg/plate)	–	–	–	–	567.3	7.18	–	–	965.3	90.50	–	–	–	–	–	–	–	–	–	–
9AA (50mg/plate)	–	–	–	–	–	–	–	–	–	–	–	–	1835.3	423.54	–	–	–	–	–	–
MMS (2mL/plate)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	594.7	17.84	–	–
2AA (2mg/plate)	–	–	1517.3	64.11	–	–	781.3	8.00	–	–	116.7	10.61	–	–	85.3	14.22	–	–	–	–
2AA (50mg/plate)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	207.0	5.31

MR: Mutation Rate; NPD: 4-Nitro-1,2-phenylenediamine; SAZ: Sodium azide; 9AA: 9-Aminoacridine; MMS: Methyl methanesulfonate; 2AA: 2-aminoanthracene
Remarks: Ultrapure water was applied as solvent of the test item and the positive control substance: SAZ and MMS; the DMSO was applied as solvent for positive control substances NPD, 9AA and 2AA. The mutation rate of the test item, SAZ, MMS and untreated control is given referring to the ultrapure water; the mutation rate of NPD, 9AA and 2AA is given referring to DMSO.

 

Table 3 Summary table of the results of the confirmatory mutation test

Confirmatory Mutation Test (Pre-Incubation Test)
Concentrations (mg/plate)	Salmonella typhimuriumtester strains																Escherichia coli
	TA 98				TA 100				TA 1535				TA 1537				WP2uvrA
	-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9
Mean values of revertants per plate Mutation rate (MR)	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR
Untreated Control	17.3	0.96	23.3	0.93	91.0	1.41	102.3	1.04	7.7	0.88	12.0	1.09	6.3	0.83	6.3	1.27	43.3	1.01	51.3	0.95
DMSO Control	18.0	1.00	22.3	1.00	–	–	93.3	1.00	–	–	12.7	1.00	7.3	1.00	6.0	1.00	–	–	51.3	1.00
Ultrapure Water Control	18.0	1.00	25.0	1.00	64.3	1.00	98.3	1.00	8.7	1.00	11.0	1.00	7.7	1.00	5.0	1.00	43.0	1.00	54.0	1.00
3200	0.0	0.00	0.0	0.00	0.0	0.00	0.0	0.00	0.0	0.00	0.0	0.00	0.0	0.00	0.0	0.00	3.3	0.08	14.3	0.27
1600	0.0	0.00	0.0	0.00	0.0	0.00	0.0	0.00	0.0	0.00	0.0	0.00	0.0	0.00	0.7	0.13	4.7	0.11	22.0	0.41
800	0.3	0.02	12.3	0.49	0.0	0.00	47.7	0.48	3.3	0.38	8.3	0.76	2.0	0.26	0.0	0.00	25.7	0.60	46.7	0.86
320	6.7	0.37	20.7	0.83	52.7	0.82	86.3	0.88	6.3	0.73	12.0	1.09	4.3	0.57	4.7	0.93	43.3	1.01	57.7	1.07
128	19.7	1.09	27.7	1.11	60.7	0.94	106.0	1.08	9.3	1.08	15.0	1.36	9.3	1.22	5.7	1.13	43.7	1.02	53.0	0.98
51.2	20.7	1.15	27.0	1.08	73.0	1.13	98.7	1.00	9.7	1.12	8.0	0.73	9.3	1.22	7.3	1.47	48.3	1.12	58.7	1.09
20.5	17.7	0.98	24.3	0.97	71.3	1.11	88.0	0.89	11.3	1.31	14.3	1.30	6.3	0.83	5.0	1.00	45.7	1.06	50.7	0.94
NPD (4mg/plate)	505.3	28.07	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
SAZ (2mg/plate)	–	–	–	–	744.0	11.56	–	–	1424.0	164.31	–	–	–	–	–	–	–	–	–	–
9AA (50mg/plate)	–	–	–	–	–	–	–	–	–	–	–	–	690.7	94.18	–	–	–	–	–	–
MMS (2mL/plate)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	1456.0	33.86	–	–
2AA (2mg/plate)	–	–	1477.3	66.15	–	–	988.0	10.59	–	–	156.0	12.32	–	–	81.0	13.50	–	–	–	–
2AA (50mg/plate)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	211.3	4.12

MR: Mutation Rate; NPD:4-Nitro-1,2-phenylenediamine; SAZ: Sodium azide; 9AA: 9-Aminoacridine; MMS: Methyl methanesulfonate; 2AA: 2-aminoanthracene
Remarks: Ultrapure water was applied as solvent of the test item and the positive control substance: SAZ and MMS; the DMSO was applied as solvent for positive control substances NPD, 9AA and 2AA. The mutation rate of the test item, SAZ, MMS and untreated control is given referring to the ultrapure water; the mutation rate of NPD, 9AA and 2AA is given referring to DMSO.

 

Table 4 Historical Control Values for Revertants/Plate (for the Period of 2016-2018)

			Bacterial strains
Historical control data of untreated control	‑S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	18.9	88.7	10.7	7.9	28.0
		SD	2.0	10.5	0.6	0.7	3.7
		Minimum	8	62	4	2	12
		Maximum	36	128	21	18	50
	+S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	24.0	106.5	11.2	8.3	35.1
		SD	1.7	10.5	0.3	0.7	3.9
		Minimum	11	71	3	2	16
		Maximum	40	152	20	17	59
			Bacterial strains
Historical control data of DMSO control	‑S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	17.5	80.6	10.7	7.5	25.9
		SD	1.2	10.6	0.8	0.7	2.6
		Minimum	9	58	4	2	12
		Maximum	35	124	21	18	52
	+S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	22.3	95.6	10.9	8.1	34.5
		SD	1.1	10.3	0.6	0.7	4.2
		Minimum	10	65	3	2	16
		Maximum	37	146	24	19	58
			Bacterial strains
Historical control data of Water control	‑S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	18.6	87.0	11.1	7.9	29.8
		SD	1.9	11.5	0.8	0.5	4.7
		Minimum	10	60	3	2	13
		Maximum	31	120	24	17	55
	+S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	23.6	105.0	11.3	8.1	36.8
		SD	1.8	11.7	0.7	0.6	4.0
		Minimum	13	76	5	3	18
		Maximum	41	148	19	17	63
			Bacterial strains
Historical control data of positive controls	‑S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	364.6	1095.3	987.7	564.4	911.6
		SD	111.2	197.3	119.3	73.8	112.8
		Minimum	182	543	409	137	361
		Maximum	844	2200	2123	2265	1995
	+S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	1419.2	1580.8	168.2	148.2	203.3
		SD	249.7	337.3	22.0	16.3	5.3
		Minimum	281	688	93	71	132
		Maximum	3421	3333	349	367	364

 

Applicant's summary and conclusion

Conclusions:

The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
In conclusion, the test item has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

Executive summary:

The test item was tested with regard to a 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) prepared from livers of Phenobarbital/β-naphthoflavone-induced rats. The study included a preliminary solubility tests, a preliminary concentration range finding test (informatory toxicity test), an initial mutation test (plate incorporation test), and a confirmatory mutation test (pre-incubation test). Based on the results of the solubility tests and the concentration range finding test the test item was dissolved in ultrapure water (ASTM Type I). At the preparation of the test item stock solution a correction (multiplier) factor of 1.953 (1/0.512=1.953) based on the purity of 51.2% was taken into consideration. Based on the cytotoxicity and solubility results of the preliminary concentration range finding test (informatory toxicity test) and based on the recommendations in OECD 471 guideline, the following concentrations of the test item were prepared and investigated in the initial and confirmatory mutation tests: 3200; 1600; 800; 320; 128; 51.2 and 20.5 μg/plate.

In the preliminary concentration range finding test strong inhibitory effect of the test item was observed at the recommended maximum test concentration of 5000 μg/plate.

No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9) throughout the study. In the initial and confirmatory mutation tests unequivocal inhibitory effect of the test item on bacterial growth was observed. The cytotoxicity was indicated by affected background lawn development (absent, reduced or slightly reduced background lawn), affected colony development (pinpoint colonies) and decreased revertant colony counts (absent revertants or revertants below the historical control data ranges and/or corresponding vehicle control data ranges). In general, 320 μg/plate (noticed following the pre-incubation procedure in S. typhimurium strains) was considered as lowest concentration showing unequivocal cytotoxicity.

The revertant colony numbers of solvent control ultrapure water (ASTM Type I) plates with and without S9 mix demonstrated the characteristic mean number of spontaneous revertants that was in line with the corresponding historical control data ranges.

The reference mutagen treatments (positive controls) showed the expected, biological relevant increases (more than 3-fold increase) in induced revertant colonies and the number of revertants fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in all experimental phases, in all tester strains.

No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 mix) in the performed experiments.

The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. In conclusion, the test item has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.