Propionamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance was considered to be non-mutagenic with and without metabolic activation in bacteria (reference 7.6.1-1).

The test item is considered to be non-mutagenic in the HPRT assay (referenc 7.6.1-2).

The test item is considered to be non-clastogenic and non-aneugenic in the in vitro micronucleus test (reference 7.6.1-3).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-08-04 to 2020-09-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

Not applicable

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: V79 cell line; supplied by Laboratory for Mutagenicity Testing; Technical University, 64287 Darmstadt, Germany
- Suitability of cells: The V79 cell line has been used successfully in in vitro experiments for many years.
- Normal cell cycle time (negative control): doubling time approximately 13 hours

For cell lines:
- Absence of Mycoplasma contamination: checked in each master cell stock
- Methods for maintenance in cell culture: Thawed stock cultures were propagated at 37 °C in 175 cm2 plastic flasks. About 5×10^5 cells were seeded into each flask with 30 mL of MEM (minimal essential medium) containing Hank’s salts, glutamine and Hepes (25 mM), supplemented with 10 % foetal bovine serum (FBS) and penicillin/streptomycin (100 U/mL/100 μg/mL). The cells were sub-cultured twice weekly. All incubations were done at 37 °C with 1.5 % carbon dioxide (CO2) in humidified air.
- Doubling time: approximately 13 hours
- Modal number of chromosomes: 22 ± 1
- Periodically checked for karyotype stability: yes
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: Please refer to “Methods for maintenance in cell culture”.

Cytokinesis block (if used):

cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: phenobarbital/β-naphthoflavone induced rat liver
- method of preparation of S9 mix: S9 mix contained MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium-ortho-phosphate-buffer (100 mM, pH 7.4).
- concentration or volume of S9 mix and S9 in the final culture medium: final protein concentration of 0.75 mg/mL in the cultures
- quality controls of S9: Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.

Test concentrations with justification for top dose:

Experiment I: 4.7, 8.3, 14.5, 22.5, 44.5, 77.9, 136, 239, 418, 731 μg/mL
Experiment II: 22.5, 44.5, 77.9, 136, 239, 418, 731 μg/mL

Based on the guideline and with regard to the molecular weight of the test item, 731 μg/mL (approx. 10 mM) were applied as top concentration for treatment of the cultures in the pre-test (=Experiment I). No cytotoxic effects were observed in Experiment I after 4 hours treatment in the absence and presence of S9 mix. Therefore, 731 μg/mL was chosen as top treatment concentration for Experiment II.

Vehicle / solvent:

- Solvent used: deionised water

- Justification for choice of solvent: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.

- Justification for percentage of solvent in the final culture medium: The final concentration of deionised water in the culture medium was 10 % due to the solubility of the test item.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Griseofulvin (continuous treatment) Dissolved in: DMSO Concentration: 7.0 μg/mL

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: Per culture approximately 5.0 – 6.0 x 10^5 cells were seeded into 25 cm2 plastic flasks.
- Test substance added: in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: Experiment I: 4 h, Experiment II: 24 h
- Harvest time after the end of treatment (sampling/recovery times): Experiment I: 20 h

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Cytokinesis blocked method used: Cytochalasin B (1.5 μg/mL), Experiment I: applied after treatment and after washing of culture in fresh medium for 20 h; Experiment II: applied together with test item for 24 h
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): Cells were detached by trypsin-EDTA-solution for approx. 5 minutes, followed by stopping the enzymatic treatment by adding complete culture medium including 10 % (v/v) FBS. The cultures were harvested and spun down by gentle centrifugation for 7 min. The supernatant was discarded and the cells were resuspended in saline G and spun down once again by centrifugation. Then the cells were resuspended in KCL solution (0.4 %) and incubated at 37 °C for 10 minutes. Ice-cold fixative mixture of methanol and glacial acetic acid (19+1 parts, respectively) was added to the hypotonic solution and the cells were resuspended carefully. After removal of the supernatant after centrifugation the cells were resuspended for 2 x 20 minutes in fixative and kept cold. The slides were prepared by dropping a small amount of the cell suspension in fresh fixative on clean, wet microscope slides and allowed to dry. The mounted cells were Giemsa-stained and, after drying, covered with coverslips. All slides were labeled with a computer-generated random code to prevent scorer bias.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): At least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. Per concentration at least 2000 binucleate cells were scored.
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): The micronuclei were counted in cells showing a clearly visible cytoplasm area. The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (1976). The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucleus.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cytokinesis-block proliferation index (CBPI)

Rationale for test conditions:

The conditions of the experiment were selected according to the recommendations of the OECD TG 487.

Evaluation criteria:

A test item is considered to be clearly negative if, in all of the experimental conditions examined:
− None of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− There is no concentration-related increase
− The results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data
The test item is then considered unable to induce chromosome breaks and/or gain or loss in this test system.
A test item is considered to be clearly positive if, in any of the experimental conditions examined:
− At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− The increase is concentration-related in at least one experimental condition
− The results are outside the range of the laboratory historical solvent control data
When all of the criteria are met, the test item is then considered able to induce chromosome breaks and/or gain or loss in this test system.
There is no requirement for verification of a clear positive or negative response.
In case the response is neither clearly negative nor clearly positive as described above and/or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations. Scoring additional cells or performing a repeat experiment possibly using modified experimental conditions could be useful.
However, results may remain questionable regardless of the number of times the experiment is repeated. If the data set will not allow a conclusion of positive or negative, the test item will therefore be concluded as equivocal.

Statistics:

Statistical significance was confirmed by the Chi square test (α < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.
A linear regression was performed using a validated test script of "R", to assess a possible dose dependency in the rates of micronucleated cells. The number of micronucleated cells obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.
Both, biological and statistical significance was considered together.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: solvent control: 7.28, test item maximum concentration without metabolic activation: 7.30
- Data on osmolality: solvent control: 282 mOsm, test item maximum concentration without metabolic activation: 303 mOsm
- Possibility of evaporation from medium: not specified
- Water solubility: The test item is sufficiently water soluble.
- Precipitation and time of the determination: No precipitation of the test item in the culture medium was observed at the end of treatment.
- Definition of acceptable cells for analysis: Please refer to “Details on test system and experimental conditions”.
- Other confounding effects: No other effects specified.

RANGE-FINDING/SCREENING STUDIES: A pre-test for toxicity was conducted. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: Please refer to “Any other information on results”.

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible: No dose dependency, tested by trend test, was observed.
- Statistical analysis: p-value, Please refer to “Any other information on results”.

Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements:
o In the case of the cytokinesis-block method: No relevant cytotoxicity could be observed in both experiments, in either experimental part, up to the highest applied concentration. Please refer to “Any other information on results” for detailed numbers.

- Genotoxicity results
o Number of cells with micronuclei separately for each treated and control culture and defining whether from binucleated or mononucleated cells: In the absence and presence of S9 mix, no relevant increase in the number of micronucleated cells was observed after treatment with the test item. In Experiment II, in the absence of S9 mix after continuous treatment, however, the value 1.60 % micronucleated cells of the lowest evaluated concentration (239 μg/mL) is statistically significant increased. Since the value is within the 95 % control limit of the historical control data (0.00 – 1.68 % micronucleated cells) and no dose dependency, tested by trend test, was observed, this finding can be considered as biologically irrelevant. Please refer to “Any other information on results” for detailed numbers.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: Please refer to “Any other information on results”.
- Negative (solvent/vehicle) historical control data: Please refer to “Any other information on results”.

Table 1 Summary of results

Exp.	Prepartion	Test item	Proliferation	Cytostasis	Micronucleated
	interval	concentration	index	in %*	cells	95 % Ctrl limit
		in µg/mL	CBPI		in %**
Exposure period 4 hrs without S9 mix
I	24 hrs	Solvent control^1	1.90		0.90	0.00 – 2.48
		Positive control^2	1.37	58.4	21.10^S
		239	1.87	2.8	1.20
		418	1.87	2.9	1.05
		731	1.87	2.7	0.85
Trend test: p-value 0.720
Exposure period 24 hrs without S9 mix
II	24 hrs	Solvent control^1/#	1.83		1.05	0.00 – 2.00
		Positive control^3	1.75	9.3	11.90^S
		239^#	1.81	2.2	1.60^S
		418^#	1.78	5.7	1.10
		731^#	1.74	10.44	0.90
Trend test: p-value 0.554
Exposure period 4 hrs with S9 mix
I	24 hrs	Solvent control^1	1.83		1.05	0.00 – 2.00
		Positive control^4	1.53	36.9	10.15^S
		239	1.80	3.8	1.05
		418	1.83	0.1	1.35
		731	1.85	n.c.	1.55
Trend test: p-value 0.058

 

*             For the positive control groups and the test item treatment groups the values are related to the solvent controls

**          The number of micronucleated cells was determined in a sample of 2000 binucleated cells

^#              The number of micronucleated cells was determined in a sample of 4000 binucleated cells

S             The number of micronucleated cells is statistically significantly higher than corresponding control values

n.c.        Not calculated as the CBPI is equal or higher than the solvent control value

1             Deion. water 10.0 % (v/v) 2 MMC 0.5 µg/mL

3             Griseofulvin 7.0 µg/mL

4             CPA 2.0 µg/mL

 

Table 2 Cytotoxicity indicated as cytokinesis-block proliferation index and cytostasis; exposure period 4 hrs without S9 mix. Experiment I

 

Treatment	Conc.	S9	Exposure/	Cell proliferation culture 1*			Proliferation	Cell proliferation culture 2*			Proliferation
group	per mL	mix	preparation	c1	c2	c4-c8	Index	c1	c2	c4-c8	Index	CBPI	Cytostasis
							CBPI				CBPI	mean	[%]
Solv. control^#	10.0 %	-	4 / 24 hrs	50	449	1	1.90	56	444	0	1.89	1.90
Pos. control^##	0.5 µg	-	4 / 24 hrs	332	165	3	1.34	303	193	4	1.40	1.37	58.4
Test item	239 µg	-	4 / 24 hrs	48	446	6	1.92	90	408	2	1.82	1.87	2.8
“	418 µg	-	4 / 24 hrs	70	425	5	1.87	69	428	3	1.87	1.87	2.9
“	731 µg	-	4 / 24 hrs	67	432	1	187	69	425	6	1.87	1.87	2.7

 

*             c1: mononucleate cells; c2: binucleate cells; c4-c8: multinucleate cells

^#              Deion. water

^##            MMC

 

Table 3 Cytotoxicity indicated as cytokinesis-block proliferation index and cytostasis; exposure period 4 hrs with S9 mix. Experiment I

 

Treatment	Conc.	S9	Exposure/	Cell proliferation culture 1*			Proliferation	Cell proliferation culture 2*			Proliferation
group	per mL	mix	preparation	c1	c2	c4-c8	Index	c1	c2	c4-c8	Index	CBPI	Cytostasis
							CBPI				CBPI	mean	[%]
Solv. control^#	10.0 %	+	4 / 24 hrs	85	407	8	1.85	93	403	4	1.82	1.83
Pos. control^##	2.0 µg	+	4 / 24 hrs	237	261	2	1.53	243	253	4	1.52	1.53	36.9
Test item	239 µg	+	4 / 24 hrs	119	375	6	1.77	94	397	9	1.83	1.80	3.8
“	418 µg	+	4 / 24 hrs	106	384	10	1.81	76	419	5	1.86	1.83	0.1
“	731 µg	+	4 / 24 hrs	74	423	3	1.886	91	401	8	1.83	1.85	n.c.

 

*             c1: mononucleate cells; c2: binucleate cells; c4-c8: multinucleate cells

^#              Deion. water

^##            CPA

n.c.        Not calculated as the CBPI is equal or higher than the solvent control value

 

Table 4 Number of micronucleated cells; exposure period 4 hrs without S9 mix. Experiment I

 

Treatment	Conc.	S9	Exposure/					Micronucleated cells
group	per mL	mix	preparation	Binucleate cells with n micronuclei culture 1			sum culture 1	Binucleate cells with n micronuclei culture 2			sum culture 2	sum in 2000 binucleate cells	[%]
				1	2	>2		1	2	>2
Solv. control^#	10.0 %	-	4 / 24 hrs	8	0	0	8	10	0	0	10	18	0.90
Pos. control^##	0.5 µg	-	4 / 24 hrs	180	28	4	212	173	25	12	210	422	21.10
Test item	239 µg	-	4 / 24 hrs	12	2	0	14	9	1	0	10	24	1.20
“	418 µg	-	4 / 24 hrs	11	0	0	11	10	0	0	10	21	1.05
“	731 µg	-	4 / 24 hrs	10	0	0	10	6	1	0	7	17	0.85

 

^#              Deion. water

^##            MMC

 

Table 5 Number of micronucleated cells; exposure period 4 hrs with S9 mix. Experiment I

 

Treatment	Conc.	S9	Exposure/					Micronucleated cells
group	per mL	mix	preparation	Binucleate cells with n micronuclei culture 1			sum culture 1	Binucleate cells with n micronuclei culture 2			sum culture 2	sum in 2000 binucleate cells	[%]
				1	2	>2		1	2	>2
Solv. control^#	10.0 %	+	4 / 24 hrs	8	1	0	9	11	1	0	12	21	1.05
Pos. control^##	2.0 µg	+	4 / 24 hrs	100	6	0	106	866	11	0	97	203	10.15
Test item	239 µg	+	4 / 24 hrs	10	1	0	11	10	0	0	10	21	1.05
“	418 µg	+	4 / 24 hrs	11	2	0	13	14	0	0	14	27	1.35
“	731 µg	+	4 / 24 hrs	14	0	1	15	16	0	0	16	31	1.55

 

^#              Deion. water

^##            MMC

Table 6 Cytotoxicity indicated as cytokinesis-block proliferation index and cytostasis; exposure period 24 hrs without S9 mix. Experiment II

 

Treatment	Conc.	S9	Exposure/	Cell proliferation culture 1*			Proliferation	Cell proliferation culture 2*			Proliferation
group	per mL	mix	preparation	c1	c2	c4-c8	Index	c1	c2	c4-c8	Index	CBPI	Cytostasis
							CBPI				CBPI	mean	[%]
Solv. control^#	10.0 %	-	24 / 24 hrs	126	361	13	1.77	76	410	14	1.88	1.83
Pos. control^##	7.0 µg	-	24 / 24 hrs	137	346	17	1.76	138	356	6	1.4	1.75	9.3
Test item	239 µg	-	24 / 24 hrs	74	418	8	1.87	132	363	5	1.75	1.81	2.2
“	418 µg	-	24 / 24 hrs	142	349	9	1.73	114	361	25	1.82	1.78	5.7
“	731 µg	-	24 / 24 hrs	153	345	2	1.70	119	372	9	1.78	1.74	10.4

 

*             c1: mononucleate cells; c2: binucleate cells; c4-c8: multinucleate cells

^#              Deion. water

^##            Griseofulvin

 

Table 7 Number of micronucleated cells; exposure period 24 hrs without S9 mix. Experiment II

 

Treatment	Conc.	S9	Exposure/					Micronucleated cells
group	per mL	mix	preparation	Binucleate cells with n micronuclei culture 1			sum culture 1	Binucleate cells with n micronuclei culture 2			sum culture 2	sum in 2000 binucleate cells	[%]
				1	2	>2		1	2	>2
Solv. control^#	10.0 %	-	24 / 24 hrs	14	3	0	17	22	2	2	26	43*	1.08
Pos. control^##	7.0 µg	-	24 / 24 hrs	97	11	2	110	113	14	1	128	238	11.90
Test item	239 µg	-	24 / 24 hrs	33	2	3	38	23	1	2	266	64*	1.60
“	418 µg	-	24 / 24 hrs	17	1	0	18	24	2	0	26	44*	1.10
“	731 µg	-	24 / 24 hrs	15	2	1	18	17	1	0	18	36*	0.90

 

#             Deion. water

##          Griseofulvin

*             Evaluation of 4000 binucleated cells

 

Table 8 Linear regression (Trend test)

Experimental group	p-value
Experiment I. exposure period 4 hrs without S9 mix	0.720
Experiment I. exposure period 4 hrs with S9 mix	0.058
Experiment II. exposure period 24 hrs without S9 mix	0.554

 

Table 9 Historical Laboratory Control Data

Micronucleus Test in V79 cells with CytB – Historical Control Data (2009-2019)

Aqueous solvents: DMEM/Ham’s F12. Deionised water (10 % v/v) Organic solvents: DMSO (0.5 or 1.0 %). Acetone. Ethanol and THF (0.5 %)

Solvent Control without S9
Micronucleated cells in %
	Pulse treatment (4/24)	Continuous treatment (24/24)	Continuous treatment (24/44)
No. of experiments	33	31	24
Mean	1.20	0.83	1.04
95 % Ctrl limit	0.00 – 2.48	0.00 – 1.70	0.00 – 2.21
1x SD	0.64	0.43	0.59
2x SD	1.28	0.87	1.18
Min – Max	0.05 – 2.85	0.30 – 2.05	0.30 – 2.55

 

Solvent Control with S9
Micronucleated cells in %
	Pulse treatment (4/24)	Continuous treatment (4/44)
No. of experiments	40	19
Mean	1.00	0.79
95 % Ctrl limit	0.00 - 2.00	0.01 – 1.57
1x SD	0.50	0.39
2x SD	1.00	0.78
Min – Max	0.20 – 2.35	0.15 – 1.75

 

Solvent Control without S9
Micronucleated cells in %
	Pulse treatment (4/24)	Continuous treatment (24/24)	Continuous treatment (24/44)
	MMC	Griseofulvin	Griseofulvin
No. of experiments	25	18	13
Mean	11.74	8.33	9.42
Min – Max	5.70 – 28.10	2.90 – 16.15	4.10 – 39.80
1x SD	5.85	4.19	9.98

 

Positive Control with S9
Micronucleated cells in %
	Pulse treatment (4/24)		Pulse treatment (4/44)
		CPA
No. of experiments	27		10
Mean	9.45		13.96
Min – Max	2.90 – 25.45		5.15 – 33.75
1x SD	4.52		9.34

 

Conclusions:

The test item did not induce micronuclei as determined by the in vitro micronucleus test in Chinese hamster V79 cells. Therefore, the test item is considered to be non-mutagenic, non-clastogenic and non-aneugenic in this in vitro micronucleus test, when tested up to the highest required concentration.

Executive summary:

The test item, dissolved in deionised water, was assessed for its potential to induce micronuclei in Chinese hamster V79 cells in vitro in the absence and presence of metabolic activation by S9 mix in a study conducted according to OECD TG 487.

Two independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure period was 24 hours without S9 mix. The cells were prepared 24 hours after start of treatment with the test item.

In each experimental group two parallel cultures were analyzed. At least 1000 cells per culture were scored for cytogenetic damage on coded slides. To determine a cytotoxic effect the CBPI was determined.

The highest treatment concentration in this study, 731 μg/mL (approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the OECD Guideline 487 for the in vitro mammalian cell micronucleus test. Dose selection of the cytogenetic experiment was performed considering the toxicity data in accordance with OECD Guideline 487. In Experiment I test item concentrations of 4.7, 8.3, 14.5, 22.5, 44.5, 77.9, 136, 239, 418 and 731 μg/mL were used, while in Experiment II 22.5, 44.5, 77.9, 136, 239, 418 and 731 μg/mL were applied.

In this study, no precipitation of the test item in the culture medium was observed at the end of treatment. No relevant influence on osmolarity or pH was observed. In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In the absence and presence of S9 mix, no relevant increase in the number of micronucleated cells was observed after treatment with the test item. In Experiment II, in the absence of S9 mix after continuous treatment, however, the value 1.60 % micronucleated cells of the lowest evaluated concentration (239 μg/mL) is statistically significant increased. Since the value is within the 95 % control limit of the historical control data (0.00 – 1.68 % micronucleated cells) and no dose dependency, tested by trend test, was observed, this finding can be considered as biologically irrelevant.

In both experiments, either Griseofulvin (7.0 μg/mL), MMC (0.5 μg/mL) or CPA (2.0 μg/mL) were used as positive controls and showed distinct increases in cells with micronuclei.  

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in Chinese hamster V79 cells. Therefore, the test item is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest required concentration. Thus, the test item is also considered as non-clastogenic and non-aneugenic.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-07-16 to 2020-08-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

HPRT (hypoxanthine-guanine phosphoribosyl transferase)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: V79 cell line; supplied by Laboratory for Mutagenicity Testing; Technical University, 64287 Darmstadt, Germany
- Suitability of cells: The V79 cell line has been used successfully in in vitro experiments for many years.
- Normal cell cycle time (negative control): doubling time 12 - 16 h in stock cultures

For cell lines:
- Absence of Mycoplasma contamination: checked in each master cell stock
- Methods for maintenance in cell culture: Thawed stock cultures were propagated at 37 °C in 75 cm2 plastic flasks. About 2-3×106 cells were seeded into each flask with 15 mL of MEM (minimal essential medium) containing Hank’s salts supplemented with 10 % foetal bovine serum (FBS), neomycin (5 μg/mL) and amphotericin B (1 %). The cells were sub-cultured once or twice weekly. All incubations were done at 37 °C with 1.5 % carbon dioxide (CO2) in humidified air.
- Doubling time: 12 - 16 h in stock cultures
- Modal number of chromosomes: 22
- Periodically checked for karyotype stability: yes
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: For seeding of the cell cultures the complete culture medium was MEM (minimal essential medium) containing Hank’s salts, neomycin (5 μg/mL), 10 % FBS, and amphotericin B (1 %). During treatment no FBS was added to the medium. For the selection of mutant cells the complete medium was supplemented with 11 μg/mL 6-thioguanine. All cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 (98.5 % air).

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Phenobarbital/β-naphthoflavone induced rat liver S9
- method of preparation of S9 mix: S9 mix contained MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium-ortho-phosphate-buffer (100 mM, pH 7.4).
- concentration or volume of S9 mix and S9 in the final culture medium: The protein concentration of the S9 preparation was 31.7 mg/mL. The final protein concentration was 0.75 mg/mL in the cultures.
- quality controls of S9: Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.

Test concentrations with justification for top dose:

22.8, 45.7, 91.4, 182.8, 365.5, 731.0 μg/mL

(731.0 μg/mL was equal to a molar concentration of approximately 10 mM)

The dose range of the main experiment was set according to data generated in the pre-experiment and according to guideline. The individual concentrations were spaced by a factor of 2.0.
To overcome problems with possible deviations in toxicity the main experiment was started with more than four concentrations. The cultures at the lowest concentration with and without metabolic activation were not continued as a minimum of only four analysable concentrations is required by the guidelines.

Vehicle / solvent:

- Solvent used: deionised water for test item

- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.

- Justification for percentage of solvent in the final culture medium: The final concentration of deionized water in the culture medium was 10 % (v/v) and is considered relative non-toxicity to the cell cultures.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration : duplicate
- Number of independent experiments: 1

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: approximately 0.7 to 1.2×10^7
- Test substance added: in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: approx. 24 h
- Exposure duration/duration of treatment: 4 h
- Harvest time after the end of treatment (sampling/recovery times):

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 7 days
- Selection time (if incubation with a selective agent): 8 days (evaluation for viability) and 9 days (mutation analysis)
- Fixation time (start of exposure up to fixation or harvest of cells): 15 or 16 days for viability and mutation analysis; 8 days for relative survival
- selective agent used: 6-thioguanine ; concentration: 11 μg/mL; duration of cell exposure: 8 or 9 days
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: mutation analysis: 4 - 5×10^5 cells each in medium containing 6-TG; evaluation for viability: approximately 500 cells per 25 cm2 flask; colonies were stained with 10 % methylene blue in 0.01 % KOH solution
- Criteria for colonies: Colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative survival (RS)

Rationale for test conditions:

The conditions of the experiment were selected according to the recommendations of the OECD TG 476.

Evaluation criteria:

A test item is classified as clearly mutagenic if, in any of the experimental conditions examined, all of the following criteria are met:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is dose-related when evaluated with an appropriate trend test,
c) any of the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95 % control limits).
A test item is classified as clearly non-mutagenic if, in all experimental conditions examined, all of the following criteria are met:
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no concentration-related increase when evaluated with an appropriate trend test,
c) all results are inside the distribution of the historical negative control data (e.g. Poisson-based 95 % control limits).

There is no requirement for verification of a clearly positive or negative response. In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations.
In rare cases, even after further investigations, the data set will preclude making a conclusion of positive or negative results, and therefore the test chemical response will be concluded to be equivocal.

Statistics:

A linear regression (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mean mutant frequencies. The mean number of mutant colonies obtained for the groups treated with the test item was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.
A t-Test was not performed since all mean mutant frequencies were well within the 95 % confidence interval of our laboratory’s historical negative control data.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: Solvent control: 7.38; 731.0 μg/mL test item: 7.42; There was no relevant shift of pH of the medium even at the maximum concentration of the test item.
- Data on osmolality: Solvent control: 324mOsm; 731.0 μg/mL test item: 297 mOsm; There was no relevant shift of osmolarity of the medium even at the maximum concentration of the test item.
- Possibility of evaporation from medium: not specified
- Water solubility: Test item is sufficiently water soluble.
- Precipitation and time of the determination: No precipitation or phase separation occurred up to the highest concentration tested at the beginning and at the end of treatment (4 hours).
- Definition of acceptable cells for analysis: Not specified
- Other confounding effects: No other effects described.

RANGE-FINDING/SCREENING STUDIES: The pre-experiment was performed in the presence and absence (4 h treatment) of metabolic activation. Test item concentrations between 5.7 μg/mL and 731.0 μg/mL (equal to a molar concentration of approximately 10 mM) were used. The highest concentration of the pre-experiment was chosen with regard to the molecular weight (73.1 g/mol) of the test item.
No relevant cytotoxic effect, indicated by a relative cloning efficiency of approx. 50 % or below was observed up to the highest concentration with and without metabolic activation.
In the pre-experiment the test medium was checked for precipitation or phase separation at the beginning and at the end of treatment (4 hours) prior to removal of the test item. No precipitation or phase separation occurred up to the highest concentration tested.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: Please refer to “Any other information on results”.

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship: No concentration-response relationship detected as p-value was > 0.05.
- Statistical analysis: Linear regression: without S9: p-value of 0.753 (mean of cultures I and II), with S9: p-value of 0.594 (mean of cultures I and II)

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
o Relative total growth (RTG) or relative survival (RS) and cloning efficiency: Please refer to “Any other information on results”.

- Genotoxicity results:
o Number of cells treated and sub-cultures for each cultures: seeded 24 h before treatment: 0.7 to 1.2×10^7; after treatment: at least 2.0×10^6 cells per experimental point were subcultivated
o Number of cells plated in selective and non-selective medium: about 4 - 5×10^5 cells per flask with 6-TG; approx.. 500 cells per flask with non-selective medium
o Number of colonies in non-selective medium and number of resistant colonies in selective medium, and related mutant frequency: Please refer to “Any other information on results”.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95 % control limits for the distribution as well as the number of data)
- Positive historical control data: Please refer to “Any other information on results”.
- Negative (solvent/vehicle) historical control data: Please refer to “Any other information on results”.

Table 1 Summary of Results

				relative	relative	rel. adjusted	mutant	95%
	conc.	P/	S9	cloning	cell	cloning	colonies/	confidence
	µg/mL	PS	mix	efficiency I	density	efficiency I	10^6 cells	interval
				%	%	%
Column	1	2	3	4	5	6	7	8
Experiment I / 4 h treatment				mean values of culture I and II
Solvent control with water			-	100.0	100.0	100.0	16.1	3.5 -31.0
Positive control (EMS)	300.0		-	98.2	102.7	101.0	231.3	3.5 -31.0
Test item	22.8	-	-	100.8	100.5	101.7	#
Test item	45.7	-	-	96.9	96.8	93.7	17.1	3.5 -31.0
Test item	91.4	--	-	104.3	108.4	112.8	13.8	3.5 -31.0
Test item	182.8	-	-	95.7	107.9	101.9	11.0	3.5 -31.0
Test item	365.5	-	-	100.6	95.1	95.6	9.5	3.5 -31.0
Test item	731.0	-	-	96.2	107.0	102.5	18.6	3.5 -31.0
Solvent control with water		-	+	100.0	100.0	100.0	8.6	4.2 -30.7
Positive control (DMBA)	2.3	-	+	92.8	99.7	92.2	122.1	4.2 -30.7
Test item	22.8	-	+	97.4	88.8	86.4	#
Test item	45.7	-	+	94.2	88.3	83.2	7.2	4.2 -30.7
Test item	91.4	-	+	92.6	99.6	92.2	15.6	4.2 -30.7
Test item	182.8	-	+	997.4	106.2	103.8	10.2	4.2 -30.7
Test item	365.5	-	+	98.4	78.6	76.9	6.4	4.2 -30.7
Test item	731.0	-	+	98.5	106.5	105.6	8.3	4.2 -30.7

P/PS = Precipitation/Phase separation
# culture was not continued as a minimum of only four analysable concentration is required

 

Table 2 Cloning Efficiency (Survival). culture I

	conc.	P /	S9	cells	number of colonies per flask			CE I	CE I	cells/mL	cell density	rel. adjusted
Test group	µg/mL	PS	mix	seeded	found			absolute	relative	at 1^st	%	CE I
				I/II	I	II	mean		%	subcultivation	of control	%
Column	1	2	3	4	5	6	7	8	9	10	11	12
Solvent control with water			-	542	301	27	294.0	0.5	100.0	1314000	100.0	100.0
Positive control with EMS	300.0		-	551	269	283	276.0	0.5	92.3	1334000	101.55	93.7
Test item	22.8	-	-	535	312	295	303.5	0.6	104.6	1444000	109.9	114.9
Test item	45.7	-	-	535	284	277	280.5	0.5	96.7	1444000	109.9	106.2
Test item	91.4	-	-	510	280	291	285.5	0.6	103.2	1802000	137.1	141.5
Test item	182.8	-	-	570	266	292	279.0	0.5	990.2	1742000	132.6	119.6
Test item	365.5	-	-	5515	2885	276	2880.5	0.5	100.4	1390000	105.8	106.2
Test item	731.0	-	-	525	273	268	70.5	0.5	95.0	1854000	141.1	134.0
Solvent control with water			+	537	284	291	287.5	0.5	100.0	1300000	100.0	100.0
Positive control with DMBA	2.3		+	503	270	265	267.5	0.5	99.3	1218000	93.7	93.1
Test item	22.8	-	+	523	269	274	271.5	0.5	97.0	1266000	97.4	94.4
Test item	45.7	-	+	535	288	254	271.0	0.5	94.6	1176000	90.5	85.6
Test item	91.4	-	+	553	273	280	276.5	0.5	93.4	1340000	103.1	96.3
Test item	182.8	-	+	504	279	262	270.5	0.5	100.2	1542000	118.6	118.9
Test item	365.5	-	+	528	253	284	268.5	0.5	95.0	1162000	89.4	84.9
Test item	731.0	-	+	519	294	279	286.5	0.6	103.1	1586000	122.0	125.8

P/PS = Precipitation/Phase separation
# culture was not continued as a minimum of only four analysable concentration is required

 

Table 3 Cloning Efficiency (Viability). culture I

	conc.	P /	S9	cells	number of colonies per flask			CE II	CE II	cells	cells
Test group	µg/mL	PS	mix	seeded	found			absolute	relative	seeded	survived
				I/II	I	II	mean		%
Column	1	2	3	4	5	6	7	8	9	10	11
Solvent control with water			-	570	361	332	346.5	0.6	100.0	419700	255133
Positive control with EMS	300.0		-	538	319	278	3998.5	0.6	91.3	397800	220712
Test item	22.8	-	-	culture was not continued^#
Test item	45.7	-	-	585	324	336	330.0	0.6	92.8	431400	243354
Test item	91.4	-	-	553	310	330	320.0	0.6	9.2	437400	253107
Test item	182.8	-	-	579	311	327	3199.0	0.6	90.6	402600	221812
Test item	365.5	-	-	519	260	271	265.5	0.5	84.2	424200	217004
Test item	731.0	-	-	528	259	255	257.0	0.5	80.1	441300	214799
Solvent control with water			+	563	381	375	378.0	0.7	100.0	42180	283198
Positive control with DMBA	2.3		+	559	326	339	332.5	0.6	88.6	407100	242148
Test item	22.8	-	+	culture was not continued^#
Test item	45.7	-	+	567	341	372	356.5	0.6	93.6	442800	278410
Test item	91.4	-	+	557	338	350	344.0	0.6	82.0	408900	252534
Test item	182.8	-	+	574	347	366	356.5	0.6	92.5	426600	264953
Test item	365.5	-	+	537	351	337	344.0	0.6	95.4	431100	276161
Test item	731.0	-	+	536	326	338	332.0	0.6	92.3	423300	262193

P/PS = Precipitation/Phase separation
# culture was not continued as a minimum of only four analysable concentration is required

 

Table 4 Mutagenicity data (Mutation rates). culture I

	conc.	P /	S9	number of mutant colonies per flask							mutant
Test group	µg/mL	PS	mix	found after plating in TG medium						standard	colonies
				I	II	III	IV	V	mean	deviation	per 10^6 cells
Column	1	2	3	4	5	6	7	8	9	10	11
Solvent control with water			-	5	6	3	5	2	4.2	1.6	16.5
Positive control with EMS	300.0		-	52	55	49	53	56	53.0	2.7	240.1
Test item	22.8	-	-	culture was not continued^#
Test item	45.7	-	-	3	4	2	3	3	3.0	0.7	12.3
Test item	91.4	-	-	2	6	3	6	4	4.2	1.8	16.6
Test item	182.8	-	-	1	2	2	3	4	2.4	1.1	10.8
Test item	365.5	-	-	4	3	0	1	2	2.0	1.6	9.2
Test item	731.0	-	-	3	5	4	5	3	4.0	1.0	18.6
Solvent control with water			+	1	5	2	2	4	2.8	1.6	9.9
Positive control with DMBA	2.3		+	38	32	299	36	37	34.4	3.8	142.1
Test item	22.8	-	+	culture was not continued^#
Test item	45.7	-	+	2	3	2	3	4	2.8	0.8	10.1
Test item	91.4	-	+	4	5	7	8	3	5.4	2.1	21.4
Test item	182.8	-	+	5	4	2	3	2	3.2	1.3	12.1
Test item	365.5	-	+	2	1	3	2	2	2.0	0.7	7.2
Test item	731.0	-	+	1	2	0	0	3	1.2	1.3	4.6

P/PS = Precipitation/Phase separation
# culture was not continued as a minimum of only four analysable concentration is required

 

Table 5 Cloning Efficiency I (Survival). culture II

	conc.	P /	S9	cells	number of colonies per flask			CE I	CE I	cells/mL	cell density	rel. adjusted
Test group	µg/mL	PS	mix	seeded	found			absolute	relative	at 1^st	%	CE I
				I/II	I	II	mean		%	subcultivation	of control	%
Column	1	2	3	4	5	6	7	8	9	10	11	12
Solvent control with water			-	531	289	286	287.5	0.5	100.0	976000	100.0	100.0
Positive control with EMS	300.0		-	502	2755	291	283.0	0.6	104.1	1014000	103.9	108.2
Test item	22.8		-	519	264	281	272.5	0.5	97.0	890000	91.2	88.4
Test item	45.7		-	510	276	260	268.0	0.5	97.1	816000	83.6	81.1
Test item	91.4		-	504	293	282	287.5	0.6	105.4	778000	79.7	84.0
Test item	182.8		-	508	267	290	278.5	0.5	101.3	8812000	83.2	84.2
Test item	365.5		-	5099	274	281	277..5	0.5	100.7	824000	84.4	85.0
Test item	731.0		-	517	268	277	272.5	0.5	97.3	712000	73.0	71.0
Solvent control with water			+	518	273	278	275.5	0.5	100.0	1140000	100.0	100.0
Positive control with DMBA	2.3		+	548	246	257	251.5	0.5	86.3	1206000	105.8	91.3
Test item	22.8		+	522	280	263	271.5	0.5	97.8	914000	80.2	78.4
Test item	45.7		+	534	259	274	266.5	0.5	93.8	982000	86.1	80.8
Test item	91.4		+	543	261	269	265.0	0.5	91.8	1096000	96.11	88.2
Test item	182.8		+	530	258	275	266.5	0.5	94.5	1070000	93.9	88.7
Test item	365.5		+	503	276	269	272.5	0.5	101.9	77200	67.7	69.0
Test item	731.0		+	514	253	260	256.5	0.5	93.8	1038000	91.1	85.4

P/PS = Precipitation/Phase separation

 

Table 6 Cloning Efficiency II (Viability). culture II

	conc.	P /	S9	cells	number of colonies per flask			CE II	CE II	cells	cells
Test group	µg/mL	PS	mix	seeded	found			absolute	relative	seeded	survived
				I/II	I	II	mean		%
Column	1	2	3	4	5	6	7	8	9	10	11
Solvent control with water			-	546	375	322	348.5	0.6	100.0	519000	331266
Positive control with EMS	300.0		-	514	311	291	301.0	0.6	91.7	583200	341524
Test item	22.8		-	culture was not continued^#
Test item	45.7		-	564	304	260	282.0	0.5	78.3	438000	219000
Test item	91.4		-	517	368	324	346.0	0.7	104.9	513600	343725
Test item	182.8		-	556	352	321	336.5	0.6	94.8	505800	306118
Test item	365.5		-	542	364	342	353.0	0.7	102.0	529200	344663
Test item	731.0		-	525	262	274	268.0	0.5	80.0	504600	257586
Solvent control with water			+	500	440	390	415.0	0.8	100.0	394500	327435
Positive control with DMBA	2.3		+	506	314	327	320.5	0.6	76.3	482700	305742
Test item	22.8		+	culture was not continued^#
Test item	45.7		+	511	318	338	328.0	0.6	77.3	503100	322929
Test item	91.4		+	500	3884	331	357.5	0.7	86.1	396300	283355
Test item	182.8		+	501	341	310	325.5	0..6	78.3	402750	261667
Test item	365.5		+	504	313	308	310.5	0.6	74.2	472200	290909
Test item	731.0		+	572	320	284	302.0	0.5	63.6	408900	215888

P/PS = Precipitation/Phase separation
# culture was not continued as a minimum of only four analysable concentration is required

 

Table 7 Mutagenicity data (Mutation rates). culture II

	conc.	P /	S9	number of mutant colonies per flask							mutant
Test group	µg/mL	PS	mix	found after plating in TG medium						standard	colonies
				I	II	III	IV	V	mean	deviation	per 10^6 cells
Column	1	2	3	4	5	6	7	8	9	10	11
Solvent control with water			-	5	6	4	7	4	5.2	1.3	15.7
Positive control with EMS	300.0		-	78	71	88	70	73	76.0	7.4	222.5
Test item	22.8		-	culture was not continued^#
Test item	45.7		-	6	3	2	88	5	4.8	2.4	21.9
Test item	91.4		-	2	4	7	3	3	3.	1.9	11.1
Test item	182.8		-	4	2	3	3	5	3.4	1.1	11.1
Test item	365.5		-	3	6	2	2	4	3..4	1.7	9.9
Test item	731.0		-	5	4	4	5	6	4.8	0.8	18.6
Solvent control with water			+	22	1	3	4	2	2.4	1.1	7.3
Positive control with DMBA	2.3		+	26	33	37	29	31	31.2	4.1	102.0
Test item	22.8		+	culture was not continued^#
Test item	45.7		+	3	0	1	2	1	1.4	1.1	4.3
Test item	91.4		+	2	4	3	1	4	2.8	1.3	9.9
Test item	182.8		+	1	4	2	1	3	2.2	1.3	8.4
Test item	365.5		+	0	2	2	3	1	1.6	1.1	5.5
Test item	731.0		+	2	3	5	1	2	2.6	1.5	12.0

P/PS = Precipitation/Phase separation
# culture was not continued as a minimum of only four analysable concentration is required

 

Table 8 Historical Laboratory Control Data

2016 – 2019
Number of mutant colonies per 10^6 cells
without metabolic activation (4 hours treatment time)
	Positive control EMS 150 and 300 µg/mL	Solvent control (medium. acetone. water. DMSO. ethanol. THF. EGDE)
Range:	70.6 – 851.6	4.1 – 40.6
Mean value:	248.6	17.2
Standard deviation:	97.5	6.9
95% confidence interval	--	3.5 – 31.0
Number of studies:	159	159
with metabolic activation (4 hours treatment time)
	Positive control DMBA 1.1 and 2.3 µg/mL	Solvent control (medium. acetone. water. DMSO. ethanol. THF. EGDE)
Range:	54.5 – 409.4	5.5 – 40.4
Mean value:	151.6	17.5
Standard deviation:	56.8	6.6
95% confidence interval	--	4.2 – 30.7
Number of studies:	160	160

The 95% confidence interval is derived from the mean value plus/minus 2 times the standard deviation.

Conclusions:

Under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test item is considered to be non-mutagenic in this HPRT assay.

Executive summary:

A study according to OECD TG 476 was performed to investigate the potential of the test item to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The treatment period was 4 hours with and without metabolic activation. The maximum test item concentration of the pre-experiment and in the main experiment (731.0 μg/mL) was equal to a molar concentration of about 10 mM. No relevant cytotoxic effect indicated by an adjusted cloning efficiency I below 50 % in both cultures occurred up to the maximum concentration with and without metabolic activation. No relevant increase in mutant colony numbers/10^6 cells was observed in the main experiment up to the maximum concentration. In the main experiment the values of the solvent controls were 16.1 mutants per 10^6 cells in the absence and 8.6 mutants per 10^6 cells in the presence metabolic activation; the range of the groups treated with the test item were from 6.4 up to 18.6 mutants per 10^6 cells. All values are well within the 95 % confidence interval of the solvent control. A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups. Appropriate reference mutagens, EMS (300 μg/mL) and DMBA (2.3 μg/mL), were used as positive controls and showed a distinct increase in induced mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test item is considered to be non-mutagenic in this HPRT assay.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-03-06 to 2019-03-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

1993

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 mix induced by ß-Naphthoflavone/Phenobarbital in livers of rats. 10% and 20% S9 in the S9 mix were used in the 1st and 2 nd test series, respectively.

Test concentrations with justification for top dose:

1st serie: 5, 15.8, 50, 158, 500, 1580 and 5000 µg/plate
2nd serie: 50, 158, 500, 1580 and 5000 µg/plate
The test material concentrations used were selected according to the EEC, OECD and Japanese guidelines for this test system.

Vehicle / solvent:

- Vehicle/solvent used: Ultra-pure water
- Justification for choice of solvent/vehicle: The selection of the solvent for this assay was based on the available information from a preliminary solubility test. Ultra-pure water showed best performance and was thus used for this experiment at a maximum concentration of 100 µL/plate.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2-AA)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Daunomycin (DAUN)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 3 replicates for test item concentrations and positive controls, 6 replicates for controls
- Number of independent experiments : 2

DURATION:
- Exposure duration: The incubation of plates was performed at 36 - 38 °C for 2 days.

OTHER:
-S9 concentration: 1st series 10%, 2nd series 20%

Evaluation criteria:

A test material was to be defined as positive or mutagenic in this assay if
- the assay is considered valid and
- a biologically relevant increase in the mean number of revertants above a threshold of 2¬fold (TA 98, TA 100, WP2 uvrA) or 3-fold (TA 1535, TA 1537) as compared to the concurrent negative controls is observed
- an increase exceeding the threshold at only one concentration is considered as biologically meaningful if reproduced in a second independent experiment
- a concentration-dependent increase is considered biologically meaningful if the threshold is exceeded at more than one concentration

A test material is defined as negative or non-mutagenic in this assay if
- the assay is considered valid and
- none of the above-mentioned criteria are met

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Following treatment with the test item, no precipitation on the agar plates occurred. No toxicity to the bacteria was observed.

Table 1: Summary 1st series

Metabolic Activation	Test material	Conc. [µg/plate]	Revertants per plate (Mean ± SD)
			TA 98	TA 100	TA 1535	TA 1537	WP2 uvrA
Without activation	H2O		38 ± 2	120 ± 11	35 ± 8	10 ± 4	36 ± 11
	Test item	5.00	27 ± 8	120 ± 15	32 ± 7	6 ± 4	44 ± 7
		15.8	33 ± 12	128 ± 10	33 ± 3	6 ± 2	33 ± 3
		50.0	42 ± 6	119 ± 17	29 ± 7	10 ± 5	37 ± 8
		158	40 ± 1	127 ± 4	36 ± 5	10 ± 2	34 ± 12
		500	33 ± 6	115 ± 4	36 ± 3	13 ± 6	40 ± 14
		1580	34 ± 7	119 ± 11	28 ± 13	14 ± 8	34 ± 1
		5000	39 ± 2	133 ± 13	37 ± 4	9 ± 4	36 ± 1
	NaN3	2.00		1687 ± 84	895 ± 109
	NQO	2.00					386 ± 54
	DAUN	5.00	565 ± 75
	9-AA	50.0				937 ± 142
With activation	H2O		47 ± 6	143 ± 11	33 ± 7	10 ± 3	47 ± 10
	Test item	5.00	34 ± 12	146 ± 2	28 ± 8	14 ± 2	48 ± 8
		15.8	46 ± 3	153 ± 7	34 ± 4	10 ± 2	50 ± 7
		50.0	47 ± 2	134 ± 18	36 ± 14	13 ± 3	51 ± 7
		158	50 ± 3	157 ± 3	32 ± 8	9 ± 2	39 ± 5
		500	58 ± 5	139 ± 18	28 ± 9	16 ± 5	45 ± 14
		1580	42 ± 3	149 ± 9	36 ± 11	8 ± 4	40 ± 7
		5000	48 ± 5	145 ± 12	27 ± 6	15 ± 7	40 ± 5
	2-AA	10.0					729 ± 18
	2-AA	2.00	594 ± 48	1074 ± 181
	2-AA	5.00			273 ± 5	337 ± 62

Table 2: Summary 2nd series

Metabolic Activation	Test material	Conc. [µg/plate]	Revertants per plate (Mean ± SD)
			TA 98	TA 100	TA 1535	TA 1537	WP2 uvrA
Without activation	H2O		34 ± 5	132 ± 8	32 ± 5	11 ± 3	42 ± 6
	Test item	5.00	34 ± 9	137 ± 8	26 ± 5	13 ± 1	42 ± 11
		158	33 ± 12	126 ± 14	27 ± 6	16 ± 3	36 ± 9
		500	42 ± 14	133 ± 25	29 ± 5	12 ± 4	41 ± 6
		1580	27 ± 6	142 ± 13	30 ± 4	10 ± 3	39 ± 8
		5000	27 ± 2	124 ± 11	34 ± 4	11 ± 0	42 ± 11
	NaN3	2.00		1733 ± 34	941 ± 47
	NQO	2.00					2212 ± 164
	DAUN	5.00	406 ± 60
	9-AA	50.0				191 ± 71
With activation	H2O		32 ± 7	172 ± 16	31 ± 6	15 ± 4	48 ± 6
	Test item	5.00	33 ± 12	160 ± 21	33 ± 4	13 ± 1	48 ± 11
		158	26 ± 4	166 ± 18	26 ± 3	21 ± 8	43 ± 7
		500	37 ± 8	179 ± 14	27 ± 7	13 ± 2	52 ± 12
		1580	30 ± 9	159 ± 16	29 ± 12	10 ± 4	47 ± 9
		5000	35 ± 9	175 ± 7	26 ± 7	12 ± 4	44 ± 9
	2-AA	2.00	136 ± 5	447 ± 10
	2-AA	5.00			249 ± 8	180 ± 14
	2-AA	10.00					248 ± 7

Conclusions:

Under the experimental conditions choosen, the test item did not induce gene mutations by base-pair or frameshift changes in the genome of the strains used. Therefore, it was concluded that with and without addition of S9 mix as the exogenous metabolizing system the test item was not mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation test.

Executive summary:

The mutagenic potential of the test item was investigated in a bacterial reverse mutation test according to OECD guidedeline 471 .
The investigations for the mutagenic potential of the test item were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 1535 and TA 1537 and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from rats pretreated with P-Naphthoflavone/Phenobarbital was used. In this study, two experimental series were performed. The S9 mix used contained 10% S9 in the 1st and 20% S9 in the 2nd series, respectively.
Solvent and positive control treatments were included for all strains. The mean numbers of revertant colonies were all within acceptable ranges for solvent control treatments, or were clearly elevated by positive control treatments, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used. Following treatment of all bacteria tester strains with the test item in the absence and presence of S9 mix, no relevant increases in revertant numbers were observed.
Under the experimental conditions choosen, the test item did not induce gene mutations by base-pair or frameshift changes in the genome of the strains used. Therefore, it was concluded that with and without addition of S9 mix as the exogenous metabolizing system the test item was not mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Bacteria Reverse Mutation Test (OECD TG 471)

The mutagenic potential of the test item was investigated in a bacterial reverse mutation test according to OECD guidedeline 471.
The investigations for the mutagenic potential of the test item were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 1535 and TA 1537 and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from rats pretreated with P-Naphthoflavone/Phenobarbital was used. In this study, two experimental series were performed. The S9 mix used contained 10% S9 in the 1st and 20% S9 in the 2nd series, respectively.
Solvent and positive control treatments were included for all strains. The mean numbers of revertant colonies were all within acceptable ranges for solvent control treatments, or were clearly elevated by positive control treatments, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used. Following treatment of all bacteria tester strains with the test item in the absence and presence of S9 mix, no relevant increases in revertant numbers were observed.
Under the experimental conditions choosen, the test item did not induce gene mutations by base-pair or frameshift changes in the genome of the strains used. Therefore, it was concluded that with and without addition of S9 mix as the exogenous metabolizing system the test item was not mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation test.

HPRT assay (OECD TG 476)

A study according to OECD TG 476 was performed to investigate the potential of the test item to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The treatment period was 4 hours with and without metabolic activation. The maximum test item concentration of the pre-experiment and in the main experiment (731.0 μg/mL) was equal to a molar concentration of about 10 mM. No relevant cytotoxic effect indicated by an adjusted cloning efficiency I below 50 % in both cultures occurred up to the maximum concentration with and without metabolic activation. No relevant increase in mutant colony numbers/10^6 cells was observed in the main experiment up to the maximum concentration. In the main experiment the values of the solvent controls were 16.1 mutants per 10^6 cells in the absence and 8.6 mutants per 10^6 cells in the presence metabolic activation; the range of the groups treated with the test item were from 6.4 up to 18.6 mutants per 10^6 cells. All values are well within the 95 % confidence interval of the solvent control. A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups. Appropriate reference mutagens, EMS (300 μg/mL) and DMBA (2.3 μg/mL), were used as positive controls and showed a distinct increase in induced mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test item is considered to be non-mutagenic in this HPRT assay.

Micronucleus assay (OECD TG 487)

The test item, dissolved in deionised water, was assessed for its potential to induce micronuclei in Chinese hamster V79 cells in vitro in the absence and presence of metabolic activation by S9 mix in a study conducted according to OECD TG 487.

Two independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure period was 24 hours without S9 mix. The cells were prepared 24 hours after start of treatment with the test item.

In each experimental group two parallel cultures were analyzed. At least 1000 cells per culture were scored for cytogenetic damage on coded slides. To determine a cytotoxic effect the CBPI was determined.

The highest treatment concentration in this study, 731 μg/mL (approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the OECD Guideline 487 for the in vitro mammalian cell micronucleus test. Dose selection of the cytogenetic experiment was performed considering the toxicity data in accordance with OECD Guideline 487. In Experiment I test item concentrations of 4.7, 8.3, 14.5, 22.5, 44.5, 77.9, 136, 239, 418 and 731 μg/mL were used, while in Experiment II 22.5, 44.5, 77.9, 136, 239, 418 and 731 μg/mL were applied.

In this study, no precipitation of the test item in the culture medium was observed at the end of treatment. No relevant influence on osmolarity or pH was observed. In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In the absence and presence of S9 mix, no relevant increase in the number of micronucleated cells was observed after treatment with the test item. In Experiment II, in the absence of S9 mix after continuous treatment, however, the value 1.60 % micronucleated cells of the lowest evaluated concentration (239 μg/mL) is statistically significant increased. Since the value is within the 95 % control limit of the historical control data (0.00 – 1.68 % micronucleated cells) and no dose dependency, tested by trend test, was observed, this finding can be considered as biologically irrelevant.

In both experiments, either Griseofulvin (7.0 μg/mL), MMC (0.5 μg/mL) or CPA (2.0 μg/mL) were used as positive controls and showed distinct increases in cells with micronuclei.  

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in Chinese hamster V79 cells. Therefore, the test item is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest required concentration. Thus, the test item is also considered as non-clastogenic and non-aneugenic.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genetic toxicity, the test item does not require classification according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighteenth time in Regulation (EU) 2022/692.