Propionamide

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Propionamide

EC Number:

201-172-1

EC Name:

Propionamide

Cas Number:

79-05-0

Molecular formula:

C3H7NO

IUPAC Name:

propanamide

Test material form:

solid

Method

Target gene:

Not applicable

Species / strain

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.

Controls

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.

Results and discussion

Test results

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”.

Any other information on results incl. tables

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

 

Applicant's summary and conclusion

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.