Sodium propionate

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.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 May 2017 - 18 July 2017

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:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

1993

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

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

Test concentrations with justification for top dose:

1st serie: 5.0, 15.8, 50.0, 158, 500, 1580, 5000 µg/plate
2nd serie: 50.0, 158, 500, 1580, 5000 µg/plate
top dose: maximum recommended concentration (according to OECD 471)

Vehicle / solvent:

- Vehicle/solvent used: ultrapure water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

sodium azide

other: 2-aminoanthracene, daunomycin

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 2 days

NUMBER OF REPLICATIONS: 3 replicates for test item concentrations and positive controls, 6 replicates for solvent controls

DETERMINATION OF CYTOTOXICITY
- Method: counting numbers of revertants

Evaluation criteria:

A test material was to be defined as negative or non-mutagenic in this assay if
- The assay was to be considered valid, and
- "no" or "weak increases" occurred in the test series performed ("weak increases" randomly occur due to experimental variation)

For valid data, the test material was considered to be positive or mutagenic if:
- a dose dependent (over at least two test material concentrations) increase in the number of revertants was induced, the maximal effect was a "clear increase", and the effects were reproduced at similar concentration levels in the same test system, or
- "clear increases" occurred at least at one test material concentration, higher concentrations showed strong precipitation or cytotoxicity, and the effects were reproduced at the same concentration level in the same test system.

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

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

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

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

Positive controls validity:

valid

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

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test material precipitate was observed on the plates any concentration.

Table 1: Summary 1st series

Metabolic Activation	Test Material	Concentr. [µg/plate]	Revertants per plate (Mean ± SD)
			TA 98	TA 100	TA 1535	TA 1537	WP2 uvrA
Without Activation	H2O		25 ± 7	116 ± 19	20 ± 5	10 ± 2	34 ± 4
	Test item	5.00	32 ± 4	105 ± 12	17 ± 4	8 ± 2	31 ± 4
		15.8	25 ± 3	109 ± 12	14 ± 3	9 ± 2	23 ± 1
		50.0	21 ± 6	108 ± 4	19 ± 3	9 ± 4	32 ± 7
		158	19 ± 4	114 ± 9	16 ± 6	9 ± 2	30 ± 2
		500	20 ± 3	111 ± 19	13 ± 3	9 ± 3	28 ± 5
		1580	19 ± 3	113 ± 11	15 ± 1	8 ± 3	33 ± 3
		5000	17 ± 4	94 ± 8	22 ± 6	7 ± 2	20 ± 10
	DAUN	1.00	779 ± 44
	NaN3	2.00		1371 ± 46	688 ± 15
	9-AA	50.0				1485 ± 390
	NQO	2.00					1924 ± 242
With Activation	H2O		33 ± 6	123 ± 10	17 ± 4	9 ± 2	35 ± 8
	Test item	5.00	29 ± 5	135 ± 25	20 ± 6	11 ± 2	41 ± 8
		15.8	21 ± 2	127 ± 20	19 ± 3	9 ± 3	33 ± 3
		50.0	25 ± 5	144 ± 6	14 ± 2	11 ± 3	35 ± 3
		158	28 ± 3	134 ± 8	13 ± 1	10 ± 4	32 ± 4
		500	25 ± 3	122 ± 3	15 ± 2	13 ± 3	39 ± 4
		1580	23 ± 5	140 ± 11	18 ± 2	14 ± 2	36 ± 3
		5000	23 ± 1	114 ± 5	19 ± 5	9 ± 5	39 ± 4
	2-AA	2.00	614 ± 50	1661 ± 160
	2-AA	5.00			147 ± 2	519 ± 30
	2-AA	10.0					406 ± 11

NaN3: Sodium azide, 2-AA: 2-Aminoanthracene, 9-AA: 9-Aminoacridine, DAUN: Daunomycin, NQO: 4-NitroquinoIine-N-oxide

Table 2: Summary 2nd series

Metabolic Activation	Test Material	Concentr. [µg/plate]	Revertants per plate (Mean ± SD)
			TA 98	TA 100	TA 1535	TA 1537	WP2 uvrA
Without Activation	H2O		40 ± 8	128 ± 7	20 ± 4	11 ± 3	24 ± 6
	Test item	50.0	39 ± 11	136 ± 7	20 ± 1	6 ± 3	28 ± 8
		158	39 ± 5	118 ± 10	21 ± 6	10 ± 3	23 ± 2
		500	28 ± 1	131 ± 8	21 ± 1	8 ± 3	26 ± 3
		1580	33 ± 6	124 ± 14	21 ± 7	8 ± 3	25 ± 3
		5000	28 ± 3	124 ± 27	17 ± 8	6 ± 5	21 ± 2
	DAUN	1.00	272 ± 7
	NaN3	2.00		1395 ± 58	770 ± 35
	9-AA	50.0				999 ± 102
	NQO	2.00					1503 ± 33
With Activation	H2O		36 ± 6	124 ± 21	17 ± 6	12 ± 5	45 ± 3
	Test item	50.0	46 ± 4	152 ± 14	20 ± 3	12 ± 5	39 ± 3
		158	36 ± 7	137 ± 2	20 ± 3	11 ± 3	39 ± 3
		500	35 ± 7	144 ± 4	13 ± 1	13 ± 2	34 ± 4
		1580	37 ± 8	149 ± 4	18 ± 6	11 ± 2	40 ± 4
		5000	35 ± 9	134 ± 21	15 ± 5	12 ± 2	39 ± 2
	2-AA	2.00	352 ± 40	670 ± 88
	2-AA	5.00			101 ± 9	128 ± 4
	2-AA	10.0					196 ± 18

NaN3: Sodium azide, 2-AA: 2-Aminoanthracene, 9-AA: 9-Aminoacridine, DAUN: Daunomycin, NQO: 4-NitroquinoIine-N-oxide

Table 3: Historical Data - Negative Controls

Strain	TA 98		TA 100		TA 1535		TA 1537*		WP2 uvrA
S9 Mix	Without	With	Without	With	Without	With	Without	With	Without	With
Compound	Solvent	Solvent	Solvent	Solvent	Solvent	Solvent	Solvent	Solvent	Solvent	Solvent
Total Plates	402	410	397	413	270	282	276	287	385	398
Number of Values	78	79	77	80	45	47	46	48	74	76
Minimum	23	25	82	101	20	18	23	20	16	17
Maximum	45	59	138	157	34	40	36	43	39	48
Mean	34	40	110	126	27	26	28	30	30	36
Standard Deviation	5.2	7.0	11.7	11.9	3.2	3.9	3.5	4.4	4.9	5.9

* The strain TA 1537 used in 2016 had a higher spontaneous revertant frequency compared to the strain used from 2017 onwards. As the historical data are generated on a yearly basis, this lower revertant frequency is not yet reflected in the historical control data presented here.

Table 4: Historical Data - Positive Controls

Strain	TA 98		TA 100		TA 1535		TA 1537		WP2 uvrA
S9 Mix	Without	With	Without	With	Without	With	Without	With	Without	With
Compound	DAUN	2-AA	NaN3	2-AA	NaN3	2-AA	9-AA	2-AA	NQO	2-AA
Total Plates	201	205	199	207	135	141	138	144	193	199
Number of Values	79	79	77	80	45	47	46	48	74	76
Minimum	117	105	412	512	583	92	122	125	395	112
Maximum	887	1632	2075	3337	1847	390	2882	1103	2286	1313
Mean	352	529	1337	1262	900	200	1175	418	1613	347
Standard Deviation	146.3	296.1	316.8	435.4	193.4	64.2	564.2	201.3	438.7	192.4

Conclusions:

The test substance was considered to be non-mutagenic with and without metabolic activation in bacteria.

Executive summary:

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 (3-Naphthoflavone/Phenobarbital was used. In this study, two experimental series were performed. The S9 mix used contained 10% S9 in the 1^st and 20% S9 in the 2^nd 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Bacterial Reverse Mutation Test

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 (3-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.

Chromosomal aberration test in mammalian cells

A chromosomal aberration test was carried out with the test item using a Chinese hamster fibroblast cell line (CHL). The test item was dissolved in physiological saline. The cells were exposed to the test item at three different doses (maximum dose: 2 mg/mL, selected by a preliminary test) for 24 and 48 hours without a metabolic activation system. Colcemid (final concentration 0.2 g/mL) was added to the culture 2 hours before cell harvesting. The cells were then trypsinised and suspended in a hypotonic KCl Solution (0.075 M) for 13 min at room temperature. After centrifugation the cells were fixed with acetic acid-methanol (1:3, v/v) and spread on clean glass slides. After air-drying, the slides were stained with Giemsa Solution (1.5%, at pH 6.8) for 12-15 min. A hundred well-spread metaphases were observed under the microscope (x600 with a nocover objective lens). The incidence of cells with structural chromosomal aberrations as well as of polyploid cells was recorded on each culture plate.

Untreated cells and solvent-treated cells served as negative controls, in which the incidence of aberrations was usually less than 3.0%.

Exposure to the test item (maximum dose: 2 mg/mL) resulted in an incidence of polyploid cells of 2.0 % after 48 hour exposure and in an incidence of aberrations of 3.0 % after 24 hour exposure. Therefore, the test item was considered to be non-clastogenic.

Justification for classification or non-classification

Classification, Labeling, 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. The results indicate that the substance is non-mutagenic. Based on available data on genetic toxicity, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EC) No 2017/776.