Sodium 2-stearoyllactate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 Sept - 02 Oct 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted in 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 2-stearoyllactate

EC Number:

246-929-7

EC Name:

Sodium 2-stearoyllactate

Cas Number:

25383-99-7

Molecular formula:

This reference substance is a UVCB of the NCS type. It is a complex mixture of compounds and therefore molecular formula, molecular weight, SMILES, InChI and structural formula cannot be given.

IUPAC Name:

sodium 2-hydroxy-2-methyl-3-oxoicosanoate

Method

Target gene:

his operon for S. typhimurium strains and trp operon for E. coli strain

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone
- concentration or volume of S9 mix and S9 in the final culture medium: 500 µL
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): 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.
- Lot no.: 060619K
- protein concentration: 29.3 mg/mL

Test concentrations with justification for top dose:

Pre-experiment/experiment I:
3, 10, 33, 100, 333, 1000, 2500, and 5000 μg/plate with and without metabolic activation

Experiment II:
Strains TA 1535 and WP2 uvrA: 33, 100, 333, 1000, 2500 and 5000 μg/plate with and without metabolic activation
Strains TA 1537 and TA 98: 10, 33, 100, 333, 1000, 2500 and 5000 μg/plate with and without metabolic activation
Strain TA 100: 3, 10, 33, 100, 333, 1000, 2500 and 5000 μg/plate with and without metabolic activation

5000 µg/plate was selected as the highest test concentration based on the results of a range-finding study (pre-experiment / experiment I). Since toxic effects were observed in the pre-experimentexperiment I, a minimum of six concentrations were tested in experiment II. The concentration range included two logarithmic decades.

Vehicle / solvent:

- Vehicle used: deionised water
- Justification for choice of solvent/vehicle: The solvent was chosen based on the solubility properties of the test substance and its nontoxicity to the bacteria.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (4-NOPD), 2-aminoanthracene (2-AA)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: two (pre-experiment/experiment I and experiment II)

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 10E08 - 10E09 cells/mL
- Test substance added in agar (plate incorporation) in pre-experiment/experiment I; preincubation in experiment II

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Inspection of the background growth inhibition

Evaluation criteria:

The test material may be considered positive in this test system if the following criteria are met:
Two-fold or above (strains TA 98, TA 100, and WP2 uvrA) or threefold or above (strains TA 1535 and TA 1537) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. An increase of revertant colonies equal or above the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels. All cultures must demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls.

Statistics:

Mean values and standard deviation were calculated.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

precipitation was observed at concentrations of 2500 µg/plate and above (experiment I + experiment II)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cytotoxicity was observed at concentrations of 5000 µg per plate (-S9/+S9, experiment I) and 1000 µg/plate and above (-S9, experiment II)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cytotoxicity was observed at concentrations of 5000 µg per plate (-S9, experiment II); precipitation at 2500 µg/plate and above in both experiments.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cytotoxicity was observed at concentrations of 333 µg per plate and above (-S9, experiment I), 2500 µg/plate and above (+S9, experiment I) and 1000 µg/plate and above (-S9, experiment II); precipitation at 2500 µg/plate and above in experimetn II.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

precipitation was observed at concentrations of 2500 µg/plate and above (experiment I + experiment II)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test substance is poorly soluble in water; therefore, it was suspended in deionised water.
- Precipitation and time of the determination: The test item precipitated in the overlay agar in the test tubes from 1000 to 5000 μg/plate in experiment I and from 2500 to 5000 μg/plate in experiment II. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 2500 to 5000 μg/plate in both experiments. The undissolved particles had no influence on the data recording.

RANGE-FINDING/SCREENING STUDY
To evaluate the toxicity of the test item a pre-experiment was performed with all strains. Eight concentrations in the range 3 - 5000 µg/plate were tested for toxicity and mutation induction with three plates each. The experimental conditions in this pre-experiment fulfilled all acceptance criteria. Therefore, the pre-experiment was also considered as experiment I. Since toxic effects were observed in the pre-experiment/experiment I, a minimum of six concentrations were tested in experiment II. 5000 μg/plate was chosen as maximal concentration.

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

Ames test:
- Individual plate counts : Please refer to 'Any other information on results incl. tables'.
- Mean number of revertant colonies per plate and standard deviation: Please refer to 'Any other information on results incl. tables'.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
Please refer to 'Any other information on results incl. tables'.

Any other information on results incl. tables

 

Table 1: Summary of experiment I

Metabolic Activation	Test Group	Dose Level (per plate)	Revertant Colony Counts (Mean ±SD)
			TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	Deionised water		10 ± 4	10 ± 2	29 ± 3	117 ± 16	47 ± 10
	Untreated		16 ± 3	14 ± 4	28 ± 8	110 ± 3	44 ± 9
	Test substance	3 µg	12 ± 1	12 ± 0	25 ± 8	109 ± 13	34 ± 7
		10 µg	14 ± 5	12 ± 3	29 ± 6	100 ± 13	45 ± 12
		33 µg	10 ± 3	12 ± 5	29 ± 9	103 ± 11	44 ± 3
		100 µg	12 ± 2	8 ± 2	33 ± 2	90 ± 11	37 ± 1
		333 µg	11 ± 4	8 ± 2	28 ± 8	39 ± 8	46 ± 12
		1000 µg	13 ± 5	6 ± 0	20 ± 1	33 ± 2	49 ± 5
		2500 µg	11 ± 3 P M	5 ± 1 P M	15 ± 2 P M	32 ± 1 P	44 ± 8 P
		5000 µg	10 ± 1 P M	3 ± 1 P M	12 ± 2 P M	22 ± 2 P M	49 ± 11 P
	NaN3	10 µg	1080 ± 47			1654 ± 91
	4-NOPD	10 µg			395 ± 8
		50 µg		72 ± 10
	MMS	2.0 µL					1051 ± 12
With Activation	Deionised water		11 ± 5	14 ± 5	34 ± 3	116 ± 7	43 ± 16
	Untreated		10 ± 1	10 ± 1	35 ± 3	100 ± 19	53 ± 9
	Test substance	3 µg	12 ± 3	14 ± 6	42 ± 7	112 ± 19	55 ± 8
		10 µg	12 ± 2	11 ± 1	44 ± 7	124 ± 4	52 ± 6
		33 µg	13 ± 3	15 ± 6	32 ± 4	109 ± 10	48 ± 4
		100 µg	13 ± 2	15 ± 2	33 ± 3	110 ± 15	45 ± 6
		333 µg	11 ± 5	17 ± 3	37 ± 6	87 ± 9	53 ± 2
		1000 µg	12 ± 3	10 ± 2	43 ± 4	69 ± 3	51 ± 6
		2500 µg	10 ± 3 P M	14 ± 2 P	32 ± 8 P	48 ± 7 P M	49 ± 9 P
		5000 µg	9 ± 3 P M	6 ± 2 P M	23 ± 4 P M	45 ± 7 P M	48 ± 9 P
	2-AA	2.5 µg	350 ± 19	415 ± 5	2845 ± 71	3939 ± 86
		10.0 µg					297 ± 29

 

Key to positive controls:		Key to plate postfix codes:
NaN3	Sodium azide	P	Precipitate
2-AA	2-Aminoanthracene	M	Manual count
4-NOPD	4-Nitro-o-phenylenediamine
MMS	Methylmethanesulfonate

 

Table 2: Summary of experiment II

Metabolic Activation	Test Group	Dose Level (per plate)	Revertant Colony Counts (Mean ±SD)
			TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	Deionised water		12 ± 2	17 ± 2	26 ± 1	118 ± 15	43 ± 9
	Untreated		12 ± 3	14 ± 5	31 ± 3	114 ± 7	51 ± 8
	Test substance	3 µg				112 ± 10
		10 µg		17 ± 3	24 ± 2	118 ± 8
		33 µg	14 ± 5	15 ± 0	29 ± 9	106 ± 2	45 ± 13
		100 µg	13 ± 3	12 ± 3	30 ± 3	102 ± 3	40 ± 4
		333 µg	14 ± 3	12 ± 2	26 ± 6	64 ± 10	38 ± 2
		1000 µg	11 ± 1	7 ± 2	24 ± 3	48 ± 3	38 ± 4
		2500 µg	14 ± 3 P	7 ± 2 P M	23 ± 8 P	49 ± 8 P	42 ± 9 P
		5000 µg	10 ± 3 P M	7 ± 3 P M	18 ± 1 P M	34 ± 6 P M	30 ± 2 P
	NaN3	10 µg	1224 ± 71			1885 ± 83
	4-NOPD	10 µg			400 ± 16
		50 µg		89 ± 1
	MMS	2.0 µL					854 ± 57
With Activation	Deionised water		12 ± 2	14 ± 3	34 ± 6	114 ± 13	49 ± 2
	Untreated		14 ± 2	14 ± 1	44 ± 5	130 ± 21	49 ± 8
	Test substance	3 µg				110 ± 19
		10 µg		14 ± 3	40 ± 4	117 ± 6
		33 µg	11 ± 5	14 ± 2	45 ± 3	110 ± 6	53 ± 4
		100 µg	11 ± 3	13 ± 2	34 ± 1	110 ± 9	52 ± 6
		333 µg	11 ± 2	14 ± 2	36 ± 4	113 ± 9	52 ± 6
		1000 µg	13 ± 3	15 ± 1	32 ± 9	87 ± 22	56 ± 9
		2500 µg	11 ± 2 P M	16 ± 2 P	29 ± 4 P M	74 ± 15 P	53 ± 10 P
		5000 µg	12 ± 2 P M	11 ± 3 P M	29 ± 4 P M	63 ± 3 P M	59 ± 4 P
	2-AA	2.5 µg	441 ± 33	455 ± 13	2763 ± 218	4196 ± 204
		10.0 µg					313 ± 13

 

Key to positive controls:		Key to plate postfix codes:
NaN3	Sodium azide	P	Precipitate
2-AA	2-Aminoanthracene	M	Manual count
4-NOPD	4-Nitro-o-phenylenediamine
MMS	Methylmethanesulfonate

 

Table 3: Historical control data

Strain			without S9 mix				with S9 mix
		Mean	SD	Min	Max	Mean	SD	Min	Max
	Solvent control	11	2.3	6	22	12	2.3	7	22
TA 1535	Untreated control	11	2.9	6	28	12	2.8	7	23
	Positive control	1245	161.4	367	1791	398	61.0	183	613
	Solvent control	10	2.2	6	19	13	3.2	7	30
TA 1537	Untreated control	10	2.7	5	21	14	3.6	6	29
	Positive control	94	30.0	48	231	170	64.8	81	421
	Solvent control	26	4.2	13	43	36	6.1	12	56
TA 98	Untreated control	27	4.7	14	40	39	6.4	12	59
	Positive control	421	176.8	196	2068	3908	815.0	223	5918
	Solvent control	160	29.3	79	214	148	31.3	76	216
TA 100	Untreated control	181	26.1	80	235	171	27.7	87	218
	Positive control	2074	262.7	511	2850	3626	981.9	553	5860
	Solvent control	39	6.4	26	60	48	7.3	28	69
WP2 uvrA	Untreated control	40	5.8	22	61	51	7.4	32	87
	Positive control	865	340.9	346	4732	426	111.2	184	1265

 

Mean: mean value of revertants/plate

SD: standard deviation

Min: minimal value

Max: maximal value

These data represent the laboratory´s historical control data from November 2016 until August 2018 representing approx. 600 experiments (for WP2 uvrA the historical data are based on approx. 400 experiments).

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative