Sodium 2-stearoyllactate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria in vitro (OECD 471): negative with and without metabolic activation

Mammalian cell micronucleus test in vitro (OECD 487): negative with and without metabolic activation

Gene mutation in mammalian cells in vitro (OECD 476): negative with and without metabolic activation

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted in 1997

Deviations:

no

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

Target gene:

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

Species / strain / cell type:

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

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.

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.

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

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

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

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

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'.

 

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).

 

Conclusions:

Interpretation of results: negative

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 Oct - 19 Dec 2019

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:

adopted in 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

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

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes: cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Suitability of cells: Human lymphocytes are the most commonly used cells in the micronucleus test and have been used successfully for a long time in in vitro experiments. They show stable spontaneous micronucleus frequencies at a low level.

For lymphocytes:
- Sex, age and number of blood donors: Experiment I: one female, 28 years; Experiment II: one male, 28 years; donors were healthy, non-smoking and did not receive medication
- Whether whole blood or separated lymphocytes were used: whole blood
- Whether blood from different donors were pooled or not: no
- Mitogen used for lymphocytes: Phytohemeagglutinine (PHA)

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature:
- Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) supplemented with 200 mM GlutaMAX™,
- penicillin/streptomycin (100 U/mL/100 μg/mL),
- PHA (3 μg/mL),
- 10 % fetal bovine serum (FBS),
- 10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid),
- heparin (125 U.S.P.-U/mL).
In the pulse exposure experiment (pre-experiment/experiment I), for the treatment with metabolic activation 50 μL S9 mix per mL culture medium was added. In both experimens (pulse exposure = pre-experiment/experiment I and continuous exposure = experiment II), after exposure to the test substance, cells were resuspended in and washed with "saline G" (pH 7.2, containing 8000 mg/L NaCl, 400 mg/L KCl, 1100 mg/L glucose.H2O, 192 mg/L Na2HPO4.2 H2O and 150 mg/L KH2PO4).

- Incubation conditions: All incubations were done at 37 °C with 5.5% CO2 in humidified air.

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 a bacterial reverse mutation assay (Ames test).
- Lot no.: 050919B
- protein concentration: 30.2 mg/mL

Test concentrations with justification for top dose:

Pre-experiment/experiment I (4 h exposure): 4.4, 7.7, 13.5, 23.7, 41.5, 72.6, 127, 222, 667, 2000 µg/mL, with and without S9 mix
Experiment II (20 h exposure): 26.0, 39.0, 58.5, 87.8, 132, 198, 296, 444, 667, 1000 µg/mL, without S9 mix

Considering the molecular weight of the test item (378.52 g/mol), 2000 μg/mL was chosen as top concentration in the pre-experiment. Precipitation was observed at 127 μg/mL and above in the absence of S9 mix and at 72.6 μg/mL and above in the presence of S9 mix. Since in the pre-experiment the cultures fulfilled the requirements for the evaluation of cytogenetic effects of the test substance, this preliminary test was designated experiment I. Due to the precipitation observed in experiment I, 1000 μg/mL was chosen as top dose for experiment II.

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 relative nontoxicity to the cell cultures..

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Demecolcine

Details on test system and experimental conditions:

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

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 h (pulse exposure = pre-experiment/experiment I); 20 h (continuous exposure = experiment II)
- Fixation time (start of exposure up to fixation or harvest of cells): 40 h

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- If cytokinesis blocked method was used for micronucleus assay: Cytochalasin B, 4 μg/mL, 20 h incubation time
- Methods of slide preparation and staining technique used including the stain used: After separation by centrifugation, cells were resuspended in fixative (ice-cold methanol and glacial acetic acid (19 parts plus 1 part, respectively)) and kept cold. The slides were prepared by dropping the cell suspension in fresh fixative onto a clean microscope slide. Cells were stained with Giemsa.
- Number of cells spread and analysed per concentration: At least 1000 binucleate cells per culture, in duplicate cultures, i.e. at least 2000 binucleate cells per concentration.
- Criteria for scoring micronucleated cells: The micronuclei were counted in cells showing a clearly visible cytoplasm area. 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. The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle, 1976 (COUNTRYMAN P.I. and HEDDLE J.A. (1976) The production of micronuclei from chromosome aberrations in irradiated cultures of human lymphocytes. Mutation Research, 41, 321-332).

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cytokinesis-block proliferation index (CBP)
- Any supplementary information relevant to cytotoxicity: The CBPI was determined in 500 cells per culture and cytotoxicity is expressed as % cytostasis.

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 (95% control limit realised as 95% confidence interval).
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 (95% control limit realized as 95% confidence interval).
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.

Statistics:

The statistical significance was confirmed by the Chi Square Test (p < 0.05) and a linear regression was performed to assess a possible dose dependency in the rates of micronucleated cells.

Key result

Species / strain:

lymphocytes: cultured peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Only in experiment II in the absence of S9 mix, moderate cytotoxicity (29.8% cytostasis) was observed at the highest evaluated concentration.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: The effect of pH was checked; pH did not impact the results.
- Data on osmolality: The effect of osmolality was checked; osmolality did not impact the results.
- Precipitation and time of the determination: Pre-experiment/experiment I: Precipitation observed at 127 μg/mL and above in the absence of S9 mix and at 72.6 μg/mL and above in the presence of S9 mix at the end of treatment. Experiment II: Precipitation observed at 198 µg/mL and above in the absence of S9 mix at the end of treatment.

RANGE-FINDING/SCREENING STUDIES:
Since the range finding pre-experiment fulfilled all validity criteria, it was consider as experiment I which is detailed in sections above.

STUDY RESULTS
Detailed study results are provided in 'Any other information on results incl. tables'.

HISTORICAL CONTROL DATA:
Historical control data are provided in 'Any other information on results incl. tables'.

 

Table 1: Summary of results

Experiment	Preparation interval	Test item concentration [µg/mL]	Proliferation index (CBPI)	Cytostasis [%]*	Micronucleated cells [%]**	95% Ctrl limit
Exposure period 4 h without S9 mix
I	40 h	Solvent control^1	1.89		0.60	0.01 – 1.20
		Positive control^2	1.66	25.5	15.00^S	2.66 – 22.74
		41.5	1.85	4.5	0.65
		72.6	1.78	12.7	0.50
		127P	1.61	31.7	1.05
Trend test: p-value 0.282
Exposure period 20 h without S9 mix
II	40 h	Solvent control1/#	1.99		0.33	0.00 – 1.14
		Positive control^3	1.57	42.7	3.50^S	1.15 – 6.44
		26.0^#	1.99	n.c.	0.65^S
		39.0^#	1.89	10.8	0.50
		58.5^#	1.70	29.8	0.85^S
		87.8	n.e.	n.e.	n.e.
Trend test: p-value 0.128
Exposure period 4 h with S9 mix
I	40 h	Solvent control^1	1.97		0.20	0.00 – 1.24
		Positive control^4	1.39	60.2	2.95^S	1.01 – 7.34
		23.7	1.96	1.6	0.55
		41.5	1.98	n.c.	0.20
		72.6^P	1.90	7.1	0.45
Trend test: p-value 0.646

 

*         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

P         Precipitation occurred at the end of treatment

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

n.e.     Not evaluable, due to strong cytotoxic effects

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 0.8 µg/mL

3         Demecolcine 150 ng/mL

4         CPA 20.0 µg/mL

 

Table 2: Historical control data (percentage of micronucleated cells in human lymphocyte cultures (2018))

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 (4/40)	Continuous treatment (20/40)
No. of experiments	69*	69**
Mean	0.60	0.53
95 % Ctrl limit	0.01 – 1.20	0.00 – 1.14
1x SD (2x SD)	0.30 (0.60)	0.31 (0.61)
Min – Max	0.00 – 1.55	0.10 – 1.30
* Aqueous solvents – 16 Experiments; Organic solvents – 53 Experiments
** Aqueous solvents – 15 Experiments; Organic solvents – 54 Experiments
Solvent Control with S9
Micronucleated cells in %
	Pulse treatment (4/40)
No. of experiments	70*
Mean	0.62
95 % Ctrl limit	0.00 – 1.24
1x SD (2x SD)	0.31 (0.62)
Min – Max	0.05 – 1.60
* Aqueous solvents – 16 Experiments; Organic solvents – 54 Experiments
Positive Control without S9
Micronucleated cells in %
	Pulse treatment (4/40)	Continuous treatment (20/40)
	MMC	Demecolcin
No. of experiments	69	79
Mean	12.70	3.80
95 % Ctrl limit	2.66 – 22.74	1.15 – 6.44
1x SD (2x SD)	5.02 (10.04)	1.32 (2.64)
Min – Max	3.95 – 28.60	1.95 – 8.80
Positive Control with S9
Micronucleated cells in %
	Pulse treatment (4/40)
	CPA
No. of experiments	80
Mean	4.18
95 % Ctrl limit	1.01 – 7.34
1x SD (2x SD)	1.58 (3.17)
Min – Max	1.80 – 8.85

 

Conclusions:

Interpretation of results: negative

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 Sept - 25 Oct 2019

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:

adopted in 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

adopted in 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

adopted in 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

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

Type of assay:

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

Target gene:

HPRT locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: V79 cells (supplied by Laboratory for Mutagenicity Testing; Technical University Darmstadt, 64287 Darmstadt, Germany)
- Suitability of cells: V79 cells exhibit a high proliferation rate and a good cloning efficiency of untreated cells (as a rule more than 50%). Both these properties are necessary for the appropriate performance of the study. Moreover, the cells have a stable karyotype with a modal chromosome number of 22.

For cell lines:
- Absence of Mycoplasma contamination: Checked regularly
- Methods for maintenance in cell culture: Thawed stock cultures were propagated at 37 °C in 75 cm2 plastic flasks. About 2 - 3 × 10E06 cells were seeded into each flask with 15 mL of minimal essential medium (MEM) containing Hank’s salts supplemented with 10% fetal bovine serum (FBS), neomycin (5 μg/mL) and amphotericin B (1%). All incubations were done at 37 °C with 1.5% carbon dioxide (CO2) in humidified air.
- Cell cycle length, doubling time or proliferation index: doubling time 12 - 16 h in stock cultures
- Modal number of chromosomes: 22
- Periodically checked for karyotype stability: yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: The complete culture medium was MEM 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 (6-TG). 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: 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: 50 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 a bacterial reverse mutation assay (Ames test).
- Lot no.: 310119
- protein concentration: 30.7 mg/mL

Test concentrations with justification for top dose:

Pre-experiment (+/-S9): 15.6, 31.3, 62.5 125, 250, 500, 1000, 2000 µg/mL
Main experiment (-S9): 0.6, 1.3, 2.5, 5.0, 10.0, 15.0, 22.5, 30.0, 45.0 µg/mL
Main experiment (+S9): 1.9, 3.8, 7.5, 15.0, 30.0, 60.0, 120.0, 240.0 µg/mL

In the pre-experiment a relevant cytotoxic effect, indicated by a relative cloning efficiency of 50% or below was observed at 31.3 μg/mL and above without metabolic activation. In the presence of metabolic activation relevant cytotoxic effects were determined at 250.0 μg/mL and above. The dose range of the main experiment was chosen according to data generated in the pre-experiment.

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 relative nontoxicity 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: one main experiment

METHOD OF TREATMENT/EXPOSURE:
- Cell density at seeding: 2 x 10E6
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 24 h
- Exposure duration/duration of treatment: 4 h

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): approx. 7 days
- Selection details : 4 - 5 x 10E5 cells were seeded in medium containing 6-thioguanine (6-TG, 11 µg/mL) and incubated at 37 ± 1.5°C in a humidified atmosphere with 1.5% ± 0.5 CO2.
- Selection time: 8 - 11 days

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cloning efficiency

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- Method: mutant frequency

Evaluation criteria:

A test item is identified as clearly mutagenic if, in any of the experiments performed, 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 identified as clearly non-mutagenic if, in all experiments performed, 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).

Statistics:

A linear regression (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies (mean values) obtained for the groups treated with the test item was compared to the solvent control groups. A trend was judged as significant whenever the p-value (probability value) was < 0.05.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Precipitation occured at 15 µg/mL without S9 mix and at 60 µg/mL with S9 mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: The effect of pH was checked; pH did not impact the results.
- Data on osmolality: The effect of osmolality was checked; osmolality did not impact the results.
- Precipitation and time of the determination: Precipitation at the end of the 4 h treatment period was observed at 15.0 μg/mL without metabolic activation, and at 60.0 μg/mL with metabolic activation.

RANGE-FINDING/SCREENING STUDIES
The pre-experiment was performed in the presence and absence (4 h treatment) of metabolic activation. Test item concentrations between 15.6 μg/mL and 2000 μg/mL were used. A relevant cytotoxic effect, indicated by a relative cloning efficiency of 50% or below was observed at 31.3 μg/mL and above without metabolic activation. In the presence of metabolic activation relevant cytotoxic effects were determined at 250.0 μg/mL and above. Precipitation occurred at 31.3 μg/mL and above after 4 h treatment without metabolic activation, and at 62.5 μg/mL and above with metabolic activation. There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.

STUDY RESULTS
Detailed study results are provided in 'Any other information on results incl. tables'.

HISTORICAL CONTROL DATA
Historical control data are provided in 'Any other information on results incl. tables'.

 

Table 2: Summary of results

	Concentration [µg/mL]	P	S9 mix	relative cloning efficiency I [%]	relative cell density [%]	rel. Adjusted cloning efficiency I [%]	mutant colonies / 10E6 cells	95% confidence interval
Main Experiment / 4 h treatment				mean values of culture I and II
Solvent control with water			-	100.0	100.0	100.0	9.9	2.8 - 30.9
Positive control (EMS)	300.0		-	87.5	104.1	91.4	170.0	2.8 - 30.9
Test item	0.6		-	97.4	100.8	98.2	10.7	2.8 - 30.9
Test item	1.3		-	101.4	100.2	102.2	8.2	2.8 - 30.9
Test item	2.5		-	99.5	92.5	92.2	17.2	2.8 - 30.9
Test item	5.0		-	88.1	113.9	101.1	8.1	2.8 - 30.9
Test item	10.0		-	89.4	87.8	78.6	14.1	2.8 - 30.9
Test item	15.0	P	-	90.2	79.8	72.3	15.2	2.8 - 30.9
Test item	22.5	P	-	culture was not continued^#
Test item	30.0	P	-	culture was not continued^#
Test item	45.0	P	-	culture was not continued^#
Solvent control with water			+	100.0	100.0	100.0	13.5	3.1 -30.7
Positive control (DMBA)	2.3		+	88.1	102.2	88.2	332.3	3.1 -30.7
Test item	1.9		+	101.8	98.3	99.8	11.7	3.1 -30.7
Test item	3.8		+	83.7	98.7	83.3	12.6	3.1 -30.7
Test item	7.5		+	92.4	115.2	106.9	12.9	3.1 -30.7
Test item	15.0		+	99.8	109.3	109.3	20.2	3.1 -30.7
Test item	30.0		+	110.7	96.4	110.9	11.8	3.1 -30.7
Test item	60.0	P	+	83.7	94.2	78.7	18.3	3.1 -30.7
Test item	120.0	P	+	culture was not continued^#
Test item	240.0	P	+	culture was not continued^#

P: precipitation at the end of treatment

^#: Culture was not continued to avoid analysis of too many precipitating concentrations

 

Table 3: Historical control data

2014 - 2018
Number of mutant colonies per 10E6 cells
without metabolic activation (4 h treatment time)
	Positive control EMS 150 and 300 µg/mL	Solvent control (medium, acetone, water, DMSO, ethanol, THF, EGDE)
Range:	53.9 – 872.3	3.4 – 41.0
Mean value:	223.3	16.9
Standard deviation:	101.1	7.0
95% confidence interval	--	2.8 – 30.9
Number of studies:	199	199
with metabolic activation (4 h treatment time)
	Positive control DMBA 1.1 and 2.3 µg/mL	Solvent control (medium, acetone, water, DMSO, ethanol, THF, EGDE)
Range:	55.6 – 739.9	2.4 – 40.4
Mean value:	188.0	16.9
Standard deviation:	98.6	6.9
95% confidence interval	--	3.1 – 30.7
Number of studies:	193	193

 

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

 

Conclusions:

Interpretation of results: negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Gene mutation in bacteria in vitro

An in vitro test was performed to investigate the potential of esterification product of C16-C18 even numbered, saturated fatty acids and lactic acid, sodium salt (sodium stearoyl lactylate, SSL, CAS No. 25383-99-7, EC No. 246-929-7) to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the E. coli strain WP2 uvrA. The study was performed according to OECD guideline 471 under GLP conditions (Chang, 2019, Ames) in two independent experiments both with and without liver microsomal activation (S9 mix). Each concentration, the positive and negative controls was tested in triplicate. The test item was tested at the following concentrations:Pre

• Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate (all strains)
• Experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate (strains TA 1535 and WP2 uvrA), 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate (strains TA 1537 and TA 98), 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate (strain TA 100)

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. The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used. Cytotoxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in experiment I in strains TA 1537, TA 98, and TA 100 and in experiment II in strains TA 1537 and TA 100. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation. There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. In conclusion, under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Mammalian cell micronucleus test in vitro

The test item esterification product of C16-C18 even numbered, saturated fatty acids and lactic acid, sodium salt (sodium stearoyl lactylate, SSL, CAS No. 25383-99-7, EC No. 246-929-7), suspended in deionised water, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in two independent experiments according to OECD guideline 487 under GLP conditions (Naumann, 2020, MNT). In each experimental group, two parallel cultures were analysed. Per culture at least 1000 binucleated cells were evaluated for cytogenetic damage. The highest applied concentration in this study (2000 μg/mL of the test item) was chosen with regard to the molecular weight of the test item and with respect to the current OECD test guideline. Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation. In Experiment I, in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed precipitation. In Experiment II, in the absence of S9 mix, moderate cytotoxicity (29.8% cytostasis) was observed at the highest evaluated concentration. The next higher tested concentration, however, which was separated by a factor of 1.5 and therefore smaller than requested by the guideline, was not evaluable due to a steep cytotoxic gradient caused by the test item. In Experiment I and II in the absence and presence of S9 mix, no relevant increases in the numbers of micronucleated cells were observed after treatment with the test item. In Experiment II in the absence of S9 mix, however, two values (0.65% and 0.85% micronucleated cells) after treatment with 26.0 and 58.5 μg/mL, respectively, were statistically significantly increased. Since both values are clearly within the range of the 95% control limit of the historical control data (0.00 - 1.14 %) and no dose-dependency, tested by a trend test, was observed, the findings can be regarded as biologically irrelevant. Appropriate mutagens were used as positive controls and induced statistically significant increases in cells with micronuclei. In conclusion, under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.

Gene mutation in mammalian cells in vitro

A study according to OECD guideline 476 and under GLP conditions was performed to investigate the potential of esterification product of C16-C18 even numbered, saturated fatty acids and lactic acid, sodium salt (sodium stearoyl lactylate, SSL, CAS No. 25383-99-7, EC No. 246-929-7) to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster (Sokolowski, 2019, HPRT). The treatment period was 4 h with and without metabolic activation. The maximum test item concentration of the pre-experiment (2000 μg/mL) was chosen with respect to the current OECD test guideline. The concentration range of the main experiment was determined by precipitation and cytotoxicity observed in the pre-experiment. No substantial and dose dependent increase of the mutation frequency was observed in the main experiment. Appropriate reference mutagens (positive controls) induced a distinct increase in mutant colonies and thus demonstrated the sensitivity of the test system and the activity of the metabolic activation system. In conclusion, under the experimental conditions reported, the test item did not induce gene mutations at the HPRT locus in V79 cells.

Justification for classification or non-classification

The available data on genetic toxicity of esterification product of C16-C18 even numbered, saturated fatty acids and lactic acid, sodium salt (sodium stearoyl lactylate, SSL, CAS No. 25383-99-7, EC No. 246-929-7) do not meet the criteria for classification according to Regulation (EC) No. 1272/2008 (CLP) and are therefore conclusive but not sufficient for classification.