Sodium 2-(1-carboxylatoethoxy)-1-methyl-2-oxoethyl isooctadecanoate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The genetic toxicity potential of the test item sodium isostearoyl lactylate was assessed with three in vitro genetic toxicity studies: One gene mutation study in mammalian cells (HPRT; performed in accordance with OECD 476 using the test item) and two genetic toxicity studies (an Ames assay in bacteria and a chromosomal aberration test in mammalian cells) using calcium stearoyl lactylate, a suitable read-across substance for the test item. In all three studies, there are no indications of genetic toxicity potential of the test item as well as its read-across substance. Taking the results in a weight-of-evidence approach, there is no genetic toxicity potential of sodium isostearoyl lactylate.

For details and justification of read-across please refer to the read-across report attached to IUCLID section 13.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

For details and justification of read-across please refer to the read-across report attached to IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not valid

Conclusions:

Interpretation of results: Negative
The test substance showed no genetic toxicity when tested on Chinese hamster lung fibroblast mammalian cell line.

Executive summary:

In a mammalian cell cytogenetics assay (chromosome aberration test), primary lung fibroblast cultures were exposed to calcium stearoyl-2-lactylate in ethanol at a concentration of 0.063 mg/ml without metabolic activation.

Calcium stearoyl-2-lactylate was tested up to cytotoxic concentrations (50 % cell-growth inhibition). Positive controls were not included.

This study is classified as partly acceptable. This study partly satisfies the requirements for test guideline OECD 473 for in vitro cytogenetic mutagenicity data.

In this study, the test item is non-mutagenic.

This information is used in a read-across approach in the assessment of the target substance. For justification of read-across please refer to the read-across report attached to IUCLID section 13.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

For details and justification of read-across please refer to the report attached in section 13 of IUCLID.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

S. typhimurium, other: TA92, TA1535, TA100, TA1537, TA94 and TA98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not valid

Conclusions:

Interpretation of results: Negative
The test substance showed no genetic toxicity when tested in S. typhimurium TA92, TA1535, TA100, TA1537, TA94 and TA98.

Executive summary:

In a reverse gene mutation assay in bacteria, strains TA92, TA1535, TA100, TA1537, TA94 and TA98of S. typhimurium were exposed to calcium stearyl-2-lactylatein benzene as solventat a concentration of 0.3 mg/plate in the presence and absence of mammalian metabolic activation.

Calcium stearyl-2-lactylate was tested up to insoluble concentrations using a co-solvent. Positive control results were not reported.

This study is classified asacceptable. This study satisfies the requirement for OECD test guideline 471 for in vitro mutagenicity (bacterial reverse gene mutation) data. The absence of strains sensitive to certain oxidising mutagens, cross-linking agents and hydrazines is not considered a major deficiency since the test substance obviously does not belong to these substance classes. The lack of positive controls is considered a minor deficiency not invalidating this study.

This information is used in a read-across approach in the assessment of the target substance. For justification of read-across please refer to the read-across report attached to IUCLID section 13.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-02-12 to 2018-10-29

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)

Version / remarks:

Adopted July 29, 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

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

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch no. of test material: Pationic ISL (Lot: 1731200025)
- Expiration date of the lot/batch: 12 months after opening

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Solubility and stability of the test substance in the solvent/vehicle: All test item solutions were freshly prepared immediately prior to use.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: A solubility test was performed with different solvents and vehicles. Based on the results of the solubility test, treatment medium (MEM + 0% FBS) was used as solvent. The solvent was compatible with the survival of the cells and the S9 activity. The pH-value detected with the test item was within the physiological range (pH 7.0 ± 0.4). Osmolality of the highest test item concentration was 324 mOsmol/kg.

Target gene:

Hprt

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: ATCC (CCL-93)
- Suitability of cells: These cells are characterised by their high proliferation rate and their high cloning efficiency of untreated cells (usually more than 50%). These features of the cells are necessary for the appropriate performance of the study.
- Cell cycle length, doubling time or proliferation index: 12 - 14 h doubling time
- Methods for maintenance in cell culture: Freshly thawed cells from stock cultures were maintained in plastic culture flasks in minimal essential medium (MEM) and cultured at a humidified atmosphere of 5% CO2 and at 37 °C.


MEDIA USED
- Type and identity of media including CO2 concentration if applicable: MEM supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/100 µg/mL penicillin/streptomycin, 2 mM L-glutamine, 25 mM HEPES and 2.5 µg/mL amphotericin B; cultured at a humidified atmosphere of 5% CO2 and at 37 °C
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 liver

Test concentrations with justification for top dose:

Cytotoxicity of the test item was determined in a pre-experiment. In the pre-experiment it was observed that cytotoxic effects occurred without metabolic activation at concentrations of 10 µg/mL or higher and with metabolic activation at concentrations of 100 µg/mL or higher. Based on these data the concentrations for the main experiment were selected.
The test item was tested for cytotoxicity at the following concentrations:
* without metabolic activation: 2, 4, 6, 8, 10, 14, 16 and 20 µg/mL
* with metabolic activation: 25, 50, 55, 60, 65, 70, 75, 80, 85 and 90 µg/mL
According to OECD test guideline 476, more than 4 concentrations may be particularly important for evaluating mutagenicity when using single cultures. Therefore, the following 5 concentrations were selected on the basis of cytotoxicity for evaluating mutagenicity.
* without metabolic activation: 4, 6, 8, 10 and 14 µg/mL
* with metabolic activation: 25, 55, 65, 75 and 85 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: treatment medium (MEM + 0 % FBS)
- Justification for choice of solvent/vehicle: A solubility test was performed with different solvents and vehicles. Based on the results of the solubility test, the treatment medium was used as solvent. The solvent was compatible with the survival of the cells and the S9 activity.

Untreated negative controls:

yes

Remarks:

Treatment medium

Negative solvent / vehicle controls:

no

Remarks:

Not required as treatment medium was used as vehicle for test item.

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding: Approximately 10–20E+06 cells per each concentration, negative and positive controls, were seeded in complete cell culture medium (MEM supplemented with 10 % FBS) in a 175 cm³ culture flask.

DURATION
- Preincubation period: 24 h after seeding
- Exposure duration: 4 h
- Expression time (cells in growth medium): 7–9 days after treatment
- Selection time (if incubation with a selection agent): 9–11 days for mutant frequency and 6–8 days for cloning efficiency

SELECTION AGENT (mutation assays): 11 µg/mL 6-thioguanine (TG)

NUMBER OF REPLICATIONS: 2 for cytotoxicity; 2–5 for mutagenicity

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Colonies were fixed with methanol, stained with Giemsa and counted.

NUMBER OF CELLS EVALUATED: 200 cells for cloning efficiency and 400000 cells for mutagenicity

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
- Any supplementary information relevant to cytotoxicity: The relative survival (indicator of cytotoxicity) was calculated based on the cloning efficiency of the cells plated immediately after treatment adjusted by any loss of cells during treatment.

Evaluation criteria:

A test chemical is considered to be clearly negative if, in all experimental conditions examined,
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend-test, and
- all results are inside the distribution of the historical negative control data.
A test chemical is considered to be clearly positive if, in any of the experimental conditions examined,
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- the increase is concentration-related when evaluated with an appropriate trend test, and
- any of the results are outside the distribution of the historical negative control data.
- if there is by chance a low spontaneous mutation rate in the corresponding negative control a concentration-related increase of the mutations within their range has to be discussed.
According to the OECD guideline, the biological relevance is considered first for the interpretation of results.

Statistics:

The non-parametric Mann-Whitney test was applied to the mutation data to determine any significant difference in mutant frequency for the concentration groups when compared to the negative control. Mutant frequencies of the negative control were used as reference. Additionally, the chi-squared test for trend was conducted to determine a statistically significant concentration-response relationship.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At the highest tested concentration for mutagenicity (75 µg/mL)

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At the highest tested concentration for mutagenicity (14 µg/mL)

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: In the first pre-experiment eight concentrations [250, 500, 600, 700, 800, 1000, 1500 and 2000 µg/mL] were tested. Due to very high toxicity, the pre-experiment had to be repeated with lower concentrations (1–250 µg/mL), which then allowed selection of the concentrations to be tested in the main experiments.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95 %): See Table 5 in “any other information on materials and methods”.

Cytotoxicity: A biologically relevant growth inhibition (i.e. reduction of relative survival below 70%) was observed after the treatment with the test item in the experiment with and without metabolic activation.

In the experiment without metabolic activation the relative survival was 10% for the highest concentration (14 µg/mL) evaluated for mutagenicity.

The highest biologically relevant concentration evaluated in the mutagenicity experiment with metabolic activation was 75 µg/mL with a relative survival of 15%. At concentration of 60 µg/mL the relative survival value was decreased (RS = 7%) but was considered to be an outlier in comparison to the next lower (55 µg/mL, RS = 77%) and the next higher (65 µg/mL; RS = 76%) tested concentrations. As the next lower and higher concentrations showed no cytotoxicity, this decrease was regarded as not biologically relevant and potentially caused by technical reasons.

Mutagenicity: In the experiments no biologically relevant increase of mutants (i.e. above the upper limit of the historical control data and no statistically relevant increase) was found after treatment with the test item (without and with metabolic activation). In the experiment without metabolic activation one mutant value (at concentration 14 µg/mL with 41.5 mutants/10⁶cells) was found to be slightly above the upper limit of the historical control data (40.2 mutants/10⁶cells). However, at this concentration, the relative survival is 10% and this positive result, according to the OECD guideline 476, should be interpreted with caution. Since this was only a slight increase and no statistically significant increase was determined, this effect was considered as not biologically relevant. All the other mutant values were within the historical control data range of the test facility.

Table 1: Cytotoxicity, without metabolic activation

Group		Concen-tration [µg/mL]	Number of cells at the		Number of colonies per flask^a			CE^b[%]	Adjusted CE^c [%]	Relative Survival (RS)^d [%]
			beginning of treatment	end of treatment	I	II	mean
NC1		0	10000000	9996000	161	156	159	79	79	100
NC2			10000000	10693000	137	167	152	76	81
1		2	10000000	10540000	192	189	191	95	100	125
2		4	10000000	10812000	162	175	169	84	91	114
3		6	10000000	10523000	169	143	156	78	82	102
4		8	10000000	8823000	127	125	126	63	56	69
5		10	10000000	6817000	63	53	58	29	20	25
6		14	20000000	6358000	46	51	49	24	8	10
7		16	20000000	4148000	70	59	65	32	7	8
8		20	20000000	2754000	77	81	79	40	5	7
EMS		300	10000000	11577000	163	170	167	83	96	120

NC: negative control

CE: cloning efficiency

EMS: ethyl methanesulfonate

a: number of cells plated: 200 cells/flask

b: cloning efficiency: CE [%] = [(number of colonies / number of cells plated) x 100]

c: adjusted CE [%] = [CE x (number of cells at the end of treatment / number of cells at the beginning of treatment)]

d: relative survival: RS [%] = [(adjusted CE in treated culture / adjusted CE in the negative control) x 100]

Table 2: Mutagenicity, without metabolic activation

		CE in non-selective medium				CE in selective medium
Group	Concen-tration [µg/mL]	Number of colonies per flask^a			CE^b[%]	Number of colonies per flask^c							CE^b[%]	Mutant Frequency per 10^6cells^d
		I	II	mean		I	II	III	IV	V	mean	SD
NC1	0	168	163	166	83	13	8	11	12	8	10.4	2.1	0.0026	31.4
NC2		150	160	155	78	8	13	13	11	14	11.8	2.1	0.0030	38.1
2	4	150	165	158	79	9	7	11	8	8	8.6	1.4	0.0022	27.3
3	6	143	161	152	76	11	14	6	16	8	11.0	3.7	0.0028	36.2
4	8	171	150	161	80	7	13	10	9	6	9.0	2.4	0.0023	28.0
5	10	161	175	168	84	9	7	8	4	6	6.8	1.7	0.0017	20.2
6	14	138	146	142	71	11	10	11	13	14	11.8	1.5	0.0030	41.5
EMS	300	155	165	160	80	60	75	76	76	77	72.8	6.4	0.0182	227.5

NC: negative control

CE: cloning efficiency

EMS: ethyl methanesulfonate

a: number of cells plated: 200 cells/flask

b: cloning efficiency:CE [%] = [(number of colonies / number of cells plated) x 100]

c: number of cells plated: 400000 cells/petri dish

d: mutant frequency (per 10⁶cells): MF = [CE of mutant colonies in selective medium / CE in non-selective medium) x 10⁶]

 Table 3: Cytotoxicity, with metabolic activation

Group		Concen-tration [µg/mL]	Number of cells at the		Number of colonies per flask^a			CE^b[%]	Adjusted CE^c [%]	Relative Survival (RS)^d [%]
			beginning of treatment	end of treatment	I	II	mean
NC1		0	10000000	13991000	113	132	123	61	86	100
NC2			10000000	13566000	148	148	148	74	100
1		25	10000000	12563000	153	141	147	74	92	99
2		50	10000000	13549000	108	114	111	56	75	81
3		55	10000000	13566000	109	103	106	53	72	77
4		60	10000000	4471000	28	29	29	14	6	7
5		65	10000000	13634000	114	93	104	52	71	76
6		70	10000000	8755000	35	38	37	18	16	17
7		75	20000000	12070000	31	63	47	24	14	15
8		80	20000000	9520000	31	41	36	18	9	9
9		85	20000000	7344000	37	37	37	19	7	7
10		90	20000000	7242000	14	21	18	9	3	3
DMBA		1.0	10000000	14484000	142	145	144	72	104	112
DMBA		1.5	10000000	14484000	146	142	144	72	104	112

NC: negative control

CE: cloning efficiency

DMBA: 7,12-dimethylbenz(a)anthracene

a: number of cells plated: 200 cells/flask

b: cloning efficiency: CE [%] = [(number of colonies / number of cells plated) x 100]

c: adjusted CE [%] = [CE x (number of cells at the end of treatment / number of cells at the beginning of treatment)]

d: relative survival: RS [%] = [(adjusted CE in treated culture / adjusted CE in the negative control) x 100]

 

Table 4: Mutagenicity, with metabolic activation

		CE in non-selective medium				CE in selective medium
Group	Concen-tration [µg/mL]	Number of colonies per flask^a			CE^b[%]	Number of colonies per flask^c							CE^b [%]	Mutant Frequency per 10^6cells^d
		I	II	mean		I	II	III	IV	V	mean	SD
NC1	0	155	137	146	73	9	7	9	13	3	8.2	3.2	0.0021	28.1
NC2		146	140	143	72	13	7	10	11	7	9.6	2.3	0.0024	33.6
1	25	143	128	136	68	11	7	13	14	5	10.0	3.5	0.0025	36.9
3	55	156	178	167	84	13	15	16	10	9	12.6	2.7	0.0032	37.7
5	65	167	181	174	87	16	20	11	12	6	13.0	4.7	0.0033	37.4
7	75	167	163	165	83	15	12	8	14	8	11.4	2.9	0.0029	34.5
DMBA	1.0	167	144	156	78	89	110	96	103	110	101.6	8.2	0.0254	326.7
DMBA	1.5	158	147	153	76	121	100	114	121	116	114.4	7.7	0.0286	375.1

NC: negative control

CE: cloning efficiency

DMBA: 7,12-dimethylbenz(a)anthracene

a: number of cells plated: 200 cells/flask

b: cloning efficiency: CE [%] = [(number of colonies / number of cells plated) x 100]

c: number of cells plated: 400000 cells/petri dish

d: mutant frequency (per 10⁶cells): MF = [CE of mutant colonies in selective medium / CE in non-selective medium) x 10⁶] 

Table 5: Historical Laboratory Control Data (January 2015 – April 2018)

	Negative Control		Positive Control
	-S9	+S9	EMS	DMBA
Mean	24	27	290	414
Min	5	8	186	117
Max	43	49	631	788
SD	7.92	8.60	71.86	170.31
RSD [%]	32.51	32.12	24.74	41.15
n =	64	70	63	62
LCL	8.5	9.6	146.7	73.2
UCL	40.2	44.0	434.2	754.4

NC: negative control

PC: positive controls (-S9 EMS; +S9 DMBA)

S9: metabolic activation

Mean: mean of mutants/10⁶cells

Min.: minimum of mutants/10⁶cells

Max.: maximum of mutants/10⁶cells

SD: standard deviation

RSD: relative standard deviation

n: number of control values

LCL: lower control limit

UCL: upper control limit

Conclusions:

In an in vitro cell gene mutagenicity test, the test item sodium isostearoyl lactylate is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese hamster.

Executive summary:

The test item sodium isostearoyl lactylate was assessed for its potential to induce mutations at the HPRT locus using V79 cells of the Chinese hamster. The selection of the concentrations was based on data from the pre-experiments. The main experiments with and without metabolic activation were performed as a 4 h short-term exposure assay. Cytotoxicity was tested in the main experiment at the ranges of 2–20 µg/mL without metabolic activation and 25–90 µg/mL with metabolic activation. The mutagenicity of the test item was investigated at the following concentrations: without metabolic activation: 4, 6, 8, 10 and 14 µg/mL and with metabolic activation: 25, 55, 65 and 75 µg/mL No precipitation of the test item was noted in the experiments.

Biologically relevant growth inhibition (i.e. relative survival < 70 %) was observed in the experiment with and without metabolic activation. In the experiment without metabolic activation the relative survival was 10 % for the highest concentration (14 µg/mL) evaluated for mutagenicity. The highest biologically relevant concentration evaluated for mutagenicity with metabolic activation was 75 µg/mL with a relative survival of 15 %.

In the experiments no biologically relevant increase of mutants (i.e. above the upper limit of the historical control data) was found after treatment with the test item (without and with metabolic activation) and no concentration-response relationship was observed. DMBA and EMS were used as positive controls and showed distinct and biologically relevant effects in mutation frequency.

Thus, in this in vitro cell gene mutagenicity test, the test item sodium isostearoyl lactylate is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese hamster.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In vivo genotoxicity testing was not necessary based on negative in vitro test results.

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The genetic toxicity potential of test item sodium isostearoyl lactylate was assessed in three different genetic toxicity tests, one performed with the test item in accordance with OECD 476 and two performed with calcium stearoyl lactylate (suitable read-across substance of the test item). All three studies showed no genetic toxicity potential of the test item as well as the read-across substance. Based on the outcomes of these studies, there is no genetic toxicity concern of sodium isostearoyl lactylate taking a weight-of-evidence approach. Therefore, classification for mutagenicity is not warranted under CLP.