Reaction product of: stearyl-diethanol-amine with C16-C18 saturated fatty acids

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 June - 11 August 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study in accordance with international guidelines.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction product of: stearyl-diethanol-amine with C16-C18 saturated fatty acids

EC Number:

943-080-0

Molecular formula:

Not applicable, UVCB

IUPAC Name:

Reaction product of: stearyl-diethanol-amine with C16-C18 saturated fatty acids

Test material form:

other: pastilles

Details on test material:

Name SABOSTAT A 300
Batch no. 20085596
Appearance withish pastilles
Composition Reaction product of stearyl-diethanol-amine with C16-C18 saturated fatty acids
CAS No. not applicable (CAS No. of main compound 2-[(2-hydroxyethyl)octadecylamino]ethyl stearate: 52497-24-2)
EINECS-No. not stated
Molecular formula not applicable
Molecular weight 668 g/mol (calculated based on average UVCB composi-tion)
Purity 100%
Homogeneity homogeneous
Vapour pressure 5.85E-21 Torr Calculated using Advanced Chemistry De-velopment (ACD/Labs) Software V11.02 (© 1994-2015 ACD/Labs) h Pa at 25°C
Stability H2O; EtOH; acetone; CH3CN; DMSO: unknown
Solubility H2O : not soluble DMSO: soluble at 50°C; EtOH; acetone; CH3CN; DMSO: unknown
Production date 16. Apr. 2015
Expiry date 10. Apr. 2016
Storage Room temperature (20 ± 5 °C), keep away from light/ hu-midity

The test item was stored in the test facility at a temperature between 18 and 26 °C pro-tected from light and humidity.

Method

Target gene:

thymidine kinase locus (Tk1)

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Cleansed and for mycoplasma contamination screened stocks of cells were stored in liquid nitrogen in the cell bank of LAUS GmbH to allow a continuous stock of cells, which guarantees similar parameters of the experiment and reproducible characteristics of the cells.
Cleansed and for mycoplasma contamination screened cells were thawed 6 - 7 days prior to treatment and cultivated in RPMI 1640 complete culture medium with 5 % HS in cell culture flasks at 37.0 ± 1.5 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2. After thawing, the cells were again screened for mycoplasma contamination before they were used in the experiments.

Metabolic activation:

with and without

Metabolic activation system:

liver S9 mix from male rats, treated with Aroclor 1254

Test concentrations with justification for top dose:

nominal concentrations in pre-test [µL/mL] 5.00 2.50 1.25 0.63 0.31 0.16 0.08
nominal concentrations in experiment I +S9 [µL/mL] 5.00 0.63* 0.31 0.16 0.08 0.04 0.02*
nominal concentrations in experiment I -S9 [µL/mL] 5.00 0.63* 0.31 0.16 0.08 0.04 0.02*
nominal concentrations in experiment II +S9 [µL/mL] 5.00 0.63* 0.31 0.16 0.08 0.04 0.02*
nominal concentrations in experiment II +S9 [µL/mL] 5.00 0.63* 0.31* 0.16 0.08 0.04 0.02

Details on test system and experimental conditions:

Test System
The L5178Y is a murine T-cell lymphoma cell line, which grows as single or aggregated round cells in suspension. This cell line is characterized by a high sensitivity to chemical mutagens, by a high proliferation rate (doubling time 10-12 hours in stock cultures), a high cloning efficiency (CE) and a stable spontaneous mutant frequency. The L5178Y consists of a stable karyotype and shows a diploid chromosome number (40 ± 2). The cells were purchased by ATCC (Wesel, Germany) and were sold under the name L5178Y TK+/- clone (3.7.2C) [TK+/- (clone 3.7.2C)] (ATCC® CRL-9518™).

Cell Cultures
Cleansed and for mycoplasma contamination screened stocks of cells were stored in liquid nitrogen in the cell bank of LAUS GmbH to allow a continuous stock of cells, which guarantees similar parameters of the experiment and reproducible characteristics of the cells.
Cleansed and for mycoplasma contamination screened cells were thawed 6 - 7 days prior to treatment and cultivated in RPMI 1640 complete culture medium with 5 % HS in cell culture flasks at 37.0 ± 1.5 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2. After thawing, the cells were again screened for mycoplasma contamination before they were used in the experiments.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

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:

valid

Additional information on results:

In total three experiments were performed of which one was invalid (experiment I) and had to be repeated. Finally, two independent valid experiments, using two parallel cultures each (replicates) were performed. Experiment I was performed with and without metabolic activation and a treatment period of 4 h. Experiment II was performed with 24 h treatment without metabolic activation. The third experiment was performed with 4 h treatment with metabolic activation.
The following concentrations of the test item were investigated in experiments I and II: 5.00 µL/mL, 0.63 µL/mL, 0.31 µL/mL, 0.16 µL/mL, 0.08 µL/mL, 0.04 µL/mL, 0.02 µL/mL.
The nominal and real test item concentrations were identical in this study. Precipitation of the test item was visible at the concentration 5 µL/mL. in both experiments in both ap-proaches. In the test item concentration 0.63 µl/mL and all further dilutions no precipitation was visible with the unaided eye during treatment but during the washing and counting step the test item clumped with the cells and those agglutinations could not be broken up also by intensively resuspension. Accordingly, the counts in those concentrations were all extremely low in comparison to the other concentrations and the controls.
MMS (19.5 µg/mL in experiment I and 12.5 µg/mL in experiment II) and CPA (4.5 µg/mL) were used as positive controls.
In experiment I (+S9 and –S9) a cytotoxic effect was observed in the concentration 0.63 µL/mlL. In experiment II (+S9 and –S9), again a cytotoxic effect was observed in the concentration 0.63 µL/mL (+S9 and -S9) as well as 0.31 µL/mL (-S9). According to the OECD 476 guideline those concentrations, that showed a cytotoxic effect (RTG <20 %) cannot be analysed for mutagenicity. Therefore, these concentrations were not considered in evaluation of the mutant frequencies.
The mutant frequencies for the solvent control DMSO in experiment I and II (+ S9 and –S9) were in the range of 50 – 170 colonies per 106 cells (experiment I: +S9: 128 and -S9: 127; experiment II: +S9: 116, -S9: 82). The mutation frequencies of the solvent controls NaCl 0.9 % (experiment I 127, experiment II 96) and MMS (experiment I 150, experiment II 119) were also in the normal range between 50 – 170 colonies per 106 cells. The positive controls MMS and CPA showed a distinct increase in total mutant frequency of more than 300 in comparison to the respective solvent control.
In all tested concentrations of the test item no substantial and reproducible dose depend-ent increase in mutant colony numbers was observed in both main experiments (see table 8-a and figures 8-a to 8-d). No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test item. The mutation frequency did not reach or exceed the threshold of 126 above the corresponding solvent control.

Any other information on results incl. tables

Main results from experiment I and II (Mean values of both cultures)

Content	Relative Total Growth [%]				Mutants per 10^6Cells
	Experiment I		Experiment II		Experiment I		Experiment II
	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9
Solvent Control of Test Item	-	-	-	-	129	127	116	82
Solvent Control of Positive Control	-	-	-	-	148	150	96	119
Positive Control	54.8	65.3	47.9	76.5	433	452	498	421
Test Item 5.00 µL/mL	98.1	90.7	102.7	88.1	155	166	150	127
Test Item 0.63 µL/mL	0.1	0.2	0.2	0.0	-	-	-	-
Test Item 0.31 µL/mL	49.1	42.1	77.0	0.0	163	168	163	-
Test Item 0.16 µL/mL	65.0	69.6	87.9	20.2	177	161	166	136
Test Item 0.08 µL/mL	74.6	82.4	86.9	58.8	176	179	218	134
Test Item 0.04 µL/mL	86.7	77.9	100.8	87.0	146	217	170	134
Test Item 0.02 µL/mL	104.9	89.9	84.1	92.0	*	*	*	150

- Cultures could not be evaluated for mutagenicity because of cytotoxicity.

* Cultures were not continued since only 4 analysable concentrations are required by the guideline.

Note: The threshold (number of mutant colonies per 10⁶cells of the respective solvent control plus 126) was 255 (+S9) and 253 (-S9) in Experiment I as well as 242 (+S9) and 208 (-S9) in Experiment II.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the L5178Y Tk+/- cell line in the absence and the presence of metabolic activation.
The recorded data in this study declare the test item as a non mutagen.

Executive summary:

This study was performed to investigate the potential of the substance to induce mutations at thethymidine kinase locus(Tk1) on chromosome 11 and/or structural chromosomal aberrations in mouse lymphoma L5178YTk^+/-cells.

The assay was performed in a pre-test and in two independent main experiments (experiment I and II) whereby the first experiment I was invalid and had to be repeated. The results of the invalid experiment will not be included in this report but will be archived with the raw data. Therefore, in total one pre-test and three experiments were performed.

The pre-test was done to detect a potential cytotoxic effect of the test item. Based on the results of this test the concentrations for the main experiments were determined.

Experiment I was performed with and without metabolic activation (liver enzyme S9 fraction / “liver S9 mix from male rats, treated with Aroclor 1254”) and a treatment period of 4 h. Experiment II was performed with a treatment period of 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation.

The highest nominal concentration that was used in the experiment was 5 µL/mL.

Not all tested concentrations could be evaluated for mutagenicity. Some of them induced a cytotoxic reaction and had to be excluded from the evaluation of the mutagenicity. However in all analysable concentrations no substantial and reproducible dose dependent increase in mutant colony numbers was observed in both experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test item.