N,N'-[(methylimino)bis(trimethylene)]bis(stearamide)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-11-02 to 2018-02-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

adopted July 2016

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-[(methylimino)bis(trimethylene)]bis(stearamide)

EC Number:

236-793-7

EC Name:

N,N'-[(methylimino)bis(trimethylene)]bis(stearamide)

Cas Number:

13483-58-4

Molecular formula:

C43H87N3O2

IUPAC Name:

N-{3-[methyl(3-octadecanamidopropyl)amino]propyl}octadecanamide

Method

Target gene:

Thymidine kinase locus (TK +/-)

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: American Type Culture Collection, Rockville,Maryland (ATCC code: CRL 9518).
- Suitability of cells: The generation time and mutation rates (spontaneous and induced) have been checked
- Modal number of chromosomes: no information

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no information
- Periodically 'cleansed' against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Experiment 1: without S9, 3 hours: 17.2, 13.7, 11.0, 6.11, 3.39 and 1.88 µg/L
Experiment 1: with S9, 3 hours: 65.5, 52.4, 41.9, 33.5, 26.8 and 21.5 µg/L
Experiment 2: without S9, 3 hours: 10.5, 6.56, 4.10, 2.56, 1.60 and 1.00 µg/L
Experiment 2: with S9, 3 hours: 30.0, 23.1, 17.8, 13.7, 10.5 and 8.08 µg/L
Experiment 2: without S9, 24 hours: 1.60, 0.890, 0.495, 0.275, 0.153 and 0.0848 µg/L

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: Vehicle was chosen based on results from solubility experiment

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

Experiment I without S9 mix

Controls 2

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Experiment I with S9 mix

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Experiment II without S9 mix

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Experiment II with S9 mix

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Experiment II 24 hours, without S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 1×1E6 cells/mL

DURATION
- Exposure duration: 3 and 24 hours
- Expression time (cells in growth medium): 2 days

SELECTION AGENT (mutation assays):
Medium (HSE 20 %), trifluorothymidine (final concentration 3.0 µg/mL)

NUMBER OF REPLICATIONS: two independent experiments.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative survival
- Any supplementary information relevant to cytotoxicity: A preliminary cytotoxicity test was performed in order to select appropriate dose levels for the mutation assays.

Rationale for test conditions:

Test conditions were chosen based on solubility and cytotoxicity testing.

Evaluation criteria:

Acceptance criteria:
The assay was considered valid if the following criteria were met:
1. The cloning efficiencies at Day 2 in the untreated/solvent control cultures fell within the range of 65-120%.
2. The untreated/solvent control suspension growth over 2 days fell within the range: 8-32 (3 hour treatment), 32-180 (24 hour treatment).
3. The mutant frequencies in the untreated/solvent control cultures fell within the range of 50−170×1E−6 viable cells.

Every assay was also evaluated as to whether the positive control met at least one of the following two acceptance criteria:
1. The positive control induced a clear increase above the spontaneous background
(induced mutent frequency = IMF) of at least 300×1E−6. At least 40% of the IMF was reflected in the small colony MF.
2. The positive control induced a clear increase in the small colony IMF of at least 150×1E−6.

Criteria for outcome of assay:
For a test item to be considered mutagenic in this assay, it is required that:
1. The induced mutant frequency (IMF) is higher than the global evaluation factor (GEF) suggested for the microwell method (126×1E−6) at one or more doses.
2. There is a significant dose-relationship as indicated by the linear trend analysis.

Results which only partially satisfy the above criteria will be dealt with on a case-by-case basis.
Similarly, positive responses seen only at high levels of cytotoxicity will require careful interpretation when assessing their biological significance.
Any increase in mutant frequency should lie outside the historical control range to have biological relevance.

Statistics:

Statistical analysis was performed according to UKEMS guidelines (RobinsonW.D., 1990).
-Test for consistency between plates
-Heterogeneity factors for replicate cultures
- Test for overall consistency
- Updated heterogeneity factors
- Comparison of each treatment with the control: one tailed Dunnett’s test.
- Test for linear trend

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The addition of the test item solution did not have any obvious effect on pH of the treatment medium.
- Effects of osmolality: The addition of the test item solution did not have any obvious effect on the osmolality of the treatment medium.
- Evaporation from medium: not expected
- Water solubility: an opaque preparation without visible precipitation, feasible for dosing, was obtained with ethanol at the concentration of 30.0mg/mL using an ultra-turrax mixer for 5 minutes and a magnetic stirrer for approximately 20 minutes.
- Precipitation: Based on precipitation a maximum dose level of 300 µg/mL was selected as the top dose level to be used in the cytotoxicity test.

RANGE-FINDING/SCREENING STUDIES: A preliminary solubility trial indicated that the maximum practicable concentration of the test item in the final treatment medium was 300 µg/mL using ethanol as solvent.
On the basis of this result, a cytotoxicity assay was performed.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Mutation frequencies per million surviving cells: range: 209 - 1103 mean 448 (+/- 142)
- Negative (solvent/vehicle) historical control data: Mutation frequencies per million surviving cells: range: 41.9 - 234 mean 81.2 (+/- 26.1)

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: In order to aid toxicity data interpretation, the relative total growth (RTG), expressed as a percentage of the concurrent negative control, was also calculated.
At low survival levels, the mutation data are prone to a variety of artefacts (selection effects, sampling error, founder effects).
Accordingly, mutation data obtained at /or below 10% RTG has been excluded from the analyses.

Applicant's summary and conclusion

Conclusions:

N-N’-[(methylimino)bis-(trimethylene)]bis-(stearamide) does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Executive summary:

The potential of the test item N,N’-[(methylimino)bis-(trimethylene)]bis-(stearamide) to induce gene mutations in mammalian cells was investigated through an Mouse Lymphoma assay according to OECD Guideline 490 (July 2016).

 

The test item N-N’-[(methylimino)bis-(trimethylene)]bis-(stearamide) was examined for mutagenic activity by assaying for the induction of 5 trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells after in vitro treatment, in the absence and presence of S9 metabolic activation, using a fluctuation method.

 

A preliminary solubility trial indicated that the maximum practicable concentration of the test item in the final treatment medium was 300 µg/mL using ethanol as solvent. 

 

On the basis of this result, a cytotoxicity assay was performed.

Both in the absence and presence of S9 metabolic activation, the test item was assayed at a maximum dose level of 300 μg/mL and at a wide range of lower dose levels: 150, 75.0, 37.5, 18.8, 9.38, 4.69, 2.34 and 1.17 µg/mL. In the absence of S9 metabolic activation, using the 3 hour treatment time, a dose level of 18.8 µg/mL yielded severe toxicity, reducing relative survival (RS) to 3% of the concurrent negative control value.

A dose related toxicity was noted over the remaining concentrations tested, reducing RS to 36% at 9.38 µg/mL. Using the 24 hour treatment time, no cells survived treatment at the seven highest dose levels. Severe toxicity was observed at the next lower concentration (RS = 4%), while moderate toxicity (RS = 21%) was seen at 1.17 µg/mL. Following treatment in the presence of S9 metabolic activation, using the short treatment time (3 hours), a concentration of 37.5 µg/mL yielded mild toxicity (RS = 51%), while no relevant toxicity was observed over the remaining dose levels tested.

 

MAIN ASSAYS

Two independent assays for mutation at the TK locus were performed.

In Main Assay I, using the short treatment time, the test item was assayed at the following dose levels:

 

Experiment 1: without S9, 3 hours: 17.2, 13.7, 11.0, 6.11, 3.39 and 1.88 µg/L

Experiment 1: with S9, 3 hours: 65.5, 52.4, 41.9, 33.5, 26.8 and 21.5 µg/L

Experiment 2: without S9, 3 hours: 10.5, 6.56, 4.10, 2.56, 1.60 and 1.00 µg/L

Experiment 2: with S9, 3 hours: 30.0, 23.1, 17.8, 13.7, 10.5 and 8.08 µg/L

Experiment 2: without S9, 24 hours: 1.60, 0.890, 0.495, 0.275, 0.153 and 0.0848 µg/L

 

In experiment I severe toxicity reducing survival below 10% was observed at higher concentrations in both treatment series, thus the number of analysable dose levels was not adequate for the evaluation of test item mutagenicity. No relevant increases in mutant frequencies were observed at any analysable concentration in the absence or presence of S9 metabolism.

Based on these results, a second Main Assay was performed both in the absence and presence of S9 metabolism, using the short treatment time. The treatment series using a longer treatment time (24 hours) in the absence of S9 metabolic activation was carried out at the same time.

 

In experiment II adequate levels of cytotoxicity, covering a range from the maximum to slight or no toxicity, were observed in all treatment series. No relevant increases in mutant frequencies were observed following treatment with the test item, in the absence or presence of S9 metabolism. Untreated, solvent/vehicle and positive control treatments were included in each mutation experiment in the absence and presence of S9 metabolism. The mutant frequencies in the solvent/vehicle control cultures fell within the normal range. Marked increases were obtained with the positive control treatments indicating the correct functioning of the assay system.

 

It is concluded that N-N’-[(methylimino)bis-(trimethylene)]bis-(stearamide) does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.