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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the results of the full set of in-vitro genotoxicity tests required by REACH regulation,N-N’-[(methylimino)bis-(trimethylene)]bis-(stearamide) does not need to be classified for germ cell mutagenicity according to CLP, EU GHS (Regulation (EC) No 1272/2008).

 

- Gene mutation in bacteria: negative in S. typhimurium TA1535, TA 1537, TA98, TA100 and E. coli WP2 (with and without metabolic activation); OECD TG 471; RL 1; GLP

- Cytogenicity/in vitro micronucleus test: negative in human lymphocytes (with and without metabolic activation); OECD TG 487; RL 1; GLP

- Gene mutation in mammalian cells: negative in mouse lymphoma L5178Y cells (with and without metabolic activation); OECD TG 490; RL 1; GLP

Link to relevant study records

Reference

Reference 1

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

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

Target gene:

Thymidine kinase locus (TK +/-)

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

Negative solvent / vehicle controls:

yes

Remarks:

Experiment I without S9 mix

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Experiment I with S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Experiment II without S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Experiment II with S9 mix

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

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.

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.

 

 

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-03-31 to 2017-04-24

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:

1997-07-21

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008-05-30

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine locus

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

liver S9 fraction from rats pre-treated with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

Main Assay I, dose levels:
Tester strain S9 Dose level (µg/plate)
TA1535 − 2500, 1250, 625, 313, 156
TA1537, TA98, WP2 uvrA − 2500, 1250, 625, 313, 156, 78.1
TA100 − 1250, 625, 313, 156, 78.1
TA1535, TA1537, TA98 + 5000, 2500, 1250, 625, 313
TA100, WP2 uvrA + 5000, 2500, 1250, 625, 313, 156

In the toxicity test, the test item N-N’-[(methylimino)bis-(trimethylene)]bis-(stearamide) was assayed at a maximum concentration of 6250 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 1980, 625, 198 and 62.5 µg/plate. Precipitation of the test item which did not interfere with the scoring was observed at the end of the incubation period at the highest or two highest dose levels, in the absence and presence of S9 metabolic activation, respectively. Toxicity was observed at higher concentrations with all tester strain/activation combinations, with the exception of TA98 tester strain in the presence of S9 metabolism.

Vehicle / solvent:

ethanol at 62.5 mg/mL

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION
in agar (plate incorporation)

DURATION
Exposure duration: approximately 72 h

NUMBER OF REPLICATIONS
Two Main Assays were performed including negative and positive controls in the absence and presence of an S9 metabolising system. Three replicate plates were used at each test point.

DETERMINATION OF CYTOTOXICITY
Method: decrease in the number of revertant colonies per plate.

Evaluation criteria:

CRITERIA FOR OUTCOME OF THE ASSAY
For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.

ACCEPTANCE CRITERIA
The assay was considered valid if the following criteria were met:
1. Mean plate counts for untreated and positive control plates should fall within 2 standard deviations of the current historical mean values.
2. The estimated numbers of viable bacteria/plate should fall in the range of 100 – 500 millions for each strain.
3. No more than 5% of the plates should be lost through contamination or other unforeseen events.

Key result

Species / strain:

S. typhimurium TA 1535

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

Key result

Species / strain:

S. typhimurium TA 1537

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

Key result

Species / strain:

S. typhimurium TA 100

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

Key result

Species / strain:

S. typhimurium TA 98

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

Key result

Species / strain:

E. coli WP2 uvr A

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:

TOXICITY TEST
The test item N-N’-[(methylimino)bis-(trimethylene)]bis-(stearamide) was assayed in the toxicity test at a maximum dose level of 6250 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 1980, 625, 198 and 62.5 µg/plate.
Precipitation of the test item, evident to the unaided eye, was observed at the end of the incubation period at the highest or two highest concentrations in the presence and absence of S9 metabolic activation, respectively.
Toxicity, as indicated by complete lack of the microbial growth, microcolony formation, thinning of the background lawn and/or reduction in revertant numbers was observed at higher dose levels both in the absence and presence of S9 metabolic activation, with all tester strains with the exception of TA98 in the presence of S9 metabolism.
No relevant increase in revertant numbers was observed with any tester strain, at any dose level, in the absence or presence of S9 metabolic activation.

EVALUATION
Results show that mean plate counts for untreated and positive control plates fell within the normal range based on historical control data. The estimated numbers of viable bacteria/plate (titre) fell in the range of 100 - 500 million for each strain. No plates were lost through contamination or cracking. The study was accepted as valid.
The test item did not induce two-fold increases in the number of revertant colonies, at any dose level, in any tester strain, in the absence or presence of S9 metabolism.

Conclusions:

N-N’-[(methylimino)bis-(trimethylene)]bis-(stearamide) does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolic activation.

Executive summary:

The potential of the test item N,N’-[(methylimino)bis-(trimethylene)]bis-(stearamide) to induce point mutations in bacteria was investigated through an Bacterial Reverse Mutation Test according to OECD Guideline 471 (21 July 1997) and according to the test method B.B.13/14"Mutagenicity: Reverse Mutation Test Using Bacteria" as described in the Council Regulation (EC) No 440/2008 of 30 May 2008.

 

The test item N-N’-[(methylimino)bis-(trimethylene)]bis-(stearamide) was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. The five tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbital and 5,6-benzoflavone.

 

VEHICLE

The test item was used as a suspension in ethanol.

 

TOXICITY TEST

The test item N-N’-[(methylimino)bis-(trimethylene)]bis-(stearamide) was assayed in the toxicity test at a maximum concentration of 6250µg/plate and at four lower concentrations spaced at approximately half-log intervals: 1980, 625, 198 and 62.5µg/plate. Precipitation of the test item which did not interfere with the scoring was observed at the end of the incubation period at the highest or two highest dose levels, in the absence and presence of S9 metabolic activation, respectively. Toxicity was observed at higher concentrations with all tester strain/activation combinations, with the exception of TA98 tester strain in the presence of S9 metabolism.

 

MAIN ASSAYS

On the basis of toxicity test results, in Main Assay I, using the plate incorporation method, the test item was assayed at the following dose levels:

Tester strain	S9	Dose level (µg/plate)
TA1535	-	2500, 1250, 625, 313, 156
TA1537, TA98, WP2 uvrA	-	2500, 1250, 625, 313, 156, 78.1
TA100	-	1250, 625, 313, 156, 78.1
TA1535, TA1537, TA98	+	5000, 2500, 1250, 625, 313
TA100, WP2 uvrA	+	5000, 2500, 1250, 625, 313, 156

Precipitation of the test item, which did not interfere with the scoring, was seen at the highest dose level tested, both in the absence and presence of S9 metabolic activation.

Toxicity was observed at the highest or two highest dose levels with all tester strain/activation combinations, with the exception of TA98 tester strain in the presence of S9 metabolism.

As no relevant increase in revertant numbers was observed at any concentration tested, a pre-incubation step was included for all treatments of Main Assay II. Due to the ethanol toxicity, the test item was assayed at the maximum practicable concentration of 1000µg/plate and at four lower dose levels spaced by a factor of two: 500, 250, 125 and 62.5µg/plate. An additional dose level of 31.3µg/plate was included in the dose range for TA100 tester strain in the absence of S9 metabolism.

Slight toxicity was observed at the highest dose level, both in the absence and presence of S9 metabolism with all tester strains, with the exception of TA98 and WP2uvrA. The test item did not induce any increase in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose level, in any tester strain, in the absence or presence of S9 metabolism.

 

It is concluded that the test item N-N’-[(methylimino)bis-(trimethylene)]bis-(stearamide) does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-05-15 to 2017-09-14

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:

2016-07-29

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: human

Cytokinesis block (if used):

Cytochalasin B (6 µg/mL)

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix (rat, S9 tissue fraction and cofactors)

Test concentrations with justification for top dose:

dose levels for short term treatment (3 h) both in the absence and presence of S9 metabolism:
313, 209, 139, 92.7, 61.8, 41.2, 27.5, 18.3 and 12.2 µg/mL

dose levels for continuous treatment (31 h) in the absence of S9 metabolism:
157, 105, 69.5, 46.4, 30.9, 20.6, 13.8, 9.15, 6.10 and 4.07 µg/mL

Top dose determined by limited solubility of the test item:
A homogeneus suspension, feasible for dosing, was obtained with ethanol at the concentration of 31.3 mg/mL after vortex mixing for approximately 40 minutes. An aliquot of this suspension added to culture medium in the ratio 1:100 gave an opaque medium without visible precipitation. On the basis of these observations a maximum dose level of 313 µg/mL was employed.

Vehicle / solvent:

Vehicle used: ethanol
Justification for the choice of the vehicle: The solvent was chosen due to the solubility properties of the test item.

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Cyclophosphamide with / Colchecin without S9 metabolism

Details on test system and experimental conditions:

Principles of the method:
The in vitro micronucleus test provides a relatively rapid method to investigate the ability of chemicals to induce chromosomal damage or damage to the mitotic apparatus. Lymphocytes in whole blood cultures are stimulated to divide by exposure to phytohaemagglutinin (PHA). After approximately 48 hours, cells are treated with the test item or control solutions.
Since cultured lymphocytes have little ability to metabolise indirect mutagens to reactive derivatives, the assay is performed both in the absence and presence of an "S9" metabolising system.
The most convenient stage to score micronuclei is the binucleate interphase stage. These cells have completed one cell division after chemical treatment and are therefore capable of expressing micronuclei. Treatment of cultures with the inhibitor of actin polymerisation cytochalasin B blocks cytokinesis and cells that have completed one cell cycle after treatment can be distinguished from non dividing cells by their binucleate appearance.

Controls:
Appropriate negative, solvent and positive control cultures were included in the experiment.
Since ethanol was selected as solvent vehicle, it was considered appropriate to include untreated cultures in the experimental scheme.
Using the short treatment time, since tests with and without metabolic activation were done concurrently, positive control cultures were treated only in the presence of S9 metabolism with Cyclophosphamide at the dose levels of 20.0 and 15.0 µg/mL.
Using the long treatment time, in the absence of S9 metabolism, the positive control cultures were treated with Colchicine at the dose levels of 80.0 and 40.0 ng/mL.

CBPI:
The cytokinesis-block proliferation index CBPI was calculated as follows:
(number of mononucleated +2×binucleated +3×multinucleated cells) divided by (total number of cells counted)

% Cytotoxicity:
% Cytotoxicity = 100 - { 100 multiplied by [ (CBPI of test item treated culture -1) divided by (CBPI of untreated / solvent control -1) ] }

Cytotoxicity:
- The CBPI was used to measure the cytotoxic effect. Five hundred cells per cell culture were analysed.
- The highest dose level for genotoxicity assessment (scoring of micronuclei) was selected on the basis of the cytotoxicity as calculated by the CBPI.
- Two lower dose levels are also selected for the scoring of micronuclei.

Scoring of micronuclei:
- For the three selected doses, for the untreated and solvent controls and the positive control Cyclohosphamide, 1000 binucleated cells per cell culture were scored to assess the frequency of micronucleated cells.
- For cultures treated with the positive control Colchicine, since it is a known mitotic spindle poison which induces mitotic slippage and cytokinesis block, a greater magnitude of response was observed in mononucleated cells. For this reason, 1000 mononucleated cells per cell culture were scored.

Criteria for identifying micronuclei:
1. The micronucleus diameter was less than 1/3 of the nucleus diameter
2. The micronucleus diameter was greater than 1/16 of the nucleus diameter
3. No overlapping with the nucleus was observed
4. The aspect was the same as the chromatin

Acceptance criteria:
The assay is considered valid if the following criteria are met:
– The incidence of micronucleated cells of the negative control is within the distribution range of our historical control values.
– Concurrent positive controls induce responses that are compatible with those generated in our historical positive control database and produce a statistically significant increase compared with the concurrent negative control.
– Adequate cell proliferation is observed in solvent control cultures.
– The appropriate number of doses and cells is analysed.

Rationale for test conditions:

Harvest time:
The harvest time used was approximately 32 hours, corresponding to approximately two cell cycle lenghts.

Controls:
Appropriate negative (untreated), solvent and positive control cultures were included in the experiment.
Using the short treatment time, since tests with and without metabolic activation were done concurrently, positive control cultures were treated only in the presence of S9 metabolism with Cyclophosphamide. Using the long treatment time, in the absence of S9 metabolism, the positive control cultures were treated with Colchicine.

Cytokinesis block:
The actin polymerisation inhibitor cytochalasin B was added prior to the targeted mitosis to allow the selective analysis of micronucleus frequency in binucleated cells.

Dose levels for scoring:
For all treatment series, dose levels were selected for the scoring of micronuclei on the basis of solubility in the final treatment medium and cytotoxicity of the test item treatments calculated by the cytokinesis-block proliferation index (CBPI).

Evaluation criteria:

Criterion for clearly positive outcome
The test item is considered as clearly positive if the following criteria are met:
– Significant increases in the proportion of micronucleated cells over the concurrent controls occur at one or more concentrations.
– The proportion of micronucleated cells at such data points exceeds the normal range based on historical control values.
– There is a significant dose effect relationship.

Criterion for clearly negative outcome
The test item is considered clearly negative if the following criteria are met:
– None of the dose levels shows a statistically significant increase in the incidence of micronucleated cells.
– There is no concentration related increase when evaluated with the Cochran-Armitage trend test.
– All the results are inside the distribution of the historical control data.

Statistics:

For the statistical analysis, a modified chi-squared test was used to compare the number of cells with micronuclei in control and treated cultures.
Cochran-Armitage Trend Test (one-sided) was performed to aid determination of concentration response relationship.

Key result

Species / strain:

lymphocytes: human

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:

The CBPI was calculated for each of the treatment series. Five concentrations were analyzed and since negligible cytotoxicity was observed, scoring of CBPI was interrupted.

On the basis of the the absence of cytotoxicity and the precipitation of test item observed in the treatment media, the dose levels selected for scoring of micronuclei were as follows:
Treatment time Harvest time Dose level
S9 (hours) (hours) (µg/mL)
± 3 32 139.0 - 92.7 - 61.8
− 31 31 69.5 - 46.4 - 30.9

Treatment time Harvest time Dose level
Experiment No. S9 (hours) (hours) (µg/mL)
1 + 3 32 Cyclophosphamide 20.0
2 − 31 31 Colchicine 0.0400

The CBPI was calculated for each of the treatment series. Five concentrations were analyzed and since negligible cytotoxicity was observed, scoring of CBPI was interrupted.

On the basis of the the absence of cytotoxicity and the precipitation of test item observed in the treatment media, the dose levels selected for scoring of micronuclei were as follows:

S9	Treatment time (hours)	Harvest time (hours)	Dose level (µg/mL)
±	3	32	139.0 - 92.7 - 61.8
−	31	31	69.5 - 46.4 - 30.9

Since negative results were obtained for the short term treatment, scoring for the frequency of micronucleated cells was performed for the long term treatment.

For the positive control, the following dose levels were selected for scoring:

Experiment No.	S9	Treatment time (hours)	Harvest time (hours)		Dose level (µg/mL)
1	+	3	32	Cyclophosphamide	20.0
2	-	31	31	Colchicine	0.0400

Summary Table / Treatment time: 3 hours / Sampling time: 32 hours

Treatment	Dose level (μg/mL)	Presence of S9 metabolism			Absence of S9 metabolism
		%Mn cells	Sig.	%Cytotox	%Mn cells	Sig.	%Cytotox
Untreated	0.00	0.35		-1	0.20		-8
Solvent	1%	0.45	NS	0	0.50	NS	0
Test item	61.8	0.30	NS	-1	0.35	NS	-7
Test item	92.7	0.50	NS	-5	0.70	NS	3
Test item	139	0.30	NS	-3	0.40	NS	2
Cyclophosphamide	20.0	3.30	***	56	-		-

 

Summary Table / Treatment time: 31 hours / Sampling time: 31 hours

Treatment	Dose level (μg/mL)	Absence of S9 metabolism
		%Mn cells	Sig.	%Cytotox
Untreated	0.00	0.50		1
Solvent	1%	0.30		0
Test item	30.9	0.35	NS	2
Test item	46.4	0.45	NS	14
Test item	69.5	0.30	NS	6
Colchicine	0.0400	2.25	***	98

Conclusions:

It is concluded that N-N’-[(methylimino)bis-(trimethylene)]bis-(stearamide) does not induce micronuclei in human lymphocytes after short term in vitro treatment in the absence and presence of S9 metabolism and after long term in vitro treatment in the absence of S9 metabolism.

Executive summary:

The potential of the test item N,N’-[(methylimino)bis-(trimethylene)]bis-(stearamide) to induce micronuclei in human lymphocytes was investigated through an In vitro Micronucleus Test in Human Lymphocytes according to OECD Guideline 487 (20 July 2016).

 

The test was performed following in vitro treatment in the absence and presence of S9 metabolic activation. Three treatment series were included in the study. A short term treatment of 3 hours, was performed in the absence and presence of S9 metabolism. The harvest time of approximately 32 hours, corresponding to approximately two cell cycle lenghts, was used. A long term (continuous) treatment was also performed, only in the absence of S9 metabolism, until harvest at approximately 31 hours.

On the basis of the results obtained in a preliminary solubility trial, solutions/suspensions of the test item were prepared in ethanol.

Dose levels of 313, 209, 139, 92.7, 61.8, 41.2, 27.5, 18.3 and 12.2 µg/mL were used for the short term treatment both in the absence and presence of S9 metabolism.

Dose levels of 157, 105, 69.5, 46.4, 30.9, 20.6, 13.8, 9.15, 6.10 and 4.07 µg/mL were used for the continuous treatment in the absence of S9 metabolism.

Each treatment series included appropriate negative controls. In addition, positive controls were included for the short term and long term treatment series. Two cell cultures were prepared at each test point. The actin polymerisation inhibitor cytochalasin B was added prior to the targeted mitosis to allow the selective analysis of micronucleus frequency in binucleated cells. For all treatment series, dose levels were selected for the scoring of micronuclei on the basis of solubility in the final treatment medium and cytotoxicity of the test item treatments calculated by the cytokinesis-block proliferation index (CBPI).

The following dose levels were selected for scoring:

S9	Treatment time (hours)	Harvest time (hours)	Dose level (µg/mL)
±	3	32	139.0 - 92.7 - 61.8
−	31	31	69.5 - 46.4 - 30.9

One thousand binucleated cells per culture were scored to assess the frequency of micronucleated cells. Following treatment with the test item, no statistically significant increase in the incidence of micronucleated cells over the control value was observed at any dose level, in any treatment series. All results were within the distribution of historical negative control data. Statistically significant increases in the incidence of micronucleated cells were observed following treatments with the positive controls Cyclophosphamide and Colchicine, indicating the correct functioning of the test system.

 

It is concluded that N-N’-[(methylimino)bis-(trimethylene)]bis-(stearamide) does not induce micronuclei in human lymphocytes after in vitro treatment, under the reported experimental conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In-vitro studies

Gene mutation in bacteria

In a reverse gene mutation assay in bacteria according to OECD guideline 471 (adopted 21 July 1997), Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA were exposed toN,N’-[(methylimino)bis-(trimethylene)]bis-(stearamide)in ethanol at the following concentrations in the absence and presence of mammalian metabolic activation (rat liver S9 mixfrom rats pre-treated with phenobarbital and 5,6-benzoflavone):

 

The test item N-N’-[(methylimino)bis-(trimethylene)]bis-(stearamide) was assayed in the toxicity test at a maximum concentration of 6250 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 1980, 625, 198 and 62.5 µg/plate. Precipitation of the test item which did not interfere with the scoring was observed at the end of the incubation period at the highest or two highest dose levels, in the absence and presence of S9 metabolic activation, respectively. Toxicity was observed at higher concentrations with all tester strain/activation combinations, with the exception of TA98 tester strain in the presence of S9 metabolism.

 

MAIN ASSAYS

On the basis of toxicity test results, in Main Assay I, using the plate incorporation method, the test item was assayed at the following dose levels:

 

Tester strain	S9	Dose level (µg/plate)
TA1535	-	2500, 1250, 625, 313, 156
TA1537, TA98, WP2 uvrA	-	2500, 1250, 625, 313, 156, 78.1
TA100	-	1250, 625, 313, 156, 78.1
TA1535, TA1537, TA98	+	5000, 2500, 1250, 625, 313
TA100, WP2 uvrA	+	5000, 2500, 1250, 625, 313, 156

 

Precipitation of the test item, which did not interfere with the scoring, was seen at the highest dose level tested, both in the absence and presence of S9 metabolic activation. Toxicity was observed at the highest or two highest dose levels with all tester strain/activation combinations, with the exception of TA98 tester strain in the presence of S9 metabolism.

 

As no relevant increase in revertant numbers was observed at any concentration tested, a pre-incubation step was included for all treatments of Main Assay II. Due to the ethanol toxicity, the test item was assayed at the maximum practicable concentration of 1000 µg/plate and at four lower dose levels spaced by a factor of two: 500, 250, 125 and 62.5 µg/plate. An additional dose level of 31.3 µg/plate was included in the dose range for TA100 tester strain in the absence of S9 metabolism.

Slight toxicity was observed at the highest dose level, both in the absence and presence of S9 metabolism with all tester strains, with the exception of TA98 and WP2uvrA. The test item did not induce any increase in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose level, in any tester strain, in the absence or presence of S9 metabolism.

 

It is concluded that the test item N-N’-[(methylimino)bis-(trimethylene)]bis-(stearamide) does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.

 

Mammalian cell gene mutation assay

In an in vitro gene mutation study in mammalian cells according to OECD guideline 490 N,N’-[(methylimino)bis-(trimethylene)]bis-(stearamide) was assessed for its potential to inducegene mutations in mammalian cells. 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.

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.

 

Cytogenicity / micronucleus assay

In an in vitro micronucleus test in human lymphocytes according to OECD Guideline 487 N,N’-[(methylimino)bis-(trimethylene)]bis-(stearamide) was investigated for the potential to induce micronuclei in human lymphocytes, in the absence and presence of S9 metabolic activation.

Three treatment series were included in the study. A short term treatment of 3 hours, was performed in the absence and presence of S9 metabolism. The harvest time of approximately 32 hours, corresponding to approximately two cell cycle lenghts, was used. A long term (continuous) treatment was also performed, only in the absence of S9 metabolism, until harvest at approximately 31 hours.

On the basis of the results obtained in a preliminary solubility trial, solutions/suspensions of the test item were prepared in ethanol.

 

Dose levels of 313, 209, 139, 92.7, 61.8, 41.2, 27.5, 18.3 and 12.2 µg/mL were used for the short term treatment both in the absence and presence of S9 metabolism. Dose levels of 157, 105, 69.5, 46.4, 30.9, 20.6, 13.8, 9.15, 6.10 and 4.07 µg/mL were used for the continuous treatment in the absence of S9 metabolism.

 

Each treatment series included appropriate negative controls. In addition, positive controls were included for the short term and long term treatment series. Two cell cultures were prepared at each test point. The actin polymerisation inhibitor cytochalasin B was added prior to the targeted mitosis to allow the selective analysis of micronucleus frequency in binucleated cells. For all treatment series, dose levels were selected for the scoring of micronuclei on the basis of solubility in the final treatment medium and cytotoxicity of the test item treatments calculated by the cytokinesis-block proliferation index (CBPI).

 

The following dose levels were selected for scoring:

 

S9	Treatment time (hours)	Harvest time (hours)	Dose level (µg/mL)
±	3	32	139.0 - 92.7 - 61.8
−	31	31	69.5 - 46.4 - 30.9

 

One thousand binucleated cells per culture were scored to assess the frequency of micronucleated cells. Following treatment with the test item, no statistically significant increase in the incidence of micronucleated cells over the control value was observed at any dose level, in any treatment series. All results were within the distribution of historical negative control data. Statistically significant increases in the incidence of micronucleated cells were observed following treatments with the positive controls Cyclophosphamide and Colchicine, indicating the correct functioning of the test system.

 

It is concluded that N-N’-[(methylimino)bis-(trimethylene)]bis-(stearamide) does not induce micronuclei in human lymphocytes after in vitro treatment, under the reported experimental conditions.

Justification for classification or non-classification

Based on the results of the full set of in-vitro genotoxicity tests required by REACH regulation,N-N’-[(methylimino)bis-(trimethylene)]bis-(stearamide)does not need to be classified for germ cell mutagenicity according to CLP, EU GHS (Regulation (EC) No 1272/2008).