N,N'-[(methylimino)dipropane-1,3-diyl]dioleamide

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

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

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,N'-[(methylimino)dipropane-3,1-diyl]dioctadecanamide

Method

Species / strain

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.

Controls

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.

Results and discussion

Test results

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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.