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

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 including read-across information form structural related source substance,N, N’-[(methylimino)bis (trimethylene)]bis(oleamide) does not need to be classified for germ cell mutagenicity according to CLP, EU GHS (Regulation (EC) No 1272/2008).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Refer to attached document

Reason / purpose for cross-reference:

read-across source

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

Conclusions:

Available data from an in vitro Micronucleus Test in Human Lymphocytes according to OECD Guideline 487 (20 July 2016) with the test item N,N’-[(methylimino)bis-(trimethylene)]bis-(stearamide) were used to evaluate the cytogenetic potential of N, N’-[(methylimino)bis (trimethylene)]bis(oleamide).
Based on this read-across information it can be concluded, that N, N’-[(methylimino)bis (trimethylene)]bis(oleamide) does not induce micronuclei in human lymphocytes in the absence and presence of mammalian metabolic activation.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Refer to attached document

Reason / purpose for cross-reference:

read-across source

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

Conclusions:

Based on a read-across there is no evidence of a mutagenic potential in mammalian cells for N, N’-[(methylimino)bis(trimethylene)] bis(oleamide) from N, N’-[(methylimino)bis(trimethylene)] bis(oleamide). 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 in a study conducted according to OECD guideline 490.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-10-19 to 2017-11-10

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:

July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

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:

rat liver S9-mix

Test concentrations with justification for top dose:

Main Test I:
all tested strains: 5000, 2500, 1250, 625 and 313 µg/plate with and without S9
Main Test II pre-incubation assay:
TA1535, TA1537, WP2 uvrA, TA98: 5000, 2500, 1250, 625, 313 µg/ plate with and without S9
TA 100 without S9: 5000, 2500, 1250, 625, 313 µg/ plate
TA 100 with S9: 5000, 2500, 1250, 625, 313, 156 µg/plate

Maximum dose level of 5000 µg/plate is tested as recommended by the guideline.

Vehicle / solvent:

DMSO
- Vehicle(s)/solvent(s) used: DMSO
This solvent was selected since it is compatible with the survival of the bacteria and the S9 metabolic activity.
The test item was found to be soluble at 100mg/mL following sonication at 37°C for approximately 20 minutes.
This result permitted a maximum concentration of 5000 µg/plate to be used in the toxicity test.

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:
Main test I plate incorporation
Main test II preincubation assay

DURATION
- Preincubation period: at 37°C for 30 minutes
- Exposure duration: 72 hours at 37°C

NUMBER OF REPLICATIONS: triplicate plates

DETERMINATION OF TOXICITY
thinning of the background lawn and/or reduction in revertant numbers

Evaluation criteria:

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.

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 event.

Statistics:

Regression lines are calculated using a minimum of the three lowest dose levels, and then including the further dose levels in turn.
The correlation co-efficient (r), the value of students "t" statistic, and the p-value for the regression lines are also given.

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

Conclusions:

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

Executive summary:

In a reverse gene mutation assay in bacteria according to OECD guideline 471, of Salmonella typhimurium TA1535, TA1537, TA98, TA100 and Escherichia coli WP2uvrA were exposed to N, N’-[(methylimino)bis(trimethylene)]bis(oleamide), at concentrations of 5000, 2500, 1250, 625 and 313 µg/plate with and without metabolic activation in experimentin experiment I.

The same concentrations were used in experiment II and an additional concentration of 156 µg/plate was included for TA 100 with metabolic activation.

The first experiment was conducted as plate incorporation assay, the second as pre-incubation test, both in the absence and presence of a mammalian metabolic activation.

 

No relevant toxicity was observed at any dose level with any tester strain, in the absence or presence of S9 metabolic activation, in experiment I, whereas dose related toxicity was observed with all tester strains at higher dose levels both in the absence and presence of S9 metabolism in experiment II.  

In both experiments precipitation of the test item was observed at the end of the incubation period at the highest dose level both in the absence and presence of S9 metabolic activation.

The test item did not induce two-fold increases 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. The reference mutagen induced a distinct increase of revertant colonies indicating the validity of the experiments.

 

It is concluded that the test item N, N’-[(methylimino)bis(trimethylene)]bis(oleamide) does not induce reverse mutation inSalmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, 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,of Salmonella typhimurium TA1535, TA1537, TA98, TA100 and Escherichia coli WP2uvrA were exposed to N, N’-[(methylimino)bis(trimethylene)]bis(oleamide), at concentrations of 5000, 2500, 1250, 625 and 313 µg/plate with and without metabolic activation in experiment I. The same concentrations were used in experiment II and an additional concentration of 156 µg/plate was included for TA 100 with metabolic activation.

The first experiment was conducted as plate incorporation assay, the second as pre-incubation test, both in the absence and presence of a mammalian metabolic activation.

No relevant toxicity was observed at any dose level with any tester strain, in the absence or presence of S9 metabolic activation, in experiment I, whereas dose related toxicity was observed with all tester strains at higher dose levels both in the absence and presence of S9 metabolism in experiment II.  

In both experiments precipitation of the test item was observed at the end of the incubation period at the highest dose level both in the absence and presence of S9 metabolic activation.

The test item did not induce two-fold increases 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. The reference mutagen induced a distinct increase of revertant colonies indicating the validity of the experiments.

 

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

 

Supporting information is available from an Ames test according to OECD guideline 471 (version 1997) in tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA with N,N’[(methylimino)bis(trimethylene)]bis(stearamide).

The substance does not induce mutations in the five tested strains, when tested up to 5000 µg/plate or cytotoxic doses in the absence and presence of a rat liver metabolic activation system.

 

Mammalian cell gene mutation assay

Available data from a OECD guideline 490 study with the test read-across substance N,N’[(methylimino)bis(trimethylene)]bis(stearamide) were used to evaluate the potential of the test item N,N’-[(methylimino)bis-(trimethylene)]bis-(stearamide) to induce gene 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.

 

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. 

 

The test item N-N’-[(methylimino)bis-(trimethylene)]bis-(stearamide) 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.

 

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 study:

Available data from a OECD guideline 490 study with the test read-across substance N,N’[(methylimino)bis(trimethylene)]bis(stearamide) were used to evaluate the potential of the test item N,N’-[(methylimino)bis-(trimethylene)]bis-(stearamide) to induce micronuclei in human lymphocytes.

 

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:

·        With and without S9/ 3 hours treatment time / 32 hours harvest time:

139.0 - 92.7 - 61.8 µg/mL

 

·        Without S9/ 31 hours treatment time / 31 hours harvest time:

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

In conclusion, based on the results of in-vitro genotoxicity tests including read-across information formN-N’-[(methylimino)bis-(trimethylene)]bis-(stearamide);N, N’-[(methylimino)bis (trimethylene)]bis(oleamide) does not need to be classified for germ cell mutagenicityaccording to CLP, EU GHS (Regulation (EC) No 1272/2008).