Amines, N'-[3-[(3-aminopropyl)amino]propyl]-N,N-di-C16-18-alkyltrimethylenedi-

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22-Jun-2015 to 16-Jul-2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

[3-({3-[(3-aminopropyl)amino]propyl}amino)propyl](hexadecyl)octadecylamine; [3-({3-[(3-aminopropyl)amino]propyl}amino)propyl]dihexadecylamine; [3-({3-[(3-aminopropyl)amino]propyl}amino)propyl]dioctadecylamine

EC Number:

941-593-4

Cas Number:

1623405-26-4

Molecular formula:

Not applicable UVCB

IUPAC Name:

[3-({3-[(3-aminopropyl)amino]propyl}amino)propyl](hexadecyl)octadecylamine; [3-({3-[(3-aminopropyl)amino]propyl}amino)propyl]dihexadecylamine; [3-({3-[(3-aminopropyl)amino]propyl}amino)propyl]dioctadecylamine

Test material form:

liquid: viscous

Details on test material:

- Name of test material (as cited in study report): di-C16-C18 (evennumbered) alkyl tripropylenetetramine
- Substance type: Viscous liquid
- Physical state: Liquid
- Storage condition of test material: At room temperature container flushed with nitrogen
- Batch/Lot number: B1; batch inspection lot no.:890000394200
- Expiration date of the lot/batch: 22 October 2017
- Purity: 100% (UVCB substance)
- pH: 8.5-10.5 (1% solution)
- specific gravity: 0.83 at 60°C

Method

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by Aroclor 1254

Test concentrations with justification for top dose:

Experiment 1
Preliminary test (without and with S9) TA100 and WP2uvrA: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
Main study: TA1535, TA1537 and TA98:
Without and with S9-mix: 17, 52, 164, 512, 1600 and 5000 µg/plate
Experiment 2:
Without and with S9-mix: 275, 492, 878, 1568, 2800 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle:
Test compound was soluble in ethanol and ethanol has been accepted and approved by authorities and international guidelines

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9 Migrated to IUCLID6: 650 µg/plate in DMSO for TA100

Controls 2

Positive control substance:

other: 2-nitrofluorene 10 µg/plate in DMSO for TA98

Remarks:

without S9

Controls 3

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9 Migrated to IUCLID6: 10 µg/plate in DMSO for WP2uvrA

Controls 4

Positive control substance:

other: ICR-191 25 µg/plate in DMSO for TA1537

Controls 5

Positive control substance:

sodium azide

Remarks:

without S9 Migrated to IUCLID6: 5 µg/plate in saline for TA1535

Controls 6

Positive control substance:

other: 2-aminoanthracene in DMSO for all tester strains

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 and TA98 is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive if:
a) A two-fold (TA100, WP2uvrA) or more or a three-fold (TA1535, TA1537, TA98) or more increase above solvent control in the mean number of revertant colonies is observed in the test substance group.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:Precipitation was observed at dose levels of 1568 µg/plate and upwards

RANGE-FINDING/SCREENING STUDIES:
dose range finding:
In tester strain TA100, toxicity was observed at dose level of 500 μg/plate in the absence of S9-mix. In tester strain WP2uvrA, no toxicity was
observed up to and including the top dose of 5000 µg/plate.

main 1:
No toxicity was observed up to and including the dose level of 1600 μg/plate. The test substance precipitated heavily on the plates at the test substance concentration of 5000 μg/plate, therefore the number of revertants of this dose level could not be determined.
main 2:
The bacterial background lawn was not reduced at any of the concentrations tested. A decrease in the number of revertants below the historical control data range was only observed in tester strain TA1535 in the absence of S9-mix at the test substance concentration of 2800 µg/plate.
No biologically relevant decrease in the number of revertants was observed in the other tester strains up to and including the dose level of 2800 μg/plate. The test substance precipitated heavily on the plates at the test substance concentration of 5000 μg/plate, therefore the number of revertants of this dose level could not be determined.

COMPARISON WITH HISTORICAL CONTROL DATA:
The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly, except for the response of the positive control for TA100 in the second experiment in the presence of S9-mix. The purpose of the positive control is as a reference for the test system, where a positive response is required to check if the test system functions correctly. Since the value was more than 3 times greater than the concurrent solvent control values, this deviation in the mean plate count of the positive control had no effect on the results of the study.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

It is concluded that di-C16-C18 (evennumbered) alkyl tripropylenetetramine is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

Di-C16-C18 (evennumbered) alkyl tripropylenetetramine was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of 5% resp. 10% S9-mix (rat liver S9-mix induced by Aroclor 1254). The study followed the most recent OECD and EU protocols and was performed under GLP.

The test substance was dissolved in ethanol. The test substance precipitated on the plates at dose levels of 1568 μg/plate and upwards. The substance was tested up to 5000 µg/plate in all strains. The bacterial background lawn was not reduced at any of the concentrations tested. Only in TA1535 cytotoxicity evidence from decrease in the number of revertants at 2800 µg/plate in absence of S9-mix. Heavy precipitation at 5000 µg/plate prohibited determination of the number of revertant colonies at this dose level.

Acceptable responses were obtained for the negative and strain-specific positive control substances indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

There was no significant or dose-related increase in the number of revertant colonies in any of the applied strains, both with and without S9-mix. This was confirmed in an independently repeated experiment.

It is concluded that di-C16-C18 (evennumbered) alkyl tripropylenetetramine is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia colireverse mutation assay.