Amines, N-(C16-18 and C18-unsatd. alkyl)trimethylenedi-, dioleates

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

key study

Study period:

16 October 2003 - 5 December 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Recent adequately performed an dreported study according to standard guideline and in compliance to GLP.

Data source

Reference

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Oleyl diamine, dioleate

IUPAC Name:

Oleyl diamine, dioleate

Test material form:

other: Fluid

Details on test material:

name: INIPOL 002
other name: N-alkyl "oleyl" propylene diamine dioleate
CAS number: 40027-38-1
batch number: 11638106
Sponsor's filing number: GRL 0023/03
description: viscous orange-colored liquid
container: one glass flask
date of receipt: 22 September 2003
storage conditions: at room temperature and protected from light
purity: 97.40%
composition: see analytical certificate
expiry date: September 2004.

Method

Target gene:

S. typhimurium: Histidine gene

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver microsomes

Test concentrations with justification for top dose:

Preliminary test: with and without S9: 10, 100, 500, 1000, 2500 and 5000 μg/plate.
Precipitate seen at ≥ 2500 μg/plate
Experiment 1 and 2: All tester strains in triplicate:
Range of dose levels up to toxicity involved:
3.91, 7.81, 15.6, 31.3, 62.5, 93.75, 125, 250, 375 and 500 µg/plate

Vehicle / solvent:

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

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2-Anthramine

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- in agar (plate incorporation) for preliminary test, both experiments without S9 mix and the first experiment with S9 mix
- preincubation method: Second experiment with S9-mix: 60 minutes at 37°C before plating

DURATION
- Exposure duration: 48 to 72 hours of incubation at 37°C,

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

DETERMINATION OF CYTOTOXICITY
- Method: Decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

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

Evaluation criteria:

Acceptance criteria:
. the number of revertants in the vehicle controls is consistent with the historical data of the testing facility,
. the number of revertants in the positive controls is higher than that of the vehicle controls and is consistent with the historical data of the testing facility.
Evaluation criteria
A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship.
Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

PRELIMINARY TOXICITY TEST
The test item was freely soluble in the vehicle (ethanol) at 100 mg/mL.
Consequently, with a treatment volume of 50 μL/plate, the dose-levels were 10, 100, 500, 1000, 2500 and 5000 μg/plate.
A marked precipitate was observed in the Petri plates when scoring the revertants at dose-levels ≥ 2500 μg/plate, toward the three strains used.
A moderate to marked toxicity was noted at dose-levels ≥ 100 μg/plate in the TA 98 and TA 102 strains without S9 mix and at dose-levels ≥ 500 μg/plate in the TA 98 and TA 102 strains with S9 mix and in the TA 100 strain, with and without S9 mix.

MUTAGENICITY EXPERIMENTS
The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.
Since the test item was toxic in the preliminary test, the choice of the highest dose-level was based on the level of toxicity, according to the criteria specified in the international guidelines.

Without S9 mix, a moderate to marked toxicity was generally noted at dose-levels ≥ 31.3 μg/plate in the TA 1537 strain, ≥ 62.5 μg/plate in the TA 1535, TA 98 and TA 102 strains, and ≥ 125 μg/plate in the TA 100 strain.
With S9 mix, a moderate to marked toxicity was generally observed at dose-levels ≥ 125 μg/plate in the TA 1537 strain, ≥ 250 μg/plate in the TA 100 and TA 102 strains, at 375 μg/plate in the TA 98 strain, and at 500 μg/plate in the TA 1535 strain.

The test item did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the five strains.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Test substance Oleyl diamine, dioleate was found to be non-mutagenic in the Salmonella typhimurium reverse mutation assay.

Executive summary:

The objective of this study was to evaluate the potential of the test item INIPOL 002 (batch No. 11638106, purity: 97.40%) to induce reverse mutation in Salmonella typhimurium.

The study was performed according to the international guidelines (OECD 471, Commission Directive No. B13/14) and in compliance with the Principles of Good Laboratory Practice Regulations.

 

Methods

A preliminary toxicity test was performed to define the dose-levels of INIPOL 002 to be used for the mutagenicity study. The test item was then tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.

Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes, 37°C).

Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to five dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored.

The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

The test item INIPOL 002 was dissolved in ethanol.

The dose-levels of the positive controls were as follows:

without S9 mix:

• 1 μg/plate of sodium azide (NaN3): TA 1535 and TA 100 strains,

• 50 μg/plate of 9-Aminoacridine (9AA): TA 1537 strain,

• 0.5 μg/plate of 2-Nitrofluorene (2NF): TA 98 strain,

• 0.5 μg/plate of Mitomycin C (MMC): TA 102 strain.

with S9 mix:

• 2 μg/plate of 2-Anthramine (2AM): TA 1535, TA 1537, TA 98 and TA 100 strains,

• 10 μg/plate of 2-Anthramine (2AM): TA 102 strain.

 

Results

Since the test item was toxic in the preliminary test, the choice of the highest dose-level was based on the level of toxicity, according to the criteria specified in the international guidelines.

Without S9 mix, the selected treatment-levels ranged from 3.91 to 500 μg/plate.

A moderate to marked toxicity was generally noted at dose-levels ≥ 31.3 μg/plate in the TA 1537 strain, ≥ 62.5 μg/plate in the TA 1535, TA 98 and TA 102 strains, and ≥ 125 μg/plate in the TA 100 strain.

With S9 mix, the selected treatment-levels ranged from 15.6 to 500 μg/plate.

A moderate to marked toxicity was generally observed at dose-levels ≥ 125 μg/plate in the TA 1537 strain, ≥ 250 μg/plate in the TA 100 and TA 102 strains, at 375 μg/plate in the TA 98 strain, and at 500 μg/plate in the TA 1535 strain.

The test item did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the five strains.

The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

 

Conclusion

Under our experimental conditions, the test item INIPOL 002 (batch No. 11638106, purity: 97.40%) did not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.