Amines, N-(3-aminopropyl)-N-(C16-18 and C18-unsatd. alkyl)trimethylenedi-

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17dec2001 / 25mar2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Cited as Directive 2000/32/EC, B.13/14

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine

EC Number:

219-145-8

EC Name:

N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine

Cas Number:

2372-82-9

Molecular formula:

C18H41N3

IUPAC Name:

N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine

Details on test material:

- Name of test material (as cited in study report): dodecyldipropylenetriamine
- Physical state at receipt: very thick white cream
- Physical state at storage conditions: light yellow liquid
- Analytical purity: 85.3%
- Lot/batch No.: FP 020010155
- Expiration date of the lot/batch: 22 August 2003
- Storage condition of test material: at room temperature and under nitrogen gas

Method

Target gene:

his

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA 1535, TA 1537, TA100, TA98, and TA102.

Metabolic activation:

with and without

Metabolic activation system:

rat S9

Test concentrations with justification for top dose:

Preliminary toxicity test: 10, 100, 500, 1000, 2500 and 5000 µg/plate, +/- S9.

Main study, with S9:
1st experiment: 6.25, 12.5, 25, 50 and 100 µg/plate (TA 98 and TA 1537), 12.5, 25, 50, 100 and 200 µg/plate (other strains).
2nd experiment: 3.125, 6.25, 12.5, 25 and 50 µg/plate (TA 98, TA 1537 and TA 1535); 12.5, 25, 50, 100 and 200 µg/plate (TA 100 and TA 102)

Main study, without S9:
1st experiment: 12.5, 25, 50, 100 and 200 µg/plate, for all tester strains
2nd experiment: 3.125, 6.25, 12.5, 25 and 50 µg/plate (TA 98, TA 1537 and TA 1535), 12.5, 25, 50, 100 and 200 µg/plate (TA 100 and TA 102).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S9: sodium azide (TA 1535 and TA 100), 9-aminoacridine (TA 1537), 2-nitrofluorene (TA 98), mitomycin C (TA 102). +S9: 2-anthramine (all strains)

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation and preincubation (experiment 2,+ S9)
The test item was dissolved in distilled water the vehicle previously heated at approximately 50°C. The preparations were made immediately before use. The direct plate incorporation method was performed as follows: test item solution (0.1 mL), S9 mix when required (0.5 mL) and bacterial suspension (0.1 mL) were mixed with 2 mL of overlay agar (containing traces of the relevant aminoacid and biotin and maintained at 45°C). After rapid homogenization, the mixture was overlaid onto a Petri plate containing minimum medium. The preincubation method was performed as follows: test item solution (0.1 mL), S9 mix (0.5 mL) and the bacterial suspension (0.1 mL) were incubated for 60 minutes at 37°C before adding the overlay agar and pouring onto the surface of a minimum agar plate.

DURATION
- Preincubation period: 60 min (experiment 2, + S9)
- Exposure duration: 48 to 72 hours

NUMBER OF REPLICATIONS: 3 plates/dose level in two independent experiments

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

Evaluation criteria:

The study is considered valid if the following criteria are fully met:
- the number of revertants in the vehicle controls is consistent with laboratory historical data;
-the number of revertants in the positive controls is higher than that of the vehicle controls and is consistent with laboratory historical data.
A reproducible two-fold increase in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. 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, other: TA 1535, TA 1537, TA100, TA98, and TA102.

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 50 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: Since the test item was toxic in the preliminary test, the choice of the highest dose-level for the main test was based on the level of toxicity, according to the criteria specified in the international guidelines.

ADDITIONAL INFORMATION ON CYTOTOXICITY: In the main study in the experiments without S9 mix, a moderate to marked toxicity was induced generally at dose-levels ≥ 50 μg/plate. In the experiments with S9 mix, a moderate to marked toxicity was induced generally at dose-levels ≥ 100 μg/plate in the first experiment and ≥ 50 μg/plate in the second experiment.
In the preliminary cytotoxicity test, with S9, toxicity was induced at dose levels ≥ 100 µg/plate in TA 98 strain and ≥ 500 µg/plate in other strains.

Applicant's summary and conclusion

Conclusions:

Under these experimental conditions, the test item does not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

Executive summary:

A Bacterial Reverse Mutation Test was performed according to OECD No. 471 and EEC B.13/14.

To assess the toxicity of the test item to the bacteria, six dose-levels (one plate/dose-level; 10, 100, 500, 1000, 2500 and 5000 µg/plate) were tested in the TA 98, TA 100 and TA 102 strains, with and without S9 mix. 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.

With dose levels based on the results of the preliminary toxicity test two independent experiments (without S9-mix: First experiment 6.25, 12.5, 25, 50 and 100 µg/plate, for the TA 98 and TA 1537 strains, 12.5, 25, 50, 100 and 200 µg/plate, for the remaining strains. Second experiment: 3.125, 6.25, 12.5, 25 and 50 µg/plate, for the TA 98, TA 1537 and TA 1535 strains, 12.5, 25, 50, 100 and 200 µg/plate, for the TA 100 and TA 102 strains. With S9 mix First experiment: 12.5, 25, 50, 100 and 200 µg/plate for all tester strains. Second experiment (preincubation method): 3.125, 6.25, 12.5, 25 and 50 µg/plate, for the TA 98, TA 1537 and TA 1535 strains, 12.5, 25, 50, 100 and 200 µg/plate, for the TA 100 and TA 102 strains) using three plates/dose-level, each strain was tested, with and without S9 mix, with at least five dose-levels of the test item, the vehicle control and the appropriate positive control.

Preliminary toxicity and First test: The direct plate incorporation method was performed as follows: test item solution (0.1 mL), S9 mix when required (0.5 mL) and bacterial suspension (0.1 mL) were mixed with 2 mL of overlay agar (containing traces of the relevant aminoacid and biotin and maintained at 45°C).

After rapid homogenization, the mixture was overlaid onto a Petri plate containing minimum medium.

Second test: The preincubation method was performed as follows: test item solution (0.1 mL), S9 mix (0.5 mL) and the bacterial suspension (0.1 mL) were incubated for 60 minutes at 37°C before adding the overlay agar and pouring onto the surface of a minimum agar plate.

After 48 to 72 hours of incubation at 37°C, revertants were scored with an automatic counter (Cardinal counter, Perceptive Instruments, CB9 7 BN, ).

This study is considered valid if the following criteria are fully met:

. the number of revertants in the vehicle controls is consistent with laboratory historical data

. the number of revertants in the positive controls is higher than that of the vehicle controls and is consistent with laboratory historical data.

A reproducible two-fold increase in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may

also be taken into account in the evaluation of the data obtained.

Preliminary toxicity test:

No precipitate was observed in the Petri plates when scoring the revertants at all dose-levels. Both with and without S9 mix, the test item was strongly toxic at dose-levels ≥ 500 µg/plate. In the TA 98 strain, the test item was also toxic at 100 µg/plate.

Mutagenicity experiments:

Without S9-mix: A moderate to marked toxicity was induced generally at dose-levels ≥ 50 µg/plate. No increase in the number of revertants was noted in all tester strains, in both experiments.

With S9-mix: A moderate to marked toxicity was induced generally at dose-levels ≥ 100 µg/plate in the first experiment and ≥ 50 µg/plate in the second experiment. No increase in the number of revertants which could be considered as relevant was induced in any of the five tester strains.

Criteria for validity were met.