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

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:

From December 17, 2001 to March 25, 2002

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

Test guideline 1

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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:

bis(3-aminopropyl)(dodecyl)amine

Details on test material:

- Lot/Batch number: FP 020010155
- Triamine content: 85.3%; Total amine content (commercial grade): 100%

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, 1,000, 2,500 and 5,000 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 substance 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 substance 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 substance 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 h

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

Key result

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 substance 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 the study conditions, the test substance was not considered to be mutagenic in the bacterial reverse mutation assay in the presence and absence of S9 mix.

Executive summary:

An in vitro bacterial reverse mutation study was conducted to determine the mutagenic potential of the test substance according to OECD Guideline 471 and EU method B.13/14, in compliance with GLP. Salmonella typhimurium strains TA 1535, TA 1537, TA100, TA98, and TA102 were exposed to the substance in the presence and absence of S9. The assay was performed in two phases, using the plate incorporation and/or pre-incubation method. The first phase (preliminary toxicity) was to establish the dose-range for the confirmatory assay and to provide a preliminary mutagenicity evaluation. The second phase (confirmatory assay) was to evaluate and confirm the mutagenic potential of the test substance. The main study was conducted in two independent experiments with at least five dose levels of the test substance using three plates/dose-level with and without S9 mix:

Without S9-mix:

First experiment (plate incorporation method): 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 (plate incorporation 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.

With S9 mix:

First experiment (plate incorporation method): 12.5, 25, 50, 100 and 200 µg/plate for all tester strains.

Second experiment (pre-incubation 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). Concurrent solvent (water) and positive controls (without S9: sodium azide (TA 1535 and TA 100), 9-aminoacridine (TA 1537), 2-nitrofluorene (TA 98), mitomycin C (TA 102); with S9: 2-anthramine (all strains), were also included.

In the preliminary toxicity test, no precipitate was observed in the Petri plates when the revertants were scored at all dose levels. Both with and without S9 mix, the test substance was strongly toxic at dose-levels ≥500 µg/plate. In the TA 98 strain, the test substance was also toxic at 100 µg/plate. In the mutagenicity assay, moderate to marked toxicity was induced generally at dose-levels ≥50 µg/plate without S9-mix and at ≥100 µg/plate with S9 mix. No increase in the number of revertants was noted in all tester strains in both the experiments. All the validity criteria were met.

Under the study conditions, the test substance was not considered to be mutagenic in the bacterial reverse mutation assay in the presence and absence of S9 mix (Haddouk, 2002).