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 mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01-jul-2009 to 11-aug-2009

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:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: recommendations of the “International Workshop on Genotoxicity Tests Workgroup” (the IWGT), published in the literature (Clive et al., 1995, Moore et al., 1999, 2000, 2002, 2003, 2006 and 2007).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 001

Cas Number:

1219458-14-6

Details on test material:

- Chemial name: Triamine C16-18, C18-unsaturated
- EC Number: 628-863-4

To the best of knowledge, the sample used is representative to the boundary composition share and agreed by each registrant.

Method

Target gene:

Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media:
-RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Dose range finding test:
Without and with S9-mix, 3 hours treatment: 0.3, 1, 3, 10, 33 and 100 µg/ml
Without S9-mix, 24 hours treatment: 0.08, 0.24, 0.8, 2.4, 8 and 26 µg/ml
Experiment 1:
Without S9-mix, 3 hours treatment: 0.01, 0.03, 0.1, 0.3, 1, 1.5, 2 and 2.5 µg/ml
With S9-mix, 3 hours treatment: 0.1, 0.3, 1, 3, 10, 16, 18 and 20 µg/ml
Experiment 2
Without S9-mix, 24 hours treatment: 0.08, 0.24, 0.5, 0.8, 1, 1.3, 1.6 and 2 µg/ml
With S9-mix, 3 hours treatment: 1, 3, 6, 10, 15, 18, 20, 22, 26, 28 and 30 μg/ml

Vehicle / solvent:

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

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9 Migrated to IUCLID6: : 15 µg/ml for the 3 hours treatment period and 5 µg/ml for the 24 hours treatment period

Controls 2

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9 Migrated to IUCLID6: : 7.5 µg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Short-term treatment
With and without S9-mix; 3 hours
Prolonged treatment period
Without S9-mix: 24 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 to 12 days

SELECTION AGENT (mutation assays): 5 µg/ml trifluorothymidine (TFT)

NUMBER OF REPLICATIONS:
- Solvent controls: Duplo cultures
- Treatment groups and positive control: Single cultures

NUMBER OF CELLS EVALUATED: 9.6 x 105 cells/concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (dose range finding test) and relative total growth (mutation experiments)

RANGE-FINDING/SCREENING STUDIES:
-The suspension growth expressed as the reduction in cell growth after approximately 24 and 48 hours or only 24 hours cell growth, compared to the cell growth of the solvent control, was used to determine an appropriate dose range for the mutagenicity tests

Evaluation criteria:

The global evaluation factor (GEF) has been defined by the IWTG as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126 (ref. 12).

A test substance is considered positive (mutagenic) in the mutation assay if:
a) It induces a MF of more then MF(controls) + 126 in a dose-dependent manner; or
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.
An observed increase should be biologically relevant and will be compared with the historical control data range.

A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Precipitation: Precipitation in the exposure medium was observed at dose levels of 100 µg/ml and above


RANGE-FINDING/SCREENING STUDIES:
- Toxicity was observed at dose levels of 3 µg/ml in the absence of S9, 3 hours treatment; at dose levels of 33 µg/ml in the presence of S9, 3 hours treatment; at dose levels of 2.4 µg/ml in the absence of S9, 24 hours treatment


COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.


ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the absence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 80 and 89% compared to the total growth of the solvent controls after the 3 and 24 hours treatment period, respectively.

In the presence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 82 and 76% compared to the total growth of the solvent controls after the 3 hours treatment period in the first and second experiment, respectively.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: L5178Y/TK+/--3.7.2C

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Tallow dipropylenetriamine is not mutagenic in the TK mutation test system under the experimental conditions described in this report.

Executive summary:

Tallow dipropylenetriamine was evaluated for its possible induction of forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells. The test was performed in two independent experiments in the absence and presence of S9-mix. The study was performed under GLP and according to the most recent OECD and EU guidelines.

Batch S001096 of Tallow dipropylenetriamine was a white paste. The test substance was dissolved in ethanol.

In the first experiment, Tallow dipropylenetriamine was tested up to concentrations of 2.5 and 20 μg/ml in the absence and presence of 8% (v/v) S9-mix. The incubation time was 3 hours. Tallow dipropylenetriamine was tested up to cytotoxic levels of 80 and 82% in the absence and presence of S9-mix, respectively.

In the second experiment, Tallow dipropylenetriamine was tested up to concentrations of 2 and 30 μg/ml in the absence and presence of 12% (v/v) S9-mix. The incubation times were 24 hours and 3 hours for incubations in the absence and presence of S9-mix, respectively. Tallow dipropylenetriamine was tested up to cytotoxic levels of 89% in the absence of S9-mix and 76% in the presence of S9-mix.

The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range

Positive control chemicals, methyl methane sulfonate and cyclophosphamide induced appropriate responses.

In the absence of S9-mix, Tallow dipropylenetriamine did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in a repeat experiment with modifications in the duration of treatment time.

In the presence of S9-mix, Tallow dipropylenetriamine did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modifications in the composition of the S9 concentration for metabolic activation.

In conclusion, Tallow dipropylenetriamine is not mutagenic in the TK mutation test system under the experimental conditions described in this report.