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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Reference substance name:
Tridecanamine, N-tridecyl-, branched and linear
EC Number:
309-798-8
EC Name:
Tridecanamine, N-tridecyl-, branched and linear
Cas Number:
101012-97-9
Molecular formula:
C24 H50 NH - C28 H58 NH
IUPAC Name:
Tridecanamine, N-tridecyl-, branched and linear
Details on test material:
- Name of test material: Tridecanamine, N-tridecyl-, branched and linear
- BASF Test Item No.: 10/0484-2
- Batch Number: 000STD77L0
- Purity: DTDA: 91.081 %, TTDA: 8.355 % (Analytical Laboratory of the Production Plant, dated March 02, 2012); (≥90 %, preliminary information at the start of the experiment, dose calculation adjusted to purity)
- Expiration Date: May 23, 2013
- Physical state, appearance: Liquid, colorless, clear
- Storage conditions: room temperature

Method

Target gene:
HPRT
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Large stocks of the V79 cell line are stored in liquid nitrogen and before freezing, the level of spontaneous mutants was depressed by treatment with HAT-medium.
- Each batch is screened for mycoplasm contamination and checked for karyotype stability and spontaneous mutant frequency.
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Experiment I
Without S9 mix (4-h exposure): 0.01, 0.02, 0.04, 0.08, 0.15, 0.3, 0.45, 0.6 µg/mL
With S9 mix (4-h exposure): 0.6, 1.2, 2.4, 4.8, 9.6, 14.4, 19.2 µg/mL

Experiment II
Without S9 mix (24-h exposure): 0.01, 0.02, 0.04, 0.08, 0.16, 0.3, 0.47, 0.63 µg/mL
With S9 mix (4-h exposure): 0.63, 1.3, 2.5, 5.0, 10.0, 12.5, 15.0 µg/mL
Vehicle / solvent:
- Solvent: acetone
- Justification for choice of solvent: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures. The final concentration of acetone in the culture medium was 0.5 % (v/v).
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24 hours
- Exposure duration: In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.
- Expression/fixation time: Three or four days after treatment 1.5E6 cells per experimental point were subcultivated in 175 cm² flasks containing 30 mL medium. Following the expression time of 7 days five 80 cm² cell culture flasks were seeded with about 3 - 5E5 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability. The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 for about 8 days. The colonies were stained with 10 % methylene blue in 0.01 % KOH solution. The stained colonies with more than 50 cells were counted.

NUMBER OF REPLICATIONS:
- The study was performed in two independent experiments, using identical experimental procedures.

DETERMINATION OF CYTOTOXICITY
- method: cloning efficiency
Evaluation criteria:
- Acceptability of the assay: The gene mutation assay is considered acceptable if it meets the following criteria: The numbers of mutant colonies per 1E6 cells found in the solvent controls falls within the laboratory historical control data. The positive control substances should produce a significant increase in mutant colony frequencies. The cloning efficiency II (absolute value) of the solvent controls should exceed 50 %.
- Evaluation of results: A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points. A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system. A positive response is described as follows: A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment. The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed. However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory's historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance was considered together.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
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
Additional information on results:
RESULTS GENOTOXICITY:
- No relevant and reproducible increase in mutant colony numbers/1E6 cells was observed in the main experiments up to the maximum concentration with and without metabolic activation. The induction factor did not reach or exceed the threshold of 3.0 at any concentration of the test item with or without metabolic activation. A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of the mutation frequency. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.

TEST-SPECIFIC CONFOUNDING FACTORS:
- Effects of pH/osmolality: There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.
- Precipitation: No precipitation or phase separation occurred up to the maximum concentration after 4 hours and 24 hours treatment with and without metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
- Three pre-tests were performed in order to determine the concentration range of the mutagenicity experiments. The highest applied concentration in the pre-test on toxicity (4240 μg/mL) was equal to approximately 10 mM with respect to the molecular weight and the purity (≥90%, preliminary information at the start of the experiment). The first pre-test was terminated and repeated for technical reasons. The second pre-test, performed in the concentration range from 33.1 to 4240 μg/mL, was not analysable in the presence and absence of metabolic activation following 4 and 24 hours treatment due to exceedingly severe cytotoxicity down to the lowest concentration. Therefore, a third pre-test was performed in a concentration range from 0.31 to 40 μg/mL (4 and 24 hours treatment) metabolic activation.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% occurred in the first experiment at 9.6 μg/mL and above with and at 0.15 μg/mL and above without metabolic activation. In experiment II cytotoxic effects as described above were noted at 10.0 μg/mL and above with or at 0.63 μg/mL without metabolic activation. The recommended cytotoxic range of approximately 10-20% relative cloning efficiency I or relative cell density was covered with and without metabolic activation.

CONCLUSION:
It can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, tridecanamine, N-tridecyl, branched and linear is considered to be non-mutagenic in this HPRT assay.

Applicant's summary and conclusion