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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Di-tridecamine did not cause gene mutations in bacteria (Ames test) or mammalian cells (HPRT assay), nor did it induce chromosomal aberrations in V79 cells. These studies were performed in the absence and presence of metabolic activation. Thus, di-tridecamine is not considered to be genotoxic.

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Referenceopen allclose all

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
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
Target gene:
HPRT
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).
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.
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.
Endpoint:
in vitro cytogenicity / chromosome aberration 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
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-naphthoflavone-induced rat liver S9
Test concentrations with justification for top dose:
First experiment
Without S9 mix (4-h exposure, 18-h sampling time): 0.16, 0.31, 0.63, 1.25, 2.5 and 5 μg/mL
With S9 mix (4-h exposure, 18-h sampling time): 1.25, 2.5, 5, 10, 20 and 40 μg/mL

Second experiment
Without S9 mix (18-h exposure, 18-h sampling time): 0.08, 0.16, 0.31, 0.63, 1.25, 2.5 and 5 μg/mL
Without S9 mix (18-h exposure, 28-h sampling time): 0.16, 0.31, 0.63, 1.25, 2.5 and 5 μg/mL
With S9 mix (4-h exposure, 28-h sampling time): 1.25, 2.5, 5, 10, 20 and 40 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol (1% v/v)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
ethanol (1% v/v)
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
ethanol (1% v/v)
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 and 18 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 18 or 28 hours

STAIN (for cytogenetic assays): After drying, the slides were stained with 7.5% (v/v) Giemsa/Titrisol solution pH 7.2 for 10 minutes. After being rinsed twice in purified water and clarified in xylene, the slides were mounted in Corbit-Balsam.

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: the first 100 consecutive well-spread metaphases of each culture were counted for all test groups, and if cells had 20 – 22 chromosomes, they were analyzed for structural chromosome aberrations. In the case of clearly increased aberration rates, the number of
metaphases to be analyzed for this test group can be reduced to at least 50 metaphases per culture.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index and cell counts

OTHER EXAMINATIONS:
- Numerical chromosome aberrations were scored and recorded:
- Chromatid gap and chromosome gap (isochromatid gap) G' and G"
- Chromatid break and chromosome break B' and B"
- Chromatid fragment and chromosome fragment F' and F"
- Chromatid deletion and chromosome deletion D' and D"
- Multiple aberrations m. A.
- Disintegration of chromosomal structure (pulverization) P
- Intrachanges at the level of chromatids and chromosomes Ex (Int' and Int'')
- Interchanges at the level of chromatids and chromosomes Ex (I' and I'')
- Aneuploidy (AP)
- Polyploidy (PP)
- Endopolyploidy (EP)

OTHER:
- Cell morphology: at the end of the treatment period, the test cultures of all test groups, were checked microscopically for cell morphology, which is an indication of attachment of the cells to the slides.
Evaluation criteria:
The test substance is considered as positive if a statistically significant, dose-related and reproducible increase in the number of cells with structural chromosome aberrations (excl. gaps) is observed and the number of aberrant cells (excl. gaps) exceeds both the concurrent negative/vehicle control value and the historical negative control data range.
A test substance generally is considered as negative if the number of cells with structural aberrations (excl. gaps) in the dose groups is not statistically significant increased above the concurrent negative/vehicle control value and is within the historical negative control data range.
Statistics:
The statistical evaluation of the data was carried out using the MUCHAN program system (BASF SE). The proportion of metaphases with structural aberrations was calculated for each group. A comparison of each dose group with the negative control group was carried out using Fisher's exact test for the hypothesis of equal proportions. This test was Bonferroni-Holm corrected versus the dose groups separately for each time and was performed one-sided.
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 applicable
Positive controls validity:
valid
Additional information on results:
ADDITIONAL INFORMATION ON CYTOTOXICITY:
Clear cytotoxicity indicated by reduced mitotic indices and/or cell numbers was observed at least in the highest applied concentrations tested for cytogenetic damage.
On the basis of the results of the present study, the test substance did not cause any biologically relevant increase in the number of structurally aberrant metaphases incl. and excl. gaps at both sampling times either without S9 mix or after adding a metabolizing system in two experiments performed independently of each other.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S-9 mix
Test concentrations with justification for top dose:
0, 20, 100, 500, 2500, 5000 µg/plate (standard plate test, all strains)
0, 3, 6, 12, 25, 50 µg/plate (standard plate test, Salmonella strains)
0, 3, 6, 12, 25, 50 µg/plate (preincubation test; Salmonella strains)
0, 4, 20, 100, 500, 2500 µg/plate (preincubation test; E. coli strain)

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: stability and solubility
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S-9 mix for strains TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-methyl-N'-nitro-N-nitrosoguanidine
Remarks:
without S-9 mix for strains TA 1535 and TA 100
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenyldiamine
Remarks:
without S-9 mix for strain TA 98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S-9 mix for strain TA 1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without S-9 mix for strain E.coli WP2 uvrA
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
Plate incorcoration method:
- Exposure duration: ca. 48-72 hours at 37°C
Preincubation method:
- Preincubation period: 20 minutes at 37°C
- Exposure duration: ca. 48-72 hours at 37°C

NUMBER OF REPLICATIONS:
- 3 test plates per dose or per control

DETERMINATION OF CYTOTOXICITY
- Decrease in the number of revertants
- Reduced his- or trp- background growth
- Reduction in the titer
Evaluation criteria:
The test chemical is considered positive when:
A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S-9 mix or after adding a metabolizing system.

A test substance is generally considered nonmutagenic when:
The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test substance precipitation was found from about 2,500 µg/plate onward

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A bacteriotoxic effect (reduced background growth, decrease in the number of revertants, reduction in the titer) was observed in the standard plate test with the Salmonella strains depending on the strain and test conditions from about 50 µg - 100 µg/plate onward. Using E. coli a bacteriotoxic effect was observed at doses >= 2,500 µg/plate.
In the preincubation assay bacteriotoxicity was observed depending on the strain and test conditions from about 6 µg - 12 µg/plate onward (Salmonella strains) or from about 4 µg - 100 µg/plate onward (E. coli).
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from male Wistar rat livers
Test concentrations with justification for top dose:
1st Exp: 0; 33; 100; 333; 1000; 2650 and 5300 μg/plate (all strains, with and without S9 mix) SPT
2nd Exp: 0; 1.0; 3.3; 10; 33; 100 and 333 μg/plate (TA strain, without S9 mix) SPT
3rd Exp: 0; 1.0; 3.3; 10; 33; 100 and 333 μg/plate (TA strains without S9 mix), 0; 3.3; 10; 33; 100; 333 and 1000 μg/plate (TA strains with S9 mix); 0; 10; 33; 100; 333; 1000 and 2650 μg/plate (E.coli with and without S9 mix) PIT
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
other: 2-aminoanthracene, N-methyl-N`-nitro-N-nitrosoguanidine, 4-nitro-o-phenylenediamine
Details on test system and experimental conditions:
Standard plate test
The experimental procedure of the standard plate test (plate incorporation method) was based on the method of Ames et al.
• Salmonella typhimurium
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution or vehicle (negative control); 0.1 mL fresh bacterial culture; 0.5 mL S9 mix (with metabolic activation) or 0.5 mL phosphate buffer (without metabolic activation).
After mixing, the samples were poured onto Minimal glucose agar plates (Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds. After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hinders the counting using the Image Analysis System.
• Escherichia coli
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution or vehicle (negative control); 0.1 mL fresh bacterial culture; 0.5 mL S9 mix (with metabolic activation) or 0.5 mL phosphate buffer (without metabolic activation).
After mixing, the samples were poured onto Minimal glucose agar plates (Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds. After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (trp+ revertants) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hinders the counting using the Image Analysis System.
Preincubation Test
The experimental procedure was based on the method described by Yahagi et al. and Matsushima et al. 0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL S9 mix (with metabolic activation) or phosphate buffer (without metabolic activation) were incubated at 37°C for the duration of about 20 minutes using a shaker. Subsequently, 2 mL of soft agar was added and, after mixing, the samples were poured onto the agar plates within approx. 30 seconds.
After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hindered the counting using the Image Analysis System.
Evaluation criteria:
The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
Species / strain:
other: TA 1535, TA 100, TA 1537, TA 98 and E.coli
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

SOLUBILITY: No precipitation of the test substance was found with and without S9 mix.

TOXICITY: A strong bacteriotoxic effect was observed depending on the strain and test conditions from about 10 μg/plate onward.

MUTAGENICITY: A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test without S9 mix or after the addition of a metabolizing system.

Conclusions:
Under the experimental conditions chosen here, it is concluded that Tridecanamine, Ntridecyl-, branched and linear (PSN 18/0019-1) is not a mutagenic test substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from male Wistar rat livers
Test concentrations with justification for top dose:
1st Exp: 0, 33, 100, 333, 1000, 2650 and 5300 µg/plate (all strains, with and without S9 mix) SPT
2nd Exp: 0; 0.33; 1; 3.3; 10; 33 and 100 μg/plate (without S9 mix); 0; 1; 3.3; 10; 33; 100 and 333 μg/plate (with S9 mix) (TA strains) SPT
3rd Exp: 0; 0.33; 1.0; 3.3; 10; 33 and 100 μg/plate (TA strains); 0; 3.3; 10; 33; 100; 333 and 1000 μg/plate (E. coli) (with and without S9 mix) PIT
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
other: 2-aminoanthracene, N-methyl-N`-nitro-N-nitrosoguanidine, 4-nitro-o-phenylenediamine
Details on test system and experimental conditions:
Standard plate test
The experimental procedure of the standard plate test (plate incorporation method) was based on the method of Ames et al.
• Salmonella typhimurium
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution or vehicle (negative control); 0.1 mL fresh bacterial culture; 0.5 mL S9 mix (with metabolic activation) or 0.5 mL phosphate buffer (without metabolic activation).
After mixing, the samples were poured onto Minimal glucose agar plates (Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds. After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hinders the counting using the Image Analysis System.
• Escherichia coli
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution or vehicle (negative control); 0.1 mL fresh bacterial culture; 0.5 mL S9 mix (with metabolic activation) or 0.5 mL phosphate buffer (without metabolic activation).
After mixing, the samples were poured onto Minimal glucose agar plates (Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds. After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (trp+ revertants) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hinders the counting using the Image Analysis System.
Preincubation Test
The experimental procedure was based on the method described by Yahagi et al. and Matsushima et al. 0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL S9 mix (with metabolic activation) or phosphate buffer (without metabolic activation) were incubated at 37°C for the duration of about 20 minutes using a shaker. Subsequently, 2 mL of soft agar was added and, after mixing, the samples were poured onto the agar plates within approx. 30 seconds.
After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hindered the counting using the Image Analysis System.
Evaluation criteria:
The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
Species / strain:
other: TA 1535, TA 100, TA 1537, TA 98 and E.coli
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

SOLUBILITY: No precipitation of the test substance was found with and without S9 mix.

TOXICITY: A strong bacteriotoxic effect was observed depending on the strain and test conditions from about 33 μg/plate onward.

MUTAGENICITY: A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test without S9 mix or after the addition of a metabolizing system.

Conclusions:
Under the experimental conditions chosen here, it is concluded that Tridecanamine, N-tridecyl-, branched and linear (PSN 18/0020-1) is not a mutagenic test substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Di-tridecamine was tested in the Ames reverse mutation assay using Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2 uvr A at 20 to 5000 µg/plate (standard plate and preincubation test) with and without metabolic activation. Di-tridecamine was cytotoxic (reduced background growth, decrease in the number of revertants, reduction in the titer) in the standard plate test with the Salmonella strains depending on the strain and test conditions from about 50 µg - 100 µg/plate and onwards. For E. coli a cytotoxic effect was observed at doses2,500 µg/plate.

In the preincubation assay cytotoxicity was observed depending on the strain and test conditions from about 6 µg - 12 µg/plate onward (Salmonella strains) or from about 4 µg - 100 µg/plate onward (E. coli). Under the conditions tested, di-tridecamine was not mutagenic in any in the S. typhimurium strains and E.coli WP2 uvr A.

 

In addition, two Ames-Tests with Tridecanamine, N-tridecyl-, branched and linear were conducted in 2018 (nonGLP). The studies were carried out to compare the toxicological properties of two batches of the UVCB DTDA.

The test substance Tridecanamine, N-tridecyl-, branched and linear (PSN 18/0020-1) was tested for ist mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay. Strains tested TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA at a dose range of 0.33 μg - 5300 μg/plate (SPT) and 0.33 μg - 1000 μg/plate (PIT). Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats). No precipitation of the test substance was found with and without S9 mix. A strong bacteriotoxic effect was observed depending on the strain and test conditions from about 33 μg/plate onward. A relevant increase in the number of his+or trp+revertants (factor2: TA 100, TA 98 and E.coli WP2 uvrA or factor3: TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test without S9 mix or after the addition of a metabolizing system. Under the experimental conditions of this study, the test substance Tridecanamine, Ntridecyl-, branched and linear (PSN 18/0020-1) is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation. 

The test substance Tridecanamine, N-tridecyl-, branched and linear (PSN 18/0019-1) was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay. Strains tested TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA at a dose range of 1.0 μg - 5300 μg/plate (SPT) and 1.0 μg - 2650 μg/plate (PIT). Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats). No precipitation of the test substance was found with and without S9 mix. A strong bacteriotoxic effect was observed depending on the strain and test conditions from about 10 μg/plate onward. A relevant increase in the number of his+or trp+revertants (factor: TA 100, TA 98 and E.coli WP2 uvrA or factorTA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test without S9 mix or after the addition of a metabolizing system. Under the experimental conditions of this study, the test substance Tridecanamine, Ntridecyl-, branched and linear (PSN 18/0019-1) is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

 

 

Induction of chromosomal aberrations was tested in an OECD 473 study, in which Chinese hamster lung fibroblasts (V79) were exposed to 0.16 to 40 µg/mL di-tridecamine with and without metabolic activation (BASF AG, 2011). Cytotoxicity was observed at least in the highest applied concentration tested and vehicle and positive controls exhibited appropriate responses. No significant increases in chromosomal aberrations upon treatment with di-tridecamine were observed.

 

 

In a GLP-compliant gene (HPRT) mutation assay, according to OECD guideline 476, chinese hamster lung fibroblast (V79) were exposed to di-tridecamine (Harlan, 2012). In the first experiment, cells were exposed for 4 hours at concentrations ranging from 0.04 to 0.45 µg/mL and 0.60 to 9.6 µg/mL, without and with metabolic activity, respectively. In the second experiment, cells were exposed for 24 hours at concentrations ranging from 0.08 to 0.63 µg/mL and for 4 hours at concentrations ranging from 1.3 to 12.5 µg/mL, without and with metabolic activation, respectively. No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation. Tridecanamine, N-tridecyl-, branched and linear is considered to be non-mutagenic in this HPRT assay.

Justification for classification or non-classification

Based on the results of the in vitro genetic toxicity studies, di-tridecamine does not need to be classified according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.