Octadecanoic acid, reaction products with triethylenetetramine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-10-26 to 2018-01-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Test material

Test material information

Constituent 1

Reference substance name:

Octadecanoic acid, reaction products with triethylenetetramine

EC Number:

270-171-6

EC Name:

Octadecanoic acid, reaction products with triethylenetetramine

Cas Number:

68412-15-7

Molecular formula:

C24H54N4O2

IUPAC Name:

N'-[2-(2-aminoethylamino)ethyl]ethane-1,2-diamine;octadecanoic acid

Method

Target gene:

Thymidine kinase locus (TK +/-)

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells:
American Type Culture Collection, Rockville,Maryland (ATCC code: CRL 9518).
- Suitability of cells:
The generation time and mutation rates (spontaneous and induced) have been checked
- Modal number of chromosomes:
no information

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no information
- Periodically 'cleansed' against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Experiment 1: without S9, 3 hours: 69.0, 34.5, 17.3, 8.63 and 4.31 µg/L
Experiment 1: with S9, 3 hours: 138, 69.0, 34.5, 17.3 and 8.63 µg/L
Experiment 2: without S9, 3 hours: 69.0, 46.0, 30.7, 20.5 and 13.6 µg/L

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: culture medium
- Justification for choice of solvent/vehicle: Vehicle was chosen based on results from solubility experiment

Controls

Controls 1

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

in the presence of S9 metabolism

Controls 2

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

in the absence of S9 metabolism

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 2 x 1E5 cells/mL
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3 and 24 hours

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 2 days
- Selection time (if incubation with a selective agent): 14 days
- Method used: microwell plates for the mouse lymphoma assay.
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure.: trifluorothymidine (final concentration 3.0 µg/mL) and an estimated 2 × 1E3 cells were plated in each well of four 96-well plates and exposed for 14 days.
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 2 x 1E3 cells were plated, wells containing viable clones will be identified and counted by eye using background illumination and colony sizing will be performed
- Criteria for small (slow growing) and large (fast growing) colonies:
Size:
Small: less than ¼ of well’s diameter; compact morphology
Large: more than ¼ of well’s diameter; diffused morphology, totally or in periphery


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: cloning efficiency, relative total growth (RTG),
- Any supplementary information relevant to cytotoxicity:

Rationale for test conditions:

Test conditions were chosen based on solubility and cytotoxicity testing.

Evaluation criteria:

Acceptance criteria:
The assay was considered valid if the following criteria were met:
1. The cloning efficiencies at Day 2 in the untreated/solvent control cultures fell within the range of 65-120%.
2. The untreated/solvent control suspension growth over 2 days fell within the range: 8-32 (3 hour treatment), 32-180 (24 hour treatment).
3. The mutant frequencies in the untreated/solvent control cultures fell within the range of 50−170×1E−6 viable cells.

Every assay was also evaluated as to whether the positive control met at least one of the following two acceptance criteria:
1. The positive control induced a clear increase above the spontaneous background
(induced mutent frequency = IMF) of at least 300×1E−6. At least 40% of the IMF was reflected in the small colony MF.
2. The positive control induced a clear increase in the small colony IMF of at least 150×1E−6.

Criteria for outcome of assay:
For a test item to be considered mutagenic in this assay, it is required that:
1. The induced mutant frequency (IMF) is higher than the global evaluation factor (GEF) suggested for the microwell method (126×1E−6) at one or more doses.
2. There is a significant dose-relationship as indicated by the linear trend analysis.

Results which only partially satisfy the above criteria will be dealt with on a case-by-case basis.
Similarly, positive responses seen only at high levels of cytotoxicity will require careful interpretation when assessing their biological significance.
Any increase in mutant frequency should lie outside the historical control range to have biological relevance.

Statistics:

Statistical analysis was performed according to UKEMS guidelines (RobinsonW.D., 1990)
Test for consistency between plates
Results of individual plates within a replicate treatment were checked for consistency by calculation of the term χ2
Heterogeneity factors for replicate cultures
Test for overall consistency
Updated heterogeneity factors
Comparison of each treatment with the control
Test for linear trend

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
The addition of the test item solution did not have any obvious effect on the osmolality or pH of the treatment medium.
- Precipitation and time of the determination: In the absence of S9 metabolic activation, slight precipitation was noted at the two highest concentrations tested, upon addition of the test item and by the end of the 3 hour treatment.


RANGE-FINDING/SCREENING STUDIES (if applicable):
Both in the absence and presence of S9 metabolic activation, the test item was assayed at a maximum dose level of 138 µg/mL and at a wide range of lower dose levels: 69.0, 34.5, 17.3, 8.63, 4.31, 2.16, 1.08 and 0.539 µg/mL.
In the absence of S9 metabolic activation, using the 3 hour treatment time, marked toxicity, reducing relative survival between 10 and 20% over the concurrent negative control value, was observed at the two highest dose levels.
Mild or no relevant toxicity was noted over the remaining concentrations tested.
Using the 24 hour treatment time, no cells survived treatment at the highest concentration tested (138 µg/mL), marked toxicity was noted at the next lower concentration (RS=19%), while no relevant toxicity was observed over the remaining dose levels tested.
Following treatment in the presence of S9 metabolic activation, using the short treatment time (3 hours), no toxic effect was seen at any concentration tested.

Applicant's summary and conclusion

Conclusions:

Octadecanoic Acid, reaction products with triethylenetetramine does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Executive summary:

The test item Octadecanoic Acid, reaction products with triethylenetetramine was examined for mutagenic activity by assaying for the induction of 5 trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells after in vitro treatment, in the absence and presence of S9 metabolic activation, using a fluctuation method.

 

A preliminary solubility trial indicated that the maximum practicable concentration of the test item in the final treatment medium was 138 µg/mL using complete culture medium as solvent.

On the basis of this result, a cytotoxicity assay was performed. Both in the absence and presence of S9 metabolic activation, the test item was assayed at a maximum dose level of 138 µg/mL and at a wide range of lower concentrations: 69.0, 34.5, 17.3, 8.63, 4.31, 2.16, 1.08 and 0.539 µg/mL.

 

In the absence of S9 metabolic activation, using the 3 hour treatment time, at the two highest dose levels of 138 and 69.0 µg/mL, marked toxicity was observed, reducing relative survival (RS) to 11% and 18% of the concurrent negative control value, respectively. Mild or no relevant toxicity was noted over the remaining concentrations tested. Using the 24 hour treatment time, no cells survived treatment at the highest dose level. Treatment at the next lower concentration (69.0 µg/mL) yielded marked toxicity (RS=19%), while no relevant toxicity was observed over the remaining dose levels tested.

Following treatment in the presence of S9 metabolic activation, using the short treatment time (3 hours), no toxic effect was seen at any concentration tested.

MAIN ASSAYS 

Based on the results obtained in the preliminary cytotoxicity assay, two independent assays for mutation at the TK locus were performed using the following dose levels:

 

Experiment 1: without S9, 3 hours: 69.0, 34.5, 17.3, 8.63 and 4.31 µg/L

Experiment 1: with S9, 3 hours: 138, 69.0, 34.5, 17.3 and 8.63 µg/L

Experiment 2: without S9, 3 hours: 69.0, 46.0, 30.7, 20.5 and 13.6 µg/L

 

In the absence of S9 metabolic activation, adequate levels of cytotoxicity, covering a range from the maximum to slight or no toxicity, were observed in both treatment series, while no relevant toxicity was seen in the presence of S9 metabolism at any concentration tested up to the maximum feasible dose level.

No relevant increase in mutant frequencies was observed following treatment with the test item, in the absence or presence of S9 metabolism.

Negative and positive control treatments were included in each mutation experiment in the absence and presence of S9 metabolism. The mutant frequencies in the solvent/vehicle control cultures fell within the normal range. Marked increases were obtained with the positive control treatments indicating the correct functioning of the assay system.

 

It is concluded that Octadecanoic Acid, reaction products with triethylenetetramine does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.