Isooctadecanoic acid, reaction products with tetraethylenepentamine

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:

16 Sep - 19 Nov, 1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study following GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1986

Report date:

1986

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of fatty acids, C14-C18 (branched and linear) and C18 (unsaturated) with tetraethylenepentamine (linear, branched, cyclic)

EC Number:

701-204-9

Cas Number:

68784-17-8

IUPAC Name:

Reaction products of fatty acids, C14-C18 (branched and linear) and C18 (unsaturated) with tetraethylenepentamine (linear, branched, cyclic)

Test material form:

liquid: viscous

Method

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Strain: 3.7.2C
Cells were cultured in Fischer's medium in a shaker incubator at 37°C in humidified 5% CO2 in air. Cultures were diluted daily to a cell density of approximately 3 x 10ˆ5 cells per ml. Each time a culture was used, it was checked for bacterial or fungal contamination.
Prior to use in the assay, L5178Y cells were treated with methotrexate to reduce the frequency of spontaneously occurring TK-/- cells.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor S-9 metabolic activation

Test concentrations with justification for top dose:

0, 3, 10, 33, 100, 333 µg/mL with S-9
0, 1, 3, 7, 10, 33 µg/mL without S-9

Vehicle / solvent:

Acetone

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Details on test system and experimental conditions:

- Test Material Exposure: Based on the data derived from the toxicity test, the test material was prepared so that the highest concentration would yield a percent total growth of approximately 10%. The test material was solubilized and diluted to produce evenly spaced dose levels which would yield approximately 90% total growth at the lowest dose. The test material was added to cells, both with and without metabolic activation and incubated for approximately 4 hours. Cells were then washed and placed into suspension culture at a density of 0.3 x 10ˆ6 cells/mL.

- Expression Time: In order for induced mutations to be expressed, the cells must undergo several divisions. This period is designated as the expression time. After the initial exposure to the test material the cells were incubated for 2 days and adjusted to 0.3 x 10ˆ6 cells/mL at 24 hours.

- Cloning: At the end of the expression period, a portion of the cells were plated in
(a) restrictive medium containing 3 ug/ml trifluorothymidine (TFT) which allowed only the TK-/- cells to grow, and
(b) non-restrictive medium which indicated cell viability.
The plates were incubated at 37°C + 1 C in humidified 5% CO2 in air for 10 days.

- Accumulation of Data: After the incubation period, both the mutagenicity (TFT restrictive medium) plates and the viability (non-restrictive medium) plates were scored for the total number of colonies per plate using an Artek 880 colony counter or by hand. The frequency of mutants induced by each test material dose was determined by comparing the average number of colonies in the mutagenicity plates to the average number of colonies in the corresponding viability plates. Induced mutagenic activity, if any, was quantified by comparing the mutant frequency of the treated plates to that of the control plates.

DETERMINATION OF CYTOTOXICITY
The test material was checked for toxicity with and without S-9 metabolic activation at dose levels of 1 ug/ml to 5000 µg/ml. No growth of the cell population was observed at >500 ug/ml with S-9 and > 100 ug/ml without S-9. Less severe toxicity was observed at 100 (+S-9) and 10 ug/ml (-S-9). Compound precipitate was present at 5000 ug/ml.

Evaluation criteria:

Criteria for an Acceptable Mouse Lymphoma Screen: All the following criteria should be met for the mutagenicity screen to be considered valid:
- The mutant frequency of the appropriate positive control (either with or without S-9 activation) should be at least twice that of the appropriate negative (solvent) control.
- The spontaneous mutant frequency of the negative (solvents control should be in the range of 20 to 200 per 10ˆ6 cells.
- The plating efficiency of the negative (solvent) control should be at or above 50%.
- The test material should be tested to the level of approximately 10% total growth, or the limits of solubility, or to a high dose of 10 mg/ml. Test materials may be tested as slurries.

The following criteria must be met for the outcome of the Mouse Lymphoma Screen (either with or without S-9 metabolic activation) to be considered mutagenic:
The mutant frequency of one or more test material concentrations, with >10% total growth, is at least two times greater than that of the negative (solvent) control.

The following result must be met for the outcome of the Mouse Lymphoma Screen (either the with or without metabolic activation) to be considered non-mutagenic:
None of the mutant frequencies of any of the test material concentrations, with a survival of >=10% total growth, are two times greater than that of the negative (solvent) control.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: No growth of the cell population was observed at >= 500 ug/ml with S-9, and > =100 ug/ml without S-9. Less severe toxicity was observed at 100 ug/ml (+S-9) and 10 ug/ml (-S-9).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

None of the cultures exhibited a mutant frequency which was two times that of the average mutant frequency of the negative (solvent) controls. Percent total growth ranged from 33% to 96% (with metabolic activation) and 90% to 97% (without activation). The positive contro (DMBA) responded satisfactorily.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Under the conditions tested, this material was considered to be nonmutagenic in this screen.

Executive summary:

The test material was evaluated for gene mutation in the L5178Y TK+/-mutagenicity screen with and without S-9 metabolic activation at concentrations ranging from 1 ug/ml to 333 ug/ml. None of the cultures exhibited a mutant frequency which was two times that of the average mutant frequency of the negative (solvent) controls. Under the conditions tested, this material was considered to be nonmutagenic.