Amides, C18-unsatd., N,N-bis(hydroxyethyl)

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:

Not available

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1999

Materials and methods

Principles of method if other than guideline:

The genotoxic potential of the test substance was determined by the induction of gene mutations in L5178Y cells in a mouse lymphoma mutagenicity test both with and without metabolic activation as per the Myhr et al., (1985) protocol. Cells deficient in thymidine kinase (TK) due to the mutation of TK+/- to TK-/- are resistant to the cytotoxic effects of trifluorothymidine (TFT). Thymidine kinase proficient cells (TK+/-) are sensitive to TFT, which causes the inhibition of cellular metabolism and halts further cell division. Thus mutant cells are able to proliferate in the presence of TFT, whereas normal cells, which contain thymidine kinase, are not able to proliferate.

GLP compliance:

no

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(9Z)-N,N-bis(2-hydroxyethyl)octadec-9-enamide

EC Number:

700-972-2

Molecular formula:

C22H43NO3

IUPAC Name:

(9Z)-N,N-bis(2-hydroxyethyl)octadec-9-enamide

Test material form:

liquid

Method

Target gene:

No data

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Supplemented Fischer’s medium (growth media) maintained at 37°C; normal cycling time was approximately 10 hours. For cloning the horse serum content was increased and noble agar was added.
- Properly maintained: Yes
- Periodically "cleansed" against high spontaneous background: Yes, by exposing to selective media containing thymidine, hypoxanthine, methotrexate, and glycine for 1 day; to medium containing thymidine, hypoxanthine, and glycine for 1 day; and to normal medium for 3 to 5 days

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 from the livers of Aroclor 1254-induced male Fischer 344 rats

Test concentrations with justification for top dose:

Without metabolic activation: Trial 1: 0, 1.25, 2.5, 5 and 7.5 nL/ mL; Trial 2: 0, 2, 3, 4, 6, 8 and 12 nL/ mL; Trial 3: 0, 3, 4, 6, 8, 12, 15 and 20 nL/ mL
With metabolic activation: Trial 1: 0, 2.5, 5, 7.5, 10 and 15 nL/ mL; Trial 2: 0, 2.5, 5, 7.5, 10, 15 and 20 nL/ mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

(solvent control)

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

-S9 : at 5 μg/mL concentration

Controls 2

Untreated negative controls:

yes

Remarks:

(solvent control)

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

+S9: at 2.5 μg/mL concentration

Details on test system and experimental conditions:

The experimental protocol is presented in detail by Myhr et al., 1985 

METHOD OF APPLICATION: in medium

DURATION
Exposure duration: 4 h
- Expression time (cells in growth medium): 48 h
- Selection time (if incubation with a selection agent): 10 to 12 d (at 37°C in 5% CO2)

SELECTION AGENT (mutation assays): Yes, Trifluorothymidine (TFT)
NUMBER OF REPLICATIONS: Triplicate (all treatment levels within an experiment, including concurrent positive and solvent controls, were replicated)
NUMBER OF CELLS EVALUATED: 6 × 10(6) cells in 10 mL medium

DETERMINATION OF CYTOTOXICITY
- Method: Cloning efficiency

Evaluation criteria:

Minimum criteria for accepting an experiment as valid and a detailed description of the statistical analysis and data evaluation are as per Caspary et al., (1988). Data was evaluated statistically for trend and peak responses.

Positive response: When there are significant differences for two responses i.e., capable of inducing TFT resistance.
Equivocal response: When a single significant response is observed.
Negative response: When there is absence of both a trend and a peak response.

Statistics:

All data was evaluated statistically for trend and peak responses as per Caspary et al., (1988).

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

Remarks:

Without metabolic activation: The highest doses of 7.5 nL/mL in trial 1 and ≥ 12 nL/mL in trial 3; With metabolic activation: 15 and 20 nL/mL in trial 1 and 2 respectively

Additional information on results:

No induction of TFT resistance was noted in L5178Y mouse lymphoma cells treated with ODEA in the presence or absence of S9 metabolic activation.

Any other information on results incl. tables

For detailed results table kindly refer to the attached background materials section of the IUCLID.

Applicant's summary and conclusion

Conclusions:

Under the study conditions, no increase in the frequency of mutant colonies of L5178Y mouse lymphoma cells was noted after exposure to test substance, with or without metabolic activation.

Executive summary:

A study was conducted to evaluate the in vitro genetic toxicity of the test substance, C18-unsatd. DEA, in a mouse lymphoma assay. The cells were exposed for 4 h to concentrations of 1.25, 2.5, 5 and 7.5 nL/ mL without metabolic activation and 2.5, 5, 7.5, 10 and 15 nL/ mL with metabolic activation in Trial 1. Cells were treated with test substance for 4 h at 52, 3, 4, 6, 8 and 12 nL/ mL without metabolic activation and 2.5, 5, 7.5, 10, 15 and 20 nL/ mL with metabolic activation in Trial 2. Cells were treated with test substance for 4 h at 3, 4, 6, 8, 12, 15 and 20 nL/ mL without metabolic activation in Trial 3. 6 X106 cells in triplicate cultures medium were exposed to the test substance (either in the presence or absence of metabolic activation), positive control and solvent control for 4 h. After a 48 h expression period, cells were plated for selection of TFT-resistant cells and cloning efficiency. The plates were scored after an incubation period of 10 to 12 d at 37±1°C in 5% CO2. Under the conditions of the study, no increase in the frequency of mutant colonies of L5178Y mouse lymphoma cells was noted after exposure to test substance, with or without S9 (NTP, 1999).