Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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
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:
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

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): Oleic acid diethanolamine condensate
- Source of the test material: Obtained from Henkel Corporation, Emery Group (Cincinnati, OH)
- Molecular weight: 387.68
- Physical state: Clear liquid
- Analytical purity: Purity of 47.5% was obtained when analyzed by HPLC
- Impurities (identity and concentrations): Analyzed by HPLC/mass spectrometry. Impurities identified were fatty acid alkanolamides (approximately 30%), other fatty acids or unidentified organic impurities, one polar nitrosamines (i.e., nitrosodiethanolamine was detected at a concentration of 68 ppb), free diethanolamine at 0.19%; no non-polar nitrosamine were found.
- Lot/batch No.: 1H01722285 (for purity determination by HPLC method) and DA-021 (for stability studies by gas chromatography)
- Stability under test conditions: Stable when stored in glass vials for 2 wk at 25 °C but unstable at 60 °C.
- Storage condition of test material: At room temperature, protected from ultraviolet light, in amber glass bottles with Teflon®-lined lids
- Other: Solubility: soluble in alcohols, glycols, ketones, chlorinated solvents, and other aliphatic hydrocarbon solvents.

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
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
(solvent control)
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Remarks:
(without metabolic activation system)
Positive control substance:
methylmethanesulfonate
Untreated negative controls:
yes
Remarks:
(solvent control)
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Remarks:
(with metabolic activation system)
Positive control substance:
3-methylcholanthrene
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.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
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 metabolic activation.
Executive summary:

A study was performed to investigate the potential of the test substance to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y, in compliance with GLP. The assay was performed both with and without rat liver microsomal (S9) activation. The substance was tested at the following concentrations:

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

With metabolic activation: Trial 1: 2.5, 5, 7.5, 10 and 15 nL/ mL; Trial 2: 2.5, 5, 7.5, 10, 15 and 20 nL/ mL         

6 X106 cells in triplicate cultures 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 (Irwin, 1999).