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Toxicological information

Genetic toxicity: in vitro

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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:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report Date:
1994

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name of test material (as cited in study report): Dipropylene Glycol Monoethyl Ether
- Substance type: Glycol ether
- Physical state: colourless liquid
- Analytical purity: 90.15%
- Impurities (identity and concentrations): Sec. ethoxy propanol (0.35%); Primary ethoxy propanol (0.06%); heavier compounds (9.39%) water (0.02%).
- Composition of test material, percentage of components:
- Isomers composition: NA
- Purity test date: 3 December 1992
- Lot/batch No.: Not specified
- Expiration date of the lot/batch: Not specified
- Stability under test conditions: Not specified
- Storage condition of test material: Room temperature in opaque plastic container/brown glass bottle
- Other:

Method

Target gene:
Thymidine kinase TK +/- locus. L5178Y Mouse Lymphoma
Species / strain
Species / strain:
mouse lymphoma L5178Y cells
Details on mammalian cell lines (if applicable):
CELLS USED
- Source of cells: MRC Cell Mutation unit, University of Surrey, UK

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:RPMI 1640 medium supplemented with 10% donor horse serum and 20mM Hepes buffer with 5% CO2 in air.
- Properly maintained: yes
- Periodically 'cleansed' against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9
Test concentrations with justification for top dose:
0, 312.5, 625, 1250, 2500, 5000 microgram/ml
Vehicle:
RPMI 1640 culture medium
Controls
Negative controls:
yes
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and conditions:
METHOD OF APPLICATION: in suspension

L5178Y TK+/- 3.7.2c mouse lymphoma cell line (MRC Cell Mutation Unit) were cultured in RPMI 1640 medium with 10% horse serum and buffer (R10) at 37 Celsius with 5% CO2.

Cells were cleansed of homozygotes by culture with thymidine etc prior to culture in THG medium before returning to R10 media.

DPGEE was weighed and dissolved in RPMI 1640 medium and appropriate dilutions were made.
Microsomal enzyme fractions (S9) were obtained from BIBRA. 10% S9 mix was prepared in R10 containing NADP (5 mM and G6P (5 mM).

Preliminary cytotoxicity was evaluated in R5 medium using 3 hr exposure.

Mutagenicity was evaluated in 5-10 x 10^5 cells/ml with duplicate replicates with and without S9 at 5 doses of DPGEE and negative and positive controls.

Doses were 0, 312.5, 625, 1250, 2500, 5000 microgram/ml.

treatment vessels were incubated at 37 Celsius for 3 hours with manual shaking at 1/2 hr intervals

On day 2 the cells were counted, diluted to 10^4 cells/ml and plated for mutant frequency in selective medium containing 4 ug/ml trifluorothymidine in microtitre plates. Cells were also diluted to 10 cells/ml and plated for viability in non-selective medium.
Evaluation criteria:
Plates were scored after 10-14 days incubation and plating efficiency (viability was computed.
Mutant frequencies were calculated per viable cell plated and the induced mutant frequency calculated by subtracting the negative control frequency.
Statistics:
no data

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Remarks:
slight cytotoxicity seen with S9
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: strain/cell type: heterozygous Thymidine kinase locus
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

DPGMEE at 312,5, 625, 1250, 2500, 5000 microg/ml was not mutagenic in this test system
Executive summary:

In a well-run guideline study to GLP DPGEE did not increase the mutant frequency at the TK +/- (heterozygous) locus in mouse L5178Y cells and is therefore considered to be non-mutagenic.