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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
Study period:
2012-08-17 to 2012-09-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

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
Test material form:
other: liquid
Details on test material:
- Name of test material: 3-methoxy- 3-methylbutan-1-ol (MMB)
- Physical state: liquid
- Analytical purity: 99.47% (GC area %)
- Lot No.: 32726
- Expiration date of the lot: June 30, 2013

Method

Target gene:
Thymidine Kinase (TK+/-)
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: complete growth medium F10P [Dulbecco’s modified Eagle’s Medium (DMEM) with 4 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate and 4.5 g/L glucose supplied with 0.1 % Pluronic, 90 % and Fetal bovine serum, 10 %]. Treatment medium F5P [Dulbecco’s modified Eagle’s Medium (DMEM) with 4 mM Lglutamine adjusted to contain 1.5 g/L sodium bicarbonate and 4.5 g/L glucose supplied with 0.1 % Pluronic, 95 % and Fetal bovine serum, 5 %].
Metabolic activation:
with and without
Metabolic activation system:
A co-factor supplemented postmitochondrial fraction (S9) from the liver of Aroclor 1254 induced Sprague-Dawley rats
Test concentrations with justification for top dose:
0.50, 0.25, 0.125 and 0.0625 mg/ml
Controls
Untreated negative controls:
yes
Remarks:
(sterile distilled water)
Negative solvent / vehicle controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 13 days

SELECTION AGENT (mutation assays): Trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: 3 x 10E6

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
The total number of colonies per TFT plate were determined for those cultures with >= 10% total growth.

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
Cytotoxicity was observed at the highest concentration tested (0.5 mg/ml).
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: The testing concentrations of the test substances for the study were selected based on the results of preliminary range-finding test using L5178Y/TK+/- mouse lymphoma cells (non- GLP study 7191037866-02 conducted from 18 to 26 July 2012). In the range-finding test, cytotoxic effect of cell growth inhibition was observed at 0.5 mg / ml and above concentrations. Based on the results of range-finding test, four concentrations were selected, which were 0.50 mg/ml, 0.25 mg/ml, 0.125 mg/ml and 0.0625 mg/ml, respectively.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
negative with and without metabolic activation

From the above study, the test item 3-methoxy-3-methylbutan-1-ol (MMB) is non-mutagenic to the L5178Y TK+/- clone, both in the absence and presence of S9 metabolic activation system.