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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

All required in vitro tests showed negative results of genetic toxicity. According to Column 2 in Annex VIII (Section 8.4), an in vivo test is not necessary given the consistently negative results in multiple in vitro studies.

Link to relevant study records
Reference
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
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine Kinase (TK+/-)
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
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.
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'.
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.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Information are available from reliable studies for all the required in vitro endpoints. The results of all the studies were in agreement.

Where there was more than one reliable study for an endpoint the most recent study was selected as key study.

Two studies are available for bacterial reverse mutation assay according to OECD 471, one study is available for mammalian cell gene mutation assay according to OECD 476 and one study is available for in vitro mammalian chromosome aberration according to OECD 473 to determine the genetic toxicity of the substance. The results show that the substance does not induce mutations in bacterial or mammalian cells, nor chromosome aberrations in mammalian cells.

Justification for selection of genetic toxicity endpoint
Data are available from reliable studies for all the required in vitro endpoints. There are results of two bacterial reverse mutation assay, one result of a in vitro mammalian chromosome aberration test and one result of a in vitro mammalian cell gene mutation test. No mutagenic effects were observed in any of the tests. The most recent study was selected.

Justification for classification or non-classification

MMB was not mutagenic in bacteria (Salmonella typhimurium TA 100, TA 1535, TA 1537, TA 1538), in vitro in mammalian cells, or in vivo in mice. The data available indicate that MMB is not genotoxic.