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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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 November to 19 December 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016

Materials and methods

Test guideline
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

Test material

Constituent 1
Chemical structure
Reference substance name:
Isopropyl 2-methylbutyrate
EC Number:
266-411-4
EC Name:
Isopropyl 2-methylbutyrate
Cas Number:
66576-71-4
Molecular formula:
C8H16O2
IUPAC Name:
isopropyl 2-methylbutyrate
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): Isopropyl 2-methylbutyrate
- Substance type: Organic
- Physical state: Liquid
- Analytical purity: 99.9%
- Purity test date:
- Lot/batch No.: H4-K-27
- Expiration date of the lot/batch: 17 August 2018
- Stability under test conditions: Detailed data regarding stability during storage and under test conditions are not available. It is however expected that no gross degradation occurs under the specified storage conditions as specified by the sponsor, or over the duration of the study when dissolved in DMSO, the selected solvent for the study (duration of treatment ca. 4 hours).
- Storage condition of test material: Ambient condition, protected from light

Method

Target gene:
hprt
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: Growth medium, media containing selective agent, media without selective agent and HAT medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Main test concentration selection (in the absence and presence of S9): 45.125, 90.25, 180.5, 361, 722 and 1444 µg/Ml (10 Mm). Concentrations based on the cytotoxicity test performed in the absence and presence of metabolic activation at the same concentrations as above
Vehicle / solvent:
Vehicle used: DMSO
- Justification for choice of solvent/vehicle: The test item was insoluble in sterile distilled water, however it was soluble in dimethyl sulfoxide at concentrations >1444 µg/mL (10Mm), the guideline limit concentration. Volume used for treatment (250 µL per 25 mL medium)
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in suspension;

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 8 days
- Selection time (if incubation with a selection agent): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 16 days

SELECTION AGENT (mutation assays): 2-amino-6-mercaptopurine (6-thioguanine) at 5 µg/mL -alpha-MEM without nucleosides

NUMBER OF REPLICATIONS: Two replicate flasks per concentration

DETERMINATION OF CYTOTOXICITY
- Method: Relative cloning efficiency.

Evaluation criteria:
Assay Acceptance Criteria

A mutation assay was considered acceptable if:
A minimum 60% absolute cloning efficiency in negative controls (DMSO) and a spontaneous mutant frequency less than 20 per 106 clonable cells.
Positive controls induce a significant increase in the mutant frequency above the concurrent negative control.

Assay Evaluation Criteria
A test item was considered positive if:
The test item caused a concentration-related biologically significant increase in mutant frequency in comparison with concurrent negative control and the test item causes a three-fold increase in the number of 6-thioguanine resistant colonies relative to concurrent negative control and such increases were statistically significant and outside the laboratory historical negative (DMSO) control range.
A net increase in mutant colonies of treated above the concurrent control was observed in at least two of the concentrations tested.
Clear negative results were not confirmed by a repeat test (short duration), as per revised OECD guideline.
Statistics:
Weighted regression analysis was performed to evaluate the dose response relationship on isopropyl 2-methylbutyrate treatment groups against the negative control group. Statistical analysis was not performed for the positive controls.

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: strain/cell type: Chinese Hamster Ovary (CHO)-K1 Cell Line’
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

No precipitation was observed at 0 and 4 hours prior to the cytotoxicity test. No biologically relevant influence of Isopropyl 2-methylbutyrate on pH or osmolality were observed both in the absence and presence of metabolic activation during the main study.

Applicant's summary and conclusion

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

Isopropyl 2-methylbutyrate does not have potential to induce gene mutations at the hprt locus of CHO-K1 cells, both in the absence and presence of metabolic activation under the experimental conditions described.