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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

From these results, it is concluded that 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 or to induce gene mutations in the bacterial reverse mutation assay (the Ames test) or the mammalian cell gene mutation assay (in the presence or absence of metabolic activation) or in the mammalian cell chromosome aberration study.

Link to relevant study records
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
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
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:
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)
Untreated negative controls:
Negative solvent / vehicle controls:
True negative controls:
Positive controls:
Positive control substance:
Details on test system and experimental conditions:

- 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

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

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.

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.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The other study available is the Ames study which uses prokaryotic cells. The CHO study uses eukaryotic mammalian cells which are more applicable.

Justification for classification or non-classification

Neither the Ames nor the mammalian cell gene mutation assay or the mammalian cell chromosome aberration study

returned a positive result (with or without activation). Therefore this substance will not be classified as genetically toxic.