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Genetic toxicity: in vitro

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in vitro gene mutation study in bacteria
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Basic data given; comparable to guideline study (deviation: only 4 strains of S. thyphimurium tested)

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
: only 4 strains of S. thyphimurium tested
GLP compliance:
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Isobutyric acid
EC Number:
EC Name:
Isobutyric acid
Cas Number:
Molecular formula:
2-methylpropanoic acid
Details on test material:
- Name of test material (as cited in study report):Propionic acid, 2-methyl (Isobuttersaeure)
- Analytical purity: 99.33 %


Target gene:
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix prepared from Dawlay rat livers after Aroclor 1254 activation
Test concentrations with justification for top dose:
20, 100, 500, 2500, 5000 ug/plate (Standard plate test)
20, 100, 500, 2500, 5000 ug/plate (Preincubation test)
4, 4, 20, 100, 500, 1000 µg/plate (Preincubation test) TA 1537/TA 98 without S9-mix
Untreated negative controls:
Negative solvent / vehicle controls:
Positive controls:
Positive control substance:
other: see details
Details on test system and experimental conditions:
Standard plate test
Test tubes containing 2 ml portions of saft agar kept in a water bath at 45'C, and the remaining companents are added in the following order:
0.1 ml test solution or vehicle
0.1 ml bacterial suspension
0.5 ml S-9 mix (in tests with metabolic activatian) or
0.5 ml phosphate buffer (in tests without metabalic activatian)
After mixing, the samples are poured onto Vogel-Bonner agar plates. After incubation at 37'C for 48 -72 haurs in the dark, the bacterial calonies (his+ revertants) are counted.

Preincubation test
0.1 ml test solution or vehicle, 0.1 ml bacterial Suspension and 0.5 ml S-9 mix are incubated at 37°C for the duration of 20 minutes. Subsequently, 2 ml of soft agar is added and, after mixing, the samples are poured onto the agar plates within approx.30 seconds.

Both Tests
In each experiment 3 Test plates per dose per control used. After incubation at 37°C for 48 -72 hours in the dark, the bacterial colonies (his+ revertants) are counted.

Posivite Control:
with S-9 mix: 10 µg 2-aminoanthracene (2-AA)(dissolved in DMSO) for the strains TA 100, TA 98, TA 1537and TA 1535

without S-9 mix: 5 µg N-methyl-N'-nitro-N-nitroso-guanidine (MNNG) (dissolved in DMSO) for the strains TA 100 and TA 1535
10 µg 4-nitro-o-phenylendiamine (NOPD) (dissolved in DMSO) for the strain TA 98
100 µg 9-aminoacridine (AAC) chloride monohydrate (dissolved in DMSO) for the strain TA 1537

The Titer was determined and in regularly measurements the strain characteristics were checked. Sterility control performed.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
Vehicle controls validity:
Untreated negative controls validity:
Positive controls validity:
Remarks on result:
other: other: Salmonella typhimurium TA1535, TA100, TA1537, TA98
Migrated from field 'Test system'.

Any other information on results incl. tables

Solubility: Complete solubility of the test substance in aqua. dest.

Toxicity: A bacteriotoxic effect was abserved only without S-9 mix in the PIT at doses >= 1000 µg/plate (TA 98) or at >= 2500 µg/plate (TA 100 and TA 1537)

Mutagenicity: An increase in the number of his+ revertants was not observed both in the standard plate test and in the preincubation test either without S-9 mix or after the addition of a metabolizing system.

The slightly enhanced values due to some single increased colony numbers observed with TA 1537 and TA 98 in the preincubation test did not show any dose dependency and were not confirmed in a 2nd preincubation assay selecting closer doses. The findings are therefore regarded as incidental.

Applicant's summary and conclusion

Interpretation of results (migrated information):

According to the results of the present study, the test substance isobutyric acid is not mutagenic in the Ames Test under the experimental conditions chosen here.
Executive summary:

In a reverse gene mutation assay in bacteria, strains TA98, TA100, TA 1535, and TA1537 of Salmonella typhimurium were exposed to isobutyric acid (purity 99.33%) at concentrations of 0 (controls), 20, 100, 500, 2500, and 5000 µg/plate in the presence and absence of mammalian metabolic activation. Tests were performed as plate incorporation and as preincubation assays.


Isobutyric acid was tested up to limit concentration (5000 µg/plate). The positive controls induced the appropriate responses. There was no evidence of induced mutant colonies over background.


Isobutyric acid was demonstrated to be not mutagenic to Salmonella typhimurium under the conditions of this test (BASF, 1989).


This study is classified as acceptable. It satisfies the requirement of Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data with minor restrictions (only 4 strains tested).