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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:
experimental study
Adequacy of study:
key study
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-study equivalent to OECD guideline 471.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
equivalent or similar to guideline
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
This study was performed according to the protocol entitled "Standard Procedures for the Salmonella/Mlcrosome Mutagenicity Assay The assay was performed according to BRRC Standard Operating Procedures.
GLP compliance:
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Valeric acid
EC Number:
EC Name:
Valeric acid
Cas Number:
Molecular formula:
pentanoic acid
Details on test material:
- Name of test material (as cited in study report): n-Valeric Acid
- Lot/batch No.: 47-167
- Storage condition of test material: Room Temperature


Target gene:
S. typhimurium histidine synthethase
Species / strain
Species / strain / cell type:
other: S. Typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 1538
Details on mammalian cell type (if applicable):
- Properly maintained: yes
Indicator organisma are stored at -80ºC. Working cultures are prepared monthly by inoculating nutrient broth from the frozen cultures and incubating with agitation overnight. Bacteria are then plated onto Vogel-Bonner Medium E agar plates (master plates) with an excess of histidine and biotin (required because of the lipopolysaccharide deficiency). After incubation for 24 hours, the strains are checked for their genetic markers to verify their identity and purity.

For testing, the broth cultures are prepared by inoculating from the master plates into nutrient broth and incubated overnight with agitation. The broth cultures are then kept on ice during the day of testing. Fresh cultures are made each day of testing.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 liver homogenate, prepared from Aroclor 1254-induced, Sprague-Dawley male rats, was purchased from Microbiological Associates, Bethesda, MD. For tests with metabolic activation, 0.5 ml of S9 mix containing 50 µl of S9 was added per plate.
Test concentrations with justification for top dose:
- Preliminary toxicity screen at 94, 30, 10, 3, 1, 0.3, 0.1, 0.03, 0.01, and 0.003 milligrams per plate with strain TA100 only.
- Definitive test: 0, 10, 3, 1 and 0.3 milligrams per plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Absolute Ethanol
Untreated negative controls:
Negative solvent / vehicle controls:
Absolute Ethanol; BRRC #47-160
True negative controls:
Positive controls:
Positive control substance:
other: 4-nitro-o-phenylenediamine; BRRC #44-71 for TA98 and TA1538; CAS #99-56-9; 9-aminoacridine for TA1537; BRRC #44-233; CAS #99-45-9; sodium azide for TA 100 and TA1535; BRRC #44-72; CAS #26628-22-8; 2-aminoanthracene; BRRC #44-67; CAS #613-13-8 for all stra
Details on test system and experimental conditions:
After a suitable period of incubation (48-72 hrs), direct revertant colony counts are made and evaluated.

METHOD OF APPLICATION: in agar (plate incorporation)

Sample Preparation:To a sterile tube containing 2 ml of top agar, 100 µl aliquot of the appropriate bacterial culture is added followed by the addition of 100 µl of the appropriate solvent control or test concentration. Either 0.5 m l of S9 mix o r 0.5 m l of phosphate-buffered saline (PBS) is added for tests with and without metabolic activation, respectively. The top agar mixture is then poured onto a VB-E p l a t e. Each dose is tested in triplicate and with all five bacterial strains. Sterility checks are done on the PBS and/or S9 mix, all solvents, and the highest concentration of each test chemical. The plates are transferred to a darkened 37ºC incubator after hardening and incubated f o r 48-72 hours.

- Method: relative total growth; Confluence was considered an indication of nontoxicity, sparse growth of moderate toxicity, and lack of growth of

Metabolic Activation
-S9 mix is prepared fresh each day of testing and kept at 0-4ºC.

Evaluation criteria:
The spontaneous reversion for the solvent controls should be within this laboratory's historical range. The positive controls should demonstrate thatthe test systems are responsive with known mutagens. A test chemical is considered to be a bacterial mutagen if the number of revertant colonies is at least twice the solvent control for at least one dose level and there is evidence of a dose-related increase in the number of revertant colonies. If a
test chemical produces a marginal or weak response that cannot be reproduced i n a second test, the test result will be considered negative. If there is no evidence of a dose-related increase in the number of revertant colonies and the number of revertant colonies is not twice the solvent control, then the test chemical is not considered to be a bacterial mutagen.
no data

Results and discussion

Test results
Species / strain:
other: S. Typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 1538
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
Vehicle controls validity:
Untreated negative controls validity:
Positive controls validity:
Additional information on results:
No evidence of mutagenicity was observed at any of the tested doses, either by evidence of a dose-response relationship or a doubling of the number of colonies over the solvent control.

RANGE-FINDING/SCREENING STUDIES: n-Valeric Acid was tested in a preliminary toxicity screen at 94, 30, 10, 3, 1, 0.3, 0.1, 0.03, 0.01, and 0.003 milligrams per plate with strain TA100 only. Because 100 µl of the solvent ethanol was toxic in a preliminary test, the test chemical was diluted so that 50 µl of the dilution would deliver the required dose. Phosphate-buffered saline was used to bring the total volume added per tube to 100 p1. The top two doses of 94 and 30 mg per plate inhibited all growth of the background lawn. Based on these results, mutagenicity testing was done with 5 doses of 30, 10, 3, 1 and 0.3 milligrams per plate in triplicate. The highest dose was expected to produce some degree of cytotoxicity based on preliminary toxicity test results, observed as either a reduction in the number of revertant colonies or an inhibition of growth of the background lawn. Gravimetric analysis of the highest test substance dilutions for each test indicated no more than a 1.1% error from the stated concentration. Subsequent dilutions had no more than a 7.4% error from the stated concentrations.


Remarks on result:
other: all strains/cell types tested
Migrated from field 'Test system'.

Any other information on results incl. tables

Dose selection appeared to be in a suitable range in the mutagenicity tests because some toxicity was evident in both tests. In the test without metabolic activation, toxicity was observed at 30 and 10 mg/plate with all strains. In the test with metabolic activation, toxicity was observed at 30 mg/plate with all strains.

All strains exhibited a positive mutagenic response with the positive controls tested both with and without S9 metabolic activation. Negative (solvent) controls were also tested with each strain, and the spontaneous reversion rates were within the historical ranges at this laboratory. All positive and negative controls were run concurrently with the test chemical. Concurrently run sterility checks showed that the S9 mix, PBS, the test chemical and all solvents and controls were sterile.

Applicant's summary and conclusion

Interpretation of results (migrated information):

n-Valeric Acid did not produce a dose-dependent mutagenic response in any of the Salmonella typhimurium strains. Under the conditions of this
assay, n-Valeric Acid was not mutagenic in the Salmonella/microsome mutagenicity assay.
Executive summary:

n-Valeric Acid was tested for potential mutagenic activity using the Salmonella/microsome bacterial mutagenicity assay (Ames test). n-Valeric Acid was tested in triplicate at five concentrations, ranging from 0.3 milligrams to 30 milligrams per plate. The highest concentration was cytotoxic in the mutagenicity test and in a preliminary test to choose appropriate doses. Mutagenic activity was not observed with any of the five bacterial strains tested with or without metabolic activation. n-Valeri Acid was not mutagenic in this screening test.