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Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

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Administrative data

in vitro gene mutation study in bacteria
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
All studies have been performed in compliance with the standard procedures of the US National Toxicology Program. However, no full reports are available form the NTP database and verly limited or no data is available about cytotoxicity by which the interpretation of the results on an individual basis is very difficult.

Data source

Referenceopen allclose all

Reference Type:
Study ID 278018
National Toxicology Program
Bibliographic source:
National Toxicology Program (
Reference Type:
other: robust study summary

Materials and methods

Test guideline
equivalent or similar to guideline
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
not specified
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Sodium naphthenate
Sodium naphthenate
Constituent 2
Reference substance name:
Naphthenic acids, sodium salts
EC Number:
EC Name:
Naphthenic acids, sodium salts
Cas Number:
sodium 3-(3-ethylcyclopentyl)propanoate
Test material form:
not specified
Details on test material:
- Name of test material (as cited in study report): Sodium naphthenate
- Molecular formula (if other than submission substance): C11-H7-Na-O2
- Molecular weight (if other than submission substance): 194.16515
- Structural formula attached as image file (if other than submission substance): see Fig.


Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
induced male Syrian hamster liver S9 and induced male Sprague Dawley rat liver S9
Test concentrations with justification for top dose:
1 - 3333 ug/L depending upon strain
Vehicle / solvent:
100% Ethanol
Details on test system and experimental conditions:
Info from NTP website:

Several strains of the S. typhimurium bacterium may be used for testing. Each is genetically different, so using several strains in a test increases the opportunity of detecting a mutagenic chemical. The most frequently used strains are TA97, TA98, TA100, TA102, TA104, TA1535, TA1537, and TA1538. In addition to the Salmonella tester strains, the NTP has recently begun to routinely employ Escherichia coli strain WP2 uvrA pKM101 as a bacterial tester strain in the Ames test. This E. coli strain is similar in mutagen detection to S. typhimurium strain TA102. All the bacterial strains used in the Ames test carry a defective (mutant) gene that prevents them from synthesizing the essential amino acid histidine from the ingredients in standard bacterial culture medium. Therefore, these "tester" strains can only survive and grow on medium that contains excess histidine. However, in the presence of a mutagenic chemical, the defective histidine gene may be mutated back to the functional state, allowing the bacterium to grow on standard medium that does not contain supplemental histidine. These mutations, which lead to a regaining of normal activity or function, are called "back" or "reverse" mutations and the process is referred to as "reversion." The mutant colonies, which can make histidine, are called "revertants." (There are other mutagenicity assays using other cell-types that measure "forward" mutations, that is, mutations that alter a functional gene in a way that causes a loss, rather than a gain, of function.)

In the standard protocol (preincubation) for conducting the Ames assay, a test tube containing a suspension of one strain of Salmonella typhimurium (or E. coli) plus S9 mix or plain buffer without S9, is incubated for 20 minutes at 37º C with the test chemical. Control cultures, with all the same ingredients except the test chemical, are also incubated. In addition, positive control cultures are prepared; these contain the particular bacterial tester strain under investigation, the various culture ingredients, and a known potent mutagen*. After 20 minutes, agar is added to the cultures and the contents of the tubes are thoroughly mixed and poured onto the surface of Petri dishes containing standard bacterial culture medium. The plates are incubated, and bacterial colonies that do not require an excess of supplemental histidine appear and grow. These colonies are comprised of bacteria that have undergone reverse mutation to restore function of the histidine-manufacturing gene. The number of colonies is usually counted after 2 days.

Several modifications of the Ames test protocol have been used over the years in special circumstances. These include standard plate incorporation (no preincubation step prior to plating onto Petri dishes), FMN reduction (use of flavin mononucleotide for reduction of test articles such as azo dyes), plate test with volatile liquids (exposure of bacteria in a sealed Petri dish), cecal reduction (use of rat cecal bacteria to provide reduction of azo compounds), and plate tests conducted within a sealed dessicator (gas chamber) for exposure to gaseous substances. The specific test protocol that was used in an Ames test is noted in the description of the assay data.

Spontaneous mutations (those that occur by chance, not by chemical treatment) will appear as colonies on the control petri dishes. If the test chemical was mutagenic to any particular strain of bacterium, the number of histidine-independent colonies arising on those plates will be significantly greater than the corresponding control plates for that strain of bacteria. The positive control plates are also counted, and the number of mutant colonies appearing on them must be significantly increased over the spontaneous control number for the test to be considered valid. Failure of the positive control chemical to induce mutation is reason to discard the experiment.

Several doses (usually at least 5) of each test chemical and multiple strains of bacteria are used in each experiment. In addition, cultures are set up with and without added liver S9 enzymes at varying concentrations. Therefore, a variety of culture conditions are employed to maximize the opportunity to detect a mutagenic chemical. In analyzing the data, the pattern and the strength of the mutant response are taken into account in determining the mutagenicity of a chemical. All observed responses are verified in repeat tests. If no increase in mutant colonies is seen after testing several strains under several different culture conditions, the test chemical is considered to be nonmutagenic in the Ames test.

*Positive control chemicals used in NTP Ames tests:

For strains tested in the absence of S9
TA98, 2-nitrofluorene or alternatively, TA98 and TA1538, 4-nitro-o-phenylenediamine
TA100 and TA1535, sodium azide
TA97 and TA1537, 9-aminoacridine
TA102, mitomycin C
TA104, methyl methanesulfonate
E.coli WP2 uvrA pKM101, methyl methanesulfonate

For strains tested with S9
All strains, 2-aminoanthracene (or occasionally, sterigmatocystin)

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:
no cytotoxicity
Vehicle controls validity:
Untreated negative controls validity:
not specified
Positive controls validity:

Any other information on results incl. tables

See original document with results

Applicant's summary and conclusion

Sodium Naphthenate is non-mutagenic in the Salmonella bacterial Mutagenicity test system.
Executive summary:

Sodium Naphthenate was non-mutagenic in the Salmonella bacterial Mutagenicity test system strains TA100, TA1535, TA97, TA98 when tested up to >333 µg/L with and without metabolic activation.