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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April - May 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
Batch no 80627X065A

Method

Target gene:
Genes involved in the functional capability of the bacteria to synthesize histidine
Species / strain
Species / strain / cell type:
S. typhimurium, other: TA 1535, TA 97a, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
5000 / 1500 / 500 / 150 / 50 µg/plate for the first experiment (plate incorporation method) and 5002 / 2501 / 1251 / 626 / 313 µg/plate (for the pre incubation method)
Vehicle / solvent:
tetrahydrofurane (THF) was chosen as vehicle, because the test item was completely soluble, and addition of 5% of this solvent doesn’t have any effects on the viability of the bacteria or the number of spontaneous revertants
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
other: 2-Amino-anthracene and 4-Nitro-1,2-phenylene diamine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) for the first experiment, then preincubation method for the second one.

DURATION:
- Preincubation period: incubation over night at 37 °C before the cultures were used in the experiment
- Exposure duration: 48h

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS: four

DETERMINATION OF CYTOTOXICITY:
- Method: background lawn visible or not and the number of revertant colonies reduced or not
Evaluation criteria:
A test item is considered to have mutagenic potential, if a significant, reproducible in-crease of revertant colonies per plate (increase factor  2) in at least one strain can be observed. A concentration-related increase over the range tested can also be taken as a sign of mutagenic activity

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium, other: TA 1535, TA 97a, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
Negative with and without metabolic activation


The test item Phosphorous acid, tri-C12-14-alkyl esters was stated as not mutagenic under the test conditions.
Executive summary:

Phosphorous acid, tri-C12-14-alkyl esters was tested in vitro in an Ames test with Salmonella typhimurium.

Five concentrations of the test item, dissolved in tetrahydrofurane (THF) (ranging from 5000 to 50 µg/plate) were used in a first plate incorporation method, then five concentrations (ranging from 5001 to 313 µg/plate) were tested in a second experiment via pre incubation method.

Five genetically manipulated strains of Salmonella typhimurium (TA 97a, TA 98, TA 100, TA 102 and TA 1535) were exposed to the test item both in the presence and in the absence of a metabolic activation system (S9) for 48 hours.

None of the concentrations in the two experiments caused a significant increase in the number of revertant colonies in the tested strains.

No signs of toxicity towards the bacteria could be observed.

The sterility control and the determination of the titre didn’t show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range. All positive controls showed mutagenic effects with and without metabolic activation.

The study was then considered as valid and the test item didn’t show mutagenic effects towards Salmonella typhimurium, strains TA 97a, TA 98, TA 100, TA 102 and TA 1535.

Therefore, no concentration-effect relationship could be determined.

The test item Phosphorous acid, tri-C12-14-alkyl esters is considered as “not mutagenic under the conditions of the test”.