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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
GLP compliance:
yes (incl. certificate)
Remarks:
Harlan Cytotest Cell Research GmbH, In den Leppsteinwiesen 19, 64380 Rossdorf, Germany
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
- Name of test material: Triisobutylphosphate
- BASF Test Item No.: 11/0642-1
- Batch Number: B722 v. 26.09.2011
- Purity: 97.8 g/100 g (± 0.1) determined by 1H-NMR-analysis (for details see analytical report No. 11L00444)
- Expiration Date: September 26, 2013
- Physical state, appearance: Liquid, colourless, clear
- Storage conditions: RT

Method

Target gene:
HPRT
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
See any other information on material and methods.
Vehicle / solvent:
- Solvent: ethanol
- Justification for choice of solvent: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures. The final concentration of ethanol in the culture medium was 0.5 % (v/v).
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24 hours
- Exposure duration: In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.
- Expression/fixation time: Three or four days after treatment 1.5×10^6 cells per experimental point were subcultivated in 175 cm² flasks containing 30 mL medium. Following the expression time of 7 days five 80 cm² cell culture flasks were seeded with about 3 - 5×10^5 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability. The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 for about 8 days.

NUMBER OF REPLICATIONS:
- The study was performed in two independent experiments, using identical experimental procedures.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
Acceptability of the assay:
The gene mutation assay is considered acceptable if it meets the following criteria: The numbers of mutant colonies per 10^6 cells found in the solvent controls falls within the laboratory historical control data. The positive control substances should produce a significant increase in mutant colony frequencies. The cloning efficiency II (absolute value) of the solvent controls should exceed 50 %.

Evaluation of results:
A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points. A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows: A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment. The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed. However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory's historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance was considered together.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS GENOTOXICITY:
- No relevant and reproducible increase in mutant colony numbers/10^6 cells was observed in the main experiments up to the maximum concentration with and without metabolic activation. In experiment I the induction factor exceeded the threshold of 3.0 at 20.8 µg/mL in the first culture without metabolic activation (induction factor of 3.9). This isolated effect however, was biological irrelevant as it was based on the rather low solvent control of just 6.7 colonies per 10^6 cells. The absolute value of the mutation frequency remained well within the historical range of solvent controls. A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of the mutation frequency. A single significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in the first culture of the second experiment with metabolic activation. Since all of the values of the mutation frequency remained within the historical range of solvent controls and the threshold described above was not exceeded the trend was judged as biologically irrelevant.

TEST-SPECIFIC CONFOUNDING FACTORS:
- In experiment I phase separation occurred at 166.0 mg/mL and above in the presence of metabolic activation.
- In the pre-test no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item was observed.

RANGE-FINDING/SCREENING STUDIES:
- In the range finding pre-experiment test item concentrations between 20.8 and 2660 µg/mL (≈10 mM) were used to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation. Relevant toxic effects were observed at 332.5 µg/mL and above following 4 hours and treatment with and without metabolic activation. After 24 hours treatment relevant toxic effects occurred at 166.3 µg/mL and above. The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item. Phase separation occurred at 1330.0 µg/mL and above after 4 hours and 24 hours treatment without metabolic activation and at 332.5 µg/mL and above with metabolic activation (4 hours treatment).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% occurred in the first experiment at 41.5 µg/mL (culture I) without metabolic activation. Exceedingly severe cytotoxic effects impeded analysis at 83.0 µg/mL and above in the first experiment without metabolic activation and at 120.0 µg/mL and above in the second experiment without metabolic activation. In the first experiment with metabolic activation phase separation of the test item limited the analysable concentration range to 249 µg/mL. In the second experiment with metabolic activation phase separation occurred together with exceedingly severe cytotoxic effects limiting the analysable concentration range to 240 µg/mL.

CONCLUSION:
It can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, Triisobutylphosphate is considered to be non-mutagenic in this HPRT assay.

Applicant's summary and conclusion