2,5-Furandione, dihydro-, mono-C15-20-alkenyl derivs.

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP- and guideline compliant

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,5-Furandione, dihydro-, mono-C15-20-alkenyl derivs.

EC Number:

272-221-2

EC Name:

2,5-Furandione, dihydro-, mono-C15-20-alkenyl derivs.

Cas Number:

68784-12-3

Molecular formula:

Due to a mixture of numerous isomers a specific molecular formular cannot be provided ( UVCB-substance) .

IUPAC Name:

2,5-Furandione, dihydro-, mono-C15-20-alkenyl derivs.

Constituent 2

Reference substance name:

ASA

IUPAC Name:

ASA

Test material form:

liquid: viscous

Details on test material:

- Name of test material (as cited in study report): ASA
- Substance type: organic
- Physical state: liquid
- Analytical purity: 97.1 %
- Lot/batch No.: 5500001727
- Expiration date of the lot/batch: October 31, 2013
- Stability under test conditions: stable
- Storage condition of test material: Room temperature

Method

Target gene:

hprt locus

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media:
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

other: selected in HAT medium to reduce the number of mutants before the test started

Metabolic activation:

with and without

Metabolic activation system:

S9 supernatant and S9 cofactor solution (below) to result in a final protein concentration of 0.75 mg/mL in the cultures 8 mM MgCl2 33 mM KCl 5 mM glucose-6-phosphate 4 mM NADP in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.

Test concentrations with justification for top dose:

The test concentrations below are in µg/mL
Experiment I
without S9 mix* 6.3 12.5 25.0 50.0 600.0 800.0 1200.0
with S9 mix* 6.3 12.5 25.0 50.0 600.0 800.0 1200.0
Experiment II
without S9 mix** 10.0 20.0 40.0 80.0 320.0 480.0 640.0
With S9 mix* 10.0 20.0 40.0 80.0 640.0 960.0 1280.0
Experiment III
without S9 mix** 100.0 200.0 400.0 600.0 800.0 1000.0
* 4 hours treatment
** 24 hours treatment

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO;
- Justification for choice of solvent/vehicle: The solvent was chosen according to its solubility properties and its relative nontoxicity to the cells.

Details on test system and experimental conditions:

METHOD OF APPLICATION:
The study was performed to investigate the potential of ASA to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.
The assay was performed in three independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. Since the recommended cytotoxic range of a relative cloning efficiency of approximately 10 - 20% was not covered in the second experiment without metabolic activation a repeat experiment was performed at higher concentrations (experiment III). The treatment time of experiment III was 24 hours without metabolic activation.
The highest concentration of the test item in the pre-experiment was 5 µL/mL. The concentration range of the main experiments was limited by cytotoxic effects and the solubility of the test item in aqueous medium.
Appropriate reference mutagens, used as positive controls, were used to demonstrate the sensitivity of the test item and the activity of the metabolic activation system.

DURATION
- Preincubation period: 24 hours
- Exposure duration: 4 hours (+/- S9) 1st experiment / 4 hours (+S9) and 24 hours (-S9) 2nd experiment / 24 hours (-S9) 3rd experiment
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): about 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): about 15 days

SELECTION AGENT (mutation assays): 6-Thioguanine
SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays):

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 3 - 5 x10^5 cells per flask (concentration, +/-S9, experiment, replicate)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: determined with a separate assay

OTHER EXAMINATIONS:
- Determination of polyploidy: no
- Determination of endoreplication: no
- Other:

OTHER:

Evaluation criteria:

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-muta¬genic 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 fre¬quency at least at one of the concen¬trations 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.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA) statistics software. 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 were considered together.
experimental group p-value
experiment I, culture I without S9 mix 0.254
experiment I, culture II without S9 mix 0.079
experiment I, culture I with S9 mix 0.670
experiment I, culture II with S9 mix 0.955
experiment II, culture I without S9 mix 0.308
experiment II, culture II without S9 mix 0.094
experiment II, culture I with S9 mix 0.853
experiment II, culture II with S9 mix 0.582

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none. The pH was checked in the solvent control (pH 7.45) and in the highest concentration (pH 7.04)
- Effects of osmolality: none. Osmolarity was checked in the solvent control (391) and in the highest concentration (322)
- Evaporation from medium: none: Vapor pressure is very low
- Water solubility: ASA is a uvcb substance. There are at least some water-insoluble fractions at the higher test concentrations.
- Precipitation: The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) before the test item was removed. Precipitation was observed by the unaided eye down to the lowest concentration of 0.039 µL/mL with and without metabolic activation following 4 hours treatment. After 24 hours treatment (without metabolic activation) precipitation occurred at 0.078 µL/mL and above.
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: A pre-test was performed in order to determine the concentration range for the mutagenicity experiments. The general culture conditions and experimental conditions in this pre-test were the same as described for the mutagenicity experiment below. In this pre-test the colony forming ability of approximately 500 single cells (duplicate cultures per concentration level) after treatment with the test item was observed and compared to the controls. Toxicity of the test item is indicated by a reduction of the cloning efficiency

COMPARISON WITH HISTORICAL CONTROL DATA: The parameters of this study are all in the range of the historical controls

ADDITIONAL INFORMATION ON CYTOTOXICITY: In the first and second experiment with and without metabolic activation the cultures at the lowest concentration with metabolic activation and at the two lowest concentrations without metabolic activation were not continued since a minimum of only four analysable concentrations is required by the guidelines. The maximum concentration with metabolic activation was not continued in both experiments due to exceedingly severe cytotoxic effects. In experiment III the three highest concentrations were not continued for the same reason.

Remarks on result:

other: all strains/cell types tested

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative

Under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.
Therefore, ASA is considered to be non-mutagenic in this HPRT assay.

Executive summary:

The test item ASA was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster.

The study was performed in three independent experiments, using identical general experimental procedures. In the main experiments of this study (with and without S9 mix) the range of the solvent controls was from 2.6 up to 30.1 mutants per 10⁶cells; the range of the groups treated with the test item was from 0.0 up to 50.8 mutant colonies per 10⁶cells.

EMS(0.15 mg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies. In 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, ASA is considered to be non-mutagenic in this HPRT assay.