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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
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-05-03 to 2012-05-31
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline Study OECD 476 with GLP compliance

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)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid, colorless, clear
Details on test material:
- Name of test material: Delta-Valerolactone

Method

Target gene:
- Hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster V79 cells
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM (Minimal Essential Medium)
- Properly maintained: yes
- Used cell batch checked for Mycoplasma contamination: yes, before freezing
- Used cell batch checked for karyotype stability: yes, before freezing
- "cleansed" against high spontaneous background: yes, HAT-medium before freezing
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital/beta-naphthoflavone induced rat liver S9 (Wistar rat (Hsd Cpb: WU))
Test concentrations with justification for top dose:
CYTOTXICITY TEST
8.1 to 1030 µg/mL with/without metabolic activation

MUTAGENICITY TEST
1030 µg/mL = equal to approximately 10 mM, recommended highest concentration for freely-soluble test items

1st Experiment, 4h exposure period:
with S9-mix: 64.4, 128.8, 257.5 515.0 and 1030.0 µg/mL
without S9-mix: 128.8, 257.5 515.0 and 1030.0 µg/mL

2nd Experiment:
4h exposure period; with S9-mix: 64.4, 128.8, 257.5 515.0 and 1030.0 µg/mL
24h exposure period; without S9-mix: 64.4, 128.8, 257.5 515.0 and 1030.0 µg/mL

In experiment I and II the cultures at the lowest concentration of 32.2 µg/mL with and without metabolic activation and additionally in experiment I the concentration of 64.4 µg/mL without metabolic activation were not continued, since a minimum of four analysable concentrations is required by the guidelines.
Vehicle / solvent:
Vehicle: deionised water
On the day of each experiment (immediately before treatment), the test item was dissolved or suspended in deionised water. The final concentration of deionised
water in the culture medium was 10 % (v/v). The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Remarks:
without S9 -mix: ethylmethane sulfonate (EMS), final concentration: 0.150 mg/mL = 1.2 mM; with S9 -mix: 7,12-dimethylbenz(a)anthracene (DMBA), final concentration: 1.1 µg/mL = 4.3 µM. Historical control data were included in the report.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24h
- Exposure duration: without S9-mix: 4/24h, with S9-mix: 4h
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): about 8 days

SELECTION AGENT (mutation assays): 6-thioguanine (11 µg/mL)
STAIN (for cytogenetic assays): 10 % methylene blue in 0.01 % KOH solution

NUMBER OF REPLICATIONS: Duplicate cultures were used for all experimental groups

NUMBER OF CELLS EVALUATED: 3 - 5×10E5 cells for assessment of mutation rates. Following seeding of approx. 500 cells, colonies with more than 50 cells were counted for viability determination.

PRE-TEST: A pre-test on toxicity was performed in order to determine the concentration range for the mutagenicity experiments.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

OTHER EXAMINATIONS:
- Osmolarity and pH value: The osmolarity and pH-value were determined in the solvent control and in the highest concentration of the
pre-experiment without metabolic activation.
- Precipitation: Precipitation was evaluated at the end of treatment by the unaided eye.
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-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 was judged as significant whenever the p-value (probability value) was below 0.05. However, both, biological relevance 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
Remarks:
Relevant cytotoxic effects occurred in experiment I at 515 μg/mL and above without metabolic activation (both cultures). No relevant cytotoxic effects were noted in all of the other experimental parts up to 1030 μg/mL or approx. 10 mM.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.
- The test item either formed an opalescent solution in water (pre-experiment) or a milky, homogeneous suspension (both main experiments). The opalescent solution or milky suspension indicated a concentration right at or slightly above the limit of solubility in water.
- In experiment I precipitation occurred at 1030 μg/mL in the presence and absence of metabolic activation. In experiment II precipitation was observed at 1030 μg/mL in the presence of metabolic activation.

PRE-TEST ON TOXICITY
- Cytotoxic effects were observed in the range-finding pre-test only without metabolic activation (4h exposure period) from a concentration of 257.5 µg/mL up to highest concentration tested, 1030 µg/mL.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The mutant frequency remained well within the historical range of solvent controls.

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 515 μg/mL and above without metabolic activation (both cultures). The recommended toxic range of approximately 10-20% relative cloning efficiency I was covered without metabolic activation. No relevant cytotoxic effects were noted in all of the other experimental parts up to 1030 μg/mL or approximately 10 mM.

POSITIVE CONTROLS:
Both positive controls showed a distinct increase in induced mutant colonies.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion