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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:
03 November 2014 - 07 January 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

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

Test material

Constituent 1
Chemical structure
Reference substance name:
2,5-thiophenediylbis(5-tert-butyl-1,3-benzoxazole)
EC Number:
230-426-4
EC Name:
2,5-thiophenediylbis(5-tert-butyl-1,3-benzoxazole)
Cas Number:
7128-64-5
Molecular formula:
C26H26N2O2S
IUPAC Name:
5-tert-butyl-2-[5-(5-tert-butyl-1,3-benzoxazol-2-yl)thiophen-2-yl]-1,3-benzoxazole
Details on test material:
- Physical state: Yellow powder
- Storage condition of test material: At room temperature

Method

Target gene:
hprt (hypoxanthine-guanine phosphoribosyl transferase)
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) containing Hank’s salts supplemented with 10% foetal bovine serum (FBS), neomycin (5 µg/mL) and amphotericin B (1%).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically checked for spontaneus mutant frequency: yes
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Experiment I, with and without S9: 0.6; 1.7; 5.0; 15.0; 45.0; (135.0) µg/ml
Experiment II, with and without S9: 0.3; 0.6; 1.7; 5.0; 15.0; (45.0) µg/ml
Numbers in parantheses: these cultures were discontinued to avoid evaluation of too many precipitating concentrations.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
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:
with S9: DMBA, 2.2 µg/mL; without S9: EMS, 150 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h (with and without S9)
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 15-16 days

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

NUMBER OF REPLICATIONS: two independent cultures were used

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency, cell density
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 fre¬quency 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 correspon¬ding 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 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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test item was observed in both experiments at 5.0 µg/mL and above with and without metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
No relevant toxic effect occurred up to the maximum concentration tested with and without metabolic activation following 4 hours of treatment. The test medium was checked for precipitation or phase separation at the end of each treatment period (4 hours) prior to removal to the test item. Precipitation occurred at 15.6 µg/mL and above after 4 hours treatment with and without metabolic activation. There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item. The dose range of the first experiment was set according precipitation observed in the pre-experiment.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No relevant cytotoxic effect indicated by a relative cloning efficiency I or cell density below 50% was observed up to the highest concentration of both experiments with and without metabolic activation.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Summary of Results

conc. (µg/ml) P S9 Mix relative cloning efficiency I (%) relative cell density (%) relative cloning efficiency II (%) mutant colonies / 106cells induction factor relative cloning efficiency I (%) relative cell density (%) relative cloning efficiency II (%) mutant colonies / 106cells induction factor
Experiment I / 4h treatment culture I culture II
solvent control (acetone) - 100 100 100 17.5 1 100 100 100 13.7 1
positive control (EMS) 150 - 90.2 99.9 89.4 182.4 10.4 97.7 92.6 98.4 227 16.6
test item 0.6 - 92 108.7 78.6 34 1.9 94 85.7 95.3 17.2 1.3
test item 1.7 - 86 87.8 80 21.1 1.2 92.2 82.6 96.1 26.4 1.9
test item 5 P - 84.2 92.6 94 10.3 0.6 100.5 88.5 94.2 28.4 2.1
test item 15 P - 91.1 67 79 27.9 1.6 96.1 88.9 97.3 30.6 2.2
test item 45 P - 82.3 114.6 73.8 23.5 1.3 95.1 74.3 84.8 28.9 2.1
test item 135 P - 66.8 culture was not continued# 87.5 culture was not continued#
solvent control (acetone) + 100 100 100 27.5 1 100 100 100 11.8 1
positive control (DMBA) 2.2 + 87.7 76.2 83.9 240.3 8.7 91.2 79.4 66.1 268.7 22.7
test item 0.6 + 91.6 72.9 104 9.1 0.3 85.9 97.9 66.5 22 1.9
test item 1.7 + 88.2 91.3 87.7 17.4 0.6 84.2 100.6 91.1 8.7 0.7
test item 5 P + 98 85.4 105.3 14.6 0.5 81 135.9 78.9 24.6 2.1
test item 15 P + 98.4 116.7 105.3 22.1 0.8 91.7 99.6 84.3 18.9 1.6
test item 45 P + 79.3 84.2 91.6 30.3 1.1 87.1 134.9 58.1 32.9 2.8
test item 135 P + 65 culture was not continued# 87.1 culture was not continued#
Experiment II / 24h treatment
solvent control (acetone) - 100 100 100 11.6 1 100 100 100 20.6 1
positive control (EMS) 150 - 92.2 121.6 106.2 85.9 7.4 93.3 101.8 106.3 80.7 3.9
test item 0.3 - 94.4 100.4 95 15.4 1.3 97 105.3 93.2 6.7 0.3
test item 0.6 - 95.1 113.7 96.7 14.9 1.3 97.5 114.7 99 17.8 0.9
test item 1.7 - 96.8 104.7 103.5 15.9 1.4 95.8 102.1 88 41.3 2
test item 5 P - 93.5 103.7 86.9 23.9 2.1 95.7 116.3 104 18.7 0.9
test item 15 P - 95.1 125.9 106 13.1 1.1 97.7 100 91.2 21.6 1.1
test item 45 P - 88.8 culture was not continued# 89.5 culture was not continued#
solvent control (acetone) + 100 100 100 18 1 100 100 100 18.4 1
positive control (DMBA) 2.2 + 91.9 123.1 104.7 123 6.8 89.9 80.5 102.3 155 8.4
test item 0.3 + 89.7 102 94.9 17.3 1 100.9 105.5 102.2 31.1 1.7
test item 0.6 + 94 149.9 112.9 24 1.3 97.8 104.2 104.5 24.3 1.3
test item 1.7 + 94.2 132 102.4 10.7 0.6 98 106.5 103.2 18.6 1
test item 5 P + 95.1 137.3 97.9 13.3 0.7 92.9 93 98.7 17.3 0.9
test item 15 P + 95.2 65.6 114.5 13.8 0.8 89.7 114.9 104.2 19.6 1.1
test item 45 P + 91.3 culture was not continued# 95.7 culture was not continued#

P = Precipitation visible at the end of treatment

# Culture was not continued to avoid evaluation of too many concentrations in the precipitating range

Applicant's summary and conclusion

Conclusions:
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 and is therefore considered to be non-mutagenic in this HPRT assay.
Executive summary:

In a GLP-compliant genotoxicity study according to OECD guideline 476 the test item was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster. The assay was performed in two independent experiments, using two parallel cultures each. The main experiments were performed with and without liver microsomal activation and a treatment period of 4 hours. The highest concentration of 1000 µg/mL in the pre-experiment was limited by the solubility of the test item in organic solvents. The concentration range of the main experiments was limited by the solubility of the test item in aqueous medium. The test item was dissolved in Acetone. The tested concentrations in the main experiment ranged from 0.6 to 135 µg/ml. Precipitation of the test item was observed in both experiments at 5.0 µg/mL and above with and without metabolic activation. No relevant cytotoxic effect indicated by a relative cloning efficiency I or cell density below 50% was observed up to the highest concentration of both experiments with and without metabolic activation. No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation. Appropriate reference mutagens (EMS and DMBA), used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. 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, the test item is considered to be non-mutagenic in this HPRT assay.