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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
Study period:
October 05, 2011 - December 09, 2011
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
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: Solid violet
- Analytical purity: >90%, dose calculation adjusted to purity
- Storage condition of test material: 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 % fetal bovine serum (FBS), neomycin (5 Pg/mL) and amphotericin B (1 %). The cell cultures were incubated at 37 °C in a 1.5 % carbon dioxide atmosphere (98.5 % air).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Without S9 mix: 10.8; 21.5; 43.0; 86.0; 172.0; 344.0 µg/ml
With S9 mix: 5.6; 10.8; 21.5; 43.0; 86.0; 172.0 µg/ml
The cultures at the lowest concentrations with metabolic activation were not continued, since a minimum of only four analysable concentrations is required by the guidelines. The cultures at the maximum concentration without metabolic activation were not continued to avoid evaluation of too many precipitating concentrations.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative nontoxicity to the cell cultures.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: with S9 mix: DMBA; 7,12-dimethylbenz(a)anthracene, 1.1 Pg/mL = 4.3 µM; without S9 mix: EMS; ethylmethane sulfonate, 0.150 mg/mL = 1.2 mM
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

Approximately 1.5×10E6 (single culture) and 5×10E2 cells (in duplicate) were seeded in plastic culture flasks. After 24 hours the medium was replaced with serum-free medium containing the test item, either without S9 mix or with 50 µL/mL S9 mix. Concurrent solvent and positive controls were treated in parallel. After 4 hours this medium was replaced with complete medium following two washing steps with "saline G". In the second experiment the cells were exposed to the test item for 24 hours in complete medium, supplemented with 10 % FBS, in the absence of metabolic activation.
The colonies used to determine the cloning efficiency (survival) were fixed and stained approximately 7 days after treatment as described below. Three or four days after treatment 1.5×10E6 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 - 5×10E5 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. The colonies were stained with 10 % methylene blue in 0.01 % KOH solution. The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

PRE-TEST ON TOXICITY
A pre-test was performed in order to determine the concentration range for the mutagenicity experiments. 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 (CE). Test item concentrations between 43.0 and 5500 µg/mL were used to evaluate cytotoxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation.
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. Precipitation occurred at 171.9 µg/mL and above without metabolic activation following 4 and 24 hours treatment and at 85.9 µg/mL and above with metabolic activation (4 hours treatment).
Based on the occurrence of precipitation in the pre-experiment, the individual concentrations of the main experiments were selected. The individual concentrations were spaced by a factor of 2.
Evaluation criteria:
The gene mutation assay is considered acceptable if it meets the following criteria:
- The numbers of mutant colonies per 10E6 cells found in the solvent controls fall 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 %.

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
Remarks:
Toxic effects only observed in the pre-test at high concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In both main experiments precipitation was observed at 86.0 µg/mL and above with metabolic activation (4 hours treatment) and without metabolic activation (4 and 24 hours treatment).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant toxic effects occurred at the maximum concentration of 5500 µg/mL without metabolic activation following 4 hours treatment. In the presence of metabolic activation (4 hours treatment) no relevant cytotoxicity were observed up to the maximum concentration. Following 24 hours treatment without metabolic activation, increasing cytotoxicity was observed at 343.8 µg/mL and above.

Any other information on results incl. tables

An increase of the induction factor exceeding the threshold of three times the mutation frequency of the corresponding solvent control was observed in the first culture of the second experiment with metabolic activation at 86.0 µg/mL. However, the increase was based on a rather low mutation frequency of the solvent control of just 6.1 colonies per 106 cells. Furthermore, the effect was not reproduced in the parallel culture under identical experimental conditions. Therefore, the increase of the induction factor was judged as biologically irrelevant fluctuation.

SUMMARY OF RESULTS

concentration (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 (water) - 100 100 100 8.6 1 100 100 100 23.8 1
positive control (EMS) 150 - 90.4 93.1 78.1 171.2 19.8 80 106.3 76.5 123.1 5.2
test item 10.8 - 96.5 139.9 92 12.7 1.5 96 112.9 89.4 16 0.7
test item 21.5 - 106.4 123.7 89.9 10.2 1.2 97.6 108.1 77.8 20.3 0.9
test item 43 - 98 100.3 69.9 13.8 1.6 99.1 68.1 90.5 11.6 0.5
test item 86 P - 93.1 112.6 68.7 21 2.4 101.6 82.9 66.9 11.5 0.5
test item 172 P - 79.8 82.6 87.5 9.8 1.1 101.6 95 68.1 18.7 0.8
test item 344 P - 77.7 culture was not continued# 91.9 culture was not continued#
solvent control (water) + 100 100 100 8.5 1 100 100 100 9.5 1
positive control (DMBA) 1.1 + 44 38.7 69.8 754.4 88.3 46.9 49.6 67.8 856.7 90.4
test item 5.6 + 91.1 culture was not continued## 98.2 culture was not continued##
test item 10.8 + 86.3 80.3 91.8 9 1.1 108.4 103.2 94.1 6.1 0.6
test item 21.5 + 85.6 88.3 119 7.4 0.9 102.2 88.3 90.3 4.8 0.5
test item 43 + 94.5 90.4 103.3 7.8 0.9 100.4 85.9 99.5 15.1 1.6
test item 86 P + 88.3 103.1 112.5 20.9 2.4 102.9 107.2 86.9 10.1 1.1
test item 172 P + 84 81.9 99.9 24.4 2.9 99.2 107.7 92.5 25.6 2.7
Experiment II / 24h treatment culture I culture II
solvent control (water) - 100 100 100 16.3 1 100 100 100 10.8 1
positive control (EMS) 150 - 83.6 60.8 107.2 410.7 25.2 98 108.7 99.6 307.2 28.5
test item 10.8 - 97.1 79.5 105.4 16 1 96.6 119.3 114.5 14.3 1.3
test item 21.5 - 91.4 83.8 102.7 18 1.1 97.4 110.7 110.2 14.1 1.3
test item 43 - 87.5 66.7 101.7 27.5 1.7 95.6 130.8 104.3 13.4 1.2
test item 86 P - 78.9 80.1 105 22.5 1.4 84.3 112 104.3 22.9 2.1
test item 172 P - 71.8 69.1 106.2 18.3 1.1 70.8 109.7 100.4 10.3 1
test item 344 P - 50.4 culture was not continued# 51.1 culture was not continued#
Experiment II / 4h treatment
solvent control (water) + 100 100 100 6.1 1 100 100 100 11 1
positive control (DMBA) 1.1 + 44 57.1 93 588.3 96 53.7 74.3 91.9 635.3 58
test item 5.6 + 111.7 culture was not continued## 100.4 culture was not continued##
test item 10.8 + 100.9 79.5 104.5 9.6 1.6 88.2 98.3 96.4 11.4 1
test item 21.5 + 110.3 105.4 99 7.7 1.3 92.7 90 92.1 15.4 1.4
test item 43 + 106.8 111.9 102.4 9.2 1.5 93 114.8 104.4 13.9 1.3
test item 86 P + 98.6 79.6 73.5 23.7 3.9 89.7 126.6 98.6 14.8 1.4
test item 172 P + 109 81.2 94.4 8.2 1.3 89 101.5 93.8 11.5 1

P = Precipitation

# culture was not continued to avoid analysis of too many precipitating concentrations

## culture was not continued as a minimum of only four analysable concentrations is required

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.
Executive summary:

A mammalian gene mutation assay compliant with GLP and in accordance with OECD guideline 476 was performed to investigate the potential of the test article to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in two 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. The highest concentration (5500 µg/mL) used in the range finding pre-experiment was chosen with respect to the current OECD guideline 476 and the purity of the test item. The test item was dissolved in deionised water. The concentration range of the main experiments was limited by the occurrence of precipitation of the test item. 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 substance is considered to be non-mutagenic in this HPRT assay.