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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
Study period:
20 Oct - 22 Nov 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted Jul 2016
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
2008
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Remarks:
Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Type of assay:
other: In vitro Mammalian Cell Gene Mutation Test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

Method

Target gene:
HPRT locus
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Laboratory for Mutagenicity Testing, Technical University, Darmstadt, Germany
- Suitability of cells: the V79 cell line has been used successfully in in vitro experiments for many years.
- Doubling time: 12 - 16 h
- Methods for maintenance in cell culture: Thawed stock cultures were propagated at 37 °C with 1.5% CO2 in 75 cm² plastic flasks. The cells were sub-cultured once or twice weekly.
- Modal number of chromosomes: 22

MEDIA USED
- Type and identity of media: MEM (minimal essential medium) containing Hank’s salts supplemented with neomycin (5 μg/mL) and amphotericin B (1%). During treatment 10% fetal bovine serum (FBS) was added.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Metabolic activation:
with and without
Metabolic activation system:
co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone
Test concentrations with justification for top dose:
Pre-test for toxicity
with and without S9 mix: 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 µg/mL (4 h)
Experiment I and II
with and without S9 mix: 62.5, 125, 250, 500, 1000 and 2000 µg/mL (4 h)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO 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
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding: 0.7 - 1.2E+07 cells per plastic flask

DURATION
- Exposure duration: 4 h exposure with and without metabolic activation.
- Expression time: 7 days
- Selection time (if incubation with a selection agent): 7 - 9 days

SELECTION AGENT (mutation assays): 11 µg/mL 6-thioguanine (6-TG)

STAIN: 10% methylene blue in 0.01% KOH solution

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
A test item is classified as positive if it induces a concentration-related increase of the mutant frequency exceeding the historical solvent control range.
A test item producing no concentration-related increase of the mutant frequency above the historical solvent control range is considered to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces with at least one of the concentrations in both parallel cultures a mutation frequency that exceeds the historical negative and solvent control data range (95% confidence interval limits).
The increase should be significant and dose dependent as indicated by statistical analysis (linear regression, least squares).
Statistics:
A linear regression (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated 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.
A t-Test was performed using a validated test script of “R”, a language and environment for statistical computing and graphics, to evaluate an isolated increase of the mutation frequency at a test point exceeding the 95% confidence interval. Again a t-test is judged as significant if the p-value (probability value) is below 0.05.

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no relevant shift of pH in the medium. The pH was 7.39 (solvent control) and 7.18 (2000 µg/mL; adjusted with 2M sodium hydroxide), respectively.
- Effects of osmolality: There was no relevant shift of osmolality in the medium. Osmolality was 392 (solvent control) and 368 (2000 µg/mL), respectively.
- Precipitation: no precipitation occured up to the maximum concentration with and without metabolic activation

RANGE-FINDING/SCREENING STUDIES: a pre-test was performed in order to determine the concentration range for the mutagenicity experiment. 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 (CE).

VALIDITY OF THE ASSAY: The 95% confidence interval was slightly exceeded at 2000 μg/mL in the first culture without metabolic activation (30.9 versus an upper limit of 29.7 mutant colonies/106 cells), and with metabolic activation (36.4 versus an upper limit of 28.7 mutant colonies/106 cells). The 95% confidence interval was also exceeded with the second culture at 250.0 μg/mL (29.8 versus an upper limit of 28.7 mutant colonied/106 cells) with metabolic activation. These isolated increases were judged as irrelevant as they were not reproduced in the corresponding parallel cultures and there was no dose dependent increase as indicated by the not-significant linear regression analysis. As the "outliers" are near to the outer border of the historical control range, the assay is considered as acceptable.

Any other information on results incl. tables

Table 2: Results of the pre-experiment

Concentration [µg/mL] Cloning efficiency [%]
- S9 + S9
0 (DMSO) 100 100
15.6 94.9 102.3
31.3 91.5 94.2
62.5 97.7 90.5
125 94.4 94.8
250 95.6 96.0
500 92.1 92.3
1000 88.6 94.6
2000 82.8 89.9

DMSO = Dimethylsulfoxide

Table 3: Experiment I - 4 h exposure - without metabolic activation

Concentration [µg/mL] Cloning efficiency [%]
Survival
Cloning efficiency [%]
Viability
Mutation colonies per 106 cells 95% confidence intervall
0 (DMSO) 100 100 13.7 0.2 - 29.7
62.5 95.0 # # 0.2 - 29.7
125 105.4 84.4 24.1 0.2 - 29.7
250 98.7 93.9 13.4 0.2 - 29.7
500 111.9 90.9 25.5 0.2 - 29.7
1000 111.3 89.5 24.6 0.2 - 29.7
2000 114.4 93.3 30.9 0.2 - 29.7
EMS 99.0 98.2 218.2 0.2 - 29.7

EMS = Ethyl methane sulphonate

DMSO = Dimethylsulfoxide

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

Table 4: Experiment I - 4 h exposure - with metabolic activation

Concentration [µg/mL] Cloning efficiency [%]
Survival
Cloning efficiency [%]
Viability
Mutation colonies per 106 cells 95% confidence intervall
0 (DMSO) 100 100 13.6 0.6 - 28.7
62.5 93.2 # # 0.6 - 28.7
125 85.6 84.4 15.2 0.6 - 28.7
250 89.3 81.3 19.0 0.6 - 28.7
500 91.6 75.5 25.3 0.6 - 28.7
1000 82.7 81.4 11.2 0.6 - 28.7
2000 92.7 95.7 36.4 0.6 - 28.7
DMBA 99.2 81.7 162.0 0.6 - 28.7

DMBA = 7,12-dimethylbenz(a)anthracene

DMSO = Dimethylsulfoxide

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

Table 5: Experiment II - 4 h exposure - without metabolic activation

Concentration [µg/mL] Cloning efficiency [%]
Survival
Cloning efficiency [%]
Viability
Mutation colonies per 106 cells 95% confidence intervall
0 (DMSO) 100 100 21.1 0.2 - 29.7
62.5 87.0 # # 0.2 - 29.7
125 82.1 77.5 19.7 0.2 - 29.7
250 90.8 88.4 10.6 0.2 - 29.7
500 86.7 79.8 25.0 0.2 - 29.7
1000 93.7 74.6 26.1 0.2 - 29.7
2000 82.7 99.8 17.0 0.2 - 29.7
EMS 88.0 80.9 191.9 0.2 - 29.7

EMS = Ethyl methane sulphonate

DMSO = Dimethylsulfoxide

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

Table 6: Experiment II - 4 h exposure - with metabolic activation

Concentration [µg/mL] Cloning efficiency [%]
Survival
Cloning efficiency [%]
Viability
Mutation colonies per 106 cells 95% confidence intervall
0 (DMSO) 100 100 21.5 0.6 - 28.7
62.5 97.1 # # 0.6 - 28.7
125 104.2 97.8 18.1 0.6 - 28.7
250 93.5 107.8 29.8 0.6 - 28.7
500 105.5 97.6 22.3 0.6 - 28.7
1000 99.6 95.4 27.4 0.6 - 28.7
2000 106.1 94.3 26.7 0.6 - 28.7
DMBA 101.3 102.1 187.9 0.6 - 28.7

DMBA = 7,12-dimethylbenz(a)anthracene

DMSO = Dimethylsulfoxide

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

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative