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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:
23 Feb - 25 Jul 2017
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)
Deviations:
not specified
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
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 gene mutation study in mammalian cells

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
HPRT locus
Species / strain
Species / strain:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell lines (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.
- Methods for maintenance in cell culture: Thawed stock cultures were propagated at 37 °C with 1.5% carbon dioxide in 76 cm² plastic flasks and sub-cultured once or twice weekly.
- Modal number of chromosomes: 22
- Normal cell cycle time: 12 - 16 h

MEDIA USED
- 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
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-experiment for toxicity:
With and without S9 mix: 7.8, 15.6, 31.3, 62.5, 125, 250, 500 and 1000 µg/mL (4 h)

The following concentrations were selected in the main experiments for the mutagenicity testing:
Experiment I
Without S9 mix: 0.98, 1.96, 3.91, 7.83 µg/mL (4 h)
With S9 mix: 0.98, 1.96, 3.91, 7.83, 15.65 µg/mL (4 h)

Experiment II (repeat experiment)
With S9 mix: 0.98, 1.96, 3.91, 7.83, 15.65 µg/mL (4 h)

Experiment III
Without S9 mix: 0.98, 1.96, 3.91, 7.83 µg/mL (4 h)
With S9 mix: 0.98, 1.96, 3.91, 7.83, 15.65 µg/mL (4h)
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures on request of the sponsor.
Controls
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding: Approximately 1.2 x 10E07 cells/mL

DURATION
- Exposure duration: 4 h with and without metabolic activation
- Expression time (cells in growth medium): Approximately 7 days
- Selection time (if incubation with a selection agent): About 8 days

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

STAIN: 10% methylene blue in 0.01% KOH solution

NUMBER OF REPLICATIONS: Duplicates each in 3 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
A test item is classified as clearly positive if it induces a significant and concentration-related increase of the mutant frequency exceeding the historical solvent control range.
When a test item cannot be classified as clearly positive, additional experiments will be performed to establish the biological relevance of the result.
A test item producing a reproducible concentration-related increase of the mutant frequency above the historical solvent control range is considered to be mutagenic in this system.
A test item producing no reproducible concentration-related increase of the mutant frequency above the historical solvent control range is considered to be non-mutagenic in this system.
Statistics:
A linear regression was performed using a validated test script of "R", a language and environment for statistical computing and graphics (p<0.05), 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.
A t-Test was performed using a validated test script of “R”, a language and environment for statistical computing and graphics, whenever the mutation frequency at a test point exceeded the 95% confidence interval. Again a t-test is judged as significant if the p-value (probability value) is below 0.05.
However, both, biological relevance and statistical significance were considered together.

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
Experiment I: ≥ 3.9 µg/mL without metabolic activation; Experiment III: ≥ 3.9 µg/mL with and without metabolic activation
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH-value was determined in culture medium of the solvent control and at the maximum concentration of the pre-experiment without metabolic activation and was 7.51 and 7.52, respectively.
- Effects of osmolality: The osmolarity [mOsm] was determined in culture medium of the solvent control and at the maximum concentration of the pre-experiment without metabolic activation and was 399 and 381, respectively.
- Precipitation: Precipitation was observed macroscopically and microscopically at the end of treatment (4 h) prior to the removal of the test substance at 15.6 μg/mL in the absence and presence of metabolic activation.

RANGE-FINDING/SCREENING STUDIES: A pre-test was performed in order to determine the toxicity of the test substance. The pre-experiment was performed in the presence and absence (4 h treatment) of metabolic activation. The test substance was tested at concentrations between 7.8 μg/mL and 1000.0 μg/mL. The highest concentration of the pre-experiment was chosen due to precipitation observed in the pre-test on solubility. Relevant cytotoxic effects indicated by a relative cloning efficiency of 50% or below were observed at 125.0 μg/mL in the presence and absence of metabolic activation following 4 hours treatment.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: The positive control values were within the range of the historical control data (please refer to Table 1 under "any other information on material and methods incl. tables").
- Negative (solvent/vehicle) historical control data: The negative and solvent control values were within the range of the historical control data (please refer to Table 1 under "any other information on material and methods incl. tables").

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the main experiments.
In experiment I, the 95% confidence interval was exceeded at 3.91 μg/mL in culture II in the absence of metabolic activation (please refer to Table 2 under "any other information on results incl. tables"). In experiment II, the 95% confidence interval was very slightly exceeded at 7.83 μg/mL and 15.65 μg/mL in culture I (please refer to Table 3 under "any other information on results incl. tables").
A t-test evaluating the data of both parallel cultures at the data points exceeding the 95% confidence interval showed only a significant response in experiment I at 3.91 μg/mL without metabolic activation. The other data points exceeding the 95% confidence interval at 7.83 μg/mL and 15.65 μg/mL in experiment II showed no significant response in a t-test. Since the significant difference was only detected at an intermediate concentration and no dose dependent trend was found no biological relvance can be stated for this effect.

Any other information on results incl. tables

Table 1: Toxicity data

Concentration [µg/mL] Cloning efficiency [%]
  - S9 + S9
0 (DMSO) 100 100
7.8 101.3 90.6
15.6 P 94.4 91.0
31.3 P 80.0 80.3
62.5 P 61.6 56.7
125 P 34.4 28.8
250 P 20.6 17.0
500 P 13.3 9.0
1000 P 47.0 19.6

Table 2: Summary of Experiment I (4 h, without metabolic activation)

Concentration [µg/mL] Cloning efficiency [%]
Culture I
Mutation colonies per 10E06 cells
Culture I
Cloning efficiency [%]
Culture II
Mutation colonies per 10E06 cells
Culture II
95% confidence intervall
Medium 154.1 21.1 76.2 19.6 1.7 - 30.2
DMSO 100 13.4 100 11.2 1.7 - 30.2
0.98 80.2 14.5 81.1 20.6 1.7 - 30.2
1.96 68.9 29.1 77.5 21.4 1.7 - 30.2
3.91 24.0 16.1 28.2 55.6 1.7 - 30.2
7.83 11.1 16.0 9.8 19.7 1.7 - 30.2
15.65 P 5.4 n.r. 5.3 n.r. 1.7 - 30.2
31.3 P 0.7 # 0.0 # 1.7 - 30.2
EMS 91.3 262.0 74.3 851.6 1.7 - 30.2

P: Precipitation (visible at the beginning and at the end of treatment)

# culture was not continued due to exceedingly severe cytotoxic effects

n.r.: not reported, due to unacceptable cytotoxicity

EMS: ethylmethane sulfonate

Table 3: Summary of Experiment II (4 h, with metabolic activation)

Concentration [µg/mL] Cloning efficiency [%]
Culture I
Mutation colonies per 10E06 cells
Culture I
Cloning efficiency [%]
Culture II
Mutation colonies per 10E06 cells
Culture II
95% confidence intervall
Medium 114.2 22.5 77.3 26.6 2.0 – 29.4
DMSO 100.0 17.4 100.0 29.3 2.0 – 29.4
0.98 104.7 28.4 88.7 15.1 2.0 – 29.4
1.96 124.6 16.7 79.5 18.9 2.0 – 29.4
3.91 111.6 22.5 72.3 21.1 2.0 – 29.4
7.83 91.1 29.6 71.3 26.8 2.0 – 29.4
15.65 P 111.0 31.2 71.0 17.1 2.0 – 29.4
31.3 P 102.2 ## 65.4 ## 2.0 – 29.4
DMBA 93.2 187.8 57.0 152.3 2.0 – 29.4

P: Precipitation (visible at the beginning and at the end of treatment)

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

DMBA: 7,12-dimethylbenz(a)anthracene

Table 4: Summary of Experiment III (4 h, with and without metabolic activation)

Concentration [µg/mL] Cloning efficiency [%]
Culture I
Mutation colonies per 10E06 cells
Culture I
Cloning efficiency [%]
Culture II
Mutation colonies per 10E06 cells
Culture II
95% confidence intervall
without metabolic activation
Medium 101.1 11.5 122.2 12.4 1.7 - 30.2
DMSO 100.0 16.1 100.0 17.9 1.7 - 30.2
0.98 72.1 14.3 107.1 13.5 1.7 - 30.2
1.96 48.1 29.3 91.7 17.1 1.7 - 30.2
3.91 25.8 16.0 43.2 21.7 1.7 - 30.2
7.83 17.4 24.0 33.0 17.6 1.7 - 30.2
15.65 P 3.6 n.r. 7.7 n.r. 1.7 - 30.2
31.3 P 1.3 # 4.5 # 1.7 - 30.2
EMS 81.2 133.6 108.1 159.4 1.7 - 30.2
with metabolic activation
Medium 117.9 13.2 142.2 20.5 2.0 – 29.4
DMSO 100.0 33.4 100.0 15.2 2.0 – 29.4
0.98 116.9 17.6 138.2 25.7 2.0 – 29.4
1.96 117.1 6.2 99.8 18.1 2.0 – 29.4
3.91 86.9 16.9 147.0 26.8 2.0 – 29.4
7.83 108.8 25.1 132.4 25.0 2.0 – 29.4
15.65 P 98.5 25.3 94.7 19.3 2.0 – 29.4
31.3 P 15.0 ## 9.1 ## 2.0 – 29.4
DMBA 97.5 216.6 118.8 152.6 2.0 – 29.4

P: Precipitation (visible at the beginning and at the end of treatment)

# culture was not continued due to exceedingly severe cytotoxic effects

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

n.r.: not reported, due to unacceptable cytotoxicity

EMS: ethylmethane sulfonate

DMBA: 7,12-dimethylbenz(a)anthracene

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative