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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 - 14 Jan 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report date:
2005

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
adopted: 29 Jul 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM
Type of assay:
other: in vitro mammalian chromosome aberration test (migrated information)

Test material

Constituent 1
Chemical structure
Reference substance name:
2-{2-chloro-4-(methylsulfonyl)-3-[(tetrahydrofuran-2-ylmethoxy)methyl]benzoyl}cyclohexane-1,3-dione
Cas Number:
473278-76-1
Molecular formula:
C20 H23 Cl O7 S
IUPAC Name:
2-{2-chloro-4-(methylsulfonyl)-3-[(tetrahydrofuran-2-ylmethoxy)methyl]benzoyl}cyclohexane-1,3-dione

Method

Target gene:
not applicable
Species / strain
Species / strain / cell type:
mammalian cell line, other: CHL/IU
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: National Institute of Health Science - Cell Bank
- Doubling time: 17 h
- Number of passages: 6(18) h: P18 -S9, P14 +S9; 24h: P16 -S9
- Methods for maintenance in cell culture if applicable: Cultures were established 16 to 72 hours prior to treatment using the appropriate number of cells per flask depending on the pre-exposure culture period. Cultures were maintained at 37°C in a humidified atmosphere of 5% CO2 in air.
- Modal number of chromosomes: 25

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Eagle's Minimal Essential Medium (MEM) with HEPES buffer and Earle's Salts supplemented with 10% foetal bovine serum and antibiotics; 5% CO2
- Properly maintained: not specified
- Periodically checked for Mycoplasma contamination: not specified
- Periodically checked for karyotype stability: not specified
- Periodically 'cleansed' against high spontaneous background: not specified
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone
Test concentrations with justification for top dose:
6 (18) h exposure: -/+S9: 276.9, 553.8,1107.5, 2215*, 3322.5* and 4430* µg/mL
24 h exposure: -S9: 276.9, 553.8,1107.5, 2215*, 3322.5* and 4430* µg/mL
* selected for metaphase analysis
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO was selected as the solvent because the test material was readily soluble in it at the required concentrations.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding: 3x10E04 cells/flask

DURATION
- Exposure duration: 6 h and 24 h
- Expression time (cells in growth medium): 18 h after short term incubation (6h)
- Fixation time (start of exposure up to fixation or harvest of cells): 2 h

SPINDLE INHIBITOR (cytogenetic assays): Colcemid 0.1 µg/mL

STAIN (for cytogenetic assays): Giemsa

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: After cells were fixed with glacial acetic acid/methanol (3:1 v/v) solution, several drops of cell suspension were dropped onto clean, wet microscope slides. Slides were dried and stained with Giemsa solution for 5 minutes, rinsed, dried and coverslipped using mounting medium.

NUMBER OF CELLS EVALUATED: 1000

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 100 (except where there were approximately 50% of cells with aberrations, then slide evaluation was terminated at 50 cells)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index, relative total growth
- Any supplementary information relevant to cytotoxicity: A preliminary toxicity test was performed on cell cultures using a 24-hour continuous exposure time without metabolic activation and a 6-h exposure period (both with and without metabolic activation) followed by an 18-h recovery period in treatment-free media. The dose range used was 17.3 to 4430 µg/mL.

OTHER EXAMINATIONS:
- Determination of polyploidy: yes, including endoreduplicated cells if they were not dose related
- Determination of endoreplication: yes

Evaluation criteria:
In all circumstances where increases in the frequency of cells with aberrations are seen, statistical comparisons will be made with the vehicle. A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.

Results and discussion

Test results
Key result
Species / strain:
mammalian cell line, other: CHL/IU
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
6 (18h) exposure: 30% growth inhibition at 4430 µg/mL +/-S9; 24 h exposure: 50% growth inhibition at 553.8 and 1107.5 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at and above 2215 µg/mL


RANGE-FINDING/SCREENING STUDIES: A preliminary cell growth inhibition test was performed at 17.3 to 4430 µg/mL. In all cases the test material showed some evidence of cell toxicity. A precipitate of the test material was seen at and above 2215 µg/mL. Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present at dose levels up to 4430 µg/mL in the 6 (18)-h without and with S9 exposure groups. The maximum dose with metaphases present in the 24-h continuous exposure group was 2215 µg/mL. The cell growth inhibition data indicated that approximately 50% inhibition was not achieved in the 6 (18)-h exposure group with metabolic activation, or in the 24-hexposure group. In the 6 (18)-h
exposure group without metabolic activation the cell growth inhibition data indicate that 74% inhibition was achieved at the 10 mM limit dose of 4430 µg/mL.


ADDITIONAL INFORMATION ON CYTOTOXICITY:
The dose levels of the test material induced optimum levels of toxicity to CHL cells in the 24-hour continous exposure group, but only modest levels of toxicity in both of the 6 (18)-hour exposure groups at dose levels up to the 10 mM limit dose.

Any other information on results incl. tables

Table 1. Historical control data

solvent control data
  6 Hour-S9 6 Hour+S9 24 Hour
% Cells with Aberrations (- gaps) % of Polyploid Cells % Cells with Aberrations (- gaps) % of Polyploid Cells % Cells with Aberrations (- gaps) % of Polyploid Cells
Minimum 0 0 0 0 0 0
Maximum 4.5 1.5 3.5 1.5 3 1.5
Mean 1.4 0.3 0.9 0.2 1 0.4
Standard Deviation 1.2 0.4 0.8 0.4 0.8 0.4
Number of experiments 28 40 23
positive control data
  6 Hour-S9 6 Hour+S9 24 Hour
% Cells with Aberrations (- gaps) % of Polyploid Cells % Cells with Aberrations (- gaps) % of Polyploid Cells % Cells with Aberrations (- gaps) % of Polyploid Cells
Minimum 14.5 0 14 0 9.7 0
Maximum 59 2 80 2.9 45 1.5
Mean 29.4 0.3 48.5 0.2 24.3 0.3
Standard Deviation 11.4 0.5 18.2 0.7 8.8 0.5
Number of experiments 28 39 22

Table 2. Results after 6 (18) h treatment with metabolic activation (+S9)

        Number and Percentages of Cells Showing Structural Chromosome Aberrations (%)   Cell Growth Index Number and Percentages of Cells Showing Numerical Aberrations (%)
Treatment Period (hours) S9 Concentration
  mix µg/mL               g Cell Count % Mitotic Index (%)        
      Observed ctb cte csb cse Others Total   Observed Polyploids Others Total
6(18) + Negative Control (DMSO) A 100 0 0 0 0 0 0 0 100 10.7 100 0 0 0
B 100 0 0 2 0 0 2 0 100 8.2 100 0 0 0
Total 200 0 0 2 0 0 2 0   9.45 200 0 0 0
% 100 0 0 1.0 0 0 1.0 0.0 100 100 0
+ 2215 A 100 0 0 0 0 0 0 0 84 5.3 100 0 0 0
  100 0 0 0 0 0 0 0 71 7.6 100 0 0 0
  200 0 0 0 0 0 0 0   6.45 200 0 0 0
% 100 0 0 0 0 0 0 0 78 68 0
+ 3322.5 A 100 0 0 0 0 0 0 0 71 5.4 100 0 0 0
B 100 0 0 1 0 0 1 0 67 8.7 100 0 0 0
Total 200 0 0 1 0 0 1 0   7.05 200 0 0 0
% 100 0 0 0.5 0 0.0 0.5 0 69 75 0
+ 4430 A 100 0 0 0 1 0 1 0 56 6 100 0 0 0
B 100 0 0 0 0 0 0 0 79 8.5 100 0 0 0
Total 200 0 0 0 1 0 1 0   7.25 200 0 0 0
% 100 0 0 0 0.5 0 0.5 0 68 77 0
+ Positive Control (CP) 5 A 50a 6 9 2 2 0 18 7 38 10.1 50 0 0 0
B 100 9 14 2 0 0 23 4 39 9.3 100 0 0 0
Total 150 15 23 4 2 0 41*** 11   9.7 150 0 0 0
% 100 10 15.3 2.7 1.3 0 27.3 7.3 39 103 0

DMSO = Dimethylsulphoxide

CP = Cyclophosphamide

*** = p <0.001

a = Slide evaluation terminated at 50 cells because approximately 50% cells with aberrations had been observed

Table 3. Results of 6 (18h) treatment without metabolic activation (-S9)

        Number and Percentages of Cells Showing Structural Chromosome Aberrations (%)   Cell Growth lndex Number and Percentages of Cells Showing Numerical Aberrations (%)
Treatment Period (hours) S9 Concentration  
  mix µg/mL               g Cell Count% Mitotic Index(%)   Polyploids Others Total
24     Observed ctb cte csb cse Others Total   Observed
- Negative Control (DMSO) A 100 0 0 0 0 0 0 0 100 5.8 100 0 0 0
B 100 0 0 0 0 0 0 0 100 8.2 100 0 0 0
Total 200 0 0 0 0 0 0 0 100 7 200 0 0 0
% 100 0 0 0 0 0 0 0 100 0
- 2215 A 100 1 0 1 0 0 2 0 75 9.2 101 1 0 1
B 100 1 0 2 0 0 3 0 73 7.8 102 2 0 2
Total 200 2 0 3 0 0 5* 0 74 8.5 203 3 0 3
% 100 1 0 1.5 0 0 2.5 0 121 1.5
- 3322.5 A 100 1 1 0 0 0 2 3 66 12.2 100 0 0 0
B 100 0 0 0 0 0 0 1 74 6.5 100 0 0 0
Total 200 1 1 0 0 0 2 4 70 9.35 200 0 0 0
% 100 0.5 0.5 0 0 0 1 2 134 0
- 4430 A 100 0 0 0 0 0 0 0 65 6 100 0 0 0
B 100 0 0 0 0 0 0 0 75 5.9 100 0 0 0
Total 200 0 0 0 0 0 0 0 70 5.95 200 0 0 0
% 100 0 0 0 0 0 0 0 85 0
- Positive Control (MMC) 0.01 A 100 3 28 3 1 0 32 15 59 7.6 100 0 0 0
B 100 14 14 2 3 0 28 15 63 6.4 100 0 0 0
Total 200 17 42 5 4 0 60*** 30 61 7 200 0 0 0
% 100 8.5 21 2.5 2 0 30 15 100 0

DMSO=Dimethylsulphoxide

MMC = Mitomycin C

* = p < 0.05

*** =p <0.001

Table 4. Results of 24 h treatment (-S9)

        Number and Percentages of Cells Showing Structural Chromosome Aberrations (%)   Cell Growth lndex Number and Percentages of Cells Showing Numerical Aberrations (%)
Treatment Period (hours) S9 Concentration  
  mix µg/mL               g Cell Count% Mitotic Index(%)   Polyploids Others Total
24     Observed ctb cte csb cse Others Total   Observed
- Negative Control (DMSO) A 100 0 0 0 0 0 0 0 100 3.1 100 0 0 0
B 100 0 0 0 0 0 0 0 100 3.9 101 1 0 1
Total 200 0 0 0 0 0 0 0 100 3.5 201 1 0 1
% 100 0 0 0 0 0 0 0 100 0.5
- 276.9 A 100 0 1 0 0 0 1 2 104 4.2 100 0 0 0
B 100 0 0 0 1 0 1 0 98 2.7 100 0 0 0
Total 200 0 1 0 1 0 2 2 101 3.45 200 0 0 0
% 100 0 0.5 0 0.5 0 1 1 99 0
- 553.8 A 100 0 0 0 0 0 0 0 94 1.9 100 0 0 0
B 94b 0 0 0 0 0 0 0 88 1.2 94 0 0 0
Total 194 0 0 0 0 0 0 0 91 1.55 194 0 0 0
% 100 0 0 0 0 0 0 0 44 0
- 1107.5 A 100 0 0 0 0 0 0 0 91 1.7 100 0 0 0
B 100 0 1 0 1 0 2 0 74 1.7 100 0 0 0
Total 200 0 1 0 I 0 2 0 83 1.7 200 0 0 0
% 100 0 0.5 0 0.5 0 1 0 49 0
- Positive Control (MMC) 0.05 A 50a 15 13 2 0 0 25 9 60 5.1 50 0 0 0
B 50a 11 14 0 1 0 24 6 53 3.7 50 0 0 0
Total 100 26 27 2 1 0 49*** 15 57 4.4 100 0 0 0
% 100 26 27 2 1 0 49 15 126 0

DMSO = Dimethylsulphoxide

MMC = Mitomycin C

*** = p <0.001

a = Slide evaluation terminated at 50 cells because approximately 50% cells with aberrations had been observed

b = Insufficient metaphases to score 100 cells

Applicant's summary and conclusion