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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
not specified
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
- Lot/batch No.: C-120

Method

Target gene:
N/A
Species / strain
Species / strain / cell type:
other: Chinese hamster lung cells CHL/IU
Details on mammalian cell type (if applicable):
- Type and identity of media: Inactivated calf serum in Eagle MEM
- Properly maintained: No data
- Periodically checked for Mycoplasma contamination: No data
- Periodically checked for karyotype stability: No data
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction (mixed function induction of rat liver)
Test concentrations with justification for top dose:
-S9 (continuous treatment): 0, 1.3, 2.5, 5.0 mg/ml
-S9 (short term treatment): 0, 1.3, 2.5, 5.0 mg/ml
+S9 (short term treatment): 0, 1.3, 2.5, 5.0 mg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone; supplied by Wako Pure Chemical Industries Co.
- Justification for choice of solvent/vehicle: Substance is poorly soluble in water; very soluble in acetone & DMSO
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
-S9; 6 & 24 hours of exposure
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
+S9; 6 hours exposure
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Preincubation period:
- Exposure duration: For continuous treatment, cells were treated for 24 hours in the absence of S9; for short-term treatment cells were treated for 6 hours with & without S9 and cultivated with fresh media for 18 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcimid 0.1 ug/mL
STAIN (for cytogenetic assays): 3% Giemsa solution

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 metaphases/slide of each replicate

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No cytotoxicity up to and including 5.0 mg/mL when inhibition of cell growth determined.

OTHER EXAMINATIONS:
- Determination of polyploidy:Negative
- Determination of endoreplication: No data or negative if equivalent to clastogenicity
Evaluation criteria:
A dose related increase or a reproducible increase in the number of cells with chromosome aberrations.
An increase in the number of polyploid cells.
An increase in the number of cells with endoreduplicated chromosomes
Statistics:
No data, thought to be Fisher's Exact Test

Results and discussion

Test results
Species / strain:
other: Chinese hamster lung cells (CHL/IU)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
5 mg/mL
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: No
- Effects of osmolality: No
- Evaporation from medium: No
- Water solubility: poor
- Precipitation: No
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES: yes; growth inhibition test (range of concentrations: 0.0-5.0 mg/mL

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No toxicity observed
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1 - Chromosome analysis of cells following short-term treatment with and without S9 mix

 

Group

Concentration (μg/mL)

S9

Exposure Time

(hours)

Cell

No. of cells

analysed

No. of structural aberrations

Number of cells with aberrations (%)

Polyploidy

mix

growth index

gap

ctb

cte

csb

cse

frg

Total

(%)

Solvent control

0

-

6

100

200

0

0

0

1

0

0

1

1 (0.5)

0.0

 

1250

-

6

107

200

0

1

0

0

0

0

1

1 (0.5)

0.0

Test substance

2500

-

6

111

200

0

0

0

1

0

0

1

1 (0.5)

0.0

 

5000

-

6

107

200

0

1

0

2

0

0

3

3 (1.5)

0.0

MMC

0.1

-

6

113

200

4

17

76

1

2

0

96

91 (45.5)

0.0

 

Solvent control

 

0

 

+

 

6

 

100

 

200

 

1

 

1

 

1

 

2

 

0

 

0

 

4

 

3 (1.5)

 

0.0

TOTM

1250

+

6

105

200

0

2

0

1

2

0

5

5 (2.5)

0.0

T0TM

2500

+

6

103

200

0

0

0

1

1

0

2

2 (1.0)

0.0

TOTM

5000

+

6

94

200

0

1

1

0

0

0

2

2 (1.0)

0.0

BP

20

+

6

133

200

2

14

144

0

2

0

160

147 (73.5)

0.0

Abbreviations:

gap: chromatid gap and chromosome gap, ctb: chromatid break, cte: chromatid exchange, csb: chromosome break, cse: chromosome exchange (dicentric and ring etc.),frg: fragment,MMC: mitomycin C,BP: benzo(a)pyrene

 

 

Table 2 - Chromosome analysis of cells following continuous treatment without S9 mix

  

Group

Concentration (μg/mL)

S9

Exposure Time

(hours)

Cell

No. of cells

analysed

No. of structural aberrations

Number of cells with aberrations (%)

Polyploidy

mix

growth index

gap

ctb

cte

csb

cse

frg

Total

(%)

Solvent control

0

-

24

100

200

1

0

1

1

0

0

2

2 (1.0)

0.0

 

1250

-

24

98

200

1

0

0

0

0

0

0

0 (0.0)

0.0

Test substance

2500

-

24

104

200

0

0

1

1

0

0

2

2 (1.0)

0.0

 

5000

-

24

107

200

2

1

0

0

0

0

1

1 (0.5)

0.0

MMC

0.03

-

24

103

200

3

20

38

2

2

0

62

61 (30.5)

0.0

 

Abbreviations:

gap: chromatid gap and chromosome gap, ctb: chromatid break, cte: chromatid exchange, csb: chromosome break, cse: chromosome exchange (dicentric and ring etc.),frg: fragment,MMC: mitomycin C

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative

In the conditions in which this study was performed the tested substance, at doses of up to 5.0 mg/mL, did not induce chromosome aberrations with or without S9 in cultured mammalian somatic cells.
Executive summary:

The substance has been assayed for its ability to cause chromosomal damage in cultured mammalian cells following in-vitro treatment in the absence and presence of S9 metabolic activation.

The substance did not induce chromosomal aberrations in human lymphocytes after in-vitro treatment