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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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
8 Feb - 15 Apr 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
adopted in 1997
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Remarks:
The Department of Health of the Government of the United Kingdom, UK
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
Not applicable
Species / strain
Species / strain / cell type:
lymphocytes: cultured peripheral human lymphocytes
Details on mammalian cell type (if applicable):
- Type and identity of media:
Eagle’s MEM medium supplemented with:
-sodium bicarbonate
-HEPES buffer
-L-glutamine
-penicillin/streptomycin
-amphotericin B
-15% foetal calf serum (FCS)

Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from livers of rats treated with Aroclor 1254
Test concentrations with justification for top dose:
First experiment:
4(20) h without and with S9: 39.06; 78.13; 156.25; 312.5; 625; 1250; 2500; 5000 µg/mL

Second experiment:
20 (20) h without S9: 156.25; 312.5; 625; 1250; 2500 and 5000 µg/mL
4(20) h with S9: 156.25; 312.5; 625; 1250; 2500 and 5000 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: cyclophosphamide, 25 µg/mL in phopsphate buffered saline, +S9; ethyl methanesulphonate , 750 µg/mL and 500 µg/mL (Experiment 1 and 2 respectively), -S9 in dimethyl sulphoxide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h (experiment 1, with and without S9); 4 and 20 h (experiment 2, with and without S9, respectively
- Fixation time (start of exposure up to fixation or harvest of cells): 20h

SPINDLE INHIBITOR (cytogenetic assays): colcemid 0.1 µg/mL
STAIN (for cytogenetic assays): Gurrs Giemsa R66 5%

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of 2000 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
Evaluation criteria:
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 (both including and excluding gaps) and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher’s Exact test or Chi-squared test.

Results and discussion

Test results
Species / strain:
lymphocytes: cultured peripheral human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: there was no observable change in pH
- Effects of osmolality: the osmolality did not increase by more than 50 mOSM
- Precipitation: a cloudy appearance of the test material was noted at all dose levels in both treatment groups, after four hours exposure

COMPARISON WITH HISTORICAL CONTROL DATA: Yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: in Experiment 1 and 2 no dose-related toxicity was observed, a 50% mitotic inhibition was not achieved and the precipitate of the test item had no effect on the toxicity response curve. Thus, 1250, 2500 and 5000 µg/mL dose levels were selected for chromosome analysis.

Any other information on results incl. tables

Table 1: Test results of experiment 1

Test item

Concentration

Mitotic Index

Aberrant cells in %

 

in µg/mL

in %

with gaps

without gaps

Exposure period 4 h, fixation time 20 h, without S9 mix

Acetone

100

3.0

2.5

EMS

750

50

20

12.5

Test substance

1250

78

3.0

2.0

2500

83

5.5

1.0

5000

70

2.0

1.5

Exposure period 4 h, fixation time 20 h, with S9 mix

Acetone

100

3.5

2.0

CP

25

29

14.5

9.0

Test substance

1250

87

3.0

3.0

2500

125

3.5

0.5

5000

117

1.5

1.0

EMS: ethyl methanesulphonate;

CP: Cyclophosphamide (positive controls)

 

Table 2: Test results of experiment 2

Test item

Concentration

Mitotic Index

Aberrant cells in %

 

in µg/mL

in %

with gaps

without gaps

Exposure period 20 h, fixation time 20 h, without S9 mix

Acetone

0

100

1.5

0.5

EMS

750

58

36.7

20.0

Test substance

1250

123

2.0

0.5

2500

110

0.5

0.0

5000

99

2.0

0.0

Exposure period 4 h, fixation time 20 h, with S9 mix

Acetone

0

100

2.0

0.5

CP

25

25

19.0

11.5

Test substance

1250

89

1.5

0.0

2500

89

1.0

0.0

5000

112

1.0

0.0

EMS: ethyl methanesulphonate;

CP: Cyclophosphamide (positive controls)

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative