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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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
06. Oct. - 16. Feb. 2004
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD). (Purity of test substance not given, evalation criteria not given)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report date:
2004

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
purity of test substance is not given (responsibility of the sponsor)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Lot/batch No.: AO-267-108
- Storage condition of test material: room temperature in the dark

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: Eagles essential medium with HEPES buffer (MEM), supplemented with:
L-glutamine, penicillin/streptomycin, amphotericin B, 15% foetel calf serum
- Properly maintained: yes
Metabolic activation:
with and without
Metabolic activation system:
co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats pretreated with phenobarbitone (80 mg/kg) and ß-naphtoflavone (100 mg/kg)
Test concentrations with justification for top dose:
Experiment I:
4 hour (with and without): 40, 80, 160, 240*, 320*, 400* µg/mL

Experiment II
4 hour (with): 40, 80, 160, 240*, 320*, 400* µg/mL
24 hour (without): 40, 80, 160, 240*, 320*, 400* µg/mL

* Dose levels (plus control dose) selected for metaphase analysis
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Mitomycin C (MMC; 0.2 and 0.4 µg/mL; -S9), cyclophosphamide (CP; 10 µg/mL; +S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h (with and without S9), 24 h (without)
- Fixation time (start of exposure up to fixation or harvest of cells): 4 h treatment: 20 h; 24 h treatment: 0 h

SPINDLE INHIBITOR (cytogenetic assays): Colcemid 0.1 µg/mL (demecolcine)
STAIN (for cytogenetic assays): 5% Gurrs Giemsa for 5 minutes

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 200 per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of 2000 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
Evaluation criteria:
no data
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
Species / strain:
lymphocytes: cultured peripheral human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: cloudy precipitates were observed at and above 40 and 80 µg/mL in the 24-hour continuous and 4-hour pulse treatment groups, respectively

RANGE-FINDING/SCREENING STUDIES:
The dose range tested was 10-320 µg/mL. The test material produced some weak toxicity in the 4-hour treatment group but not the 24-hour treatment group. Toxicity could not be reproduced in the main experiment (scorable metaphases at every dose level).
COMPARISON WITH HISTORICAL CONTROL DATA:
The results are in range with historical control data.

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 3 + 4: Test results of experiment I.

Test item

Concentration

Mitotic Index

Aberrant cells in %

 Experiment I

in µg/mL

in %

with gaps

without gaps

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

control

0

100

1

0

MMC

0.4

35

53

37

Test substance

240

98P

0.5

0

320

110P

0

0

400

91P

0.5

0.5

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

control

0

100

1

0.5

CP

10

20

35.5

28.5

Test substance

240

89P

1.5

0

320

89P

0.5

0

400

108P

3.5

1

Test item

Concentration

Mitotic Index

Aberrant cells in %

 Experiment II

in µg/mL

in %

with gaps

without gaps

Exposure period + fixation time 24h, without S9 mix

control

0

100

1.5

0

MMC

0.4

41

70

67

Test substance

240

61

0.5

0.5

320

53

0.5

0

400

83

0

0

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

control

0

100

0.5

0

CP

10

30

67

56

Test substance

240

103

1

0

320

76

1

0

400

110

1.5

0.5

Table5 +6: Mean Frequency of Polyploid Cells (%)

Experiment I

dose level µg/mL

harvest time 24 hours

4 hours without S9

4 hours with S9

0

0.0

0.0

240

0.0

0.0

320

0.0

0.0

400

0.0

0.0

MMC 0.4

0.0

NA

CP 10

NA

0.0

dose level µg/mL

harvest time 24 hours

24 hours without S9

4 hours with S9

0

0.5

0.0

240

0.0

0.0

320

0.0

0.0

400

0.0

0.5

MMC 0.4

0.0

NA

CP 10

NA

0.0

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative