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Genetic toxicity: in vitro

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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
Study period:
From January 10 to July 10 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report Date:
1992

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): P#620

Method

Target gene:
Not applicable.
Species / strain
Species / strain / cell type:
other: female Chinese Hamster lung cells.
Details on mammalian cell type (if applicable):
- Type and identity of media: Eagle's MEM powder (Eagle culture medium "Nissiu" 1 Powder, Nissiu Pharmaceutical company) was dissolved in purified water. The obtained solution was supplemented with sterile L-Glutamine, sodium bicarbonate, and with 10% heat-inactivated calf serum.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Supplemented liver preparation (S-9 mix) prepared from animals previously treated with Phenobarbital and 5,6-benzoflavone.
Test concentrations with justification for top dose:
Concentration range in the preliminary test (inhibition of cell growth): 20 ... 1000 µg/mL
Concentration range in the main test (with metabolic activation): 175 ... 700 µg/ml
Concentration range in the main test (without metabolic activation): 22.5 ... 100 µg/ml
See details in Table 7.6.1/1
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: no data.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Mitomycin C and Benzo(a)pyrene
Remarks:
Mitomycin C: 0.03 µg/mL and Benzo(a)pyrene: 20 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium.

DURATION
- Preincubation period: not applicable
- Exposure duration: Direct assay: 24 or 48 hours withour metabolic activation; 6 hours of exposure +18 h recovery in the case of metabolic activation assay (with or without S9 mix). See details in Table 7.6.1/1
- Expression time (cells in growth medium): 24 or 48h
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 24 or 48h

SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): colcemid (0.1 µg/mL)
STAIN (for cytogenetic assays): The cells were stained for 20 min using 3% Giemsa's solution diluted with 1/15 M phosphate buffer (pH 6.8).

NUMBER OF REPLICATIONS: duplicate cultures were performed for each test group. Two independant test were performed.
NUMBER OF CELLS EVALUATED: 100 from each culture. Therefore 200 cells were analyzed from each treatment group.

DETERMINATION OF CYTOTOXICITY
- Method: during the preliminary test, the cytotoxicity was analysed by counting the surviving cells. During the main test the cytotoxicity was determined with the mitotic index.

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: no
- Other: Structural chromosome aberrations (gaps, breaks, exchanges and other aberrations)
Evaluation criteria:
Any cell showing at least one chromosomal aberration was categorized as an aberrant cell. The test material was judged to have the ability to induce chromosomal aberration only when a significant difference and dose-responsiveness or reproducibility were noted in comparisons between the test groups and the negative control group.
Statistics:
Differences in the number of aberrant cells (excluding those which only had gaps) and the number of polyploid cells were statistically analysed using the Che 2 test.

Results and discussion

Test results
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
no data

RANGE-FINDING/SCREENING STUDIES: the 50% inhibitory dose for cell growth was estimated to be 84 and 85 µg/mL in the direct assay for 24 hours and 48 hours, respectively, and 93 and 632 µg/mL in the metabolic activation assay, without and with S9 mix, respectively. Based upon the results, the maximum dose level for the chromosome aberration test was set as concentration producing 50% or greater inhibition of cell growth and 3 dose levels were selected.

COMPARISON WITH HISTORICAL CONTROL DATA: the finding that the number of cells with chromosomal aberrations and the number of polyploid cells in the negative control cultures remained in the normal background range of the laboratory, while these parameters were significantly increased in the positive control cultures suggests that the chromosomal aberration test using CHL/IU cells was performed effectively.

ADDITIONAL INFORMATION ON CYTOTOXICITY: % of mitotic index was decreased at the highest tested concentrations (see details in table 7.6.1/2). At
the maximum dose level (700 µg/ml) in the first metabolic activation assay with S-9 mix, the required number of metaphase cells was not obtained due to cytotoxicity, but a full number of metaphase cells were obtained at the dose levels of 650 and 700 µg/ml dose level in the second test. So the observation of 700 µg/mL was only conducted.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

In each test the test material did not increase the number of cells with structural chromosomal aberrations or the number of polyploid cells in the assay with an exposure period of 24 or 48 hours and in the metabolic activation assay. In the negative control cultures, the number of cells with structural chromosomal aberrations was no more than 1.0%. In the positive control cultures, the number of cells with chromosomal aberrations increased significantly.

Table 7.6.1/2:Mitotic index results of the first main assay

 

Treatment

Dose (µg/mL)

Time (hour)

S9 mix

Mitotic index (%)

Direct assay

DMSO

0

24

NA

5.7

P#620

25

6.2

50

6.6

100

1.8

MMC

0.03

3.5

DMSO

0

48

6.2

P#620

22.5

4.8

45

5.2

90

4.7

MMC

0.03

2.9

Metabolic activation assay

DMSO

0

6+18

 

-

5.8

P#620

25

-

4.2

50

-

4.2

100

-

4.1

BP

20

-

4.4

DMSO

0

+

6.4

P#620

175

+

6.6

350

+

3.4

700

+

0.9*

BP

20

+

3.1

MMC: Mitomycin C

BP: Benao(a)pyrene

*: the required number of metaphase cells was not obtained because of cytotoxicity. A full number of cells was obtained at the dose levels of 650 and 700 µg/mL in the second assay. So the observation of 700 µg/mL was only conducted.

 

Table 7.6.1/3: Mitotic index results of the second main assay

 

Treatment

Dose (µg/mL)

Time (hour)

S9 mix

Mitotic index (%)

Direct assay

DMSO

0

24

NA

5.0

P#620

100

1.2

MMC

0.03

4.1

DMSO

0

48

4.2

P#620

90

1.9

MMC

0.03

3.4

Metabolic activation assay

DMSO

0

6+18

 

-

5.9

P#620

100

-

2.0

BP

20

-

5.1

DMSO

0

+

6.6

P#620

700

+

3.2

BP

20

+

0.9

 

Table 7.6.1/4:Results of chromosome analysis, Direct first assay, 24h exposure

 

DMSO

P#620

MMC

 

Low dose

Mid dose

High dose

24h exposure

Gaps

0.0

0.0

0.5

0.0

1.0

Chromatid aberrations

breaks

0.0

0.0

0.0

0.0

20.5

interchanges

0.0

0.0

0.0

0.5

18.5

Isochromatid aberrations

breaks

0.5

0.5

0.0

0.0

3.0

interchanges

0.0

0.0

0.0

0.0

0.5

Polyploidy

0.5

0.0

1.0

0.5

0.0

48h exposure

Gaps

0.5

0.5

0.0

0.0

1.0

Chromatid aberrations

breaks

0.0

2.0

0.0

0.0

23.0

interchanges

0.5

1.0

1.0

0.0

17.5

Isochromatid aberrations

breaks

0.5

1.5

0.5

0.0

4.5

interchanges

0.0

0.0

0.0

0.0

0.5

Polyploidy

0.0

0.5

0.0

0.0

1.0

 

Table 7.6.1/5:Results of chromosome analysis, Direct second assay

 

DMSO

High dose P#620

MMC

24h exposure

Gaps

0.5

0.0

0.5

Chromatid aberrations

breaks

0.0

0.0

12.5

interchanges

0.0

0.0

7.5

Isochromatid aberrations

breaks

0.0

0.0

0.0

interchanges

0.0

0.0

0.0

Polyploidy

1.0

1.5

1.0

48h exposure

Gaps

0.0

0.0

1.0

Chromatid aberrations

breaks

0.0

0.5

21.0

interchanges

0.0

0.0

18.0

Isochromatid aberrations

breaks

0.0

0.0

2.0

interchanges

0.0

0.0

0.0

Polyploidy

0.0

0.5

0.0

Table 7.6.1/6:Results of chromosome analysis, Metabolic activation first assay

 

P#620

BP

 

DMSO

Low dose

Mid dose

High dose

- S9 mix

Gaps

0.5

0.0

0.0

0.0

0.0

Chromatid aberrations

breaks

0.5

0.0

1.5

0.0

0.5

interchanges

0.0

0.5

0.0

0.0

0.0

Isochromatid aberrations

breaks

0.0

0.0

0.0

1.0

0.0

interchanges

0.0

0.0

0.0

0.0

0.0

Polyploidy

0.5

0.0

0.0

0.0

0.0

+ S9 mix

Gaps

0.0

0.0

0.0

0.0

0.5

Chromatid aberrations

breaks

0.5

0.5

0.0

0.0

23.0

interchanges

0.0

0.5

0.5

0.0

37.5

Isochromatid aberrations

breaks

0.0

0.0

0.0

0.0

1.0

interchanges

0.0

0.0

0.0

0.0

0.5

Polyploidy

0.5

0.0

1.5

0.0

0.0

Table 7.6.1/7:Results of chromosome analysis, Metabolic activation second assay

 

DMSO

High dose

BP

- S9 mix

Gaps

0.0

0.0

0.0

Chromatid aberrations

breaks

0.0

0.5

0.0

interchanges

0.5

0.0

0.0

Isochromatid aberrations

breaks

0.5

0.0

0.5

interchanges

0.0

0.0

0.0

Polyploidy

0.0

0.5

1.0

+ S9 mix

Gaps

0.0

0.0

0.0

Chromatid aberrations

breaks

0.5

0.0

23.0

interchanges

0.0

0.5

40.5

Isochromatid aberrations

breaks

0.0

0.0

1.5

interchanges

0.0

0.0

0.0

Polyploidy

0.5

2.5

0.0

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Amber Core (P#620) did not show any signs of clastogenic activity either in the presence or absence of S-9 mix, in this in vitro cytogenetic test system according to the criteria of the Annex VI of the Regulation (EC) No 1272/2008 (CLP) and the Annex VI of the Directive 67/548/EC.
Executive summary:

In a chromosomal aberration assay in mammalian cells, performed according to the OECD No.473, and in compliance with the GLP, P#620 (purity >99%) diluted in DMSO was tested in female Chinese Hamster lung cells in the presence and the absence of mammalian metabolic activation (S9)at concentrations of varying from 22.5 to 700 µg/mL.

P#620 was incubated with the cells for 6, 24 or 48 hours and the cells were analysed for the presence of chromosomal aberration 18 hours (in the case of the 6 hrs exposure period) or immediately after the end of the exposure period (in the case of the 24 or 48 hrs exposure period).

Mitomycin C and Benzo(a)pyrene were used as positive controls and induced appropriate responses.

Cytotoxicity was observed at 700 µg/mL with metabolic activation in the first assay. For the second test, the only maximum dose level (100 µg/mL direct assay, 700 µg/mL for the metabolic activation with S9) of the first test was repeated to test because the negative results were obtained in the first test. No increase in the occurence of chromatid or chromosome aberration was observed with and without metabolic activation at any tested concentration and for all exposure period tested.

Under the test conditions, P#620 did not show any cytogenic activity in the chromosomal aberration test using hamster cells according to the criteria of the Annex VI of the Regulation (EC) No 1272/2008 (CLP) and the Annex VI of the Directive 67/548/EC.

This study is considered as acceptable as it satisfied the criteria of the OECD Guideline No. 473.