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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:
29 June 1993 to 16 August 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report Date:
1993

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
other: in vitro cytogenicity / chromosome aberration study in mammalian cells

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: The Merck Institute for Therapeutic Research, West Point, PA
- Cell cycle length, doubling time or proliferation index: Doubling time 14 hours
- Number of passages if applicable: 9 and 10
- Methods for maintenance in cell culture if applicable: Master vials were stored at -80 °C. Working cultures were maintained in a 37 °C, 5 % CO2 and 95 % realtive humidity incubator in culture medium.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: 5 % CO2. Maintained in McCoy's 5A with NaHCO3 buffer, 10 % foetal bovine serum (FBS), 50 units/mL penicillin, 50 µg/mL streptomycin and 2 mL L-glutamine.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 microsome fraction
Test concentrations with justification for top dose:
0.05, 0.15, 0.5, 1.5, 5, 15 and 50 µg/mL - Nonactivated, 24 and 14 hour assays
0.015, 0.05, 0.15, 0.5, 1.5, 5, and 15 µg/mL - Activated, 24 hour assay
0.00051, 0.0015, 0.0051, 0.015, 0.051, 0.15, 0.51, 1.5, 5.1 and 15 µg/mL - Activated, 14 hour assay
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding : Seeded at a cell density of 5E+05 cells per flask

DURATION
- Exposure duration:14 or 24 hours in the nonactivated assay and 2 hours in the activated assay
- Expression time (cells in growth medium): 14 or 24 hours
- Fixation time (start of exposure up to fixation or harvest of cells): Cells were fixed at the end of the expression period

SPINDLE INHIBITOR (cytogenetic assays): Vinblastine sulfate (0.26 µg/mL added 2 hours prior to harvest)

STAIN : 5 % Giemsa

NUMBER OF REPLICATIONS: Performed in duplicate

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: At the end of the incubation period, metaphase cells were collected by treatment with trypsin (0.25 % trypsin and 0.02 % EDTA) followed by concentration of cells by centrifugation. Due to the presence of the test material and positive control in the flasks in the nonactivated assay, these cells were washed with phosphate buffered saline (PBS) solution (pH 7.3) or with medium. The cells for all assays were suspending in hypotonic solution (0.03 M KCl and 0.01 M sodium citrate) for 12 minutes at 37 °C and fixed 3 times 3:1 methanol:acetic acid by centrifugation and resuspension. Drops of the concentrated cell suspension were placed on glass slides, air dried and stained in 5 % Giemsa for approximately 5 minutes at room temperature.

NUMBER OF CELLS EVALUATED: 100

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 100

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (a minimum of 500 cells were counted)
Rationale for test conditions:
Two range finder assays were performed to determine toxic concentrations of the test material in the presence of metabolic activation in the test system. The first range finding assay was performed using concentrations of 523, 1577 and 5257 µg/mL. The second assay was performed with concentrations of 1.4 and 148 µg/mL. The test concentrations of the definitive test were selected based of the results of the two range finding tests.
Evaluation criteria:
Metaphase cells were analysed for chromosomal aberrations through a microscope using a 100 x objective. The mitotic index was determined by counting a minimum of 500 total cells. A minimum of 25 cells were analysed for each positive control flask. In the 14 hour activated assay, the highest test concentration, 15 µg/mL, was analysed uncoded due to the low number of metaphase cells (0.7 %) observed coupled with the low percentage of confluency noted (less than 10 %, toxic spots). One hundred metaphase cells per flask for at least three selected test sample concentrations and the negative control were analysed for the following types of chromosomal aberrations:
Chromatid gap
Chromatid break
Chromosome gap
Chromosome break
Quadriradial
Interstitial deletions
Double minute chromosomes
Triradial
Dicentric chromosome
Cell with at least one pulverised chromosome
Complex rearrangements
Rings
Cells with greater than 10 aberrations
A positive response was determined by a statistically significant increase in aberrations.
Statistics:
Coordinates of cells with aberrations were recorded.
% Cells with aberrations = (Number of cells with aberrations/total number of cells analysed) * 100
Number of Aberrations per Cell = Total number aberrations/Total cells analysed
% Mitotic Index (MI) = (Number of metaphase cells/Total number of cells) * 100
The number of aberrations per cell and the percent of cells with aberrations were calculated by two methods. In one calculation the number of chromatid and chromosome gaps were counted as aberrations. IN the other calculation, the more traditional method was followed in which gaps were not counted as aberrations. A cell with a large number of aberrations was counted as having 10 aberrations. No cells with pulverised chromosomes were observed.
The data were analysed statistically using the method described in Margolin et al (1986) Statistical Analyses for In Vitro Cytogenetic Assays Using Assays Chinese Hamster Ovary Cells, Environ. Mutagen. 8: 183-204.

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: The test material was found to be cytotoxic at concentrations greater than 49.3 µg/mL in the presence of metabolic activation.

Any other information on results incl. tables

Table 1: Results of the range finding studies

Concentration
(µg/mL)

1st Range Finding Study
(Mitotic Index)

2nd Range Finding Study
(Mitotic Index)

5257

Toxic

1577

Toxic

523

Toxic

148

Toxic

49.3

Toxic

14.8

11/543 (2.0 %)

5/506 (0.79 %)

4.93

18/506 (3.6 %)

16/545 (2.9 %)

1.48

13/534 (2.4 %)

17/514 (3.3 %)

1 % DMSO

35/498 (7.0 %)

14/509 (2.8 %)

28/514 (5.4 %)

10/502 (2.0 %)

 

Table 2: 14-hour assay without metabolic activation (individual results)

Treatment

No. Cells

Analysed

%

Mitotic Index

Number and type of chromosome aberration

Simple

Complex

Other

TB

SB

DM

ID

TR

QR

D

R

CR

PU

10+

TG

SG

Mitomycin C

0.5 µg/mL

25

2.6

12

3

0

1

4

3

0

0

0

0

0

8

3

25

1.8

9

5

0

1

7

4

0

0

0

0

1

6

1

DMSO

1.00%

100

10.4

1

0

0

0

0

0

0

0

0

0

0

4

1

100

6.4

0

0

0

0

0

0

1

0

0

0

0

1

0

Test material

1.5 µg/mL

100

9.4

1

2

0

0

0

0

1

0

0

0

0

2

1

100

7.2

2

0

0

0

0

0

0

0

0

0

0

4

0

5.0 µg/mL

100

11.8

0

0

0

0

0

0

0

0

0

0

0

3

0

100

4.8

0

1

0

0

0

0

0

0

0

0

0

4

0

15 µg/mL

100

3.7

0

1

0

0

0

0

0

1

0

0

0

7

0

100

8.7

0

3

0

0

0

0

0

0

0

0

0

3

0

50 µg/mL

Toxic

Chromatid gap – TG; Chromatid break – TB; Chromosome gap – SG; Chromosome break – SB; Quadriradial – QR; Interstitial deletions – ID; Double minute chromosomes – DM; Triradial – TR; Dicentric chromosome – D; Cell with at least one pulverised chromosome – PU; Complex rearrangements – CR; Rings – R; Cells with greater than 10 aberrations – 10+

 

Table 3: 14-hour assay without metabolic activation (summary)

Treatment

No. Cells Analysed

Gaps Excluded

Gaps Included

Total No. Aberrations

No. Aberrations Per Cell

% Cells with Aberrations

Total No. Aberrations

No. Aberrations Per Cell

% Cells with Aberrations

Mitomycin C

0.5 µg/mL

25

23

0.92

56

34

1.36

68

25

36*

1.44

68

43*

1.72

72

DMSO

1.00%

100

1

0.01

1

6

0.06

5

100

1

0.01

1

2

0.02

2

Test material

1.5 µg/mL

100

4

0.04

3

7

0.07

6

100

2

0.02

2

6

0.06

5

5.0 µg/mL

100

0

0

0

3

0.03

3

100

1

0.01

1

5

0.05

5

15 µg/mL

100

4

0.04

4

11

0.11

10

100

3

0.03

2

6

0.06

4

50 µg/mL

Toxic

* Data indicated pulverised cells and/or damaged cells with more than 10 chromosome aberrations per cell

 

Table 4: 24-hour assay without metabolic activation (individual results)

Treatment

No. Cells Analysed

% Mitotic Index

Number and type of chromosome aberration

Simple

Complex

Other

TB

SB

DM

ID

TR

QR

D

R

CR

PU

10+

TG

SG

Mitomycin C

0.3 µg/mL

25

1.6

10

2

0

2

3

5

0

3

9

0

1

3

1

25

1.2

20

4

0

2

9

6

0

1

5

0

0

7

1

DMSO

1.00%

100

6.0

1

2

0

0

0

0

1

0

0

0

0

3

1

100

7.0

0

3

0

0

0

0

0

1

0

0

0

0

0

Test material

1.5 µg/mL

100

6.0

1

0

0

0

0

0

0

0

0

0

0

1

0

100

5.8

0

2

0

0

0

0

0

0

0

0

0

0

0

5.0 µg/mL

100

5.9

0

2

0

0

0

0

2

0

0

0

0

1

0

100

3.9

0

2

0

0

0

0

0

0

0

0

0

1

0

15 µg/mL

100

6.0

0

1

0

0

0

0

0

0

0

0

0

1

0

100

6.3

1

2

0

0

0

0

0

0

0

0

0

1

0

50 µg/mL

Toxic

Chromatid gap – TG; Chromatid break – TB; Chromosome gap – SG; Chromosome break – SB; Quadriradial – QR; Interstitial deletions – ID; Double minute chromosomes – DM; Triradial – TR; Dicentric chromosome – D; Cell with at least one pulverised chromosome – PU; Complex rearrangements – CR; Rings – R; Cells with greater than 10 aberrations – 10+

 

Table 5: 24-hour assay without metabolic activation (summary)

Treatment

No. Cells Analysed

Gaps Excluded

Gaps Included

Total No. Aberrations

No. Aberrations Per Cell

% Cells with Aberrations

Total No. Aberrations

No. Aberrations Per Cell

% Cells with Aberrations

Mitomycin C

0.5 µg/mL

25

44*

1.76

84

48*

1.92

84

25

47

1.88

72

55

2.20

76

DMSO

1.00%

100

4

0.04

3

8

0.08

6

100

4

0.04

3

4

0.04

3

Test material

1.5 µg/mL

100

1

0.01

1

2

0.02

2

100

2

0.02

2

2

0.02

2

5.0 µg/mL

100

4

0.04

4

5

0.05

5

100

2

0.02

2

3

0.03

3

15 µg/mL

100

1

0.01

1

2

0.02

2

100

3

0.03

2

4

0.04

3

50 µg/mL

Toxic

* Data indicated pulverised cells and/or damaged cells with more than 10 chromosome aberrations per cell

 

Table 6: 14-hour assay with metabolic activation (individual results)

Treatment

No. Cells Analysed

% Mitotic Index

Number and type of chromosome aberration

Simple

Complex

Other

TB

SB

DM

ID

TR

QR

D

R

CR

PU

10+

TG

SG

Benzo(a)pyrene

15 µg/mL

50

0.6

8

1

0

0

3

1

0

0

1

0

0

4

0

50

2.9

4

7

0

5

7

0

0

2

0

0

0

6

2

DMSO

1.00%

100

4.4

0

6

0

0

0

0

0

1

0

0

0

1

0

100

2.4

1

8

0

0

1

0

0

1

0

0

0

5

0

Test material

0.51 µg/mL

100

2.8

4

9

0

0

0

0

0

1

0

0

1

3

1

100

2.2

0

6

0

0

0

0

0

2

0

0

0

3

0

1.5 µg/mL

100

4.3

0

1

0

0

0

0

0

0

0

0

0

4

0

100

2.4

3

4

0

0

1

0

0

0

0

0

0

4

0

5.1 µg/mL

100

3.8

0

2

0

0

0

0

0

1

0

0

1

5

1

100

2.0

2

3

1*

0

0

0

0

0

0

0

0

6

0

15 µg/mL

100

2.2

1

4

0

0

0

0

0

2

0

0

0

0

1

100

0.7

2

0

0

0

0

0

0

1

0

0

0

2

1

Chromatid gap – TG; Chromatid break – TB; Chromosome gap – SG; Chromosome break – SB; Quadriradial – QR; Interstitial deletions – ID; Double minute chromosomes – DM; Triradial – TR; Dicentric chromosome – D; Cell with at least one pulverised chromosome – PU; Complex rearrangements – CR; Rings – R; Cells with greater than 10 aberrations – 10+
* 4 pairs of double minutes in one cell

 

Table 7: 14-hour assay with metabolic activation (summary of results)

Treatment

No. Cells Analysed

Gaps Excluded

Gaps Included

Total No. Aberrations

No. Aberrations Per Cell

% Cells with Aberrations

Total No. Aberrations

No. Aberrations Per Cell

% Cells with Aberrations

Benzo(a)pyrene

15 µg/mL

50

14

0.28

24

18

0.36

30

50

25

0.50

36

33

0.66

44

DMSO

1.00%

100

7

0.07

5

8

0.08

6

100

11

0.11

7

16

0.16

11

Test material

0.51 µg/mL

100

25*

0.25

6

29*

0.29

7

100

8

0.08

2

11

0.11

5

1.5 µg/mL

100

1

0.01

1

5

0.05

5

100

9

0.09

7

13

0.13

11

5.1 µg/mL

100

13*

0.13

4

19*

0.19

9

100

7

0.07

5

13

0.13

10

15 µg/mL

100

7

0.07

6

8

0.08

7

100

4

0.04

4

7

0.07

5

* Pulverised cells/and or damaged cells with more than ten chromosome aberrations per cell.

 

Table 8: 24-hour assay with metabolic activation (individual results)

Treatment

No. Cells Analysed

% Mitotic Index

Number and type of chromosome aberration

Simple

Complex

Other

TB

SB

DM

ID

TR

QR

D

R

CR

PU

10+

TG

SG

Cyclophosphamide

50 µg/mL

25

1.1

20

3

0

3

10

2

1

1

3

0

0

4

1

25

1.9

12

1

0

1

13

4

1

2

10

0

2

0

2

Benzo(a)pyrene

15 µg/mL

25

5.0

0

1

0

0

0

0

0

0

0

0

0

0

1

25

8.5

0

1

0

0

1

0

0

0

0

0

0

0

0

DMSO

1.00%

100

4.8

3

3

0

0

0

0

1

0

0

0

0

1

1

100

7.7

1

5

0

0

0

0

0

1

0

0

0

1

1

Test material

1.5 µg/mL

100

4.9

1

0

0

0

0

0

0

0

0

0

0

0

0

100

5.0

1

3

1*

0

0

0

0

0

0

0

0

1

0

5.1 µg/mL

100

3.8

2

6

0

0

0

0

0

0

0

0

0

1

2

100

5.9

2

2

0

0

0

0

0

0

0

0

0

0

0

15 µg/mL

100

5.1

1

5

0

0

0

0

0

0

0

0

0

1

1

100

3.7

3

4

0

0

0

0

0

0

0

0

0

1

0

Chromatid gap – TG; Chromatid break – TB; Chromosome gap – SG; Chromosome break – SB; Quadriradial – QR; Interstitial deletions – ID; Double minute chromosomes – DM; Triradial – TR; Dicentric chromosome – D; Cell with at least one pulverised chromosome – PU; Complex rearrangements – CR; Rings – R; Cells with greater than 10 aberrations – 10+
* 7 pairs of double minutes in one cell

 

Table 9: 24-hour assay with metabolic activation (summary of results)

Treatment

No. Cells Analysed

Gaps Excluded

Gaps Included

Total No. Aberrations

No. Aberrations Per Cell

% Cells with Aberrations

Total No. Aberrations

No. Aberrations Per Cell

% Cells with Aberrations

Cyclophosphamide

50 µg/mL

25

43

1.72

64

48

1.92

68

25

64*

2.56

92

66*

2.64

92

Benzo(a)pyrene

15 µg/mL

25

1

0.04

4

2

0.08

8

25

2

0.08

8

2

0.08

8

DMSO

1.00%

100

7

0.07

6

9

0.09

8

100

7

0.07

2

9

0.09

4

Test material

1.5 µg/mL

100

1

0.01

1

1

0.01

1

100

5

0.05

4

6

0.06

5

5.1 µg/mL

100

10

0.10

5

13

0.13

6

100

4

0.04

3

4

0.04

3

15 µg/mL

100

7

0.07

6

9

0.09

7

100

8

0.08

6

9

0.09

7

* Pulverised cells/and or damaged cells with more than ten chromosome aberrations per cell.

 

Applicant's summary and conclusion

Conclusions:
The test material was evaluated for its ability to induce chromosomal damage in vitro using CHO cells. The test material was tested with and without metabolic activation. The positive controls were found to be valid under the conditions of the test. Under the conditions of the test, there were no increases in the percentage of aberrant cells (including and excluding gaps) with or without metabolic activation.
The test material was therefore concluded to not be clastogenic under the conditions of the test.
Executive summary:

The clastogenicity of the test material was evaluated in an in vitro mammalian chromosome aberration test conducted to a method comparable to OECD 473. Chinese hamster ovary cells were exposed to the test material in the presence and absence of metabolic activation (S9) up to cytotoxic concentrations. Under the conditions of the test, the test material was concluded to be non-clastogenic in the presence and absence of metabolic activation.