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Genetic toxicity: in vitro

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Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 December 1994 to 28 February 1995
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report date:
1995

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: The Notification on Partial Revision of Testing Methods Relating to New Chemical Substances
Version / remarks:
Notification No. 700 of Kanpogyo, No.1039 of Yakuhatsu and No.1014 of 61 Kikyoku, December 5, 1986
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
439-910-1
EC Name:
-
Cas Number:
93705-66-9
Molecular formula:
C23H28O4
IUPAC Name:
2,2'-(methylenebis((2,6-dimethyl-4,1-phenylene)oxymethylene))bis oxirane
Test material form:
solid
Details on test material:
- Appearance: light yellow solid
- Storage conditions: A cold and dark place

Method

Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: National Institute of Hygienic Sciences. Cells of passage number 18 were supplied.
- Suitability of cells: Chinese hamster lung fibroblasts (CHL cells, clone No. 11) were used in this test because they are widely employed, for in vitro chromosomal aberration tests, show quite high sensitivity to chemical mutagens, and a large amount of data is available about their chromosomal aberrations.
- The modal number of chromosomes is 25 per cell. The time required for doubling of cell number is about 15 hours.
- CHL cells preserved by freezing were defrosted and cultured. Two or three days after incubation, a subculture was made, and the cells in logarithmic growth phase were used for the test.
- The passage number of cells used in the chromosomal aberration test was 40 for 24 hours treatment, 40 for 48 hours treatment by the direct method, and 23 by the metabolic activation method.

MEDIA USED
- Eagle's minimum essential medium was supplemented with new-born calf serum at a rate of 10 v/v %. This medium is referred to hereafter as 10 % NCS/MEM.
- Purified water for preparation of the medium was obtained from a water ultra purifier, through which distilled water prepared with a glass still was passed.
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix
Test concentrations with justification for top dose:
24 h treatment by the direct method: 20, 25 and 30 μg/mL
48 h treatment by the direct method: 20, 25 and 30 μg/mL
Metabolic activation method: 200, 215 and 230 μg/mL
The highest concentration decided in the dose determination test was employed as the maximum dose for the chromosomal aberration test. The test was carried out at three dose levels prepared by diluting the maximum dose using a arithmetical progression.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethylsulfoxide
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
CELL CULTURE
- Five mL of 10 % NCS/MEM containing 1.5 x 10^4 or 0.5 x 10^4 cells/mL was seeded in a 60 mm diameter Petri dish and cultured for two or three days, respectively. Monolayer cells in logarithmic growth phase were exposed to the test material two or three days after culturing.
- Throughout the study, the incubation of the cells was conducted in a CO2 incubator controlled at 37+0.5°C, 5% of CO2 and nearly 100% relative humidity.

PREPARATION OF THE TEST MATERIAL AND THE POSITIVE CONTROL MATERIAL SOLUTIONS
- Test Material: The test material was dissolved in dimethylsulfoxide. The original solution was diluted with the same solvent to make the required concentrations. Test material solutions were prepared immediately before use and used within 2 hours. To the medium, 1% solution was added.
- Positive Control Material: Positive control materials were dissolved in pure water, and diluted with the same solvent to make the required concentrations. Positive control material solutions were prepared immediately before use and used within 2 hours.

PREPARATION OF MICROSCOPE SLIDES
- Cells were removed from each dish with 0.25% trypsin, and collected by centrifugation in a tube. Then, 0.075 M KCl was added to the tube, and hypotonic treatment was carried out at 37°C for 15 minutes. Methanol-acetic acid (3:1) solution was then poured into the tube to fix the cells and make a slightly cloudy cell suspension, which is then plated onto microscope slides and spread. The cells on each slide were finally dried and stained with 2% Giemsa solution. Two slides per dish were prepared.

DOSE DETERMINATION TEST
- Preliminary tests for cell growth inhibition and cell division inhibition were performed to determine the dose levels for the chromosomal aberration test. In the former test, the 50% growth inhibition concentrations of the test material were determined. In the latter test, the highest concentration at which metaphase cells were practically scorable was determined as the maximum dose for the chromosomal aberration test.
- Cell growth inhibition test: In the test without metabolic activation (direct method), 5 mL of the medium including the test material was added to each dish after removal of the medium from two- or three-day-old cultures, followed by culture for 24 or 48 hours. In the test with metabolic activation, 3 mL of S9 Mix/MEM including the test material was added to each dish after removal of the medium, and treated for 6 hours. After S9 Mix/MEM including the test material had been removed, the dishes were rinsed three times with 2 mL of Ca2+, Mg2+ -free phosphate-buffered saline [PBS(--)]. Then, 5 mL of 10% NCS/MEM was added to each dish, and the cells were cultured for 18 hours. After removal of the medium at the end of incubation, the cells were detached from each dish with 2 mL of 0.25% trypsin, and counted using a Microcell Counter F-500. Two dishes were used for each dose, and the cells were counted in each respective dish.
- Cell division inhibition test: Cell cultures and treatments with the test material for both the direct and metabolic activation methods were done using the same procedure as that for the cell growth inhibition test. Two hours before the end of incubation, colcemid was added to the medium to give a concentration of 0.1 μg/mL. The chromosome preparations were made and observed microscopically for the incidence of mitotic cells.

CHROMOSOMAL ABERRATION TEST
- The highest concentration decided in the dose determination test was employed as the maximum dose for the chromosomal aberration test. The test was carried out at three dose levels prepared by diluting the maximum dose using an arithmetical progression.
- The following concentrations were used in the positive controls, based on background data: Direct method 24 and 48 hour treatment: MMC 0.05 µg/mL. Metabolic activation method: CPA 10 μg/mL.
- Direct Method: After removal of the medium from three-day-old cultures, 5 mL of the medium including the test material was added and incubated for 24 or 48 hours. Two hours before the end of incubation, colcemid was added to the medium to give a concentration of 0.1 μg/mL. Then chromosome preparations were made. A solvent-treated group and a MMC-treated group were used as a negative and a positive control, respectively.
- Metabolic activation method: After removal of the medium from three-day-old cultures, 3 mL of S9 Mix/MEM including the test material was added, followed by incubation for 6 hours. After removal of the S9 Mix/MEM, the dishes were rinsed three times with 2 mL of PBS(-), and 5 mL of 10% NCS/MEM was added. The cells were cultured for 18 hours. Two hours before the end of incubation, colcemid was added to give a concentration of 0.1 μg/mL. Then, chromosome preparations were made. A solvent-treated group and a CPA-treated group were used as a negative and a positive control, respectively. To clarify the effect of metabolic activation, a culture without S9 Mix was treated with the test material at the same dose levels and the same treatment time as those for the control of this test.
- Two dishes were chosen at random to minimise any variation in results, and used for each dose. Preparation was conducted in each respective dish. The test code number, treatment method and dose level were noted on the dishes for identification of each culture.

OBSERVATION AND SCORING OF CHROMOSOMAL ABERRATIONS
- The numbers of cells with chromatid or chromosome-type structural aberrations (such as gaps, breaks, exchanges, etc.) and with numerical aberrations (polyploid, endo-reduplication) were checked by observing 100 metaphases for each dish. The incidences of cells with numerical and/or structural aberrations were obtained by observing 200 metaphases for each group. The incidence of structural aberration was calculated with and without gaps, respectively.
- A gap was scored when a clear discontinuity (larger than a chromatid width) was evident, and when the distal part of the chromatid or chromosome showed no dislocation. Slides were all coded and blind-tested by microscopy.
Evaluation criteria:
EVALUATION OF RESULTS
- The results obtained were assessed as follows. The incidence of cells (mean value for two dishes) with aberrations including gaps was:
less than 5 % = negative ( - )
5% or more and less than 10% = suspect positive (±)
10% or more and less than 20% = positive (+)
20% or more and less than 50% positive (++)
50% or more = positive (+++)
- The test material is judged to be positive for induction of chromosomal aberration when the incidence of 10% or more was dose related or reproducible. If it was judged to be positive, the D20 value, which meant the dose being inducible the chromosomal aberration of 20%, was calculated. Re-examination was performed whenever the result was suspect positive, positive at only one dose level, or not evidently dose-related.

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
VALIDITY OF THE TEST
- The values for two dishes were not markedly different, the incidence of cells with any aberration did not exceed 5% in the negative control, the incidence of cells with structural aberrations excluding gap was 10% or more in the positive control. There were no fluctuations in the test conditions and no contamination with microorganisms in the cultures which might have affected the results. The test results were therefore considered to be valid.

CELL GROWTH INHIBITION TEST AND CELL DIVISION INHIBITION
- The growth rate in the solvent control was considered to be 100%. Fifty percent growth inhibition concentrations of the test material were about 28 μg/mL in the 24 hour treatment, about 16 μg/mL in the 48 hour treatment by the direct method, and about 205 μg/mL by the metabolic activation method with S9 Mix. Mitotic metaphases of chromosomes sufficient for assessing chromosomal aberration were observed at doses of 30 μg/mL or less in 24 and 48 hour treatment by the direct method, and 230 μg/mL or less by the metabolic activation method with S9 Mix.
- Cells and their induction of chromosomal aberration were observed microscopically. Based upon the results, the following 3 dose levels were employed in each method of chromosomal aberration test, in order to examine dose-dependent reaction of chromosomal aberration:
24 h treatment by the direct method: 20, 25 and 30 μg/mL
48 h treatment by the direct method: 20, 25 and 30 μg/mL
Metabolic activation method: 200, 215 and 230 μg/mL

CHROMOSOMAL ABERRATION TEST
Direct method
- 24 hour treatment: The incidences of cells with structural aberrations including gap were 1.5% at 20 μg/mL, 9.5% at 25 μg/mL and 18.5% at 30 μg/mL. The incidence in the solvent control was 1.5%, i.e. within the normal range. The positive control treated with MMC showed the structural aberrations of 39.5%. The incidences of polyploid cells at any doses were less than 5%.
- 48 hour treatment: The incidences of cells with structural aberrations including gap were 1.5% at 20 μg/mL, 3.5% at 25 μg/mL, and 3.5% at 30 μg/mL. The incidence in the solvent control was 0.5%, i.e. within the normal range. The positive control treated with MMC showed the structural aberration of 51. 5%. The incidences of polyploid cells at any doses were less than 5%.

Metabolic activation method
- With S9 Mix: The incidences of cells with structural aberrations including gap were 2.5% at 200 μg/mL, 6.5% at 215 μg/mL and 11.0% at 230 μg/mL. The incidence in the solvent control was 1.0%, i.e. within the normal range. The positive control treated with CPA showed the structural aberration of 27.5%. The incidences of polyploid cells at any doses were less than 5%.
- Without S9 Mix: The incidences of cells with structural aberrations including gap at 200, 215 and 230 μg/mL of the test material were unable to be observed due to cytotoxicity. On the other hand, the appearance rates in the solvent and positive control groups were 0 and 1.5%, respectively, which were within a normal range. The incidences of polyploid cells at any doses were less than 5%.

Any other information on results incl. tables

Table 1: Results of Chromosomal Aberration Test (Without Metabolic Activation)

Treatment

Time (h)

Concentration (µg/mL)

Observed

No. of Cells

No. and % of cells showing structural chromosomal aberrations

No. of Polyploids

Judgement

Gap (g)

Chromatid type

Chromosome type

Others

Total

Evaluation

ctb

cta

csb

cse

-g

+g

Solvent (DMSO)

24

0

100

1

 

0

0

0

0

2

0

2

2

 

100

2

0

0

0

0

1

0

1

1

200

3 (1.5)

0 (0.0)

0 (0.0)

0 (0.0)

0 (0.0)

3 (1.5)

0 (0.0)

3 (1.5)

3 (1.5)

48

0

100

1

 

0

0

0

0

0

0

0

0

 

100

0

0

0

1

0

0

0

1

1

200

1 (0.5)

0 (0.0)

0 (0.0)

1 (0.5)

0 (0.0)

0 (0.0)

(0.0)

1 (0.5)

1 (0.5)

Test Material

24

20

100

0

-

0

1

0

0

0

0

1

1

-

100

1

0

0

2

0

0

0

2

2

200

1 (0.5)

0 (0.0)

1 (0.5)

2 (1.0)

0 (0.0)

0 (0.0)

0 (0.0)

3 (1.5)

3 (1.5)

25

100

0

-

1

4

3

1

2

0

9

10

±

100

1

3

4

3

1

0

0

6

9

200

1 (0.5)

4 (2.0)

8 (4.0)

6 (3.0)

2 (1.0)

2 (1.0)

0 (0.0)

15 (7.5)

19 (9.5)

30

100

0

-

2

7

5

1

0

0

12

14

+

100

0

3

13

11

0

0

0

22

23

200

0 (0.0)

5 (2.5)

20 (10.0)

16 (8.0)

1 (0.5)

0 (0.0)

0 (0.0)

34 (17.0)

37 (18.5)

48

20

100

0

-

0

0

1

0

0

0

1

1

-

100

0

0

0

1

0

1

0

2

2

200

0 (0.0)

0 (0.0)

0 (0.0)

2 (1.0)

0 (0.0)

1 (0.5)

0 (0.0)

3 (1.5)

3 (1.5)

25

100

1

-

1

0

0

0

1

0

1

2

-

100

0

2

0

2

1

0

0

3

5

200

1 (0.5)

3 (1.5)

0 (0.0)

2 (1.0)

1 (0.5)

1 (0.5)

0 (0.0)

4 (2.0)

7 (3.5)

30

100

1

-

1

2

1

0

0

0

2

3

-

100

0

1

0

2

0

1

0

3

4

200

1 (0.5)

2 (1.0)

2 (1.0)

3 (1.5)

0 (0.0)

1 (0.5)

0 (0.0)

5 (2.5)

7 (3.5)

Positive Control (MMC)

24

0.05

100

0

-

0

9

33

1

0

0

40

40

++

100

0

0

5

36

0

0

0

39

39

200

0 (0.0)

0 (0.0)

14 (7.0)

69 (34.5)

1 (0.5)

0 (0.0)

0 (0.0)

79 (39.5)

79 (39.5)

48

0.05

100

1

-

3

9

44

0

2

0

50

53

+++

100

0

3

14

37

0

4

0

49

50

200

1 (0.5)

6 (3.0)

23 (11.5)

81 (40.5)

0 (0.0)

6 (3.0)

0 (0.0)

99 (49.5)

103 (51.5)

 

 

Table 2: Results of Chromosomal Aberration Test (With Metabolic Activation)

Treatment

S9 Mix

Concentration (µg/mL)

Observed

No. of Cells

No. and % of cells showing structural chromosomal aberrations

No. of Polyploids

Judgement

Gap (g)

Chromatid type

Chromosome type

Others

Total

Evaluation

ctb

cta

csb

cse

-g

+g

Solvent (DMSO)

-

0

99

1

 

0

0

0

0

0

0

0

0

 

100

0

0

0

0

0

0

0

0

0

199

1 (0.5)

0 (0.0)

0 (0.0)

0 (0.0)

0 (0.0)

0 (0.0)

0 (0.0)

0 (0.0)

0 (0.0)

+

0

100

2

 

0

0

0

0

0

0

0

0

 

100

0

1

0

1

0

0

0

1

2

200

2 (1.0)

1 (0.5)

0 (0.0)

1 (0.5)

0 (0.0)

0 (0.0)

(0.0)

1 (0.5)

2 (1.0)

Test Material

-

200

Mitotic metaphases were not obtained because of cytotoxicity

215

Mitotic metaphases were not obtained because of cytotoxicity

230

Mitotic metaphases were not obtained because of cytotoxicity

+

200*

100

3

-

2

1

0

0

0

0

1

3

-

100

2

0

1

0

0

1

0

2

2

200

5 (2.5)

2 (1.0)

2 (1.0)

0 (0.0)

0 (0.0)

1 (0.5)

0 (0.0)

3 (1.5)

5 (2.5)

215*

100

1

-

0

3

1

0

0

0

4

4

±

100

3

0

4

6

0

1

0

9

9

200

4 (2.0

0 (0.0)

7 (3.5)

7 (3.5)

0 (0.0)

1 (0.5)

0 (0.0)

13 (6.5)

13 (6.5)

230*

100

1

-

4

1

13

0

0

0

13

14

+

100

0

1

0

6

0

1

0

7

8

200

1 (0.5)

5 (2.5)

1 (0.5)

19 (9.5)

0 (0.0)

1 (0.5)

0 (0.0)

20 (10.0)

22 (11.0)

Positive Control (CPA)

-

10

100

1

-

0

0

0

0

3

0

3

3

-

100

0

0

0

0

0

0

0

0

0

200

1 (0.5)

0 (0.0)

0 (0.0)

0 (0.0)

0 (0.0)

3 (1.5)

0 (0.0)

3 (1.5)

3 (1.5)

+

10

100

1

-

0

11

19

2

1

0

30

30

++

100

1

0

2

24

0

0

0

25

25

200

2 (1.0)

0 (0.0)

13 (6.5)

43 (21.5)

2 (1.0)

1 (0.5)

0 (0.0)

55 (27.5)

55 (27.5)

* Precipitation of the test material was observed in culture medium.

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study the test material induced chromosomal aberrations in Chinese hamster lung fibroblasts.
Executive summary:

The genetic toxicity of the test material was investigated in a chromosomal aberration test using Chinese hamster lung fibroblasts (CHL cells). The test was performed in accordance with ‘The Notification on Partial Revision of Testing Methods Relating to New Chemical Substances’ (Notification No. 700 of Kanpogyo, No.1039 ofYakuhatsu and No.1014 of61 Kikyoku, December 5, 1986). The testing was performed under GLP conditions.

Mitomycin C (MMC) and cyclophosphamide (CPA) were employed as the positive controls for the tests without and with metabolic activation, respectively. Metabolic activation was provided in the form of S9-mix.

A cell growth inhibition test and cell division inhibition test were carried out to determine the appropriate dose levels of the test material. From the results of these tests, chromosomal aberration tests were carried out using 20, 25 and 30 µg/mL of the test material for 24 and 48 hour treatment by the direct method (without metabolic activation), and 200, 215 and 230 µg/mL by the metabolic activation method. As the positive control, MMC was employed at 0.05 µg/mL for 24 and 48 hour treatment by the direct method, and CPA at 10 µg/mL by the metabolic activation method.

The test material induced structural aberrations dose-dependently within the dose range of 25 - 30 μg/mL in the 24 hour treatment, and within the dose range of 215 - 230 μg/mL by the metabolic activation method. D20 value of 24 hour treatment group in the direct method and with S9 Mix treatment group by the metabolic activation method were 34, and 280 μg/mL, respectively. MMC and CPA induced evident chromosomal aberrations.

Under the conditions of this study the test material induced chromosomal aberration in Chinese hamster lung fibroblasts.