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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
Testing Method Concerning New Chemical Substances etc
(November 21, 2003, Joint Notice, Director of Pharmaceutical and Food Safety Bureau,
the Ministry of Health, Labour and Welfare, Yakushokuhatsu No. 1121002, Director of
Manufacturing Industries Bureau, the Ministry of Economy, Trade and Industry,
Heisei 15-11-13 Seikyoku No. 2 and Director of Environmental Policy Bureau, the
Ministry of the Environment, Kanpokihatsu No. 031121002)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
Lithium phosphorodifluoridate
EC Number:
643-080-8
Cas Number:
24389-25-1
Molecular formula:
LiPO2F2
IUPAC Name:
Lithium phosphorodifluoridate
Test material form:
solid: particulate/powder
Details on test material:
Batch number: 250407K2

Method

Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: DS Pharma Biomedical Co., Ltd.
- Doubling time: 15.5 hours
- Number of passages:
At purchasing: 14
At thawing: 17
At using: 18 to 23 (3 to 22 days after thawing, date of thawing is Day 0)
- Methods for maintenance in cell culture if applicable:
DMSO was added to the medium at the final ratio of 10 v/v%. Cells were suspended in
the medium, subdivided to approximately 1 mL, frozen and transferred to liquid
nitrogen storage vessel on April 17, 2007. The sample was thawed prior to the test or
during test period, cultured and shared with other tests.
- Modal number of chromosomes: 25

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
MEM ((Eagle MEM Liquid Nissui (Nissui Pharmaceutical Co., Ltd.))
MEM medium ((Inactivated (heat treatment at 56°C for 30 minutes) bovine serum
Invitrogen Corp.) was added to MEM at the ratio of 10 v/v%.)
Vessel: Plastic plate (diameter: 6 cm and 10 cm; Beckton Dickinson and
Company)
Temperature: 37°C
CO2 concentration: 5%
Humidity: Under humidified condition
Incubator: Carbon Dioxide Cell Incubator (Jouan S.A., type 6301C and 7300)
- Periodically checked for Mycoplasma contamination: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 rat liver
Test concentrations with justification for top dose:
-S9 mix: 156, 313, 625, 1250, 2500 and 5000 μg/mL
+S9 mix: 156, 313, 625, 1250, 2500 and 5000 μg/mL
24-hour treatment: 39.1, 78.1, 156, 313, 625 and 1250 μg/mL

No cytotoxicity was seen in the short-term treatment and therefore the max. dose of 5000 µg/mL was applied in the mian test. Cytotoxicity was seen in the 24-hour treatment at 1100 µg/mL and therefore 1250 µg/mL was selected as the top dose for this test.
Vehicle / solvent:
DMSO
Physiological saline was not used in the vehicle examination because hydrolyzation of the test substance was likely. As the result of vehicle examination, the test substance was not suspended in DMSO at 500 mg/mL but suspended at 250 mg/mL. Exothermic, bubbling and fading were not observed. Therefore, DMSO was selected as the vehicle (negative control material) for the test substance.
Controls
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: Each 5 mL of cell suspension prepared at 4 x 10^3 cell/mL was inoculated onto 6 cm plate and pre-incubated for 3 days. Two plates were used for each treatment conditions and each treatment doses.
- Exposure duration: Cells were treated for 6 hours in short term treatment and for 24 hours in continuous treatment. For short term treatment, cell surface was washed for three times with MEM after 6-hour treatment, and 5 mL of fresh MEM medium was added and incubation was continued for further 18 hours.

STAIN (for cytogenetic assays):
cells were stained with 3 v/v% Giemsa solution for 10 minutes.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
(1) At 2 hours before the end of treatment, colcemid was added to each plate to make the final dose of 0.1 μg/mL and metaphase cells were accumulated.
(2) After treatment was completed, surface of the cells were washed with PBS(-).
(3) Cells were separated by treatment of 0.25 w/v% trypsin.
(4) Cell suspension was taken into centrifuge tube and cells were collected by centrifugation (1000 rpm, 5 minutes; following procedure was done under the same condition).
(5) After removal of supernatant, 4 mL of 0.075 mol/L potassium chloride solution was added into each centrifuge tube and hypotonically treated (37°C, 15 minutes).
(6) A 0.5 mL of cooled fixative (mixture of methanol and acetic acid [3:1, v/v]) was added, mixed and centrifuged to remove supernatant.
(7) A 4 mL of fixative was added, mixed and centrifuged to remove supernatant.
(8) The operation of (7) was repeated.
(9) Cells were suspended in appropriate volume of fixative.
(10) The suspension was dropped onto 2 sites on the slide glass which was placed on wet towel and the slide glass was dried. Two specimens were prepared per plate.
(11) The specimen was stained with 3 v/v% Giemsa solution for 20 minutes, washed with water and dried.
(12) The slide glass was enclosed with cover glass and sealing agent.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells):
More than 50 metaphase cells in total are obtained from 2 specimens which are prepared from each plate.

DETERMINATION OF CYTOTOXICITY
- Method: measurement of cell growth
Cell growth rate was determined using monolayer cultured cell densitometer (monocellater, Olympus Optical Co., Ltd.,) for each plate which was washed with water, dried and stained.
Rationale for test conditions:
In accordance with guideline
Evaluation criteria:
Negative: Frequency of cells with structural or numerical chromosome aberration is below 5% in all test substance treatment groups.
False-positive: Frequency of cells with structural or numerical chromosome aberration is over 5% and below 10% in any of test substance
treatment groups.
Positive: Frequency of cells with structural or numerical chromosome aberration is over 10% in any of test substance treatment groups and dose-dependent increase tendency is noted.

Evaluation criteria for confirmatory test
Negative: When no reproducibility is noted in clastogenicity.
Positive: When reproducibility is noted in clastogenicity.
Statistics:
None.

Results and discussion

Test resultsopen allclose all
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Remarks on result:
other:
Remarks:
confirmation test

Any other information on results incl. tables

Table 1: Result of Cell Growth Inhibition Test

       Cell growth rate (%)
      Short-term treatment Continuous treatment 
   - S9 mix + S9 mix  24-hour treatment 

 Negative control

(DMSO)

 100.0 100.0  100.0 
 39.1  -  - 104.5 
 78.1 99.0 
 156 100.0  100.0  101.0 
 313  97.8 83.2  61.7 
 625  84.5  71.1 45.8 
 1250  70.2  13.4 38.8 
 2500  56.9 0.0 
 5000 43.1  0.0 

-

Table 2: Results of chromosomal aberration test (short-term treatment)

Treatment/recovery period  S9 mix (-/+) Dose of test item (ug/mL)  No. cells showing structural chromosome aberration (incidence, %)                     No. of gaps Cell proliferation rate (%)  No. of cells showing numerical chromosome aberration (incidence, %)          
       No. cells observed Chromatid break   Chromatid exchange Chromosome break  Chromosome exchange  Fragment   Total no. of aberrations       No. of cells observed Polyploid   Endoreduplication Total no. of aberrant cells (%) 

 6 -18

 -

 Negative control (DMSO)

 100

100

200

0

0

0 (0.0) 

 0

1

1 (0.5)

 0

1

1 (0.5

0

0

0 (0.0) 

0

0

0 (0.0) 

0

2

2 (1.0) 

0

0

101.8

98.2

100.0 

100

100

200 

0

0

0 (0.0) 

0

0

0 (0.0) 

0

0

0 (0.0) 

  

6 -18

 -

 313

 -

 98.2

105.3

101.8

  

6 -18

 -

 625

 -

 -

 -

 94.7

83.0

88.9

  

6 -18

 -

 1250

100

100

200 

1

2

3 (1.5) 

0

1

1 (0.5) 

0

0

0 (0.0) 

0

0

0 (0.0) 

0

0 (0.0)

1

3

4 (2.0) 

0

0

0

87.7

79.5

83.6 

100

100

200 

 0

0

0 (0.0)

0

0

0 (0.0) 

0

0

0 (0.0) 

  

6 -18

 -  2500

100

100

200 

1

1

2 (1.0) 

1

2

3 (1.5) 

0

0

0 (0.0) 

0

0

0 (0.0) 

0

0

0 (0.0) 

2

5 (2.5)

0

0

0

69.0

62.0

65.5 

100

100

200 

0

0

0 (0.0) 

0

0

0 (0.0) 

0

0

0 (0.0) 

  

6 -18

 -

 5000

 100

100

200

15

23

38 (19.0) 

28

40

68 (34.0) 

1

0

1 (0.5) 

1

0

1 (0.5) 

0

0

0

33

45

78 (39) 

0

0

0

46.8

46.8

46.8 

100

100

200 

0

0

0 (0.0) 

0

0

0 (0.0) 

0

0

0 (0.0) 

  

6 -18

-

 Positive control (MMC 0.1)

 100

100

200

28

18

46 (23.0) 

30

25

55 (27.5) 

0

1

1 (0.5) 

0

0

0 (0.0) 

0

0

0 (0.0) 

55

38

93 (46.5) 

0

0

 -

100

100

200 

0

0

0 (0.0) 

0

0

0 (0.0) 

0

0

0 (0.0) 

  

6 -18

 +

Negative control (DMSO)

 100

100

200

0

0

0 (0.0) 

0

0

0 (0.0) 

1

0

1 (0.5) 

0

0

0 (0.0) 

0

0

0 (0.0) 

1

0

1 (0.5) 

0

0

0

 100.0

100.0

100.0

100

100

200 

0

0

0 (0.0) 

0

0

0 (0.0) 

0

0

0 (0.0) 

  

6 -18

 +

 156

 -

 -

 100.0

103.3

101.7

  

6 -18

 +

 313

 -

 -

75.6

78.9

77.2 

  

6 -18

 +

 625

75.6

78.9

77.2 

-

-

  

6 -18

 +

 800

 100

100

200

0

1

1 (0.5) 

2

0

2 (1.0) 

0

0

0 (0.0) 

0

0

0 (0.0) 

0

0

0 (0.0) 

2

1

3 (1.5) 

0

0

0

68.9

72.2

70.6 

100

100

200 

0

1

1 (0.5) 

3

1

4 (2.0)

 3

2

5 (2.5)

  

6 -18

 +

 1000

 100

100

200

 0

2

2 (1.0)

2

1

3 (1.5) 

0

0

0 (0.0) 

0

1

1 (0.5) 

0

0

0 (0.0) 

2

4

6 (3.0) 

0

0

0

51.1

58.9

55.0 

 100

100

200

 2

0

2 (1.0)

 0

0

0 (0.0)

 2

0

2 (1.0)

  

6 -18

 +

1250

100

100

200 

3

3

6 (3.0)

9

4

13 (6.5)

1

0

1 (0.5) 

0

0

0 (0.0)

 0

0

0 (0.0)

13

7

20 (10.0) 

0

0

 41.1

41.1

41.1

100

100

200 

0

0

0 (0.0) 

0

0

0 (0.0) 

 0

0

0 (0.0)

6 -18   Positive control (BP20)

100

100

200 

 13

19

32 (16.0)

 73

73

146 (73.0)

1

1

2 (1.0) 

0

0

0 (0.0) 

0

0

0 (0.0)

74

76

150 (75.0) 

0

0

0

100

100

200 

 0

0

0 (0.0)

0

0

0 (0.0) 

0

0

0 (0.0) 

Table 3: Results of chromosomal aberration test (short-term treatment, confirmatory test, -S9 mix)

Treatment/ recovery S9 mix (-/+) Dose of test item (ug/mL)  No. cells showing structural chromosome aberration (incidence, %)                     No. of gaps Cell proliferation rate (%)  No. of cells showing numerical chromosome aberration (incidence, %)          
       No. cells observed Chromatid break   Chromatid exchange Chromosome break  Chromosome exchange  Fragment   Total no. of aberrations       No. of cells observed Polyploid   Endoreduplication Total no. of aberrant cells (%) 

 6 -18

 -

 Negative control (DMSO)

 100

100

200

0

0

0 (0.0) 

 1

0

1 (0.5)

 0

0

0 (0.5)

0

0

0 (0.0) 

0

0

0 (0.0) 

1

0

1 (0.5) 

0

0

97.9

102.1

100.0

100

100

200 

0

0

0 (0.0) 

0

0

0 (0.0) 

0

0

0 (0.0) 

  

6 -18

 -

 2500

 100

100

200

1

1

2 (1.0)

1

0

1 (0.5) 

0

0

0 (0.0)

0

0

0 (0.0) 

0

0

0 (0.0) 

2

1

3 (1.5)

0

0

 58.5

58.5

58.5

100

100

200

0

2

2 (1.0)

0

0

0 (0.0)

0

2

2 (1.0)

  

6 -18

 -

 3000

100

100

200 

3

4

7 (3.5)

2

6

8 (4.0)

0

0

0 (0.0)

0

0

0 (0.0)

0

0

0 (0.0) 

5

9

14 (7.0) 

0

0

0

51.1

51.1

51.1

100

100

200

0

0

0 (0.0)

0

0

0 (0.0) 

0

0

0 (0.0) 

 6 -18  -  4000

 100

100

200

3

7

10 (5.0) 

10

4

10 (7.0) 

0

0

0 (0.0) 

0

0

0 (0.0) 

0

0

0 (0.0) 

13

11

24 (12.0) 

0

0

13

11

24 (12.0) 

100

100

200

0

0

0 (0.0) 

0

0

0 (0.0)

0

0

0 (0.0)

 6 -18  -  5000

 100

100

200

15

16

31 (15.5)

23

21

44 (22.0)

 0

0

0 (0.0)

0

0

0 (0.0 

0

0

0 (0.0)

28

27

55 (27.5) 

1

0

1

42.6

51.1

46.8

100

100

200 

0

0

0 (0.0) 

0

0

0 (0.0)

0

0

0 (0.0) 

 6 -18  -  Positive control (MMC 0.1)

100

100

200 

36

41

77 (38.5) 

38

41

79 (38.5) 

1

0

1 (0.5)

0

0

0 (0.0)

0

0

0 (0.0)

51

59

110 (55)

0

1

1  

100

100

200 

0

0

0 (0.0) 

0

0

0 (0.0) 

0

0

0 (0.0) 

Applicant's summary and conclusion

Conclusions:
It was concluded that the test substance is clastogenic to CHL/IU cells under the conditions of this test.