Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a GLP-compliant in vitro cytogenicity assay conducted with a method equivalent to the OECD guideline, the test substance was considered to be clastogenic to V79 cells. In a standard bacterial reverse mutation assay (Ames test), the substance was not mutagenic (negative).

Link to relevant study records

Referenceopen allclose all

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
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
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.
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.
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

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) 

Conclusions:
It was concluded that the test substance is clastogenic to CHL/IU cells under the conditions of this test.
Endpoint:
in vitro gene mutation study in bacteria
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
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
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
0, 313, 625, 1250, 2500, 5000 ug/plate

As the result of the dose-finding study, increase of the number of revertant colonies or growth inhibition was not noted in any tester strains either with or without S9 mix.
Therefore, the main test was conducted at the highest concentration of 5000 μg/plate and following concentrations:
Vehicle / solvent:
DMSO
The test substance was soluble in distilled water and DMSO at 50 mg/mL as the result of solvent examination. No exothermic, discoloration and bubbling were observed when the test substance was mixed with any solvent. However, it was noted that the test substance was hydrolysable. Therefore, DMSO selected as the solvent (negative control material) for this test substance. DMSO was used after dehydration by molecular sieve.
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylaic amide, 2-aminoanthracene
Details on test system and experimental conditions:
Selection of the test method
The test was conducted by preincubation method with and without S9 mix.

Preincubation method
1) A 0.1 mL of the test substance solution or negative control material, or positive control material was added into sterilized tube.
2) For without S9 mix, 0.5 mL of 0.1 mol/L of sodium/phosphoric acid buffer (pH 7.4) was added and mixed, then 0.1 mL of bacterial suspension was further added.
3) For with S9 mix, 0.5 mL of S9 mix was added and mixed instead of 0.1 mol/L of sodium/phosphoric acid buffer.
4) The mixture was gently shaken at 37°C for 20 minutes (frequency: 90 times/minute) and incubated (preincubation).
5) After preincubation, 2 mL of top agar was added to this mixture and multilayered onto minimum glucose agar plate medium.
6) After the multilayered top agar was solidified, the plate was incubated at 37°C for 48 hours.

Sterility test
Sterility test was conducted at for each test using one plate each for the test substance solution and S9 mix to check the contamination.
1) A 2 mL of top agar was added to 0.1 mL of the highest dose of test substance solution or 0.5 mL of S9 mix, and mixed.
2) Each mixture was multilayered onto minimum glucose agar plate medium.
3) After the multilayered top agar was solidified, the plate was incubated at 37°C for 48 hours, and then contamination was visually inspected.

Colony counting
Number of revertant colonies on the plate was counted using automated colony counter (Nissui Pharmaceutical Co., Ltd., NeQCS). Area correction and counting loss correction were conducted for the instrumental measurement. Visual counting was not performed.

Number of plates used
Dose-finding test: 2 plates/concentration
Main test: 2 plates/concentration

Calculation of the results
Mean value of the counted colonies was calculated for each dose of the negative control group, positive control group and test substance group. Mean values were round off to the nearest integer.
Rationale for test conditions:
Standard guideline requirements
Evaluation criteria:
Experiment was considered to be valid if it met all the following criteria

1) Both values of negative control (mean) and positive control (mean) are within the appropriate range of the historical data of the test facility.
2) Positive control value (mean) apparently increases more than twice in comparison with the negative control value (mean) of the relevant tester strain.
3) There are more than 4 concentrations without growth inhibition and also there are more than 5 evaluable doses in the main test.
4) No contamination is observed as the result of the sterility test.
5) Test plate is not in uncountable status or lost due to contamination or other unexpected event.

Judgement of the test result
The test substance was judged as mutagenic (positive) when the number of revertant colonies (mean) increased dose-dependently more than twice of the mean value of the negative control in any of the tester strains either with or without S9 mix, and eproducibility was noted in the increase. Other cases were judged as negative. If the test substance was judged as positive according to above criteria, specific activity (the number of induced revertant colonies per 1 mg) was calculated.
Statistics:
None.
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Dose range finding study = negative at every concentration
Main study = negative at every concentration

Main Study results

 Metabolic activation

 test substance conc.

(ug/plate)

No of reverse mutations (No. of colonies/plate)             

           Base pair substitution type     Frameshift type
     TA100 TA1535 

  WP2 uvrA/

pKM101

TA98  TA1537 
   S9 mix (-)   Negative

103

100 (107)

12

13 (13) 

71

65 (68) 

21

18 (20)

8

7 (8)

    S9 mix (-)   313

99

106 (103)

11

8 (10)

61

70 (66) 

18

24 (21)

7

10 (9)

    S9 mix (-)   625

109

107 (108)

13

10 (12)

64

62 (63)

22

18 (20) 

12

9 (11)

    S9 mix (-)   1250

94

104 (99) 

 8

10 (9)

70

75 (73)

20

19 (20) 

7

8 (8) 

    S9 mix (-)   2500

112

94 (103) 

13

8 (11) 

63

70 (67)

24

22 (23) 

7

8 (8)

    S9 mix (-)   5000

99

97 (98)

11

8 (10) 

62

60 (61) 

18

19 (19)

11

9 (10) 

    S9 mix (+)   Negative

106

109 (108) 

11

13 (12) 

82

75 (79)

24

26 (25)

 9

12 (11)

      S9 mix (+)  313

105

116 (111)

11

14 (13)

71

76 (74)

21

26 (24)

13

10 (12)

      S9 mix (+)  625

116

108 (112)

8

10 (9) 

86

78 (82)

25

30 (28)

 9

11 (10)

     S9 mix (+)   1250

106

114 (110)

10

12 (11) 

93

84 (89) 

23

23 (23)

10

10 (10)

      S9 mix (+)  2500

102

106 (104) 

13

11 (12) 

83

71 (77)

27

29 (28) 

 12

10 (11)

      S9 mix (+)

 5000

113

103 (108)

 8

10 (9)

74

75 (75)

24

23 (24)

 8

8 (8)

Positive control

(S9 + only)

Name  AF-2 NaN3  AF-2 AF-2  9 -AA 

Positive control

(S9 + only)

 Dose (ug/plate)

 0.01

0.5 

 0.005

 0.1

80 

Positive control

(S9 + only)

 No. of colonies/plate

807

789 (789)

363

376 (370) 

806

662 (734) 

801

839 (820) 

497

455 (476) 

Positive control

(S9 - only)

Name  

 2 -AA

 2 -AA

2 -AA 

2 -AA 

2 -AA 

Positive control

(S9 - only)

Dose (ug/plate) 

1.0 

2.0

2.0 

0.5 

2.0 

Positive control

(S9 - only)

 No. of colonies/plate

861

839  (850)

249

261 (255) 

573

563 (255)

359

320 (340) 

171

157 (164) 

Conclusions:
Under the condtions of the test, it was concluded that the test substance was not mutagenic.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In an OECD guideline, GLP-compliant in vivo micronucleus test, no induction of micronuclei in bone marrow erythrocytes was observed following administration of the test item to mice at up to and including 100 mg/kg bw/day; thus, there was no evidence of any genotoxic activity of the test item under the conditions of this study.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
Due to the observed mortality during the main experiment, an additional two replacement animals were used in the 100 mg/kg body weight dose group (24-hour sampling time point) to examine the required number of animals.
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian erythrocyte micronucleus test
Species:
mouse
Strain:
NMRI
Remarks:
RjHan
Details on species / strain selection:
The NMRI mouse is one of the standard animals used internationally in this type of mutagenicity testing
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Elevage Janvier, Route des Chènes Secs B.P. 4105, 53940 LE GENEST-ST-ISLE, France
- Age at study initiation: approx. 7 weeks at treatment
- Weight at study initiation: 34.7 – 38.0 g (males, preliminary experiment), 28.2 – 31.8 g (females, preliminary experiment), 33.3 – 37.9 g (males, main test)
- Assigned to test groups randomly: yes, based on body weights SPSS/PC+ software was used in order to verify homogeneity/variation among/within groups.
- Fasting period before study:
- Housing: Group caging (5 animals/cage or 2 animals/cage) to allow social interaction, and with deep wood sawdust bedding, to allow digging and other normal rodent activities. Cage type: II. type polypropylene/polycarbonate
- Diet (e.g. ad libitum): Animals received ssniff® SM R/M "Autoclavable complete diet for mice and rats breeding and maintenance" (Batch number: 523 7816, Expiry date: January 2013; and Batch Number: 445 8440; Expiry date: May 2013) produced by ssniff Spezialdiäten GmbH (D-59494, Soest, Germany)
- Water (e.g. ad libitum): tap water from municipal supply, as for human consumption from 500 ml bottle ad libitum.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.2 - 23.5°C
- Humidity (%): 31 - 70%
- Air changes (per hr): 15 - 20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12h dark, 12h light
Route of administration:
oral: gavage
Vehicle:
Name: Distilled water
Supplier: TEVA
Batch number: 3450611
Expiry date: 30 June 2014
Storage conditions: Room temperature
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The necessary amount of the test item was weighed into a calibrated volumetric flask; the appropriate amount of vehicle was added and stirred to obtain homogenous formulations. The concentrations of the test item formulations were chosen to assure the same dosing volumes in mice for all dose levels (10 mL/kg bw). The test item was used for treatment in the main test at concentrations of 10, 5 and 2.5 mg/mL. The formulations were prepared immediately before the treatment

PRELIMINARY TOXICITY TESTING
Two preliminary toxicity tests were performed to identify the appropriate maximum dose level for the main test. The preliminary toxicity tests also determined whether there were large differences in toxicity between the sexes. Groups of two male and female mice were treated on one occasion by oral gavage at the dose levels of 300, 200, 100 and 50 mg/kg body weight (Preliminary Experiment I). Based on the observed mortality in the 300 and 200 mg/kg body weight dose groups, additional doses of 150 and 125 mg/kg body weight were also examined in an additional experiment (Preliminary Experiment II.)

MAIN TEST
Based on the results of the preliminary toxicity tests, 100 mg/kg body weight was selected for the highest dose level of the test item in the main micronucleus test. Two lower dose levels separated by a factor of two (50 and 25 mg/kg body weight) were also included in the main test. The dose levels were expressed in terms of the test item as received.
Duration of treatment / exposure:
single dose, oral gavage
Frequency of treatment:
once
Post exposure period:
48 hours
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Negative control (vehicle)
Dose / conc.:
25 mg/kg bw/day (nominal)
Remarks:
Test item (dose conc: 2.5 mg/mL)
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Test item (dose conc.: 5 mg/mL)
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Test item (Dose conc.: 10 mg/mL)
No. of animals per sex per dose:
Negative control (vehicle) = 10 males
25 mg/kg bw/day = 5 males
50 mg/kg bw/day = 10 males
100 mg/kg bw/day = 10 + 7 males
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclphosphamide (Dose conc. 6 mg/mL)
Tissues and cell types examined:
bone marrow
polychromatic erthrocytes (PCEs)
normochromatic erythrocytes (NCEs)
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
negative control (vehicle) = sampling 24h and 48h after treatment
25 mg/kg bw/day = sampling 24h after treatment
50 mg/kg bw/day = sampling 24h after treatment
100 mg/kg bw/day = sampling 24h and 48h after treatment

DETAILS OF SLIDE PREPARATION:
At the scheduled sacrifice time in the main test, mice were euthanized by asphyxiation with ascending doses of carbon dioxide. Deep anaesthesia was confirmed before cervical dislocation or transection of the major cervical blood vessels before confirming death and discarding carcasses. Bone marrow was obtained from two exposed femurs of mice* immediately after sacrifice.

The bone marrow was flushed out of each pair of femurs with foetal bovine serum (5 mL) using a syringe and needle into a sterile centrifuge tube. After mixing, the cell suspension was concentrated by a gentle centrifugation and the supernatant was discarded. Smears of the cell pellet were made on standard microscope slides (2 slides / animal). Slides were air-dried at room temperature for approximately 24 hours.

Dried slides were fixed in methanol for a minimum of 5 minutes and allowed to air-dry. Two slides per animal were stained with 10 % Giemsa solution for 20 minutes then thoroughly rinsed with distilled water, and then air-dried at room temperature for at least 12 hours. After staining, coverslips were mounted on them.

METHOD OF ANALYSIS:
Prior to microscope analysis, the stained slides were given unique code numbers by a person who was not involved in the analysis. The code labels covered all unique identification markings on the slides to ensure that they were scored without bias.
Two thousand polychromatic erythrocytes (PCEs) were scored per animal to assess the incidence of the micronucleated cells. The frequency of micronucleated cells was expressed as percent of micronucleated cells based on the first 2000 PCEs counted in the optic field. Fewer than 2000 PCEs may be counted in the event of a clear positive effect.
The proportion of immature among total (immature + mature) erythrocytes was be determined for each animal by counting a total of at least 1000 cells (immature erythrocytes, PCEs plus mature normochromatic erythrocytes, NCEs), in which the number of micronuclei were recorded in both types of erythrocytes.

Evaluation criteria:
Criteria for a positive response:
The test item is considered to have shown genotoxic activity if statistically significant increases in the frequency of micronucleated polychromatic erythrocytes are observed in treated animals compared to the corresponding negative controls, and the increases are dose-related. Historical control data are taken into consideration when evaluating the biological significance of small increases.

Criteria for a negative response:
The test item is concluded to have given a negative response if no reproducible, statistically significant increases are observed above the concurrent and historical control values.

Equivocal response:
Results which do not meet the criteria for a positive or negative response are considered to be equivocal. Further investigations or scoring of additional cells may be necessary in case of an equivocal result.
Statistics:
Kruskal Wallis test
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
Based on the available information, the test item was soluble in Distilled water. As this vehicle was compatible to the test system, it was selected as vehicle for the study. Groups of two male and female mice were treated on one occasion by oral gavage at the dose levels of 300, 200, 100 and 50 mg/kg body weight (Preliminary Experiment I). The treatment volume was 10 mL/kg body weight. Animals were examined regularly for toxic signs and mortalities. Based on the observed mortality in the 300 and 200 mg/kg body weight dose groups, additional doses of 150 and 125 mg/kg body weight were also examined in an additional experiment (Preliminary Experiment II.). All the surviving mice were euthanized 48 hours after treatment.

There were some individual variability in the time of the appearance or severity of the symptoms, but both male and female animals died or were considered moribund in the 300, 200, 150 and 125 mg/kg body weight dose groups. Decreased activity was detected for all animals of the 100 mg/kg body weight dose group, in some animals hunched back, prone position and/or piloerection was also observed.

All animals were free of clinical signs in the 50 mg/kg body weight dose group. No treatment related effect on body weight was observed in the 100 and 50 mg/ kg body weight group.

Based on the results of the preliminary toxicity test, dose levels of 100, 50 and 25 mg/kg body weight were selected for the micronucleus test. As the toxicity of the test item was similar in both sexes in the preliminary toxicity tests, the main experiment was performed using male mice only.

RESULTS OF DEFINITIVE STUDY
Marked body weight loss (>10%) was detected for 1 of 7 surviving animals in the high dose group at the 24-hour sampling time point and for 2 of 6 surviving animals in the high dose group at the 48-hour sampling time point in the main test. No effect on the body weight was observed in the mid and low dose groups, or in the negative or positive control groups.

Mortality was observed in the high dose group (100 mg/kg body weight) in the main test: 4 animals were found dead from the 17 treated animals at different time points. Signs of systemic toxicity (decreased activity, hunched back, prone position or intermittent tremors) and piloerection were also observed for some animals in this dose group. The animals in the negative (vehicle) control, positive control and low and mid dose groups were symptom-free during the whole observation period (with the exception of one animal in the mid dose group (50 mg/kg body weight) where piloerection was observed).

Two thousand polychromatic erythrocytes (PCEs) were scored per animal to assess the micronucleated cells. The frequency of micronucleated cells was expressed as percent of micronucleated cells based on the first 2000 PCEs counted in the optic field. The proportion of immature among total erythrocytes was determined for each animal by also counting mature erythrocytes (NCEs) until a total of 1000 PCEs+NCEs had been observed.

The group treated for 24 hours with 100 mg/kg bw/day, which gave the highest number of micronuclei, was compared with the corresponding negative (vehicle) control group using the Kruskal Wallis test. This gave a value of H = 0.561 which is non-significant, giving a negative response. All other groups showed the same or lower numbers of micronuclei than the corresponding negative (vehicle) control group.

The positive and negative control results were also compared, and gave a value of H = 6.991 (p<0.01). The positive control treatment therefore caused a very substantial increase, demonstrating the sensitivity of the test system. The positive and negative control data were considered to give adequate data to confirm the validity of the study.

The frequency of micronucleated polychromatic erythrocytes of the negative (vehicle) control group was within the range of historical laboratory control data; the positive control item produced a biologically and statistically relevant increase in the number of micronucleated polychromatic erythrocytes; each treated and control group included at least 5 analysable animals; therefore, the test was considered to be valid.

Table 1: Micronucleus data (24 hours after dosing)

 Treatment  Animal number  MNPCE/2000 PCE

 PCE/1000

PCE + NCE

 Negative control

(vehicle)

 

 

 

 

 

 

 

 

 7

 0

558

 22

 2

601

 24

 2

535

 47

 2

397

 42

 3

400

 Mean

 

 1.8

498.2

 S.D

 

 1.10

94.05

 Treatment  Animal number  MNPCE/2000 PCE

 PCE/1000

PCE + NCE

 Test item

25 mg/kg bw

 

 

 

 

 

 

 9

 1

333

13

1

397

34 

603

36

2

490

39

2

569

 Mean

 

 1.8

478.4

 S.D

 

0.84

113.62

 Treatment  Animal number  MNPCE/2000 PCE

 PCE/1000

PCE + NCE

 Test item

50 mg/kg bw

 

 

 

 

 

 

 

 

 6

1

282

12

 2

505

16

0

594

20

2

592

33

0

505

 Mean

 

1

495.6

 S.D

 

1.00

127.26

Treatment  Animal number  MNPCE/2000 PCE

 PCE/1000

PCE + NCE

Test item

100 mg/kg bw

 

 

 

 

 

 

 

 

 3

 3

 615

 8

5

 550

 23

0

536

 28

1

592

 47

5

608

 Mean

 

2.8

 580.2

 S.D

 

 2.28

35.32

Treatment  Animal number  MNPCE/2000 PCE

 PCE/1000

PCE + NCE

 Positive control

(cyclophosphamide)

 

 

 

 

 

 

 

 

11 

 36

 427

 15

 56

514

 29

49

425

 46

 51

463

49 

56

474

 Mean

 

49.6

460.6

 S.D

 

 8.20

36.86

MNPCE: Number of Micronucleated Polychromatic Erythrocytes referring to counts of 2000 PCE.

PCE: Polychromatic Erythrocyte

NCE: Normochromatic Erythrocyte

Table 2: Micronucleus data (48 hours after dosing)

Treatment  Animal number  MNPCE/2000 PCE

 PCE/1000

PCE + NCE

Negative control

(vehicle)

 

 

 

 

 

 

 

 

 14

 3

486

 18

 3

447

 40

3

435

43

2

458

45

2

384

 Mean

 

2.6

442.0

 S.D

 

 0.55

 37.52

Treatment  Animal number  MNPCE/2000 PCE

 PCE/1000

PCE + NCE

 Test item

100 mg/kg bw

 

 

 

 

 

 

 

 

 2

0

276

30

1

523

32

429

35

3

406

41

0

520

 Mean

 

1.4

430.8

 S.D

 

 1.52

101.29

Conclusions:
In conclusion, no induction of micronuclei in bone marrow erythrocytes was observed following administration of the test item to mice at up to and including 100 mg/kg bw/day; thus, there was no evidence of any genotoxic activity of the test item under the conditions of this study.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

The test item was negative in a in vitro gene mutation study conducted on bacteria and negative in an in vivo micronucleus assay to assess cytogenicity therefore the substance does not fulfil the criteria within the CLP Regulation (EC1272/2008, as amended) to be classified as a mutagenic. Therefore the substance is not classified for this endpoint.