Registration Dossier

Administrative data

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
From 29 NOV 2012 to 31 JAN 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD 486), according to GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.39 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells In Vivo)
Deviations:
no
GLP compliance:
yes
Type of assay:
unscheduled DNA synthesis

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 8 - 11 weeks
- Weight at study initiation: mean value 266.9 g (SD ± 11.8 g)
- Assigned to test groups randomly: yes
- Housing: group
- Diet (e.g. ad libitum): pelleted standard diet ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2 °C
- Humidity (%): 45 - 65 % (20-90% for several hours)
- Photoperiod (hrs dark / hrs light): 6.00 a.m. - 6.00 p.m.

INLIFE DATES: From: 2012-11-29 To: 2013-01-31

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
The vehicle of the test item was used as vehicle control.
Identity: 1% Carboxymethylcellulose (CMC)
Supplier: FLUKA Chemie AG, 9471 Buchs, Switzerland
Batch: BCBD7651V

Details on exposure:
Route and frequency of administration: orally, once
Volume administered: 15 mL/kg b.w.
Duration of treatment / exposure:
single exposure
Frequency of treatment:
once
Post exposure period:
The animals were examined for acute toxic symptoms at intervals of approx. 1 h, 2 h and 4 h for the 4 hours treatment, and 1 h, and 16 h for the 16 hours treatment after administration of the test item.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 1000, 2000 mg/kg b.w.
Basis:
actual ingested
No. of animals per sex per dose:
2 males and 2 females were used in the pre-experiment
32 males were used in the main experiment, 4 males per dose and exposure time
Control animals:
yes, concurrent vehicle
Positive control(s):
Positive Control Substances
4 hours preparation interval:
Name: DMH; N,N´-dimethylhydrazinedihydrochloride (sym.)
Supplier: Sigma-Aldrich Vertriebs GmbH
82041 Deisenhofen, Germany
Batch: ST BB 7661V
Purity: > 99 %
Dissolved in: 0.9 % NaCl solution
Dosing: 80 mg/kg b.w.
Route and frequency
of administration: orally, once
Volume administered: 10 ml/kg b.w.

16 hours preparation interval:
Name: 2-AAF; 2-acetylaminofluorene
Supplier: Sigma-Aldrich Vertriebs GmbH
82041 Deisenhofen, Germany
Batch: 127K1581
Purity: > 99 %
Dissolved in: dimethylsulfoxide/polyethylene glycol 400 (1 + 9)
Dosing: 100 mg/kg b.w.
Route and frequency
of administration: orally, once
Volume administered: 10 mL/kg b.w.
Solutions prepared on day of administration.
The stability of the positive control substances in vehicle is unknown, but a DNA repair response in the expected range (> 5 net grains [3]) means biological stability demonstration.

Examinations

Tissues and cell types examined:
liver
hepatocytes
Details of tissue and slide preparation:
Isolation of the Primary Hepatocytes
After anaesthetising the rats with 46% Ketamin (Ketavet 100, Pharmacia GmbH, 76139 Karlsruhe, Germany), 23% Xylazin (Rompun 2%, Bayer HealthCare, 51368 Leverkusen, Germany) and 31% Midazolam (Dormicum, Hoffmann LaRoche, 79639 Grenzach-Wyhlen, Germany) (approx. 2 mL/kg body weight) the liver was perfused through the vena portae with Hanks' balanced salt solution (HBSS, Invitrogen, 76344 Eggenstein, Germany) supplemented with collagenase (0.05 % (w/v), Roche Diagnostics, 68305 Mannheim, Germany) adjusted to pH 7.4 and maintained at 37° C (8).
The isolated hepatocytes were washed twice with HBSS. The crude cell suspension was filtered through a stainless steel mesh to yield a single cell suspension. The quality of the performed perfusion was determined by the trypan blue dye exclusion method for cell viability. In addition, the number of the cells was determined.

Culture Conditions
The washed hepatocytes were centrifuged and transferred into Williams medium E (WME, Invitrogen, 76344 Eggenstein, Germany) supplemented (1) with:
Hepes 2.38 mg/mL L-Glutamine 0.29 mg/mL
Penicillin 100 units/mL Insulin 0.50 µg/mL
Streptomycin 0.10 mg/mL Fetal calf serum (FCS) 100 µl/mL
This complete medium was adjusted to pH 7.6.
At least three cultures were established from each animal. Aliquots of 2.5 mL with freshly isolated hepatocytes in complete culture medium (2.0  105 viable cells/ml) were added to 35 mm six-well dishes (Greiner, 72603 Nürtingen, Germany) containing one 25 mm round plastic coverslip (Thermanox, Nunc, 65203 Wiesbaden, Germany) per well coated with gelatine.
After an attachment period of approximately 1.5 h in a 95 % air/ 5 % CO2 humidified incubator at 37° C the culture medium was discarded. Then, the cell layer was rinsed once with PBS to remove non-adherent cells (9). Subsequently, 3HTdR (5 µCi/mL, specific activity 70 90 Ci/mmol; Perkin Elmer, 63110 Rodgau, Germany) in 2.0 mL culture medium (WME, 1 % (v/v) FCS) was added to the cultures. After a labelling time of 4 h the cells were washed twice with WME supplemented with 1 % (v/v) FCS and 0.25 mM unlabelled thymidine. Cultures were incubated overnight using the same medium (2). To prepare for autoradiography the medium was replaced by a hypotonic solution of 1 % (w/v) sodium citrate for 10 minutes to swell the nuclei for better grain detection (9). The cells on the coverslips were then fixed by three changes of methanol:acetic acid (3+1 v/v) for 20 minutes each, rinsed with 96 % (v/v) ethanol, and air-dried.

Autoradiographic Processing
The cover slips were mounted the side carrying the cells up on glass slides and coated with KODAK NTB (Carestream Health, VWR, Germany) photographic emulsion in the dark. The coated slides were stored in light-proof boxes in the presence of a drying agent for 12 - 14 days (excepted the reserves slides for 7 days) at 4° C. The photographic emulsion was then developed with Ilford Phenisol (Firma Hobbylab, 3303 Jegenstorf, Switzerland) at room temperature, fixed in Rapid Fixer (Firma Hobbylab, 3303 Jegenstorf, Switzerland) and stained with hematoxylin/eosin.

Quantification of UDS
Evaluation was performed microscopically on coded slides using NIKON microscopes with oil immersion objectives. The cells for scoring were randomly selected according to a fixed scheme. The number of silver grains above the nucleus was counted automatically using the Sorcerer UDS device version 2.0 DT3152 (Perceptive Instruments). In addition, the number of grains of the most heavily labelled nuclear-sized cytoplasm area adjacent to the nucleus was counted (2). At least two slides per animal and 50 cells per slide were evaluated. Heavily labeled S-phase cells were excluded from counting.

Data Recording
The data generated were recorded in the raw data. The results were presented in tabular form, including experimental groups with the test item, vehicle and positive controls.
The nuclear and cytoplasmic grain counts, the net grain counts (nuclear minus cytoplasmic grains) as well as the mean and percentage of cells in repair (cells with a net grain count larger than 5) is reported separately (5). Individual slide and animal data are provided. The mean counts with standard deviation are used to describe the distribution of 3HTdR incorporation in the nucleus, the cytoplasm and for the net grains, respectively.

Evaluation of Results
Nuclear and net grain counts are estimated together. Increased net grains should be based on enhanced nuclear grain counts rather than on decreased cytoplasmic grain counts.
A test item is classified as positive if the mean number of net grains is higher than 5 per nucleus at one of the test points.
A group average between 0 and 5 net grains is considered as a marginal response. A dose-related increase in nuclear and net grains and/or a substantial shift of the percentage distribution of the nuclear grain counts to higher values provide additional informations to confirm a positive response with less than 5 net grains.
Statistical significance may give further evidence for a positive evaluation.
Statistical significance can be evaluated by means of the non-parametric Mann-Whitney test (4).
A test item producing net grains not greater than 0 or not significantly greater than the concurrent control, at anyone of the test points is considered non-effective in this system.
However, the primary point of consideration is the biological relevance of the results.
Statistics:
A statistical evaluation of the results was not necessary to perform as the number of net grain counts of the groups treated with the test item were in the range of the corresponding controls.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid

Any other information on results incl. tables

Viability and Number of the Hepatocytes

Treatment

Period

Animal no.

Viability*
[%]

Number of isolated cells
[´ 106]

1% CMC

4 h

1

83

212

2

66

233

3

81

292

4

64

210

1000 mg/kg b.w.

test item

4 h

5

67

296

6

69

333

7

74

344

8

71

238

2000 mg/kg b.w.

test item

4 h

9

85

516

10

68

243

11

70

287

12

72

268

80 mg/kg b.w.

DMH

4 h

13

81

115

14

76

424

15

80

332

16

74

237

1% CMC

16 h

17

70

205

18

81

142

19

83

272

20

70

231

1000 mg/kg b.w.

test item

16 h

21

82

209

22

79

247

23

86

269

24

62

158

2000 mg/kg b.w.

test item

16 h

25

87

198

26

84

210

27

78

166

28

80

174

100 mg/kg b.w.

2-AAF

16 h

29

79

233

30

69

205

31

83

183

32

78

160

* Viability determined by means of trypan blue dye exclusion assay

Mean Nucleus, Cytoplasmic Area, and Net Grains

Preparation Interval: 4 h

Test Group

Animal No.

Mean Nuclear Grain Count

Mean Cytoplasmic Grain Count

Mean Net Grain Counts

Mean Nuclear Grains of Cells in Repair

% Cells in Repair

 

 

Mean

 S.D. 

Mean

 S.D. 

Mean

 S.D. 

Mean

 S.D. 

Vehicle control
(1% CMC)

1

19.19

8.75

27.18

10.41

-7.99

9.81

11.29

6.85

7

2

15.28

9.32

27.86

10.29

-12.58

10.98

12.17

10.80

6

3

12.76

4.91

21.82

8.25

-9.06

7.09

6.33

1.53

3

4

13.12

6.38

17.90

8.41

-4.78

8.19

7.92

3.23

13

1000 mg/kg b.w.
test item

5

25.26

9.37

40.77

14.39

-15.51

12.27

8.80

4.44

5

6

23.97

11.55

37.93

13.03

-13.96

11.78

11.60

4.83

5

7

20.05

7.73

33.39

11.45

-13.34

9.65

5.50

0.71

2

8

28.24

10.86

46.28

12.61

-18.04

12.07

47.00

0.00

1

2000 mg/kg b.w.
test item

9

34.46

14.57

55.50

16.90

-21.04

12.05

5.00

0.00

1

10

39.71

18.22

65.79

27.48

-26.08

20.08

15.67

6.66

3

11

23.49

9.56

41.92

14.48

-18.43

11.76

6.00

0.00

1

12

31.68

11.37

53.37

19.63

-21.69

15.37

9.25

4.57

4

Positive control
(DMH
80 mg/kg b.w.)

13

40.91

15.34

20.84

8.44

20.07

13.86

22.83

12.34

88

14

38.56

16.81

20.74

8.44

17.82

13.01

20.33

11.77

88

15

49.00

14.70

19.30

8.90

29.70

14.31

30.64

13.45

97

16

48.84

20.22

20.89

8.87

27.95

16.74

28.52

16.42

98

SD = Standard deviation. The Standard deviation shown for a each animal is the deviation between the 100 analysed cells. The deviation shown for the mean of each group is the standard deviation between the results obtained for each test group consisting of three animals.

Preparation Interval: 16 h

Test Group

Animal No.

Mean Nuclear Grain Count

Mean Cytoplasmic Grain Count

Mean Net Grain Counts

Mean Nuclear Grains of Cells in Repair

% Cells in Repair

 

 

Mean

 S.D. 

Mean

 S.D. 

Mean

 S.D. 

Mean

 S.D. 

Vehicle control
(1% CMC)

17

34.67

18.45

51.35

24.36

-16.68

12.57

9.00

3.46

3

18

12.45

5.75

23.83

6.78

-11.38

7.26

6.00

0.00

1

19

19.87

9.02

30.24

7.94

-10.37

9.48

10.00

4.86

6

20

18.20

8.31

31.74

12.90

-13.54

10.23

7.67

3.06

3

1000 mg/kg b.w.
test item

21

19.88

9.19

32.36

10.42

-12.48

11.56

12.67

9.56

6

22

19.64

8.66

36.05

12.58

-16.41

11.10

8.20

3.35

5

23

27.27

10.67

38.49

16.25

-11.22

14.18

8.64

2.73

11

24

20.68

8.52

36.34

12.42

-15.66

11.08

6.00

0.00

1

2000 mg/kg b.w.
test item

25

18.58

8.64

27.76

6.96

-9.18

8.89

9.80

2.86

5

26

23.16

9.82

36.03

10.80

-12.87

10.48

9.50

4.12

4

27

18.20

6.86

32.80

7.52

-14.60

9.17

0.00

0.00

0

28

16.03

6.57

26.50

7.35

-10.47

7.90

8.50

5.74

4

Positive control
(2-AAF
100 mg/kg b.w.)

29

40.36

17.86

23.58

10.88

16.78

13.55

20.02

11.94

85

30

68.17

24.27

35.55

15.11

32.62

21.04

33.43

20.43

98

31

41.26

12.45

29.95

9.47

11.31

11.59

16.84

8.78

70

32

64.00

14.70

33.88

9.54

30.12

15.85

31.11

15.01

97

SD = Standard deviation. The Standard deviation shown for a each animal is the deviation between the 100 analysed cells. The deviation shown for the mean of each group is the standard deviation between the results obtained for each test group consisting of three animals.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
In conclusion, it can be stated that under the experimental conditions reported, i.e. oral administration up to the Maximal Tolerated Dose of 2000 mg/kg the test item did not induce DNA-damage leading to increased repair synthesis in the hepatocytes of the treated rats.
Executive summary:
The test item was assessed in thein vivoUDS assay for its potential to induce DNA repair (UDS) in the hepatocytes of male rats at two different sampling times (4 h and 16 h).

The test item was suspended in1% carboxymethylcellulose,which was used as vehicle control. The volume administered orally was 15 mL/kg body weight. Four and sixteen hours after a single oral administration, the animals were anaesthetised and sacrificed by liver perfusion. Primary hepatocyte cultures were established and exposed for 4 hours to3HTdR (methyl-3H-thymidine), which is incorporated if UDS occurs.

The highest dose was estimated in a pre-experiment to be the maximum applicable dose.

Dose levels of 1000 and 2000 mg/kg b.w. of the test item were investigated.

For each experimental group including the controls, hepatocytes from four animals were assessed for the occurrence of UDS.

The viability of the hepatocytes was not substantially affected due to thein vivotreatment with the test item at any of the treatment periods or dose groups. The interindividual variations obtained for the numbers and the viabilities of the isolated hepatocytes are in the range of our historical laboratory control (3).

No dose level of the test item revealed biologically relevant UDS induction in the hepatocytes of the treated animals as compared to the current vehicle controls. The net grain counts were not distinctly increased afterin vivotreatment of the animals with the test item at 4 hours or 16 hours, respectively. Net grain counts obtained after treatment with the test item remained consistently negative.

In addition, no substantial shift to higher values of percentage of cells in repair was reported.

Appropriate reference mutagens [DMH (10), 80 mg/kg b.w. and 2-AAF, 100 mg/kg b.w.] were used as positive controls.In vivotreatment with DMH or 2-AAF revealed distinct increases in the number of nuclear and net grain counts.