Registration Dossier

Administrative data

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-02-20 to 2019-09-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
According to final decision on a testing proposal of 12 September 2018 (decision number: TPE-D-2114440060-69-01/F).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No 2017/735, Annex Part B, B.62: In vivo Mammalian Alkaline Comet Assay
Version / remarks:
2017
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian comet assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: in house breed (TOXI-COOP ZRT. Cserkesz u. 90.H-1103 Budapest Hungary)
- Age at study initiation: 49 - 51 days (young adult rats, less than 10 weeks old at the commencement of the treatment)
- Weight at study initiation: 228 - 246 g (weight variation in animals involved at the start of the study did not exceed +/- 20 %
- Assigned to test groups randomly: yes (based on body weight)
- Fasting period before study: not applicable
- Housing: 3 animals / cage (test item and control group) 2 animals per cage (positive control group)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 3 °C
- Humidity: 30 - 70 %
- Air changes: > 10 per hr
- Photoperiod: 12 / 12 hrs dark / hrs light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle/solvent used: sunflower oil (Helianthii annui oleum raffinatum); physiological saline (positive control)
- Justification for choice of solvent/vehicle: the test item was well soluble and stable in the vehicle. The vehicle has been proven to be well tolerated by the animals
- Concentration of test material in vehicle: 400, 200 and 100 mg/mL (nominal)
- Amount of vehicle: 5 mL/kg bw (test item and control groups); 10 mL/kg bw (positive control)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in Sunflower oil at the adequate concentrations. The formulations were prepared in the laboratory of the Test Facility before each treatment.
Duration of treatment / exposure:
2 consecutive days (test item and control goups); 1 day (positive control)
Frequency of treatment:
once a day
Post exposure period:
3 - 4 hours after the second treatment
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
vehicle control
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
6 males (5 + 1 back-up) for test item and negative control groups;
4 males (3 + 1 back-up) for the positive control group
Control animals:
yes, concurrent vehicle
yes, historical
Positive control(s):
Ethylmethanesulphonate

- Justification for choice of positive control: according to the respective guideline and well historical control data available at the performing CRO
- Route of administration: oral (gavage)
- Doses: 200 mg/kg bw

Examinations

Tissues and cell types examined:
site of first contact:
Stomach, Duodenum

Site of (potential) metabolism:
Liver
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Based on Acute oral toxicity study (LD50 > 2000 mg/kg bw)

TREATMENT AND SAMPLING TIMES:

DETAILS OF SLIDE PREPARATION:
The slide preparation was done within one hour after single cell preparation.

Pre-treatment of slides:
Conventional (superfrost) slides were dipped in hot 1 % normal melting point agarose in water. Thereafter the underside of the slides was wiped in order to remove the excess of agarose. The slides were then laid on a flat surface and were allowed to dry.

Embedding the cells:
Before the use, a volume of 130 μL of 0.5 % normal melting point agarose (NMA) was added on a microscope slide pre-layered with 0.5 % NMA (see above) and covered with a glass coverslip. The slides were placed on a tray until the agarose hardens (~ 5 minutes). After the cell isolations each cell suspension was mixed with 0.5 % or 1.0 % Low Melting Point Agarose (LMPA). Thereafter 85-165 μL (~1-9 x 104 cells) of this mixture was added on the microscope slide after gentle slide off the coverslip. The microscope slides were covered with a new coverslip. After the LMPA-cell mixture hardens an additional 70 μL of NMA was dropped on the microscope slide after a gentle slide off the (second) coverslip and an additional new coverslip was laid on the slide. After the repeated NMA layer hardens the coverslip was removed.

Lysis
After the top layer of agarose had solidified and the last glass coverslip was removed and the slides were immersed in chilled lysing solution. The slides were kept overnight in lysing solution at 2-8 °C (in a refrigerator) in the dark. After the incubation period, the slides were rinsed to remove residual detergents and salts prior to the alkali unwinding step. This rinsing procedure was performed in electrophoresis buffer.

Unwinding and Electrophoresis
The slides were removed from the lysing solution and randomly placed on a horizontal gel electrophoresis unit. The unit was filled up with freshly prepared electrophoresis solution until the surfaces of the slides were completely covered with the solution (to about 1-2 mm above the slides). During the unwinding and electrophoresis a balanced design was used to place slides in the electrophoresis tank to mitigate the effects of any trends or edge effect within the tank and to minimise batch to batch variability. The slides were left in the electrophoresis tank for 30 min. for the DNA to unwind. Thereafter the electrophoresis was conducted for 30 min. by applying a constant voltage of 25V and an electric current of about 300 mA (270-300 mA). The current was recorded at the start and end of the electrophoresis period. The same volume of the electrophoresis solution was used at every run, therefore at constant voltage slight change in the electric current was noticed. This slight change in the electric current had no influence on the results of the test. All of these steps were sheltered from the daylight to prevent the occurrence of additional DNA damage. The electrophoresis solution was kept in a refrigerator until use (before the unwinding and electrophoreses). Its temperature was noticed to be 4.1-5.2 °C. During the unwinding and electrophoresis procedures it was maintained at a low temperature (5 °C) using a special cooler designed for Comet electrophoresis tank. The temperature of the electrophoresis solution was recorded once during the procedure.

Neutralisation and Preservation of Slides
After electrophoresis, the slides were removed from the electrophoresis unit, covered with neutralisation solution, left stand for about 5 minutes, thereafter blotted and covered again with neutralisation solution. This procedure was repeated twice. Subsequently the slides were exposed for additional 5 minutes to absolute ethanol in order to preserve all of the slides.

Staining
The slides were air dried and then stored at room temperature until they were scored for comets. Just prior the scoring the DNA, the slides were stained using 50 μL of 2 μg/mL Ethidium bromide.


METHOD OF ANALYSIS:

Evaluation of Slides (Visualisation and Analysis):
Every animal was euthanised and cells of the defined organs were isolated. For each animal and each tissue 4 slides were prepared (12 slides per animal, 72 slides per test item dose and negative control and 48 slides per positive control). Three of the four slides from five of six animals per vehicle control and test item treatment were stained and analysed (9 slides per animal, 45 slides per dose, per vehicle control) and three slides of three animals per positive control were stained and analysed (9 slides per animal, 27 slides per dose). Coded slides were stained and blind scored. The slides were examined with an appropriate magnification (200x) using fluorescent microscope equipped with an appropriate excitation filter and with an Alpha DCM 510B CMOS camera. For image analysis the Andor Kinetic Imaging Komet 6.0 was used. For each tissue sample fifty cells per slide were randomly scored i.e. 150 cells per animal (750 analysed cells per test item treatment, per vehicle control and 450 per positive control). DNA strand breaks in the comet assay were measured by independent endpoints such as % tail DNA, olive tail moment (OTM) and tail length. The tail % DNA (also known as tail intensity) was applied for the evaluation and interpretation of the results and determined by the DNA fragment intensity in the tail expressed as a percentage of the cell’s total intensity. The OTM is expressed in arbitrary units and is calculated by multiplying the percentage of DNA (fluorescence) in the tail by the length of the tail in μm. The tail length is measured between the center of the comet head and the end of the comet tail. In addition, each slide was examined for presence of ghost cells. Ghost cells results from a total migration of the DNA from the nucleus into the comet tail, reducing the size of the head to a minimum. Ghost cells, also known as clouds or hedgehogs, are morphological indicative of highly damaged cells and their presence is often associated with severe genotoxicity, necrosis and apoptosis. Ghost cells were excluded from the image analysis data collection, however determining of their frequency is useful for the data interpretation. The ghost cells were recorded for each slide per animal, per treatment and per tissue.

Evaluation criteria:
The test chemical is clearly negative if:
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control;
- there is no concentration-related increase when evaluated with an appropriate trend test;
- all results are inside the distribution of the historical negative control data for given species, vehicle, route, tissue and number of administration;
- direct or indirect evidence supportive of exposure of, or toxicity to, the target tissue(s) is demonstrated.

The test chemical is clearly positive if:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control;
- the increase is dose-related when evaluated with an appropriate trend test;
- any of the results are outside the distribution of the historical negative control data for given species, vehicle, route, tissue and number of administration;

There is no requirement for verification of clearly negative or positive response.
Statistics:
The statistical significance of % tail DNA values, tail length, OTM values and number of ghost cells was carried out using the appropriate statistical method, using SPSS PC+ software. The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. In case of a positive analysis, Duncan's Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. If the data were not normal distributed, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. In case of a positive analysis result, the inter-group comparisons were performed using Mann-Whitney U-test.

Results and discussion

Test results
Key result
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

Table 1. Tail DNA % of Medians per Slide, Means per Animal, per Dose (Stomach)

Dose (treatment)

Tail DNA %

Per slide

Per animal

Per dose

(Median of 50 cells)

(Mean Median of 150 cells; n=3 á 50 cells)

(Mean Median of 750 cells)

Sunflower oil (x2)

9.76

10.13

13.83

SD: 3.97


9.29

11.33

11.58

10.99

11.15

10.25

12.11

13.08

11.40

15.73

15.13

14.80

13.93

15.33

17.54

20.15

18.21

24.69

Test item
(500 mg/kg bw/day)x2

15.25

16.90

14.41

SD: 1.98

NS

16.78

18.67

15.62

14.18

14.63

12.31

14.19

12.87

11.51

12.92

8.63

12.21

12.39

15.62

16.17

15.89

17.01

14.48

Test item
(1000 mg/kg bw/day)x2

20.72

19.70

12.83

SD: 3.91


NS

18.71

19.67

13.11

11.96

9.59

13.19

10.31

11.67

14.16

10.56

13.14

10.66

9.16

9.68

12.77

10.16

10.60

7.13

Test item
(2000 mg/kg bw/day)x2

14.67

13.94

13.55

SD: 1.51

NS

12.00

15.14

12.78

13.38

12.85

14.51

17.53

15.81

15.37

14.54

13.48

11.71

10.23

11.44

11.60

12.91

13.26

13.89

EMS
(200 mg/kg bw/day) x1

27.24

27.94

33.70

SD: 9.33

*

29.12

27.46

38.96

44.47

45.36

49.09

28.92

28.71

29.56

27.65

 EMS      :   Ethyl methanesulfonate

SD         :    Standard deviation

1)             :              In the case of EMS positive control 450 cells/tissue.

The possible slight differences (in the second decimals) between the mean median values of this summary table and corresponding Appendix 3 data are originated from rounding.

Statistically not significant: NS

Statistically significant:

           *     : p<0.05

           **  : p<0.01

Mann-Whitney U-test Versus Control

 

 

 

Table 2. Tail DNA % of Medians per Slide, Means per Animal, per Dose (Duodenum)

Dose (treatment)

Tail DNA %

Per slide

Per animal

Per dose

(Median of 50 cells)

(Mean Median of 150 cells; n=3 á 50 cells)

(Mean Median of 750 cells)

Sunflower oil (x2)

7.07

8.65

16.05

SD: 4.34


9.89

8.99

16.03

19.96

23.87

19.97

15.74

17.52

18.10

18.71

21.80

17.80

16.64

14.96

20.18

16.32

14.73

14.06

Test item
(500 mg/kg bw/day)x2

5.45

6.90

17.79

SD: 8.11

NS

8.88

6.37

28.11

27.61

24.77

29.95

22.18

21.19

21.43

19.97

8.33

12.51

14.40

14.79

21.83

20.73

19.66

20.70

Test item
(1000 mg/kg bw/day)x2

20.36

21.19

16.33

SD: 6.62


NS

22.23

21.00

10.24

7.59

5.85

6.70

11.18

10.90

11.09

10.43

19.65

19.75

17.71

21.91

23.94

22.19

20.64

21.99

Test item
(2000 mg/kg bw/day)x2

10.55

11.24

15.53

SD: 7.09

NS

11.86

11.32

20.29

17.60

18.07

14.44

26.82

27.10

26.25

28.24

12.14

11.41

10.05

12.03

9.61

10.30

10.60

10.70

EMS
(200 mg/kg bw/day) x1

29.19

28.71

35.60

SD: 6.40

**

28.50

28.46

35.61

36.74

36.56

38.06

43.59

41.35

37.28

43.19

EMS      :   Ethyl methanesulfonate

SD         :    Standard deviation

1)             :              In the case of EMS positive control 450 cells/tissue.

Statistically not significant: NS

Statistically significant:

           *     : p<0.05

           **  : p<0.01

Duncan's multiple range test

 

 

 

Table 3. Tail DNA % of Medians per Slide, Means per Animal, per Dose (Liver)

Dose (treatment)

Tail DNA %

Per slide

Per animal

Per dose

(Median of 50 cells)

(Mean Median of 150 cells; n=3 á 50 cells)

(Mean Median of 750 cells)

Sunflower oil (x2)

5.54

4.10

6.19

SD: 1.54

4.28

2.48

6.30

6.15

5.60

6.54

6.60

5.35

4.96

4.50

7.84

7.55

7.68

7.12

9.82

7.80

6.11

7.47

Test item
(500 mg/kg bw/day)x2

3.30

4.94

7.05

SD: 3.00

NS

6.64

4.88

9.63

9.76

9.70

9.96

4.63

7.16

7.42

9.42

9.78

10.16

10.34

10.35

3.24

3.25

3.20

3.32

Test item
(1000 mg/kg bw/day)x2

3.12

5.08

5.94

SD: 1.56

NS

5.32

6.80

8.65

8.44

9.29

7.39

7.71

5.88

5.77

4.16

4.82

6.04

7.22

6.09

3.30

4.28

5.34

4.21

Test item
(2000 mg/kg bw/day)x2

6.36

7.56

5.87

SD: 1.84

NS

9.14

7.18

8.30

7.44

6.54

7.50

7.38

5.34

3.55

5.09

6.45

5.99

5.56

5.98

3.98

3.05

2.17

3.00

EMS
(200 mg/kg bw/day) x1

25.21

23.14

26.63

SD: 7.97

*

23.49

20.72

19.58

21.00

21.12

22.30

35.31

35.75

35.45

36.51

EMS      :   Ethyl methanesulfonate

SD         :    Standard deviation

1)             :              In the case of EMS positive control 450 cells/tissue. Statistically not significant: NS

Statistically significant:

           *     : p<0.05

           **  : p<0.01

Mann-Whitney U-test Versus Control

 

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions, the test item did not induce statistically significant increases in DNA strand breaks at any of the tested dose levels in stomach, duodenum or liver cells. The investigated test item did not show genotoxic activity in the examined tissues.
Executive summary:

In this in vivo alkaline comet assay on the rat stomach, duodenum and liver with the test item the purpose was to evaluate the mutagenic potential of the test item by measuring its ability to induce DNA damage in the target organ tissues by single cell gel electrophoresis assay. Six Male Wistar rats were treated with doses of 2000, 1000 and 500 mg/kg bw/day. A negative control (vehicle) and positive control (ethylmethanesulfonate, EMS at 200 mg/kg bw) was run concurrently. Formulations were prepared before each treatment. In the analytical measurements the measured concentration values remained within the ± 10 % of nominal range in the test item suspensions at all concentrations. The animals of the test item dose groups and the negative control animals were treated by oral gavage twice, once on the day 0 and once 24 hours thereafter. The positive control animals were treated by oral gavage once during the experiment on the day 1. 3-4 hours after the last treatment the animals were euthanised and the cells of the target tissues were isolated. Cytotoxicity was determined on a small sample of each isolated cell suspension following the Trypan blue dye exclusion technique, directly after sampling. Embedding the cells; Lysis (pH=10); Unwinding (pH>13; for 30 min.); Electrophoresis (pH>13; for 30 min. at 25V and about 300 mA). After neutralisation (pH=7.5; 3 times for 5 min.) preservation (abs. ethanol for 5 min. and air dried) of slides. Prior the scoring the DNA was stained with 50 μL of 2 μg/mL Ethidium bromide. The comets were measured via a digital camera linked to an image analyzer system using a fluorescence microscope equipped with an appropriate excitation filter at a magnification of 200X. For image analysis the Komet 6.0 F was used. In addition, each slide was examined for presence of ghost cells (possible indicator of toxicity and/or apoptosis). Ghost cells were excluded from the image analysis data collection. For each tissue sample fifty cells per slide were randomly scored i.e. 150 cells per animal (750 analyzed cells per test item treatment and per vehicle control; 450 per positive control). All of the validity criteria regarding the negative and positive control treatments as well as the number of analysed cells, and the investigated dose levels were met. No mortality was observed during the treatments and expression period in any dose group up to the limit dose of 2000 mg/kg bw/day and in the controls. Toxic symptoms or any clinical signs were not observed during the treatments in any dose or control group.

At the tissue isolation normal appearance and anatomy of stomach and duodenum was noticed in all dose levels and controls. The appearance of liver was normal at the vehicle control, at EMS control and at the 500 mg/kg bw/day dose; however, mosaic like appearance of the liver was noticed at the dose levels of 1000 and 2000 mg/kg bw/day. In the liver cell suspension fibres were observed at 100X magnification at 1000 mg/kg bw/day. Easily disintegrated liver structure, granulose coagulated blood and, at the stomach opening intensive test item smell were described at 2000 mg/kg bw/day.

 

The average body weights increased in all dose groups as well as in the negative and positive control groups. The body weight increases were in the range of 0.92 – 2.29 % when comparing values measured just before the first treatment and measured just before the sacrifice. At the cytotoxicity screening measurements (using Trypan blue dye exclusion method) no cytotoxicity was noticed in any test item and control item treatments at any target tissue.

 At the examined test item treated groups, the number of ghost cells in the duodenum and liver samples remained nearly in the same range and did not differ statistically significantly from that of the vehicle control.

In the stomach the percentage of ghost cells differed statistically significantly from that of the of the vehicle control at the dose level of 1000 mg/kg bw/day. The percentage of ghost cells was ~9 % at the vehicle control group and ~13 % at 1000 mg/kg bw/day. Both values were well within the laboratory’s experience regarding the percentage of ghost cells at the stomach preparations. Furthermore, dose dependent increase at the stomach samples was not established in the percentage of ghost cells (i. e. at 2000 mg/kg bw/d ghost cell percentage was 10.07 and not statistically significant). Therefore, the slightly higher percentage of ghost cells in the 1000 mg/kg bw/d group was considered as acceptable and, being within the biological variability range of the test. A statistically significant increase of ghost cells was noticed in all tissues after EMS treatment. The ghost cells are a possible indicator of cytotoxicity and/or apoptosis. According to the referred literature increased frequency of ghost cells may also indicate cells with severe DNA damage (genotoxicity). To be conscious of the mutagenicity results and laboratory’s earlier experience, the relatively higher number of ghost cells at the positive control, EMS treatment (mean = 19.31 %) are considered being a possible indicator of genotoxicity.

The mean median % tail DNA values of each dose remained in the vehicle control range at the examined tissues, and the slightly different (higher or lower) values did not differ statistically significantly from that of the vehicle control up to the highest dose of 2000 mg/kg bw/day. The mean median % tail DNA values of the vehicle control and test item doses in the stomach, duodenum and liver samples fell within the corresponding historical control data ranges within the 95 % confidence intervals, C-charts.

Additionally, the tail length values and Olive Tail Moment (OTM) of the vehicle control and each treatment were compared. The tail length values of the stomach, duodenum and liver samples did not differ statistically significantly from that of the vehicle control in whole examined dose range. The Olive Tail Moment values in the stomach, duodenum and liver of the test item treated groups did not differ statistically significant from that of the vehicle control.

Additionally, all of the tail length and OTM values (vehicle control and dose groups) remained well within the established historical control data ranges, within the 95 % confidence intervals, C-charts.

 

In conclusion, under the experimental conditions, the test item did not induce statistically significant increases in DNA strand breaks at any of the tested dose levels in stomach, duodenum or liver cells. The investigated test item did not show genotoxic activity in the examined tissues.