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Toxicological information

Genetic toxicity: in vivo

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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:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
R & D Grade intermediate, considered acceptable for use on genetox tests This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labeling and/or risk assessment.

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:
OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
Deviations:
no
Remarks:
The study was conducted according to the guideline in effect at the time of study conduct.
Qualifier:
according to
Guideline:
EU Method B.39 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells In Vivo)
Deviations:
no
Remarks:
The study was conducted according to the guideline dated 1997.
Qualifier:
according to
Guideline:
other: ICH S2A document April 24, 1996
Qualifier:
according to
Guideline:
other: ICH S2B document November 21, 1997
GLP compliance:
yes
Type of assay:
unscheduled DNA synthesis

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Purity: 82.6%

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 9 weeks, 3 days (9 weeks 4 days - Pilot Toxicity Assay)
- Weight at study initiation: 256.4 - 275.2 g (284.6 - 304.0 g - Pilot Toxicity Assay)
- Fasting period before study: no
- Housing: up to five rodents per Micro-Barrier cage placed in racks equipped with Micro-VENT full ventilation, HEPA filtered system with heat-treated hardwood chips for bedding
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: no less than 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72±3 ºF
- Humidity (%): 50±20%
- Air changes (per hr): At least 10 changes of fresh HEPA-filtered air
- Photoperiod (hrs dark / hrs light): 12


Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: distilled water was the only vehicle evaluated when the test substance was observed to be soluble at 200 mg/mL to yield the highest concentration for maximum dosing
- Concentration of test material in vehicle: 2 g/kg bw
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw
Details on exposure:
PREPARATION OF TEST SUBSTANCE FORMULATION:

-Solvent used: distilled water
-Preparation frequency: not reported
-Preparation details: not reported
-Adjusted for purity: not reported


Duration of treatment / exposure:
single exposure
Frequency of treatment:
single exposure
Post exposure period:
2-4 hours or 12-16 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
500, 1000, 2000 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
10
Control animals:
yes
Positive control(s):
- Positive Control: Dimethylnitrosamine (DMN)
- Justification for choice of positive control(s): not reported
- Route of administration: single oral gavage injection
- Doses / concentrations: 35 mg/kg

Examinations

Tissues and cell types examined:
hepatocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
In the Pilot Toxicity Assay, the test substance was administered via oral gavage to male rats at 1, 10, 100, 1000, and 2000 mg/kg body weight (bw) in a total volume of 10 mL/kg bw. All animals appeared normal less than four hours following dosing, and 1, 2, and 3 days following dosing except for two of five 2000 mg.kg-treated animals that were observed to be lethargic only on Day 1. Based on the information obtained from the Pilot Toxicity Assay, the high dose for the UDS assay was set at the maximum tolerated dose which was 2000 mg/kg bw, accompanied by two lower doses of 500 and 1000 mg/kg bw.

TREATMENT AND SAMPLING TIMES:
The liver was perfused with 0.5 mM ethylene glycol-bis(β-amino ethyl ether) N,N,N’,N’-tetra acetic acid (EGTA) solution followed by collagenase solution (80-100 units Type I collagenase/mL culture medium). The liver was removed, transected, and shaken in a dilute collagenase solution to release the hepatocytes. The cells were pelleted by centrifugation, resuspended in complete Williams’ medium E (WME). Approximately 5x 100,000 cells were seeded into each of six 35mm tissue culture dishes containing 25mm cover slips and preconditioned complete WME. A minimum of 6 cultures were set up for each rat. The hepatocytes cultures were maintained in a humidified atmosphere of 5±1% CO2 and 37±1ºC. Ninety to 180 minutes after plating, the cells were washed once with complete WME and refed with serum-free WME containing 10 µCi 3H-thymidine/mL. Four hours later, the radioactive medium was removed; the cultures were washed three times in serum-free WME containing 0.25 mM thymidine, and then refed with serum-free WME containing 0.25 mM thymidine and incubated for 17-20 hours. Seventeen to twenty hours after exposure to thymidine, the cover slips bearing cultures were washed in serum-free WME.

DETAILS OF SLIDE PREPARATION:
The nuclei were swelled in 1% sodium citrate solution and the culture fixed in three changers of ethanol–glacial acetic acid fixative (3:1, v/v). The cover slips were allowed to air dry for at least 1 hour before mounting cell side up on glass slides. The slides were labeled with the study number and a code to identify the animal number. At least three of the six slides for each rat were dipped in photographic emulsion (diluted 1:1 in deionized water), fixed in Kodak fixer and stained with hematoxylin-eosin stain.

METHOD OF ANALYSIS:
The slides were viewed microscopically under a 100X oil immersion lens. A computer equipped with image analyzing software was interfaced through a video camera with the microscope so that silver grains within each nuclei and the surrounding cytoplasm could be counted. ProtoCOL system Version 3.07 with accompanying support software was used for grain counts. First the number of grains in a nucleus was counted. Then the number of grains in three separate nuclear-size cytoplasmic areas was counted. The counts were captured directly by the software and stored as raw data in an electronic data file created by the software. Fifty nuclei were scored from each of three replicate cultures for a total of 150 nuclei from each rat. Replicative DNA synthesis is evidenced by nuclei completely blackened with grains, and such cells were not counted. Cells exhibiting toxic effects of treatments, such as irregularly shaped or very darkly stained nuclei were not counted.
Evaluation criteria:
All conclusions are based on sound scientific judgment; however, the following is offered as a guide to interpretation of the data. Any mean net nuclear count which was increased by at least five counts over the vehicle control was considered significant. The test substance was judged positive if it induced a dose-related increase with no less than one dose significantly elevated above the vehicle control. A significant increase in the mean net nuclear grain count in at least two successive doses in the absence of a dose response was considered positive. A significant increase in the mean net nuclear grain count at the high dose group only with no evidence of a dose response was considered suspect. A significant increase in the mean net nuclear grain count at one dose with no evidence of a dose response was judged to be equivocal. The test substance was considered negative if no significant increase in the mean net nuclear grain counts was observed. The criteria for a valid test is the proportion of cells in repair in the vehicle control group must be less than 15% and the mean net nuclear grain count of the vehicle control must be less than one. The mean net nuclear grain count of the positive control group must be at least 5 counts over that of the vehicle control group.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 1, 10, 100, 1000, 2000 mg/kg body weight
- Solubility: no data
- Clinical signs of toxicity in test animals: none except two of the five 2000 mg/kg-treated animals exhibited lethargy on day 1 only.
- Evidence of cytotoxicity in tissue analyzed: no data
- Rationale for exposure: to determine an upper toxicity limit for the main study
- Harvest times: no data
- High dose with and without activation: no data

RESULTS OF DEFINITIVE STUDY
- Appropriateness of dose levels and route: no data
- Statistical evaluation: no data
- See Tables 1 and 2 below for Summary of UDS Assay with Test Substance

Any other information on results incl. tables

Table 1: Summary of UDS Assay with Test Substance

2 to 4 Hour Exposure

Group

Ear

Tag ID

Slide Code

Cells Scored

Per Animal

Per Treatment Group

Mean Grain Counts ± Standard Deviation

Mean Net

± SD

Cells in Repair

Nuclear

Cyto –

Plasmic

Net per Nucleus

Cells in Repair

Distilled Water (10 mL/kg)

Vehicle

326

18

150

7.8 ± 3.7

10.5 ± 3.7

-2.6 ± 3.7

3%

-4.2 ± 1.5

2%

327

11

150

6.6 ± 3.3

12.1 ± 4.8

-5.5 ± 4.4

1%

328

16

150

6.3 ± 3.0

10.7 ± 3.1

-4.3 ± 3.3

1%

Test Substance

500 mg/kg

331

32

150

8.8 ± 4.4

12.8 ± 5.2

-4.0 ± 4.3

1%

-3.7 ± 0.2

4%

332

21

150

7.7 ± 4.5

11.4 ± 4.8

-3.7 ± 4.5

5%

333

20

150

8.8 ± 4.3

12.3 ± 4.8

-3.5 ± 4.4

4%

1000 mg/kg

336

33

150

9.9 ± 4.9

15.8 ± 4.9

-6.0 ± 4.3

2%

-4.3 ± 1.5

2%

337

14

150

9.4 ± 4.2

13.3 ± 4.7

-3.9 ± 3.9

3%

338

34

150

8.3 ± 4.0

11.3 ± 4.4

-3.0 ± 3.4

3%

2000 mg/kg

341

38

150

10.8 ± 4.4

15.9 ± 5.5

-5.1 ± 4.7

1%

-3.9 ± 2.0

1%

342

26

150

10.8 ± 4.5

15.7 ± 5.6

-4.9 ± 4.7

1%

343

30

150

6.4 ± 3.3

8.0 ± 3.3

-1.6 ± 2.9

1%

Positive Control: Dimethylnitrosamine

35 mg/kg

346

27

150

19.9 ± 7.4

5.8 ± 2.1

14.2 ± 6.9

93%

15.0 ± 0.8

93%

347

24

150

27.9 ± 9.7

12.1 ± 6.9

15.8 ± 7.8

95%

348

17

150

24.5 ± 9.4

9.4 ± 5.8

15.1 ± 7.8

92%

 

 

Table 2: Summary of UDS Assay with Test Substance

12 to 16 Hour Exposure

Group

Ear

Tag ID

Slide Code

Cells Scored

Per Animal

Per Treatment Group

Mean Grain Counts ± Standard Deviation

Mean Net

± SD

Cells in Repair

Nuclear

Cyto –

plasmic

Net per

Nucleus

Cells in Repair

Distilled Water (10 mL/kg)

Vehicle

301

15

150

9.0 ± 4.8

13.1 ± 4.7

-4.0 ± 4.6

4%

-4.3 ± 0.7

2%

302

39

150

7.9 ± 3.9

13.0 ± 4.6

-5.1 ± 4.3

2%

303

40

150

7.1 ± 3.4

10.9 ± 4.0

-3.8 ± 3.3

0%

Test Substance

500 mg/kg

306

12

150

7.4 ± 3.4

11.5 ± 3.7

-4.1 ± 3.2

0%

-4.3 ± 0.6

1%

307

37

150

9.5 ± 5.9

13.4 ± 5.9

-3.9 ± 5.5

2%

308

19

150

9.2 ± 4.0

14.3 ± 4.5

-5.1 ± 4.0

0%

1000 mg/kg

311

29

150

9.4 ± 4.3

14.4 ± 4.9

-5.0 ± 4.5

2%

-4.0 ± 0.9

2%

312

25

150

8.3 ± 3.6

12.1 ± 4.1

-3.8 ± 3.5

1%

313

35

150

7.7 ± 5.0

10.9 ± 4.9

-3.2 ± 4.5

4%

2000 mg/kg

316

22

150

6.6 ± 3.5

10.1 ± 4.6

-3.5 ± 3.6

2%

-4.8 ± 1.2

1%

317

36

150

9.3 ± 4.1

14.6 ± 5.3

-5.3 ± 3.9

1%

318

13

150

9.8 ± 4.3

15.5 ± 5.4

-5.7 ± 4.4

1%

Positive Control: Dimethylnitrosamine

35 mg/kg

321

31

150

22.8 ± 13.0

6.7 ± 3.6

16.1 ± 11.5

95%

16.0 ± 1.2

95%

322

28

150

25.6 ± 11.7

8.4 ± 4.4

17.2 ± 10.9

95%

323

23

150

20.1 ± 8.9

5.3 ± 2.6

14.8 ± 8.0

95%

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
All criteria for a valid study were met. The positive and negative controls were within the historical control value ranges from 2004-2006 data. The results of this assay indicate that, under the test conditions, the test substance did not induce a significant increase in the mean number of net nuclear grain counts in hepatocytes isolated from test substance-treated animals, and was concluded to be negative in the Unscheduled DNA Synthesis (UDS) Test in Mammalian Cells In Vivo.
This study and the conclusions which are drawn from it fulfill the quality criteria (validity, reliability, repeatability).
Executive summary:

The test substance was tested in the Unscheduled DNA Synthesis (UDS) Test in Mammalian Cells In Vivo. The UDS assay was used to evaluate the potential of the test substance to induce unscheduled DNA synthesis in primary cultures of hepatocytes of test substance exposed male rats. 

For the Pilot Toxicity Assay, the test substance was administered via oral gavage to male rats at 1, 10, 100, 1000, and 2000 mg/kg body weight (bw) in a total volume of 10 mL/kg bw. All animals appeared normal less than four hours following dosing, and 1, 2 and 3 days following dosing except for two of the five 2000 mg/kg-treated animals were observed to be lethargic only on day 1. 

Based on the information obtained from the Pilot Toxicity Assay, the high dose for the UDS assay was set at the maximum tolerated dose which was 2000 mg/kg bow, accompanied by two lower doses of 500 and 1000 mg/kg bow. 

In the UDS test, the test substance was administered to 5 male rats per dose for 2 to 4 hours and 12 to 16 hours at doses of 500, 1000 and 2000 mg/kg bw. Two additional groups of 5 rats each, for each time point, received a single oral dose of distilled water or 35 mg/kg Dimethylnitrosamine (DMN) which served as the vehicle and positive control, respectiviely. The vehicle control, positive control and test substance were administered at a constant volume of 10 mL/kg bw by a single oral gavage injection. One male rate from the 12-16 hour treatment at 2000 mg/kg bw was observed to be normal less than four hours after dose administration and displayed lethargy and piloerection at the time of harvest. No mortality or clinical signs were observed in any of the remaining test substance- or control-treated harvested animals immediately following dosing or prior to harvest. 

The group mean net nuclear grain (NG) counts for animals treated with the test substance were not increased when compared to the vehicle control. For the 2 to 4 hour time point, the group mean NG counts for the test substance-treated animals were -3.7, -4.3, and -3.9 with ≤4% of cells in repair (cells with ≥ 5 NG) for the 500, 1000 and 2000 mL/kg bw animals, respectively. The group mean NG count for the vehicle control group was -4.2 with 2% of cells in repair. For the 12 to 16 hour time point, the group mean NG counts for the test substance-treated animals were -4.3, -4.0 and -4.8 with ≤2% of cells in repair (cells with ≥ 5 NG) for the 500, 1000 and 2000 mL/kg bw animals, respectively. The group mean NG count for the vehicle control group was -4.3 with 2% of cells in repair. The positive control group mean NG counts were 15.0 and 16.0 for the 2 to 4 hour and 12 to 16 hour, respectively. The percentage of cells in repair for the positive control group was 93% and 95% for the 2 to 4 hour and 12 to 16 hour, respectively. Results are summarized in Tables 1 and 2. 

The test substance did not induce a significant increase in the mean number of net nuclear grain counts (i.e., an increase of at least 5 counts over the vehicle control group) in hepatocytes isolated either 2 to 4 hours and 12 to 16 hours after dose administration. The test substance was concluded to be negative in the Unscheduled DNA Synthesis (UDS) Test in Mammalian Cells In Vivo.