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Toxicological information

Genetic toxicity: in vivo

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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: Guideline study to GLP without any deviations.
Cross-reference
Reason / purpose for cross-reference:
read-across: supporting information
Reference
Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Justification for type of information:
See read across justification document
Reason / purpose for cross-reference:
read-across source
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
reduction of activity, ruffled fur all animals both doses and treatment times
Vehicle controls validity:
valid
Positive controls validity:
valid

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.39 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells In Vivo)
Deviations:
no
Principles of method if other than guideline:
Not applicable.
GLP compliance:
yes (incl. QA statement)
Type of assay:
unscheduled DNA synthesis

Test material

Constituent 1
Reference substance name:
Proban STi
IUPAC Name:
Proban STi
Constituent 2
Reference substance name:
-
EC Number:
436-230-7
EC Name:
-
Cas Number:
359406-89-6
Molecular formula:
This substance is a UVCB formed from the multiple reactions. It is not possible to provide a molecular formula.
IUPAC Name:
Phosphonium, tetrakis(hydroxymethyl)-, chloride (1:1), reaction products with 1-tetradecanamine and urea
Details on test material:
- Name of test material (as cited in study report): Proban STi

Test animals

Species:
rat
Strain:
other: Wistar Hanlbm: WIST (SPF)
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd, Animal Breeding Services, CH-4414 Fuellinsdorf and Harlan Winkelmann GmbH, D-33178 Borchen
- Age at study initiation: 6-10 weeks
- Weight at study initiation: 177.2-205.6 g
- Fasting period before study: 6 hours or overnight
- Housing: individually
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 3
- Humidity (%): 30-70
- Air changes (per hr): not available
- Photoperiod (hrs dark / hrs light): 12:12

IN-LIFE DATES: from 4 October 2004 to 14 December 2004

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: acceptable solubility and non-toxicity to the animals
- Concentration of test material in vehicle: 10 ml/kg bw
Duration of treatment / exposure:
2 and 16 hours
Frequency of treatment:
once
Post exposure period:
not applicable
Doses / concentrations
Remarks:
Doses / Concentrations:
175 and 350 mg/kg
Basis:
actual ingested
based on dry content (65.8%)
No. of animals per sex per dose:
4 (males only)
Control animals:
yes
Positive control(s):
2 hours preparation interval: DMH (N,N'-dimethylhydazinedihydrochloride)
- Justification for choice of positive control(s): validated for this assay
- Route of administration: oral gavage
- Doses / concentrations: 40 mg/kg bw / 10 ml/kg bw

16 hours preparation interval: 2-acetylaminofluorene
2 hours preparation interval: DMH (N,N'-dimethylhydazinedihydrochloride)
- Justification for choice of positive control(s): validatd for this assay
- Route of administration: oral gavage
- Doses / concentrations: 100 mg/kg bw / 10 ml/kg bw

Examinations

Tissues and cell types examined:
primary hepatocytes
Evaluation criteria:
A test item is classified as positive if the mean number of net grains is higher than five per nucleus at one of the test points (based on historical data)
A group average between 0 and 5 net grains is considered a marginal response. A dose-related increase in nuclear and net nuclear grains and/or a substantial shift of the percentage distribution of the nuclear grain counts to higher values provide additional information to confirm a positive result with less than 5 net grains.
Statistics:
not required.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
reduction of activity, ruffled fur all animals both doses and treatment times
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 100-500 mg/kg
- Solubility: soluble in water at all dose levels
- Clinical signs of toxicity in test animals:
500 mg/kg: Mortality of 1/2 males and 2/2 females. Reduction of spontaneous activity, abdominal position, eyelid closure, ruffled fur, apathy in all animals 6 h post dosing.
350 mg/kg: Mortality of 2/2 females. Reduction of spontaneous activity, abdominal position, ruffled fur, apathy in all animals 6 h post dosing.
250 mg/kg: Reduction of spontaneous activity, abdominal position, ruffled fur, apathy in all animals 6 h post dosing.
100 mg/kg: Reduction of spontaneous activity in all animals 6 h post dosing.

Any other information on results incl. tables

The change in the urine colour of the treated animals could be due to the systemic distribution of the test substance, thus demonstrating its bioavailability.

The viability of the hepatocytes was not substantially affected by the in vivo treatment with the test substance at any of the treatment periods or dose groups. The inter-individual variations obtained for the numbers and the viabilities of the isolated hepatocytes are in the range of historical controls.

No dose level of the test substance revealed UDS induction in the hepatocytes of the treated animals compared to the current vehicle controls. Neither the nuclear grains nor the resulting net grains were distinctly enhanced due to the in vivo treatment of the animals with the test substance for 2 hours or 16 hours. Therefore the net grain values obtained after treatment with the test substance were consistently negative. No substantial shift to higher values was obtained in the percentage of cells in repair.

Appropriate reference mutagens [DMHat 40 mg/kg and 2-AAF at 100 mg/kg] were used as positive controls.In vivotreatment withDMHor 2-AAF revealed distinct increases in the number of nuclear and net grain counts.

Toxic symptoms in the Main Experiment

2 hour exposure

Toxic reactions

Hours post-treatment 175 / 350 mg/kg bw

 

1 h

2 h

Reduction of spontaneous activity

4/4

4/4

Ruffled fur

4/4

4/4

 

16 hour exposure

Toxic reactions

Hours post-treatment 175 / 350 mg/kg bw

 

1 h

16 h

Reduction of spontaneous activity

4/4

4/4

Ruffled fur

4/4

4/4

Urine colour

No observations

Orange

Viability and number of hepatocytes

Treatment

Period

Animal No.

Viability (%)

Number of isolated cells (x 106)

Deionised water

2 h

1

71

530

2

79

300

4

85

468

175 mg/kg bw Proban STi

2 h

5

73

226

6

74

266

7

71

309

350 mg/kg bw Proban STi

2 h

9

78

312

10

72

281

11

74

289

40 mg/kg bwDMH

2 h

13

70

375

14

75

375

15

73

314

Deionised water

16 h

17

75

225

19

72

241

20

71

249

175 mg/kg bw Proban STi

16 h

21

74

363

22

81

267

23

75

315

350 mg/kg bw Proban STi

16 h

25

72

317

26

70

361

27

81

271

100 mg/kg bw 2-AAF

16 h

29

76

235

30

74

252

31

75

296

 

Mean Nucleus, Cytoplasmic Area and Net Grains

Preparation interval: 2 hours

Test group

Animal No.

Mean Nuclear Grain Count

Mean cytoplasmic Grain Count

Mean Net Nuclear Grain counts

Mean Nuclear Grains of Cells in Repair

% cells in repair

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Deionised water

1

8.4

3.8

14.4

6.0

-6.0

5.5

0.0

0.0

0

2

11.9

7.3

17.1

7.5

-5.2

5.4

8.8

4.9

4

4

13.9

6.8

19.9

9.1

-5.9

6.2

5.3

0.6

3

Mean

11.4

2.8

17.1

2.7

-5.7

0.5

4.7

4.4

2

175 mg/kg bw Proban STi

5

15.9

9.9

27.1

12.7

-11.3

9.8

13.0

9.9

2

6

11.8

5.9

20.5

8.5

-8.7

7.7

7.0

3.5

3

7

24.1

14.2

39.5

20.8

-15.3

14.2

17.5

3.5

2

Mean

17.3

6.3

29.0

9.6

-11.8

3.3

12.5

5.3

2

350 mg/kg bw Proban STi

9

18.3

8.4

28.6

12.4

-10.3

8.7

7.3

1.5

3

10

11.4

7.2

21.6

10.6

-10.3

8.6

10.0

0.0

1

11

16.2

6.5

24.7

8.9

-8.5

7.1

5.7

1.2

3

Mean

15.3

3.6

25.0

3.5

-9.7

1.0

.7

2.2

2

40 mg/kg bwDMH

13

26.5

16.9

22.1

15.4

4.4

11.1

13.4

7.7

45

14

22.8

10.2

20.0

8.9

2.8

11.2

12.2

6.8

46

15

34.0

15.9

20.3

8.0

13.8

13.2

20.0

10.6

70

Mean

27.8

5.7

20.8

1.1

7.0

5.9

15.2

4.2

54

SD=Standard Deviation. SD for each animal is deviation between the analysed cells. SD for means is the deviation between results for the animals in group.

 

Mean Nucleus, Cytoplasmic Area and Net Grains

Preparation interval: 16 hours

Test group

Animal No.

Mean Nuclear Grain Count

Mean cytoplasmic Grain Count

Mean Net Nuclear Grain counts

Mean Nuclear Grains of Cells in Repair

% cells in repair

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Deionised water

17

11.5

5.3

16.4

6.2

-4.9

5.3

6.7

2.9

3

19

17.8

7.4

29.0

11.5

-11.3

9.6

9.5

2.9

4

20

14.2

6.0

21.8

8.1

-7.6

7.3

6.0

0.0

2

Mean

14.4

3.1

22.4

6.3

-8.0

3.2

7.4

1.9

3

175 mg/kg bw Proban STi

21

10.3

4.6

21.0

9.2

-10.7

8.2

0.0

0.0

0

22

16.9

7.1

28.8

11.5

-11.9

9.9

7.0

0.0

1

23

17.8

6.9

30.3

11.2

-12.5

8.8

5.0

0.0

1

Mean

15.0

4.1

26.7

5.0

-11.7

0.9

4.0

3.6

1

350 mg/kg bw Proban STi

25

20.4

9.7

28.6

10.9

-8.1

8.8

11.4

5.6

7

26

18.0

8.4

29.1

13.3

-11.2

10.9

6.4

1.1

5

27

28.2

11.1

36.4

15.1

-8.2

11.3

9.8

6.2

12

Mean

22.2

5.3

31.4

4.4

-9.2

1.7

9.2

2.6

8

100 mg/kg bw 2-AAF

 

29

26.2

10.3

22.5

6.4

3.7

9.5

11.9

7.2

43

30

42.1

18.2

31.2

12.5

10.9

15.0

19.1

12.0

64

31

27.3

9.5

24.0

8.0

3.3

10.3

12.5

6.4

45

Mean

31.9

8.9

25.9

4.6

6.0

4.3

14.5

4.0

51

SD=Standard Deviation. SD for each animal is deviation between the analysed cells. SD for means is the deviation between results for the animals in group.

Historical Control Data (1995-2002)

Test group

Net Grains

Treatment period

Number of animals

Range

Mean

Vehicle controls

1.82 to -13.51

-5.64 +/- 2.27

2, 3 or 16 hours

326

Positive controls (DMH)

0.270 to 47.46

15.69 +/- 9.57

2 or 3 hours

125

Positive controls (2-AAF)

2.60 to 80.18

20.44 +/- 10.98

16 hours

200

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under the experimental conditions reported, the test substance did not induce DNA damage leading to increased repair synthesis in the hepatocytes of treated rats.
Executive summary:

Proban STi was assessed in the in vivo UDS assay for its potential to induce DNA repair (UDS) in the hepatocytes of rats. The test substance was formulated in deionised water, which was used as the vehicle control. The test substance was administered orally by gavage at 175 and 350 mg/kg bw (dose volume 10 ml/kg bw). After a treatment period of 2 and 16 hours respectively, 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 of the test substance administered in the main experiment (350 mg/kg) was estimated in pre-experiments to be close to the maximum tolerated dose. The main experiment was performed using male rats only, since the males could be dosed higher than females. At a dose of 350 mg/kg of the test substance, the treated females died. However, due to the close proximity of the maximum tolerated dose for the females (250 mg/kg) to that of the males (350 mg/kg) and the limited number of animals assessed, the difference in the toxicity observed in males and females is not considered significant.

The change in the urine colour of the treated animals could be due to the systemic distribution of the test substance, thus demonstrating its bioavailability.

The viability of the hepatocytes was not substantially affected by the in vivo treatment with the test substance at any of the treatment periods or dose groups. The inter-individual variations obtained for the numbers and the viabilities of the isolated hepatocytes are in the range of historical controls.

No dose level of the test substance revealed UDS induction in the hepatocytes of the treated animals compared to the current vehicle controls. Neither the nuclear grains nor the resulting net grains were distinctly enhanced due to the in vivo treatment of the animals with the test substance for 2 hours or 16 hours. Therefore the net grain values obtained after treatment with the test substance were consistently negative. No substantial shift to higher values was obtained in the percentage of cells in repair.

Appropriate reference mutagens [DMH at 40 mg/kg and 2 -AAF at 100 mg/kg] were used as positive controls. In vivo treatment with DMH or 2 -AAF revealed distinct increases in the number of nuclear and net grain counts.

It was concluded that under the experimental conditions reported, the test substance did not induce DNA-damage leading to increased repair synthesis in the hepatocytes of the treated rats.

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