Registration Dossier

Toxicological information

Carcinogenicity

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Administrative data

Endpoint:
carcinogenicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 Nov 1996 - 27 Nov 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Version / remarks:
adopted 1981
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.33 (Combined Chronic Toxicity / Carcinogenicity Test)
Version / remarks:
adoption not stated
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPP 83-5 (Combined Chronic Toxicity/Oncogenicity Study) adopted 1984
Qualifier:
according to
Guideline:
other: JMAFF (Combined Chronic Toxicty/Oncogenicty Study) adopted 1985
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
other: Sprague Dawley (CRL:CD (IGS) BR)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Ltd., Margate, Kent, UK
- Weight at receipt (measured for 10% of the animals): 55-79 g (males), 55-84 g (females)
- Age at receipt: 28 days
- Housing: Polycarbonate cages with grid floor in groups of 5 animals
- Diet: Modified SQC Expanded Ground Rat and Mouse Maintenance Diet No. 1 (Special Diet Services Ltd., Witham, UK), ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±2
- Humidity (%): 45-65
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: diet
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly by two identical mixes.
- Mixing appropriate amounts: For the highest dose level, the required amount of test material and a small amount of diet were mixed. This mix was then blended with the remaining diet. Subsequent dietary concentrations were prepared by serial dilution.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Aliquots of freshly prepared test diets were taken were taken weekly from all concentration. Samples from Weeks 1, 4, 6, 11, 14, 23, 34, 47, 71, 83, 95 and 106 (all concentrations) were analysed. The results were within the range +10% to -15% of nominal.
Duration of treatment / exposure:
104 weeks (interim sacrifice of 20 animals/sex/dose at 52 weeks)
Frequency of treatment:
Continuously
Post exposure period:
None
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
20, 200, 2000 and 4000 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
1.0, 10, 101 and 210 mg/kg bw/day
Basis:
other: calculated based on 'Group mean food consumption' and 'Group mean body weight'
No. of animals per sex per dose:
70
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Dose levels were based on the results of an earlier 90-day dietary rat study (M-141124-01-1).
Positive control:
None.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Observation for abnormal behavior, including neuro-muscular coordination and physical appearance were done each morning and as well in the afternoon on Mondays to Fridays (except on holidays).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once weekly prior to weighing.

BODY WEIGHT: Yes
- Time schedule for examinations: 10% of the animals upon receipt. Each animal at randomisation, at the start of treatment, at weekly intervals to Week 14, every second week thereafter and at necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- For each sex, the weekly food consumption of each cage of animals was measured at weekly intervals for the first 13 weeks of treatment and approx. every four weeks thereafter.
- Compound intake calculated as time-weighted averages from the consumption and body weight data: Yes

FOOD EFFICIENCY: Yes
- Body weight gain in % calculated as time-weighted averages from the consumption and body weight gain data

WATER CONSUMPTION: Yes
- Time schedule for examinations: Weekly for each cage of the numerical last 20 animals from each group over 5-day periods (Monday to Friday) during Weeks 9, 17, 33 and 49.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to the start of treatment in all animals and prior to the interim (Month 12) and terminal kill (Month 24) in the control and high dose group.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: After 3, 6, 12, 18 and 24 months of treatment.
- Anaesthetic used for blood collection: Yes (ether anaesthesia (Months 3, 6 and 12) or isoflurane anaesthesia (Months 18 and 24))
- Animals fasted: Yes
- How many animals: First 10 surviving animals/sex/dose group with the lowest identification number.
- Parameters checked: Haematocrit (HCT), Haemoglobin (HB), Red blood celIs (RBC), Mean cell volume (MCV), Mean cell haemoglobin (MCH), Mean cell haemoglobin concentration (MCHC), Platelets (PLT), Prothrombin time (PT), White blood cells (WBC), Neutrophils (NEUT), Lymphocytes (LYMP), Monocytes (MONO), Eosinophils (EOS), Basophils (BASO), Large unstained cells (LUC), Reticulocyte count (RET), Activated partial thromoplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: After 3, 6, 12, 18 and 24 months of treatment.
- Anaesthetic used for blood collection: Yes (ether anaesthesia (Months 3, 6 and 12) or isoflurane anaesthesia (Months 18 and 24))
- Animals fasted: Yes
- How many animals: First 10 surviving animals/sex/dose group with the lowest identification number.
- Parameters checked: Total protein (PROT), Albumin (ALB), Total globulin (GLOB), A/G ratio (A/G), Calcium (CA), Phosphate (PO4), Sodium (NA), Potassium (K), Urea (UREA), Creatinine (CREAT), Glucose (GLUC), Total cholesterol (CHOL), Total bilirubin (TBIL), Chloride (CL), Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Alkaline phosphatase (AP), G-glutamyl transpeptidase (GGT), Creatine kinase (CPK)

URINALYSIS: Yes
- Time schedule for collection of urine: After 3, 6, 12, 18 and 24 months of treatment.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- How many animals: First 10 surviving animals/sex/dose group with the lowest identification number.
- Parameters checked: Appearance (APP), pH (PH), Protein (PROT), Glucose (GLUC), Specific gravity (SG), Colour of Spun deposit (SDEP), Bacteria (BACT), Red blood cells (RBC), Epithelial cells (EPTH), Ketones (KET), Urobilinogen (UBIL), Bilirubin (BIL), Blood (BLD), Phosphate crystals (PO4), Urate crystals (URAT), White blood cells (WBC), Sperm (SPER), Volume (VOL), Casts (CAST)

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Time schedule: After approx. 52 weeks the first 20 animals/sex/group (interim kill); the remaining animals after 104 weeks (terminal kill)
- Organ weights: Adrenals, brain, heart, kidneys, liver, ovaries, spleen, testes with epididymides
- List of organs: Adrenals, aorta (abdominal), bile duct*, brain, caecum, colon, diaphragm, duodenum, epididymides, eyes, femur and joint, Harderian gland, head, heart, ileum, jejunum, kidneys, lacrimal gland (exorbital), liver, lungs (inflated), lymph nodes (cervical and mesenteric), mammary gland, muscle (skeletal), nerves (optic and sciatic), oesophagus, oviducts, ovaries, pancreas, pituitary, prostate, rectum, salivary glands (submaxillary, sublingual and parotid), seminal vesicles, skin, spinal cord (3 levels), spleen, sternum, stomach, testes, thymus, thyroid (with parathyroids), tongue, trachea, urinary bladder, uterus, vagina, any other tissue showing macroscopic abnormalities

* Taken from animals killed from Week 62 of treatment onwards.

HISTOPATHOLOGY: Yes
Histopathology was conducted on organs/tissues indicated above from all animals.
Other examinations:
None.
Statistics:
The significance of differences between control and treated groups was analysed by either parametric or non-parametric statistical tests as appropriate. A maximum 2-tailed probability value of 5% (p <0.05) was considered statistically significant.
The following convention has been used to indicate statistical significance:
* p < 0.05
** p < 0.01
*** p < 0.001

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
4000 ppm: Slight increased incidence of urogenital staining for males (non-adverse).
Mortality:
mortality observed, treatment-related
Description (incidence):
4000 ppm: Slight increased incidence of urogenital staining for males (non-adverse).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
4000 ppm: Reduced body weight gain in females (non-adverse).
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
4000 ppm: Slight but consistent increase for females.
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
2000 ppm: Increased incidence of ketones in urine of males.
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
2000 ppm Decreased absolute and relative kidney and increased relative liver weights in males (non-adverse).
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
4000 ppm: Increased incidence of centrilobular hepatocytic hypertrophy in males and progressive nephropathy in females.
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
There was no treatment-related effect on mortality during the course of the study.
The incidence of mortality after 12 months of treatment was 2/70, 1/70, 3/70, 1/70 and1/70 for males and 3/70, 5/70, 1/70, 2/70 and 5/70 for females, at 0, 20, 200, 2000 and 4000 ppm respectively.
The incidence of mortality at 24 months was 23/50, 27/50, 24/50, 19/50 and 17/50 for males and 33/50, 28/50, 27/50, 34/50 and 31/50 for females, at 0, 20, 200, 2000 and 4000 ppm respectively.

At 4000 ppm there was a slight increased incidence of urogenital staining for males from approx. Week 41 onwards.

BODY WEIGHT AND WEIGHT GAIN (see Table 'Group mean body weights')
At 4000 ppm body weight gain of females was reduced by 8%. This was mainly due to a marked reduction during Week 1 (38%) although a trend was seen throughout the study. Males of the same group showed as well a reduction in Week 1 (19%) but body weight gain in general was unaffected.

FOOD CONSUMPTION AND COMPOUND INTAKE (see Tables 'Group mean food consumption' and 'Group mean test material intake')
At 4000 ppm food intakes of both sexes were reduced during Week 1 but reached the control level thereafter.

FOOD EFFICIENCY
At 4000 ppm food conversion efficiency of females were reduced by 31% during Week 1 but reached the control level thereafter.

WATER CONSUMPTION (see Table 'Group mean water consumption')
At 4000 ppm a slight (18-27%) but consistent increase in water consumption was seen throughout the study for females.

URINALYSIS (see Table 'Group incidence of ketones present in urine')
At Month 3, 6 and 12 an increased incidence of ketones present in the urine was seen in males treated at 2000 and 4000 ppm and also at Month 18 in males given 4000 ppm.

ORGAN WEIGHTS (see Table 'Treatment-related effects on organ weights at interim and terminal kill')
At interim kill relative liver weights were slightly increased for males (16%)and females (14%) treated at 4000 ppm. At 2000 ppm, relative liver weights were 8% higher for males.

At terminal kill both absolute and relative kidney weights were decreased for males at 4000 ppm (12% and 9%, respectively) and at 2000 ppm (10% and 13%, respectively). Furthermore, relative liver weights were increased (8%) for males given 4000 ppm.

HISTOPATHOLOGY: NON-NEOPLASTIC (see Table 'Histopathological treatment-related effects at interim and terminal kill')
At interim and terminal kill an increased incidence of centrilobular hepatocytic hypertrophy was seen in males receiving 4000 ppm.
In addition, at terminal kill there was also an increased incidence and severity of progressive nephropathy in females treated at 4000 ppm.
Relevance of carcinogenic effects / potential:
No carcinogenic effects found.

Effect levels

open allclose all
Dose descriptor:
LOEL
Effect level:
> 4 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No oncogenic effects observed.
Remarks on result:
other: Effect type: carcinogenicity (migrated information)
Dose descriptor:
LOEL
Effect level:
> 210 mg/kg bw/day (actual dose received)
Based on:
other: calculated based on 'Group mean food consumption' and 'Group mean body weight'
Sex:
male/female
Basis for effect level:
other: No oncogenic effects observed.
Remarks on result:
other: Effect type: carcinogenicity (migrated information)
Dose descriptor:
NOEL
Effect level:
4 000 ppm
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: Effect type: carcinogenicity (migrated information)
Dose descriptor:
NOEL
Effect level:
210 mg/kg bw/day (nominal)
Based on:
other: calculated based on 'Group mean food consumption' and 'Group mean body weight'
Sex:
male/female
Remarks on result:
other: Effect type: carcinogenicity (migrated information)
Dose descriptor:
LOAEL
Effect level:
2 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Increased incidence of ketones in the urine as well as decreased absolute and relative kidney and increased relative liver weights in males.
Remarks on result:
other: Effect type: toxicity (migrated information)
Dose descriptor:
LOAEL
Effect level:
101 mg/kg bw/day (actual dose received)
Based on:
other: calculated based on 'Group mean food consumption' and 'Group mean body weight'
Sex:
male/female
Basis for effect level:
other: Increased incidence of ketones in the urine as well as decreased absolute and relative kidney and increased relative liver weights in males.
Remarks on result:
other: Effect type: toxicity (migrated information)
Dose descriptor:
NOAEL
Remarks:
= NOEL
Effect level:
200 ppm
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: Effect type: toxicity (migrated information)
Dose descriptor:
NOAEL
Remarks:
= NOEL
Effect level:
10 mg/kg bw/day (actual dose received)
Based on:
other: calculated based on 'Group mean food consumption' and 'Group mean body weight'
Sex:
male/female
Remarks on result:
other: Effect type: toxicity (migrated information)

Any other information on results incl. tables

Table 1: Group mean bodyweights (g)

Weeks

Dose level ppm)

Males

Females

0

20

200

2000

4000

0

20

200

2000

4000

0-14

347

(−)

338

(97)

338

(97)

328

(95)

316

(91)

151

(−)

151

(100)

150

(99)

146

(97)

132

(87)

14-28

108

(−)

110

(102)

107

(99)

108

(100)

107

(99)

39

(−)

45

(115)

43

(110)

37

(95)

37

(95)

28-52

99

(−)

107

(108)

99

(100)

103

(104)

88

(89)

70

(−)

80

(114)

81

(116)

72

(103)

60

(86)

52-78

69

(−)

76

(110)

60

(87)

62

(90)

61

(88)

81

(−)

85

(105)

64

(79)

66

(81)

45

(56)

0-104

606

(−)

650

(107)

618

(102)

635

(105)

593

(98)

359

(−)

385

(107)

365

(102)

345

(96)

332

(92)

() Percentage of control values

 

Table 2: Group mean food consumption (g/animal/day)

Weeks

Dose level ppm)

Males

Females

0

20

200

2000

4000

0

20

200

2000

4000

1-12

27

(−)

27

(100)

27

(100)

27

(100)

26

(96)

21

(−)

21

(100)

21

(100)

21

(100)

20

(95)

13-26

26

(−)

27

(104)

26

(100)

26

(100)

26

(100)

20

(−)

22

(110)

21

(105)

22

(110)

21

(105)

30-51

27

(−)

27

(100)

27

(100)

27

(100)

27

(100)

22

(−)

23

(105)

22

(100)

23

(105)

22

(100)

54-74

27

(−)

27

(100)

27

(100)

27

(100)

26

(96)

22

(−)

24

(109)

23

(105)

23

(105)

23

(105)

79-103

26

(−)

27

(104)

26

(100)

27

(104)

27

(104)

23

(−)

24

(104)

24

(104)

24

(104)

24

(104)

1-103

27

(−)

27

(100)

27

(100)

27

(100)

26

(96)

22

(−)

22

(100)

22

(100)

22

(100)

22

(100)

() Percentage of control values

 

Table 3: Group mean test material intake (mg/kg bw/day)

Weeks

 

Dose level ppm)

20

200

2000

4000

1-104

Males

0.8

8

84

171

Females

1.1

12

118

249

Combined sexes

1.0

10

101

210

 

Table 4: Group mean water consumption (g/animal/day)

Week

Dose level ppm)

Males

Females

0

20

200

2000

4000

0

20

200

2000

4000

9

35

(−)

32

(91)

32

(91)

34

(97)

35

(100)

28

(−)

28

(100)

30

(107)

31

(111)

33

(118)

17

31

(−)

31

(100)

29

(94)

32

(103)

33

(106)

27

(−)

29

(107)

29

(107)

28

(104)

33

(122)

33

29

(−)

30

(103)

28

(97)

31

(107)

32

(110)

34

(−)

31

(91)

34

(100)

30

(88)

41

(121)

49

28

(−)

32

(114)

31

(111)

29

(104)

33

(118)

33

(−)

35

(106)

38

(115)

31

(94)

42

(127)

() Percentage of control values

 

Table 5: Group incidence of ketones present in urine

Month

Dose level ppm)

Males

Females

0

20

200

2000

4000

0

20

200

2000

4000

3

1/10

0/10

3/10

9/10

10/10

2/10

0/10

0/10

0/10

2/10

6

0/10

0/9

2/10

9/10

10/10

3/10

0/10

1/10

1/10

2/10

12

0/10

1/9

0/10

5/10

9/10

0/10

0/10

0/10

0/10

0/10

18

0/10

0/10

0/10

2/10

5/10

0/10

0/10

0/10

0/10

0/10

24

0/10

0/10

0/10

2/10

2/10

1/10

0/10

1/9

0/10

0/10

 

Table 6: Treatment-related effects on organ weights at interim and terminal kill

 

Dose level ppm)

Males

Females

0

20

200

2000

4000

0

20

200

2000

4000

Interim kill

 

Relative liver weight (%)

2.67

(−)

2.76

(103)

2.68

(100)

2.88

(108)

3.10

(116)

2.78

(−)

2.97

(107)

2.77

(100)

2.95

(106)

3.16

(114)

Terminal kill

 

Absolute kidney weights (g)

4.11

(−)

4.18

(102)

4.29

(104)

3.68

(90)

3.60

(88)

No treatment-related effects

Relative kidney weight (%)

0.53

(−)

0.51

(96)

0.56

(106)

0.46

(87)

0.48

(91)

Relative liver weight (%)

2.56

(−)

2.54

(99)

2.70

(105)

2.53

(99)

2.77

(108)

() Percentage of control values

Table 7: Histopathological treatment-related effects at interim and terminal kill

 

Dose level ppm)

Males

Females

0

20

200

2000

4000

0

20

200

2000

4000

Interim kill

Liver

 

Centrilobular hepatocytic hypertrophy

1/19

2/20

0/19

5/20

15/20

No treatment-related effects

Terminal kill

 

Liver

 

Centrilobular hepatocytic hypertrophy

0/26

0/22

0/25

0/31

12/33

No treatment-related effects

Kidney

 

Progressive nephropathy

No treatment-related effects

25/50

22/50

23/50

17/50

31/50

Minimal

16

13

16

12

11

Slight

8

7

4

5

10

Moderate

0

2

2

0

6

Sever

1

0

1

0

4

Applicant's summary and conclusion