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Diss Factsheets

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
chronic toxicity: oral
Remarks:
combined chronic toxicity and carcinogenicity study
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:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
carcinogenicity: oral
Remarks:
combined chronic toxicity and carcinogenicity study
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:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Version / remarks:
Current version adopted in 2018
Deviations:
yes
Remarks:
Thyroid and uterus weights were not measured.
Qualifier:
according to guideline
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Version / remarks:
Guideline in place during study conduct: adopted in 1981
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.33 (Combined Chronic Toxicity / Carcinogenicity Test)
Version / remarks:
Adoption not stated
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPP 83-5 (Combined Chronic Toxicity/Oncogenicity Study)
Version / remarks:
Adopted in 1984
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: JMAFF (Combined Chronic Toxicty/Oncogenicty Study) adopted 1985
GLP compliance:
yes
Species:
rat
Strain:
Sprague-Dawley
Remarks:
CRL:CD (IGS) BR
Details on species / strain selection:
The rat was chosen as the test system according to regulatory guidelines.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Ltd., Margate, UK
- Age at study initiation: 33 days
- Body weight at receipt (measured for 10% of the animals): male: 55 - 79 g, female: 55 - 84 g
- Fasting periods during study: overnight during urine collection
- Housing: group housed, 5 animals per cage by sex and dose group, in suspended polycarbonate cages with grid floor
- Diet: powdered laboratory rodent diet, Modified SQC Expanded Ground Rat and Mouse Maintenance Diet No. 1 (Special Diet Services Ltd., Witham, UK), ad libitum
- Water: tap water in drinking water quality, ad libitum
- Acclimation period: 15 days

DETAILS OF FOOD AND WATER QUALITY: At their respective levels in the diet and water, none of the contaminants known or expected to be present was considered likely to influence the outcome of the study.

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

IN-LIFE DATES: From: 14 Nov 1996 To: 13 - 27 Nov 1998
Route of administration:
oral: feed
Vehicle:
other: powdered laboratory rodent diet
Remarks:
Modified SQC Expanded Ground Rat and Mouse Maintenance Diet No. 1
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:
52 weeks = 12 months (interim sacrifice)
104 weeks = 24 months (terminal sacrifice)
Frequency of treatment:
Continuously
Dose / conc.:
20 ppm
Remarks:
actual test substance intake: males: 0.8 mg/kg bw/day, females: 1.1 mg/kg bw/day, combined sexes: 1.0 mg/kg bw/day
Dose / conc.:
200 ppm
Remarks:
actual test substance intake: males: 8 mg/kg bw/day, females: 12 mg/kg bw/day, combined sexes: 10.0 mg/kg bw/day
Dose / conc.:
2 000 ppm
Remarks:
actual test substance intake: males: 84 mg/kg bw/day, females: 118 mg/kg bw/day, combined sexes: 101 mg/kg bw/day
Dose / conc.:
4 000 ppm
Remarks:
actual test substance intake: males: 171 mg/kg bw/day, females: 249 mg/kg bw/day, combined sexes: 210 mg/kg bw/day
No. of animals per sex per dose:
20 (interim sacrifice)
50 (terminal sacrifice)
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).
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), and 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), and 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), and 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, and 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, and 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
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 4000 ppm a slight increased incidence of urogenital staining for males from approx. Week 41 onwards (non-adverse).
Mortality:
mortality observed, non-treatment-related
Description (incidence):
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.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 4000 ppm, body weight gain of females was reduced by 8% (non-adverse). 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 (see Table 1 under "Any other information on results incl. tables").
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
At 4000 ppm food intakes of both sexes were reduced during Week 1 but reached the control level thereafter (see Table 2 and 3 under "Any other information on results incl. tables").
Food efficiency:
no effects observed
Description (incidence and severity):
At 4000 ppm food conversion efficiency of females were reduced by 31% during Week 1 but reached the control level thereafter.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
At 4000 ppm a slight (18 - 27%) but consistent increase in water consumption was seen throughout the study for females (see Table 4 under "Any other information on results incl. tables").
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
Single parameters of treated animals distributed through all dose levels were statistically significantly changed compared to control. However, all changes were without any dose-response dependence, therefore, they are of no toxicological relevance.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significantly increased urea and creatinine levels were observed at several time points at 2000 and 4000 ppm in both sexes (for details see attached result tables under "Attached background material"). However, all values were within the physiological range of this rat strain.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
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 (see Table 5 under "Any other information on results incl. tables").
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
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 increased (8%) for males; however, this is considered not toxicologically relevant as there was no histopathological correlate at terminal kill.
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 (see Table 6 under "Any other information on results incl. tables").
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
At interim and terminal kill, an increased incidence of centrilobular hepatocytic hypertrophy (minimal to slight) was seen in males receiving 4000 ppm.
At terminal kill, an increased incidence and severity of progressive nephropathy was observed in females treated at 4000 ppm (see Table 7 under "Any other information on results incl. tables").
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
Administration of the test substance did not result in any alterations to indicate a carcinogenic potential from animals killed after 52 or 104 weeks of treatment (for details see attached data tables under "Attached background material").
Other effects:
no effects observed
Relevance of carcinogenic effects / potential:
No carcinogenic effects found.
Key result
Dose descriptor:
NOAEL
Remarks:
carcinogenicity
Effect level:
4 000 ppm
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: actual test substance intake: males: 171 mg/kg bw/day, females: 249 mg/kg bw/day, combined sexes: 210 mg/kg bw/day
Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
200 ppm
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: actual test substance intake: males: 8 mg/kg bw/day, females: 12 mg/kg bw/day, combined sexes: 10 mg/kg bw/day
Key result
Dose descriptor:
LOAEL
Remarks:
systemic toxicity
Effect level:
2 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
urinalysis
Remarks on result:
other: actual test substance intake: males: 84 mg/kg bw/day, females: 118 mg/kg bw/day, combined sexes: 101 mg/kg bw/day
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
2 000 ppm
System:
other: urinary and hepatobiliary
Organ:
kidney
liver
other: increased ketone content in urine in males, decreased absolute and relative kidney weights in males; increased relative liver weights and hepatocytic hypertrophy in males at 4000 ppm
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Table 1: Group mean body weights (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 substance 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
Conclusions:
Under the conditions of the present study, after chronic oral exposure to the test substance, a LOAEL of 101 mg/kg bw/day for combined sexes was identified based on urinary/renal findings. Hence, no classification for specific target organ toxicity is required according to Regulation (EC) No. 1272/2008.
No effects indicating a carcinogenic potential of the test substance were observed.

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
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Version / remarks:
Current version adopted in 2018
Deviations:
yes
Remarks:
Thyroid and uterus weights were not measured.
Qualifier:
according to guideline
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Version / remarks:
Guideline in place during study conduct: adopted in 1981
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.33 (Combined Chronic Toxicity / Carcinogenicity Test)
Version / remarks:
Adoption not stated
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPP 83-5 (Combined Chronic Toxicity/Oncogenicity Study)
Version / remarks:
Adopted in 1984
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: JMAFF (Combined Chronic Toxicty/Oncogenicty Study)
Version / remarks:
adopted in 1985
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
443-870-0
EC Name:
-
Cas Number:
163520-33-0
Molecular formula:
C18H17NO3
IUPAC Name:
ethyl 5,5-diphenyl-4,5-dihydro-1,2-oxazole-3-carboxylate

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
CRL:CD (IGS) BR
Details on species / strain selection:
The rat was chosen as the test system according to regulatory guidelines.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Ltd., Margate, UK
- Age at study initiation: 33 days
- Body weight at receipt (measured for 10% of the animals): male: 55 - 79 g, female: 55 - 84 g
- Fasting periods during study: overnight during urine collection
- Housing: group housed, 5 animals per cage by sex and dose group, in suspended polycarbonate cages with grid floor
- Diet: powdered laboratory rodent diet, Modified SQC Expanded Ground Rat and Mouse Maintenance Diet No. 1 (Special Diet Services Ltd., Witham, UK), ad libitum
- Water: tap water in drinking water quality, ad libitum
- Acclimation period: 15 days

DETAILS OF FOOD AND WATER QUALITY: At their respective levels in the diet and water, none of the contaminants known or expected to be present was considered likely to influence the outcome of the study.

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

IN-LIFE DATES: From: 14 Nov 1996 To: 13 - 27 Nov 1998

Administration / exposure

Route of administration:
oral: feed
Details on route of administration:
The dietary route was selected as a major potential route of exposure to man.
Vehicle:
other: powdered laboratory rodent diet
Remarks:
Modified SQC Expanded Ground Rat and Mouse Maintenance Diet No. 1
Details on oral 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:
52 weeks = 12 months (interim sacrifice)
104 weeks = 24 months (terminal sacrifice)
Frequency of treatment:
Continuously
Doses / concentrationsopen allclose all
Dose / conc.:
20 ppm
Remarks:
actual test substance intake: males: 0.8 mg/kg bw/day, females: 1.1 mg/kg bw/day, combined sexes: 1.0 mg/kg bw/day
Dose / conc.:
200 ppm
Remarks:
actual test substance intake: males: 8 mg/kg bw/day, females: 12 mg/kg bw/day, combined sexes: 10.0 mg/kg bw/day
Dose / conc.:
2 000 ppm
Remarks:
actual test substance intake: males: 84 mg/kg bw/day, females: 118 mg/kg bw/day, combined sexes: 101 mg/kg bw/day
Dose / conc.:
4 000 ppm
Remarks:
actual test substance intake: males: 171 mg/kg bw/day, females: 249 mg/kg bw/day, combined sexes: 210 mg/kg bw/day
No. of animals per sex per dose:
20 (interim sacrifice)
50 (terminal sacrifice)
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).

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 4 weeks thereafter.
- Compound intake calculated from the consumption and body weight data: Yes

FOOD EFFICIENCY: Yes
- Body weight gain in % calculated 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), and 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), and 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), and 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, and 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, and 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.
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):
At 4000 ppm a slight increased incidence of urogenital staining for males from approx. Week 41 onwards (non-adverse).
Mortality:
mortality observed, non-treatment-related
Description (incidence):
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.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 4000 ppm, body weight gain of females was reduced by 8% (non-adverse). 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 (see Table 1 under "Any other information on results incl. tables").
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
At 4000 ppm food intakes of both sexes were reduced during Week 1 but reached the control level thereafter (see Table 2 and 3 under "Any other information on results incl. tables").
Food efficiency:
no effects observed
Description (incidence and severity):
At 4000 ppm food conversion efficiency of females were reduced by 31% during Week 1 but reached the control level thereafter.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
At 4000 ppm a slight (18 - 27%) but consistent increase in water consumption was seen throughout the study for females (see Table 4 under "Any other information on results incl. tables").
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
Single parameters of treated animals distributed through all dose levels were statistically significantly changed compared to control. However, all changes were without any dose-response dependence, therefore, they are of no toxicological relevance.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significantly increased urea and creatinine levels were observed at several time points at 2000 and 4000 ppm in both sexes (for details see attached result tables under "Attached background material"). However, all values were within the physiological range of this rat strain.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
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 (see Table 5 under "Any other information on results incl. tables").
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
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 increased (8%) for males; however, this is considered not toxicologically relevant as there was no histopathological correlate at terminal kill.
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 (see Table 6 under "Any other information on results incl. tables").
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
At interim and terminal kill, an increased incidence of centrilobular hepatocytic hypertrophy (minimal to slight) was seen in males receiving 4000 ppm.
At terminal kill, an increased incidence and severity of progressive nephropathy was observed in females treated at 4000 ppm (see Table 7 under "Any other information on results incl. tables").
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
Administration of the test substance did not result in any alterations to indicate a carcinogenic potential from animals killed after 52 or 104 weeks of treatment (for details see attached data tables under "Attached background material").

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
200 ppm
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: actual test substance intake: males: 8 mg/kg bw/day, females: 12 mg/kg bw/day, combined sexes: 10 mg/kg bw/day
Key result
Dose descriptor:
LOAEL
Effect level:
2 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
urinalysis
Remarks on result:
other: actual test substance intake: males: 84 mg/kg bw/day, females: 118 mg/kg bw/day, combined sexes: 101 mg/kg bw/day

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

Table 1: Group mean body weights (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 substance 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

Severe

1

0

1

0

4

Applicant's summary and conclusion

Conclusions:
Under the conditions of the present study, after chronic oral exposure to the test substance, a LOAEL of 101 mg/kg bw/day for combined sexes was identified based on urinary/renal findings.