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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1993
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report Date:
1993

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 452 (Chronic Toxicity Studies)
GLP compliance:
yes
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
dog
Strain:
Beagle
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Ridgian Farms, Mt. Horeb, Wisconsin
- Number of animals per group: 4 males and 4 females per concentration group
- Control animals: 4 males and 4 females
- Age at study initiation: Approximately 5 months
- Weight at study initiation: 10.6-14.3 kg (males) and 7.2-12.3 kg (females)
- Housing: The dogs were individually housed in stainless steel cages (animals rooms were changes every two weeks)
- Diet: Food consumption per day ad libitum
- Water: available ad libitum.
- Acclimation period: 28 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): The temperature was maintained at 23 ± 3 °C
- Humidity (%): The humidity was maintained at 50 ± 20 %
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark cycle.

IN-LIFE DATES: From March 28 and April 4, 1991 (arrive in the laboratory): To May 13-14, 1992 (date of sacrifice)

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): Fresh diet was prepared and offered to the animals each week
- Mixing appropriate amounts with Test diet were prepared by direct addition of PBO to Certified Canine Chow® #5007, Ralston Purina Company, St. Louis, Missouri.
- Storage temperature of food: Diets were stored at room temperature
- Frequency of exposure: Daily
- Postexposure period: Treatment continued through the end of the terminal sacrifice

VEHICLE
- Justification for use and choice of vehicle: the substance was mixed in the diet for “ad libitum” administration. A potential route of exposure to humans is oral. Therefore, the peroral route of administration was employed in this study.
- Diet: Certified Canine Chow® #5007, Ralston Purina Company, St. Louis, Missouri
- Concentration in vehicle: Mixtures were offered at actual Piperonyl Butoxide concentrations corresponding to 100, 600, 2000 ppm.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For homogeneity analyses, 100 gram test substance: diet mixture samples were collected from test diets prepared at the upper (2000 ppm) and lower (100 ppm) ends of the range of dietary concentrations scheduled to be used in this study.
The samples was collected from the top, middle and bottom portions of the test diets while being dispensed from the blender.

For stability analyses, a single 100 gram random sample was collected from the 100 and 2000 ppm test diets at the time the homogeneity samples were collected. These samples were stored under expected use conditions for 10 days and then analysed.

For concentration verification, 100 grams samples were taken from the top, middle and bottom of each diet at the time test substance: diet mixtures were prepared. The three samples (top, middle and bottom) were mixed to form a composite sample. For study weeks 1 through 4, and every four weeks thereafter, samples were analysed in duplicate for determination of the test substance concentration.
Duration of treatment / exposure:
1 year
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0 (control), 100, 600, 2000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
4/male/0; 4/male/100; 4/male/600; 4/male/2000;
4/female/0; 4/female/100; 4/female/600; 4/female/2000;
Control animals:
yes
Details on study design:
- Dose selection rationale: Diet mixtures were prepared at nominal concentrations of 100, 600 and 2000 ppm. These dietary concentrations were selected based on the results of the Report study n. 542-004 (Evaluation of PBO in an eight week toxicity study in dogs.) reported in this IUCLID Dossier.
- Rationale for animal assignment: Sixteen males (weighing from 10.6 to 14.3 kg) and sixteen females (weighing from 7.2 to 12.3 kg) were selected on the basis of the present data and assigned to the control group or to one of the three other treated groups by the use of a computerized randomization procedure (See the table "GROUP ASSIGNMENTS" attached in backgorund material).

Each dog was identified by cage, group and individually by ear tattoos

Examinations

Observations and examinations performed and frequency:
- Clinical signs Mortality: Observations were conducted two times daily to assess mortality, morbidity and signs of overt toxicity. Detailed observations were recorded once a week to record pharmacotoxic signs and the occurrence, size, location and description of palpable masses. Clinical laboratory tests were conducted during the pretest period and at 6 and 12 months.
- Physical examinations: A physical examination of all test animals were conducted during pre-initiation and at 3, 6, 9 and 12 months. A complete necropsy and histopathological examinations were performed on all animals after terminal sacrifice.
- Clinical findings: The dogs were observed twice daily for signs of overt toxicity. Detailed observation were conducted at least once a weekly.
- Body weight : Individual body weights were recorded pretest and thereafter just like food consumption recorded weekly for the first 14 weeks of the study and once every 2 weeks thereafter (See table "MEAN BODY WEIGHTS" attached in background material).
- Food consumption : Individual food consumption was recorded weekly. Test article consumption was calculated from food consumption and body weight values.
- Water consumption : Not recorded.
- Ophthalmoscopic examination: Ophthalmoscopic examinations were conducted on all dogs by a veterinary ophthalmologist once during the pretest and at termination of the study period.
- Haematology Clinical Chemistry: Clinical laboratory studies were conducted on all animals once prior to the study initiation and at 6 and 12 months of study. Blood samples were obtained from the jugular vein following a 24 hour fasting period.
- Urinalysis: Urine was collected on dry ice during a 24 hour fasting period.
Sacrifice and pathology:
- Organ Weights: Organs: liver, kidneys, adrenals, testes, ovaries, brain (with stem), heart, pituitary, thyroid/parathyroid
- Gross and histopathology: All animals/sex/dose groups received a complete post-mortem examination under the direct supervision of a pathologist. All animals were euthanised by intravenous sodium pentobarbital followed by exsanguination. All macroscopic abnormalities were recorded. Representative samples of protocol designated organs and tissues were collected and placed in phosphate buffered neutral formalin and hematoxylin- and eosin-stained paraffin sections were processed for microscopic examinations. A full complement of organs and tissues was prepared for all animals.
Statistics:
Body weights, food and compound consumption, hematological, biochemical and urological parameters and absolute and relative organ weights were analyzed using the one- way variance analysis and Bartlett’s test for homogeneity of variance. Treatment groups were compared to the control group by sex, using the appropriate t-statistic. Dunnett’s multiple comparison tables were used to determine the significance of differences. Total bilirubin, urine specific gravity and volume were analyzed using a nonparametric approach, by transforming the data into ranks prior to analysis. All statistical tests were two-trailed, with p<0.05 and p<0.01 used as levels of significance.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
All dogs survived to study termination
Mortality:
no mortality observed
Description (incidence):
All dogs survived to study termination
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Reduction in body weight gains occurred for males and females receiving 600 and 2000 ppm, these changes were not statistically significant (See the table "MEAN BODY WEIGHTS" attached in background material).
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Not biologically and statistically significant (See the table "FOOD CONSUMPTION" attached in background material)
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related eye abnormalities.
Haematological findings:
no effects observed
Description (incidence and severity):
No treatment related changes in haematology were observed.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
See "Details on results" and the table "CLINICAL CHEMISTRY" attached in background material.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No findings
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
See "Details on results" and the table "CLINICAL CHEMISTRY" attached in background material.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
See "Details on results"
Details on results:
- Clinical signs: Clinical signs were unremarkable at all dosage levels.
- Mortality: All animals survived to study termination.
- Body weight gain: Reduction in body weight gains occurred for males and females receiving 600 and 2000 ppm, these changes were not statistically significant. Reduction in body weight gains occurred for males and females receiving 600 and 2000 ppm, these changes were not statistically significant. (See the table "MEAN BODY WEIGHTS" attached in background material).
- Food consumption and compound intake: Reduction in food consumption occurred for males receiving 600 and 2000 ppm but were not statistically significant. Small reductions in food consumption was noted for females in all dose groups as compared to controls, but these findings were not dose related and thus, considered not biologically significant. Average compound intake was 0, 2.9, 15.5 and 53 mg/kg bw/d for males and 0, 2.7, 16.3 and 71 mg/kg bw/d for females. (See the table "FOOD CONSUMPTION" attached in background material).
- Ophtalmoscopic examination: There were no treatment-related eye abnormalities.
- Haematology: No treatment related changes in haematology were observed.
- Clinical chemistry: Increases in alkaline phosphatase values were observed at 6 and 12 months for males and females in the 2000 ppm group. Cholesterol levels were decreased at these same intervals for females in the 2000 ppm group. No other changes in biochemistry parameters were observed. Not biologically and statistically significant (See the table "CLINICAL CHEMISTRY" attached in background material)
Urinalysis: No findings.
Organ weights: Increased liver /gallbladder weights were observed fro both sexes in the 2000 ppm group and small increases in thyroid/parathyroid weights were observed for females in this high dose group. (See the table "ORGAN WEIGHTS" attached in background materia)
Gross and histopathology: Diffuse, mild hypertrophy of the hepatocytes occurred in three of four males and in all four females in the 2000 ppm group.

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
600 ppm
Sex:
male/female
Basis for effect level:
other: under the conditions of this study
Key result
Dose descriptor:
LOAEL
Effect level:
2 000 ppm
Sex:
male/female
Basis for effect level:
other: hepatotoxicity and reduced body weight gain

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Mean Body Weights Pre-test and at Week 52 in a 52 Week Dog Feeding Study

Dosage level (ppm)

Group Mean Body Weights (kg)

Male

Female

pretest

week 52

pretest

week 52

 

0

13.3

16.6

11.0

13.4

 

100

13.2

16.0

10.9

13.6

 

600

13.1

15.2

10.6

12.7

 

2000

13.1

13.5

10.2

10.0

 

Average food consumption and compound intake values for 52 weeks

Dosage level (ppm)

Average Food Consumption g/animal/day

Average Compound Consumption

(mg/kg bw/day)

 

Male

Female

Male

Female

0

435

363

0

0

100

429

327

2.90

2.69

600

368

315

15.50

16.30

2000

350

338

52.83

71.03

Clinical Chemistry-Selected Parameters In A 52 Week Dog Feeding Study

Parameter

sex

0 ppm control

100 ppm

600 ppm

2000

month

 

0

6

12

0

6

12

0

6

12

0

6

12

Alkaline Phosphatase

(IU/L)

M

93

45

36

98

55

47

112

72

59

114

152*

194*

F

108

60

49

133

90

71

98

46

44

99

221*

300*

Aspartate Aminotransferase

(IU/L)

M

27

29

21

22

27

23

23

23

22

24

22

19

F

24

23

19

23

23

20

27

24

18

29

24

22

Alanine Aminotransferase

(IU/L)

M

33

39

36

32

39

41

29

33

34

27

29

35

F

30

30

27

32

38

44

33

34

32

37

32

40

Cholesterol (mg/dL)

M

183

162

161

171

153

134

186

154

136

189

143

129

F

171

192

206

170

160

174

138

158

129

168

126

100

*significantly different from the control group: p < 0.05

Selected organ weights and pathologies in a 52 week dog feeding study

ppm

Sex

Body weight (kg)
Liver/Gall-bladder
(g)
Relative Liver/Gall-bladder (%)
Thyroid left (mg)
Relative Thyroid left (mg))
Thyroid right (g)
Relative Thyroid right (%)

0

m

15,9

362

2,29

1,0

6,21

0,85

5,18

 

f

13,0

293

2,26

0,59

4,63

0,63

4,90

100

m

15,5

354

2,34

0,82

5,27

0,76

4,82

 

f

13,2

286

2,22

0,72

5,35

0,67

5,06

600

m

14,5

375

2,58

0,85

5,86

0,81

5,56

 

f

12,2

329

2,69

0,75

6,08

0,68

5,56

2000

m

12,9

442

3,49*

0,87

6,83

0,83

6,53

 

f

9,6

397

4,21**

0,79

8,22**

0,85

8,99**

*significantly different from the control group: p < 0.05

** significantly different from the control group: p < 0.01

Applicant's summary and conclusion

Conclusions:
MATERIALS AND METHODS
According to OECD Guideline 452, groups of 4 beagle dogs/sex/dosage level were offered diets containing 0, 100, 600 or 2000 ppm actual Piperonyl Butoxide for a period of 52 weeks, equal to 0, 2.9, 15.5 and 53 mg/kg bw/d for males and 0, 2.7, 16.3 and 71 mg/kg bw/d for females.
Observations were reported on mortality, clinical signs, body weights and food consumption. Ophthalmoscopic examinations were conducted prior to study initiation and at termination. Physical examinations were conducted on all animals pretest and quaterly throughout the study. Hematological, biochemical and urological evaluations were conducted on all animals prior to study and at 6 and 12 months of study. At study termination, a thorough post-mortem examination was conducted on all dogs and a complete set of organs was harvested, and examined histologically for all animals. Selected organ weights were determined.

RESULTS AND DISCUSSION
No mortalities occurred.
Reduction in body weight gains occurred for males and females receiving 600 and 2000 ppm, for males of these two higher dose groups a reduction in food consumption was observed.
No other treatment related findings in body weight or food consumption and no effects in ophthalmological and physical examinations were noted.
No treatment related changes in haematology were observed. Increases in alkaline phosphatase values were observed at 6 and 12 months for males and females in the 2000 ppm group. Cholesterol levels were decreased at these same intervals for females in the 2000 ppm group. No other changes in biochemistry parameters were observed.
Increased liver /gallbladder weights were observed for both sexes in the 2000 ppm group and small increases in thyroid/parathyroid were observed for females in the high dose group. These latter changes were not associated with microscopic changes in the thyroid and were considered of questionable biological significance. No other changes in organ weights and no macroscopic pathology changes were noted.
Microscopic pathology changes were limited to diffuse, mild hypertrophy of the hepatocytes for both sexes of the 2000 ppm dietary concentration. These findings are consistent with increases in alkaline phosphatase and increased liver/gallbladder weight in the 2000 ppm dose group.

CONCLUSION
The NOAEL under the conditions of this study is 600 ppm corresponding to 15.5 mg/kg bw/d for males and 16.3 mg/kg bw/d for females.
LO(A)EL: The LOAEL for hepatotoxicity and reduced body weight gain is assumed to be 2000 ppm, corresponding t 53 mg Piperonyl Butoxide /kg bw/d for males and 71 mg Piperonyl Butoxide /kg bw/d for females.
NO(A)EL: NOAEL was 600 ppm, equal to 16 mg Piperonyl Butoxide /kg bw per day.
Reliability: 1
Deficiencies: None.