Registration Dossier

Administrative data

Description of key information

Key value for chemical safety assessment

Toxic effect type:
concentration-driven

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1993
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 452 (Chronic Toxicity Studies)
GLP compliance:
yes
Limit test:
yes
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)
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
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
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.
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.

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
Critical effects observed:
not specified

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

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.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
15.5 mg/kg bw/day
Study duration:
subchronic
Species:
dog
Quality of whole database:
Klimish score: 1
System:
other: mild hypertrophy of the hepatocytes for both sexes. These findings are consistent with increases in alkaline phosphatase and increased liver/gallbladder weight.
Organ:
blood
liver

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1992
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
EPA OPP 82-4 (90-Day Inhalation Toxicity)
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
GLP compliance:
yes
Limit test:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Kingston, New York, USA.
- Age at study initiation:Approx. 9 weeks .
- Weight at study initiation: 311-357 g (males) and 202-262 g (females).
- Number of animals per group: 15 per sex.
- Control animals: 15 per sex.
- Housing: During the non-exposure periods the animals were doubly housed in suspended stainless stell wire mesh cages during the first week of the equilibration period and individually housed during the remainder of the equilibration period and all other non-exposure pperiods. During the exposure periods the animals were individually housed in wire mesh, stainless stell cages within a 1000 liter glass and stainless steel exposure chamber.
- Diet: During the non-exposure periods ad libitum. During the exposure periods none. (See the diagram "EXPOSURE CHAMBER" attached in background material)
- Water: During the non-exposure periods ad libitum (by automated watering system). During the exposure periods none.
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): During the non-exposure periods 16-24 °C. During the exposure periods 20-24 °C.
- Humidity (%): During the non-exposure periods 10-86. During the exposure periods 26-74%.
- Air changes (per hr): 12,4 (approximatly every 4.8 minutes)

IN-LIFE DATES: From 09 September 1991 (receipt of Animals) To : 19 December 1991
Route of administration:
inhalation
Type of inhalation exposure:
whole body
Vehicle:
clean air
Remarks on MMAD:
MMAD / GSD: MMAD (mass median aerodynamic diameter) (+ GSD (geometric standard deviation) [µm]
Mean 2.7 +/- 1.7 µm. (See the table "DAILY MEAN EXPOSURE LEVELS" reported in "Any other information on materials and methods incl. tables)
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure procedure: For groups II and III the appropriate amounts of PBO were placed in a glass syringe covered in foil and mounted on a syringe infusion pump. For the groups IV and V the appropriate amounts of PBO were placed into an Erlenmeyer flask covered in foil and connected to an FMI fluid metering pump.
For groups from II to V the test substance was fed via 1/8’’ Teflon from the pump tubing, directly into the liquid inlet of an air atomizing nozzle. House-line air was delivered through a regular and backpressure gauge via ¼” plastic tubing to a metering valve, flow meter and backpressure gauge into the air inlet of the atomizer to generate the aerosol.
The test atmosphere was directed into the inlet turret of the exposure chamber which housed the animals. he animals remained in the chamber for 30 minutes following the exposure to allow the chamber to clear, using clean air at the same airflow rate used during exposure.
Group I is for control
- Temperature, humidity, pressure in air chamber: Temperature (°C): During the non-exposure periods 16-24 °C. During the exposure periods 20-24 °C. Humidity (%): During the non-exposure periods 10-86. During the exposure periods 26-74%.
- Air change rate: Air changes (per hr) = 12,4 (approximatly every 4.8 minutes)
- Method of particle size determination. For group I: Particle size distribution measurements were performed once during each exposure for chamber and room air using a TSI Aerodynamic Particle Sizer. Sample were withdrawn at rate of 5 liters per minute for 20 seconds. The particle size were calculated by mean of a computer system. For groups to II to V : Particle size distribution measurements were performed once during each exposure for chamber and room air using a TSI Aerodynamic Particle Sizer. Sample were withdrawn at rate of 5 liters per minute for 20 seconds. The particle size were calculated by mean of a computer system. Samples for particle size distribution assessment were drawn once during each exposure using a Delron DCI-6 cascade impactor. The mass median aerodynamic diameter (MMAD), geometric standard deviation and percent of particles ≤1.0 and ≤ 10 microns were calculated based on the amount of material collected on the impactor stages (stainless steel slides and final filter) using a graphical analysis of an assumed lognormal distribution. (see the table "DAILY MEAN EXPOSURE LEVELS" reported in "Any other information on materials and methods incl. tables")

TEST ATMOSPHERE
- Samples taken from breathing zone: yes.
- Brief description of analytical method used: Sample for gravimetric determination of the PBO exposure levels were withdrawn from the breathing zone in the exposure chamber through glass fiber filter paper mounted open-face in a filter holder. Samples were withdrawn approximately every 90 minutes from the sampling portals. The filter papers were weighed before and after sample collection and the gravimetric concentration in mg/mc was calculated by dividing the weight difference in milligrams by the volume of air sampled.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Nominal concentration of the test substance: The nominal concentration (mg/mc) was determined by measuring the flow of air (lpm) through the chamber and the volumetric flow (µl/min) of test substance into the chamber (flowrate times total exposure time) to give a nominal concentration.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hours per day, 5 days per week (whole-body exposure) per 13 weeks
Remarks:
Doses / Concentrations:
0 (control), 15, 74, 155 and 512 mg/m3
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
0 (control), 38, 68, 230, 827 mg/m3
Basis:
nominal conc.
No. of animals per sex per dose:
15/male/0; 15/male/15; 15/male/74; 15/male/155 and 15/male/512 [mg/m³];
15/female/0; 15/female/15; 15/female/74; 15/female/155 and 15/female/512 [mg/m³]
Control animals:
yes
Details on study design:
- Dose selection rationale: The exposure concentrations were selected on the basis of results of an acute inhalation toxicity study in rats in which no mortality or signs of systemic toxicity were observed at PBO concentration as high as 5.9 mg/l.
- Route: Inhalation, administered into the breathing zone of the animals as a respirable aerosol during whole-body exposures.
- Justification of route of administration: whole-body inhalation exposure was chosen as the route of administration since it is a potential route of human exposure to this test substance.
- Number of exposures: all animals received at least 65 exposures. In order to sacrifice the animals the day after the exposure and because of staggered sacrifice intervals, over three days, some animals received more exposures.
Observations and examinations performed and frequency:
CLINICAL SIGS: Observations in mortality and clinical signs were observed daily commencing on exposure day 35. Body weights and food consumption were also examined. In addition haematology, clinical chemistry, gross pathology and histopathology were examined.
Mortality: Yes
BODY WEIGHT: Individual body weights were determined three times pre-test, weekly during treatment and terminally (after fasting).
OPHTHALMOSCOPIC EXAMINATION: Ophthalmoscopic examinations were performed pretest and prior to the terminal sacrifice.
HAEMATOLOGY: Clinical laboratory studies were conducted on 15 animals at study termination.
CLINICAL CHEMISTRY: Hematology studies were conducted on 15 animals at study termination.
URINALYSIS: Analysis of urine was not performed.
FOOD CONSUMPTION: Individual food consumption was measured weekly beginning one week prior to treatment. None during the test periods
FOOD EFFICIENCY: no data
WATER CONSUMPTION: none during the test periods
Sacrifice and pathology:
GROSS PATHOLOGY and HISTOPATHOLOGY: Examinations were performed on all animals which died during the study as well as all survivors at study termination. All animals/sex/dose group received a complete post-mortem examination under the direct supervision of a pathologist after exsanguination under carbon dioxide anaesthesia.
Histopathological examination of controls and high dose level groups.
Lungs, liver and larynx were examined in all groups.
Other examinations:
ORGAN WEIGHTS: liver, kidneys, adrenals, testes, epididymides, ovaries, , brain, heart, lungs.
Statistics:
Statistical evaluation of equality of means was made by the appropriate one way analysis of variance technique, followed by a multiple comparison procedure if needed. For the comparison of variance between groups Bartlett’s test was used. If the variance of the test groups were equal, parametric procedures like one way ANOVA was used using the F distribution to assess significance. Differences from the control were determined by using Dunnett’s test. If a nonparametric test was needed, the Kruskal-Wallis Test was used.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No animals died as a result of exposure to PBO.
Mortality:
mortality observed, treatment-related
Description (incidence):
No animals died as a result of exposure to PBO.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No food during the test period
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No food during the test period
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No water during the test period
Ophthalmological findings:
no effects observed
Description (incidence and severity):
See "Details on results"
Haematological findings:
no effects observed
Description (incidence and severity):
See "Details on results"
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
See "Details on results"
Urinalysis findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
See "Details on results"
Gross pathological findings:
no effects observed
Description (incidence and severity):
See "Details on results"
Details on results:
CLINICAL SIGNS AND MORTALITY: Dose related increases in clinical signs included secretory signs in both males and females such as nasal discharge and dried material in the facial area ad extremities and anogenital staining in the two highest dose groups. No animals died as a result of exposure to PBO.

BODY WEIGHT AND WEIGHT GAIN: The absolute body weights and body weight gains were not affected

FOOD CONSUMPTION: The mean food consumption values were not affected

FOOD EFFICIENCY

WATER CONSUMPTION

OPHTHALMOSCOPIC EXAMINATION: There were no signs of any Piperonyl Butoxide related ocular effects.

HAEMATOLOGY: There was no indication of Piperonyl Butoxide related hematologic changes

CLINICAL CHEMISTRY: Significant differences in clinical chemistry parameters were seen in group V: Serum levels of SGOT, SGPT and glucose were decreased while BUN, total protein and albumin were increased. The differences were not always statistically significant in both sexes. (See the table "CLINICAL CHEMISTRY-SELECTED PARAMETERS" reported in "Any other information on results incl. tables")

URINALYSIS: not done

NEUROBEHAVIOUR

ORGAN WEIGHTS: Significant increases in organ weights or ratios were observed for liver and kidneys of group V males and females. Relative liver weights were also increased for group IV males and females. (See the table "SELECTED ORGAN WEIGHTS AND PATHOLOGIES" reported in "Any other information on results incl. tables")

GROSS AND HISTOPATHOLOGY: Changes noted in the larynx were indicative of local irritation rather than systemic and were minimal to slight in severity and were seen in control animals as well.
In the liver, vesiculation/vacuolation of hepatocellular cytoplasm (minimal to slight) was seen in animals of all groups but was slightly more pronounced in the highest dose group males.
Key result
Dose descriptor:
NOAEL
Effect level:
155 mg/m³ air
Sex:
male/female
Basis for effect level:
other: under the conditions of this study
Key result
Dose descriptor:
LOAEL
Effect level:
>= 512 mg/m³ air
Sex:
male/female
Basis for effect level:
other: Based upon the hepatotoxicity (decreased serum enzymes activity, increased liver weight and increased kidney weight), together with a range of qualitatively different minimal to slight laryngeal lesions.
Critical effects observed:
not specified

The tables reported below have been extracted by the full study report Project number 91-8333

Clinical Chemistry-Selected Parameters

Parameter

n = 15/sex

0 control

15 (mg/m3)

74 (mg/m3)

155 (mg/m3)

512 (mg/m3)

AST (SGOT)

(IU/l)

M

62

60

57

53

51*

F

53

53

48

60

47

ALT (SGPT)

(IU/l)

M

29

31

28

27

25*

F

29

28

27

33

21*

Total protein (g/dL)

M

6.5

6.6

6.6

6.7

7.1**

F

7.2

7.0

7.2

7.4

7.6

Globulin (g/dL)

M

2.5

2.5

2.6

2.5

2.7

F

2.5

2.4

2.5

2.6

2.6

Albumin (mg/dL)

M

4.0

4.1

4.0

4.2

4.4**

F

4.8

4.6

4.7

4.7

5.0

Glucose (mg/dL)

M

165

166

160

146

149

F

162

158

159

151

133*

BUN (mg/dL)

M

13.0

12.4

12.9

13.2

14.9**

F

13.3

13.8

14.8

13.2

14.6

AST- Aspartate Amino Transferase (SGOT = Serum Glutamic Oxaloacetic Transaminase)

ALT - Alanine Amino Transferase (SGPT = Serum Glutamic Pyruvic Transaminase)

*significantly different from the control group: p < 0.05

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

Selected Organ Weights and Pathologies

mg/m3

Sex
(n = 15)

Body weight (g)
Liver weight (g)
Relative liver weight (%)
Kidney weight (g)
Relative kidney weight (%)

0

m

563

14.83

2.63

5.00

0.73

f

330

9.12

2.76

2.37

0.72

15

m

555

14.84

2.66

4.09

0.74

f

334

9.08

2.73

2.49

0.76

74

m

541

14.76

2.73

4.03

0.75

f

331

9.23

2.79

2.38

0.72

155

m

558

15.78

2.83*

4.11

0.74

f

319

9.58

3.01*

2.41

0.76

512

m

538

18.20**

3.39**

4.40

0.82*

f

319

10.90**

3.43**

2.52

0.80*

*significantly different from the control group: p < 0.05

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

TOXIC RESPONSE

At 512 mg/m3 (males/females) differences were noted from control in clinical Chemistry parameters. Serum levels of SGOT, SGPT and glucose were decreased, while BUN, total protein and albumin were increased. These differences were not always statistically significant in both sexes. However, a similar trend was evident in both sexes. Therefore, a relationship to test substance exposure could not be excluded. Statistically significant increases in absolute or relative organ weights were seen in the liver and kidneys. The relative kidney weights were increased by about 11%. Absolute and relative liver weights were both increased by about 25%. In the liver, vesiculation/vacuolation of hepatocellular cytoplasm was slightlymore pronounced than in the other dose groups, especially for the males.

Conclusions:
MATERIALS AND METHODS
Chronic inhalation toxicity study according to US EPA Pesticide Assessment Guideline, Subdivision F, 82-4.
Groups of 15 Sprague Dawley rats of each sex were whole body exposed to analytical concentrations of 0, 15, 74, 155, and 512 mg Piperonyl Butoxide /m³ for 13 weeks, 6 hours/day, generally 5 days/week and a minimum of 65 exposures in total. Determinations of size distribution showed an overall mass median aerodynamic diameter of 1.7 µm.

RESULTS AND DISCUSSION
No animals died. Clinical signs were observed from 74 mg/m³ onwards and included secretory signs.
There were no ocular effects.
At the highest dose (512 mg/m³) significant differences in clinical chemistry parameters were seen, however, the differences were not consistent between sexes.
Significant increases in liver weights and relative organ were observed for liver and kidneys.
At a dose level of 155 mg/m³ minimal increase was noted for the relative liver weights.
Morphological abnormalities in the larynx, observed by light microscopy were considered to be localized responses indicative of a treatment-related effect.

CONCLUSION
Based upon hepatotoxicity at 512 mg/m³, a NOAEL of 155 mg/m³ is considered for systemic toxicity under the conditions of this study.

The rat is highly susceptible to develop squamous laryngeal metaplasia due to its anatomy, airflow and epithelial pattern . There were no severe grade effects in the larynx of high dose level rats. In fact, all the findings of squamous metaplasia were essentially graded minimal to slight in all treated groups. The occurrence of additional qualitative changes at the high dose indicated 0.512mg/l to be the LOAEL. The weight of evidence, including current pathological diagnostic criteria do not classify minimal or slight effects as adverse.


LO(A)EL: 512 mg/m³. Based upon the hepatotoxicity (decreased serum enzymes activity, increased liver weight and increased kidney weight), together with a range of qualitatively different minimal to slight laryngeal lesions.

NO(A)EL: 155 mg/m³ is considered to be the NOAEL for systemic and local toxicity under the conditions of this study.

Reliability: 1
Deficiencies: No
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
155 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
Klimish score: 1

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rabbit

Additional information

Justification for classification or non-classification