Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study according to internationally accepted guideline. Only minor deviation from guideline protocol.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Report Date:
1995

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
functional observations were not performed; the epididymides were not weighed.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Batch no.5A7990
White crystalline powder
Purity: 99.48% w/w
Stability: Analytical confirmation of the stability of the substance in the vehicle was performed by the Sponsor and communicated to the Study Laboratory: Stability of the test material in the vehicle resulted to be about 20 days

Test animals

Species:
rat
Strain:
other: Sprague-Dawley Crl: CD (SD) BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Italia
- Age at study initiation: 4 week old
- Weight at study initiation: Males 75-85 g, Females 60-70 g

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% (w/v) methylcellulose 400 cps aqueous solution
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Concentration in vehicle: 5, 10 and 30 mg/mL
- Amount of vehicle (if gavage): 10 mL / b.w.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Test duration: 90 days
Frequency of treatment:
Dosing regime: 7 days/week
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
50 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
100 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
300 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
Male: 20 animals at 0 mg/kg bw/day
Male: 10 animals at 50 mg/kg bw/day
Male: 10 animals at 100 mg/kg bw/day
Male: 20 animals at 300 mg/kg bw/day
Female: 20 animals at 0 mg/kg bw/day
Female: 10 animals at 50 mg/kg bw/day
Female: 10 animals at 100 mg/kg bw/day
Female: 20 animals at 300 mg/kg bw/day
Control animals:
yes, concurrent vehicle
Details on study design:
Total volume applied: 10 ml/kg b.w.

Examinations

Observations and examinations performed and frequency:
Clinical signs and mortality: Daily

Body weight: Day 0 before the treatment and weekly throughout the treatment period

Food consumption: Weekly throughout the treatment and the recovery periods

Water consumption: Weekly throughout the treatment and the recovery periods

Ophthalmoscopic examination: 4 and 6 days before the start of the treatment in females and males, respectively. During week 7 and week 13 of the treatment period and at the end of the recovery.

Haematology: yes
- number of animals: all animals
- time points: weeks 6, weeks 12 and at the end of the recovery period
- parameters: Haematocrit, mean corpuscular volume, haemoglobin, mean corpuscular haemoglobin concentration, mean corpuscular haemoglobin, erythrocyte count, total leukocyte count, WBC differential count, platelet count, prothrombin time

Clinical Chemisty: yes
- number of animals: all animals
- time points: weeks 6, weeks 12 and at the end of the recovery period
- parameters: sodium, potassium, calcium, inorganic phosphorus, chloride glucose, total cholesterol, urea, total bilirubin, creatinine, total protein, albumin, globulin, alpha 1 globulin, alpha 2 globulin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase, gamma globulins, Lactic dehydrogenase (LDH).

Urinalysis: yes
- number of animals: all animals
- time points: weeks 6, weeks 12 and at the end of the recovery period
- parameters: appearance, volume, specific gravity, pH, protein, glucose, urobilinogen, nitrites, leukocytes, ketone bodies, bilirubin, blood
Sacrifice and pathology:
Organ Weights : yes
- organs: liver, kidneys, adrenals, prostate, testes, uterus, ovaries, spleen, brain, heart, thymus, brain, pituitary

Gross and histopathology: yes
- Gross pathology was performed in all dose groups. Histopathology was performed in high dose group and controls, and in other dose groups only when some effects was found at the high dose level. Liver in males and adrenals in females were also examined in the mid- and low-dose groups, since they were considered as potential target organs of the substance.
- organs: brain, spinal cord, pituitary, thyroid with parathyroid, thymus, oesophagus, salivary glands, stomach, small and large intestines, liver, pancreas, kidneys, adrenals, spleen, heart, trachea, lungs, aorta, gonads, uterus, prostate, urinary bladder, epydidimes, female skin and mammary gland, lymph nodes, peripheral nerve, bone marrow, eyes,
Statistics:
The following parameters were subjected to statistical analysis:
Body weight, body weight gain, food intake (abs. and rel.), haematology, blood chemistry, urinalysis (only quantitative parameters), organ weights
A preliminary analysis of the homogeneity of the variance was carried out by the Bartlett’s analysis. Homogeneous data were subjected to ANOVA and Dunnett’s multiple component test analysis.
Non-homogeneous data were subjected to Kruskal-Wallis non parametric ANOVA and Mann Whitney’s “U” test.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No mortality; high-dose group: episodes of salivation, an increased incidence of fur loss
Mortality:
mortality observed, treatment-related
Description (incidence):
No mortality; high-dose group: episodes of salivation, an increased incidence of fur loss
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
no treatment related effects were observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No mortality was observed during the treatment and recovery period.
Episodes of salivation were observed in the high-dose group animals generally within 20 minutes from the administration, starting from the 4th week of treatment but not observed in the recovery period. An increased incidence of fur loss possibly caused by increased grooming was also observed in the high-dose group animals during the second half of the treatment period with fur loss still evident in some animals during the recovery period.

BODY WEIGHT GAIN:
A slight but not significant decrease (-3%) in body weight gain over the 90 day treatment was recorded in males from the high-dose group when compared with controls. The difference in body weight gain started from week 3-4 upwards but was not observed in the last two weeks of treatment. With the exception of the first week after treatment bodyweight gain in the recovery period was comparable for both treated and control males. Other than a significant increase (9%, P<0.05) in mean body weight (at day 90) in females from the mid-dose group, no differences in body weight and body weight gain was observed in treated females when compared to controls during the treatment and recovery period.

FOOD CONSUMPTION AND COMPOUND INTAKE
No adverse effects on food consumption and feed efficiency were observed in any of the treated animals when compare to controls. Slightly increased water consumption (~12% over the treatment period) was observed in the mid-and high-dose groups during the treatment period in comparison to controls.

HAEMATOLOGY :
No treatment related effects were observed.

CLINICAL CHEMISTRY:
A dose-related decrease in AST activity, recorded in both sexes at week 6 and 12 of treatment, achieving statistical significance at weeks 6 only in males and also at weeks 12 in females. No statistically significant changes were observed at the end of the recovery period.

A dose-related increase in lactic dehydrogenase (LDH) activity recorded at week 12 in both sexes, achieving statistical significance at ≥ 100 mg/kg bw/day. No statistically significant changes were observed at the end of the recovery period.

A dose-related increase in creatinine levels measured at week 6 in females only and at week 12 in males only. The effect achieved statistical significance at ≥ 100 mg/kg/day in both sexes. No significant changes were observed after the recovery period.
Total bilirubin was found to slightly increase without dose-response relationship in males from all treated groups at week 12. A statistically significant increase was noted in the high-dose group females at week 12. A statistically significant decrease was observed in males of the high-dose group at the end of the recovery period in comparison to control. No statistically significant changes were observed in females at the end of the recovery period.

A reduction in total cholesterol was observed in males of the high-dose group at week 6 (statistically significant) and week 12 and in females at week 6 and 12. A reduction but not statistically significant was also observed in treated males and females at the end of the recovery period.

A dose related increase in urea reaching statistical significance in males of the high-dose group was observed at week 12. No statistically or biologically significant changes were observed at the end of the recovery period.

A not dose related increase in glucose levels was observed at ≥ 100 mg/kg bw/day in females at week 6, achieving statistical significance at week 12. A statistically significant increase was observed in treated females at the end of the recovery period.

A not statistically significant increase in alkaline phosphatase levels was observed in females of the high-dose group at week 6 and 12 when compared to controls. No similar increase was observed at the end of the recovery period.

URINALYSIS:
A dose related increase in frequency of animals with positive results for bilirubin was observed in females of the high-dose group ( 8/10 and 5/10 at week 6 and week 12, respectively). No positive results were found at the end of the recovery period. No other treatment related changes from control values were observed.

ORGAN WEIGHTS:
A statistically significant increase in relative liver weights was observed in males of the high-dose group in comparison to controls with a similar increase but not statistically significant observed in females of the same group. No biologically or statistically significant differences were observed at the end of the recovery period.

A statistically significant increase in absolute and relative adrenal weight was observed in most treated female groups compared to controls. No biologically or statistically significant differences were observed at the end of the recovery period.

GROSS AND HISTOPATHOLOGY:
No findings were recorded after macroscopic pathology examination.

Histopathology revealed a dose-related increase in the frequency of centrilobular hypertrophy of the liver in males of the mid-dose (4/10) and high-dose groups 4/10 and 5/10, respectively). These changes were not observed in treated males at the end of the recovery period.

Slight hypertrophy of the zona glomerulosa of adrenal glands was observed in females of all groups (3/10, 4/10 and 4/10 at 50, 100 and 300 mg/kg bw/day, respectively). No effects on adrenals were observed at the end of the recovery period.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: original NCD unit is mg/kg/day.
Dose descriptor:
NOEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: original NCD unit is mg/kg/day.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Results of repeated dose toxicity study:

Parameter

Control

low dose

medium dose

high dose

dose-response

+/-

 

m

f

m

f

m

f

m

F

m

f

number of animals examined

10

10

10

10

10

10

10

10

 

 

Mortality

0

0

0

0

0

0

0

0

-

-

clinical signs*

-

-

-

-

-

-

Salivation, fur loss

+

+

body weight

No effects

-

-

food consumption

No effects

-

-

clinical chemistry*

Described in table A6_4_1-1

haematology*

No effects

-

-

urinalysis*

-

-

-

-

-

-

-

Bilirubin in urine of 8 and 5 animals after 6 and 12 weeks of treatment, respectively

-

+

Liver

organ relative weight*

-

-

-

-

-

-

+ 13%*

+10%

+

+

gross pathology*

-

-

-

-

-

-

-

-

-

-

microscopic pathology*

-

-

-

-

Hepatic centrilobular hypertrophy in 4/10

-

Hepatic centrilobular hypertrophy in 5/10

-

+

-

Adrenals

organ absolute weight

-

-

-

+20%*

-

+29%**

-

+28%*

-

-

organ relative weight*

-

-

-

+22%**

-

+16%

-

+29%**

-

-

gross pathology*

-

-

-

-

-

-

-

-

-

-

microscopic pathology*

-

-

-

Slight hypertrophy of zona glumerulosa in 3/10

 

Slight hypertrophy of zona glumerulosa in 4/10

-

Slight hypertrophy of zona glumerulosa in 4/10

-

+/-

 

 

 

 

 

 

 

 

 

 

 

Results of clinical chemistry, haematology and urinalysis:

parameter changed

Unit§

low dose

medium dose

high dose

weeks after start of treatment

 

6

12

6

12

6

12

20 (recov)

MALES

 

 

 

 

 

 

 

 

AST

%

-12

-10

-15

-6

-23**

-18

-

Total cholesterol

%

-

+9

-

-

-25**

-23

-19

Urea

%

-

-

-

-

-

+16*

 

Creatinine

%

-

+16

-

+58**

-

+59**

-

Bilirubine

%

-

+35*

-

+16*

-

+35*

-22

Total protein

%

-

-

-4**

+7**

-5*

-

-

LDH

%

-

+14

-

+46**

-

+89

-

FEMALES

 

 

 

 

 

 

 

 

Creatinine

%

+20

 

+31*

 

+31*

 

 

AST

%

-17*

-14**

-25**

-18**

-25**

-21**

-

LDH

%

-

 

-

+52**

-

+44**

-

Total cholesterol

%

-

-

-

-

-

-9

-12

Bilirubine

%

-

-

-

+7

-

+46**

-

Alkaline phosphatase

%

-

-

-

-

+31

+47

-

Glucose

%

-

12

+16

+28**

+10

+16*

+15**

Applicant's summary and conclusion

Conclusions:
LO(A)EL:
300 mg/kg bw/day: increased liver weight, associated to increased incidence of slight hepatocyte hyperthrophy and altered clinical chemistry and urinalysis parameters, possibly indicative of altered hepatic functionality. All the effects were not present after the recovery period.

NO(A)EL
50 mg/kg bw/day may be regarded as the absolute NOEL of the study. 100 mg/kg bw/day can be considered as the study NOAEL in view of the low severity and complete reversibility of the observed effects.
Executive summary:

Study setup:

10 rats/sex/group were exposed to 0, 50, 100, 300 mg/kg/day PAC by gavage for 13-consecutive weeks. Further groups of 10 rats/sex/groups were exposed to 0 and 300 mg/kg/day PAC for 13 weeks and allowed for a recovery period of 6 weeks.

Clinical signs, mortality, body weights, food and water consumption were monitored regularly during the study. Ophtalmological examinations were performed at week 7 of treatment and at the end of the treatment and recovery periods.

Blood chemistry analysis, haematology and urinalysis, were performed at week 6 and at the end of the treatment and recovery periods.

Gross pathology and histopathology examinations were carried out at the end of the treatment period for the main groups and at the end of the recovery period for the recovery groups.

Study results:

Administration of the active substance’s main degradation product for 90 consecutive days in rats induced changes in clinical chemistry and liver.

At the highest dose tested (300 mg/kg be day) several changes in chemical chemistry parameters,possibly related to altered hepatic functionality, were observed:

·      Increased LDH activity in both sexes

·      Increased creatinine levels in both sexes

·      Increased bilirubine levels in both sexes

·      Decreased total cholesterol levels in both sexes

·      Increased urea levels in males

·      Increased alkaline phosphatase activity in females

·      Increased glucose levels in females

 

Increased LDH and creatinine levels were observed also at 100 mg/kg bw/day, and increased bilirubine levels were observed at 50 and 100 mg/kg bw/day.

 

All the measured parameters were judged to be of low severity. The increase in total bilirubine was judged as incidental by the study director, since it was not dose-related and all the individual values were considered to fall under the physiological norm.

With the exception of reduced total cholesterol values in both sexes and increased glucose levels in females, none of the differences observed in the clinical chemistry parameters during treatment were observed at the end of the recovery period.

 

The urinalysis showed an increased incidence of animals with positive results for bilirubin was observed in females of the high-dose group during the exposure period.

 

Hepatic effects were limited to a slight increase in organ weight relative to body weight observed in both sexes treated at 300 mg/kg bw/day, and to an increased incidence of centrilobular hepatic hypertrophy of low severity observed at ≥ 100 mg/kg bw/day.

No effects in liver weight and histopathology were observed at the end of the recovery period.

Increased adrenals weight, accompanied by findings of incidence of slight hypertrophy of thezona glomerulosa of the adrenals were observed in females from all the treatment groups. However, since the increase in adrenal weight were reported to be still within the range of historical controls, and the incidence of slight hypertrophy were not dose related, this effect was considered as incidental. In addition, no effects were seen at the end of the recovery period.