Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 October 1994 to 22 November 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline and GLP compliant study reported with no apparent deviations
Cross-reference
Reason / purpose:
reference to same study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): PTH-decahydroamide

- Substance type:
- Physical state:colourless to pale yellow solid
- Analytical purity: 99.8% (GC)
- Impurities (identity and concentrations): no data
- Composition of test material, percentage of components: no data

- Purity test date:
- Lot/batch No.: 25927
- Expiration date of the lot/batch: 30 June 1995

- Stability under test conditions: Stable
- Storage condition of test material:room temperature in the dark
- Other: stability in chosen vehicle (1% aq. carboxymethylcellulose) was confirmed over a 48 hour period

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BRL Ltd, Basel, Switzerland (albino Wistar, SPF and outbred)
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: mean weight in a range of 143-147g for males and 121 to 125g for females
- Fasting period before study: not applicable
- Housing: group housed in fives by sex in stainless steel suspended caging
- Diet (e.g. ad libitum): Kliba 343, supplied by Klingentalmuhle AG, Kaiseraugst, Switzerland ad libitum
- Water (e.g. ad libitum): ad libitum access to tap water
- Acclimation period:at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): circa 21°C
- Humidity (%): 50% RH
- Air changes (per hr):15
- Photoperiod (hrs dark / hrs light): 12h/12h

IN-LIFE DATES: From: 25 October 1994 To: 22 November 1994

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared freshly for daily administration, immediately prior to dosing. Any interim storage was at room temperature.

VEHICLE
- Justification for use and choice of vehicle (if other than water): test material was analytically confirmed as stable in 1% aqueous carboxymethylcellulose
- Concentration in vehicle: 0, 0.6%, 3.0% and 12%
- Amount of vehicle (if gavage): 5 ml/kg bw
- Lot/batch no. (if required): no data
- Purity: no data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the formulation were prepared, after study termination, and analysed for homogeneity and accuracy. The formulations were confirmed as homogeneous and accurate.
Duration of treatment / exposure:
Up to 28 days daily gavage administration.
Frequency of treatment:
Daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 30, 150 and 600 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
Five males and five females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: selected on basis of range-finding study results, a five day study with 3 rats/sex/group dosed at 50, 200 or 800 mg/kg bw/d (NOTOX study number 130725). Mortalities occurred at 800 mg/kg bw/da together with adverse clinical signs, reduced weight gain and food consumption. No macroscopic abnormalities were evident. No toxicologically significant changes were apparent in the 200 or 50 mg/kg bw/d groups. Dose levels of 30, 150 and 600 mg/kg bw/d were chosen.
- Rationale for animal assignment (if not random): Random
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random): random
Positive control:
Not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twicedaily mortality and viability checks

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: time of onset, degree and duration of signs recorded. Clinical signs of reaction to treatment were recorded at least once daily from Day 1 to termination

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly weights recorded from day of initial dose administration. Weights also recorded prior to fasting in preparation for termination.

FOOD CONSUMPTION :
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes based on weekly consumption recordings per cage

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION A: Yes
- Time schedule for examinations: subjective visual assessments completed throughout the study, no quantitative measurements included

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during last week of treatment. Animals were pre-treated with tropicamide to aid ocular examinations
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: samples collected immediately prior to scheduled termination (samples were also collected where possible from animals killed on welfare grounds, shortly before they were euthanetised)
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: all
- Parameters examined: erytrocyte count (T/l); haemoglobin (mmol/l); haematocrit (l/l); mean corpuscular volume (fl); mean corpuscular haemoglobin (fmol); mean corpuscular haemoglobin concentration (mmol/l); platelet count (g/l); red cell distribution width (%); total leucocyte count (g/l); diferential leucocyte count - neutrophils, eosinophils, basophils, lymphocytes, monocytes; prothrombin time (s); partial thromboplastin time (s).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: samples collected immediately prior to scheduled termination (samples were also collected where possible from animals killed on welfare grounds, shortly before they were euthanetised)
- Animals fasted: Yes
- How many animals: all
- Parameters examined: alanine aminotransferase (ALT, ukat/l); aspartate aminotransferase (AST, ukat/l); bilirubin (umol/l); creatinine (umol/l); glucose (umol/l); urea (umol/l); total protein (g/l); albumin (g/l); alkaline phosphatase (ukat/l); sodium(umol/l); potassium(umol/l) ; chloride (umol/l); calcium (umol/l); phosphorus(umol/l).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No


OTHER: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Necropsy was completed for all rats surviving to the end of treatment (Day 29) and for those animals terminated on grounds or welfare or moribundity during the study. Decedents were also subject to necropsy where possible.
Organ weights were recorded for all surviving rats at termination: adrenal glands; heart; kidneys; liver; spleen and testes
Sampes of the following tissues were taken from all necopsied rats: adrenal glands; heart; kidneys; liver; spleen, stomach and testes plus all gross lesions for possible histopathology


HISTOPATHOLOGY: Yes
slides of adrenal glands; heart; kidneys; liver; spleen and testes prepared for control, intermediate and high dose animals and gross lesions examined for all affected animals.
Statistics:
The following statistical methods were used to analyse the data:
Univariate one-way analysis of variance was used to assess the significance of intergroup differences.
If variables could be assumed to follow a normal distribution, the Dunnett test based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
The Steel-test was applied when the data could not be assumed to follow a normal distribution.

The Fisher exact test was applied to the ophthalmoscopic data.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.

Group means were calculated for continuous data and medians were calculated for discrete data (scores).

(Clinical laboratory results of animals killed in extremis were not analysed statistically.)

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Severe signs of toxicity were observed in the majority of rats dosed at 600 mg/kg bw/day. All of the rats dosed at 600 mg/kg bw/day died or were killed on humane grounds between Day 6 and 18. No signs for rats dosed at 150 or 30 mg/kg bw/d.
Mortality:
mortality observed, treatment-related
Description (incidence):
Severe signs of toxicity were observed in the majority of rats dosed at 600 mg/kg bw/day. All of the rats dosed at 600 mg/kg bw/day died or were killed on humane grounds between Day 6 and 18. No signs for rats dosed at 150 or 30 mg/kg bw/d.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Bodyweight and weight gains were similar to controls for the treated rats in the low and intermediate groups. Bodyweight recordings in the high dose group were disrupted by mortalities but the bodyweights for survivors were also similar to controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Variable values were obtained for food consumption in the high dose group for weeks 1, 2 and 3. Given the number of mortalities in this group the values have little meaning. No effects on food consumption in the 30 or 150 mg/kg bw/d groups.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Assessed visually not quantitatively throughout the study. No apparent effects on water consumption.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No findings in any group during the week 4 examinations.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At 600 mg/kg bw/d lower red blood cell counts and haemoglobin levels. MCHC was low and MCV increased in males. One rat had a higher differential neutrophil count and low platelets. No haematological changes were evident in the 150 or 30 mg/kg bw/daygroups
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related findings at 600 mg/kg bw/d - reduced total protein and albumin concentrations and increased inorganic phosphate in males and females. One rat had high levels of AST, ALT and AP. No changes evident at 30 or 150 mg/kg bw/d.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No effects on absolute or relative organ weights in 30 or 150 mg/kg bw/d groups. No effects clearly attributable to treatment at 600 mg/kg bw/day among decedents.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic changes were evident for the rats dosed at 150 or 30 mg/kg bw/day.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Slight degenerative changes in the liver of 4 males and 1 female in the high dose group. No microscopic effects apparent in rats dosed at 150 or 30 mg/kg bw/day. All microscopic findings considered to be within normal background ranges.
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
All rats dosed at 600 mg/kg bw/day died. Five died between Day 6 and 14 and the remainder were killed on welfare grounds on Day 18. Clinical signs recorded for this group included clonic spasms, tremors, convulsions, increased activity, abnormal gait or posture, ventro-lateral recumbency, laboured respiration and brown staining of the fur. Typically the signs were more intense directly after dose administration and became less severe after approximately one hour.
No signs of reaction to treatment at 150 or 30 mg/kg bw/day were observed.

BODY WEIGHT AND WEIGHT GAIN
Bodyweight and weight gains were similar to controls in all groups, including the high dose group where weights could be recorded.

FOOD CONSUMPTION
Food consumption values for the high dose group were highly variable and, given the sporadic mortalities, of dubious validity.
No effects on food consumption were evident in the low and intermediate dose groups.

WATER CONSUMPTION
Assessed visually throughout study no effects were observed that merited a quantitative assessment of water consumption.

OPHTHALMOSCOPIC EXAMINATION
The examinations completed during week 4 of treatment revealed no ocular changes attributable to PTH-decahydroamide.

HAEMATOLOGY
Changes observed in blood samples obtained from the high dose animals included decreased red blood cell counts and reduced haemoglobin levels. The MCHC was low and mean corpuscular volume increased in the males and one rat (no. 18) had a higher differential neutrophil count and low platelet numbers.
No haematological changes were evident in the 150 or 30 mg/kg bw/daygroups that were considered attributable to treatment with PTH-decahydroamide.

CLINICAL CHEMISTRY
In the samples obtained from rats dosed at 600 mg/kg bw/day, reduced total protein and albumin concentrations were commonly observed, together with increased inorganic phosphate in males and females. Rat no. 18 also had high levels of AST, ALT and AP.
No clinical pathology changes were evident in the 150 or 30 mg/kg bw/daygroups that were considered attributable to treatment with PTH-decahydroamide


ORGAN WEIGHTS
Generally no effects on organ weights were detected. The mean bodyweight for the low dose males was greater than for controls and so the relative organ weights were considered. The organ weights and relative organ weights showed no significant variation between treated and control animals.


GROSS PATHOLOGY
No specific changes were observed in the high dose rats found dead or killed after becoming moribund that could account for in-life clinical observations. One male had a pale liver.
No macroscopic changes were evident for the rats dosed at 150 or 30 mg/kg bw/day.

HISTOPATHOLOGY: NON-NEOPLASTIC
Slight degenerative changes noted in the liver for four males and one female dosed at 600 mg/kg bw/day. Vacuolation in two males and the female and centrilobular necrosis in two males.
No microscopic changes, other than those commonly observed as background pathology, were observed in the rats dosed at 150 or 30 mg/kg bw/day.

Effect levels

Dose descriptor:
NOEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The NOEL of 150 mg/kg bw/d was based on the absence of toxicologically signficant findings at this level, compared with mortality, marked clinical signs and various clinical pathology changes observed at 600 mg/kg bw/d.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the results of the repeated administration of PTH-decahydroamide at 30, 150 or 600 mg/kg bw/day, the no observed effect level (NOEL) was 150 mg/kg bw/day and the LOAEL was 600 mg/kg bw/day.
The marked effects observed at 600 mg/kg bw/day including mortality, functional disturbances and toxicologically significant effects (e.g. convulsions) indicate consideration of the risk phrase R48 "Danger of serious damage to health by prolonged exposure". However, this is only appropriate where effects are observed in rats following oral administration at 50 mg/kg bw/day in a repeated administration study or at levels circa three-fold higher in a subacute study. Since the effects were not apparent at 150 mg/kg bw/day and only observed at the next higher dose level, classification as R48 is not considered appropriate for PTH-decahydroamide and no STOT-RE classification is appropriate.
Executive summary:

A 28-day repeated oral administration study of subacute toxicity in the rat was conducted with PTH-decahydroamide. The main study dose levels were set using data from 5-day preliminary toxicity investigation. Range-finding results indicated suitable dose levels were 0, 30, 150 and 600 mg/kg bw/day. The study design was based on the test guidelines set out in EEC Directive 92/69/EEC, B.7 subacute toxicity - oral, 1992 and OECD Test Method No. 407, Repeated Dose Oral Toxicity - Rodent, 1981.

PTH-Decaydroamide was administered daily by oral gavge to groups of five male and five female Wistar rats. The test guideline parameters - clinical signs, bodyweights, food consumption, ophthalmoscopy, clinical pathology, haematology, organ weights, terminal necropsy and subsequent histopathology were completed in accordance with the test methods.

Formulations of PTH-decahydroamide were confirmed by analysis to be accurate, stable and homogeneous.

There were no teatment-related findings of any toxicological significance in the animals dosed at 30 or 150 mg/kg bw/day.

In the 600 mg/kg bw/day group, five of the rats died (on treatment days 6, 7 or 14) and the remaining five were humanely terminated on welfare grounds on Day 18. The marked signs of toxicity, observed ante-mortem, included clinical signs of convulsive movements, abnormal posture, abnormal gait, recumbency and laboured respiration. Decreased red blood cell counts and reduced haemoglobin content were recorded in the males and females; decreased mean corpuscular haemoglobin concentration and an increased mean corpuscular volume were noted among the males only. Total protein and albumin levels were reduced for males and females and the inorganic phosphate levels were increased in both sexes. Increased liver enzyme activities were recorded for one male rat. Necropsy of the high dose rats revealed vacuolation or centrilobular necrosis in the liver of four males and one female.

Based on the results of the repeated administration of PTH-decahydroamide at 30, 150 or 600 mg/kg bw/day, the no observed effect level (NOEL) was 150 mg/kg bw/day and the LOAEL was 600 mg/kg bw/day.

The marked effects observed at 600 mg/kg bw/day including mortality, functional disturbances and toxicologically significant effects (e.g. convulsions) indicate consideration of the risk phrase R48 "Danger of serious damage to health by prolonged exposure". However, this is only appropriate where effects are observed in rats following oral administration at 50 mg/kg bw/day in a repeated administration study or at levels circa three-fold higher in a subacute study. Since the effects were not apparent at 150 mg/kg bw/day and only observed at the next higher dose level, classification as R48 or STOT-RE Cat 1 or 2 is not considered appropriate for PTH-decahydroamide.