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Description of key information

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 January 1998 - 08 June 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD guideline 407 and EU Method B.7 without significant deviations from the guidelines and conducted according to GLP protocols.
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: approximately 6 weeks
- Housing: Group housing of 5 animals per sex per cage in stainless steel suspended cages with wire mesh floors. During activity monitoring, animals were individually housed overnight in Macrolon plastic cages with sterilised sawdust.
- Diet (e.g. ad libitum): Free access to standard pelleted laboratory animal diet (from Carfil Quality BVBA, Oud-Turnhout, Belgium).
- Water (e.g. ad libitum): Free access to tap water
- Acclimation period: 5 days prior to study Initiation

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

IN-LIFE DATES: From: To: 12 January 1998 - 09 February 1998
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily immediately prior to dosing. Adjustment was made for specific gravity of vehicle.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Using the validated GC method (see NOTOX project 217384), the EPYRROL concentration could not be determined in the study formulation, due to the combination of the required vehicle and low dose levels used in this study. Therefore no chemical anaylysis could be performed.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily for 28 days, approximately the same time each day, 7 days per week
Remarks:
Doses / Concentrations:
1 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
5 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
25 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
5/sex/dose level
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: Based on the results of two 5-day range finding studies (See Notox projects 217349 and 221131), the dose levels for the 28-day toxicity study were selected to be 0, 1, 5, and 25 mg/kg/day.
- Rationale for animal assignment (if not random): Randomization: At least 5 days before study start, by computer-generated random algorithm accoring to body weight, with all animals within +/- 20% of the sex mean. A helath inspection was performed prior to commencement of treatment to ensure that the animals were in good state of health.
Positive control:
N/A
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations checked: Mortality and Viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily for all animals. Once prior to the start of treatment and on days 8, 15, 22, and 28, this was also performed outside the home cage in a standard arena. The time of onset, degree and duration were recorded. All symptoms were recorded and graded according to fixed scales: Maximum grade 4: grading slight (1) to very severe (4); Maximum grade 3: drading slight (1) to severe (3); Maximum grade 1: presence is scored (1).

BODY WEIGHT: Yes
- Time schedule for examinations: On days 1, 8, 15, 22, and 28.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/week: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

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: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations: N/A
- Dose groups that were examined: N/A

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Prior to post mortem examination between 7:30 and 9:30 am
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: All animals/sex/dose level
- Parameters checked: Erythrocytes count (RBC), Haemoglobin (HB), Haematocrit (HTC), Mean corpuscular volume (MCV), Mean corpuscular haemoglobin concentration (MCHC), Platelet count, Red cell distribution width (RDW), Total leucocyte count (WBC), Differential leucocyte count (SEG) (Neutrophils, Eosinophils, Basophils, Lymphocytes, Monocytes), Prothrombin time (PT), Partial thromboplastin time (PTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Prior to post mortem examination between 7:30 and 9:30 am
- Animals fasted: Yes, overnight
- How many animals: All animals/sex/dose level
- Parameters checked: Alanine aminotransferase (ALAT/GPT), Aspartate aminotransferase (ASAT/GOT), Bilirubin total (BILI T), Cholesterol total (CHOLEST T), Creatinine, Glucose, Urea, Protein total (PROTEIN T), Protein albumin (ALBUMIN), Alkaline phosphatase (ALP), Sodium, Potassium, Chloride, Calcium, Phosphorus (INORG PHOSPH)

URINALYSIS: No Data
- Time schedule for Collection of urine: N/A
- Metabolism cages used for collection of urine: N/A
- Animals fasted: N/A
- Parameters checked: N/A

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During week 4 of treatment
- Dose groups that were examined: all animals
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, grip strength (Score 0= normal/present, Score 1= abnormal/absent), activity test (based on hourly data per animal, using a computerized motor activity monitoring system)

OTHER: None
Sacrifice and pathology:
GROSS PATHOLOGY: Yes; All animals surviving to the end of the observation period and all animals killed for humane reasons were deeply anaesthetised using ether vapour and subsequently exsanquinated. All animals assigend to the study were necropsied and descriptions of all macroscopic abnormalities recorded). Samples of the following tissues and organs were collected from all animals at neropsy and fixed in neutral phosphate buffered 4% formaldehyde solution, including the following: Adrenal glands, Aorta, Brain, Caecum, Cervix, Clitoral gland, Colon, Duodenum, Epididymides, Eyes with optic nerve and Harderian gland, Female mammary gland area, Femur including joint, heart, Ileum, Jejunum, Kidneys, Larynx, Lacrimal gland (exorbital), Liver, Lung infused with formalin, Lymph nodes (mandibular and mesenteric), Nasopharynx, Oesophagus, Ovaries, Pancreas, Peyer's patches (jejunum and ilenum if detectable), Pituitary gland, Preputial gland, Prostate gland, Rectum, Salivary glands (mandibular and sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin, Spinal cord (cervical, midthoracic, and lumbar), Spleen, Sternum with bone marrow, Stomach, Testes, Thymus, Thyroid including parathyroid, Tongue, Trachea, Urinary bladder, Uterus, Vagina, and all gross lesions.


HISTOPATHOLOGY: Yes; All organs and tissues collected for histopathology were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin, at the scheduled scarifice from all aniamls of the control and high dose group as well as from all animals of all dose groups which were terminated for humane reasons, and all gross lesions of all animals were exsamined by a pathologists based on the treatment related morphologic changes. Livers were also examined from all rats in the intermediate dose group. All Abnormalities were described adn included int eh report. All organs and tissues collected for histopathology include: Cervix, Clitoral gland, Eyes with optic nerve and Harderian gland, Female mammary gland area, Femur including joint, Larynx, Lacrimal gland (exorbital), Nasopharynx, Preputial gland, Salivary glands (mandibular and sublingual), Seminal vesicles, Skeletal muscle, Skin, Tongue, and Vagina.
Other examinations:
Organ weights and terminal body weights were taken and recorded for the surviving animals on the scheduled day of necropsy, including the following organs: Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Spleen, Testes, and Thymus.
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 the variable could be assumed to follow a normal distribution, the Dunnett-test (many-to-one-t-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 (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- 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).
- Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Not considered to be a dose response relationship
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Not considered to be a dose response relationship
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Not considered to be a dose response relationship
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
liver
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No treatment related mortality occurred during the study period. One female, receiving 1 mg/kg/day was sacrificed on humane grounds on day 14, due to causes unrelated to the test substance treatment.

There were no clinical signs of toxicity or behavioural changes over the 28-day observation period that were considered to be related to the treatment. Excessive salivation was observed in control and treated animals. This sign is often noted in rats this age and strain following oral gavage and considered to be related to multiple intra-oesophageal intubation and/or the taste of the test substance. Therefore, this finding was considered not to be a sign of systemic toxicity. Other findings that were noted among control and treated animals included diarrhea, red or brown staining of the skin, scabs, alopecia and broken lower incisor. These findings were considered to be within the biological variation for rats of this age and strain.

BODY WEIGHT AND WEIGHT GAIN
Body weights and body weight gain of treated animals remained in the same range as controls over the 4-week study period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
There were no differences in food consumption before or after allowance for body weight between treated and control animals.

FOOD EFFICIENCY
N/A

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
N/A

OPHTHALMOSCOPIC EXAMINATION
N/A

HAEMATOLOGY
Haematological parameters of treated rats were considered not to have been affected by treatment. Minor statistically significant differences arising between controls and males receiving 25 mg/kg/day were considered to have arisen by chance (haemoglobin) or as a result of slightly high control values (mean corpuscular haemoglobin) and not to represent a change or biological significance, since all values remained within the range of historical background data from rats of this age and strain.

CLINICAL CHEMISTRY
There were no differences noted between control and treated rats that were considered to be related to treatment with the test substance. Values in treated animals achieving a level of significance when compared to controls were considered to have arisen by chance, as all values remained within the range of background data for rats of this age and strain. In the absence of a treatment-related distribution or corroborative findings in the opposite sex, these values were considered to be of no toxicological significance.

URINALYSIS
N/A

NEUROBEHAVIOUR
The number and distribution of abnormalities noted during the functional observation tests did not indicate a relation with treatment.

ORGAN WEIGHTS
Organ weights and organ weight:body weight ratios of treated animals were considered to be similar to those of control animals. Statistically significant changes between testes weight and spleen:body weight ratios of 5 mg/kg/day treated males and control males were considered not to be a sign of toxicity as no dose response relationship could be detected.

GROSS PATHOLOGY
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment. A variety of findings noted among treated and control animals were considered to be within the range of biological variation for rats of this age and strain not to represent a change of toxicological significance, since no microscopic correlates were found.

Microscopic findings due to treatment within the test substance were seen in the liver of rats treated with 25 mg/kg/day. These findings took the form of a minimal to moderate degree of bile duct proliferation recording in two males and in three females. In the females a minimal to slight degree of Cholangiofibrosis was also present in three females. One female treated at 1 mg/kg/day and two females treated at 25 mg/kg/day had a moderate to severe degree of lymphoid inflammation of the urinary bladder which was associated with inflammation of the renal pelves. These findings may be found in untreated rats from time to time and were not considered to be due to treatment. There were no microscopic findings noted in rats treated at 1 or 5 mg/kg/day, that were considered to be treatment-related. The remainder of microscopic findings recorded were considered to be spontaneous in nature and within the range of background morphological alterations encountered in Wistar rats of this age and strain. They occurred at similar incidences and severity in both control and treated rats.


HISTOPATHOLOGY: NON-NEOPLASTIC
N/A

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
N/A

HISTORICAL CONTROL DATA (if applicable)
N/A

OTHER FINDINGS
N/A
Dose descriptor:
NOAEL
Effect level:
5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Dose descriptor:
LOAEL
Effect level:
25 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Morphological findings indicative of toxicity in the liver. Minimal to moderate degree of bile duct proliferation recorded in two males and in three females. Additionally, in the females, a minimal to slight degree of cholangiofibrosis was present.
Critical effects observed:
not specified
Conclusions:
The administration of Epyrrol by one daily gavage to Wistar rats for 28 days led to morphological findings indicative of toxicity in the liver of some rats which had received the highest dose of 25 mg/kg/day. These consisted of bile duct proliferation or cholangiofibrosis.

There were no changes in clinical appearance, functional observations, body weights, food consumption, clinical laboratory investigations, macroscopic examination and organ weights that were considered to be an effect of treatment.

From the results presented in this report a definitive No Observed (Adverse) Effect Level (NO(A)EL) of 5 mg/kg/day was established.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
25 mg/kg bw/day
Study duration:
subacute
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The administration of Epyrrol by one daily gavage to Wistar rats for 28 days led to morphological findings indicative of toxicity in the liver of some rats which had received the highest dose of 25 mg/kg/day. These consisted of bile duct proliferation or cholangiofibrosis, both of which are considered significant, adverse effects.

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver

Justification for classification or non-classification

In the absence of a 90-day study the equivalent guidance values for 28-day studies according to Haber's rule are:

Acute Oral, Category 1: ≤ 30 mg/kg bw d

Acute Oral, Category2 : ≤ 300 mg/kg bw d

Thus, because the observed adverse effects from a 28 -day study occur at a dose less than the criteria value for classification, Epyrrol is classified as STOT RE 1 according to EU CLP.