Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
adopted in 1995
Deviations:
yes
Remarks:
(animals not fasted prior to sampling, endocrine activity not evaluated, prostate and seminal vesicles not weighed, no histology of peripheral nerve and bone marrow)
GLP compliance:
yes (incl. certificate)
Remarks:
Ministerium für Umwelt, Raumordnung und Landwirtschaft des Landes Nordrhein-Westfalen, Düsseldorf, Germany
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Details on strain: HsdCpb:WU (SPF)
- Age at study initiation: 6 to 8 weeks
- Weight at study initiation: 143.7 to 188.9 g (variation did not exceed 20% of the mean body weight)
- Housing: 5 animals/cage
- Diet: Ssniff R 10 diet in pelletform (Ssniff Spezialfutter GmbH, Soest, Germany); ad libitum
- Water: tap water; ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 06 April 1998 To: 20 May 1998

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Before start of the study instability of the test substance formulations were examined after a period of one week. Therefore the dosing formulations for administration were prepared daily.
The test subsance was melted in a beaker and a corresponding weight of corn oil, calculated for the target dosages was filled into the beaker and was stirred by means of a magnetic stirrer for 15 minutes.

VEHICLE
- Concentration in vehicle: 10, 30 and 60% (w/v)
- Amount of vehicle (if gavage): 5 mL/kg bw

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity and concentration of the test substance formulations were determined. Samples of all dose levels were taken repeatedly and analyzed by gas chromatography. The analytical results showed a good stability and satisfactory concordance between measured and nominal concentration. The analysis also revealed, that the samples are homogenous.
Duration of treatment / exposure:
28 days (control and test groups)
28 days and 14 days post-exposure observation period (recovery control and high-dose group)
Frequency of treatment:
once daily, 7 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
50, 150 and 300 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
5 (main study)
5 (recovery control and recovery high dose group)
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale:
The dose levels were chosen based on the results of a range-finding study at the same testing laboratory.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Parameters: general health state, behavioural changes, toxicosis, and mortality
A general clinical observation was performed once a day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

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/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

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 AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: daily

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: week 4
- Dose groups that were examined: all groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of the treatment period/recovery period
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: No
- How many animals: all animals
- Parameters examined: red blood cell count (RBC), total white blood cell count (WBC), platelet count (PLT), haemoglobin (HGB), haematocrit (HCT), erythrocyte indices: mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), differential white blood cell count (granulocyte, lymphocyte, monocyte, plasmacyte)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of the treatment period/recovery period
- Animals fasted: No
- How many animals: all animals
- Parameters examined: sodium, potassium, calcium, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (AP), glucose, triglycerides (TRIG), cholesterol, total bilirubin, blood urea nitrogen (BUN), creatinine, total protein, albumin

URINALYSIS: Yes
- Time schedule for collection of urine: weekly
- Parameters examined: volume, specific gravity, pH, colour, protein, glucose, keton, urobilinogen, blood ingredients.
A microscopical urine sediment analysis was performed on all samples and the following parameters were determined: leucocytes, erythrocytes, bacteria, epithelial cells squamous and renal, oxalate crystals, triple phosphate crystals, carbonate, granular cylinder, urate crystals.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: weekly
- Dose groups that were examined: all dose groups
- Battery of functions tested: Home Cage Observation (HCO): posture of the animals when they move or rest, coordination of movement/gait, tremor and convulsion, abnormal behaviour; Open Field Observation (OFO): behaviour when taken out of the cage, autonomic functions, fur, skin and external appearance, muscle tone, posture of the animal when they move or rest, coordination of movement/gait, respiration, activity/attention, tremor and convulsion, abnormal behaviour, eyes and palpepral closure, faeces (appearance/consistency) released during OFO, urine (volume/colour) released during OFO.
In the 4th week additional observations were carried out: motor activity, rearing, landing foot-splay and grip strength and extended OFO including vision test, pupillary reflex, winking reflex, pinna-reflex, hearing test, sense of smell, examination of catalepsy, coordination of movement and sensitivity to pain
Sacrifice and pathology:
Necropsy:
On completion of the dosing period and recovery period all animals were killed by CO2 asphyxiation and exsanguination. A complete autopsy
including a macropathological examination was performed.

Organ weights:
The following organs from animals killed at the end of the study, dissected free from fat, were weighed before fixation: adrenals, brain,
epididymides, heart, kidneys, liver, spleen, testes and thymus.

Histopathology:
Samples from the following tissues were removed from animals of all groups (including recovery group) and preserved in 10% formalin:
adrenals, aorta (thoracic), anus, brain, caecum, coagulation gland, colon, concha (tattooed), duodenum, epididymides, eyes, exorbital lacrimal, glands, gross lesions, heart, ileum, jejunum, kidneys, larynx, liver, lungs, lymphnodes (skin, cervical and mesenteric), mammary gland, muscle (skeletal), ovaries, oesophagus, pancreas, pituitary, prostate, rectum, salivary glands, sciatic nerve, seminal vesicles, skin, spinal cord, spleen, stomach, sternum, testes, thymus, thyroid/parathyroid, tongue, trachea, urinary bladder, uterus and vagina.
A limited number of organs of all animals of the control group and of the high dose group were prepared as paraffin blocks, sectioned and stained
for a subsequent microscopic examination.
Statistics:
Haematological data/clinical chemistry: ANOVA, Kruskal Wallis, Scheffe Test
Rearing, motoractivity, bodyweight, organ weights, urine analysis: Kruskal Wallis, Wilcoxon, Mann, Whitney U-Test
Significance levels: * p < 0.05, ** p < 0.01

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
mid dose group: convulsions, squatting position, one individual animal each showed vocalisation and was walking on tiptoes on one day; high dose groups: convulsions, prone and squatting position, straub tails, salivation and vocalisation, individual animals were walking on tiptoes; no effects in control and low dose groups or during recovery period.
Mortality:
mortality observed, treatment-related
Description (incidence):
mid dose group: convulsions, squatting position, one individual animal each showed vocalisation and was walking on tiptoes on one day; high dose groups: convulsions, prone and squatting position, straub tails, salivation and vocalisation, individual animals were walking on tiptoes; no effects in control and low dose groups or during recovery period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
male high dose recovery group: significant reduction in absolute body weight on day 28 and group mean body weight changes in week 3 and 4; females high dose recovery group (week 2): stastically significant increase of group mean weekly body weight;
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
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
symptoms like ataxia, fasciculations, paddling movements, aggressiveness, squatting position, hyperactivity, straub tail, piloerection in individual animals predominantly of high dose groups. Recovery period: no dose-related functional changes
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
slight effects (increased relative adrenal and epididymides weights) in high dose males
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
alpha-2 micro globulin nephropathy syndrome in the kidneys of 5 high dose males and one control male; slight vacuolation in the kidney of 2 females (control and high dose group)
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
Two animals of the male high dose group died because of application failure (day 20 and 26). The severe defense against application of the test substance especially in the high dose groups could explain the application failure.
Moderate to severe clinical symptoms were observed in the mid and high dose groups. In the mid dose group convulsions (5/5 males and females), squatting position (5/5 females, 3/5 males), vocalisation and walking on tiptoes (1/5 females each) were observed. In the high dose groups convulsions (5/5 males and females; recovery: 5/5 males and females), squatting position (5/5 males and females; recovery: 5/5 males and females), vocalisation (1/5 males and females; recovery: 3/5 females, 4/5 males), prone position (5/5 males and females; recovery: 5/5 males and females), straub tail (1/5 females), walking on tiptoes (2/5 females; recovery: 1/5 females) and salivation (1/5 males; recovery: 2/5 females) were noted. The peak period of clinical effects was approx. 15 min after dosing. Convulsions disappeared after a while and the animals began to recover. Individual animals predominantly of the high dose group still showed mild clinical symptoms in the afternoon. No effects in control group, low dose group or during the recovery period were noted.

BODY WEIGHT AND WEIGHT GAIN
The male and female low, medium and high dose groups showed no differences of toxicological importance concerning body weight and body weight changes compared to control animals.
In the male high dose recovery group statistically significant reductions of group mean weekly body weight change were noted in the final weeks of the treatment period (week 3: 300 mg/kg bw/day: 19.2 ± 0.95 g, control: 30.1 ± 2.22 g; week 4: 300 mg/kg bw/day: 11.6 ± 7.16 g, control: 25.8 ± 3.33 g). This finding correlates with the significant reduction of body weight (277.5 ± 12.38 g) in the male high dose recovery group in the final treatment week (day 28) compared to the recovery control group (310.3 ± 11.00 g). The reduction of the mean weekly body weight change and of the absolute body weight in the high dose male recovery group at the end of the treatment period might have been caused by the test substance.
The female high dose recovery group showed a statistically significant increase of group mean weekly body weight change at the beginning of the study compared to the control recovery group (week 2: 300 mg/kg bw/day: 27.7 ± 6.86 g; control: 13.4 ± 2.81 g).
During and also at the end of the recovery period no statistically significant differences concerning group mean weekly body weight or body weight change in the male and female high dose recovery group were observed.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
A clear pattern in these changes was lacking and examination of the both sexes gave contradictory results. Therefore, all these differences were considered to be of minor toxicological importance.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
No intergroup differences were observed.

OPHTHALMOSCOPIC EXAMINATION
No effects were observed.

HAEMATOLOGY
At the end of the treatment and recovery period no statistically significant differences to controls were found in treated females.
High dose males showed a statistically significant increase of total white blood count (WBC). Also high dose females showed a slight statistically not significant increase of WBC when compared with control. At the end of the recovery period the high dose recovery males showed a slight statistically non-significant decrease of WBC in comparison with the control recovery animals, indicating evidence of reversibility.
Statistically significant decreases of red blood cell count (RBC), haemoglobin (HGB) and platelet count (PLT) were found in high dose recovery males in comparison to recovery control animals.
All changes in high dose males from controls at the end of treatment period and at the end of the recovery period were within the normal range of the historical background data. Therefore, all changes were considered to be of minor toxicological importance.

CLINICAL CHEMISTRY
The male medium and high dose group showed a statistically not significant decrease of glucose compared to controls. Creatinine was statistically increased in the male medium dose group. Aspartate aminotransferase (AST) was statistically significant increased in high dose males. Treated females showed a decrease of AST compared to control. Glucose values of treated males and females were slightly above the control values at the end of the recovery period. The female high dose recovery group showed a statistically significant decrease of cholesterol, trigycerides, total bilirubin and alanine aminotransferase (ALT) when compared with recovery control.
The significant differences from controls at the end of the treatment period and at the end of the recovery period were minor and within the normal range of the historical background data. Thus, they are not considered substance-related. Examination of the two sexes concerning AST and creatinine gave contradictory results. Evidence of reversibility was found at the end of the treatment period. Therefore, all the differences were considered to be of minor toxicological importance. Furthermore, variabilitiy in clinical parameters can be due to the fact that the animals were not fasted before sampling.

URINALYSIS
No significant changes were noted in males compared to controls.
In the female medium dose group a statistically significant decrease of specific gravity (SPGR) was noted compared to control values. The female high dose recovery group showed a statistically significant increase of SPGR and a statisticaly significant decrease of pH when compared to control values. The significant differences from control at the end of the treatment and recovery period was minor and not dose-related and therefore considered to be of minor toxicological importance.

NEUROBEHAVIOUR
HCO and OFO (week 1 - week 6):
In the examination of week 2, 3 and 4 clinical symptoms were noticed in individual animals predominantly of the high dose groups. Ataxia, fasciculation, paddling movement, defence against touching, aggressiveness, hunchback/squatting position, reduced respiration, hyperactivity, straub tail, piloerection and slight convulsions were noticed. No significant findings were observed during the recovery period in week 5 and 6.
Extended OFO (4th week):
Animals of the control, low and medium dose groups showed a normal response to different stimuli. Individual animals of the high dose groups showed minimal or high sensitivity of pain. One male rat of the high dose group showed positive response when examined for catalepsy.
Rearing, landing foot-splay and grip strength (4th week):
Female and male animals of all dose groups and recovery high dose females showed no significant findings compared to control groups. High dose recovery males showed statistically significant increase of group mean values for landing foot-splay and rearing and decrease of group mean values for grip strength when compared with recovery control group. The described statistically significant differences from control in high dose male recovery group were minor. A clear pattern in these changes was lacking and examination of the two sexes gave contradictory results. Therefore all these differences were considered to be of minor toxicological importance.
Motoractivity:
The group mean values of activity counts of all intervals (IVACX) showed no statistically significant increase in low, medium and high dose groups and high dose male recovery group when compared with control. However, the assessment of the motoractivity showed an increase of group mean motoractivity values (statistically not significant) in the high dose groups, especially in the female high dose group, when compared to control.

ORGAN WEIGHTS
At the end of the treatment period a statistically increase in relative (% of body weight) weight of adrenals was noted in the high dose male group (0.022%; control: 0.016%). The increase in relative adrenal weight is considered to be caused by stress in connection with the application of the test substance. The absolute weight of the adrenals showed no statistically significant increase in high dose males.
At the end of the recovery period, the high dose male group showed a statistically significant increase in relative (% of body weight) weight of epididymides (0.411%; control: 0.326%).

GROSS PATHOLOGY
No abnormalities were detected.

HISTOPATHOLOGY: NON-NEOPLASTIC
The kidneys of 5 high dose and one control male showed a slight number of eosinophilic hyaline droplets in the epithelial cell cytoplasm of the proximal tubules (alpha-2 micor globulin nephropathy syndrome). These findings are specific to male rats and of no concern to man.
The kidney of one control and one high dose female showed a slight vacuolation of the epithelial cells of the proximal tubules.
Histological examination of the adrenals showed no significant changes.
The livers of two animals of the male and female control group and of two males and one female of the high dose group showed slight multifocal vacuolation predominantly in the periphery of the liver. However, these lesions were not dose-related and considered of minor toxicological importance.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Dose descriptor:
LOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: effects on neurobehaviour, motoractivity, body weight

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In this 28-day oral toxicity study in the rat a NOAEL of 150 mg/kg bw/day was found, as the clinical signs observed in the mid dose groups (150 mg/kg bw/day) are considered non-adverse.