Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study (OECD test guideline 407)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report Date:
1993

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
not specified
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
not specified
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Description: blue powder
- Expiration date of the batch: 01-Apr-1997
-Stability under storage conditions: stable

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BRL Ltd., Basel, Switzerland
- Age at study initiation: approximately 6 weeks old
- Housing: 5 animals/sex/cage in stainless steel suspended cages with wire mesh floors
- Diet: standard pelleted laboratory animal diet (Kliba, Klingentalmühle AG, 4303 Kaiseraugst, Switzerland), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21 °C
- Humidity: 55 %
- Air changes: 15 changes/hour
- Photoperiod: 12 hours dark / 12 hours light

In-Life phase: 08 June 1993 (delivery of animals) - 27 July 1993 (termination recovery group)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on oral exposure:
Method: oral gavage
Frequency: once daily, approximately the same time each day, 7 days per week
Dose Levels: 0, 50, 200 and 1000 mg/kg bw/day
On days 12 and 13, the mid dose group was dosed with 1191 mg/kg bw (instead of 200 mg/kg bw) and the high dose group with 161 mg/kg bw (instead of 1000 mg/kg bw), due to an error in labelling of test formulation containers. Considering the duration of treatment this represented an error of less than 10 %. Evaluation of the results indicated no effect on the outcome of this study.
Dose volume: 5 mL/kg bw
Duration of treatment: 28 days (followed by a 14-day recovery period for some animals)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of dose formulations prepared during week 2 and after the treatment period, using identical procedures as during the study period, were analysed to check stability, homogeneity and concentration.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 50, 200, 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
- 5 animals/sex/dose level
- additional 5 animals/sex at 0 and 1000 mg/kg bw/day
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on data from previous studies in rats.
- Post-exposure recovery period in satellite groups: 14 days
Positive control:
not required

Examinations

Observations and examinations performed and frequency:
Clinical signs:
At least once daily. The time of onset, degree and duration was recorded daily.

Mortality/Viability:
Twice daily

Body weights:
Weekly and on the day preceding termination, prior to overnight fasting.

Food consumption: Weekly.

Water consumption:
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

Ophthalmoscopic examinations:
Both eyes were examined following instillation of tropicamide solution (5 mg/mL) at week 4 (all animals) and at week 6 (recovery animals).

Blood sampling for clinical laboratory investigations:
Blood samples were collected under light ether anaesthesia between 8.00 and 10.00 a.m. from main and recovery group animals on days 29 and 43, respectively. The animals were fasted overnight before blood sampling, but water was provided. Blood samples were drawn from the retro-orbital sinus of all rats/sex/group and collected into tubes prepared with EDTA for determination of haematological parameters (0.6 mL), tubes prepared with citrate for blood clotting times (1.0 mL) and untreated tubes for determination of clinical biochemistry parameters (>2.0 mL).

Haematology:
The following haematology parameters were determined:
erythrocyte count, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelet count, red cell distribution width, total leucocyte count, differential leucocyte count, neutrophils, eosinophils, basophils, lymphocytes, monocytes, prothrombin time, partial thromboplastin time

Clinical biochemistry:
The following clinical biochemistry parameters were determined:
alanine aminotransferase, aspartate aminotransferase, bilirubin total, cholesterol, triglycerides, creatinine, glucose, urea, protein total, albumin, globulin, albumin/globulin, alkaline phosphatase, sodium, potassium, chloride, calcium, phosphorus
Sacrifice and pathology:
All animals surviving to the end of the observation period (day 29 for the main groups and day 43 for the recovery groups) were deeply anaesthetised using ether vapour and subsequently exsanguinated. All animals assigned 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 necropsy and fixed in neutral phosphate buffered 4 % formaldehyde solution:
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, liver, lung, lymph nodes, nasopharynx, oesophagus, ovaries, pancreas, pituitary gland, preputial gland, prostate gland, rectum, salivary glands, sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord, spleen, sternum with bone marrow, stomach, testes, thymus, thyroid (including parathyroid), tongue, trachea, urinary bladder, uterus, vagina, all gross lesions
Other examinations:
The following organ weights (and terminal body weight) were recorded on the scheduled dates of necropsy:
adrenal glands, brain, heart, kidneys, liver, spleen, testes, ovaries

All organ and tissue samples were processed, embedded and cut at a thickness of 2-4 µm and stained with haematoxylin and eosin.
Slides of adrenals, heart, kidneys, liver, spleen, stomach and testes, collected at termination from all animals of the control group and the highest surviving dose group, as well as from all gross lesions of all animals were examined. Based on the treatment related morphologic changes, livers were also examined from all rats at 50 and 200 mg/kg bw/day. All abnormalities were described and included in the report.
Statistics:
The following statistical methods were used to analyse the body weight, food consumption, organ weights and clinical laboratory data:
Univariate one-way analysis of variance was used to assess the significance of intergroup differences. If the variables 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. The Steel-test (many-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. The exact Fisher-test was applied to the ophthalmoscopic examination data.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
transient clinical signs, no mortality
Mortality:
mortality observed, treatment-related
Description (incidence):
transient clinical signs, no mortality
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
white blood cell counts
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
alanine aminotransferase and aspartate aminotransferase activity and in cholesterol and triglyceride levels
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
liver
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
blue discoloration of fat tissue
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Very slight to slight centrilobular hypertrophy of hepatocyte
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS
A high incidence of alopecia was noted among males and females receiving 1000 mg/kg bw/day during the course of the 6-week study period. Although this finding is commonly seen in untreated animals of this age and strain, a possible relationship with the test item treatment could not be excluded. Signs of ill health, observed during the treatment period were hunched posture and rough coat. Hunched posture was noted in 5/5 males and 1/5 females receiving 50 mg/kg bw/day between days 16 and 26, in 4/5 males and 5/5 females receiving 200 mg/kg bw/day between days 11 and 29 and in 10/10 males and females receiving 1000 mg/kg bw/day between days 11 and 40. Rough coat was noted on day 27 in 1/5 females receiving 50 mg/kg bw/day, in 2/5 males receiving 200 mg/kg bw/day between days 11 and 29 and in 3/10 males receiving 1000 mg/kg bw/day between days 25 and 37. In addition, lethargy was noted in 5/10 males receiving 1000 mg/kg bw/day on days 20 to 24.
Dark appearance of the faeces and blue appearance of the skin were attributed to the staining properties of the test item and were considered not of toxicological significance. In addition, the brown appearance of the skin, as noted in the genital area of a few males and females treated at 1000 mg/kg bw/day, was associated to the occurrence of dark faeces. Salivation, sometimes with rales, was seen on a few occasions in control animals and during the treatment period among most treated animals. This finding is commonly noted following oral dosing with a stomach tube. After ceasing the oral treatment, salivation had stopped immediately. This finding was considered related to an irritant effect or bad taste of the test item and was not a sign of toxicity.

MORTALITY
No mortality occurred during the 4-week treatment and 2-week recovery period.

BODY WEIGHT
Body weights and body weight gain of animals receiving 1000 mg/kg bw/day were consistently lower than control animal weights during the treatment period, although not all differences achieved a level of statistical significance. This decrease was greater for males than for females and for males only remained also low during the recovery period. For males receiving 200 mg/kg bw/day, body weights and body weight gain were considered as slightly lower than controls during the 4-week treatment period without showing statistical significance. Body weight and body weight gain of animals receiving 50 mg/kg bw/day were in the same range as controls during the treatment and recovery period.

FOOD CONSUMPTION
During the treatment period, food consumption was lower than controls in animals receiving 1000 mg/kg bw/day. No such difference was seen in these animals during the recovery period and in animals receiving 50 or 200 mg/kg bw/day during the treatment period. Relative food consumption was low in comparison with control animals in males and females treated at 1000 mg/kg bw/day over the first week of treatment. This value was high for males and females previously treated at 1000 mg/kg bw/day over the first week and for males also over the second week of recovery. Relative food consumption values of animals treated at 50 or 200 mg/kg bw/day were similar to those of control animals during the treatment period.

OPHTHALMOSCOPIC EXAMINATION
There were no ophthalmoscopic findings at weeks 4 and 6 that could be attributed to treatment.
Focal lens opacity, as noted in 1 male receiving 1000 mg/kg bw/day at the end of the treatment period, was the only abnormality found in treated animals and considered of no toxicological significance.

CLINICAL LABORATORY INVESTIGATIONS
HAEMATOLOGY
The only change that was considered of possible toxicological significance was noted in white blood cell counts. After 4 weeks of treatment, white blood cell values of females receiving 1000 mg/kg bw/day were increased with statistical significance when compared to controls. In males of this group, the white blood cell numbers remained in the same range as controls. White blood cell numbers of animals receiving 50 or 200 mg/kg bw/day were comparable to those of controls, as were the white blood cell numbers of animals previously treated at 1000 mg/kg bw/day at the end of the 2-week recovery period. Fluctuations in differential white blood cell counts as noted in treated females at week 4 of treatment did not exceed the range of normal biological variation for rats of this age and strain.
Multiple statistically significant changes in red blood cell parameters of treated males at week 4 of treatment, and of males and females previously treated at 1000 mg/kg bw/day at week 6 (week 2 of recovery), remained within the range normally expected for rats of this age and strain. Since there
was no clear coherence between the sexes and between the red blood cell parameters that were changed with statistical significance, these changes were considered to have occurred fortuitously.
The statistically significant increase in number of platelets in males receiving 1000 mg/kg bw/day and decrease in prothrombin and partial thromboplastin time of females receiving 1000 mg/kg bw/day at the end of week 4 were considered to be of no biological significance.

CLINICAL BIOCHEMISTRY
Statistically significant differences between control and treated animals that were considered toxicologically significant were observed in alanine aminotransferase and aspartate aminotransferase activity and in cholesterol and triglyceride levels. Alanine aminotransferase activity was increased in males and females receiving 1000 mg/kg bw/day at the end of the 4-week treatment period and for males of this group also after the subsequent 2-week recovery period. Aspartate aminotransferase was increased in females receiving 1000 mg/kg bw/day after 4 weeks treatment. Cholesterol was noted as increased in females receiving 200 mg/kg bw/day and in males and females receiving 1000 mg/kg bw/day at week 4 of treatment. In females previously treated at 1000 mg/kg bw/day cholesterol was still slightly increased at the end of recovery, although this difference was not statistically significant. Triglyceride levels were high in males and females receiving 1000 mg/kg bw/day at week 4 and in females of this group also after the subsequent recovery period. All other biochemical parameters attaining statistical significance when compared to corresponding controls were considered the result of normal biological variation, as no corroborative changes were seen between the sexes and in other parameters examined in this study.

MACROSCOPIC EXAMINATION
There were no treatment-related findings observed in this study. Macroscopic examinations revealed blue appearance of fat tissue, the gastro-intestinal tract and the subcutis of the skin in most treated animals. In addition, the liver of 5/10 males receiving 1000 mg/kg bw/day had a dark appearance at the end of the 4-week treatment period. In the absence of any microscopic correlates, these changes were attributed to the physico-chemical properties of the test item and considered not to be changes of toxicological significance. All other findings did not exceed the normal incidence of biological variation.

ORGAN WEIGHTS
Differences between organ weights of control and treated animals that were considered of possible toxicological significance were noted in the liver.
At week 4 of treatment, liver weights of males and females receiving 1000 mg/kg bw/day were increased with statistical significance before and after allowance for body weight. Liver weights and liver weight:body weight ratios of animals receiving 50 or 200 mg/kg bw/day were comparable to controls. After 2 weeks of recovery, liver weights of animals previously receiving 1000 mg/kg bw/day were still slightly increased in females only.
Other statistically significant differences between control and treated animals were considered not to be of toxicological significance.

MICROSCOPIC EXAMINATION
Treatment-related changes were detected in the liver. Very slight to slight centrilobular hypertrophy of hepatocytes was observed in most male and all female rats receiving 1000 mg/kg bw/day after 4 weeks of treatment. At the end of the recovery period no such changes were apparent in these rats. Examination of the liver of animals receiving 50 or 200 mg/kg bw/day at the end of the treatment period also revealed no differences with the livers of controls.
All other findings recorded were within the normal range of background alterations which may be seen in untreated rats of this age and strain.

Effect levels

Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on hunched posture (males and females), decreased body weight and body weight gain (males only) and increased cholesterol levels (females only) noted at 200 mg/kg bw/day.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion