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: see 'Remark'
Remarks:
Well documented and reported study fully adequate for assessment. The study was conducted according to an internationally accepted technical guideline and in compliance with GLP in a recognized contract research organization. Study according to OECD guideline no. 407 under GLP.

Data source

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

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 (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: solid
- Analytical purity: >99%

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Wistar rats, strain: HanIbm:Wist (SPF)) with appropriate range of bodyweight at study start.
- Source: RCC Ltd, Biotechnology & Animal Breeding Division, CH-4414 Füllinsdorf, Switzerland
- Age at delivery: 6 weeks.
- Weight at acclimatization: males: minimum 179 g, maximum 196 g, mean 188 g
females: minimum 118 g, maximum 129 g, mean 124 g
- Identification: Cage card and individual ear tattoo
- Randomization: Computer-generated random algorithm
- Acclimatization: Under test conditions after health examination for 7 days. Only animals without any visible signs of illness were used for the study.
Deviations from these target ranges were not evident.

HUSBANDRY
- Conditions: Standard laboratory conditions, air-conditioned with 10 - 15 air changes per hour, and continuously monitored environment
- Temperature: 22 +/- 3 deg-C
- Relative humidity: 40 - 70 %
- Photoperiod (fluorescent light): 12 h day / 12 h night (light period between 06:00 and 18:00 h, music during the light period)
- Accomodation: in groups of 5 in Makrolon type-4 cages with wire mesh tops and standardized softwood bedding
- Diet (ad libitum): Pelleted standard Provimi Kliba 3433 rat maintenance diet, the feed batch was analyzed for contaminants
- Fasting (diet withheld): Before blood sampling, animals were fasted for approx. 18 h
- Water (ad libitum): Community tap-water from Itingen, chemical and contaminant analyses of representative samples have been performed
Routine analysis of the batch of diet used and water, chew blocks and wood bedding did not provide evidence of contamination that might have prejudiced the study.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
PEG300
Details on oral exposure:
- Concentration in vehicle: The concentration of the test material in vehicle varied between dose groups thus allowing constant dosage volume in terms of ml/kg bw/day.
- Dose volume: 5 ml/kg bw/day
- Duration of treatment: 28 days

CONCENTRATION OF TEST MATERIAL IN VEHICLE
Group Concentration of Test Substance Treatment Volume Dose Level of Test Substance
(mg/ml) (ml/kg bw/day) (mg/kg bw/day)
(1) Vehicle Control 0 5 0
(2) Low Dose 10 5 50
(3) Mid Dose 40 5 200
(4) High Dose 200 5 1000

RATIONALE FOR DOSE LEVEL SELECTION
Based upon the results of a non-GLP 5-day dose-range-finding study in which the test article was administered by gavage to 2 rates per group and sex. Animals showed no changes which were related to the treatment with the test article.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Mean concentrations of the homogeneity were found to be 90.7% to 101.5% (dose group dependeent) of the nominal concentrations.
The test item was found to be stable in PEG 300 for two hours at test conditions.
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily, Dosing regime: 7 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
0 (vehicle control) 50, 200, 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
Male: 5 animals at 0 mg/kg bw/day
Male: 5 animals at 50 mg/kg bw/day
Male: 5 animals at 200 mg/kg bw/day
Male: 5 animals at 1000 mg/kg bw/day
Female: 5 animals at 0 mg/kg bw/day
Female: 5 animals at 50 mg/kg bw/day
Female: 5 animals at 200 mg/kg bw/day
Female: 5 animals at 1000 mg/kg bw/day
Control animals:
yes, concurrent vehicle
Positive control:
not required

Examinations

Observations and examinations performed and frequency:
OBSERVATIONS
- Mortality/Viability: Twice daily
- General cageside: Animals were observed for clinical signs once before commencement of administration, twice daily on days 1 - 3 (ca. 1 and 3 h after administration); as well as once daily on days 4 - 28 (ca. 1 h after administration)
- Detailed clinical observations: Animals were observed in their home cages, outside their home cages in a standard arena and in the hand. These observations were performed in random sequences once before commencement of administration and once weekly thereafter.
- Food consumption: Food consumption was recoreded once during the pretest period and weekly thereafter, using an on-line electronic recording system connected to the testing facility's computer system.
- Body weights: Body weights were recorded weekly during pretest, treatment and recovery and before each necropsy, using an on-line electronic recording system connected to the testing facility's computer system.
- Functional Observational Battery (FOB*): During week 4

* FOB: Including grip strength and locomoter activity

CLINICAL LABORATORY INVESTIGATIONS
Blood samples for hematology and clinical biochemistry were collected from all animals under light ether anesthesia. Animals were fasted for approx. 18 h before blood sampling but allowed access to water ad libitum. Blood samples were collected early in the working day to reduce biological variaton caused by circadian rhythms. Blood samples were drawn from the retro-orbital plexus using a micro-hematocrit glass capillary tube.

- Hematological examinations: erythrocyte count; hemoglobin; hematocrit; mean corpuscular volume; mean corpuscular hemoglobin; mean corpuscular hemoglobin concentration; platelet count; reticulocyte count; reticulocyte fluorescence ratios; nucleated erythrocytes (normoblasts); Heinz bodies; methemoglobin; total leukocyte count; differential leucocyte count; red blood cell morphology; thromboplastin time; activated partial thromboplastin time
- Clinical biochemistry: glucose; urea; creatinine; uric acid; bilirubin, total; cholesterol, total; triglycerides; phopholipids; aspartate aminotransferase; alanine aminotransferase; lactate dehydrogenase; creatine kinase; alkaline phophatase; gamma-glutamyl transferase; calcium; phosphorus; sodium; potassium; chloride; albumin; protein, total; globulin; albumin/globulin ratio
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, see below
WEIGHING OF ORGANS: Yes, see below
HISTOPATHOLOGY: Yes, see below

TERMINAL SACRIFICY
All animals were killed after 4 weeks

NECROPSY
All animals were weighed and necropsied after 4 weeks.
Samples of the following tissues and organs were collected from all animals at necropsy:

(adrenal glands); aorta; bone (sternum, femur incl. joint); (bone marrow (femur)); (brain (cerebrum, cerebellum, pons)); (cedum); (colon); (duodenum); (epididymides); esophagus; eyes with optic nerve; Harderian gland; (heart); (ileum, with Peyer's patches); (ovaries); pancreas; pituitary gland; (prostate gland); (rectum); salivary glands (mandibular, sublingual); (sciatic nerve); (seminal vesicles); skeletal muscle; skin; (spinal cord (cerical, midthoracic, lumbar)); (spleen); (stomach); (testes); (jejunum with Peyer's patches); (kidneys); larynx; lacrimal gland (exorbital); (liver); (lungs); (lymph nodes (mesenteric, mandibular)); mammary gland aera; nasal cavity; (thymus); (thyroid incl. parathyroid gland); tongue; (trachea); (urinary bladder); (uterus); (vagina); (gross lesions)

ABSOLUTE AND RELATIVE ORGAN WEIGHTS
- The following organ weights were recorded on the scheduled dates of necropsy: brain; heart; liver; thymus; kidneys; adrenals; spleen; testes; epididymides
- The organ to terminal body weight ratios as well as organ to brain weight ratios were determined.
- The determination of the termnal body weight was performed immediately prior to necropsy.

HISTOPATHOLOGY
Slides of all organs and tissues listed in brackets under NECROPSY which were collected at scheduled sacrifices from the animals of control and high-dose groups were examined by a pathologist. All gross lesions from all animals were processed as described under HISTOTECHNIQUE and examined by light microscopy.

HISTOTECHNIQUE
All organ and tissue samples defined under HISTOPATHOLOGY were processed, embedded and cut at an approx. thickness of 2 - 4 µm, and stained with hematoxylin and eosin.
Statistics:
Dunnett- and Steel-test
Fisher's exact test

The following statistical methods were used to analyze grip strength, locomotor activity, body weights, organ weights and all ratios, as well as clonical laboratory data:
- Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Fisher's exact- test was applied to macroscopical findings.

REFERENCES
- C. W. Dunnett: A Multiple Comparison Procedure for Comparing Several Treatments with a Control, J. Amer. Stat. Assoc. 50, 1096-1121 (1955).
- R. G. Miller: Simultaneous Statistical Inference, Springer Verlag, New York (1981)
- R. A. Fisher: Statistical Methods for Research Workers, Oliver and Boyd, Edinburgh (1950)

Results and discussion

Results of examinations

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):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL OBSERVATIONS:
-----------------------

MORTALITIY:
All animals survived the scheduled study period.

GENERAL CAGESIDE OBSERVATIONS (DAILY):
No clinical signs were evident during daily observations throughout the entire study period.

DETAILED CLINICAL OBSERVATIONS (WEEKLY):
Slight to moderately increased exploratory behavior was observed in one male treated with 1000 mg/kg/day and in one female treated with 50 mg/kg/day. This finding was considered to be incidental.

FUNCTIONAL OBSERVATIONAL BATTERY:
No qualitative or quantative differences to the control animals were evident in any test article-treated animal.

- GRIP STRENGTH: A statistically significant increase in the grip strength of forelimbs was recorded in females treated with 1000 mg/kg/day (p < 0.01), and of both fore- and hindlimbs in females treated with 200 mg/kg/day (p < 0.01) when compared with the control females. Because similar differences to the control values were not evident in any test article-treated male, this finding was considered to be incidental.
- LOCOMOTER ACTIVITY: Although a dose-related inhibition of locomoter activity was noted in males treated at 200 or 1000 mg/kg/day, the differences did not attain statistical significance when compared with the control animals. This finding was not noted in the test article-treated females and it was therefore considered to be fortuitious.

FOOD CONSUMPTION:
The mean daily food consumption of the test article-treated animals compared favorably with those of the control animals. Evaluation of the relative food consumption did not indicate test article-related differences between groups.

BODY WEIGHTS:
The mean body weight development of the test article-treated animals was considered to be unaffected when compared with those of the control animals. Although a slight reduction of body weight was noted in the males treated with 1000 mg/kg/day, similar findings were not noted in the females and therefore considered to be incidental.

LABORATORY INVESTIGATIONS:
----------------------------

HEMATOLOGY AND CLINICAL BIOCHEMISTRY:
No test article related differences to the hematology and clinical biochemistry parameters of the control values were ascertained at any dose level. A few differences to the control values were considerecd to be unrelated to the test article. All hematology and clinical biochemistry parameters are summarised in the attached document "Hematology_Clinical_Chemistry_Summary.pdf".

PATHOLOGY:
------------

ORGAN WEIGHTS:
No test article related difference in the relative organ weights were noted when compared with the control values. The heart to body weight ratio of the males treated with 1000 mg/kg/day was reduced with statistical significance (p < 0.05) when compared with the control males. This finding was not noted in the females of this group and was therefore considered to be incidental.


MACROSCOPICAL FINDINGS:
A number of macroscopic findings were recorded. These findings did not distinguish test article related treated rats from those of the control group. Findings included renal pelvis dilation, uterine horn dilation or discoloration, thickening of the thyroid gland or focal discoloration of thymus, and were noted in animals of all groups. A summary of the macroscopical findings is given in the attached document "Macroscopical_Findings_Summary.pdf".

MICROSCOPICAL FINDINGS:
A number of findings were noted in organs and tissues examined. Their incidence, severity or morphologic appearance did not distinguish test article treated rats from those of the controls. A summary of the microscopical findings, their incidence, severity or morphology is given in the attached document "Microscopical_Findings_Summary.pdf".

Effect levels

Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on these results and according to the EC criteria for classification and labelling according to DSD (Directive 67/548/EEC) or GHS/CLP (Regulation (EC) No. 1272/2008), the test item does not have to be classified and has no obligatory labelling requirement for repeated dose toxicity.

The no-observed-effect-level (NOEL) of the test item was determined to be 1000 mg/kg bodyweight/day.
Executive summary:

GENERAL

In this subacute toxicity study, the test article was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 50, 200 and 1000 mg/kg body weight/day for a period of 28 days. The groups comprised 5 animals per sex which were sacrificed after 28 days of treatment. A control group comprised of 5 animals per sex was treated similarly with the vehicle. PEG 300, at a dose volume of 5 ml/kg body weight.

Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during the pretest and treatment periods. Functional observational battery, locomoter activity and grip strength were performed during week 4.

At the end of the dosing period, blood samples were withdrawn for hematology and plasma chemisry analyses. All animals were killed, necropsied and examined post mortem. Histological examinations were performed on organs and tissues from all control and high dose animals, and all gross lesions.

MORTALITY / VIABILITY

All animals survived until scheduled necropsy.

CLINICAL SIGNS

No test article-related clinical signs were evident during daily or weekly observations (weeks 1 -3).

FUNCTIONAL OBSERVATIONAL BATTERY

No differences between test article-treated and control animals were ascertained (week 4).

GRIP STRENGTH AND LOCOMOTER ACTIVITY

No test article-related differences between treated and control animals were noted during week 4 of treatment.

FOOD CONSUMPTION AND BODY WEIGHT

The mean daily food consumption and the body weight development of test article-treated animals were similar to those of the control animals.

CLINICAL LABORATORY INVESTIGATIONS (HEMATOLOGY AND BIOCHEMISTRY)

No test article-related effects upon the hematology or clinical biochemistry parameters were noted when compared with the control values.

ORGAN WEIGHTS

No test article-related changes in organ weights or ratios were noted.

MACROSCOPIC / MICROSCOPIC FINDINGS

There were no macroscopic or microscopic findings wihch were considered to distinguish rats treated with 1000 mg/kg/day from those of the control group.

All macroscopic and microscopic findings noted in this study were considered to be incidental findings commonly occuring in rats of this strain and age.