Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: other route
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
29. May to 26. June 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test procedure in accordance with generally accepted standards and described in sufficient detail

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guideline
Qualifier:
no guideline available
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 4-Androstene- 3,17 -dione
- Lot/batch No.: JC9410
- Lot/batch No. of formulation: N/7277C-1 to 13, N/7277B-1 to 13, N/7277A -1 to 13
- Stability under test conditions: The substance in the formulation was analytically verified to be stable for at least 4 days.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: HAN: WIST (SPF)
- Source: Schering AG
- Age at study initiation: not reported
- Weight at study initiation: males 168-189 g, females 170-199 g
- Housing: singly in conventional housing conditions (Makrolon type III cages)
- Diet and water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-24
- Humidity (%): 50-58
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
subcutaneous
Vehicle:
other: physiological saline containing 0.085 % Myrj 53
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
28/29 days
Frequency of treatment:
once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
5, 15, and 50 mg/kg bw
No. of animals per sex per dose:
6
Control animals:
yes, concurrent vehicle
Details on study design:
Administration volume: 10 mL/kg

The dosage was established taking the results of previously performed acute toxicity studies into account. In this studies the LD50 was between 500 and 1000 mg/kg and clinical signs of intoxication were still seen at the dose of 250 mg/kg. In the German regulations concerning the classification and labelling of dangerous substances ("Chemikaliengesetz, Gefahrstoffverordnung") dosages of 150 mg/kg and 15 mg/kg are relevant for classification of substances after repeated administration over aperiod of 28 days. In a previous study intragastric application of dosages ranging from 15 to 150 mg/kg revealed no androgenic effects even after the high dose. This might be due to a first pass effect. Since no first pass effect and 100 % bioavailability are given after subcutaneous administration, the present study was performed in order to obtain data more appropriate for assessment of possible risks of the substance at the work place.
The high dose of 50 mg/kg applied subcutaneously corresponds to approximately 500 mg/kg given orally. As the dose of 500 mg/kg provoked distinct clinical signs of toxicity after single administration, no higher dose than 50 mg/kg s.c. was used.

The content of the test substance in the formulation was analytically verified for all doses at least at beginning and termination of the study.
Method of analysis: HPLC with UV absorption at 237 nm.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes, all animals
All signs of weak health, reactions to treatment and behavioural changes were recorded.
- Time schedule: Animals were checked regularly twice daily, also on weekends.

BODY WEIGHT: Yes, all animals
- Time schedule for examinations: once weekly

FOOD CONSUMPTION:
- Time schedule for examinations: once weekly
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION: Yes, all animals
- Time schedule for examinations: once weekly

OPHTHALMOSCOPIC EXAMINATION: Yes, all animals
- Time schedule for examinations: Ophthalmoscopic examinations were carried out in week 4.

HAEMATOLOGY: Yes, in 5-6 males and 5-6 females per group
- Time schedule for collection of blood: Day 28, blood for coagulation studies sampled on Day 26
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes, no food was offered to the animals for at least 16 hours prior to blood sampling.
- Parameters checked: Hematological investigations including determination of the following parameters were performed on Day 28: erythrocyte and leucocyte count, hemoglobin, hematocrit (packed cell volume), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), reticulocyte count, platelets, and differential count [neutrophils (myelocytes, immature, band types l and II, segmented), Iymphocytes, eosinophils, basophils, monocytes].
Coagulation studies: The following parameters were determined in citrated plasma from 4-6 male and 5-6 female animals per group on Day 26: thrombin time, thromboplastin time, activated partial thromboplastin time and fibrinogen.

CLINICAL CHEMISTRY: Yes, in 5-6 males and 6 females per group
- Time schedule for collection of blood: Day 2, Day 28
- Animals fasted: Yes, no food was offered to the animals for at least 16 hours prior to blood sampling except for sampling on Day 2.
- Parameters checked: glutamic pyruvic transaminase (GPT) and alkaline phosphatase (AP) on Day 2 (approximately 24 hours after the first treatment) and Day 28, total cholesterol, glucose, urea nitrogen, total protein, protein electrophoresis, sodium, potassium, calcium and chloride on Day 28 only.

URINALYSIS: Yes, all animals
- Time schedule for collection of urine: Day 23
- Animals fasted: Yes, no food was offered to the animals for at least 16 hours prior to urine sampling
- Parameters checked: pH, specific gravity and volume as weil as protein, glucose, blood, ketones, urobilinogen, bilirubin and sediment of urine.

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, all animals
At autopsy in all terminally sacrificed animals the following organs were dissected free of surrounding fat and connective tissue, removed and weighed, the paired organs being weighed together: liver, kidneys, heart, lung, pituitary gland, thyroid with parathyroid glands, adrenal glands, ovaries, uterus including both horns and cervix, testes, prostate, seminal vesicle, thymus, spleen, iliac Iymph nodes, brain (cerebrum, cerebellum, medulla oblongata), submandibular salivary glands and pancreas.
At terminal sacrifice (Day29/30) bone marrow was sampled from the femur of all animals. The number of nucleated cells/mg bone marrow was determined and bone marrow smears were prepared. If the determination of nucleated bone marrow cells/mg bone marrow and the histological examination of the bone indicated any signs of effects, the bone marrow smears were evaluated.

HISTOPATHOLOGY: Yes
The following organs or representative specimens thereof were collected from each animal and fixed in Lillie's buffered formalin (F) or Bouin's fixative (B):
liver (F), kidneys (F), urinary bladder (F), heart including atrium (F), aorta thoracalis (F), vena cava caudalis (F), trachea (F), lung (F), pituitary gland (B), thyroid with parathyroid glands (F), adrenal glands (F), ovaries (F), oviducts (F), uterus including both horns and cervix (F), vagina (F), mammary glands (F), skin (F), testes (B), epididymides (F), prostate (F), seminal vesicle (F), spleen (F), thymus (F), iliac Iymph nodes (F), mesenteric Iymph nodes (F), bone including femur and sternum (F), brain including cerebrum, cerebellum and medulla oblongata (F), spinal cord [cervical] (F), nervus saphenus (F), eyes (F), lacrimal glands (F), tongue (F), submandibular salivary glands (F), esophagus (F), stomach (F), duodenum (F), jejunum (F), ileum (F), cecum (F), colon (F), rectum (F), pancreas (B), skeletal muscle [M. gastrocnemius] (F) and all organs/tissues with macroscopic findings, if necessary for evaluating a diagnosis.
After fixation, specimens of these organs/tissues were embedded in Paraplast-Crosssections of 4 - 6 microns were routinely processed and stained with hematoxylin-eosin for histological examination.
Microscopic examination was performed for all animals on samples of liver, pituitary gland, thyroid with parathyroid glands, ovaries, vagina, mammary glands, testes, epididymides, seminale vesicle, thymus, submandibular salivary glands, and pancreas. Moreover, all organs/tissues from the low- and intermediate-dose group which showed treatment-related necropsy findings and/or relevant alterations in organ weights (absolute and/or relative) were histopathologically assessed.
Statistics:
For all parametric measurements except urinanalysis, the Dunnett-test was used to assess statistically significant differences between the group mean values of the control and the dose groups. All values which differ significantly from those of the control group were marked by *(p < 0.05) or **(p < 0.01). Qualitative findings as well as non-parametric results were evaluated by visual comparison (clinical observations), necropsy and histopathological findings, as well as certain parameters of urinalysis.

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS AND MORTALITY
No compound-related death was observed. Compound-related findings were observed after administration of 15 mg/kg (enlargement of the mamillas and slight swelling of mammary complexes; local intolerance reactions such as slight swellings, indurations and white deposits at the application sites) and after administration of 50 mg/kg (additionally moderate swellings at the application sites).

BODY WEIGHT AND WEIGHT GAIN
Regarded over the whole study period a non-significant, but dose-dependent decrease in body weight gain was observed after administration of 5, 15, and 50 mg/kg in male animals. This decrease in body weight gain was statistically significant (p < 0.05) in the high-dose group in week 3. In addition, the male animals of all dose groups (5, 15 and 50 mg/kg) showed a slight decrease in body weight in week 4. Body weights determined on the day of sacrifice in fasted animals showed a more distinct non-significant to significant (p < 0.01) decrease in body weight.
In two female animals dosed with 50 mg/kg a decreased body weight gain was detected from week 3 onwards. However, since no clear dose-reponse relationship was observed this effect is only suspected to be compound-related.

FOOD CONSUMPTION
A decrease in food consumption (n.s. to p < 0.05) was observed in male animals of the high-dose group (50 mg/kg) from week 3 onwards.

WATER CONSUMPTION
An increase in water consumption (n.s. to p < 0.05) was observed in female animals from the mid dose (15 mg/kg) onwards.

OPHTHALMOSCOPIC EXAMINATION
No compound-related findings were observed.

HAEMATOLOGY
Suspected compound-related effects were a tendency to a slight increase in leucocyte count in male animals from the low-dose group onwards (n.s.), as well as an increase in leucocyte count in female animals in the high-dose group (p < 0.05) associated with slightly increased values in the low- (n.s.) and mid-dose group (n.s.). These increases of leucocytes are reflected in an augmentation of relative and absolute neutrophil count in male and female animals from the low-dose group onwards (n.s. to p < 0.01 ).
For coagulation studies except for a slight increase in plasma fibrinogen in male (p < 0.01) and female animals (n.s.) in the high-dose group, no other compound-related effect was observed.

CLINICAL CHEMISTRY
Interpretation of the serum protein electrophoresis based on the relative serum protein distribution revealed the following effects that are considered compound-related: Slight non-significant decrease in serum total cholesterol in female animals from the mid dose onwards.
A slight increase in total alpha-globulin (p < 0.01) due to an increase of alpha 1- (p < 0.05) and alpha 2-globulin (p < 0.01) and a slight increase in total ß-globulin (p < 0.01) in male animals of the high-dose group. An increase of alpha 2-globulin (p < 0.01) and ß-globulin (p < 0.05) was also observed in males of the mid-dose group. Together with a slight decrease in serum albumin in males of the high-dose group (p < 0.01) the changes in alpha- and ß-globulin content resulted in a slightly decreased albumin/globulin-quotient in males of the high-dose group (p < 0.01 ).

URINALYSIS
Except for very small changes such as the slight to moderate appearance of protein in urine in female animals from the mid dose onwards and in male animals treated with the highest dose and a slight decrease in urinary volume in male animals of the high-dose group, no other compound-related effect was observed.

ORGAN WEIGHTS
The increase in liver weight in female animals of the high-dose group was not correlated with morphological alterations. It is rather to be interpreted as an adaptation to the metabolie burden with the substance.

GROSS PATHOLOGY
- reproductive organs, males: At >/= 15 mg/kg reduction of the testicular weights (absolute) and increase of the absolute and relative weight of seminal vesicle tissue.
- reproductive organs, females: Enlargement of the mammary glands and nipples observed from 15 mg/kg onwards and, also at >/= 15 mg/kg increase of the absolute and relative weight of the uterus.
- extragenital organs: Reduction of the absolute and relative thymus organ weight detected in female animals from 5 mg/kg onwards and in male animals from 15 mg/kg onwards.
The reduction of the absolute weights of the pituitary glands in male animals at 15 and 50 mg/kg was not correlated with altered morphology at histopathological examination. Nevertheless, the finding was considered treatment-related (steroid hormone effect).
Increased absolute and relative weights of the kidneys in animals of either sex were not accompanied by a morphological alteration at histopathology. However, due to the dose dependence the weight changes were considered to be treatment-related from a dose of 15 mg/kg onwards.

HISTOPATHOLOGY
- systemic effects on reproductive organs, males: Atrophy of the interstitial cells of Leydig in the testes (>/= 5 mg/kg); increased secretory activity of the seminal vesicles and prostate from 5 mg/kg onwards accompanied with a simple diffuse hyperplasia of the epithelia (trom 5 mg/kg onwards in the prostate and from 15 mg/kg onwards in the seminal vesicles).
The occurrence of unilateral spermatogenic granulomas in the epididymides of one animal of the 15 and 50 mg/kg group was suspected to be treatment-related, since this was an obvious effect to the male reproductive organs. On the other hand, spermatogenic granulomas are a common spontaneous alteration in untreated laboratory rats.
- systemic effects on reproductive organs, females: Increased rate of follicular atresia In the ovaries (>/= 5 mg/kg); cyst formation in the functional structures (follicular cysts and lutein cysts) and, in single animals, a reduction in the number of corpora lutea also from 5 mg/kg onwards. However, the presence of follicles and corpora lutea at 50 mg/kg points against a complete interruption of the cyclic activity of the ovaries. In the uterus and the vagina diffuse hyperplasia of the epithelia of the luminal mucosa apparent from 5 mg/kg onwards; the uterine epithelia underwent mucification at 15 and 50 mg/kg. In the vagina, the degree of mucification exceeded the physiological mucification normally occurring at the proestric stage of the cycle; the condition, detected from 5 mg/kg onwards, was reported as hypermucification. From 15 mg/kg onwards, epithelial hyperplasia of the luminal uterine epithelia accompanied by a stromal hyperplasia; in addition, in single animals at 15 and 50 mg/kg accumulation of fluid in the uterine cavity led to a dilatation. The presence of a small number of ectopic uterine glands in the muscular layer of the vaginal wall (adenomyosis), observed in one animal at 50 mg/kg is suspected to be treatment-related, as the condition occurred in context with other alterations to the female reproductive system. However, adenomyosis also occurs spontaneously in our colony of rats.
In the mammary gland lobular hyperplasia leading to formation of acini with secretory activity was detected in animals of either gender trom a dose of 5 mg/kg onwards. The lobular growth and hyperplasia led to a precocious maturation of the gland in female animals.
- systemic effects on extragenital organs: Atrophy of the thymus and the reduction of the absolute and relative organ weight was detected in female animals from 5 mg/kg onwards and in male animals from 15 mg/kg onwards. If a substantial reduction of the size of the organ, detectable by reduced width of the thymic cortex was detectable at histopathological examination, it was diagnosed as "atrophy", whereas the underlying loss of lymphoid cells ("lymphoid depletion") which occurred in all examined animals was reported separately. All regressive changes in the thymus were considered treatment-related.
- local effects at application site: At the application sites of the skin and in the adjacent subcutaneous tissue, various inflammatory reactions including the formation of granulation tissue and inflammatory granulomas were detected with a higher incidence and severity in treated animals than in the controls. These reactions were presumably due to the property of the formulation (microcrystalline suspension) and were therefore considered not to be related to an intrinsic effect of the substance.

The assessment of bone marrow samples (number of nucleated cells/mg bone marrow) revealed no compound-related changes.

n.s.: non-significant

Effect levels

Dose descriptor:
LOAEL
Effect level:
5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Steroid hormone-related effects predominantly on male/female fertility starting in this dose group, i.e. findings in histopathology and/or effects on organ weights of respective organs (testes, prostate, ovaries, uterus, vagina).

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

The daily subcutaneous application of the substance over approx. 4 weeks led to dose-dependent effects such as decrease of thymus and testes weight, increase of the organ weights of prostate, seminal vesicles or uterus and related histological alterations, which can be attributed to the endocrine action of the test compound, with partial androgenic, estrogenic and progestagenic action. Additionally signs of general toxicity like decreased body weight gain, decreased food and increased water consumption together with occurence of protein in the urine and increased kidney weight were observed at the higher doses. Due to the onset of some of the described hormonal effects already in the low-dose group, a NOAEL could not be established.

Applicant's summary and conclusion

Conclusions:
A NOAEL could not be established; LOAEL: 5 mg/kg
Executive summary:

The once daily subcutaneous administration of the substance to male and female rats over 4 weeks at doses of 0, 5, 15 and 50 mg/kg led to dose-dependent effects such as decrease of thymus and testes weights, increase of organ weights of prostate, seminal vesicles or uterus and related histological alterations, which can be attributed to the endocrine action of the test compound with partial androgenic, estrogenic and progestagenic action. Additional signs of general toxicity like decreased body weight gain, decreased food and increased water consumption together with occurence of protein in the urine and increased kidney weight were observed at higher doses. Due to the onset of some of the described hormonal effects already at the low dose, a NOAEL could not be established.