Estradiol

Endpoint summary

Description of key information

Study report: Test substance was administered to 3 Sprague Dawley rats each group daily via gavage for 5 days. Doses applied were 20 and 300 mg/kg bw/d.

Spraque-Dawley rats revealed transient decrease in body weight gain and water consumption at 20 and 300 mg/kg, but no liver toxicity (Research Report No. AG69).

Study report: Test substance was administered in combination with Levonorgestrel to 10 female Jcl: Sprague Dawley SDrats/dose in 0.5% CMC-Na + 0.04% Tween 80 by gavage at dose levels of 0 (control), 0.02, 0.4 and 20 mg/kg bw/day for 28 consecutive days. Additionally, Estradiol was administered to 10 female Jcl:SD rats at a dose of 18.2 mg/kg bw for 10 days. Control, Estradiol and high dose groups included 6 additional animals per sex to be sacrificed after 2 weeks of recovery. No mortality occurred. No clinical signs and no changes were observed at the weekly detailed clinical observations. Some changes on body weight and food consumption were noted. Body weight gains were significantly inhibited in the 20000 µg/kg and E2 groups during the treatment period. Significant decreases in RBC, Hb, PCV and MCHC were observed in the 20000 µg/kg and E2 groups. Reversible effects in coagulation were seen in the highest dose group. On the basis of the results obtained in this study, the dose level of 0.4 mg/kg bw/day was considered the LOEL (Lowest Observed Effect Level).

 

Other studies revealed no effects during a subacute treatment period.

Application of Estradiol (1.28 mg/kg or 2.4 mg/kg every third day or 1.28 mg/kg once a week) to beagle dogs showed clinical, haematological, biochemical and histopathological effects but not indication of neoplastic or anaplastic processes (Research Report No. 3102).

Several clinical studies are available, indicating different effects after long-term exposure to Estradiol.

Only few real repeated dose studies were found for Estradiol in the literature, most reports focussed on carcinogenic effects. Nevertheless, effects on several organs, hematopoiesis, centrilobular hepatocellular hypertrophy, diffuse hyperplasia of the pituitary gland, feminization of the male mammary gland, mammary gland hyperplasia in females, cystic ovarian follicles, hypertrophy of the endometrium and endometrial glands in the uterus, degeneration of the seminiferous epithelium, and atrophy of the testes and accessory sex glands were reported. The toxic effects of Estradiol are an exaggeration of the normal pharmacological effects and result in an increase of female characteristics. Estradiol is widely used for oral contraception and in post-menopausal hormonal therapy. Therefore a wide base of case reports is available pointing out various types of adverse effects. The most frequent adverse dermatologic reaction associated with estrogen therapy is chloasma, melasma and erythema. An increased risk of thromboembolic and thrombotic disorders including thrombophlebitis, pulmonary embolism, stroke, subarachnoid hemorrhage, and myocardial infarction was reported for the use as oral contraceptive as well as breakthrough bleeding, spotting, changes in menstrual flow, missed menses (during use), or amenorrhea (after use). Dysmenorrhea and a premenstrual-like syndrome also occurred.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: Guidelines for Toxicity Studies of Drugs (PAB/ERD-1, Notification no. 24, dated 11 Sep 1989)

Version / remarks:

1989

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Jcl:SD

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: CLEA Japan, Inc., No.2 INARI Bldg., 20-14 Aobadai-2, Meguro-ku, Tokyo, Japan
- Age at study initiation: 8-9 weeks
- Weight at study initiation: 204-234 g
- Fasting period before study: no
- Housing: Bracket cages with wire-bottom for rats (CL-2315), One animal per cage
- Diet (e.g. ad libitum): Rodent diets (CE-2, Nippon Formula Feed Mfg Co., Ltd., Japan)
were provided ad libitum
- Water (e.g. ad libitum): Tap water via an automatic watering system was provided ad
libitum
- Acclimation period: 10 days

DETAILS OF FOOD AND WATER QUALITY: contaminants were confirmed to be within the limits of the test facility

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.5 - 23.0
- Humidity (%): 51-66
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: 0.5% CMC-Na + 0.04% Tween 80

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The required amount of estradiol, multiplied by a factor of 1.03 to compensate for its 3.15% water content, was suspended in the vehicle (a solution of 0.5% CMC-Na + 0.04% Tween 80).

DIET PREPARATION
- Rate of preparation of diet (frequency): Dosing suspensions were prepared at least once a week.

VEHICLE
- Concentration in vehicle: 0.5% CMC-Na + 0.04% Tween 80
- Amount of vehicle (if gavage): 5 mL

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily

Dose / conc.:

18.2 mg/kg bw/day (nominal)

Remarks:

estradiol alone

Dose / conc.:

20 mg/kg bw/day (nominal)

Remarks:

Combination: Estradiol + Levonorgestrel

Dose / conc.:

0.4 mg/kg bw/day (nominal)

Remarks:

Combination: Estradiol + Levonorgestrel

Dose / conc.:

0.02 mg/kg bw/day (nominal)

Remarks:

Combination: Estradiol + Levonorgestrel

No. of animals per sex per dose:

10for the treatment and in dose group 0.4, 20, and 18.2 mg/kg 6 additional animals as recovery group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were determined based on the results of the 4-week repeated-dose oral toxicity study of a combination of gestodene and estradiol (combination ratio 1:20) in female rats (report no. AX25). In that study, no abnormalities were observed at the doses of 1 and 20 µg/kg, while some abnormalities in body weight changes, food consumption, clinical examinations and pathological examinations were observed at the dose of 20000 µg/kg. On the basis of those results, the highest dose was set at 20000 µg/kg, as it was expected to have some toxic effects, the medium dose at 400µg/kg and the lowest dose at 20 µg/kg.
- Post-exposure recovery period in satellite groups: 14 days

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): The food consumption of each animal was measured weekly using an electronic balance.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

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

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmoscopic examinations were performed in 6 animals from the control, 400 µg/kg, 20000µg/kg and E2 groups on day 25 in the treatment period. In the 20 µg/kg group, the examinations were not performed because no compound-related effects were observed at 400 µg/kg or more.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Hematological examinations were performed in 10 animals per group on day 25 of the treatment period and 6 animals per group on day 39 after the commencement of treatment ( withdrawal period).
- Anaesthetic used for blood collection: Yes : anesthesia by ethyl ether
- Animals fasted: Not specified

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood chemical investigations were performed in 10 animals per group after the treatment period and 6 animals per group after the withdrawal period.
- Animals fasted: Yes, 18.5 h

PLASMA/SERUM HORMONES/LIPIDS: Yes , blood coagulation
- Time of blood sample collection: Blood coagulation examinations were performed in 10 animals per group after the treatment period and 6 animals per group after the withdrawal period. The following parameters were determined in citrated plasma samples collected from the jugular vein of the animals which were fasted for about 18.5 hours prior to blood sampling and anesthetized by ethyl ether:
prothrombin time, activated partial thromboplastin time and thrombin time, fibrinogen concentration
- Animals fasted: Yes , 18.5 h

URINALYSIS: Yes
- Time schedule for collection of urine: Urinalysis was performed in 8-10 animals from each group on day 23 in the treatment period.
- Metabolism cages used for collection of urine: No
- Animals fasted: Not specified

OTHER:
Bone marrow examinations were performed in 8-10 animals per group after the treatment period and 6 animals per group after the withdrawal period.
The following parameters were determined in bone marrow cells collected from femur at necropsy: nucleated cell count/mg bone marrow, myelogram (myeloblasts and promyelocytes, myelocytes and metamyelocytes, neutrophils, eosinocytes, lymphocytes, erythroblasts, orthochromatic erythroblasts and other cells) - examined bone marrow smears with May-Giemsa stain under a microscope - myeloid / erythroid cell (M/E) ratio - calculated

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes

Statistics:

The student’s t-test was used for parametric values to assess the statistical significance of
differences between the control group and the treatment groups. When the p-value was less
than 0.05, the parameters were reassessed by a Dunnett-test (each one performed at a 5% or
1% -level), using the SAS GLM procedure.

Clinical signs:

no effects observed

Description (incidence and severity):

No animals died in any group. No compound-related findings were observed.
Alopecia and scab (crust) was noted in one animal in the 400 µg/kg group on days 9 - 13.
These findings were not considered to be compound-related, since similar findings were also
observed in the control group on days 15 - 28 and no dose-dependency was noted. In the E2
group, hair loss or alopecia was noted in 3 animals during the treatment period. Subsequently, hair loss was noted in 2 animals during the withdrawal period. The changes were not considered to be compound-related, since they occurred only sporadically and these findings were known as sporadic lesions.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gains were significantly inhibited in the 20000 µg/kg and E2 groups during the
treatment period. In the withdrawal period, body weight changes in the 20000 µg/kg were lower than the corresponding control values, however, significant inhibition of body weight gains disappeared in week 6. In the E2 group, body weight gains were significantly inhibited during the withdrawal period.
In other treated groups, body weight gains were similar to those in the control group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A significant decrease in food consumption was recorded in the 20000 µg/kg group in weeks 1
and 4, and in the E2 group in week 1.
In the withdrawal period, no significant difference from the control group was observed in either
the 20000 µg/kg or E2 groups.
In other treated groups, food consumption was similar to that in the control group.

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Significant decreases in RBC, Hb, PCV and MCHC were observed in the 20000 µg/kg and E2
groups. In the withdrawal period, significant decreases in RBC and Hb were still observed in the 20000 µg/kg group, but there were trends toward improvement by comparison with the values in the treatment period. In addition, a significant increase in reticulocyte ratio was noted in the 20000 µg/kg and E2 groups.
A significant increase in segmented cell ratio was observed in the 20000 µg/kg and E2 groups,
and a significant increase in neutrophil ratio was noted in the E2 group, but WBC showed no
changes in either group. In the E2 group, a decrease in WBC was noted in the withdrawal period, but the change was within the limits of normal variation. Therefore, the changes in
leukocytic parameters were not considered to be biologically significant.
In other treated groups, no significant changes were observed.

Description (incidence and severity):

In the 20000 µg/kg group, significant increases in GPT (ALT), ALP, glucose, total protein and
1 -globulin, and significant decreases in total cholesterol, phospholipid, chloride and A/G ratio
were observed. Similar changes were noted in the E2 group. In the 400 µg/kg group, decreases in total cholesterol and phospholipid, and a tendency for ALP to increase were noted.
After the withdrawal period, an increase in glucose in the 20000 µg/kg group, and increases in
total protein and the percentage of α1 -globulin in the 20000 µg/kg and E2 groups were still noted, but there were trends toward improvement by comparison with the values after the treatment period. Significant increases in total cholesterol and phospholipid, which were contrary to the changes at the end of the treatment, were also noted in both groups. In the E2 group, a significant increase in glucose was noted.
Significant decreases in the percentage of albumin and β-globulin were observed in the 20000
µg/kg and E2 groups. However, these changes were only apparent changes based on an
increase in total protein correlating to an increase in the percentage of α1 -globulin, therefore, the changes were not considered to be biologically significant. A significant increase in NEFA in the 20 µg/kg group was not considered to be compound-related, since no dose-dependency was observed.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Increases in the weights of the uterus, liver, adrenal and pituitary, and decreases in the weights of the ovary and thymus were observed in the 20000 µg/kg group. Similar changes were noted in the E2 group.
After the withdrawal period, increased liver weight was still observed in the 20000 µg/kg and E2
groups, and decreased thymus weight was observed in the E2 group. However, there were trends toward improvement by comparison with the values after the treatment period. In the E2 group, decreased ovary weight was noted. Although significant increased pituitary weights in
the 20000 µg/kg and E2 groups and adrenal weight in the E2 group were noted, these changes
were considered to be less biologically significant because there were no relevant microscopic
findings.
Statistically significant changes were noted in other organs (thyroid, lung, heart, kidney and brain) in the 20000 µg/kg and E2 groups. These changes were not considered to be compound-related, because they were only apparent changes related to the difference from control values in body weight and no compound-related microscopic findings were observed in these organs.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Compound-related changes were noted in the vagina, uterus, ovary, liver, adrenal, pituitary,
spleen, thymus, femur and mammary gland.
Vagina:
An increased incidence of pseudopregnant changes was found in the 400 µg/kg, 20000 µg/kg and E2 groups. After the withdrawal period, this finding was detected with comparable
incidence in the control and treatment groups.
Uterus:
Increased incidence of squamous metaplasia, thickening of the muscular layer and edema in
endometrium, and increased severity of hydrometra were found in the 20000 µg/kg and E2
groups. Polyp in the endometrium was also observed in the 20000 µg/kg group. After the
withdrawal period, these changes were not found, or the incidence was lower than that after the treatment period. In addition, slight hydrometra was found in the 20 µg/kg, 20000 µg/kg and E2 groups after the treatment period, and in the 400 µg/kg and 20000 µg/kg groups after the withdrawal period. The change was not considered to be compound-related, since it was noted in each group infrequently and interpreted as a spontaneous finding. Marked hydrometra in the 400 µg/kg group after the withdrawal period was not considered to be compound-related, since no dose-dependency was observed and it was not observed after the treatment period.
Ovary:
Decreases in corpora lutea and growing follicles, an increase in atretic follicles, and hypertrophy of corpus luteum were found in the 20000 µg/kg and E2 groups.
After the withdrawal period, the incidence of decreased corpora lutea was lower than that after the treatment period. Other findings were detected with comparable incidence in the control and treatment groups.
Liver:
Centrilobular hypertrophy and scattered fatty degeneration were observed in the 20000 µg/kg and E2 groups. After the withdrawal period, the incidence of centrilobular hypertrophy was lower than that after the treatment period. The scattered fatty degeneration was still observed in the 20000 µg/kg and E2 groups. However, this change in the E2 group showed a trend toward regression. The incidence of decreased peripheral fatty degeneration in the 400 µg/kg, 20000 µg/kg and E2 groups were lower than that of the control group after the treatment and withdrawal periods. This change was considered to be related to treatment but not toxicologically significant because of the reduced incidence of animals with degenerative changes.
Adrenal:
Hypertrophy of zona fasciculata and zona reticularis, and lipid depletion in zona fasciculata were noted in the 20000 µg/kg and E2 groups. After the withdrawal period, these changes
disappeared.
Pituitary:
Hyperplasia of vacuolar cells (mammotrophs) containing eosinophilic substances in the anterior lobe was noted in the 20000 µg/kg and E2 groups. In the observation of cell types in the anterior lobe, the frequency of somatotrophs and gonadotrophs were lower, and the frequency of mammotrophs was higher in the 20000 µg/kg and E2 groups than in the control group. After the withdrawal period, these changes were not observed.
Spleen:
There was a higher incidence of brown pigmentation and increased hematopoiesis in the 20000 µg/kg and E2 groups. After the withdrawal period, these changes were still noted in both groups, but the increased hematopoiesis in the E2 group showed a trend toward regression.
Thymus:
Atrophy associated with a decrease in lymphocytes was observed in the 20000 µg/kg and E2
groups. After the withdrawal period, the change showed a trend toward regression in both
groups.
Femur:
Hyperostosis of spongy bone was observed in the 20000 µg/kg and E2 groups. After the
withdrawal period, the change showed a trend toward regression in both groups.
Mammary gland:
Hyperplasia and lactation of the mammary gland and brown pigmentation in alveolar cells were observed in the 20000 µg/kg and E2 groups. After the withdrawal period, hyperplasia was still observed in all animals in both groups, however, a decrease in the severity of the changes was observed. Decreased incidence and severity of lactation were also observed in both groups. However, increased incidence of brown pigmentation in alveolar cells was noted in all animals in both groups.

All other microscopic findings in the vagina, uterus, ovary, liver, adrenal, pituitary, kidney, urinary bladder, pancreas, heart, lung, stomach and skin were not considered to be compound-related, because the incidence or severity of these changes were comparable to those in the control group, and they were interpreted as spontaneous lesions.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Key result

Dose descriptor:

LOEL

Effect level:

0.4 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical biochemistry

gross pathology

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

0.4 mg/kg bw/day (nominal)

System:

female reproductive system

Organ:

vagina

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

presumably yes

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

0.4 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Relevant for humans:

presumably yes

Conclusions:

On the basis of the results obtained in this study, the dose level of 0.4 mg/kg bw/day was considered the LOEL.

Executive summary:

In a subacute toxicity study according to Guidelines for Toxicity Studies of Drugs (PAB/ERD-1, Notification no. 24, dated 11 Sep 1989) Estradiol was administered in combination with Levonorgestrel to 10 female Jcl: Sprague Dawley SDrats/dose in 0.5% CMC-Na + 0.04% Tween 80 by gavage at dose levels of 0 (control), 0.02, 0.4 and 20 mg/kg bw/day for 28 consecutive days. Additionally, Estradiol was administered to 10 female Jcl:SD rats at a dose of 18.2 mg/kg bw for 10 days. Control, Estradiol and high dose groups included 6 additional animals per sex to be sacrificed after 2 weeks of recovery.

No mortality occurred. No clinical signs and no changes were observed at the weekly detailed clinical observations. Some changes on body weight and food consumption were noted. Body weight gains were significantly inhibited in the 20000 µg/kg and E2 groups during the treatment period. In the withdrawal period, body weight changes in the 20000 µg/kg were lower than the corresponding control values, however, significant inhibition of body weight gains disappeared in week 6. In the E2 group, body weight gains were significantly inhibited during the withdrawal period.

In other treated groups, body weight gains were similar to those in the control group.

The decrease in body weight gain is considered to be related to the reduced food consumption. A significant decrease in food consumption was recorded in the 20000 µg/kg group in weeks 1

and 4, and in the E2 group in week 1. In the withdrawal period, no significant difference from the control group was observed in either the 20000 µg/kg or E2 groups.

In other treated groups, food consumption was similar to that in the control group.

Significant decreases in RBC, Hb, PCV and MCHC were observed in the 20000 µg/kg and E2

groups. In the withdrawal period, significant decreases in RBC and Hb were still observed in the 20000 µg/kg group, but there were trends toward improvement by comparison with the values in the treatment period.  In addition, a significant increase in reticulocyte ratio was noted in the 20000 µg/kg and E2 groups. A significant increase in segmented cell ratio was observed in the 20000 µg/kg and E2 groups, and a significant increase in neutrophil ratio was noted in the E2 group, but WBC showed no changes in either group.  In the E2 group, a decrease in WBC was noted in the withdrawal period, but the change was within the limits of normal variation.  Therefore, the changes in leukocytic parameters were not considered to be biologically significant.

In other treated groups, no significant changes were observed.

Reversible effects in coagulation were seen in the highest dose group. No differences were reported in relative organ weights between treated and control animals; total organ weights changed but were considered to be not treatment related due to the absence of compound-related microscopic findings and no treatment-related changes were noted at microscopic observations. At necropsy changes were noted in the vagina, uterus, ovary, liver, adrenal, pituitary, spleen, thymus, femur and mammary gland. However, most of these changes were reversible and/or not dose-dependent and therefore not considered to be treatment related

 

On the basis of the results obtained in this study, the dose level of 0.4 mg/kg bw/day was considered the LOEL (Lowest Observed Effect Level).

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

other information

Study period:

Jan 1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

no guideline available

Principles of method if other than guideline:

Test substance was administered to rats daily via gavage for 5 days.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Sprague-Dawley rats were selected as test species because this strain was used in the original
rat liver foci bioassay. Since the present study is performed in order to choose the appropriate
dosage of the test compounds for upcoming bioassay the identical rat strain and route of
administration (intragastric) were selected.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Schering AG
- Weight at study initiation: 32 - 72 g male and 30 - 66 g female animals
- Housing: individually in Makrolon@ type II with straw under conventional conditions
- Diet (e.g. ad libitum): pulverized Altromin® R ad libitum 24 hours per day
- Water (e.g. ad libitum): tap water in water bottles ad Iibitum 24 hours per day
- Acclimation period: none, since the animals were raised at the Institute for Experimental Toxicology

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23
- Humidity (%): 42-54
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To: 22 Dec. 1994 to 24 Jan. 1995

Route of administration:

oral: gavage

Details on route of administration:

The animals were treated once daily in the morning intragastrically by gavage over aperiod of
5 days according to the treatment schedule. Doses were adjusted daily to changes in body weight.

Vehicle:

other: 0.225 g NaCI, 0.0213 g Myrj 53® ad 25 ml bidest. water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The formulations were prepared on the day of application or one or two days before, pH ranged trom 5.4 - 6.08, measured on day 1 - 3. They were kept in multivials at room temperature and pratected fram light.

VEHICLE
- Concentration in vehicle: 0.225 g NaCI, 0.0213 g Myrj 53® ad 25 ml bidest. water
- Amount of vehicle (if gavage): 10 mL/kg

Analytical verification of doses or concentrations:

yes

Remarks:

The accuracy of preparing formulations was checked by the analysis of the content of the stock solutions as weil as the diluted samples.

Details on analytical verification of doses or concentrations:

not reported

Duration of treatment / exposure:

5 days treatement afterwards 7 days recovery before necropsy

Frequency of treatment:

daily for 5 days

Dose / conc.:

20 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

No. of animals per sex per dose:

3/sex/group

Control animals:

other: 0.9% (w/v) NaCl

Details on study design:

- Dose selection rationale: The high dosages of all tested compounds were envisaged in view of an expected maximum systemic and liver exposure without profound disturbance of homeostasis. Since available data on the pharmacokinetics were insufficient, these dosages were orientated to LD50 values if available, considering the repeated administration (5x) of every compound. Moreover, limiting resorption capacities in the g.i. tract were also taken into consideration.
- Rationale for animal assignment (if not random): by lot
- Fasting period before blood sampling for clinical biochemistry: not reported
- Post-exposure recovery period in satellite groups: 7 days

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: daily for the first 6 days and at the end of the study at day 12.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
The amount of food consumed was not recorded since the the pulverized food was spread
araund the whoie cage.

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 / No / Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: The quantity of water consumed by the individual animal was recorded over the treatment period days 1 - 5 and over the reversibility period days 5 - 12.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: Yes
- Animals fasted: Not specified
- How many animals: All
The following parameters were determined in 12 male and 12 female contraI animals and in
3 male and 1 to 3 female treated animals per group on day 6:
in serum:
glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT) and alkaline
phosphatase (ALP).

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Not for the test substance

Statistics:

The Dunnett-test was used for parametric values to assess differences between group 1
(control) and the treatment groups. Group mean values which differ significantly from the
control group are marked by *(p < 0.05) or **(p < 0.01).

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A transient decrease in body weight gain occurred during the repeated administration of estradiol (20 mg/kg onwards).

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

To summarize, a transient decrease of water consumption could be detected during the
treatment with estradiol (20 mg/kg onwards). Since the amount of water consumption is generally correlated with food consumption a decreased intake of food can be suspected. However, the food consumption could not be determined. Therefore, a definite interpretation for the reduced water intake is not possible.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

A decrease of Glutamic-oxaloacetic-transaminase and Glutamic-pyruvic-transaminase in male animals and female and male animals, respectively. However, only an increase in these parameters indicate a liver damage.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

> 300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

300 mg/kg bw/day (nominal)

System:

other: decreased body weight in female and male animals

Organ:

other: not specified

transient decrease in water consumption (male) and body weight gain (male and female)

Compound-related decreases in water consumption over the treatment period (days 1 - 5)

Group	Substance	Dose (mg/kg bw)	Sex	Statitical significance
4	Test item	20	M	p < 0.01
5		300	M	p < 0.05

M = male

Compound-related decreases in body weight gain over the first 6 days (including treatment period days 1 - 5)

Group	Substance	Dose (mg/kg bw)	Sex	Statitical significance
4	Test item	20	M	p < 0.05
			F	n.s.
5		300	M	p < 0.01
			F	p < 0.01

 

Statistically significant differences to the control group on day 6 which are not considered as compound-related hepatotoxic effects

Parameter	Increase ↑/ Decrease ↓	Group	Sex	Statistical significance	Reason
GOT	↓	4	M	p < 0.05	only an increase in this parameter indicates a liver damage
	↓	5	M, F	p < 0.01	only an increase in this parameter indicates a liver damage
GPT	↓	5	M, F	p < 0.05, p < 0.01	only an increase in this parameter indicates a liver damage

The obtained data showed that treatment with up to 249 mg/kg bw estradiol was tolerated well without detectable liver cell damage.

 

 

 

Conclusions:

no liver toxicity observed

Executive summary:

In a subacute toxicity study Estradiol was administered to 3 female and 3 male Sprague Dawley rats by gavage at dose levels of 20 and 300 mg/kg bw/day for 5 days.

A transient decrease in body weight gain occurred during the repeated administration of estradiol (20 mg/kg onwards) in male animals (20 mg/kg bw/d) and male and female animals (300 mg/kgbw/d). Furthermore, a transient decrease of water consumption could be detected during the treatment with estradiol (20 mg/kg onwards) in male animals. Since the amount of water consumption is generally correlated with food consumption a decreased intake of food can be suspected. However, the food consumption could not be determined. Therefore, a definite interpretation for the reduced water intake is not possible. A decrease of Glutamic-oxaloacetic-transaminase and Glutamic-pyruvic-transaminase in male animals and female and male animals (at 20 and 300 mg/kg bw/d), respectively. However, only an increase in these parameters indicate a liver damage. Thus, these changes are considered non-treatment related. No Mortality occurred. Also, no changes were observed during necropsy.

Based on the results obtained from this study a NOAEL of > 300 mg/kg bw/d can be derived.

Endpoint conclusion

Dose descriptor:

LOAEL

0.4 mg/kg bw/day

Study duration:

subacute

Experimental exposure time per week (hours/week):

5

Species:

rat

Quality of whole database:

comparable to guideline study Klimisch score 2

System:

other: decreased food consumption and body weight gain, decrease of clinical blood parameters

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

In the following sections information about the effects of repeated dose administration is provided for animals as well as shown in human data:

Animal Data

 

Test system	Substance	Application	Test concentration	End point/Effect	Literature
Spraque- Dawley rats, 3 male and 3 female	Estradiol	Intragastric application on 5 consecutive days.	20 and 300 mg/kg	Transient decrese in water consumption (males) and body weight gain (both sexes) was observed in both doses.   No liver toxicity	Research Report No. AG69, Schering AG, Ethinylestradiol, estradiol, megastrol acetate, drospirenone, chlormadinone acetate, dexamethasone, flutamide, tamoxifen citrate ¿ Systemic tolerance study with special regard to liver toxicity in juvenile Spraque-Dawley rats after daily per os (intragastric) administration over 5 days, dated 05 Nov. 1997.
Crl:CD Rat, male and Female	Ethinyl Estradiol (EE), Estradiol valerate (EV)	Oral	EE: 0.005 ¿ 0.09 mg/kg bw/d EV: 1.2 - 12 mg/kg bw/d for 80-105 weeks	Increased incidence of adenocarcionoma, nodular hyperplasia, cervix epithelial carcinoma, mammary gland cancer	Seibert, 1996. Endocrinically active Chemicals in the environment, UBA Texte, 88-93.
Crl:CD BR rat, female	Estradiol	Oral	0. 0.003, 0.17, 0.69, or 4.1 mg/kg bw/d for 90 d	The end-points were chosen to evaluate both short-term and reproductive toxicity and several mechanistic and biochemical parameters. Doses > 0.17 produced dose- dependent increases in body weight, food consumption, and feed efficiency. Evidence of ovarian malfunction (reduced corpora lutea and large antral folicles) was found.   At 0.69 and 4.1 mg/kg bw/d, minimal to mild non- regenerative anaemia, lymphopenia, decreased serum cholesterol (at the high dose only), and altered splenic lymphocyte subtypes were observed. Changes in the weights of several organs were noted. Centrilobular hepatocellular hypertrophy, diffuse hyperplasia of the pituitary gland, feminization of the male mammary gland, mammary gland hyperplasia in females, cystic ovarian follicles, hypertrophy of the endometrium and endometrial glands in the uterus, degeneration of the seminiferous epithelium, and atrophy of the testes and accessory sex glands.	Biegel et al., 1998b. Toxicol. Sci., 44, 143-154 as cited by Toxicological Evaluation of certain Veterinary Drug Residues in Food. WHO Food Additives Series: 43, prepared by the Fifty-second meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA), 2000.
Syrian hamsters, male, castrated	various steroidal and non-steroidal estrogens	Subcutan implantation	Release rate: 110 µg/d for 9 months	Good correlation among the hormonal parameters progesterone receptor induction and serum prolactin and relative estrogenic potency (estrogen receptor binding) in hamster kidney. All animals trested with estradiol developed renal tumours.	Li et al., 1995. Cancer Res., 55, 4347-4351 as cited by Toxicological Evaluation of certain Veterinary Drug Residues in Food. WHO Food Additives Series: 43, prepared by the Fifty-second meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA), 2000.
Syrian hamsters, male, castrated	Estradiol	Subcutaneous pellets	Release rate: Estradiol, 96 µg/d for 8 months	tumour incidence of 100%, completely abolished by concurrent treatment with ethinylestradiol	Li et al., 1998 Carcinogenesis, 19, 471-477 as cited by Toxicological Evaluation of certain Veterinary Drug Residues in Food. WHO Food Additives Series: 43, prepared by the Fifty-second meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA), 2000.
Syrian hamsters, male	Estradiol	subcutaneous implantation	25 mg,sacrified after 175 d	Renal tumours in 4/5 animals	Liehr et al., 1986 J. Steroid Biochem.,24, 353-356as cited by Toxicological Evaluation of certain Veterinary Drug Residues in Food. WHO Food Additives Series: 43, prepared by the Fifty-second meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA), 2000.
Syrian hamsters, male, castrated	Estradiol	subcutaneous implantation	released 100- 210 µg/d for 9 months	Renal tumour incidence: 75- 100%,	Li et al., 1983. Cancer Res., 43, 5200-5204356 as cited by Toxicological Evaluation of certain Veterinary Drug Residues in Food. WHO Food Additives Series: 43, prepared by the Fifty-second meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA), 2000.
B6C3F1mice, both sexes	Catechol estrogens	intraperitoneal injection	Treatment on days 12-15 after birth, monitored for 18 months	Estrone-3,4-quinone was significantly carcinogenic in the livers of male mice. It was also highly toxic, as most of the mice died from unknown causes shortly after treatment. Estrone was protective against liver tumour formation in this system, and few tumours were induced in female mice	Cavalieri et al., 1997. Proc. Natl Acad. Sci. USA, 94, 10937- 10942 as cited by Toxicological Evaluation of certain Veterinary Drug Residues in Food. WHO Food Additives Series: 43, prepared by the Fifty-second meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA), 2000.
C3H/HeJ mice, female	Estradiol	Oral	0.015, 0.15, or 0.75 mg/kg bw/d from week 6 to week 110.   Sacrified after 52 weeks	Preneoplastic and neoplastic findings in mice sacrificed after up to 104 weeks on the estrogenic diets.   High doses of estradiol increased the incidence of adenosis but did not affect the incidence of ovarian tubular adenomas. After 66- 91 weeks of treatment, high doses of estradiol also increased the incidence of mammary gland hyperplastic alveolar nodules	Highman et al., 1980. J. Environ. Pathol. Toxicol., 4, 81-95 as cited by Toxicological Evaluation of certain Veterinary Drug Residues in Food. WHO Food Additives Series: 43, prepared by the Fifty-second meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA), 2000.
C3H/HeJ mice, female	17ß-Estradiol	In drinking- water	0.5 mg/l for 1 y	17ß-Estradiol caused tumours	Welsch, 1976. J. Toxicol. Environ. Health, Suppl. 1, 161- 175as cited by Toxicological Evaluation of certain Veterinary Drug Residues in Food. WHO Food Additives Series: 43, prepared by the Fifty-second meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA), 2000.
Mouse	Estradiol			Adverse affect on hematopoiesis, resulting in marrow hypocelluarity and depressed numbers of colony-forming units. Decreased erythropoiesis, although a compensatory splenic increase occurs several days following cessation of estrogen administration.	Thomas et al., 1985. Toxicology. New York,: Raven Press, Ltd., p. 72 as cited by the Hazardous Substances Data Bank (HSDB), Estradiol on November 23, 2009.
C3H HeJ (MTV + ) mice	17ß-Estradiol	Diet	0, 100, 1000 and 5000 µg/kg  for 24 months starting at 6 weeks of age	Mammary adenocarcinomas after 52 weeks and other malignant tumors in high doses	Highman et al., 1977. J. Environ. Pathol. Toxicol., 1, 1-30 as cited by IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Geneva: World Health Organization, International Agency for Research on Cancer, 1972- PRESENT. (Multivolume work), V21 (1979).
Strong A Mice, male	estradiol 3- benzoate	Subcutaneous injection	16.6 or 50 µg for at least 6 months	Interstitial-cell tumors of the testis occurred	Hooker & Pfeiffer,1942. Cancer Res. 2, 759-769 as cited by IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Geneva: World Health Organization, International Agency for Research on Cancer, 1972- PRESENT. (Multivolume work), V21 (1979).
Marsh Buffalo mice, female	17ß-Estradiol	Subcutaneous or Intramuscul ar injection	80 pg for twice weekly for 6 months	Lymphosarcomas occurred earlier (between 3 and 10 months) and in a higher incidence (28% in intact, 47% in ovariectomized) than in controls.	Bischoff et al, 1942. Cancer Res. 2, 52- 55769 as cited by IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Man.: World Health Organization, International Agency for Research on Cancer, 1972- PRESENT. (Multivolume work), V21 (1979).
C3H x RIII F1 mice (MTV+), male	17ß-Estradiol	subcutaneous implantation	0.5-1 mg	Mammary cancer.	Rudali er al., 1971. Rev. Eur. Etud. Clin. Biol., 16, 425-429 as cited by IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Geneva: World Health Organization, International Agency for Research on Cancer, 1972- PRESENT. (Multivolume work), V21 (1979).
(C3H x RIII) Fl mice, male	17ß-Estradiol	subcutaneous implantation	0, 1, 2.5, 5, 10 or 100 ug	Mammary tumors	Rudali er al., 1971. Biomedicine, 29, 45- 46 as cited by IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Geneva: World Health Organization, International Agency for Research on Cancer, 1972- PRESENT. (Multivolume work), V21 (1979).
C3H/MS mice, neonatally	17ß-Estradiol			Increased mammary tumorigenesis, hyperplastic nodules or metaplastic lesions have also been found in various accessory sex organs, including prostatic lobes.	Mori, 1967. Annot. Zool. Jpn., 41, 43-52 as cited by IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Geneva: World Health Organization, International Agency for Research on Cancer, 1972- PRESENT. (Multivolume work), V21 (1979).
guinea-pigs, female, ovaritectomized	Etradiol 3- benzoate	Subcutaneous injection	20-80 µg	Multiple tumors, described as fibromas or fibromyomas, arose in the uterus and mesentery at several locations.	Lipschütz & Iglesias, 1938. C.R. Soc.Biol. 129, 519-524 as cited by IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Geneva: World Health Organization, International Agency for Research on Cancer, 1972- PRESENT. (Multivolume work), V21 (1979).
Beagle dog, 24 female	Estradiol valerate or 17ß Estradiol	Intramuscular application	1.28 or 2.4 mg/kg every third day and 1.28 mg/kg once weekly for a period of 52 or 66 weeks	Morphological changes do not indicate neoplastic or anaplastic processes. Benign proliverative lesions of the mesothelial lining of genital organs together with other changes known to be related to Estrogen effects (clinical, haematological, biochemical and histopathological indices).	Research Report No. 3102, Schering AG, Systemic tolerance study of ZK Nr. 5018 and ZK Nr. 5104 in beagle dogs by repeated intramuscular application over a period of 65 weeks, with special regards to ovarian carcinoma, dated 03March 1978.
Macaca mulatta, female	17ß-Estradiol	subcutaneous implantation	Total doses of 575-825 mg at intervals of 5-6 weeks	Cystic hyperplasia of the mammary gland but no tumors was found.	Engle et al., 1943. Cancer Res., 3, 858- 866429 as cited by IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Man.: World Health Organization, International Agency for Research on Cancer, 1972- PRESENT. (Multivolume work), V21 (1979).

 

Human data

 

 

End point/Effect	Literature
The most frequent adverse dermatologic reaction associated with estrogen therapy is chloasma or melasma. Other dermatologic reactions include erythema multiform, erythema nodosum, and hemorrhagic eruption. Hirsutism and alopecia have also occurred. Porphyria cutanea has reportedly been adversely affected in some women receiving estrogen therapy.	McEvoy, G.K. (ed.).Formulary Service - Drug Information 1999.,: American Society of Health-System Pharmacists, Inc. 1999 (Plus Supplements)., p. 2689 as cited by the Hazardous Substances Data Bank (HSDB), Estradiol on November 23, 2009.
Oral contraceptive use is associated with an increased risk of thromboembolic and thrombotic disorders including thrombophlebitis, pulmonary embolism, stroke, subarachnoid hemorrhage, and myocardial infarction. Retinal thrombosis and mesenteric thrombosis also have been reported in women receiving oral contraceptives. An increased risk of postsurgery thromboembolic complications has also been reported in patients receiving oral contraceptives.	McEvoy, G.K. (ed.).Formulary Service - Drug Information 1999.,: American Society of Health-System Pharmacists, Inc. 1999 (Plus Supplements)., p. 2689 as cited by the Hazardous Substances Data Bank (HSDB), Estradiol on November 23, 2009.
Estrogens may cause some degree of fluid retention and edema. Estrogen therapy should therefore be used with caution in patients with conditions that might be aggravated by fluid retention.	McEvoy, G.K. (ed.).Formulary Service - Drug Information 1999.,: American Society of Health-System Pharmacists, Inc. 1999 (Plus Supplements)., p. 2690 as cited by the Hazardous Substances Data Bank (HSDB), Estradiol on November 23, 2009.
Decreased glucose tolerance has occurred in women receiving estrogen-containing oral contraceptives and may occur in patients receiving large dosages of estrogens.	McEvoy, G.K. (ed.).Formulary Service - Drug Information 1999.,: American Society of Health-System Pharmacists, Inc. 1999 (Plus Supplements)., p. 2690 as cited by the Hazardous Substances Data Bank (HSDB), Estradiol on November 23, 2009.
Breakthrough bleeding, spotting, changes in menstrual flow, missed menses (during use), or amenorrhea (after use) may occur in women receiving estrogen therapy. Dysmenorrhea and a premenstrual-like syndrome also have been reported.	McEvoy, G.K. (ed.).Formulary Service - Drug Information 1999.,: American Society of Health-System Pharmacists, Inc. 1999 (Plus Supplements)., p. 2690 as cited by the Hazardous Substances Data Bank (HSDB), Estradiol on November 23, 2009.
Changes in cervical erosion and secretions may occur during estrogen therapy. In addition, preexisting uterine leiomyoma may increase in size in women receiving estrogens. A cystitis-like syndrome has been reported but has not been definitely attributed to estrogens. An increased incidence of Candida vaginitis has been associated with estrogen therapy.	McEvoy, G.K. (ed.).Formulary Service - Drug Information 1999.,: American Society of Health-System Pharmacists, Inc. 1999 (Plus Supplements)., p. 2690 as cited by the Hazardous Substances Data Bank (HSDB), Estradiol on November 23, 2009.
Headache, especially migraine headache, may occur during estrogen therapy.	McEvoy, G.K. (ed.).Formulary Service - Drug Information 1999.,: American Society of Health-System Pharmacists, Inc. 1999 (Plus Supplements)., p. 2690 as cited by the Hazardous Substances Data Bank (HSDB), Estradiol on November 23, 2009.
Breast changes, including tenderness, enlargement, and secretion, may occur during estrogen therapy. The incidence of breast pain may be increased in patients receiving estrogens in conjunction with progestins compared with those receiving estrogens alone; breast pain was reported in about 33% of women receiving conjugated estrogens concomitantly with medroxyprogesterone acetate compared to 12% of women receiving unopposed conjugated estrogen therapy.	McEvoy, G.K. (ed.).Formulary Service - Drug Information 1999.,: American Society of Health-System Pharmacists, Inc. 1999 (Plus Supplements)., p. 2690 as cited by the Hazardous Substances Data Bank (HSDB), Estradiol on November 23, 2009.
Use of estrogens, especially in large dosages, may be associated with an increased risk of several serious conditions including thromboembolism, stroke, myocardial infarction, liver tumor, gallbladder disease, visual disturbances, fetal abnormalities, malignancy, and hypertension. If a progestin is administered concomitantly with estrogen therapy, potential risks may include adverse effects on lipid metabolism, glucose tolerance, or possible enhancement of mitotic activity in breast epithelial tissue.	McEvoy, G.K. (ed.).Formulary Service - Drug Information 1999.,: American Society of Health-System Pharmacists, Inc. 1999 (Plus Supplements)., p. 2690 as cited by the Hazardous Substances Data Bank (HSDB), Estradiol on November 23, 2009.

 

 

 

The following studies are described in more detail in the respective IUCLID section and are used to determine an effect level:

In a subacute toxicity study Estradiol was administered to 3 female and 3 male Sprague Dawley rats by gavage at dose levels of 20 and 300 mg/kg bw/day for 5 days.

A transient decrease in body weight gain occurred during the repeated administration of estradiol (20 mg/kg onwards) in male animals (20 mg/kg bw/d) and male and female animals (300 mg/kgbw/d). Furthermore, a transient decrease of water consumption could be detected during the treatment with estradiol (20 mg/kg onwards) in male animals. Since the amount of water consumption is generally correlated with food consumption a decreased intake of food can be suspected. However, the food consumption could not be determined. Therefore, a definite interpretation for the reduced water intake is not possible. A decrease of Glutamic-oxaloacetic-transaminase and Glutamic-pyruvic-transaminase in male animals and female and male animals (at 20 and 300 mg/kg bw/d), respectively. However, only an increase in these parameters indicate a liver damage. Thus, these changes are considered non-treatment related. No Mortality occurred. Also, no changes were observed during necropsy.

Based on the results obtained from this study a NOAEL of > 300 mg/kg bw/d can be derived.

 

 

In a subacute toxicity study according to Guidelines for Toxicity Studies of Drugs (PAB/ERD-1, Notification no. 24, dated 11 Sep 1989) Estradiol was administered in combination with Levonorgestrel to 10 female Jcl: Sprague Dawley SDrats/dose in 0.5% CMC-Na + 0.04% Tween 80 by gavage at dose levels of 0 (control), 0.02, 0.4 and 20 mg/kg bw/day for 28 consecutive days. Additionally, Estradiol was administered to 10 female Jcl:SD rats at a dose of 18.2 mg/kg bw for 10 days. Control, Estradiol and high dose groups included 6 additional animals per sex to be sacrificed after 2 weeks of recovery.

No mortality occurred. No clinical signs and no changes were observed at the weekly detailed clinical observations. Some changes on body weight and food consumption were noted. Body weight gains were significantly inhibited in the 20000 µg/kg and E2 groups during the treatment period. In the withdrawal period, body weight changes in the 20000 µg/kg were lower than the corresponding control values, however, significant inhibition of body weight gains disappeared in week 6. In the E2 group, body weight gains were significantly inhibited during the withdrawal period.

In other treated groups, body weight gains were similar to those in the control group.

The decrease in body weight gain is considered to be related to the reduced food consumption. A significant decrease in food consumption was recorded in the 20000 µg/kg group in weeks 1 and 4, and in the E2 group in week 1. In the withdrawal period, no significant difference from the control group was observed in either the 20000 µg/kg or E2 groups.

In other treated groups, food consumption was similar to that in the control group.

Significant decreases in RBC, Hb, PCV and MCHC were observed in the 20000 µg/kg and E2 groups. In the withdrawal period, significant decreases in RBC and Hb were still observed in the 20000 µg/kg group, but there were trends toward improvement by comparison with the values in the treatment period.  In addition, a significant increase in reticulocyte ratio was noted in the 20000 µg/kg and E2 groups. A significant increase in segmented cell ratio was observed in the 20000 µg/kg and E2 groups, and a significant increase in neutrophil ratio was noted in the E2 group, but WBC showed no changes in either group.  In the E2 group, a decrease in WBC was noted in the withdrawal period, but the change was within the limits of normal variation. Therefore, the changes in leukocytic parameters were not considered to be biologically significant.

In other treated groups, no significant changes were observed.

Reversible effects in coagulation were seen in the highest dose group. No differences were reported in relative organ weights between treated and control animals; total organ weights changed but were considered to be not treatment related due to the absence of compound-related microscopic findings and no treatment-related changes were noted at microscopic observations. At necropsy changes were noted in the vagina, uterus, ovary, liver, adrenal, pituitary, spleen, thymus, femur and mammary gland. However, most of these changes were reversible and/or not dose-dependent and therefore not considered to be treatment related

 

On the basis of the results obtained in this study, the dose level of 0.4 mg/kg bw/day was considered the LOEL (Lowest Observed Effect Level).

Justification for classification or non-classification

Due to the outcome of reported repeated dose studies classification is not required according to Regulation (EC) 1272/2008 (CLP).