Estradiol

Repeated dose toxicity: oral

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Estradiol

EC Number:

200-023-8

EC Name:

Estradiol

Cas Number:

50-28-2

Molecular formula:

C18H24O2

IUPAC Name:

estra-1,3,5(10)-triene-3,17-diol

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

18.2 mg/kg bw/day (nominal)

Remarks:

estradiol alone

Doses / concentrations 2

Dose / conc.:

20 mg/kg bw/day (nominal)

Remarks:

Combination: Estradiol + Levonorgestrel

Doses / concentrations 3

Dose / conc.:

0.4 mg/kg bw/day (nominal)

Remarks:

Combination: Estradiol + Levonorgestrel

Doses / concentrations 4

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

Examinations

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.

Results and discussion

Results of examinations

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

Effect levels

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

Target system / organ toxicity

1

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

2

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

Applicant's summary and conclusion

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).