Ethynyl estradiol was evaluated for sub-acute oral toxicity in a repeated 28-day study using rats in accordance with OECD 407 guidelines. Rats were divided into four groups that received daily doses of either an olive oil solution (control), 10 μg/kg, 50 μg/kg, or 200 μg/kg of ethynyl estradiol by oral gavage. Parameters evaluated included body weight, food consumption, hematology, histopathology, spermatology, and estrous cycling. Results showed reduced food consumption and body weight gain in males at 200 μg/kg. Hematological changes and abnormal estrous cycling were also observed at higher doses.

1. Sub-acute oral toxicity study
Prepared by, Arghya Chowdhury
Roll No.:18162027
M.Pharm 2nd Semester
Department of Pharmaceutical Engineering and Technology
IIT(BHU), Varanasi

2. Ethynyl estradiol
• ANDA : 207081 on 05/17/2017 from AUROBINDO PHARMA LTD
• CAS No. : 57-63-6
• An agonist of the estrogen receptors.
• Used as contraception in combined oral contraceptives (COC)
• Bioavailability : 38–48%
• Protein binding : 97–98% (to albumin)
• Elimination half-life : 7–36 hours
• Excretion : Feces: 62% and Urine: 38%
2

3. Evaluate the safety of ethynyl estradiol
• The experimental protocol was approved by the IAEC (Institutional Animal
Ethical Committee).
• Care of the experimental animals taken according to the CPCSEA
guidelines.
• Sub-acute toxicity study was carried out in rats by using OECD 407
guidelines.
3

4. Repeated Dose 28-Day Oral Toxicity Study
• The test substance is orally administered daily in graduated doses to several groups, one dose
level per group for a period of 28 days.
• During this period the animals are observed closely, each day for signs of toxicity.
• Endpoints : clinical observations, blood analysis, whole body gross necropsy, and micros-
copic examination of all organs and tissues (histopathology).
• Significance : the characterization of the test substance toxicity, for an indication of the
dose response relationship and the determination of the No-Observed Adverse Effect Level
(NOAEL).
4

5. Animal handling and care
• Temperature : 22°C (± 3°C).
• Relative humidity : at least 55±5% with 12-h light/dark cycle (0600–1800
hours).
• Animals: SD rats aged 6 weeks(130 gms-200 gms) of both sexes.
• Access to food and water were maintained under standard laboratory
conditions.
• Housed individually in stainless steel, wire-mesh cages during the study.
5

6. Study design
• Grouping : Both sexes of rats (130-200g) were divided into four groups with 10 animals (5
males plus 5 females in each).
• Dosing :
 Group I (control)-olive oil solution (5 ml/kg)
 Group II – 10 μg/kg EE
 Group III – 50 μg/kg EE
 Group IV – 200 μg/kg EE
• Dosing frequency : Daily from 7 weeks of age for at least 28 days.
6

7. Parameters
• Euthanasia : exsanguination under ether anesthesia.
• Blood samples collection : from the abdominal aorta.
• Parameters considered :
General observations
Body weight and food consumption
Hematology, Histopathological findings
Spermatology, Estrous cycling
7

8. General observations
8
• Autonomic activity (e.g. lacrimation, pupil size, respiratory pattern),
• Changes in gait, posture,
• Presence of clonic or tonic movements.
• Sensory reactivity to stimuli of different types (e.g. auditory, visual),
• Assessment of grip strength,
• Motor activity assessment.

9. Body weight and food consumption
9
• Individual body weight was recorded ;
twice weekly
immediately before necropsy.
• Food consumption was measured weekly.

10. Hematology, Histopathological findings
10
• Hematology examination : RBC count, WBC count, hemoglobin concentration,
hematocrit value , platelet count, reticulocyte count, prothrombin time and
differentiation of leukocytes.
• Histopathological examination : organs were fixed in 10% neutral buffered
formalin and examined: prostates(ventral prostate and dorsolateral prostate),
seminal vesicles, ovaries, uterus, vagina, mammary gland, brain, thyroids, adrenals,
liver, spleen, kidneys, heart, lungs, pancreas, thymus, parathyroids and pituitary
glands.
• The epididymes and testes were fixed in Bouin’s solution and examined.

11. Spermatology, Estrous cycling
• Spermatology :
Sperm motility (100 spermatozoa in Ham’s F10 tissue culture
medium at 37°C).
Viability (decrease in motility during 5 min).
• Estrous cycling : determined daily by vaginal smears stained with Gimsa
from day 15 to the day 21.
1111

12. Results
• General observations : no abnormalities in rats given EE.
• Food consumption was reduced in both males and females ; body weight gain
significantly depressed only in the males of the 200 µg/kg group.
• RBC counts decreased in females of 50 and 200 µg/kg groups ; haemoglobin
concentration and haematocrit values decreased in males of 200 µg/kg group.
• Abnormal estrous cycling was observed in female rats of the 50 and 200 µg/kg groups.
• Adrenal cortical hyperplasia was detected in males ; Splenic hemosiderosis were
detected in females of 200 µg/kg group.
12

13. Analysis
• Data obtained was analyzed by using one way ANOVA followed by
Dunnett’s test and p<0.05 was considered as statistically significant.
• Ethynyl estradiol taken orally exerts following toxic effects;
Anemia,
Breast tenderness,
Atrophy of the prostates,
Weight change.
1313

14. References
• Arch Toxicol (2002) 76: 65–74
• McLachlan JA, Korach KS (1995) Estrogens in the environment: global
health implications. Environ Health Perspect 103: 3–4
14

15. Thank You