SUB ACUTE ORAL TOXICITY AS PER OECD GUIDELINES

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%
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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.
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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).
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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.
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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.
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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
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8. General observations
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• 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
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• Individual body weight was recorded ;
twice weekly
immediately before necropsy.
• Food consumption was measured weekly.

10. Hematology, Histopathological findings
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• 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.
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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.
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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.
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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
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15. Thank You