Subacute toxicity testing as per oecd guidelines tulsi 407

The guidelines describe about the subacute toxicity studies in rodents with a comparison with the previous guideline.it also includes the comparison of all three subacute toxicity studies OECD 407, OECD 410, and OECD 412

1. SUBACUTE TOXICITY TESTING AS PER OECD GUIDELINES Tulsi patil M. PHARMACOLOGY, SEM2 SSRCP

2. Toxicity studies -introduction Toxicology is the branch of science that deals with toxins and poisons, their effects and treatment. Toxicological screening is very important for development of new drugs Significance: It helps to calculate the no observed adverse effects level(NOAEL)dose and it is helpful for clinical trials.

3. What is toxicity testing? General Toxicity testing : Is referred to as series of toxicity testing required by International regulatory for compliance to G ood L aboratory P ractices (GLP) for proof of safety in experimental animal prior to their testing in human.

4. It comprises of : Acute, Sub-acute and chronic toxicity . These studies are conducted based on Guidelines given by the regulatory agencies, ICH FDA OECD WHO EPA Schedule Y

5. What is subacute toxicity? The effect of exposure is generally for 28 days. OECD Test Guidelines describing short‐term repeat‐dose toxicity test item administered via different routes are: Goal: To estimate safety margin of test item ROUTE TG(test guideline) Oral 407 Dermal 410 Inhalation 412

6. History of subacute toxicity The original OECD Test Guideline 407 was adopted in 1981. In 1995 a revised version was adopted, to obtain additional information from the animal used in the study, in particular on neurotoxicity and immunotoxicity. Amended in 1998, to obtain information related to endocrine disruptors.

7. Allows to determination of the No-Observed Adverse Effect Level (NOAEL) and provide information on selection of doses for long term studies

8. OECD Guidelines No. 407: Repeated Dose 28-day Oral Toxicity Study in Rodents

9. INITIAL CONSIDERATIONS and limitations Determination of oral toxicity using repeated doses may be carried out after initial information on toxicity has been obtained by acute toxicity testing. This TG is intended to investigate effects on the nervous, immune and endocrine systems. It should identify chemicals with neurotoxic potential and chemicals that interfere with thyroid physiology. It may also provide data on chemicals that affect the male and or female reproductive organs in young adult animals and may give an indication of immunological effects.

10. The results from the TG 407 should be used for hazard identification and risk assessment. The results obtained by the endocrine related parameters should be seen in the context of the “OECD Conceptual Framework for Testing and Assessment of Endocrine Disrupting Chemicals”. The international program conducted on the validation of parameters suitable to potentially detect endocrine activity of test substance. A variety of parameters were found to be indicative of endocrine-related toxicity and have been incorporated in the TG. Parameters for which insufficient data were available to prove usefulness or which showed only weak evidence in the validation programme of their ability to help in detection of endocrine disrupters are proposed as optional endpoints.

11. Endpoints recommended for the detection of endocrine disrupters (EDs) in TG 407 Mandatory endpoints Optional endpoints Weight Testes Ovaries Epididymides Uterus, Including cervix Adrenals Thyroid Prostate + seminal vesicles Histopathology Gonads Vaginal smears -Testes Male mammary glands -Ovaries Pituitory Accessory sex organs : - Epididymides , -Prostate + seminal vesicles Uterus, including cervix -adrenal -thyroid -vagina Hormonal Measurements T3 and T4 TSH

12. On the basis of data generated in the validation process, it must be emphasized that the sensitivity of this assay is not sufficient to identify all substances with (anti)androgenic or (anti) oestrogenic modes of action. The TG nevertheless, during the validation process identified compounds weakly and strongly affecting thyroid function, and strong and moderate endocrine active substances acting through oestrogen or androgen receptors, but in most cases failed to identify endocrine active substances that weakly affect oestrogen or androgen receptors. Thus it can’t be described as a screening assay for endocrine activity.

13. Principle The test substance is orally administered daily in graduated doses to several groups of experimental animals, one dose level per group for a period of 28 days. During the period of administration the animals are observed closely, each day for signs of toxicity. At the end of the study, the experimental animals are sacrificed. Gross Pathological changes are observed and all the tissues are subjected to histopathological analyses. The data obtained by this TG Allows to determination of the No-Observed Adverse Effect Level (NOAEL) and provide information on selection of doses for long term studies

14. Subacute (Guidelines 407) SD Rats, male and Female Control Low dose Medium dose High dose

15. DESCRIPTION OF THE METHOD

16. Selection of Animals The preferred rodent species is the rat, although other rodent species may be used (Young healthy adult animals should be employed) If the parameters specified within this TG 407 are investigated in another rodent species a detailed justification should be given. Other species should respond to toxicants in a similar manner to the rat.

17. The use of smaller species may result in increased variability due to technical challenges of dissecting smaller organs. In the international validation program for the detection of endocrine disrupters, the rat was the only species used. Female rats should be nulliparous and non pregnant. When a repeated oral dose is conducted as a preliminary to a longer-term study, it is preferable that animals from the same strain and source should be used in both studies.

18. Dosing should begin as soon as feasible after weaning, and, in any case, before the animals are nine weeks old. weight variation of animals used should be minimal and not exceed ± 20%

19. Housing Husbandry conditions : Temperature : 22°C (± 3°C) RH : 30%- 70% Lighting should be artificial, 12 hours light, 12 hours dark. Feeding: Conventional laboratory diets may be used with an unlimited supply of drinking water. For group caging, not more than five animals should be housed per cage. The feed should be regularly analysed for contaminants. A sample of the diet should be retained until finalisation of the report.

20. Preparation of animals Healthy young adult animals grouped in test and control. Cages should be arranged in such a way that possible effects due to cage placement are minimized. The animals are identified uniquely. Kept in their cages for at least five days prior to the start of the treatment study to allow for acclimatisation to the laboratory conditions.

21. Preparation of doses The test compound is administered by gavage or via the diet or drinking water. Test substance is dissolved or suspended in a suitable vehicle. Vehicles examples Water Methylcellulose or carboxymethylcellulose Oil(corn, peanut, sesame)

22. Number and sex of animals At least 10 animals (five female and five male) in each group If interim euthanasia are planned, the number should be increased by the number of animals scheduled to be euthanised before the completion of the study. Satellite group: in the control and in the top dose group for observation of reversibility, persistence, or delayed occurrence of toxic effects, for at least 14 days post treatment.

23. Grouping and number of animals

24. Dosage Generally, at least three test groups and a control group should be used, but if from assessment of other data, no effects would be expected at a dose of 1000mg/kg, a limit test may be performed. 1000 mg/kg no toxicity effect Toxicity effect No need for subacute toxicity studies Subacute toxicity studies

25. If there are no suitable data available, a range finding study (animals of the same strain and source) may be performed to aid the determination of the doses to be used. Control group should receive the vehicle.

26. Dose levels should be selected taking into account any existing toxicity and ( toxico -) kinetic data available for the test compound or related materials. Two to four fold intervals are frequently optimal for setting the descending dose levels and addition of a fourth test group is often preferable to using very large intervals Intervals (e.g. more than a factor of 10) between dosages.

27. Administration of doses The animals are dosed with test substance daily 7 days each week for a period of 28 days. When the test substance is administered by gavage, this should be done in a single dose The volume should not exceed 1 ml/100g body weight except in the case of aqueous solutions where 2 ml/100 g body weight may be used. Ensure a constant volume at all dose levels. Substances administered via the diet or drinking water it is important to ensure that test substance do not interfere with normal nutrition or water balance. Where a repeated dose study is used as a preliminary to a long term study, a similar diet should be used in both studies.

28. Observations The observation period should be 28 days Animals in a satellite group observed for 14 days without treatment to detect delayed occurrence, or persistence of, or recovery from toxic effects. Observations should be made at least once a day, preferably at the same time. At least twice daily, all animals are observed for morbidity and mortality.

29. Before the first exposure : Detailed clinical observations should be made in all animals. These observations should be made outside the home cage in a standard arena and preferably at the same time of day on each occasion. They should be carefully recorded, preferably using scoring systems, explicitly defined by the testing laboratory . Signs noted should include, Changes in skin, fur, eyes, mucous membranes. Autonomic activity (e.g. lacrimation , piloerection , pupil size, unusual respiratory pattern).

30. In the fourth exposure week:(different types of stimuli are observed e.g. auditory and visual ) Assessment of grip strength and motor activity At necropsy, the oestrus cycle of all females could be determined (optional). In the presence of observed general toxicity (e.g. reduced body weight, liver , heart, lung or kidney effects, etc.) or other changes that may not be toxic responses (e.g. reduced food intake, liver enlargement) Observed effects on immune, neurological or endocrine sensitive endpoints should be interpreted with caution.

31. Observation : Body weight/body weight changes. All animals should be weighed at least once a week. Food/water consumption. Measurements of food consumption should be made at least weekly. If the test substance is administered via the drinking water, water consumption should also be measured at least weekly.

32. Observation : Haematology :Blood samples should be collected just prior to euthanasia of the animals and stored under appropriate conditions. The following haematological examinations : Haematocrit, Haemoglobin concentrations, Erythrocyte count and Reticulocytes, Total and differential leucocyte count, platelet count and a measure of blood clotting time.

33. Haematological Data of Rat Rat RBC(x10v /mm³) 7-10 PCV(%) 36-18 Hb (g/dl) 11-18 WBC(X10³/m³) 6-17 Neutrophils (%) 9-34 Lymphocytes(%) 65-85 Eosinophils (%) 0-6 Monocytes (%) 0-5 Basophils (%) 0-1.5 Platelets(X10³/mm³) 500-1300

34. Clinical biochemistry Investigations of plasma or serum shall include sodium, potassium, glucose, total cholesterol, urea, creatinine , total protein and albumin. Optionally, the following urinalysis determinations could be performed during the last week of the study using timed urine volume collection; Appearance, volume, osmolality , pH, protein, glucose and blood/blood cells.

35. Clinical Data Of Rat Rat Protein (g/dl) 5.6-7.6 Albumin (g/dl) 2.8-4,8 Globulin (g/dl) 1.8-3 Glucose (mg/dl) 50-135 Urea nitrogen 15-21 Creatinine (mg/dl) 0.2-0.8 Bilirubin (mg/dl) 0.2-0.55 Cholesterol (mg/dl) 40-130

36. CLINICAL SIGNS OF TOXICITY RESPIRATORY : Blockage in the nostrils, changes in rate & depth of breathing, changes in color of body surfaces Signs like dyspnea, abdominal breathing, Apnea.

37. MOTOR ACTIVITY Changes in frequency & nature of movements Signs like decrease/increase in spontaneous motor activity, Anesthesia Ataxia Tremors

38. OCULAR SIGNS Lacrimation, Chromodacryorrhea ( red lacrimation ) Miosis and Mydriasis.

39. CARDIO-VASCULAR SIGNS Bradycardia, tachycardia, Arrhythmias. All signs indicates involvements of CNS, Sensory, autonomic and neuromuscular systems.

40. OTHER SIGNS Salivation Piloerection Analgesia Muscle tone - hypotonia - hypertonia GIT signs: Emesis

41. Although in the international evaluation of the endocrine related endpoints a clear advantage for the determination of thyroid hormones (T3, T4) . It may be helpful to retain plasma or serum samples to measure T3, T4 and TSH (optional) if there is an indication for an effect on the pituitary-thyroid axis. These samples may be frozen at -20° for storage. Plasma samples specifically intended for hormone determination should be obtained at a comparable time of the day. The numerical values obtained when analysing hormone concentrations differ with various commercial assay kits.

42. Gross necropsy All animals in the study shall be subjected to a full, detailed gross necropsy which includes careful examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents. The liver, kidneys, adrenals, testes, epididymides , prostate + seminal vesicles ,thymus, spleen, brain and heart of all animals (apart from those found moribund and/or euthanised prior to the termination of the study) In addition, two other tissues could be optionally weighed as soon as possible after dissection, to avoid drying: paired ovaries (wet weight) and uterus, including cervix . The thyroid weight (optional) could be determined after fixation.

43. Histopathology Full histopathology should be carried out on the preserved organs and tissues of all animals in the control and high dose groups.

44. DATA AND REPORTING Data should be summarised in tabular form. All the possible, numerical results should be evaluated by an appropriate and generally acceptable statistical method.

45. Schedule y 14-28 days Group Rodents Non Rodents M F M F Low dose 6-10 6-10 2-3 2-3 Moderate dose 6-10 6-10 2-3 2-3 High dose 6-10 6-10 2-3 2-3 Numbers of animals required for Repeated dose toxicity studies

46. REference OECD Guideline for the Testing of Chemicals, 407 Adopted 3 October 2008 Repeated Dose 28-day Oral Toxicity Study in Rodents. CARE V. CPCSEA guidelines for laboratory animal facility. Indian Journal of Pharmacology. 2003;35:257-74.

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