OECD Guidelines

The Slides present guidelines for Acute oral testing of drugs as well as other OECD guidelines in short.

1. O rganisation for E conomic C o-operation and D evelopment Guidelines for Toxicity Testings Dr Urmila M. Aswar, Poona College of Pharmacy, BVDU, PUNE, INDIA

2. Drug: Safe and Effective Drug (D & C Act 1940) : All medicines for internal or external use of human beings or animals and all substances intended to be used for or in the diagnosis, treatment, mitigation or prevention of any disease or disorder in human beings or animals, including preparations applied on human body for the purpose of repelling insects like mosquitoes. The objective of  toxicity testing  in the laboratory is to  elucidate the toxic properties of drugs.

3. What are OECD guidelines ??? The OECD Guidelines are a unique tool for assessing the potential effects of chemicals on human health and the environment. Accepted internationally as standard methods for safety testing. Used by professionals in industry, academia and government involved in the testing and assessment of chemicals (industrial chemicals, pesticides, personal care products, etc.). These Guidelines are regularly updated with the assistance of thousands of national experts from OECD member countries. Covered by the Mutual Acceptance of Data, implying that data generated in the testing of chemicals in an OECD member country, or a partner country having adhered to the Decision, in accordance with OECD Test Guidelines and Principles of Good Laboratory Practice (GLP), be accepted in other OECD countries and partner counties having adhered to the Decision, for the purposes of assessment and other uses relating to the protection of human health and the environment.

4. Purpose Hundreds of new chemicals, such as industrial chemicals, pesticides, food additives, biotechnology products and pharmaceuticals, flood the world market each year and may require safety testing in most parts of the world. Purpose: To enhance the validity and international acceptance of test data; Make the best use of available resources in both governments and industry; Avoid the unnecessary use of laboratory animals; Minimise non-tariff trade barriers.

5. Website

6. Common considerations The Globally Harmonized System (GHS),defines  TOXICITY  as  "those adverse effects occurring  following oral or dermal administration of a single dose of a substance, or multiple  doses given within 24 hours, or an  inhalation exposure of 4 hours"  The preferred species for oral and inhalation testing is the rat, and for dermal testing, the rat or rabbit. Oral administration is the most common form of acute systemic toxicity testing.

7. Toxicological investigation Acute toxicity study Repeated dose toxicity study Dermal toxicity test Mutagenicity test Carcinogenicity test Reproductive and development toxicity tests

8. Toxicological investigation Paracelsus (1493-1541) stated that “All substances are poisons; The right dose differentiates a poison and remedy” this concept is the fundamental principle of toxicology. WHO- guidelines have given the important criteria to establish the safety profile of the drugs. Toxicological study results play an important safety assessment for Herbal Drugs, Pharmaceuticals, food additives , pesticides and other chemicals.

9. Toxicity study Essential for any compounds having biological activity. Acute toxicity: Adverse effects occurring within a short time following administration of single dose. Sub- acute toxicity Chronic toxicity- Period of 90 days or more. Special toxicity studies- Period of 2 years or more Carcinogenicity Mutagenicity Teratogenicity Reproductive toxicity

10. Initial Considerations The testing laboratory should consider all available  information on the test substance prior to conducting  the study.  The identity and chemical structure of the substance Its  physico ‐chemical properties The results of any other in vitro or in vivo toxicity tests on the substance Toxicological data on structurally related substances; The anticipated use(s) of the substance

11. Cont… Animal species: Rat or Mice Age : Nine weeks old. Housing condition Temperature: 22 ± 3 ° C Humidity: 50-60 % Lighting: 12 hr light 12 hr dark light Feed : laboratory feed with water ad libitum House: individually, small groups in same sex, not more than five/cage Preparation of doses: Administration by gavages or via the diet or drinking water in volume of 1ml/100gm for suspension and 2ml/100gm b.w for aqueous.

12. Necessities of Toxicological Studies Benefit –risk ratio can be calculated Prediction of therapeutic index Therapeutic index= Maximum tolerated dose/ Minimum curative dose Smaller ratio, better safety of the drug.

13. Important OECD guideline 420 Acute oral Toxicity- fixed dose method 423 Acute oral Toxicity –acute toxic class method 425 Acute oral Toxicity-Up and Down method 402 Acute dermal Toxicity 404 Acute dermal Irritation/ Corrosion 403 Acute inhalation Toxicity 405 Acute Eye Irritation /Corrosion Skin sensitization 28 days repeated oral Toxicity studies in rodents 408 90 days repeated oral Toxicity studies in rodents 409 90 days repeated oral Toxicity studies in non rodents

14. Important OECD guidelines 410 90 days repeated Dermal Toxicity 411 90 days inhalation Toxicity study 412 28/14 days repeated dose inhalation Toxicity study 413 90 days repeated dose inhalation Toxicity study 414 Prenatal Developmental Toxicity study Reproduction /Development toxicity screening test Combined Repeated dose toxicity study with Reproduction/Developmental Toxicity screening test Neurotoxicity study in rodents Carcinogenicity studies Chronic Toxicity studies Combined chronic toxicity/carcinogenic studies

15. Test  Guidelines- 402, 403, 420, 423, and 425 .              Fixed Dose Procedure-OECD 420, Acute Toxic Class method -OECD 423, Up‐and‐Down Procedure -OECD 425, 28 days repeated oral Toxicity studies in rodents- OECD 407 90 days repeated oral Toxicity studies in rodents OECD 408 Acute Dermal Toxicity-OECD  402, Acute inhalation toxicity-OECD 403.

16. OECD- 420: Acute Oral Toxicity - Fixed Dose Procedure Groups of animals of a single sex are dosed in a stepwise procedure using the fixed doses of 5, 50, 300 and 2000 mg/kg (exceptionally an additional fixed dose of 5000 mg/kg may be considered). The initial dose level is selected on the basis of a sighting study as the dose expected to produce some signs of toxicity without causing severe toxic effects or mortality. The preferred rodent species is the rat , although other rodent species may be used. Normally females are used. This is because literature surveys of conventional LD50 tests show that usually there is little difference in sensitivity between the sexes

17. Sighting study(1rat)- 420

18. Cont..

19. Main Study(5 rats)- 420

20. AOT 420- Important points

21. Observations Animals are observed individually after dosing at least once during the first 30 minutes , periodically during the first 24 hours , with special attention given during the first 4 hours, and daily thereafter, for a total of 14 days , except where they need to be removed from the study and humanely killed for animal welfare reasons or are found dead. However, the duration of observation should not be fixed rigidly.

22. Acute Oral Toxicity 423 – Acute Toxic Class Method Principle: It is based on a stepwise procedure with the use of a minimum number of animals per step , sufficient information is obtained on the acute toxicity of the test substance to enable its classification. The substance is administered orally to a group of experimental animals at one of the defined doses. The substance is tested using a stepwise procedure, each step using three animals of a single sex (normally females). Absence or presence of compound-related mortality of the animals dosed at one step will determine the next step. No further testing is needed, Dosing of three additional animals , with the same dose Dosing of three additional animals at the next higher or the next lower dose level.

23. Acute Oral Toxicity 423 – Acute Toxic Class Method

24. Cont…

25. Acute Oral Toxicity 425- Up-and-Down The first animal dose below best preliminary estimate of LD 50 . If the animal survives. The dose for second animal is increased by (a factor of) 3.2 times the original dose. If the first animal dies or appears morbid than dose for second animal is decrease by (a factor of) 3.2 times the original dose. 3.2 is default factor corresponding to a dose progression of one half log unit. Dosing would be select from the sequence dose 1.75, 5.5, 17.5, 55, 175, 550, 2000, (or 1.75, 5.5, 17.5, 55, 175, 550, 1750 and 5000 for specific regulatory needs). If no information available regarding lethality of drug than dosing start from 175 mg/kg, bw .

26. OECD 425- Limit test Limit test carryout at 2000 mg/kg, orally (Exceptionally 5000 mg/kg) when information indicating test material is likely to be nontoxic or toxicity above regulatory limit dose. The LD 50 less than the test dose (2000 mg/kg) when 3 or more animals die. LD 50 is greater than the test dose (2000 mg/kg) when 3 or more animal survive. Observation: 14 days same as OECD 423

27. 28 day repeated dose oral Toxicity study (OECD- 407) Number of animal: 1 0 animals per group (5 female and 5 male) Dosage: At least three test groups and the control group should be used. Limit test: 1000 mg/kg/day for 28 days daily

28. Observation : 28 days General clinical observation at least once a day Morbidity and mortality - at least twice daily Changes in skin, eyes, mucous membrane, secretion and excretion. ANS activity- lacrimation , piloerection , pupil size, respiratory pattern, grip strength, motor activity assessment. Body weight: weekly Food consumption: weekly Water consumption: weekly

29. Cont.. Hematology parameters: Haematocrit , Hb , RBC, WBC, DC, platelet , clotting time. Clinical Biochemistry Liver and kidney function test Plasma or serum Na, K, glucose, cholesterol, urea, creatinine , SGOT, SGPT, total protein, albumin, ALP, bilirubin , Gamma glutamyl transpeptidase , Lipid profile. Urine analysis Last week of the study: Volume, appearance, specific gravity, pH, protein glucose and blood cells.

30. Cont.. Metabolic profiles Calcium, phosphate and triglycerides. Pathology: Liver, kidney, adrenals, testes, thymus, spleen, brain, stomach, intestine, breast, lungs, urinary bladder, peripheral nerve, bone marrow etc.

31. 90 day repeated dose oral Toxicity study (OECD- 408) Preparation of doses: Administration by gavages or via the diet or drinking water in volume of 1ml/100gm for suspension and 2ml/100gm b.w for aqueous. If Vehicles use other than water than toxic properties of the vehicle must be known. Number of animal: 1 0 animals per group (5 female and 5 male) Dosage: At least three test groups and the control group should be use. Limit test: 1000 mg/kg/day for 90 days daily Observation: Same as OECD 407

32. Acute Dermal Toxicity Study OECD-402 Prerequisites Solid or liquid test substance Chemical identification of test substance Purity (impurities) of test substance Solubility characteristics Melting point/boiling point PH

33. Cont.. C.S.M.D.R.I.A.S Acute dermal toxicity is the adverse effects occurring within a short time of dermal application of a single dose of a test substance. Principle : The test substance is applied to the skin in graduated doses to several groups of experimental animals, one dose being used per group.

34. Cont.. C.S.M.D.R.I.A.S Preparations 24 hours before the test, fur should be removed from the dorsal area of the trunk of the test animals by clipping or shaving. Care must be taken to avoid abrading the skin, which could alter its permeability. Not less than 10 per cent of the body surface area should be clear for the application of the test substance. The weight of the animal should be taken into account when deciding on the area to be cleared and on the dimensions of the covering

35. Reproductive toxicity 421, 422

36. Introduction The occurrence of biologically adverse effects on the reproductive systems of females or males that may result from exposure to environmental agents. The toxicity may be expressed as alterations to the female or male reproductive organs, the related endocrine system, or pregnancy outcomes.

37. Effects on onset of puberty, Gamete production and transport, Reproductive cycle normality, Sexual behavior , Fertility, gestation, Parturition, lactation, developmental toxicity Premature reproductive senescence

38. Developmental toxicity Exposure prior to conception (either parent), during prenatal development, or postnatally to the time of sexual maturation. The major manifestations of developmental toxicity include (1) death of the developing organism, (2) structural abnormality, (3) altered growth, and (4) functional deficiency

39. Experiment Adequate dosing For example, damage to spermatogonial stem cells will not appear in samples from the cauda epididymis or in ejaculates for 8 to 14 weeks. Chlordimeform develops the compensatory mechanism. Knowledge of the relevant pharmacokinetic and pharmacodynamic data can facilitate selection of dose levels and treatment duration. Proper timing of examination of treated animals relative to initiation and termination of exposure to the agent.

40. Length of Mating Period Traditionally, pairs of rats or mice are allowed to cohabit for periods ranging from several days to 3 weeks. Given a 4- or 5-day estrous cycle, each female that is cycling normally should be in estrus four or five times during a 21-day mating period.

41. Cont… Reproductive toxicity studies in laboratory animals generally involve continuous exposure to a test substance for one or more generations. The objective is to detect effects on the integrated reproductive process as well as to study effects on the individual reproductive organs.

42. Number of animals {Use of 20 males and enough females to produce at least 20 pregnancies for each dose group in each generation in the multigeneration reproduction test.} 20 pregnancies may have been achieved by mating two females with each male and using fewer than 20 males per treatment group.

43. The single-generation reproduction test evaluates effects of subchronic exposure of peripubertal and adult animals. In the multigeneration reproduction protocol, F1 and F2 offspring are exposed continuously in utero from conception until birth and during the preweaning period. Animals producing the first generation of offspring should be considered the parental (P) generation, and all subsequent generations should be designated filial generations (e.g., F1, F2). Only the P generation is mated in a single-generation test, while both the P and F1 generations are mated in a two-generation reproduction test.

44. Single generation testing In the P generation, both females and males are treated prior to and during mating , with treatment usually beginning around puberty. Cohabitation can be allowed for up to 3 weeks, females are monitored for evidence of mating. Females continue to be exposed during gestation and lactation.

45. Two-generation reproduction test Randomly selected F1 male and female offspring continue to be exposed after weaning (day 21) and through the mating period. Treatment of mated F1 females is continued throughout gestation and lactation.

46. Parameters evaluated Visual examination of reproductive animals. Weights of the testes, epididymides , and accessory sex glands from males, and histopathology of these organs. Histopathology of the vagina, uterus, cervix, ovaries, and mammary glands from females. In addition, litters (individual pups) are weighed at birth and examined for number of live and dead offspring, gender, gross abnormalities, and growth and survival to weaning.

47. Cont.. Maturation and behavioural testing may also be performed on the pups.

48. Results If adverse effects are observed on litters in a study using one of these protocols: Male/ Female parent Therefore, male (histopathology or sperm evaluations) and female (ovarian and reproductive tract histology or changes in estrous cycle) evaluation parameters will help to decide.

49. Endpoints

50. Mating Index = Pregnancy Index =

51. Genotoxicity - Micronucleus test-OECD 474 A  micronucleus test  is a test used in toxicological screening for potential  genotoxic . The mammalian in vivo micronucleus test is used for the detection of damage induced by the test substance to the chromosomes or the mitotic apparatus of erythroblasts by analysis of erythrocytes as sampled in bone marrow and/or peripheral blood cells of animals, by evaluating the presence of micronuclei. There are two major versions of this test, one  in vivo  and the other  in vitro.

52. The  in vivo  test normally uses mouse bone marrow or mouse peripheral blood. When a bone marrow erythroblast develops into a polychromatic erythrocyte, the main nucleus is extruded; any micronucleus that has been formed may remain. Visualisation of micronuclei is facilitated in these cells because they lack a main nucleus. An increase in the frequency of micronucleated  polychromatic erythrocytes in treated animals is an indication of induced chromosome damage

53. Procedure In vivo (0ECD 474) : Animals are exposed to the test substance by an appropriate route. If bone marrow is used, the animals are sacrificed at appropriate times (24 h) after treatment, the bone marrow extracted, and preparations made and stained.

54. In vitro (OECD 487) : The test can be performed in primary human peripheral blood lymphocytes (HPBLs) or established cell lines. Cultures are incubated with several concentrations of the test compound for three to four hours in the presence and absence of metabolic activation (S9) and for 21 to 24 hours in the absence of S9. A positive outcome is characterized by a statistically significant, dose-dependent increase in micronucleated cells that exceeds historical negative control limits.

55. In Vitro Mammalian Chromosome Aberration Test The purpose of the in vitro chromosome aberration test is to identify agents that cause structural chromosome aberrations in cultured mammalian cells.

56. Consequence Chromosome aberrations and related events are the cause of many human genetic diseases and there is substantial evidence that chromosome mutations and related events causing alterations in oncogenes and tumour suppressor genes of somatic cells are involved in cancer induction in humans and experimental animals.

57. Principle behind Cell cultures are exposed to the test substance both with and without an exogenous source of metabolic activation unless cells with an adequate metabolizing capability are used. At predetermined intervals after the start of exposure of cell cultures to the test substance, they are treated with a metaphase-arresting substance (e.g. Colcemid ® or colchicine ), harvested, stained and metaphase cells are analysed microscopically for the presence of chromosome aberrations.