Regulatory guidelines for conducting toxicity studies

OECD guidelines for acute and chronic toxicity studies

1. REGULATORY GUIDELINES FOR CONDUCTING TOXICITY STUDIES PRESENTED BY:- HIMIKA RATHI MPHARM SEMESTER2

2. CONTENT Introduction to OECD guidelines List of guidelines included in health effects Annex-1(definitions) What is toxicity studies? Acute toxicity studies Subacute and chronic toxicity studies Carcinogenicity Summary references

3. INTRODUCTION TO OECD GUIDELINES OECD guidelines for the testing of chemicals are a set of internationally accepted specifications for testing of chemicals decided on by the Organization for economic Co-operation and development(OECD). They are split into five sections: Section 1: physical chemical properties Section 2: effects on biotic system Section 3 : degradation and accumulation Section 4: health effects Section 5: other test guidelines

4. INTRODUCTION TO OECD GUIDELINES Here our main focus is to study the regulatory guidelines for conducting toxicity studies, therefore we would focus mainly on health effects. Guidelines are under constant review, with guidelines being periodically updated, new guidelines being adopted. Following is the list of guidelines included in health effects. Number Title 401 Acute Oral Toxicity 402 Acute Dermal Toxicity 403 Acute Inhalation Toxicity 404 Acute Dermal Irritation/Corrosion 405 Acute Eye Irritation/Corrosion 406 Skin Sensitisation 407 Repeated Dose 28-day Oral Toxicity Study in Rodents 408 Repeated Dose 90-Day Oral Toxicity Study in Rodents

5. NUMBER TITLE 409 Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents 410 Repeated Dose Dermal Toxicity: 21/28-day Study 411 Subchronic Dermal Toxicity: 90-day Study 412 Subacute Inhalation Toxicity: 28-Day Study 413 Subchronic Inhalation Toxicity: 90-day Study 414 Prenatal Development Toxicity Study 415 One-Generation Reproduction Toxicity Study 416 Two-Generation Reproduction Toxicity 417 Toxicokinetics 418 Delayed Neurotoxicity of Organophosphorus Substances Following Acute Exposure

6. NUMBER TITLE 419 Delayed Neurotoxicity of Organophosphorus Substances: 28-day Repeated Dose Study 420 Acute Oral Toxicity – Fixed Dose Procedure 421 Reproduction/Developmental Toxicity Screening Test 422 Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test 423 Acute Oral toxicity – Acute Toxic Class Method 424 Neurotoxicity Study in Rodents 425 Acute Oral Toxicity: Up-and-Down Procedure 426 Developmental Neurotoxicity Study 427 Skin Absorption: In Vivo Method 428 Skin Absorption: In Vitro Method 429 Skin Sensitisation 430 In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test Method (TER) 431 In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method

7. NUMBER TITLE 432 In Vitro 3T3 NRU Phototoxicity Test 435 In Vitro Membrane Barrier Test Method for Skin Corrosion 436 Acute Inhalation Toxicity – Acute Toxic Class Method 437 Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage 438 Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage 439 In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method 440 Uterotrophic Bioassay in Rodents 441 Hershberger Bioassay in Rats 442A Skin Sensitization 442B Skin Sensitization 442C In Chemico Skin Sensitisation 442D In Vitro Skin Sensitisation

8. NUMBER TITLE 443 Extended One-Generation Reproductive Toxicity Study 451 Carcinogenicity Studies 452 Chronic Toxicity Studies 453 Combined Chronic Toxicity/Carcinogenicity Studies 455 Draft Performance-Based Test Guideline for Stably Transfected Transactivation In Vitro Assays to Detect Estrogen Receptor Agonists and Antagonists 456 H295R Steroidogenesis Assay 457 BG1Luc Estrogen Receptor Transactivation Test Method for Identifying Estrogen Receptor Agonists and Antagonists 460 Fluorescein Leakage Test Method for Identifying Ocular Corrosives and Severe Irritants 471 Bacterial Reverse Mutation Test 473 In Vitro Mammalian Chromosomal Aberration Test 474 Mammalian Erythrocyte Micronucleus Test 475 Mammalian Bone Marrow Chromosomal Aberration Test 476 In Vitro Mammalian Cell Gene Mutation Tests using the Hprt and xprt genes

9. NUMBER TITLE 477 Genetic Toxicology: Sex-Linked Recessive Lethal Test in Drosophila melanogaster 478 Rodent Dominant Lethal Test 479 Genetic Toxicology: In vitro Sister Chromatid Exchange Assay in Mammalian Cells 480 Genetic Toxicology: Saccharomyces cerevisiae, Gene Mutation Assay 481 Genetic Toxicology: Saacharomyces cerevisiae, Miotic Recombination Assay 482 Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells in vitro 483 Mammalian Spermatogonial Chromosomal Aberration Test 484 Genetic Toxicology: Mouse Spot Test 485 Genetic toxicology, Mouse Heritable Translocation Assay 486 Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo 487 In Vitro Mammalian Cell Micronucleus Test

10. NUMBER TITLE 488 Transgenic Rodent Somatic and Germ Cell Gene Mutation Assays 489 In Vivo Mammalian Alkaline Comet Assay 490 In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene 491 Short Time Exposure In Vitro Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage 492 Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage 493 Performance-Based Test Guideline for Human Recombinant Estrogen Receptor ( hrER ) In Vitro Assays to Detect Chemicals with ER Binding Affinity

11. ANNEX 1 DEFINITIONS :- Acute oral toxicity refers to those adverse effects occurring following oral administration of a single dose of a substance, or multiple doses given within 24 hours. Delayed death means that an animal does not die or appear moribund within 48 hours but dies later during the 14-day observation period. Dose is the amount of test substance administered. Dose is expressed as weight of test substance per unit weight of test animal (e.g. mg/kg). Evident toxicity is a general term describing clear signs of toxicity following the administration of test substance ,

12. GHS: Globally Harmonised Classification System for Chemical Substances and Mixtures. A joint activity of OECD (human health and the environment), UN Committee of Experts on Transport of Dangerous Goods (physical–chemical properties) and ILO (hazard communication) and co-ordinated by the Interorganisation Programme for the Sound Management of Chemicals (IOMC). Impending death: when moribund state or death is expected prior to the next planned time of observation. Signs indicative of this state in rodents could include convulsions, lateral position, recumbence, and tremor. LD50 (median lethal oral dose) is a statistically derived single dose of a substance that can be expected to cause death in 50 per cent of animals when administered by the oral route. The LD50 value is expressed in terms of weight of test substance per unit weight of test animal (mg/kg).

13. Limit dose refers to a dose at an upper limitation on testing (2000 or 5000 mg/kg). Moribund status: being in a state of dying or inability to survive, even if treated. Predictable death: presence of clinical signs indicative of death at a known time in the future before the planned end of the experiment, for example: inability to reach water or food .

14. What is toxicity studies? Toxicity is the degree to which a chemical substance or a particular mixture of substances can damage an organism . Toxicity studies in the animal models are done to determine the dose level recommended for the treatment of disease as drug. This guideline enables the characterization of adverse effects following repeated daily exposure to a test . The US-FDA states that it is essential to screen new molecules for pharmacological activity and toxicity potential in animals. Toxicity testing also helps calculate the No observed adverse effect level(NOAEL) dose and is helpful for clinical trials .

15. The following tests are conducted for toxicity detection. Acute toxicity tests (14 DAYS) Sub acute toxicity tests ( daily dose :- 14 to 28 days) Sub chronic toxicity tests (daily dose :- up to 90 days) Chronic toxicity tests (daily dose :- 6 months and more)

16. Acute toxicity studies The globally harmonized system (GHS), defines it as “ those adverse effects occurring following oral or dermal administration of a single dose of a substance ,or a multiple dose 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 is the rat or rabbit. OBJECTIVES:- To determine the median Lethal dose (LD50 ) after a single dose administered through one or more routes, one of which is the intended route of administration in humans. To determine Maximum tolerated dose(MTD) and no observable effect level (NOEL). To identify potential target organs for toxicity. To help select doses for repeated dose toxicity tests.

17. OECD TEST GUIDELINES WHICH INCLUDE ACUTE SYSTEMIC TESTING Fixed dose procedure (OECD TG-420) Acute toxic class method (OECD TG-423) Up and down procedure (OECD TG- 425) Acute oral toxicity (OECD TG- 401) Acute dermal toxicity (OECD TG- 402) Acute inhalation toxicity (OECD TG-403 )

18. A simple flow of all the guidelines INTRODUCTION INTIAL 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. PRINCIPLE OF THE TEST

19. DESCRIPTION OF METHOD:- Selection of animal species:- Healthy young adult animals of commonly used laboratory strains should be employed . Females should be nulliparous and non-pregnant . Each animal, at the commencement of its dosing, should be between 8 and 12 weeks old and its weight should fall in an interval within + 20 % of the mean weight of any previously dosed animals. Housing and feeding conditions:- The temperature of the experimental animal room should be 22ºC ( + 3ºC). Although the relative humidity should be at least 30% and preferably not exceed 70% other than during room cleaning the aim should be 50-60 %.

20. Lighting should be artificial, the sequence being 12 hours light, 12 hours dark. For feeding, conventional laboratory diets may be used with an unlimited supply of drinking water . Animals may be group-caged by dose, but the number of animals per cage must not interfere with clear observations of each animal . Preparation of animals Preparation of doses . PROCEDURE Administration of doses:- The test substance is administered in a single dose by gavage using a stomach tube or a suitable intubation canula . In the unusual circumstance that a single dose is not possible, the dose may be given in smaller fractions over a period not exceeding 24 hours.

21. Animals should be fasted prior to dosing (e.g. with the rat, food but not water should be withheld over-night; with the mouse, food but not water should be withheld for 3-4 hours). Following the period of fasting, the animals should be weighed and the test substance administered. After the substance has been administered , food may be withheld for a further 3-4 hours in rats or 1-2 hours in mice . Where a dose is administered in fractions over a period of time, it may be necessary to provide the animals with food and water depending on the length of the period. Limit test at 2000mg/kg Limit test at 5000mg/kg Main test

22. 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. Body weight:- Individual weights of animals should be determined shortly before the test substance is administered and at least weekly thereafter. Weight changes should be calculated and recorded . At the end of the test surviving animals are weighed and then humanely killed.

23. Pathology All test animals (including those that die during the test or are removed from the study for animal welfare reasons) should be subjected to gross necropsy . All gross pathological changes should be recorded for each animal. Microscopic examination of organs showing evidence of gross pathology in animals surviving 24 or more hours after the initial dosing may also be considered because it may yield useful information.

24. DATA AND REPORTING:- Data Calculation of LD50 Test report The test report must include the following information, as appropriate:- Test substance:- Physical nature, purity, physicochemical properties. Identification data Vehicle (if appropriate): justification for choice of vehicle, if other than water .

25. Test animals: species/strain used; microbiological status of the animals, number, age and sex of animals source, housing conditions, diet etc. Test conditions:- Details of test substance formulation details of the administration of the test substance including dosing volumes and time of dosing . details of food and water quality (including diet type/source, water source); the rationale for the selection of the starting dose .

26. Results: tabulation of response data and dose level for each animal (i.e. animals showing signs of toxicity including mortality, nature, severity and duration of effects); tabulation of body weight and body weight changes; individual weights of animals at the day of dosing, in weekly intervals thereafter, and at time of death or sacrifice; date and time of death if prior to scheduled sacrifice; time course of onset of signs of toxicity and whether these were reversible for each animal ; Discussion and interpretation of results . Conclusions .

27. ACUTE ORAL TOXICITY (OECD TG401) (Conventional acute toxicity method) In a study of toxic characteristics of substance, acute oral toxicity testing is initial step, gives information on health hazards. Test substance is admistered orally in graduated doses to several group of experimental animals. At least 5 rodents at each dose level of same sex are used. Following the OECD council decision, the test 401’ acute oral toxicity was deleted on 17 th December 2002.

28. Fixed dose procedure (OECD TG-420) A principle of the method is that in the main study only moderately toxic doses are used, and the administration of doses that are expected to be lethal should be avoided. This Guideline is intended primarily for use with rat . Groups of animals of a single sex (normally females) are dosed in a stepwise procedure using the fixed doses of 5, 50, 300 and 2000 mg/kg (exceptionally 5000 mg/kg ). 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 . Further groups of animals may be dosed at higher or lower fixed doses, depending on the presence or absence of signs of toxicity or mortality .

29. This procedure continues until the dose causing evident toxicity or death is identified, or when no effects are seen at the highest dose or when deaths occur at the lowest dose . The test substance is administered in a single dose by gavage using a stomach tube or a suitable intubation cannula. Animals should be fasted prior to dosing. A total of five animals of one sex will normally be used for each dose level investigated. The results of this study include: measurements (weighing at least weekly) and daily detailed observations, as well as gross necropsy . The method provides information on the hazardous properties and allows the substance to be classified for acute toxicity according to the Globally Harmonised System of classification and labelling of chemicals.

30. PROCEDURE Procedure involves two steps 1) Sighting study 2) Main study

31. Acute toxic class method ( OECD TG-423) It is the principle of the test that based on stepwise procedure with the use of minimum number of animals. 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 i.e. - 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

32. PROCEDURE Three animals are used for each step . The dose level to be used as the starting dose is selected from one of four fixed levels , 5, 50, 300 and 2000 mg/kg body weight. The starting dose level should be that which is most likely to produce mortality in some of the dosed animals. The flow charts of Annex 2 describe the procedure that should be followed for each of the starting doses. When available information suggests that mortality is unlikely at the highest starting dose level (2000 mg/kg body weight), then a limit test should be conducted. When there is no information on a substance to be tested, for animal welfare reasons it is recommended to use the starting dose of 300 mg/kg body weight. The time interval between treatment groups is determined by the onset, duration, and severity of toxic signs. Treatment of animals at the next dose, should be delayed until one is confident of survival of the previously dosed animals.

33. Up and down procedure ( OECD TG- 425) PRINCIPLE OF THE MAIN TEST The main test consists of a single ordered dose progression in which animals are dosed, one at a time, at a minimum of 48-hour intervals. The first animal receives a dose a step below the level of the best estimate of the LD50 . If the animal survives , the dose for the next animal is increased by [a factor of] 3.2 times the original dose ; if it dies , the dose for the next animal is decreased by a similar dose progression . (Note: 3.2 is the default factor corresponding to a dose progression of one half log unit) Each animal should be observed carefully for up to 48 hours before making a decision on whether and how much to dose the next animal. That decision is based on the 48-hour survival pattern of all the animals up to that time.

34. Acute dermal toxicity ( OECD TG- 402) This method provides information on health hazard likely to arise from short-term exposure to a test chemical by dermal route. Test chemicals should not be administered at doses that are known to cause marked pain and distress due to potential corrosive or severely irritant actions. Groups of animals, of a single sex, are exposed via the dermal route to the test chemical in a stepwise procedure using the appropriate fixed doses . The initial dose level is selected at the concentration expected to produce clear signs of toxicity without causing severe toxic effects or mortality

35. Further groups of animals may be tested at higher or lower fixed doses, depending on the presence or absence of signs of toxicity or mortality. This procedure continues until the dose causing toxicity or no more than one death is identified, or when no effects are seen at the highest dose or when deaths occur at the lowest dose.   Subsequently, observations of effects and deaths are made. Animals which die during the test are necropsied, and at the conclusion of the test the surviving animals are sacrificed and necropsied.

36. Acute inhalation toxicity (OECD TG-403) PRINCIPLE OF THE TEST :- This Guideline offers two test methods:- The first method is a Traditional protocol in which groups of animals are exposed to a limit concentration (limit test) or a series of concentrations in a stepwise procedure for a predetermined duration of usually 4 hours. Other durations of exposure may apply to serve specific regulatory purposes . The second method is a (C x t) protocol in which groups of animals are exposed to one (limit concentration) or a series of multiple concentrations over multiple durations

37. EXPOSURE CONDITIONS Administration of concentrations Nose-only exposures may be any duration up to 6 hours in rats . If mice are exposed nose-only, exposures generally should not exceed 4 hours . Animals exposed to aerosols in whole-body chambers should be housed individually to prevent ingestion of test article due to grooming of cage mates. Feed should be withheld during the exposure period. Water may be provided throughout a whole-body exposure. Animals are exposed to the test article as a gas, vapour, aerosol, or a mixture thereof .

38. The physical state to be tested depends on the physico -chemical properties of the test article, the selected concentration, and/or the physical form most likely present during the handling and use of the test article. Hygroscopic and chemically reactive test articles should be tested under dry air conditions. Care should be taken to avoid generating explosive concentrations

39. SUBACUTE AND CHRONIC TOXICITY TESTING The Globally Harmonized System (GHS)defines it as "specific target organ/systemic toxicity arising from a repeated exposure " Repeated dose toxicity testing using oral administration of a test substance for 28 and 90 days is used to evaluate chronic toxic effects, primarily effects on various organ systems. Chronic toxicity testing consists of oral, dermal, and inhalation subacute repeated dose studies (28‐day) and subchronic repeated dose studies (90‐day ). The endpoints for repeat dose testing consist of an evaluation of clinical observations, blood analysis, whole body gross necropsy, and microscopic examination of all organs and tissues (histopathology)

40. The objectives of chronic toxicity studies The identification of the hazardous properties of a chemical . The identification of target organs Characterisation of the dose : response relationship , Identification of a no‐observed‐adverse‐effect level (NOAEL) or point of departure for establishment of a Benchmark Dose (BMD), The prediction of chronic toxicity effects at human exposure levels

41. Repeated Dose 28‐day Oral Toxicity Study in Rodents (TG407) Repeated Dose 90‐Day Oral Toxicity Study in Rodents (TG 408) Repeated Dose 90‐Day Oral Toxicity Study in non -Rodents (TG 409) Repeated Dose Dermal Toxicity: 21/28‐day Study (TG 410) Subchronic Dermal Toxicity: 90‐day Study (TG 411) Repeated Dose Inhalation Toxicity: 28‐day or 14‐day Study (TG 412) Subchronic Inhalation Toxicity: 90‐day Study (TG 413) OECD Test Guidelines describe short‐term repeat‐dose toxicity testing:

42. Initial considerations:- The 90- day study provides information on the possible health hazards likely to arise from repeated exposure over a prolonged period of time covering post-weaning maturation and growth into adulthood. Description of the method:- selection of animal species:- Commonly used laboratory strains of young healthy adult animals should be employed. The females should be nulliparous and non-pregnant . Dosing should begin as soon as possible after weaning and, in any case, before the animals are nine weeks old . At the commencement of the study the weight variation of animals used should be minimal and not exceed ± 20 % of the mean weight of each sex. Where the study is conducted preliminary to a long term chronic toxicity study, animals from the same strain and source should be used in both studies.

43. Housing and feeding conditions Preparation of animals:- Healthy animals, which have been acclimated to laboratory conditions for at least 5 days and have not been subjected to previous experimental procedures, should be used . The test animals should be characterised as to species, strain, source, sex, weight. Animals should be randomly assigned to the control and treatment groups. Cages Preparation of doses:- The test compound is administered by gavage ,via the diet or drinking water . The method of oral administration is dependent on the purpose of the study and the physical/chemical properties of the test material. Where necessary, the test substance is dissolved or suspended in a suitable vehicle .

44. It is recommended that, wherever possible, the use of an aqueous solution/suspension be considered first, followed by consideration of a solution/emulsion in oil (e.g., corn oil) and then by possible solution in other vehicles. For vehicles other than water , the toxic characteristics of the vehicle must be known. The stability of the test substance under the conditions of administration should be determined . PROCEDURE Observations:- The observation period should be at least 90 days. General clinical observations should be made at least once a day, preferably at the same time(s) each day, taking into consideration the peak period of anticipated effects after dosing.

45. At least twice daily, usually at the beginning and end of each day, all animals are inspected for signs of morbidity and mortality . Signs noted should include, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g., lacrimation, piloerection , pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g., excessive grooming, repetitive circling ) or bizarre behaviour (e.g., self-mutilation, walking backwards) should also be recorded. Towards the end of the exposure period and in any case not earlier than in week 11,sensory reactivity to stimuli of different types (e.g., auditory, visual and proprioceptive stimuli) assessment of grip strength and motor activity assessment should be conducted . Further details of the procedures that could be followed are given in the respective references. However, alternative procedures than those referenced could also be used

46. Ophthalmological examination:- Ophthalmological examination, using an ophthalmoscope or equivalent suitable equipment , should be made prior to the administration of the test substance and at the termination of the study, preferably in all animals but at least in the high dose and control groups. If changes in the eyes are detected all animals should be examined. Body weight, food/water consumption and food efficiency:- All animals should be weighed at least once a week . Measurements of food consumption should be made at least weekly .

47. If the test substance is administered via the drinking water, water consumption should also be measured at least weekly. Water consumption may also be considered for dietary or gavage studies during which drinking activity may be altered . Haematology:- T he following haematological examinations should be made at the end of the test period and when any interim blood samples may have been collected :- haematocrit, haemoglobin concentration, erythrocyte count, total and differential leukocyte count, platelet count a measure of blood clotting time/potential

48. Clinical biochemistry:- Clinical biochemistry determinations to investigate major toxic effects in tissues and , specifically, effects on kidney and liver, should be performed on blood samples obtained from each animal just prior to or as part of the procedure for killing the animals. Optionally, the following urinalysis determinations could be performed during the last week of the study using timed urine volume collection: appearance, volume, osmolality or specific gravity, pH, protein, glucose and blood/blood cells . In addition, studies to investigate serum markers of general tissue damage should be considered . Other determinations that should be carried out if the known properties of the test substance may, or are suspected to, affect related metabolic profiles include calcium , phosphorus, fasting triglycerides, specific hormones, methaemoglobin and cholinesterase.

49. The following factors might influence the variability and absolute concentration of the hormone determinations : time of sacrifice because of diurnal variation of hormone concentration. method of sacrifice to avoid undue stress to the animals that may affect hormone concentrations. test kits for hormone determinations that may differ by their standard curves

50. Pathology:- 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 with coagulating glands as a whole, uterus, ovaries, thymus, spleen, brain, pituitary gland, and heart of all animal. The following tissues may give valuable indication for endocrine-related effects: gonads (ovaries and testes), accessory sex organs (uterus, cervix, vagina, epididymides, seminal vesicles with coagulation glands, dorsolateral and ventral prostate), pituitary, mammary gland, thyroid gland and adrenal gland

51. Histopathology:- Full histopathology should be carried out on the preserved organs and tissues of all animals in the control and high dose groups . These examinations should be extended to animals of all other dosage groups, if treatment-related changes are observed in the high dose group. All gross lesions should be examined . DATA AND REPORTING:- Data:- Individual data should be provided . Additionally, all data should be summarised in tabular form showing for each test group the number of animals at the start of the test; number of animals found dead during the test or killed for humane reasons and the time of any death or humane kill ; number of animals showing signs of toxicity,

52. a description of the signs of toxicity observed, including time of onset, duration, and severity of any toxic effects; and the number of animals showing lesions, the type of lesions and the percentage of animals displaying each type of lesion. Test report The test report must include the following information : Test substance :- physical nature, purity and physico-chemical properties; identification data. Vehicle :- justification for choice of vehicle, if other than water.

53. Test animals :- species and strain used; number , age and sex of animals; source , housing conditions, diet, etc .; individual weights of animals at the start of the test. Justification for species if not rat . Test conditions :- rationale for dose level selection; details of test substance formulation/diet preparation, achieved concentration, stability and homogeneity of the preparation; details of the administration of the test substance;

54. actual doses (mg/kg body weight/day), and conversion factor fromdiet /drinking water test substance concentration (ppm) to the actual dose, if applicable; details of food and water quality Results :- body weight and body weight changes; food consumption, and water consumption, if applicable; toxic response data by sex and dose level, including signs of toxicity; nature , severity and duration of clinical observations (whether reversible or not ); results of ophthalmological examination; sensory activity, grip strength and motor activity assessments (when available ); haematological tests with relevant base-line values ;

55. clinical biochemistry tests with relevant base-line values; terminal body weight, organ weights and organ/body weight ratios; necropsy findings; a detailed description of all histopathological findings; absorption data if available; statistical treatment of results, where appropriate . Discussion of results . Conclusions .

56. Test Guideline 453: Combined Chronic Toxicity\Carcinogenicity Studies This Test Guideline focuses on exposure via the oral route, the route most commonly used in carcinogenicity studies. The majority of chronic toxicity/carcinogenicity studies are carried out in rodent species. The use of non-rodent species may be considered when available data suggest that they are more relevant for the prediction of health effects in humans. The objectives of chronic toxicity/carcinogenicity studies covered by this Test Guideline include: the identification of the carcinogenic properties of a chemical, namely its potential to induce neoplastic lesions, resulting in an increased incidence of malignant neoplasms or an appropriate combination of benign and malignant neoplasms, and its chronic toxicity,

57. the identification of target organs characterisation of the dose : response relationship, identification of a no-observed-adverse-effect level (NOAEL) or departure point for establishment of a Benchmark Dose (BMD), the prediction of the health effects of a chemical at human exposure levels, provision of data to test hypotheses regarding mode of action

58. SUMMARY Acute toxicity tests (14 DAYS) Sub acute toxicity tests ( daily dose :- 14 to 28 days) Sub chronic toxicity tests (daily dose :- up to 90 days) Chronic toxicity tests (daily dose :- 6 months and more) BASIC FLOW:- INTRODUCTION INITIAL CONSIDERATIONS PRINCIPLE DESCRIPTION PROCEDURE OBSERVATIONS DATA COLLECTION RESULTS CONCLUSION

59. REFERENCES https:// read.oecd-ilibrary.org http:// www.srmuniv.ac.in/sites/default/files/downloads/Acute_Subacute_And_Chronic_ToxicityAnimals.pdf https:// www.slideshare.net/ShitalMagar2/oecd-guidline-on-acute-and-chronic-toxicity https:// outage.niehs.nih.gov/iccvam/suppdocs/feddocs/oecd/oecd_gl420.pdf