Genotoxicity studies according to oecd guildline.

Detail about Genotoxicity studies according to oecd guildline.

1. KARNATAKA COLLEGE OF PHARMACY (Department of pharmacology) PHARMACOLOGICAL AND TOXICOLOGICAL SCREENING METHODS PRESENTATION TOPIC : Genotoxicity studies SUBMITTED TO : DR NAGARATNA P.K.M PREPARED BY : DIANA MORIA

2. Genotoxicity Genotoxicity tests can be defined as in vitro and in vivo tests designed to detect compounds that induce genetic damage by various mechanisms. These tests enable hazard identification with respect to damage to DNA and its fixation.

3. Genotoxins can be of the following category depending on its effects 1 ) Carcinogens or cancer causing agents 2)Mutagens or mutation causing agents 3)Teratogens or birth defect causing agents

4. Agents that can cause direct or indirect damage to the DNA Reactive oxygen species. UV and ionizing radiations. Nucleoside analogues . Topoisomerase inhibitors . Protein synthesis inhibitors .

5. History & Background Origin of genetic toxicology in 1900 , Genetic toxicity independent branch of science started in 1927 . OECD Genetic Toxicology TGs was first published in 1987. Major test guidelines: OECD ,ICH, SCHEDULE Y (D&C India).

6. OECD GUIDELINES Genetic Toxicology : was first published in 1987 .Following a global update of the Genetic Toxicology [1997,2013,2014,2015,2016] Latest revision provides : ( 1) general background and historical information on the OECD genetic toxicology. ( 2) a brief overview of the important types of genetic damage evaluated by these tests. ( 3) a description of the specific tests.

7. SCHEDULE –Y Gene mutation in bacteria An in-vitro test with cytogenic evaluation of chromosomal damage . An in-vivo test for chromosomal damage using rodent hematopoietic cells (chromosomal aberration , micronucleus ). DNA adduct tests , DNA strands break , DNA repair /recombination .

8. ICH • S2A:Guidance on Specific aspects of Regulatory Tests for Pharmaceuticals . • S2B: Standard Battery for Testing of Pharmaceuticals • M3:Timing of Pre-Clinical Studies in Relation to Clinical Trials.

9. Importance Genotoxicity assays have become an integral component of regulatory requirement. C ompounds which are positive in these tests, have the potential to be human carcinogens and/or mutagens. so it’s used in prediction.

10. Aim T o identify substances that can cause genetic alterations in somatic and/or germ cells. To identify substances that causes genetic alterations and thus use this information in regulatory decisions.

11. Mechanism of G enotoxicity The damage to the genetic material is caused by the interactions of the genotoxic substance with the DNA structure and sequence. These genotoxic substance interact at a specific location or base sequence of the DNA structure causing lesions, breakage, fusion, deletion, mis -segregation or non- disjunction leading to damage and mutation .

12. Standard test battery for genotoxicity

13. TG 471 : AMES TEST (Bacterial reverse mutation test ) Bacteria : Salmonella typhimurium or strains E.coli .

14. Ames test was brought forward by Bruce Ames in 1970 . He is professor in university of California , berkely . In department of biochemistry. He developed this method because previous methods were expensive and time consuming.

15. Principle Identifies substances that induce gene mutations by base substitutions or frame-shifts. Two species of bacteria Salmonella typhimurium and Escherichia coli with identified mutations in an amino acid i.e. His or Trp as the reporter locus. It detects mutations which revert mutations present in the test strains and restore the functional capability of the bacteria to synthesize an essential amino acid

16. PROCEDURE 2 methods : 1.Plate incorporation method. 2. pre-incubation method.

17. STEPS OF AMES TEST: Prepare the culture of Salmonella histidine auxotroph's (His-). Mix the bacterial cells and test substance in dilute molten top agar with a small amount of histidine in one set, and control with complete medium plus large amount of histidine . Pour the molten mixture on the top of minimal agar plates and incubate at 37°C for 2-3 days.

18. Until histidine is depleted all the His- cells will grow in the presence of test mutagen. When the histidine is completely exhausted only the revertants will grow on the plate. High number of colonies represent the greater mutagenicity

19. There are some chemicals that are non mutagenic , but they become mutagenic when they come in contact in body metabolism. Bacteria does not have metabolizing capacity, therefore, the liver extract is added to this test, to promote transformation And then the bacterial sample is inoculated. But from the idea the negative test will not show any growth, but the growth can occur due to spontaneous mutation , this is the only limitation of this test.

20. Pre-incubation method Pre-incubated with the test strain . 0.05-0.1ml (approx. 108 cells) & sterile buffer or the metabolic activation system (s9 0.5 ml) usually for 20 min @30-37°c [aeration+ shaker – 48 to 72hrs ] Mix overlay agar (2ml) and pouring onto the surface of a minimal agar plate Report number of revertant colonies per plate ( with + ve & - ve coloies nos ) Standard deviation

21. Spontaneous mutation can give us false positive result. So to understand the difference between spontaneous mutation and mutagenicity. If the bacterial colony are obtained scattered they show that bacteria is there because of spontaneous mutation. And if the colony are aggregated they show that the mutagenicity is brought forward by the chemical mutagens.

22. REPORTING Test substance. Solvent/Vehicle . Strains. Test conditions .

23. Results: S igns of toxicity . signs of precipitation . individual plate counts . the mean number of revertant colonies per plate and standard deviation . dose-response relationship, where possible. Statistical analyses

24. TG 487 : INVITRO MAMALIAN CELL MICRONUCLEUS TEST -2010 Micronuclei is the small nucleus that forms whenever a chromosome or its fragment is incorporated with daughter nuclei during cell division .

25. principle Micronuclei are the product of fragmented chromosomes or mitotic spindle failure in a cell. Micronuclei are formed by condensation of acentric chromosomes that are not included in the main nuclei following the anaphase. Micronuclei are formed in the cytoplasm through the following events: In anaphase, a chromatid and chromosomal fragments lag behind when the centric elements move towards the spindle poles. Micronucleus arises from chromosomal fragments or acentric chromosomes that are not incorporated into daughter nuclei at mitosis because they lack a centromere .

26. Micronuclei appear as separate, small nucleus in the cytoplasm in addition to the main nucleus of the cell. Since micronuclei cannot be observed until after the first cell cycle, the frequencies of micronuclei within a cell population are highly dependent on the kinetics of cell proliferation.

27. D etection of the frequency of micronuclei: Cell cultures of human or other mammalian origin are exposed to the test chemical, formation of micronuclei in interphase cells. Harvested and stained interphase cells are analysed for the presence of micronuclei , treated with a cytokinesis blocker. assay detects the activity of clastogenic and aneugenic chemicals .

28. REPORTING • Report should include : Percentage of vehicle in the final culture medium should also be indicated . Cells: - type and source of cells used.

29. TG 474:Mammalian Erythrocyte Micronucleus Test Animals are exposed to the test substance by an appropriate route If bone marrow > the animals are sacrificed, bone marrow extracted, and preparations made and stained If peripheral blood > the blood is collected at appropriate times after treatment and smear preparations are made and stained. Preparations are analysed for the presence of micronuclei

30. Principle For the detection of damage induced by the test substance to the chromosomes or the mitotic apparatus of erythroblasts (rodents) Identifies micronuclei containing lagging chromosome fragments or whole chromosomes. An increase in the frequency of micronucleated polychromatic erythrocytes in treated animals is an indication of induced chromosome damage because they lack main nucleus

31. TG 473: INVITRO MAMMALIAN CHROMASOMAL ABBERATION TEST PRINCIPLE After exposure of cell cultures , treated with a metaphase-arresting substance colchicine . with and without metabolic activation harvested, stained and metaphase cells are analysed microscopically for the presence of chromosome aberrations .

32. Cell lines: CHO, CHL, V79, TK6. Structural aberrations may be of two types: chromosome or chromatid. Observed only in metaphase of 1st or 2nd mitotic division after treatment. Damage induced pre-S-phase ---------- chromosome aberration . Damage induced post-S-phase ---------- chromatid aberration .

33. Treatment of test with lymphocytes started at about 48 hours after mitogenic stimulation. negative/solvent control cultures Cells should exposed to the test substance both with and without metabolic activation for 3-6hrs sampled at a time equivalent to about 1.5 normal cell cycle length after the beginning of treatment.

34. Chromosome preparation: culture treated with Colcemid ® or colchicine 3 hr prior to harvesting , process involves hypotonic treatment of the cells, fixation and staining.

35. Analysis: should be independently coded before microscopic analysis. . At least 200 well-spread metaphases should be scored per concentration and control. it is important to record polyploidy and endoreduplication when these events are seen. Treatment of results: % of cells with structural chromosome aberration(s) .

36. TG 475: MAMMALIAN BONE MARROW CHROMOSOME ABERRATION TEST principle For the detection of structural chromosome aberrations induced by test compounds only in bone marrow cells of animals (rodents). Animals are exposed to the test substance , metaphase-arresting agent , sacrificed at appropriate times after treatment.

37. Bone marrow cells are usually obtained from the femurs or tibias immediately after sacrifice , and stained using established methods. Blood :tail vein or other appropriate blood vessel , smear preparations are made and then stained DNA specific stain [ e.g acridine orange or Hoechst 33258 plus pyronin -Y]

38. Prior to sacrifice, animals are injected i.p with an appropriate dose of a metaphase arresting agent , sampled thereafter. Cells are harvested from the bone marrow and analysed from chromosome aberrations. Chromosome preparation: bone marrow in hypotonic solution , spread on slides and stained

39. Analysis The mitotic index should be determined as a measure of cytotoxicity in at least 1000 cells per animal.

40. scoring • Different types of structural chromosome aberrations should be listed with their numbers and frequencies for treated and control groups. • Biological relevance of the results should be considered first. Statistical methods may be used as an aid in evaluating the test results • An increase in polyploidy may indicate that the test substance has the potential to induce numerical chromosome aberrations • An increase in endoreduplication may indicate that the test substance has the potential to inhibit cell cycle progression

41. REFERENCE 1 . Essential concepts in toxicology by prof.Dr.Gupta . 2 . Shaily Umang Shah . Importance of Genotoxicity & S2A guidelines for genotoxicity testing for pharmaceuticals .IOSRJPBS.2012 1(2) 43-54 . 3. OECD website , www.oecd.org , genotoxicity test guidelines : 2016 OECD Test guidelines; 471,473,474,475,483,487 .

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