This document discusses genotoxicity, which is the study of how chemical and physical agents affect the integrity of genetic material in cells. It covers the history, importance, and regulatory guidelines for conducting genotoxicity tests, including various methodologies and their applications. The content emphasizes the significance of identifying potential human carcinogens and mutagens as part of regulatory requirements for new chemical entities.

1. GENOTOXICITY STUDIES
1
PREPARED BY:SINDHOORA D
FIRST MPHARM
DEPT OF PHARMACOLOGY
SCP,MANGALORE

2. CONTENTS
1. INTRODUCTION
2. HISTORYAND BACKGROUND
3. AIM AND IMPORTANCE
4. REGULATORY GUIDELINES
5. CONCLUSION

3. INTRODUCTION
• Genotoxicity is defined as the study of effects of chemical and physical agents
on a cell's genetic material affecting its integrity.
• Genotoxic agents have intrinsic affinity for DNA the nucleophilicity facilitates
interaction with nucleic acids.
• Unscheduled DNA synthesis, sister chromatid exchanges, DNAstrands
breakup genotoxic measures :not transmissible
• The production of transmissible genetic alteration mutagenicity
• Genotoxicity tests are in vitro and in vivo tests conducted to detect compounds
which induce genetic damage directly or indirectly.
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History & Background
• Origin of genetic toxicology in 1900 , Genetic toxicity independent branch of science started in1927.
• OECD Genetic Toxicology TGs was first published in 1987.
• Major test guidelines: OECD ,ICH, SCHEDULE Y (D&Cindia)
• Tests are rarely used in the various legislative jurisdictions ,better performance methods& new
mammalian cell based tests lead to deletion of few test guidelines.
• Most guidelines are devoid of recommendations for genotoxic-compound act by non-DNAtargets.

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Importance & AIM
• Genotoxicity assays have become an integral component of regulatoryrequirement.
• Compounds which are positive in these tests, have the potential to be human carcinogens and/or mutagens.
• Aim to identify substances that can cause genetic alterations in somatic and/or germcells.
REGULATORYGUIDELINES
• OECD (The Organization for Economic Cooperation and Development) -1961
• ICH ( International Council for Harmonisation ) – 1990
• SCHEDULE Y of Drug and Cosmetic act-1940

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

7. TG TITLE Adopted Revised Deleted
471 Bacterial reverse mutation test (also named Ames test) 1983 1997
472 Genetic toxicology : E.coli: reverse 1983 1997
473 In vitro mammalian chromosomal aberration test 1983 1997/2014/2016
474 Mammalian erythrocyte micronucleus test 1983 1997/2014/2016
475 Mammalian bone marrow chromosomal aberration test 1984 1997/2014/2016
476 In vitro mammalian cell gene mutation test using the hprt orxprt locus 1984 1997/2016
477 Sex-linked recessive lethal test in Drosophila
melanogaster
1984 2013
478 Rodent dominant lethal assay 1984 2015/2016
479 In vitro sister chromatid exchange assay in mammaliancells 1986 2013
480 Saccharomyces cerevisiae, gene mutation assay 1986 2013
481 Saccharomyces cerevisiae, mitotic recombination assay 1986 2013
482 Unscheduled DNA synthesis in mammalian cells in vitro 1986 2013
483 Mammalian spermatogonialchromosome aberration test 1997 2015/2016
484 Mouse spot test 1986 2013
485 Mouse heritable translocation assay 1986
486 Unscheduled DNA synthesis test with mammalian liver cells in vivo 1997
487 In vitro mammalian cell micronucleus test 2010 2014/2016
488 Transgenic rodent somatic and germ cell genemutation
assays
2011
489 In vivo alkaline Comet assay
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2014
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490 In-vitro gene mutation assays using thetk locus 2015

8. TG 471 : AMES TEST (Bacterial reverse mutation
test) R.1997
• Bacteria : 1.Salmonella typhimurium :TA1535; TA1537 or TA97a or TA97; TA98; and TA100
) strains 2. E.coli :WP2 uvrA, WP2 uvrA(pKM101)
• Mutagens cause reverse mutations and bacterial cells there by get the property to grow on the
medium without histidine and form colonies.
Principle
• Suspensions of bacterial cells are exposed to the test substance in the presence and in the
absence of an exogenous metabolic activation system.
• Treatment mixture is incubated and then mixed with an overlay agar plating onto minimal
medium.
• after two or three days of incubation, revertant colonies are counted and compared.
PROCEDURE :2 methods ;
1.Plate incorporation method 2. preincubation method07-11-2017 8

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Pre-incubation method
Pre-incubated with the test strain .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 72 hrs]
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

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• REPORTING
• Test report
• Test substance: Solvent/Vehicle: Strains: Test conditions
• Results:
• - signs 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, if any;
• - concurrent negative (solvent/vehicle) and positive control data, with ranges,
means
• and standard deviations;
• - historical negative (solvent/vehicle) and positive control data, with e.g.
ranges, means and standard deviations

13. TG 487 : INVITRO MAMALIAN CELL
MICRONUCLEUS TEST -2010
Micronuclei is either the whole chromosome or a chromosomal fragment as a
small body outside nucleus
Principle: detection 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, this is easily achieved by scoring only
binucleate cells.
assay detects the activity of clastogenic and aneugenic chemicals
PR07-1O1-2C017EURE : 12

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16. Micronuclei in peripheral lymphocytes
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REPORTING
• cytokinesis-block technique is used, only the frequencies of binucleate cells with
micronuclei: evaluation of micronucleus induction .
• Concurrent measures of cytotoxicity and/or cytostasis for all treated, negative and
positive control cultures should be determined
• Report should include
• Test substance:
• - Solvent/Vehicle: - justification for choice of solvent/vehicle;
• - solubility and stability of the test substance in solvent/vehicle;
• - percentage of vehicle in the final culture medium should also be indicated
• Cells: - type and source of cells used; - suitability of the cell type used;

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TG 474:Mammalian Erythrocyte Micronucleus
Test
PRINCIPLE
• 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.

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PROCEDURE
• Number and sex of animals: 1.treated and 2.control group must include at least 5
analysable animals per sex
• 1, 2, or more treatments at 24 h intervals
•the limit dose has been used, and dosing continued until the time of sampling
also be administered as a split dose.
 2 ways
1.) treated with once. 2.) 2 or more daily treatments
• 1st : Samples of bone marrow =24hr , peripheral blood twice=36hr
• 2nd : bone marrow samples collected once between 18 and 24 hours ,
peripheral blood: sampling between 36 and 48 hours

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• 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]
• Analysis
• at least 200 erythrocytes
• At least 2000 immature erythrocytes per animal are scored for the incidence of
micronucleated immature erythrocytes.
• Result: presented in tabular form , dose-related increase ,
• Statistical methods may be used : statistical significants

21. TG473: INVTROMAMMALIAN CHROMASOMAL ABBERATION
TEST
PRINCIPLE
• with and without metabolic activation
• After exposure of cell cultures , treated with a metaphase-arresting substance
Colcemid® or colchicine
• harvested, stained and metaphase cells are analysed microscopically for the
presence of chromosome aberrations
PROCEDURE
• 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
acti0v7-1a1-t20i1o7 n for 3-6 hours 20

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• sampled at a time equivalent to about 1.5 normal cell cycle length after the
beginning of treatment
• Chromosome preparation: culture treated with Colcemid® or colchicine 3 hr
prior to harvesting , process involves hypotonic treatment of the cells, fixation
and staining.
• 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)

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TG 475: MAMMALIAN BONE MARROW
CHROMOSOME ABERRATION TEST
PRINCIPLE
• Animals are exposed to the test substance , metaphase-arresting agent , sacrificed at
appropriate times after treatment
• Chromosome preparations are then made from the bone marrow cells and stained, and
metaphase cells are analysed for chromosome aberrations.
PROCEDURE
• Number and sex of animals:treated and control group must include at least 5 analysable
animals per sex

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• Treatment schedule: Test substances are preferably administered as a single treatment
also be administered as a split dose ;scientifically justified.
• Samples should be taken at two separate times , first sampling interval is 1.5
normal cell cycle (12-18 hr) , later sample collection 24 hr after the first sample
time
• Prior to sacrifice, animals are injected i.p with an appropriate dose of a
metaphase arresting agent ( Colcemid® or colchicine), 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.

25. potential to inhibit cell cycle progression
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• Analysis: The mitotic index should be determined as a measure of cytotoxicity
in at least 1000 cells per animal.
2n +2.• Scoring :number of centromeres equal to
REPORT
• each animal the number of cells scored, the number of aberrations per cell and
the percentage of cells with structural chromosome aberration(s)
• 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

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TG 483: DRAFT MAMMALIAN
SPERMATOGONIAL CHROMOSOMAL
ABERRATION TEST
PRINCIPLE
• Animals are exposed to the test chemical by an appropriate route of exposure
and are treated with a metaphase-arresting agent then euthanized at appropriate
times after treatment.
• Chromosome preparations are then made from germ cells and stained, and
metaphase cells are analysed for chromosome aberrations.
PROCEDURE
• Number of animals : 5 male animals
• Treatment schedule : Test chemicals are usually administered once.
• one early and one late sampling time approximately 24 and 48 hours after
treatment are used

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• A repeat dose treatment regimen can be used, such as in conjunction with a test on
another endpoint that uses a 28 day administration period.
• metaphase arresting chemical injected intraperitoneally , euthanasia , Animals are
sampled at an appropriate interval thereafter.
• The highest dose may also be defined as a dose that produces some indication of
toxicity in the spermatogonial cells
• Observations: General clinical observations , once a day. health condition , morbidity
and mortality
• Chromosome preparation: After euthanasia, cell suspensions are obtained from
testes,(hypotonic solution ) .The cells spread on slides and stained [blind coded ].
• Analysis :200 well spread metaphases should be scored , Chromosome and chromatid-
type aberrations should be recorded separately and classified

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SCHEDULE –Y
1. Gene mutation in bacteria
2. An in-vitro test with cytogenic evaluation of chromosomal damage
(mammalian cell, in-vitro mouse lymphoma tk assay )
3. An in-vivo test for chromosomal damage using rodent hematopoitic cells
(chromosomal aberration , micronucleus )
4. DNA adduct tests , DNA strands break , DNA repair/recombination

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ICH
• S2A:Guidance on Specific aspects of Regulatory Tests for Pharmaceuticals
• S2B:Genotoxicity: a Standard Battery for Testing of Pharmaceuticals
• M3:Timing of Pre-Clinical Studies in Relation to Clinical Trials.
• TESTS
I. A test for gene mutation in bacteria
II. An invitro test for cytogenetic evaluation of chromosomal damage with
mammalian cells OR An in vitro mouse lymphoma TK assay.
III. An in vivo test for chromosomal damage using rodent haematopoietic
cells

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CONCLUSION
• The development of broad range of short-term assays for genotoxicity serves
to identify many mutagens and their relationship with cancer causing agents
• Genetic toxicology data is essential for regulatory approval of new chemical
entity
• OECD, ICH guidelines are widely used for regulatory approval of new
chemical Entity, internationally. In India SCHEDULE Y , periodic revision of
test Guidelines provides sophisticated methods .

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REFERENCE
1. Essential concepts in toxicology by prof.Dr.Gupta , page no. 139-151
2. Importance of Genotoxicity & S2A guidelines for genotoxicity testing for pharmaceuticals
by Shaily Umang Shah. IOSR Journal of Pharmacy and Biological Sciences (IOSRJPBS)
ISSN : 2278-3008 Volume 1, Issue 2 (May-June 2012), PP 43-54 www.iosrjournals.org.
3. Requirements And Guidelines For Permission To Import And / Or Manufacture Of New
Drugs For Sale Or To Undertake Clinical Trials. Schedule Y(ammended version) – CDSCO
[rules 122A, 122B, 122D, 122DA, 122DAA and 122E] .
file:///G|/RGCB%20IHEC/Schedule%20Y(ammended%20version)%20-
%20CDSCO.htm[08-05-2014 10:53:51]
4. OECD website , www.oecd.org , genotoxicity test guidelines :29 july 2016
OECD Test guidelines; 471,473,474,475,483,487
• Karthik Yamjala, Meyyanathan Subramania Nainar, Nageswara Rao Ramisetti. Ultra high performance liquid
chromatography with tandem mass spectrometry screening and mutagenicity evaluation of photodegradation
products of Carmoisine (E122) dye in a beverage. Journal of Separation Science. 2016; 39:2246-51.