This document discusses genetic toxicology and genotoxicity testing. It defines genetic toxicology as the study of agents that can damage DNA and chromosomes. It also defines genotoxicity tests as in vitro and in vivo tests to detect compounds that induce genetic damage. The document outlines the importance of genotoxicity testing and describes some standard tests used, including the Ames test, chromosome aberration test, and micronucleus test. It provides details on procedures for these common genotoxicity assays and discusses recommendations for interpreting genotoxicity test results.

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2. CONTENTS
•DEFNITION
•HISTORY
•IMPORTENCE OF GENOTOXICITY
•GENOTOXICITY TESTS
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3. DEFINITIONS
A. Genetic toxicology is the branch of science that deals with the study of
Agents or substances that can damage the cell’s DNA and chromosomes.
B. Genotoxins are mutagens that can cause genotoxicity leading to the damage
Of DNA or chromosomal material thus causing mutation. Genotoxins can
Include chemical substance as well as radiation.
C. Genotoxicity tests can be defined as in vitro and in vivo tests designed to
Detect compounds that induce genetic damage by various mechanisms.
D. These tests enable hazard identification with respect to damage to DNA and
Its fixation. Fixation of damage to DNA in the form of gene mutations,
Larger scale chromosomal damage.
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4. Genotoxins can be of the following category depending on its effects :
o Carcinogens or cancer causing agents
o Mutagens or mutation causing agents
o Teratogens or birth defect causing agents
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5. HISTORY
i. S2A: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for
Pharmaceuticals
ii. S2B: Genotoxicity: A Standard Battery for Genotoxicity Testing of
Pharmaceuticals
iii. S2(R1): Revision of the S2A and S2B Guidelines which have been merged
As part of the revision
IMPORTENCE OF GENOTOXICITY
a. Genetic toxicology is the branch of science that deals with the study of
Agents or Substances that can damage the cell’s DNA and chromosomes.
b. Genotoxicity tests can be defined as in vitro and in vivo tests designed to
Detect Compounds that induce genetic damage by various mechanisms.
c. These tests enable hazard identification with respect to damage to DNA and
Its Fixation. Fixation of damage to DNA in the form of gene mutations,
Larger scale Chromosomal damage.
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6. AGENTS THAT CAN CAUSE DIRECT OR INDIRECT DAMAGE TO THE
DNA
Reactive oxygen species are known to be genotoxic in nature, thus any chemical or
Substance that may increase the reactive oxygen species (ROS) production.The
Following agents are capable of damaging the DNA Directly or indirectly.
o Electrophilic species that form covalent adducts to the DNA
o Reactive oxygen species
o Ultra violet and ionizing radiations.
o Nucleoside analogues( eg:Abacavir,Adefovir,Didanosine,Stavudine,Zidovudin )
o Topoisomerase inhibitors (eg: Etopodise, Topotecan, Irinotecan)
o Protein synthesis inhibitors (eg:Chloramphenicol.Erythromycin,Clindamycin)
o Some herbal plants like Aconite, Alfa-alfa, Calamus, Aloe vera, Isabghol etc.
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7. ANTIMUTAGENS
Anti-mutagen is any agent that decreases the effect of spontaneous and
Induced mutations. There are mainly two mechanisms of anti-mutagenesis.
•Desmutagenesis
•Bio-antimutagenesis,
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8. The five major pathways through which the cell repair the damaged DNA are
• Direct repair
• Base excision repair( BER)
• Nucleotide excision repair (NER)
•Mismatch repair
•Single/ double strand break repair
GENOTOXICITY STUDIES
THE STANDARD TEST BATTERY FOR GENOTOXICITY
i. Registration of pharmaceuticals requires a comprehensive assessment of their
Genotoxic potential.
ii. It is clear that no single test is capable of detecting all relevant genotoxic
Agents. Therefore, the usual approach should be to carry out a battery of in
Vitro and in vivo tests for genotoxicity.
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9. The general features of a standard test battery are as follows:
i. Assessment of mutagenicity in a bacterial reverse gene mutation test. This test
has been shown to detect relevant genetic changes and the majority of
genotoxic rodent and human carcinogens.
ii. Genotoxicity should also be evaluated in mammalian cells in vitro and/or in
vivo as follows.
The standard test battery for genotoxicity recommends the following for genotoxicity
Evaluation.
TG 471 Bacterial Reverse Mutation Test (Ames Test)
TG 472 Genetic Toxicology: Escherichia coli, reverse assay
TG 473 In-Vitro Mammalian Chromosome Aberration Test
TG 474 Mammalian Erythrocyte Micronucleus Test
TG 475 Mammalian Bone Marrow Chromosome Aberration Test
TG 476 In-Vitro Mammalian Cell Gene Mutation Test
TG 477 Genetic Toxicology: Sex-linked Recessive Lethal Test in Drosophila
Melanogaster
TG 478 Genetic Toxicology: Rodent Dominant Lethal Test
TG 479 Genetic Toxicology: In-Vitro Sister Chromatid Exchange Assay in
Mammalian Cells
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10. 1.Test Repetition and Interpretation
2.Recommended Protocol for the Bacterial Mutation Assay
Selection of Top Dose Level
Study Design/Test Protocol
3.Recommended Protocols for the Mammalian Cell Assays
•Selection of Top Concentration
•Study Design/Test Protocols
 RECOMMENDATIONS FOR IN VITRO TESTS
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11. RECOMMENDATIONS FOR IN VIVO TESTS
Tests for the Detection of Chromosome Damage In Vivo
Other In Vivo Genotoxicity Tests
Dose Selection for In Vivo Assays
Short-Term Studies
Multiple Administration Studies
Testing Compounds that are Toxic for Blood or Bone Marrow
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12. When In Vitro Genotoxicity Tests are Negative
oSampling Times for In Vivo Assays
oNumber of Animals Analyzed
oUse of Male/Female Rodents in In Vivo Genotoxicity Tests
oRoute of Administration
oUse of Positive Controls for In Vivo Studies
When an In Vitro Genotoxicity Test is Positive (or not done)
Demonstration of in vivo exposure should be made by any of the following
Measurements:
i. Cytotoxicity
ii. Exposure
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13. TESTING METHODS
IN-VITRO TESTING METHODS
I. Bacterial reverse mutation test which is otherwise called as Ames test
Whose endpoint is the gene mutations in the bacterial cell
II. Mammalian chromosome aberration test with the end point of
Chromosome aberration
III. Mammalian cell gene mutation test or the mouse lymphoma test whose
End point is the gene mutations
IN-VIVO GENOTOXICITY TESTING METHODS
I. In-vivo comet assay
II. In-vivo micronuclei test/In-vivo chromosome aberration test
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AMES TEST
1.PRE INCUBATION METHOD
Pre incubated with test strain 0.5-0.1ml & sterile buffer or the metabollic activation
system
Usually for 20minutes at 30 to37oc(aeration + shaker 48-72hrs).
Mix overlay agar(2ml) and pouring onto to the surface of a minimal agar plate.
Find number of retardant colonies per plate

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2.PLATE INCORPORATION METHOD

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CHROMOSOMALABBREATION TEST
Each control and test group contain 5 male animals
Animals are exposed to the test substances once or two times in a day
By an appropriate route.
The animals are treating with metaphase arresting agents
Metaphase cells are analyzed for chromosomal abbreation

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MICRONUCLEUS TEST

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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 ways1) treated with once,2.) 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
PROCEDURE

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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.

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REFEENCES
 Guidance for Industry,S2(R1) Genotoxicity Testing and Data Interpretation
For Pharmaceuticals Intended for Human Use.
Guideline for Industry Specific Aspects of Regulatory Genotoxicity Tests
For Pharmaceuticals April 1996.
Guidance for Industry S2B Genotoxicity: A Standard Battery for Genotoxicity Testing of
Pharmaceuticals.
Guidance on genotoxicity testing and data interpretation for pharmaceutical intended
For Human use S2(R1).
 Mohamed SAKS etal.,Genotoxicity: Mechanisms, Testing Guidelines and Methods,
Glob J Pharmaceu Sci ,Volume 1 Issue 5 - April 2017DOI:10.19080/GJPPS.2017.02.555575.

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