The document provides information about genotoxicity testing. It discusses: 1) The origins and development of genetic toxicology as a field of science and the major guidelines for testing like OECD, ICH, and Schedule Y. 2) Standard tests for genotoxicity including the Ames test, micronucleus assay, and comet assay. 3) Mechanisms of action by which genotoxic substances can damage DNA and mechanisms like point mutations and chromosomal mutations. 4) The importance of genotoxicity studies in identifying substances that can damage genetic material.

1. GENOTOXICITY
MINIATURES : LIFE

2. GROUP MEMBERS:
UKA TARSADIA UNIVERSITY
HIMANSHU PATEL 78
VISHAL RATIYA 85
ZEEL DHOLAKIA 100
BRINDA DAVE 21
ABHISHEK GANDHI 22
KHUSHALI NAIK 35
MEGHA MODI 13
JEET DESAI
54
ADITI MISTRY
56
SHREYA PARIKH 108
FREYA PATEL 111
DEEP GORASIYA 132
CLASS- I
CLASS- II

3. HISTORY AND
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.
• OECD, ICH, SCHEDULE Y (D&C India)
3

4. MAJORTESTS &
GUIDELINES:
• OECD (The Organization for Economic Cooperation
and Development)-1961
• ICH (International Council for Harmonization)-1990
• SCHEDULE Y OF Drug and Cosmetics Act-1940
4

5. INTRODUCTION:
• Genotoxicity is a word used in genetics
that describes the possession of
substance that has destructive effect on
the genetic material of the cell (DNA,
RNA), thus affecting the integrity of the
cell.
• Genotoxins are mutagens that can
cause genotoxicity leading to damage
of DNA or chromosomal materials thus
causing mutations.
• Genetic Toxicology is the branch of
science that deals with study of agents
or substance that can damage the cell’s
DNA chromosome.
5

6. RISK ASSOCIATIONS:

7. IMPORTANCE OF GENOTOXIC STUDIES:
7
• Genotoxicity studies can be defined as various in-vitro and in-vivo tests designed
to identify any substance or compounds which may induce damage to genetic
material either directly or indirectly by various mechanisms.
• These tests should enable the identification of hazard with respect to DNA
damage and fixation.

8. GENOTOXINS CAN BE OF THE FOLLOWING CATEGORY
DEPENDING ON ITS EFFECTS:
1. Reactive Oxygen
Species
2. UV and Ionizing
Radiations
3. Nucleoside Inhibitors
4. Topoisomerase
Inhibitors
5. Protein Synthesis
Inhibitors
8
1. Carcinogens or Cancer
Causing Agents.
2. Mutagens or Mutation
Causing Agents.
3. Teratogens or Birth
Defect Causing Agents.
AGENTS WHICH CAN CAUSE DIRECT OR INDIRECT
DAMAGE TO THE DNA:

9. MECHANISM OF ACTION:
9
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 as a specific location
or base sequence of the DNA structure
causing lesions, breakage, fusion,
deletion, mis-segregation or non-
disjunction leading to damage or
mutation.

10. 10

11. STANDARDTEST BATTERY FOR GENOTOXICITY:
• 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 Gene Mutation Test
• TG 477 Genetic Toxicology Sex-Linked Recessive Lethal Test in Drosophila Melanogaster
• TG 479 Genetic Toxicology: In-Vitro Sister Chromatid Exchange Assay in Mammalian Cells
• TG 480 Genetic Toxicology: Saccharomyces cerevisiae, Gene Mutation Assay
11

12. • TG 481 Genetic Toxicology: Saccharomyces cerevisiae, Mitotic Recombination Assay
• TG 482 Genetic Toxicology: DNA damage and Repair, Unscheduled DNA Synthesis in
Mammalian Cells In-Vitro
• TG 483 Mammalian Spermatogonial Chromosome Aberration Test
• TG 484 Genetic Toxicology: Mouse Spot Test
• TG 485 Genetic Toxicology: Mouse Heritable Translocation Assay
• TG 486 Unscheduled DNA Synthesis (UDS) Test with Mouse Liver Cells In-Vitro
• TG 487 In-Vitro Mammalian Cell Micronucleus Test.
12

14. TG 471: AMES
TEST
(BACTERIAL
REVERSE
MUTATION
TEST)
14

15. • Ames Test was brought forward
by Bruce Ames in 1970
• He was a professor in University
of California, Berkley in
department of Biochemistry
• He developed this method
because previous methods were
expensive and time consuming.
15

16. PLATE INCORPORATION METHOD:
(STEPS FOR AMESTEST)
• Prepare the culture of Salmonella histidine Auxotrophs (His-)
• Mix the bacterial cells and the 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.
• 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.

17. 17

18. • 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.
• 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.
18

19. PRE-INCUBATION METHOD:
• Pre-incubated with the test strain.
• 0.05-0.1 ml (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 colonies nos)
• Standard deviation
19

20. Negative Positive
Ames Test Ames Test
20

21. COMET
ASSAYTEST:

22. PRINCIPLE:
The basis for this assay is that loops of DNA
containing a break, lose their super coiling
and become free to extend toward the anode
when exposed to current during
electrophoresis at high pH. The results
appear as structures resembling comets
observed by fluorescence microscopy.

23. 23

25. TG 474: MAMMALIAN ERYTHROCYTE
MICRONUCLEUS TEST:
25

26. • 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 analyzed for the presence of micronuclei.

27. 27

28. 28
Animals are exposed to the test substance by an appropriate route.
Bone marrow and/or blood cells are collected, prepared and stained
Preparations are analyzed for the presence of micronuclei.
Each treated and control group must include at least 5 analyzable
animals per sex.
Administration of the treatments consists of a single dose or two daily
doses (or more).
The limit dose is 2000 mg/kg/body weight/day for treatment up to 14
days, and 1000 mg/kg/body weight/day for treatment longer than 14
days.

29. FISH AS A MODEL FORTHE AQUATIC GENOTOXICITY:
• Aquatic animal specially fishes are most susceptible to genotoxic effect caused by
the pollutants usually agricultural wastes, chemicals, heavy metal etc.
• The selection of fishes as a model in the eco-genotoxicological studies
could be made since fish is a very sensitive bio- indicator of water
quality and can highlight the potential danger of new chemical
introduced in the aquatic environment and also respond in a manner
similar to higher vertebrates.
• They have greater ability to metabolise xenobiotics and accumulate pollutants.
29

30. • They are capable of inhabiting practically all zones of the aquatic habitat and
have great Commercial and recreational value.
• They play different roles in the tropic web such as undergoing
bio-accumulation of environment pollutants and biotransformation
of xenobiotics through cytochrome 450- dependent oxidative metabolism like mammals,
Besides, they respond to mutagens at low concentration.
• In Addition as compared to mammalian cells, they have been shown to be more
sensitive for the induction of DNA damage, there for they can used as sentinel
organism for bio-monitoring studies.
30

31. GENOTOXICITY DUETO HEAVY METALS:
31
• Aquatic ecosystem receive a number of toxic substances, among which heavy metals released from
domestic, industrial and other man-made activities are the significant importance due to their toxicity,
their bioaccumulation potential and their ability to induce damage in DNA.
• The studies carried out on various fishes have
shown that
these metals alter the physiological activities and
biochemical parameters both in tissues and blood.
The mercury toxicity for clarias batrachus resulted
in marked decrease in haemoglobin and
Erythrocyte count.

32. GENOTOXICITY
DUETO
MICROBIAL
TOXINS:
Ricardo et.al (2010) evaluated
the toxicity and genotoxicity in
Astyanax bimaculatus ‚as
induced by an extract of
cyanobacterial microcystins
,using two administration routes
and different end points, such as
micronucleus and Apoptosis-
necrosis testing and comet
assaying.
32

33. The genotoxicity caused by the
microcystins LR and LA
from a bloom collected in a
eutrophic lake LC50 (72 h) was
determined as 242.81 Mg L-1 and
LD50 (72 h) as 49.19 mg kg-1 bw.
There observed a significant
increase of DNA damage
in peripheral erythrocytes
LC50 was determined as
242.81.
33

34. MUTAGENICITY:

35. • Mutagenicity refers for the induction of mutations by mutagens through permanent transmissible
changes in the amount or structure of the genetic material of cells or organism.
• These changes may involve a single gene or gene segment, a block or genes and chromosomes.
The genetic change is referred to as a mutation and the agent causing the change as a mutagen.
• Mutagens: The agents which cause mutation:
1. Radioactive substance
2. Radiation
3. Chemicals (Benzopyrene , Pyrolysed Proteins)
4. Certain anticancer drugs

36. TARGET CELLS FOR MUTATION:
• Mutations can either occur in Germ cells or Somatic cells
• If the mutation occurs in the germ cell (sperm and ova)the effect is heritable. There is no
the exposed person; rather the effect is passed on to future generations. Toxicity to germ
cells can cause effects on the developing foetus (such a birth defects , abortions).
• If the mutation occurs in somatic cell, it can cause altered cell growth (e.g cancer) or cell
death (e.g. teratogenesis) in the exposed person. Toxicity to somatic cells causes a variety
of toxic effects to the exposed individual (such as dermatitis , death ,and cancer).
36

37. CLASSES OF MUTATION:
• Spontaneous mutation: They are
mainly caused during DNA replication or
by incorporation of incorrect nucleotide
in the growing DNA chain. They occur by
change in DNA sequence.
• Induced mutation: They are caused by
the changes in DNA brought by some
environmental factors called mutagens.
37

38. CHROMOSOMAL
MUTATION:
Changes in structure by either gain or loss
• Deletion: Due to breakage, a chromosome is
lost
• Inversion: Chromosomes segment breaks offs
and reattaches.
• Duplication: Occurs when a gene sequence is
repeated.
• Translocation: Involves two chromosomes
that are not homologous and a part of one is
transferred to another chromosome.
• Nondisjunction: Failure of chromosomes to
separate during meiosis.
38

39. • Point Mutation: Change in single nucleoside e.g.
sickle cell anaemia.
• Frame Shift: Insertion or deleting one or more
nucleotides.
Changes the reading frame like changing a sentence.
39

40. THANK YOU