Presentation by : Shital Magar Department of Pharmacology

M.Pharm (II-Sem)R.C.P. I.P.E.R

INVITRO GENOTOXICITY- OECD Guidelines

Objectives

To achieve sustainable economic growth and employment and

rising standards of living

To maintain financial stability

To assist sound economic expansion

To contribute to growth in world trade on a multilateral, non-

discriminatory basis 2

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Introduction to Genotoxicity

Genotoxicity is a word in genetics defined as a destructive effect on a

cell's genetic material (DNA, RNA) affecting its integrity. Genotoxins are

mutagens; they can cause mutations. Genotoxins include both

radiation and chemical genotoxins. A substance that has the property

of genotoxicity is known as a genotoxin.

Genetic information, encoded chemically in DNA, is maintained,

replicated and transmitted to successive generations with high fidelity.

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History of Genotoxicity

The role of radiation in producing heritable changes in a

living organism was first reported by Muller (Muller, 1927).

Auerbach was the first to report the ability of chemicals

to cause mutations (Auerbach et al.,1947).

“Gregor Mendel, Father of Genetics”

Discovered that plants receive Genetic

information from its parents passed down

through generations, on pea plant in 1856.

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In-Vitro Genetic Toxicology

Tests for gene mutation

TG 471: Bacterial reverse mutation test

TG 476: Mammalian cell gene mutation test using Hprt /

Xprt / Thymidine kinase Test for chromosomal abnormalities

TG 473: Mammalian chromosomal aberration

test

TG 487: Mammalian cell micronucleus test

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Identifies substances that induce gene mutations by base

substitutions or frameshifts.

Two species of bacteria Salmonella typhimurium and Escherichia

coli with identified mutations in an amino acid i.e His or Trp as

the reporter locust.

Detects mutations which revert mutations present in the test

strains and restore the functional capability of the bacteria to

synthesize an essential amino acid.

TG 471: Bacterial Reverse Mutation Test PRINCIPLE

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Ames test procedure

Maximum test concentration is 5 mg/plate or 5 ml/plate.

Two methods: The plate incorporation method and The pre-incubation method

For both techniques incubate at 37°C for two or three days

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Large number of cell can be exposed

There have been developments to

automate the test and minimize the use of

test substances

Highest induced mutant frequency can

be detected

ADVANTAGES

Relativetly sensitive and reliable detection of

compound

Parameters

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In the cell lines the genetic endpoints measure mutation at

thymidine kinase (TK) and hypoxanthine-guanine

phosphoribosyl transferase (HPRT) and a transgene of

xanthineguanine phosphoribosyl transferase (XPRT).

Hprt is present on X-chromosome, various cell lines of Hprt

are CHO, CHL, V79 of Chinese hamster cells, etc.

Xprt coded by gpt transgene, AS52 cell lines are used.

TG 476: Mammalian cell gene mutation test

PRINCIPLE

cell Suspension Culture

With metabolic activation

Without metabolic activation

Expose for 4 hrs

Sub CultureFor determining Genotoxicity

Growth medium

Mutation by phenotypic Expression

Known No. of cell in medium

With selective medium

Without selective medium

Mutantcell

Clonning Efficacy

Procedure

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The cell density in culture plate should be limited in order to avoid metabolic co-operation between mutant and non-mutant cells which would alter mutant selection.

This is particularly important for cells growing monolayer rather than suspension.

PARAMETER

LIMITATIONS

Tests for Chromosomal Aberrations

Chromosomal Aberrations

Endpoints

Micronuclei

Cells are not viable and aberrations are not transmitted to daughter cells.

Micronuclei are visualized in cells following the first cell division

Visualized under a microscope.

Nonviable chromosomal aberrations arethe basis for dominant lethal mutations resulting in fetal loss

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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

G-2 active substance induces chromatid aberration at 1st mitosis but

many of its events get converted into chromosome aberration in 2nd

mitosis

Damage induced pre-S-phase ---------- chromosome aberration Damage induced post-S-phase ---------- chromatid aberration

TG 473: Mammalian Chromosomal Aberration Test

PRINCIPLE

Cell strains

Test substance

3 concentration of test substance

Duplicate culture during each concentration

Finally treated with m-phase arresting

substances

Harvestd and

stained

1.5 normal cell cycle lenght

Cell Culture

Cell lines

Procedure

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Detection of polyploidy including endoreduplication which indicates cell cycle perturbation.

This test is not optimized for detection of aneuploidy

PARAMETER

LIMITATIONS

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Cell lines: CHO, V79, CHL, L5178Y, TK6 or primary human or other mammalian peripheral blood lymphocyte.

For the detection of micronuclei in the cytoplasm of interphase cells.

Micronuclei may originate from acentric chromosome fragments (i.e. lacking a centromere), or whole chromosomes that are unable to migrate to the poles during the anaphase stage of cell division.

The assay detects the activity of clastogenic and aneugenic test substances in cells that have undergoes cell division during or after exposure to the test substance.

TG 487: Mammalian cell micronucleus test PRINCIPLE

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Cytochalasin B used to block cyotokinesis and generate binucleate cells during or after test substance exposure.

Procedure

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Automated system that measures micronucleated cell frequencies

Does not allow identification of translocation and other complex chromosomal rearrangement

PARAMETERS

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Softwares For Genotoxicity

Lazar

CAESAR

TOPKAT

HazardExpert

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REFERENCES

Combes RD, Gaunt, I, Balls M (2004). A Scientific and Animal Welfare Assessment of

the OECD Health Effects Test Guidelines for the Safety Testing of Chemicals under

the European Union REACH System. ATLA 32, 163-208.

NRC (2007).

Toxicity Testing in the 21st Century: A Vision and a Strategy. Washington, DC: The

National Academies Press.

www.oecd.org

Genetic toxicology guidance document,2015 Hacettepe University, Faculty of

Pharmacy, Department of Toxicology, Ankara

Department of Environmental Biotechnology, University of Warmia and Mazury in

Olsztyn, ul. Sloneczna 45G, 10–712 Olsztyn, Polan

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