OECD Guidlines By Genotoxicity
-
Upload
shital-magar -
Category
Health & Medicine
-
view
32 -
download
0
Transcript of OECD Guidlines By Genotoxicity
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
3
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.
4
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.
5
6
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
7
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
8
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
9
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
10
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
11
12
13
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
15
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
17
18
Detection of polyploidy including endoreduplication which indicates cell cycle perturbation.
This test is not optimized for detection of aneuploidy
PARAMETER
LIMITATIONS
19
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
20
Cytochalasin B used to block cyotokinesis and generate binucleate cells during or after test substance exposure.
Procedure
21
22
23
Automated system that measures micronucleated cell frequencies
Does not allow identification of translocation and other complex chromosomal rearrangement
PARAMETERS
24
25
Softwares For Genotoxicity
Lazar
CAESAR
TOPKAT
HazardExpert
26
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
27