Shital Magar presented on in vitro genotoxicity testing based on OECD guidelines. The presentation covered the objectives of genotoxicity testing, introduction to genotoxicity, history, and details of key in vitro tests including bacterial reverse mutation assay, mammalian cell gene mutation tests, mammalian chromosomal aberration test, and mammalian cell micronucleus test. Parameters and limitations of each test were discussed along with examples of software used to analyze genotoxicity results.

1. Presentation by : Shital Magar
Department of Pharmacology
M.Pharm (II-Sem)
R.C.P. I.P.E.R
INVITRO GENOTOXICITY-
OECD Guidelines

2. 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.
3

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.
4

5. 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
6

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.
7
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
8

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
9

10. 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

11. cell Suspension
Culture
With metabolic
activation
Without
metabolic
activation
Expose
for 4
hrs
Sub Culture
For determining
Genotoxicity
Growth
medium
Mutation by
phenotypic
Expression
Known No. of
cell in medium
With
selective
medium
Without
selective
medium
Mutant
cell
Clonning
Efficacy
Procedure
11

12. 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
13

14. 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 are
the 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
15
TG 473: Mammalian Chromosomal Aberration Test
PRINCIPLE

16. 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. 17

18.  Detection of polyploidy including endoreduplication which
indicates cell cycle perturbation.
 This test is not optimized for detection of aneuploidy
18
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.
19
TG 487: Mammalian cell micronucleus test
PRINCIPLE

20. Cytochalasin B used to block
cyotokinesis and generate binucleate
cells during or after test substance
exposure. 20
Procedure

21. 21

22. 22

23. Automated system that measures
micronucleated cell frequencies
Does not allow identification of
translocation and other complex
chromosomal rearrangement
23
PARAMETERS

24. 24

25. Softwares For Genotoxicity
 Lazar
 CAESAR
 TOPKAT
 HazardExpert
25

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 26

27. 27