It is a brief power point presentation about the types of Mutagenicity tests and about AMES Test.

1. S. Archana
(Reg no. 22701502)
I MSc Biochemistry
V.V.Vanniapermual college for
women, Virudhunagar.

2. contents
 Introduction
 Types of mutagenicity tests
 Molecular level
 Gene level
 Chromosomal level
 Three important tests
 AMES Test
 HPRT Test
 Mouse Micronucleus Test

3. Introduction
 The purpose of mutagenicity testing is to
identify substances that can cause genetic
alterations in somatic and/or germ cells and use
this information in regulatory decisions.
 Mutagenicity testing is the first step to screen the
chemicals for their potential to be a pesticide,
food additive, or drug.
 The most widely used mutation test is Ames test,
developed by Ames, which is performed in different
strains of Salmonella typhimurium and in Escherichia
coli.

4.  The most widely used mutation test is Ames test,
developed by Ames, which is performed in different
strains of Salmonella typhimurium and in Escherichia
coli.

5. Types of mutagenicity tests
 It can be separated on the basis of
1)Molecular level
2)gene level
3) chromosomal level
1)Molecular level:
Comet assay –
The comet assay (single-cell gel electrophoresis) is a
simple method for measuring deoxyribonucleic acid (DNA)
strand breaks in eukaryotic cells.
Cells embedded in agarose on a microscope slide are
lysed with detergent and high salt to form nucleoids containing
supercoiled loops of DNA linked to the nuclear matrix.

7. 2) Gene level test
TK Gene Mutation test :
The in vitro mammalian cell gene mutation
test (OECD 490), also referred to as the mouse
lymphoma assay, is used to detect a spectrum of
genetic events denoting gene mutations induced by
chemical substances in the cell lines that measure
mutation at thymidine kinase (TK).

9. In vivo pig gene mutation assay:
The Pig-a gene mutation assay has emerged
as a valuable tool for quantifying in vivo and in
vitro mutational events. The Pig-a locus is located at
the X-chromosome, giving the advantage that one
inactivated allele can give rise to a mutated phenotype,
detectable by multicolour flow cytometry.

11. 3)Chromosomal level test
In Vivo Micronucleus Assay:
The micronucleus test (MNT) is used to determine if a
compound is genotoxic by evaluating the presence of micronuclei.
Micronuclei may contain chromosome fragments produced from
DNA breakage (clastogens) or whole chromosomes produced by
disruption of the mitotic apparatus (aneugens).
SCE assay:
It is a powerful technique to visually detect the physical
exchange of DNA between sister chromatids. SCEs could result
as a consequence of DNA damage repair by homologous
recombination (HR) during DNA replication.

12. Micronucleus assay SCE

13. Important tests
The three most commonly conducted assays are:
1)The Ames Salmonella typhimurium reverse mutation
assay,
2)The Chinese hamster ovary (CHO) hypoxanthine-
guanine phosphoribosyltransferase (HGPRT) in vitro
cytogenetics assay, and
3)The mouse micronucleus test .

15. AMES TEST
Ames test it is a biological assay to assess the
mutagenic potential of chemical compounds. It
utilizes bacteria to test whether a given chemical can
cause mutations in the DNA of the test organism. The
test was developed by Bruce N. Ames in 1970s to
determine if a chemical at hand is a mutagen.
Objective
To determine the mutagenic activity of
chemicals by observing whether they cause mutations
in sample bacteria.

16. Principle
 Ames test uses several strains of bacteria (Salmonella, E.coli) that
carry a particular mutation.
 Point mutations are made in the histidine (Salmonella
typhimurium) or the tryptophan (Escherichia coli) operon,
rendering the bacteria incapable of producing the corresponding
amino acid.
 These mutations result in his- or trp- organisms that cannot
grow unless histidine or tryptophan is supplied.
 But culturing His- Salmonella is in a media containing certain
chemicals, causes mutation in histidine encoding gene, such
that they regain the ability to synthesize histidine (His+). This is
to say that when a mutagenic event occurs, base substitutions or
frameshifts within the gene can cause a reversion to amino acid
prototrophy. This is the reverse mutation.
 These reverted bacteria will then grow in histidine- or
tryptophan-deficient media, respectively.

17.  A sample’s mutagenic potential is assessed by exposing
amino acid-requiring organisms to varying
concentrations of chemical and selecting for the
reversion event. Media lacking the specific amino acid
are used for this selection which allow only those cells
that have undergone the reversion to histidine /
tryptophan prototrophy to survive and grow. If the test
sample causes this reversion, it is a mutagen.

18. Method:
I ) Isolate an auxotrophic strain of Salmonella Typhimurium for
histidine. (ie. His-ve)
II) Prepare a test suspension of his-ve Salmonella Typhimurium in a
plain buffer with test chemical (eg. 2-aminofluorene). Also add a
small amount of histidine.
Note: small amount of histidine is required so bacteria starts growing.
Once histidine is depleted only those bacteria mutated to gain the
ability to synthesize histidine form colonies.
III) Also prepare a control suspension of His-
ve Salmonella Typhimurium but without test chemicals.
IV) Incubate the suspensions at 37°C for 20 minutes
V) Prepare the two agar plate and spread the suspension on agar plate.
VI) Incubate the plates at 37°C for 48 hours.
VII) After48 hours count the number of colonies in each plate.

20. Result Interpretation
 The mutagenicity of chemicals is proportional to number of
colonies observed.
 If there is a large number of colonies on the test plate in
comparison to control, then such chemical are said to be
mutagens.
 Very few numbers of colonies can be seen on control plate also.
This may be due to spontaneous point mutation on hisidine
encoding gene.
Uses
1)While Ames test is used to identify the revert mutations
which are present in strains, it can also be used to detect the
mutagenicity of environmental samples such as drugs, dyes,
reagents, cosmetics, waste water, pesticides and other substances
which are easily solubilized in a liquid suspension.
2)AMES test are used in pharmacy research before a
compound given to a animal.

21. 3)Smoker Urine test for mutagenicity is possible by the
use of AMES test.
4)Primary DNAdamage test can be done through
AMES test.
5)This test is highly sensible for testing the
mutagnicity of water and solid soil sample from the
nuclear.
Merits:
1)Simple, rapid and robust bacterial assay.
2)Ease and low cost of the test make it invaluable for
screening substances in our environment for possible
carcinogenicity.
3)Ames test can detects suitable mutants in large
population of bacteria with high sensitivity.

22. limitation
1)Some substances that cause cancer in laboratory
animals (dioxin, for example) do not give a positive
Ames test (and vice-versa)
2)Ames assay consists of Salmonella
typhimurium strains and so it is not a perfect model
for human.
3)Some sample shows false postive test. That is
scattered large population microbes integates only
spontaneous mutation not mutation due to the
compound taken as test.Aggregated population of
microbes only positive for test compound.

23. HPRT GENE TEST
In Vitro Mammalian Cell Gene Mutation Test
(HPRT Gene)
The in vitro mammalian cell gene mutation test is used
to detect mutations of the hypoxanthine-guanine
phosphoribosyltransferase (HPRT) gene in Chinese
hamster ovary (CHO) or lung (V79) fibroblasts.
Screening and Regulatory Support
1)Cultures are incubated with several concentrations of the
test compound for three to four hours in the presence
and absence of metabolic activation (S9).

24. 2) Cells are subcultured for seven to eight days after
treatment to allow expression of the mutant
phenotype, and then plated in media with and
without 6-thioguanine (TG) to select for mutants
and determine cloning efficiency.
3) A positive outcome is characterized by a
statistically significant, dose-dependent increase
in mutant frequency that exceeds historical
negative control limits.

25. When To Perform
Screening
 Reduced volume and/or abbreviated formats available
REACH requirement
 As part of Annex VIII testing
Mutagenicity
 To confirm presumed mutagenic activity arising from
limited/small genetic damage (e.g., positive Ames or
large colony MLA)
 To assess mutagenicity when the Ames assay may not be
appropriate (e.g., antibiotics, nanomaterials)

27. The Mouse Micronucleus Test
Screening and Regulatory Support
 Male and/or female rats or mice are treated with the test compound at
three dose levels, usually two or three times at 24-hour intervals.
 Approximately 24 hours after the last dose, bone marrow or peripheral
blood is collected to determine the frequency of micronucleated
polychromatic erythrocytes (MN-PCEs) or micronucleated
reticulocytes (MN-RETs), respectively.
 A positive outcome is characterized by a statistically significant, dose-
dependent increase in MN-PCEs or MN-RETs that exceeds historical
control limits.
 Can be combined with standard toxicology tests, the comet assay and
the Pig-a assay.
 Administration routes include: oral, intravenous, infusion and
inhalation

28. When to Perform
 Screening
 Abbreviated formats available or can be added to non-GLP
tolerability studies
 IND-enabling
 As part of the ICH S2(R1) standard battery (Option 1 or 2)
 REACH requirement
 To follow up a positive result in any of the Annex VII or VIII
genotoxicity tests

30. REFERENCE:
 https://www.criver.com/
 https://microbiologyinfo.com/
 https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-
biology/mutagen-testing
 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4352214/
 https://www.slideshare.net/manoahram1/ames-test#
 https://www.slideshare.net/krupasagar12/ames-test-109209493
 https://www.nature.com/articles/s41596-020-0398-1
 https://www.mbbiosciences.com/genotoxicity
 https://www.sciencedirect.com/science/article/pii/B9780128007648000057
 https://onlinelibrary.wiley.com/doi/10.1002/em.20627
 https://www.mdpi.com/1422-0067/21/4/1534/htm
 https://currentprotocols.onlinelibrary.wiley.com/doi/abs/10.1002/0471143030.c
b2207s25