ADVERSE DRUG REACTIONS: TERATOGENICITY, MUTAGENICITY AND CARCINOGENICITY.pptx

ADVERSE DRUG REACTIONS:
TERATOGENICITY,
MUTAGENICITY AND
CARCINOGENICITY
DR AISHWARYA NIKOSE
FIRST YEAR RESIDENT,
DEPARTMENT OF PHARMACOLOGY,
LTTMMC & GH, SION, MUMBAI
DATE: 30-04-2021
1

OVERVIEW
• Introduction to Adverse Drug Reactions:
Definition
Serious adverse event
Classifications of Adverse drug reactions
• Teratogenicity:
Introduction
Teratogen:
 Definition
 Types of Teratogens
 Factors affecting effect of teratogens
 Effects of drug on foetus during pregnancy
 Effects of teratogens on pregnancy
Mechanism of teratogenicity
Drug induced teratogenicity 2

OVERVIEW
Drugs causing teratogenicity:
 Thalidomide
 Warfarin
 Phenytoin
 Valproate
 Methotrexate
 Retinoids
 Alcohol
 Tetracycline
 Penicillamine
Pregnancy and lactation labelling rule
Schedule Y:
 Segment II
3

OVERVIEW
In-vitro tests:
 Whole embryo culture test
 Micro-mass teratogen test
 Embryonic stem cell test
 Dictyostelium discoideum based assay
• Mutagenicity:
Mutagens
Classes of mutations
Drugs causing mutagenicity
Mechanism of mutagenicity
Tests for mutagenicity
 Ames test
4

OVERVIEW
 Variants of Ames test:
Ames spot test
Host mediated assay
Coliform assay
 In-vitro tests:
Saccharomyces forward mutation assay
DNA damage /repair
Forward mutations in Chinese hamster cells
Mouse lymphoma cell assay
Chromosomal aberrations
 In-vivo tests:
Micronuclei test
Dominant lethal assay
Comet assay
Muta-ChromoPlate Ames Test
ISO and Modified ISO Ames Tests
5

OVERVIEW
• Carcinogenicity:
Carcinogen
Carcinogen vs mutagen
Drugs causing carcinogenicity
Mechanism of carcinogenicity
Tests for carcinogenicity:
 BALB/c 3T3 cell Transformation assay
 Bhas 42cell transformation assay
 Syrian Hamster assay
Regulatory authorities
• Summary
6

ADVERSE DRUG REACTION
• According to WHO, Adverse drug reaction is
“A response to a drug that is noxious, unintended
and occurs at doses normally used in humans for
prophylaxis, diagnosis or therapy of a disease or
modification of physiological function”.
• FDA regulations define Adverse drug reaction as
“an undesirable effect, reasonably associated with
the use of a drug, that may occur as part of the
pharmacological action of the drug or may be
unpredictable in its occurrence”.
7
Brunton L, Hilal-Dandan R, Knollmann B. Goodman & Gilman's the pharmacological basis of therapeutics. 13th ed. new york: Mcgraw Hill.

SERIOUS ADVERSE EVENT
Adverse event which
• Results in death
• Is life threatening
• Resulting in hospitalization or prolongs existing
hospitalization
• Resulting in persistent or significant incapacitation
or disability
• A congenital anomaly or birth defect
• Requires intervention to prevent above or
permanent impairment or damage
8

CLASSIFICATION OF ADVERSE
DRUG REACTIONS
• Type A – Augmented effects
• Type B – Bizarre effects
• Type C – Chronic effects
• Type D – Delayed effects
• Type E – End of treatment effects
• Type F – Failure of a drug to produce the desired
effects
9

TERATOGENICITY
MUTAGENICITY
CARCINOGENICITY
10
TYPE D ADR

TERATOGENICITY
• TERAS : Greek word meaning Malformation
• The ability of a exogenous agents to
cause foetal abnormalities when administered
to the mother at any stage of pregnancy.
• The ability of formation abnormal embryo
David E. Golan., Ehrin J. Armstrong., April W. Armstrong. Principles of Pharmacology: The Pathophysiologic Basis of Drug Therapy, 4e. 4th ed.
Baltimore, MD: Lippincott, Williams & Wilkins; 2017. 12

TERATOGEN
• TERAS: Malformation
• Any substance, agent or exposure given to
pregnant woman that can induce anatomical,
structural or developmental defects in foetus
• Different type of teratogens are present which are
as follows

TYPES OF TERATOGEN
• Physical agents:
e.g. Ionizing radiations, Heat, etc.
• Environmental chemical agents:
e.g. Organic mercury compounds, PCB, etc.
• Infections:
e.g. Rubella, Cytomegalovirus, Varicella, HSV, etc.
• Drugs:
e.g. Methotrexate, Lithium, Phenytoin, etc.
• Metabolic conditions:
e.g. Maternal PKU, Diabetes mellitus, PIH, etc.

SOME COMMON TERATOGENS
• Alcohol
• Hormones
• Smoking
• Teratogenic drugs
15
FOETAL ALCOHOL SYNDROME
VIRILIZATION OF FEMALE
FOETUS
MALIGNANCY
CONGENITAL MALFORMATIONS
David E. Golan., Ehrin J. Armstrong., April W. Armstrong. Principles of Pharmacology: The Pathophysiologic Basis of Drug
Therapy, 4e. 4th ed. Baltimore, MD: Lippincott, Williams & Wilkins; 2017.

FACTORS AFFECTING EFFECT OF
TERATOGENS
• Placental barrier
• Dose reached foetus
• Duration of exposure
• Time of pregnancy during which drug exposure
occurs
16

EFFECT OF TERATOGEN ON
FOETUS DURING PREGNANCY
• FERTILIZATION AND IMPLANTATION:
(From conception to 17 days)
e.g. Failure of pregnancy
• ORGANOGENESIS:
(18 to 55 days)
e.g. Congenital malformations
• GROWTH AND DEVELOPMENT:
(56 days and onwards)
e.g. Developmental & Functional abnormalities
MOST VULNERABLE PERIOD
17
Tripathi K. Essentials of medical pharmacology. 8th ed. New Delhi: Jaypee Brothers medical publisher.

18
Mazzu-Nascimento T, Melo DG, Morbioli GG, Carrilho E, Vianna FS, Silva AA, Schuler-Faccini L. Teratogens: a public health issue–a
Brazilian overview. Genetics and molecular biology. 2017 Jun;40(2):387-97.

EFFECTS OF TERATOGEN ON
PREGNANCY
• Spontaneous abortion
• Congenital malformations
• Intrauterine growth retardation
• Intellectual disability
• Carcinogenesis
• Mutagenesis
• Specific syndromes related to drugs
19
Beckman DA, Brent RL. Mechanisms of teratogenesis. Annual review of pharmacology and toxicology. 1984 Apr;24(1):483-500.

MECHANISM OF
TERATOGENICITY
Environmental agents or factors that interact with an
embryo during the period of development
Errors in genetic programming based on deviations in
genotype of the embryo OR low probability for error
of normal genotype
Beckman DA, Brent RL. Mechanisms of teratogenesis. Annual review of pharmacology and toxicology. 1984 Apr;24(1):483-500. 20

TERATOGENIC MECHANISMS
• Folate Antagonism: Methotrexate, Carbamazepine
• Neural crest cell disruption: Retinoic acid
• Endocrine disruption: Diethylstilboestrol
• Oxidative stress: Thalidomide, Phenytoin,
Valproate
• Vascular disruption: Aspirin, Ergotamine
• Enzyme-mediated teratogenesis: ACE Inhibitors
21
Beckman DA, Brent RL. Mechanisms of teratogenesis. Annual review of pharmacology and toxicology. 1984 Apr;24(1):483-500.

GHS CLASSIFICATION CRITERIA
FOR TERATOGENICITY
Category Criteria
Category 1A Chemicals known to have carcinogenic potential to
humans - largely based on human evidence (Mixtures
containing ≥ 0.1% of such a category 1A carcinogen)
Category 1B Chemicals presumed to have carcinogenic potential to
humans - largely based on animal evidence (Mixtures
containing ≥ 0.1% of such a category 1B carcinogen)
Category 2 Suspected human carcinogen - evidence from human
and/or animal studies is limited (Mixtures containing ≥
0.1% of such a category 2 carcinogen)
22
Carcinogenicity [Internet]. Chemsafetypro.com. 2021 [cited 22 April 2021]. Available from:
https://www.chemsafetypro.com/Topics/CRA/Carcinogenicity.html

DRUG INDUCED
TERATOGENICITY
Drug induced teratogenicity can be described as
“The ability of a drugs to cause foetal abnormalities
when administered to the mother at any stage of
pregnancy.”
23

DRUGS CAUSING
TERATOGENICITY
• Thalidomide
• Penicillamine
• Warfarin
• Phenytoin
• Valproate
• Methotrexate
• Retinoids
• Androgens
24

THALIDOMIDE: PHOCOMELIA
• Phocomelia - limb
malformations :
absence of
development of long
bones of limbs
• Also known as Seal
limbs
25
Brunton L, Knollmann B, Hilal-Dandan R. Goodman & Gilman's :. 13th ed. New York, N.Y.: McGraw-Hill Education LLC.; 2018.

26
Gao S, Wang S, Fan R, Hu J. Recent advances in the molecular mechanism of thalidomide teratogenicity. Biomedicine &
Pharmacotherapy. 2020 Jul 1;127:110114.
THALIDOMIDE INDUCED
TERATOGENICITY

THALIDOMIDE INDUCED
PHOCOMELIA
28
Gao S, Wang S, Fan R, Hu J. Recent advances in the molecular mechanism of thalidomide teratogenicity. Biomedicine & Pharmacotherapy.
2020 Jul 1;127:110114.

WARFARIN: FOETAL WARFARIN
SYNDROME
• Saddle nose deformity
• Mental retardation
• Bone stippling
(Chondrodysplasia
punctata)
• Also known as
Warfarin
embryopathy
syndrome
29

30
Vitale N, De Feo M, De Santo LS, Pollice A, Tedesco N, Cotrufo M. Dose-dependent fetal complications of warfarin in pregnant
women with mechanical heart valves. Journal of the American College of Cardiology. 1999 May;33(6):1637-41.

PHENYTOIN: FOETAL HYDANTOIN
SYNDROME
• Cleft lip/ palate
• Microcephaly
• Mental retardation
• Hypoplasia of distal
Phalanges with small
nails
31

PHENYTOIN: FOETAL HYDANTOIN
SYNDROME
32
David E. Golan., Ehrin J. Armstrong., April W. Armstrong. Principles of Pharmacology: The Pathophysiologic Basis of Drug Therapy, 4e.
4th ed. Baltimore, MD: Lippincott, Williams & Wilkins; 2017.

VALPROATE: FOETAL VALPROATE
SYNDROME
• Neural tube defects
• Flat nasal bridge
• Trigonocephaly
• Thin upper lip
• Broad forehead
• Thin arched eyebrows
• Small eyes
• Distal digital hypoplasia
33

METHOTREXATE: NEURAL TUBE
DEFECTS
Neural tube defects
34
Anti-folate agent
which inhibit Folic
acid utilization
causing neural tube
defects
• First trimester –
Ancephaly
• Second trimester
– Spina Bifida

35
Kozub, P & Simaljaková, Maria. (2011). Systemic therapy of psoriasis: Methotrexate. Bratislavské lekárske listy. 112. 390-4.

RETINOIDS:
• Mental retardation &
Learning disability
• Cranio-facial
dysmorphism
• Neural tube defects
• Thymic aplasia
• Cleft lip and cleft palate
• Heart defects
• Eye and ear deformities
36

FOETAL ALCOHOL SYNDROME
• Spectrum of disorders
• Irreversible damage to brain and growth
• No known safe amount of alcohol to consume
while pregnant and no known safe time during
pregnancy to consume alcohol to prevent birth
defects
• Abnormal appearance, Short height, Low body
weight, head size, behavioral problems, learning
disabilities, etc.
37

38

TETRACYCLINE:
• Yellow, brown staining
of teeth
• Enamel hypoplasia
• Caries and
hypopigmentation of
permanent teeth
39

PREGNANCY & LACTATION
LABELLING RULE
41
CATEGORY RISK EXAMPLES
A Controlled human studies
shows no risk
e.g. Folic acid,
Thyroxine
B No confirmatory evidence of
risk in humans
e.g. Amoxicillin,
Ondansetron
C Risk cannot be rule out e.g. Fluconazole,
Metoprolol
D Positive evidence of risk e.g. Phenytoin,
Lithium
X Contraindicated in
pregnancy
e.g. Methotrexate,
Warfarin
Pernia S, DeMaagd G. The new pregnancy and lactation labeling rule. Pharmacy and therapeutics. 2016 Nov;41(11):713.

NEW PREGNANCY & LACTATION
LABELLING RULE, 2015
• Pregnancy risk letter categories eliminated
• Sections for Pregnancy & Lactation combined
• New section FEMALES AND MALES OF
REPRODUCTIVE POTENTIAL added
42
Pernia S, DeMaagd G. The new pregnancy and lactation labeling rule. Pharmacy and therapeutics. 2016 Nov;41(11):713.

TESTS FOR TERATOGENICITY
• Teratogenicity testing came into being since the
Thalidomide tragedy of 1961
(2000 deaths, >10000 birth defects and > 50% were
in Germany)
• High demand for a rapid, reliable and cost-
effective method for detection of teratogenic
toxicity
43
Gao S, Wang S, Fan R, Hu J. Recent advances in the molecular mechanism of thalidomide teratogenicity. Biomedicine & Pharmacotherapy.
2020 Jul 1;127:110114.

SCHEDULE Y
• Requirements and guidelines for permission to
import and / or manufacture of new drugs for sale
or to undertake clinical trials
• Female reproduction and Developmental toxicity
Studies
– Appendix I, Item 4.4
– Segment I – Female Fertility Study
– Segment II – Teratogenicity Study
– Segment III – Perinatal Study
44
https://rgcb.res.in/documents/Schedule-Y

Animal toxicity requirements for
clinical trials
and marketing of a new drug
45
TOXICITY STUDIES Human phase for which study
is proposed to be conducted
Segment II
(Teratogenicity studies)
Phase II, III involving female
patients of child-bearing age
Segment III
(Perinatal studies)
Phase III
• Drugs to be given to pregnant
or nursing mothers for long
periods
• Drugs with indications of
possible adverse effects on
foetal development

Teratogenicity Study (Segment II)
46
One rodent (preferably
rat)
One non-rodent (rabbit)
Route of administration
same as intended for human
therapeutic use
Given
throughout
pregnancy
Three graded doses
• Highest dose –
minimum maternal
toxicity
• Lowest dose –
proportional to the
proposed dose
for clinical use in
humans
• Intermediate dose –
logarithmically
between two
other doses

47
At least 20 pregnant rats
(or mice) and 12 rabbits,
on each dose level
All fetuses subjected to
gross examination
Skeletal and visceral
abnormalities
CONTROL
GROUP
TEST
GROUP

STUDY PARAMETERS
MATERNAL:
• Signs of intoxication
• Effect on body weight
• Examination of genital
system and
abnormalities
FOETAL:
• Gender
• Body weight
• Body length
• Gross/visceral/skeletal
abnormalities
48

IN-VITRO TECHNIQUES
• Simple and cost-efficient
• Can reduce pressure imposed by society to
decrease or replace the number of animals
used in research and testing strategies
• Simple, easy to perform, yield of interpretable
results
• Having relevance to mechanisms of teratogenesis
• Usable with various types of agents
49
Lindhout D, Omtzigt JG. Pregnancy and the risk of teratogenicity. Epilepsia. 1992 Jul;33:41-8.

IN-VITRO TECHNIQUES
• Whole embryo culture test
• Micro-mass teratogen test
• Embryonic stem cell test
• Dictyostelium discoideum based assay
50

WHOLE EMBRYO CULTURE TEST
Rodent embryo culture (rat or mouse)
• Culturing of whole embryos at early stage of
organogenesis
• Exposing of these to a potential teratogen
• Endpoints generally used
– Mortality
– Malformation
– Growth inhibition
51

WHOLE EMBRYO CULTURE TEST
Zebrafish embryo:
• Easy to maintain in large stocks due to their high
fecundity
• Develop rapidly ex utero, with most organs
becoming functional between 3 and 5 days post-
fertilization
• Transparency of larval zebrafish
• Reasonably tolerant to concentration of
dimethylsulphoxide (DMSO) generally used as a
solvent at drug screening
• High concordance with mammalian data
52

MICRO-MASS TERATOGEN TEST
• Chick, mouse and rat embryo midbrain or limb cell
culture
• Exposed to test compounds for varying times and
concentrations
• A phase of in vivo embryo exposure will act as a
control for the effects of drug metabolism and
pharmacokinetics
• Using cells from different organs and species to
reproduce the in vivo sensitivity of particular
embryonic tissues or species to teratogenic agents
53

MICRO-MASS TERATOGEN TEST
54
In vitro culturing of embryo limb or rather
central neural cells
Differentiation of the cells into chondrocytes or
neurons starting from numerous small
aggregates or foci of cells
Observation of cell adhesion, movement,
communication, division and differentiation
involving the new synthesis of tissue specific
patterns of enzymes and structural proteins

EMBRYONIC STEM CELL TEST
• Pluripotent embryonic stem cells (ESC) isolated
from mouse blastocysts
• ESC are able to differentiate into a variety of
embryonal tissues, retain the euploid chromosome
constitution, and proliferate very rapidly
• Based on the resemblance between early
embryonic stages and the differentiation of
embryonic stem cells in vitro
• Cell lines are used to identify cytotoxic, mutagenic,
embryo toxic and teratogenic effects of chemical
compounds
• Evaluation of cell differentiation is performed both
morphological and via molecular techniques
55

56
EMBRYONIC STEM CELL TEST

DICTYOSTELIUM DISCOIDEUM
BASED ASSAY
• Cell differentiation leading to the formation of stalk
and spore cells has been shown to be similar, to
some extent, to the development of mammals
• Reporter genes serve as highly specific indicators
for the developmental fate of a certain cell at a
given time point
• Easier to handle and less expensive
• Rapid large-scale screening of chemicals
57

DIAGNOSTIC TESTS FOR
TERATOGENICITY
• Screening tests:
First trimester screening
Second trimester screening
• Diagnostic tests
• Post-natal testing
58
Cunningham F, Leveno K, Bloom S, Gilstrap L, Cunningham F. Williams Obstetrics (23rd Edition). 23rd ed. New York, USA: McGraw-
Hill Professional Publishing; 2010.

FIRST TRIMESTER SCREENING
• Done between 11 to 13 weeks of pregnancy
• Maternal blood test:
-Human chorionic gonadotropin (hCG)
-Pregnancy associated plasma protein A (PAPP-A)
• Ultrasound:
-Nuchal translucency
59
Cunningham F, Leveno K, Bloom S, Gilstrap L, Cunningham F. Williams Obstetrics (23rd Edition). 23rd ed. New York, USA:
McGraw-Hill Professional Publishing; 2010.

SECOND TRIMESTER SCREENING
• Done between 15-20 weeks of pregnancy
• Maternal serum screen:
-Triple (AFP, hCG, Estriol)
-Quadruple (AFP, hCG, Estriol, Inhibin A)
• Foetal echocardiogram:
-For heart defects
• Ultrasound:
-Also known as Anomaly scan
60

DIAGNOSTIC TESTS
• Chorionic villus sampling:
-10 to 12 weeks of pregnancy
• High resolution ultrasound:
• Amniocentesis:
61

POST-NATAL TESTING
• Seen after few days of birth
• Congenital heart diseases
• On evaluation for other conditions
• Sometimes unnoticed
62

PREVENTION OF
TERATOGENICITY
• Patient education:
Educate about planning of pregnancy and hazards of
teratogens and preventable causes of teratogenicity.
e.g. Alcohol and smoking.
• Past and family history:
History of diabetes mellitus, hypertension, hereditary
disorders, past history of congenital birth defects,
miscarriage leading to high risk.
63

PREVENTION OF
TERATOGENICITY
• Management of pre-existing conditions:
e.g. Treatment of Hypertension to prevent IUGR,
prematurity, etc.
• Medications:
e.g. Isotretinoin used for Acne treatment may result
into pregnancy loss.
64

VACCINES CONTRAINDICATED IN
PREGNANCY
• All live vaccines are contraindicated in pregnancy
except Yellow fever vaccine.
e.g. MMR, Hep B, HPV, Influenza, Varicella, etc.
• Vaccines for prevention of COVID-19 are
contraindicated in pregnancy.
e.g. COVAXIN, COVISHIELD, etc.
65

MUTAGENICITY
• Mutagenicity is ability of induction of permanent
changes in the information content of genetic
material
• It is replacement of nitrogen base with another in
one or both the strands or addition or deletion of
base pair in DNA molecule
67
David E. Golan., Ehrin J. Armstrong., April W. Armstrong. Principles of Pharmacology: The Pathophysiologic Basis of Drug Therapy, 4e.
4th ed. Baltimore, MD: Lippincott, Williams & Wilkins; 2017.

MUTAGEN
• Any agent which can induce mutations are
collectively known as mutagens
• It can be any substance, chemicals, physical agents
or environmental agents
• Different types of mutagens are there
68
David E. Golan., Ehrin J. Armstrong., April W. Armstrong. Principles of Pharmacology: The Pathophysiologic Basis of Drug
Therapy, 4e. 4th ed. Baltimore, MD: Lippincott, Williams & Wilkins; 2017.

69
MUTAGEN
PHYSICAL CHEMICAL BIOLOGICAL
• UV light
• Heat
• Base analogs
• Intercalating
agents
• Alkylating
agents
• Deaminating
agents
• Metals
• Transposons
• Virus
• Bacteria
David E. Golan., Ehrin J. Armstrong., April W. Armstrong. Principles of Pharmacology: The Pathophysiologic Basis of Drug Therapy,
4e. 4th ed. Baltimore, MD: Lippincott, Williams & Wilkins; 2017.

CLASSES OF MUTATIONS
• Spontaneous mutation:
-mainly caused during DNA replication or by
incorporation of incorrect nucleotide in the growing
DNA chain
-occur by changes in DNA sequence
• Induced mutation:
-caused by changes in DNA brought by
environmental factors called mutagens
70
Liu B, Xue Q, Tang Y, Cao J, Guengerich FP, Zhang H. Mechanisms of mutagenesis: DNA replication in the presence of DNA damage. Mutation
Research/Reviews in Mutation Research. 2016 Apr 1;768:53-67.

DRUGS CAUSING MUTAGENICITY
• Metronidazole
• Furan
• Acetylaminofluorene
• Tiaprofenic acid
• Chlorothalonil
71

MECHANISM OF MUTAGENICITY
72
Cytotoxic
Inhibit protein synthesis
Bhardwaj, Niti & Saraf, Shailendra & Sharma, Pankaj & Kumar, Pradeep. (2009). Syntheses, Evaluation and Characterization
of Some 1, 3, 4-Oxadiazoles as Antimicrobial Agents. E-Journal of Chemistry. 8. 240-244.

TESTS FOR MUTAGENICITY
• Beginning as early as 1944, shortly after Charlotte
Auerbach published her finding that allyl
isothiocyanate and, later mustard gas were
mutagenic in the fruit fly Drosophila
• Number of laboratories began looking for a
connection between mutagens and carcinogens
using a variety of organisms, including E. coli,
Drosophila, and Neurospora and plants
73
Liu B, Xue Q, Tang Y, Cao J, Guengerich FP, Zhang H. Mechanisms of mutagenesis: DNA replication in the presence of DNA damage.
Mutation Research/Reviews in Mutation Research. 2016 Apr 1;768:53-67.

AMES TEST
• Named after it’s developer in 1970s - Bruce Ames
• Back mutation: Mutation can be reversed, with a
gene regaining its function and Revertant can grow
on media lacking Histidine
• Strain used – Salmonella typhimurium strain (His -)
Cannot grow without Histidine
• Animal used – Rat
• Mutation – Histidine gene synthesis
74
Liu B, Xue Q, Tang Y, Cao J, Guengerich FP, Zhang H. Mechanisms of mutagenesis: DNA replication in the presence of DNA
damage. Mutation Research/Reviews in Mutation Research. 2016 Apr 1;768:53-67.

75
Hogendorf AS, Hogendorf A, Kurczab R, Kalinowska-Tłuścik J, Popik P, Nikiforuk A, Krawczyk M, Satała G, Lenda T, Knutelska J, Bugno R. 2-
Aminoimidazole-based antagonists of the 5-HT6 receptor–A new concept in aminergic GPCR ligand design. European journal of medicinal
chemistry. 2019 Oct 1;179:1-5.

76
AMES SALMONELLA ASSAY
Mortelmans K, Zeiger E. The Ames Salmonella/microsome mutagenicity assay. Mutation research/fundamental and molecular
mechanisms of mutagenesis. 2000 Nov 20;455(1-2):29-60.

77
AMES TEST
AMES TEST
Mortelmans K, Zeiger E. The Ames Salmonella/microsome mutagenicity assay. Mutation research/fundamental and molecular
mechanisms of mutagenesis. 2000 Nov 20;455(1-2):29-60.

AMES SPOT TEST
• It consist of the incubation of
a suitable tester strain of
salmonella typhimurium and test
agent place on the agar.
• Chemical is tested on one
petri plate.
• The zone of inhibition is
indicated of the toxicity for
the bacterial growth.
78
Mortelmans K. A perspective on the development of the Ames Salmonella/mammalian-microsome mutagenicity assay.
Mutation Research/Genetic Toxicology and Environmental Mutagenesis. 2019 May 1;841:14-6.

HOST MEDIATED ASSAY
• Salmonella are injected intraperitoneally into rat or
a hamster.
• The animal is treated with the test substance orally.
• Afterwards sample is withdrawn from peritoneal
cavity and mutation in salmonella is measured
COLIFORM ASSAY:
• Tester strain of E.coliPQ37 + test substance
incubation with or with out S-9 liver fraction, may
show control for protein synthesis
79

IN-VITRO METHODS
• Saccharomyces forward mutation assay
• Mammalian cell test systems:
1)DNA damage/repair
2)Forward mutations in Chinese hamster cells.
3)Mouse lymphoma cell assay
4)Chromosomal aberrations
80

Saccharomyces forward mutation
assay
• The test uses strain of S.cervisiae carrying a
defective mutation of the adenine-1 and 2 genes
and growth in yeast culture results in production of
red colored colonies as a consequence of the
accumulation of intracellular pigment.
• Colonies that grow in culture being white when
grow on a low adenine medium.
81

82
ISOLATED
HEPATOCYTES
PRIMARY CULTURE
WASH EXTENSIVELY
Incorporation of H-
thymidine into DNA
Autoradiography of
exposed cells,
scintillation counting
of extracted DNA
WASH
DNA DAMAGE AND REPAIR
Incubate with
Thymidine
Incubate with test
drug

83

MOUSE LYMPHOMA CELL ASSAY
84

IN VIVO METHODS
• Micronuclei test
• Dominant lethal assay
• Comet assay
86

MICRONUCLEI ASSAY
• A mutagenic test system for the detection of
chemicals which induce the formation of small
membrane bound DNA fragments i.e. micronuclei
in the cytoplasm of interphase cells.
• These micronuclei may originate from acentric
fragments (chromosome fragments lacking a
centromere) or whole chromosomes which are
unable to migrate with the rest of the
chromosomes during the anaphase of cell division.
87

MICRONUCLEI ASSAY
• Micronuclei arise from chromosomal fragments
that are not incorporated into daughter cell nuclei
at mitosis because they lack a centromere and are
not pulled to the appropriate pole of the spindle.
• The purpose of the micronucleus assay is to detect
those agents which modify chromosome structure
and segregation is such a way as to lead to
induction of micronuclei in interphase cells.
88

DOMINANT LETHAL ASSAY
Indicate that genetic damage has occurred in the
form of structural or numerical chromosome
aberrations.
METHODOLOGY
• Male mice or rats are treated with test agent
• Males mated with groups of untreated females
• Females are killed 14 days after mating, dissected
and scored for corpora lutea, early fatal deaths and
total implantations.
90

COMET ASSAY
• First introduced by OSTLING and JOHANSON in 1984.
PRINCIPLE:
• Strand breakage of the supercoiled duplex DNA leads to
the reduction of the size of the large molecule and
these strands can be stretched out by electrophoresis.
• DNA migration is a function of both size and the
number of broken ends of the DNA
• Tail length increases with damage initially and then
reaches a maximum that is dependent on the
electrophoretic conditions, not the size of fragments.
91

APPLICATIONS OF COMET ASSAY
• Genetic toxicology (DNA damage):
-In vivo & in vitro evaluation of genotoxic chemicals
• DNA damage:
-SSB’s, DNA crosslinking, alkali labile sites
• DNA repair:
-Strand break repair
-Excision repair
92

APPLICATIONS OF COMET ASSAY
• Eco-toxicology:
-Assay has been used to monitor soil and aquatic
toxicology
• Nutrition
• Environmental biomonitoring:
-Evaluation of genotoxic pollutants from hazardous
waste sites
• Hypoxia assessment
93

Muta-ChromoPlate Ames Test
•Ideal for direct assessment of
environmental samples
•Educational uses for classroom projects (easy to
preform)
•More endpoints/ sample can improve significance
• Extremely user friendly
•Excellent to test volatile materials
•No preconcentration steps required, large sample
volumes used
•More sensitive
94

95
ISO and Modified ISO Ames Tests
•Ideal for pharmaceutical and novel molecule
discoveries
•HIGH THROUGHPUT METHOD - Many samples or
dilution levels can be tested rapidly
•Smaller sample sizes required
•Quantification is simple and test scoring is quick and
easy
•More sensitive than the plate-incorporation assay
•Utilizes pre-exposure period to enhance bacterial
uptake
Mortelmans K. A perspective on the development of the Ames Salmonella/mammalian-microsome mutagenicity assay. Mutation
Research/Genetic Toxicology and Environmental Mutagenesis. 2019 May 1;841:14-6.

CARCINOGENICITY
• Carcinogenicity is the ability to induce unregulated
growth processes in cells or tissues of multicellular
animals, leading to malignancy
• Cancer causing property
97

CARCINOGEN
• Any substance or agent that is capable of causing
cancer or increases it’s incidence
• Chronic toxins
• Cause damage after repeated or long-duration
exposure
• May have not immediate apparent harmful effects,
with cancer developing only after a long latency
period
98

MUTAGEN VS CARCINOGEN
MUTAGEN
• A substance or agent
that induces heritable
change in cells or
organisms
CARCINOGEN
• A substance that
induces unregulated
growth processes in
cells or tissues of
multicellular animals,
leading to the disease
called cancer
99

DRUGS CAUSING
CARCINOGENICITY
• Chloramphenicol - Leukemia
• Mitomycin C – Warts and Epithelial tumors
• Progesterone – Genital malignancies
• Adriamycin – Bladder and Mammary tumors
• Azacitidine – Hematopoietic, lung and liver tumors
• Phenobarbital – Hepatic lesions
100

MECHANISM OF
CARCINOGENICITY
101
CARCINOGEN
REACTIVE
INTERMEDIATE
INACTIVE PRODUCT
ERROR FREE DNA
DNA ADDUCT CANCER
DNA MUTATION
PHASE 1 & 2
METABOLSIM

TESTS FOR CARCINOGENICITY
• Epidemiological studies - relationship between a
cancer suspect chemical and a human population
over a long period of time
• Animal studies - induce cancer in test animals,
usually of two or more species with varying dose
and time parameters
• Most known human carcinogens produce cancer in
experimental animals
103

RODENT BIOASSAY
• Design:
- Rat or Mice aged 6 to 8 weeks
- Number used : 60 per gender
- Dose : maximum tolerable dose
- Route : mostly oral
• Assessment:
-Fasted overnight
-Necropsy: Gross examination
-Blood samples for haematology
104

RODENT BIOASSAY
• Analysis:
Tissue specific tumours:
- Incidence of tissue specificity
Stratified tumours:
- Survival weighted analysis
Overall analysis:
- Analysed for benign as well as malignant
tumours
105

CRITERIA FOR CARCINOGENICITY
TESTING
• Genotoxic
• Structurally related to carcinogens
• Pharmacological class
• Repeat dose toxicity studies
• Used for infrequent or short duration in humans
106

TESTS FOR CARCINOGENICITY
• BALB/c 3T3 cell Transformation assay
• Bhas 42 Cell Transformation Assay
• Syrian Hamster Embryo Assay
• Transgenic models
107

VACCINES FOR PREVENTION OF
CANCERS
108
Vaccine
name
Brand name Approved for Year of
FDA
approval
HPV
vaccine
1. Gardasil
2. Cervarix
Cervical
malignancy
2006
Hep B
vaccine
Hepatocellular
mailgnancy
Prostate
cancer
Sipuleucel-T Metastatic,
symptomatic,
castration
resistant prostate
cancer
2010
Melanoma Talimogene
laherparepve
c (T-vec)
Unresectable,
cutaneous nodal
lesions,
Melanoma
2016

REGULATORY AUTHORITIES
OECD guidelines
• 451- Carcinogenicity studies
• 453- Combined chronic toxicity/carcinogenicity
ICH guidelines
• S1A- Guideline on the need for carcinogenicity
studies of pharmaceuticals
• S1B- Testing for carcinogenicity of pharmaceuticals
• S1C- Dose selection for carcinogenicity studies of
pharmaceuticals
109

SUMMARY
• Patient education and proper selection of drugs
• New test for securely identifying teratogenic,
mutagenic and carcinogenic effects of drugs
110

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