CARCINOGENICITY

Long term toxicity tests : Carcinogenicity
Complete assessment method for carcinogenicity

1. LONG TERM TOXICITY
STUDY
(CARCINOGENICITY)

RVS Chaitanya Koppala
Assistant Professor
Vignan Institute of Pharmaceutical Technology
Visakhapatnam

2. CARCINOGENICTIY
CHEIMCAL CARCINOGENESIS
Cancer is a disease of uncontrolled manipulation and growth of body’s own cells
A carcinogen may be defined as a physical and chemical agent that cause autonomous growth
of tissue. Carcinogens are of two types, genotoxic and non-genotoxic. Genotoxic carcinogens
produce cancer by directly damaging or changing the DNA structure.
They are further divided into direct carcinogens and indirect carcinogens. The direct acting
carcinogens are highly reactive electrophile molecules that do not require metabolic activation
etc. they directly bind to the DNA and cause tumors at the exposure site. Examples dimethyl
carbamoyl chloride, ethylamine and dimethyl sulphate. While indirect acting carcinogens or
procarcinogens are substance that require metabolism activation. The reactive molecules that
are formed from procarcinogen after metabolism is known as ultimate carcinogens. The
intermediate metabolites formed between procarcinogens and ultimate carcinogens are called
proximate carcinogens. Procarcinogen produce tumors at the target tissue and not at all
exposure site.
While non-genotoxic carcinogens represent chemical that produce cancer by some secondary
metabolism not related to direct DNA damage.
Carcinogenesis:
The process of development of cancer is called carcinogenesis. It takes place in 3 stages.
1.Initiation: this phase is characterized by carcinogen induced DNA damage. Damaged DNA
either undergoes repair or become permanently changed (mutated). The cell undergoes division
leading to proliferation of initiated cell.
2.Promotion: in this phase, cellular proliferation is stimulated which allow cancer cell
transformation to occur
3.Progression: this phase involves conversion of benign preneoplastic lesion into neoplastic
cancer. It is characterized by wide spread invasion, angiogenesis and growth factor stimulation
resulting in metastasis and a state of androgen independence.

3. MECHANISM OF ACTION:
Carcinogenesis by chemical is called chemical carcinogenesis. Some examples of chemical
carcinogenesis include, benzene, vinyl chloride, 2-naphthylamine and few metals. It occurs due
to impairment of various repair mechanism such as, failure of DNA repair, failure of apoptosis
and failure to terminate cell proliferation.
1. Failure of DNA repair
a) Genotoxic carcinogens covalently bind with DNA and alter its structure preventing
accurate replication. If the DNA damaged is not repaired or injured cell is not eliminated
than a mutation is induced in the daughter strand during the replication process. If the
mutation effects a gene. Controlling cell cycle then neoplastic transformation is
initiated. Mutations in at least two types of genes (proto oncogenes and tumor
suppressor genes) contribute to the development of cancer.
Protooncogenes: they encode proteins that help to regulate cell growth. Any
change(mutation) in the DNA sequence of proto oncogenes leads to formation of
oncogene which encodes for different protein that force cell into the division cycle.
Example: Ras is a oncogene protein, belonging to GTPase family. When it is stimulated,
it stimulates gene involved in growth, differentiation and survival of cells, mutations in
genes result is permanent activation of Ras proteins, which in turn leads to uncontrolled
proliferation and transformation.
Tumor suppressor genes: they encode proteins that retard progression of cells in
division cycle or encourage DNA repair or apoptosis in case of irreparable DNA
damage. Any mutation in tumor suppressor genes lead to uncontrolled cell division.
Example: the P53 tumor suppressor gene encodes 53 KDa protein which trans activates
genes that arrest the cell cycle, repair damaged DNA or promotes apoptosis. Any
mutation in P53 gene eventually leads to cancer.
b) Non genotoxic carcinogens do not cause DNA damage but still produce cancer after
prolonged exposure. They cause cancer by promoting carcinogenesis initiated by
genotoxic agents. They affect the expression of proto oncogenes and tumor suppressor
genes. They enhance the production of normal proto oncogenes proteins and decrease
the production of normal tumor suppressor genes. They also increase the incidence of
mutations which increases the number of cells with DNA damage and mutations.

4. Examples of non-genotoxic carcinogens include,
i) Endogenous mitogens (E.g. Growth factors)
ii) Exogenous mitogens that enhance proliferation
iii) Ethionine and diethanolamine
iv) Toxicants and produce sustained cell injury
2. Failure of apoptosis: the apoptotic activity in mutated cells (preneoplastic) is higher than
that of normal cells. So as to prevent clonal expansion of the initiated cells and tumor cells.
Inhibition of apoptotic mutations and clonal expansion of pre-neoplastic cells.
3. Failure to terminate proliferation:
Normal cells transform into malignant cells due to
a) Accumulation of genetic damage
b) Increased transcription and or translation of normal proto oncogenes
c) Decreased transcription and or translation of tumor suppressor genes
Uncontrolled proliferative activity occurs due to imbalance between apoptosis and mitosis
a) Increase mitotic activity increase mutations. When mitotic activity (cell division) is
augmented, G1 phase shortens due to which damaged DNA is not repaired and this damage
is transferred to daughter cells, and as a result a greater number of mutated cells are formed.
b) Increased mitotic activity compromises DNA methylation. The methylation pattern of
parenteral DNA strand is copied to daughter DNA strand, the DNA methyltransferase
(DNMTS). This is necessary for normal cell development dysregulation of this process
reads to cancer.
c) Mitosis promotes clonal expansion of initiated cells which form nodules and tumors
d) During mitosis, communication and adhesion between cells via, gap junctions and
cadherins respectively is temporarily disrupted which contributes to invasiveness of tumor
cells.
TEST SYSTEMS FOR CARCINOGENICITY ASSESSMENT:
The potential of the chemical to cause cancers is assessed by a number of in vivo and in vitro
experimental tests, these tests are classified into short term tests, intermediate tests and chronic
long-term tests

5. Short term test for mutagenicity
These tests typically last from a few days to a few weeks. They aim to identify carcinogens on
the basic of their ability to cause mutations in DNA either in vitro or in vivo; these tests fail to
detect non-genotoxic carcinogens
1. In vitro gene mutation assays:
a) Ames assay
This is most widely used short term test. It uses salmonella typhimurium strains
deficient in DNA repair and histidine synthesis. The capability of test substance to
reverse to histidine positive phenotype is determined.
b) Mouse lymphoma assay
In this test, thymidine kinase proficient cells are exposed to trifluorothymidine (TFT)
which inhibits cellular metabolism and halts cell division. Cells deficient in thymidine
kinase develop resistance to cytotoxic effects of TFT and thus proliferate, forming
colonies. An increase in the number in response to test substance indicates mutagenic
potential of the test substance.
2. In vivo gene mutation assays
The commonly used in vivo models include transgenic rodent mutation assay based on the
genes of the big blue, muta mouse and Lac operon. Mutations is any rodent tissue can be
measured after exposure to test chemicals the ratio of mutants in DNA of tissue to the total
population gives frequency of mutation for each chemical and each organ tested.
3. Chromosomal alternations:
Chromosomal alternations are common feature of malignant neoplasms. To determine
chemical induced chromosomal alternations, cells are harvested in their first mitotic
division post chemical exposure. Cells are then stained with Giemsa and scored for
karyotype completeness (21+2 chromosomes). Cells in their second mitotic division are
harvested and scored to determine the frequency of sister chromatid exchanges (SCE) cell
for each dose level. SCE are a measure of DNA damage caused due to mutation induction
and cancer.
4. DNA damage:
Unscheduled DNA synthesis (UDS) assay is commonly employed to measure the potential
of a chemical to cause DNA Lesions. An increase in DNA repair is indicative of DNA
damage

6. 5. Transformation assay:
The C3H/10T1/2 cell line is extensively used in the transformation assays. They are derived
from fibroblasts of the prostate of C3H mouse embryo. They are chromosomally abnormal
and have undergone few stages involved in the transformation of normal cells into
neoplastic cell. When these cells are plated, they stop growing (contact growth inhibition).
But open exposure to xenobiotics the contact inhibition fails and transformed colonies are
formed. Based on the percentage of transformed colonies, the carcinogenesis potential is
assessed.

7. CHRONIC TESTS FOR CARCINOGENICITY:
Chronic 2-year bioassay: this is the primary method by which carcinogenic potential of physical
and chemical agents is determined. A minimum of 50males and 50females animals (mice and
rats) of 8 weeks age are treated with two or three dose levels on a test chemical and vehicle
control through out their life period. Food consumption weight gain are monitored during the
study. At necropsy, the number and location of tumor and pathological diagnosis is done for
each animal.
Organ specific bioassays:
These assays are commonly employed to assess carcinogenic activity of chemicals in different
organs.
1. Liver: major target organ of toxicity. almost half of the chemicals tested in chronic 2 year
bioassay demonstrated increased incidence of hepatic cancer. Carcinongenicity testing in
the liver helps to identify chemicals that influence the initiation or promotion stage of
hepatic cancer.
2. Skin: carcinogenicity testing in the skin is performed using mouse skin model. This model
offer advantages like, development or neoplasia can be visually seen, the number and size
of papillomas and carcinomas can be qualified. This model assess the initiating and
promoting activities of carcinogens. Repeated application of tumor promoters causes clonal
expansions of initiated keratinocytes followed by development into papillomas and
carcinomas.
3. Other organs: different models are available for carcinogenicity testing in the kidney,
bladder, lung, stomach, colon, pancreas, intestine and oral cavity. These models differ with
respect to initiating carcinogen used, site, duration and frequency of application and
duration of promoting chemical exposure.
Chemical carcinogenesis in humans:
Major factor that contribute to the development of cancer in humans include lifestyle, infectious
agents, environmental and occupational exposure and medical treatments.
1. Life style: the component contributing the most to carcinogenesis in human is lifestyle.
Usage of tobacco, alcohol and dietary intake of fat, protein calories all contribute to cancer.
a) Tobacco: use (smoking, chewing or sniffing) accounts for 25-40% of all human cancers.
There is a strong correlation between tobacco use and development of lung, mouth ,

8. larynx, oesophageal and bladder cancer. Tobacco use is responsible for 85-90% of all
lung cancer cases in U.S.
b) Alcohol: use is estimated to be responsible for 2-4% of all cancer cases of larynx, liver
and oesophagus.
c) Diet: poor dietary intake of antioxidants, vitamins( A and E) contribute to onset of
cancer. High fat and high calorie intake has been associated with colon, breast and gall
bladder cancer in human. The method of cooking influence the production of
carcinogen. For instance, during boiling and grilling of meat carcinogenic heterocyclic
amines and polycyclic aromatic hydrocarbons are formed.
2. Environmental and occupational exposure:
Exposure to certain environmental and occupational pollutant contribute to onset of cancer
a) Environmental pollutants
i) Exposure to radon gas is the leading cause of lung cancer
ii) Consumption of high levels of arsenic in drinking water increase the risk of
lungs, bladder and skin cancer.
iii) Indoor smoke emissions from coal have been classified by IARC as human
carcinogenic and from other types of solids fuels as probable carcinogens
iv) Exposure to fine particulate matter in outdoor air (E.g. Diesel exhaust) increases
the risk of lung cancer.
b) Occupational pollutants
Exposure to numerous substances in the work place are known to produce cancer.
Agent Use Neoplasm
4-aminobiphenyl Rubber manufacture Bladder
Benzidine Dye/ pigment synthesis Bladder
Beryllium and beryllium Aerospace industry/metals Lung
Cadmium and cadmium Dye/ pigment synthesis Lung
Benzene Solvent, fuel Leukemia
Wood dust Wood industry Nasal cavity
Mineral oil, shale oil Lubricant Skin
Mustard gas War gas Pharynx and lung
Asbestos Insulation, textiles Lung
Chromium and nickel Metallurgy Lung

9. 3. Medical therapy and diagnosis:
Medical tests and treatment are an important part of getting healthy but there are certain
treatments which increases a person’s risk of developing cancer.
Chemical Neoplasm
Phenytoin Lymphoma, neuroblastoma
Estrogens Skin,liver cell adenoma
Chloramphenicol Leukemia
Alkylating agents Bladder and leukemia
Azathioprine Skin, kaposi’s sarcoma, lymphoma
Thorotrast Liver (angiosarcoma)

4. Infectious agents:
Infections with certain bacteria, viruses and parasites increases a person’s task of developing
cancer. They contribute to 15-20% of all cancer cases. However they alone cannot lead to
cancer, other factors also contribute.
Agent Neoplasm
Human papilloma virus Cervical
Epstein barr virus Nasopharyngeal Burkitt lymphoma
Hepatitis B and C virus Liver
HIV virus Kaposi sarcoma, cervical
Hpylori Stomach
Chlamydia trachomotis Cervical
Opisthorchis viverrini Bile duct
Schistosoma haemotobium Bladder