TOXICITY STUDIES

Toxicity testing of new compounds is essential for drug development

process. The preclinical toxicity testing on various biological systems
reveals the species-, organ- and dose- specific toxic effects of an
investigational product.
The toxicity of substances can be observed by:
(a) studying the accidental exposures to a substance
(b) in vitro studies using cells/ cell lines
(c) in vivo exposure on experimental animals.
This mainly focuses on the various experimental animal models and
methods used for toxicity testing of substances. The pre-clinical toxicity
testing helps to calculate “No Observed Adverse Effect Level” which is
needed to initiate the clinical evaluation of investigational products.

INTRODUCTION
Toxicology is a branch of science that deals with toxins and poisons and
their effects and treatment. Toxicological screening is very important for
the development of new drugs and for the extension of the therapeutic
potential of existing molecules.
The US Food and Drug Administration (FDA) states that it is essential
to screen new molecules for pharmacological activity and toxicity
potential in animals.
Toxicity tests are mostly used to examine specific adverse events or
specific end points such as cancer, cardiotoxicity, and skin/eye irritation.
Toxicity testing also helps calculate the No Observed Adverse Effect
Level (NOAEL) dose and is helpful for clinical studies.

HISTORY OF TOXICITY STUDIES

Paracelsus, pictured here, was a 16th century physician and is considered
to be the “Father of Toxicology.” who determined specific chemicals
responsible for the observed toxicity of plants and animals.
He demonstrated the harmless and beneficial effects of toxins and proved
dose-response relationships for the effects of drugs.
Toxicological screening methods and toxicological research on individual
substances developed in the mid-1900s, and environmental toxicological
studies developed in the mid-20th century.
The use of animals in toxicity studies began in 1920, when J. W. Trevan
proposed the use of the 50% lethal dose (LD50) test to determine the lethal
dose of individual chemicals.
After the introduction of LD50, a FDA scientist John Draize developed a
method for testing eye and skin irritation using rabbits, and this method
was widely accepted for testing the effects of chemicals and
pharmaceuticals on the eye and skin.

EYE IRRITATION SKIN IRRITATION

Institute ethics committee
Before conducting any toxicological testing in animals or collecting
tissue/cell lines from animals, the study should be approved by the
Institute Animal Ethics Committee (IAEC) or the protocol should
satisfy the guidelines of the local governing body.
The guidelines for conducting experiments and regulatory
requirements vary from region to region. In India, the Committee
for the Purpose of Control and Supervision of Experiments on
Animals (CPCSEA) guidelines should be followed for the
maintenance of experimental animals.
An approved fluid withdrawal method should be used, and schedule
Y (India) should be complied with to fulfill the regulatory
requirements.

TYPES OF TOXICITY AND THEIR TESTING

Acute toxicity
The study aim to determine acute effect of chemical produced after
administration. Dosing is done either once in a day or multiple time
but from day 1 and the total study duration which might extend to
2weeks .
Example: HYDROGEN CYANIDE: A highly toxic gas that can
cause death
ARSINE: One of the major industrial causes of sudden
extensive hemolysis
ANIMAL SPECIES FOR ACUTE TOXICITY STUDIES
 Rodent – Rat, mice (5 to 10) in numbers
 Non rodent – Dog, monkey (2 to 3) in numbers

Acute Toxicity Testing

Acute toxicity testing is carried out to determine the effect of a single dose
on a particular animal species. In general, it is recommended that acute
toxicity testing be carried out with two different animal species (one
rodent and one non-rodent).

The table below shows GHS classification criteria for acute toxicity for different routes.
 In this testing, the investigational product is administered at different dose
levels, and the effect is observed for 14 days.
All mortalities caused by the investigational product during the
experimental period are recorded and morphological, biochemical,
pathological, and histological changes in the dead animals are
investigated.
Acute toxicity testing permits the 50% lethal dose (LD50) of the
investigational product to be determined. The LD50 was used as an
indicator of acute toxicity previously.
The determination of the LD50 involves large numbers of animals, and the
mortality ratio is high. Because of these limitations, modified methods
were developed:

Sub-Acute toxicity
It can be defined as repeated exposure to a chemical for one to three
months. It focus on administration of single or multiple dose of test
samples per day and given for a period 14 to 28 days.
These studies are conducted for duration of 2 to 4 weeks inorder evaluate
the potential ADR of new drug. It helps in initial clinical study trail.
ANIMAL SPECIES FOR SUB ACUTE TOXICITY STUDIES
 RODENT – Rat (Preferred Model)
Mice ( As an Alternative)

NOAEL - Non Observed Adverse Effect Level

The greatest concentration or amount of a substance at which no
detectable Adverse Effect occurs in a exposed population.
LOAEL – Low Observed Adverse Effect Level
The lowest concentration of a substance at which minimum
detectable Adverse Effect occurs in a exposed population.
Chronic toxicity
It can be defined as exposure to a chemical for more than 3 months (6
months to 2 years) NOTE: Specific Organ damages.
EXAMPLE: 1. Long term Ethanol Ingestion in alcoholic results liver
cirrhosis

2. Several years of lead exposure in mine working leads to kidney fail ure.
These toxicity studies are performed with minimum one Rodent and one
Non Rodent species.
The test sample is administered to the animal for over more than 90
days and periodically observer for the result.
ANIMAL SPECIES FOR CHRONIC TOXICITY STUDIES
 RODENT – Rat and Mice ( 6 to 24 Months )
 NON RODENTS – Dogs and Monkey ( 12 Months or Longer
10% of their Life Span )
Mechanism of Toxicity :
1) Delivery from the site of exposure to the target :
Theoretically, the intensity of a toxic effect depends primarily on the
concentration and persistence of the toxicant at its site of action.

 Life cycle of a toxicant

 Absorption
 Distribution
 Degradation
 Reabsorption
2) Reaction of the toxicant with the target molecule:
 Types of reactions:
 Noncovalent binding – temporary
 Covalent binding – permanent.
3) Inappropriate repair:

Many toxicants alter macromolecules, which eventually cause

damage at higher levels of the biological hierarchy in the organism.
This toxic lesions can be interfere by repair mechanisms operating
at molecular, cellular, and tissue levels.

4) Inappropriate adaptation:
The organism can resist the noxious chemical is by increasing its
own readiness to cope with it and with its harmful effects. This
phenomenon is called adaptation.
Mutagenicity testing

Mutagenicity testing is used to assess submicroscopic changes in the base sequence of DNA,
chromosomal aberrations, and structural aberrations in DNA including duplications, insertions,
inversions, and translocations. Certain types of mutations result in carcinogenesis (alteration in
proto-oncogenes of tumor suppressor gene mutation), and so the determination of the
mutagenicity is essential in the drug development process. In vitro testing is carried out in two or
three different bacteria and mammalian cells to cover the end points of gene mutations,
clastogenicity, and aneuploidy. The test generally includes a bacterial reverse mutation assay.
The choice of an additional test depends on the chemical structure/class of the substance. In vivo
mutagenicity which is dose dependent is used to determine the case-by-case basis risk
assessment of the test substances. Mutagenicity studies with transgenic animals are more
appropriate assay techniques to determine the toxicity of a test substance,19]

Subchronic oral toxicity testing (repeated dose 90-day oral toxicity testing)

Rodents and nonrodents are used to study the subchronic toxicity of a substance. The test
substance is administered orally for 90 days, and weekly body weight variations, monthly
biochemical and cardiovascular parameters changes, and behavioral changes are observed. At the
end of the study, the experimental animals are sacrificed. Gross pathological changes are
observed, and all the tissues are subjected to histopathological analyses. There should be little
individual variation between the animals, and the allowed weight variation range is ±20%. A
satellite group may be included in the study protocol, and this group has both a control group and
a high-dose group.[20,21]

Chronic oral toxicity testing

Chronic toxicity studies are conducted with a minimum of one rodent and one nonrodent species.
The test compound is administered over more than 90 days, and the animals are observed
periodically. A chronic toxicology study provides inferences about the long-term effect of a test
substance in animals, and it may be extrapolated to the human safety of the test substance. The
report on chronic oral toxicity is essential for new drug entities. There should be little individual
variation between the animals, and the allowable weight variation range is ±20%. A satellite
group may be included in the study protocol. This group has both a control group and high-dose
group. During the study period, the animals are observed for normal physiological functions,
behavioral variations and alterations in biochemical parameters. At the end of the study, tissues
are collected from all parts of the animal and subjected to histological analyses.[22]

Carcinogenicity testing

Both rodents and nonrodent animal species may be used in carcinogenicity testing. The tests are
carried out over the greater portion of an animal's lifespan. During and after exposure to test
substances, the experimental animals are observed for signs of toxicity and development of
tumors. If these are not found, a test may be terminated after 18 months in the case of mice and
hamsters and after 24 months with rats. If the animals are healthy, hematological analysis is
performed after the 12 months and the 18 months, respectively, and the study is terminated. The
animals are sacrificed, and gross pathological changes are noted and histopathological studies are
carried out on all the tissues.[23]

One-generation reproduction toxicity testing

The test compound is administered to both male and female animals. Administration is for the
duration of one complete spermatogenic cycle in male animals and for two complete estrous
cycles for female animals. Rodents are preferred for the one-generation reproduction toxicity
testing. After the completion of the specified duration of drug administration, the animals are
allowed to mate. The test compound is administered to the female animals during the period of
pregnancy and nursing. The sperms of male animals are collected, and the sperm morphology
and motility are analyzed. During the study period, the animals are observed for signs of toxicity.
Parturition, the number of offspring and their sexes are recorded. The number of dead and live
pups are noted, and live pups are weighed in the morning and evening each day during the first 4
days. After the termination of the study, the animals and pups are sacrificed and subjected to a
histopathological examination.[24]

Two-generation reproduction toxicity studies

Both male and female rodents are administered the test substance. The duration of administration
extends to one complete spermatogenic cycle for males and two complete estrous cycles for
females. After the administration period, the animals are intertwined (parental mating), after
which the female animals are separated. Sperms are collected from male animals, and the sperm
morphology and motility are analyzed. The test substance is administered continuously to
pregnant female animals, which are monitored regularly for mortality and signs of toxicity. After
parturition, nursing rats are administered the test drug, and the mortality of the pups (F1
generation) is observed. From the F1 generation, one male and one female animal are selected.
The same procedure is repeated to get the F2 generation offspring. F1 offsprings are not allowed
to mate until they have attained full sexual maturity, and pairs without a pregnancy are evaluated
for infertility. Necropsies and histological examinations are carried out. At the end of the study,
the animals are sacrificed and gross pathological and histological examinations are carried out on
all the animals.[24–26]

Toxicokinetics

Toxicokinetics which is an extension of pharmacokinetics deals with the kinetic patterns of

higher doses of chemicals/toxins/xenobiotics. Toxicokinetics helps study the metabolism and
excretion pattern of xenobiotics. Animal toxicokinetic data help extrapolate physiologically
based pharmacokinetics in humans. In toxicological testing, pharmacokinetic studies are usually
carried out in rodents, rabbits, dogs, nonhuman primates and swine using many routes of
administration. Blood samples are collected at various time points to analyze pharmacokinetic
data such as the area under the curve, drug distribution ratio, Cmax, tmax, and other
pharmacokinetic parameters. Toxicokinetic studies may be performed using in vitro cell lines
also.[27,28]

Neurotoxicity studies in rodents

The effects of a test substance on the central nervous system can be studied through
neurotoxicity studies. The peripheral nervous system is further divided into the somatic and
autonomic nervous systems. Neurotoxic studies may be employed to evaluate the specific
histopathological and behavioral neurotoxicity of a chemical and are used to characterize
neurotoxic responses such as neuropathological lesions and neurological dysfunctions (loss of
memory, sensory defects, and learning and memory dysfunctions). Usually neurotoxicological
studies are carried out in adult rodents. The test substance may be administered for 28 days or
even more than 90 days, and neurological changes are evaluated. In 1998, the in vitro model for
neurotoxicity was developed, and various regulatory agents now recommend in vitro
neurotoxicity testing.[24]

Developmental toxicity/embryotoxicity studies

Embyrotoxicity can be studied using both in vivo and in vitro methods. Rodents are preferred for
in vivo toxicity screening. The compound is administered between the 8thand 14thday of
pregnancy, and embryolethal effects are studied. At the end of the study or on the 21st day of the
study, a caesarean section is performed and parameters such as fetuses with hemorrhagic bullae,
limb malformations, exencephaly, cleft palates, open eyelids, and tail deformities as well as the
mortality and the numbers of dead and live pups are noted. Embryotoxicity studies can be
performed using in vitro methods such as the embryonic stem cell test (EST) for embryotoxicity,
micromass embryotoxicity assay, and whole rat embryo embryotoxicity assay.[29–31]

Genetic toxicity testing

Genetic toxicity tests are used to identify gene mutations, chromosome changes, and alterations
in the DNA sequencing. These tests are usually conducted in various species including whole
animals, plants, micro-organisms, and mammalian cells. In the whole animal model, rodents are
preferred. Genetic toxicity is assessed using the rodent chromosome assay, dominant lethal
assay, mouse-specific locus test, micronucleus test, heritable translocation assay, and sister
chromatid exchange assay.[32,33]

Regulatory requirements

Before conducting any clinical study, the safety of the test substance should be assessed using
animals. The target organ toxicity, relationship between the dose and response, relevant human
effects, and any complications arising during treatment (adverse drug reactions) should be
established through preclinical evaluations. The toxicity study should be carried out with a
minimum of three doses viz. low, medium, and high doses in the experimental animals and the
toxic effect compared with data from a control group of animals. The Committee for Proprietary
Medicinal Products (CPMP) has set guidelines on the toxicological experiment on various
animal species. The guideline instructs that the maximum selected dose should be sufficient to
identify the target organ toxicity. From the toxicological evaluation, the no observed effect level
(NOEL) or NOAEL, which may be useful for human studies, may be established. The low dose,
intermediate dose, and high dose used in the toxicity test provide the NOEL, dose–response
relationship, and target organ toxicity in animals, respectively.[34]
LABORATORY ANALYSIS OF TOXINS
Toxins may be evaluated qualitatively or quantitatively. Qualitative analysis provides
information about the nature of toxins, but quantitative analysis gives information about the
chemistry of the toxins and their concentration. Nonspecific instrumental analyses such as
colorimetric and UV-visible spectrophotometric analyses may be used for qualitative analysis of
toxins. Sophisticated techniques such as infrared spectroscopy, gas chromatography, High
Pressure Liquid Chromatography, and immunoassay techniques may be employed to quantify the
toxins.

Footnotes
Source of Support: Nil

Conflict of Interest: None declared

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