Principles of Toxicology

PRINCIPLES OF TOXICOLOGY
Dr.R.Vadivelan M.Pharm.PhD,FIC
Professor in Pharmacology
JSS College of Pharmacy
JSS Academy of Higher Education and Research,
Ooty,The Nilgiris,Tamilnadu-643001
vadivelanr@jssuni.edu.in
July 24, 2020 Principles of Toxicology 1

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 Introduction
 Acute, subacute and chronic toxicity
 Genotoxicity, carcinogenicity, teratogenicity and
mutagenicity
 General principles of treatment of poisoning
 Clinical symptoms and management of barbiturates,
morphine, organophosphosphates
 Lead, arsenic and mercury poisoning
Principles of Toxicology 2July 24, 2020

Introduction
Toxicology – Study of poisons
Study of the adverse effects of chemical substances on
living organisms and the practice of diagnosing and
treating exposures to toxins and toxicants.
Relationship between dose and its effects
Factors -Dosage, duration of exposure (acute or
chronic), route of exposure, species, age, sex, and
environment.
Toxicologists-Experts on poisons and poisoning
Evidence-based toxicology (EBT)

History
Pedanius Dioscorides was a a Greek physician in
the court of the Roman emperor Nero made the first
attempt to classify plants according to their toxic and
therapeutic effect.(Der materia medica)
Mathieu Joseph Bonaventure Orfila was a Spanish
toxicologist and chemist, the founder of the science of
toxicology. First formal treatment in 1813 in his Traité
des poisons, also called Toxicologie générale.
Jean Stas (1850) became the first person to
successfully isolate plant poisons from human tissue.
(Nicotine)
Theophrastus von Hohenheim (1493–1541) (also
referred to as Paracelsus, Roman physician) is also
considered "the father" of toxicology. “The dose makes
the poison”

ToxicantsVsToxinVs Poisons
Poison is a toxicant that
cause immediate death or
illness in very small amount

Key concept in toxicology
Quantitative relationship
between the concentration of
a xenobiotic in the body and the
magnitude of the biological
effect it produces.
The Dose Makes the Poison
Beneficial Dose Toxic Dose
Aspirin 300 – 1,000 mg 1,000 – 30,000 mg
Vitamin A 5000 units/day 50,000 units/day

Types of toxicology
Mechanistic
toxicology
Descriptive
toxicology
Regulatory
toxicology
Forensic
toxicology
Environmental
toxicology
Clinical
toxicology
Reproductive
toxicology
Developmental
toxicology

Types of exposure & effect
Acute - A single
exposure lasting less
than 24 hours
Subacute- Exposure for
1 month or less.
Subchronic – Repeated
exposures of less than a
lifetime (e.g.1- 3 months)
Chronic – (Long term) – Exposures are
essentially for the lifetime of the species
(more than 3 months)
Local
Systemic
Cumulative
Poisoning

Route of Exposure &Time
How long an organism is
exposed to a chemical is
important
Duration and frequency
contribute to dose. Both may
alter toxic effects.
Acute Exposure- usually
entails a single exposure
Chronic Exposures -multiple
exposures over time
(frequency)

Regulatory guidelines for
conducting toxicity studies
OECD
• Organization
for Economic
Co-operation
and
Development
ICH
• International
Conference
on
Harmonization
• QSEM
EPA
• Environmental
Protection
agency (USA)
ScheduleY
• The current
regulator
(CDSCO –
Central Drugs
Standard
Control
Organization)
enforced law
in India has
been
established
under Drugs
and Cosmetic
Act 1945
07/02/2020 ETHICS GUIDELINES IN AT- KP 10

Animal toxicity studies
Acute toxicity study- 14 days
Sub acute toxicity studies- 28 days
Sub Chronic toxicity studies- 3 months
Chronic toxicity studies- 6 months and 2yrs
Special toxicity studies- e.g Carcinogenicity

Acute toxicity studies
LD50 – used as an indicator for acute toxicity previously, involved
large no. of animals and mortality ratio is high(24 hrs
-Graphical method (Miller & Tainter Method)
-Arithmetical method (Karbers’s) method, when number is small

Acute toxicity study
To study the effect of a single dose on a particular animal species.
Acute toxicity testing be carried out with two different animals
species (one rodent and one non-rodent).
In acute toxicological testing, the investigational product is
administered at different dose levels, and the effect is observed
for14 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 study
Acute toxicity studies provide
information on:
 The potential for acute
toxicity in humans;
 An estimate of safe acute
doses for humans;
 The potential target organs
of toxicity;
 Time course of drug-induced
clinical observations;
 The appropriate dosage for
multiple-dose toxicity
studies; and
 Species differences in
toxicity.
Introduction with purpose of the test
Description of test procedure
Principle of the test
Preparations
Experimental animals
Selection of species
Number and sex
Housing and feeding conditions
Testing conditions
Dose levels ,Limit test
Observation period
Procedure
Clinical examinations
Pathology
Assessment of toxicity in other sex
Data and reporting
Treatment of results
Evaluation of report
Test report

OECD- Acute oral toxicity methods
Principles of three alternative methods

S.No. Parameters 0 min 30 hour 1 hour 2
hour
4
hour
24 hour
1 Hyperactivity
2 Piloerection
3 Twitching
4 Rigidity
5 Irritability
6 Jumping
7 Clonic convulsions
8 Tonic convulsion
9 Ptosis
10 Sedation
11 Sleep(Loss of R.R)
12 Loss of traction
13 Loss of pinna reflex
14 Catatonia
15 Ataxia
16 Loss of muscle tone
17 Analgesia
18 Straub’s tail
19 Labored respiration
20 Cyanosis
21 Blanching
22 Reddening
23 Abnormal secretion
24 Death
16

Subacute toxicity study
Subacute toxicity studies are conducted to evaluate a
new drug's potential adverse effects following a treatment
period of 2–4 weeks' duration.
Subacute toxicity studies are conducted as range-finding
studies (very important in helping determine the
maximum tolerable dose (MTD) of a drug product) .
To choose dosage levels to be used in subsequent
subchronic and chronic toxicity studies.

Repeated Dose 28 Days OralToxicity study
in Rodents (OECD 407)
• In the assessment and evaluation of the toxic
characteristics of a chemical, the determination
of oral toxicity using repeated doses may be
carried out after initial information on toxicity
has been obtained by acute testing.
Purpose of
the study
• The test substance is orally administered daily in
graduated doses to several groups of experimental
animals, one dose level per group for a period of 28 days.
• During the period of administration the animals are
observed closely, each day for signs of toxicity.
• Animals which die or are killed during the test are
necropsied and at the conclusion of the test surviving
animals are killed and necropsied
Principle of
the study

Repeated Dose 28 Days OralToxicity study
 Introduction & Purpose
 Initial considerations and Limitations
 Principle of test
 Description of the method
◦ Number & sex of animals
◦ Housing & Feeding
◦ Preparation of animals
◦ Preparation of doses
 Procedure
◦ No. & Sex of animals (10 rats /each dose level (5M and 5 F) and additional satellite group)
◦ Dosage- Control group and at least three test groups
◦ Limit test - One dose level of 1000 mg/kg
◦ Administration of doses
◦ Observations
◦ Body weight and food/water consumption
◦ Hematology
◦ Clinical Biochemistry
 Pathology
◦ Gross necroscopy
◦ Histopathology
 Data & Reporting
July 24, 2020 Oral Toxicity studies 19

Sub chronic toxicity study -Repeated Dose 90 Days
OralToxicity in Rodents (OECD 408)
Purpose
 Possible health
hazards likely to arise
repeated exposure
over a prolonged
period of time .
 Provide information on the
major toxic effects,indicate
target organs and the
possibility of accumulation,
and can
 Provide an estimate of a
NOAEL of exposure -
selecting dose levels for
chronic studies and for
establishing safety criteria
for human exposure
Principle
The test substance is orally
administered daily in graduated
doses to several groups of
experimental animals, one dose
level per group for period of 90
days.
Observed closely for signs of
toxicity.
Animals which die or are killed
during the test are necropsied
and, at the conclusion of the test,
surviving animals are also killed
and necropsied.

Repeated Dose 90 Days OralToxicity
 Procedure
◦ No. & Sex of animals (20 rats /each dose level (10M and 10 F) and additional satellite group)
◦ Observations
◦ Hematology
 Pathology
◦ Histopathology

Chronic toxicity studies
(OECD 452)
The identification of the chronic toxicity of a
chemical
The identification of target organs
Characterisation of the dose-response
relationship
Identification of a no-observed-adverse-effect
level (NOAEL) or point of departure for
establishment of a Benchmark Dose (BMD)
The prediction of chronic toxicity effects at
human exposure levels
Provision of data to test hypotheses regarding
mode of action
Principle
 Orally Administered daily in
graduated doses to several
groups of 12 months, depending
on regulatory requirements
 Allow any effects of cumulative
toxicity.
 Deviations from exposure
duration of 12 months should be
justified.
 One or more interim kills, e.g. at
3 and 6 months, and additional
groups
 Observed for signs of toxicity.
 At end of test of the test,
surviving animals are killed and
necropsied.

Chronic toxicity studies
(OECD 452)
 Procedure
◦ No. & Sex of animals (40 rats /each dose level (20M and 20F) and additional satellite group)
◦ Observations
◦ Hematology
 Pathology
◦ Histopathology

Genotoxicity
 Property of chemical agents that damages the genetic information within a
cell causing mutations, which may lead to cancer.
 Genotoxic agent is a chemical or another agent that damages cellular DNA,
resulting in mutations or cancer.
◦ E.g.Alkylating agents, polycyclic aromatic hydrocarbons, Griseofulvin
 Potentially mutagenic/ carcinogenic when inhaled, ingested or penetrate the
skin.

Mutagenicity
Mutagenicity refers to a chemical or
physical agent's capacity to cause
mutations (genetic alterations).
Agents that damage DNA causing lesions
that result in cell death or mutations are
genotoxins.

Carcinogenicity
 A carcinogen is any substance,
radionuclide or radiation, that is an
agent directly involved in the
exacerbation of cancer or in the
increase of its propagation.
 May be due to the ability to damage
the genome or to the disruption of
cellular metabolic processes.
 E.g Arsenic, cadmium,virus, fungi

Teratogenicity
Production or induction of malformations or monstrosities,especially of a developing embryo
or fetus.
Teratogen is an agent that can disturb the development of the embryo or fetus.Teratogens
halt the pregnancy or produce a congenital malformation (a birth defect).
Classes of teratogens include radiation,maternal infections,chemicals, and drugs.
(Thalidomide)

General principles of treatment of poisoning
Identify the poison
Ensure and maintain a clear airway
Ensure adequate ventilation
Suppress convulsions
Fluid and electrolyte therapy
Decontamination
Specific antidotes
Non specific pharmacological antidotes like anticonvulsants
Promote elimination of drug
Haemodialysis or peritoneal dialysis
General supportive and nursing measures

• Use of a selective antagonist may be helpful.
Identify the
poison
• Remove debris (vomitus, mucus, dentures),suck away
secretions consider endo tracheal intubation or
tracheostomy, if necessary.
Ensure and
maintain a clear
airway
• Tidal volume of about 400 ml and minute volume about
4 litres/minute for adults by mechanical ventilators, if
necessary, should be maintained.
• Underventilation leads to hypoxemia, and
overventilation may cause alkalosis and hypotension.
Ensure
adequate
ventilation.

• Not controlled by adequate ventilation, diazepam
10 mg should be administered in adults.
Suppress
convulsions
• Restoration of venous return and cardiac output.
• For this isotonic saline /isotonic glucose solution per day
must be administered.
• The urine volume and fluid balance should be recorded
to avoid or manage renal failure.
Fluid and
electrolyte therapy
• Gastric lavage in a conscious patient. .
• Vomiting -apomorphine or syrup of ipecac
• Activated charcoal 10-30 g suspended in 100 to 200 ml of
water
• Universal antidote : (2 parts activated charcoal, 1 part
tannic acid, and 1 part magnesium oxide) Orally
Decontamination

Non specific
pharmacological
antidotes
Analeptics
• Forced diuresis,
Ionization,increasing and
reducing the the pH of
urine.
Promote
elimination of
drug
• Patient’s condition
deteriorates inspite of
conventional treatment
Haemodialysis
or peritoneal
dialysis

 General supportive and nursing measures have to be simultaneously
instituted, specially in the comatosed or semicomatosed patient.
The principles of management of drug poisoning: (ABC)
*never done when corrosive agents have been ingested; never done in
unconscious patients, unless cuffed endotracheal tube in position.
Maintenance of vital functions Airway
circulation
Fluid and electrolytes
Hypothermia(gradual rewarming if rectal
temperature low)
Elimination of drug Induced vomiting/gastric lavage*
Other techniques- Forced diuresis,dialysis
Antidotes Specific
Pharmacological(symptomatic treatment)
34

Barbiturates Poisoning

Morphine Poisoning

Organophosphates Poisoning

Heavy metals

Heavy metal symptoms

Heavy metal poisoning

Lead Arsenic Mercury
Form of
absorption
Metallic lead, inorganic
or organic salts
Inorganic salt or
arsine gas
Metal
inorganic salts
Route of
absorption
Lungs, GIT(skin) GIT, lungs(arsine),
skin
Lungs, (elemental
Hg)GIT
Distribution Bone,teeth,hair,RBC,
liver, kidneys crosses
placenta
RBC(24 h)
liver,kidneys,lungs,
spleen, muscles
CNS(4 wk),bone,
skin, hair, crosses
placenta
CNS,kidneys
blood
Excretion Very very
slow,urine,faeces
Very slow
urine(faeces)
Slow,urine,faeces
42
Lead,Arsenic & Mercury Poisoning

Lead Arsenic Mercury
Mechanism
of toxicity
Lead binds to SH-
enzymes
Arsenic(trivalent)
binds to SH-enzymes
Hg2+ binds to SH
enzymes
Targets of
toxicity
Haeme synthesis,
neuropathy(motor)
kidneys,liver,intestine
reproduction
Bone marrow
neuropathy(sensory),
kidneys, liver, GIT,
Haemolysis (arsine)
CNS, kidneys, GIT,
gingivitis
Treatment
of poisoning
Stop exposure,
Ca-disodium edetate i.v.
dimercaprol i.m(children),
Dimercaptosuccinic Acid
(DMSA)
less severe cases)
Stop exposure,
decontamination,
supportive therapy,
dimercaprol i.m.or
DMSA (pencilamine
oral) exchange
transfusion and
haemodialysis(arsine)
Stop exposure,
Dimercaprol i.m.N-
acetyl-pencilamine,
DMSA,
Dimercapto-1-
propanesulfonic
acid (DMPS).
Hemodialysis in
severe cases
43

Metal Acute Chronic Toxic Concn Treatment
Lead
Nausea, vomiting,
encephalopathy
(headache, seizures,
ataxia, obtundation)
Encephalopathy, anaemia,
abdominal pain,
nephropathy,foot-drop/
wrist-drop
Pediatric:
symptoms or [Pb]
≥45 µ/dL (blood);
Adult: symptoms
or [Pb] ≥70 µ/dL
BAL, CaNa2 ,
EDTA, Succimer
Arsenic
Nausea, vomiting,
“rice-water” diarrhoea,
encephalopathy,
MODS, LoQTS,
painful neuropathy
Diabetes, hypo
pigmentation/
hyperkeratosis
cancer: lung, bladder,
skin, encephalopathy
24-h urine: para,
≥50 µg/L urine,
100 µg/g
creatinine
BAL (acute,
symptomatic)
Succimer
DMPS (Europe)
Mercury
Elemental (inhaled):
fever, vomiting,
diarrhea, Inorganic salts
(ingestion): caustic
gastroenteritis
Nausea, metallic taste,
gingivo-stomatitis,
tremor, neurasthenia,
nephrotic syndrome;
hypersensitivity (Pink
disease)
Background
exposure “normal”
limits: 10 µg/L
(whole blood); 20
µg/L (24-h urine)
BAL, Succimer
DMPS (Europe)

Need of toxicity study
Useful drug only if it is having relevant
pharmacological and therapeutic activity.
Free from short and long term
toxicity
Superior in any way to existing
drugs.
To be certain that a new drug is safe
and detail studies are made to know the
effects of varying doses and their
prolonged administration.

Principles of Toxicology

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