Islamic university in Uganda

Faculty of management studies

Advanced diploma in health service management

Course code; AHM 1202

Course unit; principles of environmental Health

Lecturer: Mr Sydney Nsubuga

Group members. Reg No.s

1. Kissa Scovia 120-032123- 22788

2. Kissa sisco 120-032123-22971

3. Chebet Frida 120-032123-22481

4. Cherotwo Justine 120-032123-22658

5. Chelogoi Robert 120-032123-23206

6. Chesang Dennis 120-032123-22115.

7. Ayub Maiga Hussein 120-032123-22587

Question

A desirable first step in toxicity tests is to estimate median lethal time for each of a series of

concentrations. Describe the various methods of measuring acute and chronic toxicity

including carcinogens.
Objectives.

• Define toxicology
• Explain the meaning of common terms used in toxicology.
• Describe the subdivisions of toxicology.
• Describe toxic kinetics and toxic dynamics.
• Describe the various methods/test procedure of measuring acute and chronic toxicity
including carcinogens.
• Illustrate various mechanisms of toxicity testing and how they are performed.
• Perform simple toxicological experiments.
Introduction

• Toxicology is the study of the adverse effects of chemicals on living organisms or

cells.
• Toxicity is the inherent capacity of a chemical to cause injury.
• All chemicals, including drugs, have some degree of toxicity
• Poisoning is an adverse effect from a chemical that has been taken in excessive
amounts.
• The body is able to tolerate and, in some cases, detoxify a certain dose of a chemical;
• However, once a critical threshold is exceeded, toxicity results.
Poisoning can produce minor local effects that can be treated readily in the outpatient
setting, or systemic life-threatening effects that require intensive medical intervention
• Virtually any chemical can become a poison when taken in sufficient quantity.
• The potency of some compounds leads to serious toxicity with small quantities.
• Poisoning by chemicals includes exposure to drugs, industrial, chemicals, household
products, plants, venomous animals, and agrochemicals.
• Virtually any chemical can become a poison when taken in sufficient quantity.
• The potency of some compounds leads to serious toxicity with small quantities.
• Poisoning by chemicals includes exposure to drugs, industrial, chemicals, household
products, plants, venomous animals, and agrochemicals.
 Sub-divisions of toxicology
• Mechanistic toxicology is concerned with identifying and understanding the cellular,
biochemical, and molecular mechanisms by which chemicals exert toxic effects on
living organisms
• Descriptive toxicology- involves carrying out the appropriate tests in experimental
animals which are designed to yield information that can be used to evaluate the risks
posed to humans and the environment by exposure to specific chemicals.
• Regulatory toxicologist has the responsibility for deciding, on the basis of data
provided by descriptive and mechanistic toxicologists, whether a drug or other
chemical poses a sufficiently low risk to be marketed for a stated purpose or
subsequent human or environmental exposure resulting from its use
• Clinical Toxicologist:
Deals with toxicity of drugs used in Medicine for diagnostic, preventive & therapeutic
purpose
• Forensic Toxicologist:

Deals mainly with medicolegal aspects of drugs and poisons to establish and explain the
circumstances of legal cases
• Occupational Toxicologist:

Concerned with both potential toxicity of industrial chemicals & toxicity of products
that contend them & any waste from them to human health.
• Eco-Toxicologist:

Study the toxic effects of chemicals on the environment. I.e. population & on
ecosystem.
• Environmental Toxicologist:

The source of toxicant, their transport, degradation. & bio concentration. In the environ.
And their effects on human

Common terms used in toxicology.

 Median lethal time, after exposure of an organism to a toxic substance or stressful

condition. Median lethal time is used in toxicology studies to quantify amount of
stressor necessary to kill an organism.
  Toxicity-the intrinsic quality of a chemical to produce an adverse effect.
• Hazard- the ability of a chemical agent to cause injury in a given situation or
setting.
Risk is the expected frequency of the occurrence of an undesirable effect arising from
exposure to a chemical or physical agent

 Safety is the practical certainly that injury will not result from a substance when used
in quantity and manner proposed.
 Teratogen- a chemical or argent which acts during pregnancy to produce a physical
or physiological defect on the offspring.
 Mutagen- a chemical capable of producing inheritable defect.
 Carcinogen- a chemical capable of causing cancer.
 Safety is the practical certainly that injury will not result from a substance when used
in quantity and manner proposed.

 Teratogen- a chemical or argent which acts during pregnancy to produce a physical

or physiological defect on the offspring.
 Mutagen- a chemical capable of producing inheritable defect.

• Carcinogen- a chemical capable of causing cancer.

• Toxin: generally refers to toxic substances that are produced by biological systems
such as plants, animals, fungi, or bacteria.

• Toxicant: refers to toxic substances that are produced by or are a by-product of

anthropogenic (human-made) activities.

Lethal dose concentrations of test drugs/plant extract result in 50% mortality.

Confidence intervals are determined from the 24hour and 48hour and the dose-
response data are transformed into a straight line by means of a trend line fit linear
regression.

Toxico-kinetics

Pharmacokinetics parameters including liberation, absorption, distribution,

metabolism and excretion dictates how much dose reaches the target tissues for
 drugs ingested for therapeutic purpose. These parameters are also used to calculate or
determine how much toxin gets to its target tissue.

• Toxic kinetic parameters can be affected by age, gender, body composition in terms
of fat-to- muscle ratio, disease state, nutritional value, hormonal changes

Toxico- dynamics.

• Refers to mechanism by which toxic substances produce their toxicity.

• Dose-response principles are relevant in estimating the potential severity of

intoxication.

• Toxic substances are metabolised to products that produce direct toxicity to cells
(paracetamol) or inactivate enzymes directly (organophosphates).

• Same as pharmacodynamics (only toxicity parameters considered)

Toxicogenetics.

• Genetic polymorphisms are responsible for many of the differences observed among
individuals concerning toxin metabolism
• It can be used to explain some differences observed among individuals exposed to the
same toxin at the same dose under the same conditions.
Duration and frequency of exposure.

• For many toxins, the length of time over which exposure occurs and frequency of
exposure determines the onset and severity of symptoms observed.
• This relationship gives rise to 2 different types of toxicology; acute & chronic.
Acute Toxicity.

• This refers to the ability of the chemical to cause harm as a result of a one-time
exposure.

E.g. when a child ingests some medications or house hold products, the exposure is sudden,
often involves large doses and results into medical emergency
• In toxicity testing, a single dose is administered and the animal observed for 24 hours
and up to 14 days after. BASIC TOX\2. experiment.mp4
 TOXICITY RATING.

DOSE LEVELS COMMENTS

15g/kg > Practically non toxic

5-15g/kg Slightly toxic

0.5g-5g/kg Moderately toxic

50-500mg/kg Very toxic

5-50mg/kg Extremely toxic

<5mg/kg Super toxic

Why do acute toxicity test?

• Identify lethal/toxic doses of chemicals for humans (primarily for the regulatory
purposes of classification and labeling).
• Indicate the mode of toxicity in humans, including the susceptibility of key target
organs.
• Provide a rough guide for dose selection in repeat-dose test in animals.
• Select doses for short-term and sub chronic toxicity tests when no other toxicology
information is available.
• To deal with cases of accidental ingestion of a large amount of the material, especially
for poison control information.
Sub chronic toxicity.

• Sub-chronic toxicity tests are employed to determine toxicity likely to arise from
repeated exposures for several weeks to several months (1-3months)

Chronic toxicity.

• Is when exposure occurs repeatedly over a period of 6 month or greater?

• The dose is usually lower than necessary to cause acute responses.

• Here the chemicals are metabolized slowly (long elimination ½ life) and are deposited
in organs. E.g. lead toxicity.

• Can be noted in patients exposed at work places (occupational hazards).

Routes of exposure.

• The route of exposure to a toxin determines how much of it enters the body and which
organs are exposed to the largest dose.

The 3 primary routes of exposure are; the lungs (inhalation), the skin (dermal), the G.I.T
(oral)

The route of exposure influences

 Absorption and distribution of toxins
Potentials of toxicity.

 All substances are poisons; it is the dose that makes a thing not a poison.

 What becomes clear as one studies toxicology is that everything is potentially toxic.

 the potential of toxicity depends on the dose and the conditions under which a person
is exposed to the substance

COMMON TOXICOLOGICAL TESTS.

• Carcinogenesis tests: - test the ability of a chemical to cause cancer specific animals
are used.
• Mutagenesis- tests the ability of a chemical to cause mutation that can be inherited
(Ames test).
 • Reproductive effect- effect on reproduction ability of individuals e.g. teratogenicity,
spermatogenesis and even libido.

Measurement/tests of acute and chronic toxicity including carcinogens

The majority of chronic and acute toxicity including carcinogenicity studies are carried out
mainly in rodent species. Should such studies be required in non-rodent species, the
principles and procedures outlined may also be applied, with appropriate modifications,
together with those outlined in Organisation for economic cooperation and development tests
(OECD TG 4093). The three main routes of administration used in chronic and acute
toxicity/carcinogenicity studies are oral, dermal and inhalation. The choice of the route of
administration depends on the physical and chemical characteristics of the test chemical and
the predominant route of exposure of humans.Oral routes mainly measure chronic toxicity
and carcinogenicity studies. Exposure via the dermal or inhalation routes may also be
necessary for human health risk assessment and/or may be required under certain regulatory
regimes, both routes of exposure involve considerable technical complexity. Such studies
will need to be designed on a case-by-case basis though it may constitute acute toxicity.

The objectives of chronic and acute toxicity/carcinogenicity tests include.

-The identification of the carcinogenic properties of a chemical, resulting in an increased

incidence of neoplasms, increased proportion of malignant neoplasms or a reduction in the
time to appearance of neoplasms, compared with concurrent control groups

- The identification of the time to appearance of neoplasms.

-The identification of the chronic toxicity of the chemical.

-The identification of target organ(s) of chronic toxicity and carcinogenicity.

- 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).

- Extrapolation of carcinogenic effects to low dose human exposure levels.

- Prediction of chronic toxicity effects at human exposure levels

-Provision of data to test hypotheses regarding mode of action.

To carry ot the tests, the following may be necessary/concidered:

-selection of animal species population and control group matters which are rodents.

-Housing and feeding conditions,Preparation of animals,Preparation of doses based on

OECD test guidelines on targgeted organ and must be in cages.
Observations Ophthalmological examination. Body weight, food/water consumption and foo
d efficiency. Haematology and clinical biochemistry, Pathology gross necropsy and
deductions with reportings.

PROCEDURE

Both sexes should be used. A sufficient number of animals should be used so that a thorough
biological and statistical evaluation is possible. For rodents, each dose group and concurrent
control group intended for the carcinogenicity phase of the study should therefore contain at
least 50 animals of each sex. Depending on the aim of the study, it may be possible to
increase the statistical power of the key estimates by differentially allocating animals
unequally to the various dose groups, with more than 50 animals in the low dose groups, e.g.,
to estimate the carcinogenic potential in low doses. However it should be recognised that a
moderate increase in group size will provide relatively little increase in statistical power of
the study. Each dose group and concurrent control group intended for the chronic or acute
toxicity phase of the study should contain at least 10 animals of each sex, in the case of
rodents. It should be noted that this number is lower than in the chronic toxicity study TG
452. The interpretation of the data from the reduced number of animals per group in the
chronic toxicity phase of this combined study will however be supported by the data from the
larger number of animals in the carcinogenicity phase of the study. In studies involving mice,
additional animals may be needed in each dose group of the chronic toxicity phase, to
conduct all required haematological determinations. OECD guidline be followed.

Other procedures given below can also be used to measure acute toficity based on
Five Organisation for Economic Cooperation and Development (OECD) Test Guidelines set
also are used to describe acute systemic testing and they are

-Fixed Dose Procedure

-Acute Toxic Class method

-Up‐and‐Down Procedure

-Acute Dermal Toxicity

-Acute inhalation toxicity

Dose groups and dosages that may be used in the experimental tests

-At least three dose levels and a concurrent control should be used based on specific
guidline, for both the chronic and carcinogenicity phases.

- levels will generally be based on the results of shorter-term repeated dose or range finding
studies and should take into account any existing toxicological and toxicokinetic data
available for the test chemical or related materials.

-In the dose selection the investigator should also consider and ensure that data generated is
adequate to fulfil the regulatory requirements across OECD countries as appropriate (e.g.,
hazard and risk assessment, classification and labelling, ED assessment, etc.)

-For the chronic toxicity phase of the study, a full study using three dose levels may not be
considered necessary, if it can be anticipated that a test at one dose level, equivalent to at
least 1000 mg/kg body weight/day, is unlikely to produce adverse effects. This should be
based on information from preliminary studies and a consideration that toxicity would not be
expected, based upon data from structurally related substances. A limit of 1000 mg/kg body
weight/day may apply except when human exposure indicates the need for a higher dose level
to be used.

-Unless limited by the physical-chemical nature or biological effects of the test chemical, the
highest dose level should be chosen to identify the principal target organs and toxic effects
while avoiding suffering, severe toxicity, morbidity, or death. The highest dose level should
be normally chosen to elicit evidence of toxicity, as evidenced by, for example, depression of
body weight gain (approximately 10%). However, dependent on the objectives of the study
(see paragraph 6), a top dose lower than the dose providing evidence of toxicity may be
chosen, e.g., if a dose elicits an adverse effect of concern, which nonetheless has little impact
on lifespan or body weight.
Duration of study.

The period of dosing and duration of the chronic and acute phases of this study is normally
12 months, although the study design also allows for and can be applied to either shorter (e.g.
6 or 9 months) or longer (e.g., 18 or 24 months) duration studies, depending on the
requirements of particular regulatory regimes or for specific mechanistic purposes.
Deviations from an exposure duration of 12 months should be justified, particularly in the
case of shorter durations. All dose groups allocated to this phase will be terminated at the
designated time for evaluation of chronic toxicity and non-neoplastic pathology. Satellite
groups included to monitor the reversibility of any toxicological changes induced by the
chemical under investigation should be maintained without dosing for a period not less than 4
weeks and not more than one third of the total study duration after cessation of exposure.

OBSERVATIONS (CARCINOGENICITY PHASE)

- All animals should be checked for morbidity or mortality, usually at the beginning and the
end of each day, including at weekends and holidays. Animals should additionally be
checked once a day for specific signs of toxicological relevance. In the case of gavage
studies, animals should be checked in the period immediately following dosing. Particular
attention should be paid to tumour development; and the time of tumour onset, location,
dimensions, appearance, and progression of each grossly visible or palpable tumour should be
recorded as acute or chronic.

-All animals should be weighed at the start of treatment, at least once a week for the first 13
weeks and at least monthly thereafter. Measurements of food consumption and food
efficiency should be made at least weekly for the first 13 weeks and at least monthly
thereafter. Water consumption should be measured at least weekly for the first 13 weeks and
at least monthly thereafter when the substance is administered in drinking water. Water
consumption measurements should also be considered for studies in which drinking activity
is altered.

Results

Based on specific timings, results can be deduced based on the following:

Survival data, body weight food consumption, calculations of food efficiency and water con
sumption toxic response data by sex and dose level, including signs of toxicity,
nature, incidence (and, if scored, severity), and duration of clinical observations ophthalmolo
gical examination,haematological tests,clinical biochemistry tests; urinalysis tests; Investiga
tions of neurotoxicity or immunotoxicity,Terminal body weight, organ weights (and their rati
os, if applicable); necropsy findings and detailed description of all treatment‐
related histopathologicalfindings.

Report

The test report should include the following information:

Test chemical( physical nature, purity, and physicchemical properties)

-Identification data, source of substance,batch number, certificate of chemical analysis.

-Vehicle (if appropriate, justification for choice of vehicle (if other than water).

-Test animals, species/strain used and justification for choice made, number, age, and sex of
animals at start of test, source, housing conditions, diet, etc. individual weights of animals at
the start of the test.

-Test conditions, rationale for route of administration and dose selection, when applicable,
the statistical methods used to analyse the data, details of test chemical formulation/diet
preparation, analytical data on achieved concentration, stability and homogeneity of the
preparation, route of administration and details of the administration of the test chemical,for
inhalation studies, whether nose only or whole body,actual doses (mg/kg body weight/day),
and conversion factor from diet/drinking water test chemical concentration (mg/kg or ppm) to
the actual dose, if applicable, details of food and water quality.

Results (summary may be tabulated data and individual animal data presented):

General,Survival data, Body weight/body weight changes, Food consumption, calculations of

food efficiency, if made, and water consumption if applicable,Toxicokinetic data if available,
Opthalmoscopy (if available), Haematology (if available), Clinical chemistry (if available)
Clinical findings, Signs of toxicity, Incidence (and, if scored, severity) of any abnormality,
Nature, severity, and duration of clinical observations (whether transitory or permanent),

Necropsy data, Terminal body weight, Organ weights and their ratios, if applicable, Necropsy
findings; Incidence and severity of abnormalities.
Histopathology, Non neoplastic histopathological findings, Neoplastic histopathological
findings, Correlation between gross and microscopic finding, Detailed description of all
treatment-related histopathological findings including severity gradings, Report of any peer
review of slides

Statistical treatment of results, as appropriate

Discussion of results including. a discussion of any modelling approaches, dose:response

relationships,Historical control data ,Consideration of any mode of action informationand a
of determination and Relevance for human
AMES TEST METHOD FOR DETERMINATION OF MUTAGENICITY
POTENTIAL OF A SUBSTANCE/CHEMICAL

Teratogenesis

This is the production of gross foetal anatomical malformation e.g. loss of eye, limb
etc. modern teratogenesis involves behavioural, enzymatic and internal organ
malformation. It is now mandatory for all drugs to be interested for teratogenesis
promoted by the thalidomide disaster.
SOURCES OF TERATOGENES

• Environmental- pesticides, drugs, radioactive substances etc.

st
• Drugs- especially during the 1 trimester.
TEST PROCEDURES

• Rats are caged over night and vaginal plug identified. This marks the “0” points of
pregnancy.

• Drugs are administered between 0-5 days (1st trimester) since gestation is 18-20 days.
 • The rat is dissected and the foetus examined on day 19.

• The rat is not allowed to deliver as it will eat any dead foetus before the observer
arrives.

• The 1st trimester is used because organogenesis occurs within this period. After this
the drug will have no effect.

• This result in the pregnancy categorisation or labelling of drugs i.e. according to the
risks involved.

Category A

• Drugs in which a well-controlled study has not demonstrated any teratogenic effect
e.g. penicillin’s
Category B

• Animal data have not exhibited any teratogenic effect but there is no data on human
beings.

Category C

• A drug indicated as teratogenic in both man and animals but their use is still
warranted e.g. phenytoin is an anticonvulsant which can still be used on epileptic
pregnant women.
PREGNANCY CATEGORY D

• There is positive evidence of risk to the human fetus.

• Investigational or post marketing data show risk to the fetus.

• However, potential benefits may outweigh the risk to the fetus. If needed in a life-
threatening situation or a serious disease, the drug may be acceptable if safer drugs
cannot be used or are ineffective.

Category X

• Studies in animals or human beings have demonstrated foetal abnormalities, or there

is evidence of foetal risks based on human experience or both, and
• the risk of the use of the drug in pregnant women clearly out weighs any possible
benefit.

• The drug is contraindicated in women who are or may become pregnant. Eg

efavirenz, most anticancer drugs.
 References.
1. Betrum .G. Katzung, Basic & clinical pharmacology 10thed.
2. Laurence & Bennet; Clinical Pharmacology. 7th ed.
3. Casarett and Doull's Toxicology. The Basic Science of Poisons 7th ed,
OECD, (1995). Report of the Consultation Meeting on Sub-chronic and Chronic
Toxicity/Carcinogenicity Testing Rome, (1995), internal working document.

4. EPA (2005). Guidelines for Carcinogen Risk Assessment Risk -Assessment Forum
U.S. -Environmental Protection Agency Washington.
5. Combes RD, Gaunt, I, Balls M (2004). A Scientific and Animal Welfare Assessment
of the OECD Health Effects Test-guidelines for the Safety Testing of Chemicals under
the European Union REACH System.

6. Barlow S.M, Greig J.B, Bridges J.W.et, al (2002). Hazard identification by methods of
animal based toxicology.