Toxicity testing methods

“In all things there is a poison, and there is nothing

without a poison. It depends only upon the dose
whether a poison is poison or not.”

Paracelsus (1493 - 1541)

 Toxicity testing
■ To make judgements on risks and appropriate control
measures information needs to be obtained on the hazardous
properties of the substance

– This may be from studies of human exposures (epidemiological studies)

– however, these often do not provide the full range of information
required

– A range of other approaches have been developed which toxicologists

use to investigate the toxicity of a substance
■ Physico-chemical properties
■ Similarities in chemical structure to other substances
■ Usually more information is required and some experimental
testing will be needed
 Toxicity testing
■ Toxicity testing on animals remains main approach
– Potentially controversial issue - progress has been made to replace animal
tests, reduce numbers used and refine testing methods to reduce suffering

– Most toxicity tests use rodents (specially bred rats, mice, guinea pigs, rabbits)

– Animal testing usually carried out to established international guidelines to

ensure they meet agreed standards and that results are acceptable to
regulatory authorities

■ Other approaches use cells or tissues in ‘culture’ (e.g. test tubes or

Petri dishes)
– Known as in vitro (‘in glass’) methods
 Toxicokinetic studies

■ Undertaken to study how a substance is absorbed,

distributed, metabolised and excreted from the body
– Studies involve administering a substance (often tagged with
radioactive atom) to animals via one of the routes of entry

– Presence of substance (or metabolite) measured in various

tissues over a period of up to a few weeks

– Considerable recent progress in computer aided modelling of

toxicokinetics, particularly as a means of extrapolating from
one species or route of exposure to another, and across a
range of doses
Methods

■ ACUTE TOXICITY STUDIES

■ SHORT-TERM TOXICITY STUDIES
■ LONG-TERM TOXICITY STUDIES
 Acute toxicity studies

■ Aim is to provide information on what general toxicity

might be seen if someone was exposed to a single, relatively
high dose of a substance e.g. accidental poisoning

■ Substance administered to groups of animals as single doses

– May be by inhalation, ingestion or dermal route

■ Animals observed for up to two weeks after administration

of dose for signs of toxicity
– After this period, organs of the animals examined for
signs of damage as markers of toxic effects
 Acute toxicity studies

■ Historically these studies designed to derive LD50 or LC50 values

– These values are relatively crude measures of toxicity
– However, they are useful to make relative comparisons of acute toxicity of
substances in order to rank them in terms of severity
– Have been used to derive hazard classifications
– Still find these values on Material Safety Data Sheets

■ However, studies often overlooked more critical information e.g.

toxic signs that might indicate how a substance exerts its effects
– Newer methods have been developed which use fewer animals and depend
more on signs of toxicity, rather than just mortality
– These methods are becoming more common and are now part of regulatory
schemes with established international guidelines
 Acute toxicity studies
■ Irritation studies
– Some substances can cause localised inflammation on contact
with skin, eyes or respiratory tract
■ In worst cases damage can be severe leading to corrosive
destruction of the tissue

– Traditionally animals, usually rabbits, used for skin and eye

irritation tests

– Significant efforts have been made to find alternative non-

animal test methods for assessing potential for skin or eye
irritation
■ Internationally validated methods have been developed which use in
vitro systems for skin irritation and corrosion
Experimental Design

■ Selection of Species of Animal

– In general, the rat, and sometimes the mouse,
– They are economical, readily available, and easy to handle.
– There are more toxicologic data on these species of animals, a fact that facilitates
comparisons of toxicities to other chemicals.
– Sometimes a nonrodent species is desirable.
■ The LD50 determination is preferably done in animals of both genders, and
also in adult and young animals, because of their differences
Route of Administration
■ Generally, the toxicant is administered by the route by which humans
would be exposed.
■ The oral route is most commonly used.
■ The dermal and inhalation routes are used increasingly, not only for
chemicals that are intended for human use by such routes but also for
chemicals whose health hazards to personnel handling these chemicals
are to be assessed
Dosage and Number of Animals
■ Untuk menentukan LD50 yang tepat:
– Perlu dipilih dosis yang akan membunuh sekitar separuh jumlah hewan
– Dosis yang membunuh lebih dari separuh, kalau bisa kurang dari 90%
– Dosis ketiga, yang membunuh kurang dari separuh, kalua bisa lebih dari 10%
■ Secara umum LD50 akan lebih tepat bila menggunakan banyak hewan untuk tiap dosis
■ Banyak peneliti menggunakan 40-50 hewan per penelitian dan menggunakan ratio 1.2 – 1.5
■ Disarankan juga menggunakan 4 hewan untuk tiap dosis dan rasio antar dosis 2.0
■ Saat ini diusulkan uji sederhana yang menggunakan hanya 6-9 untuk sekali uji (Bruce,
1985).
Observations and Examinations

■ Untuk memperkirakan LD50, setelah perlakuan, jumlah hewan yang mati dan waktu
kematianya harus diamati, yang terpenting tanda-tanda toksisitasnya harus dicatat
■ Jangka waktu pengamatan biasanya 7 – 14 hari, tapi bisa lebih
■ Autopsi kasa harus dilakukan pada semua hewan yang mati dan yang masih hidup;
terutama yang tampak sakit pada akhir percobaan
■ Autopsi dapat memberikan informasi penting tentang organ sasaran terutama bila
kematian tidak terjadi segera setelah pemberian toksikan
■ Mungkin juga diperlukan pemeriksaan histopatologik organ tubuh dan jaringan tertentu
Evaluation of the Data
■ Dose–Response Relationship
Bila frekwensi dihubungkan dengan dosis dalam skala logaritmik, akan diperoleh suatu
kurva berbentuk S
■ Potensi suatu toksikan sangat beragam/toksisitas relatif
 Dose response curves

■ Particular points on dose response curves include:

– LD50 (Lethal dose, 50%) the dose that kills 50% of the test population
– LC50 (Lethal concentration, 50%) the concentration that kills 50% of
the test population

– TD50 (Toxic dose, 50%) the dose that causes a particular effect in 50%
of the test population
– TC50 (Toxic concentration, 50%) the concentration that causes a
particular effect in 50% of the test population
 The dose-response curve
100% LD50 = 50% of species exposed
to dose die (Oral route)
measured in mg/Kg

threshold? LC50 = 50% of species exposed

% mortality

to concentration die (Inhalation

route)
50 measured in ppm or mg/m3
%

NOAEL – Highest dose at

which there is No Observed
Adverse Effect Level. Some
dose response curves may not
have a threshold, starting at
zero.

Dose
Slope of the curve
Uses of LD50 Values and Signs of Toxicity

■ Klasifikasi zat kimia sesuai dengan toksisitas relatifnya

■ Berguna dalam mengevaluasi dampak keracunan yang tidak disengaja

■ Perencanaan penelitian toksisitas sub-akut dan kronik
■ Memberikan informasi tentang: mekanisme toksisitas, pengaruh umur, jenis kelamin, factor pejamu,
factor lingkungan, variasi respon antar spesies
SHORT-TERM TOXICITY STUDIES

■ Manusia lebih sering terpajan zat kimia dengan dosis yang jauh lebih rendah dari dosis
yang menyebabkan kematian, namun pajananya lebih lama
■ Untuk menilai efek toksik dalam situasi yang lebih realistic, dilakukan penelitian jangka
pendek dan panjang
Experimental Design

Spesies dan Jumlah

■ Umumnya dipakai dua atau lebih spesies hewan, biasanya mencit dan tikus, dipilih yang
metabolisme zat kimianya sama dengan manusia
■ Alasan lain karena ukuran sesuai, kemudahan mendapatkanya, banyaknya informasi
toksikologi berbagai zat kimia dari hewan tsb
■ Hewan jantan dan betina harus sama jumlahnya, umumnya 10-30 tikus dalam setiap
kelompok dosis dan kelompok pembanding
■ Bila menggunakan anjing, jumlahnya sekitar 4-8 ekor
Cara perlakuan
■ Zat kimia yang diuji harus diberikan lewat jalur yang sama dengan
penggunaan atau pajanannya pada manusia
■ Jalur yang biasa adalah oral melalui pencampuran dengan makanan atau
kadang air minum.
■ Kalau zat kimia tsb reaktif thd zat makanan, rasa tidak enak sering
dilakukan dengan sonde
■ Bisa juga melalui inhalasi
■ Dosis dan Jangka Waktu
■ Kalau tujuan ini adalah menentukan sifat dan tempat efek, disamping menentukan
“kadar tanpa efek”, disarankan untuk memakai tiga dosis
■ Dosis tinggi, menengah, rendah
■ Dosis dipilih berdasar informasi yang diperoleh dari uji toksisitas akut baik berupa
LD50, kemiringan kurva dosis respon, informasi metabolisme terutama tentang
bioakumulasi
■ Lama perlakuan 7 – 90 hari, pada anjing sering diperpanjang sampai 6 bulan
■ Pengamatan dan pemeriksaan
■ Berat badan dan konsumsi makanan
■ Pengamatan umum
■ Uji laboratorium
LONG-TERM TOXICITY STUDIES

■ Untuk menentukan sifat toksisitas zat kimia dan menetapkan NOEL

■ Dilakukan dalam rentang waktu 2 tahun
■ Biasanya digunakan 40-100 tikus pada setiap dosis
 No observed adverse effect level
(NOAEL)
■ Experimentally derived value – highest level/dose at which no
adverse effect observed in the test species

■ Lowest observed adverse effect level (LOAEL)

– Lowest dose/concentration that causes any observed effect

■ Other terms include:

– LD/LCLo (Lowest dose/concentration at which death occurs)
– TD/TCLo (Lowest dose/concentration at which particular effect occurs) –
effectively the same as LOAEL

■ As values usually established in species other than humans, various

safety or uncertainty factors are applied before this data is used in
the establishment of workplace exposure standards
 Acute toxicity studies
■ Traditional irritation studies
– Skin irritation tests (Draize Tests)
■ Substance applied to skin, covered for a few hours under a
dressing which is then removed
■ Skin assessed by looking for redness (erythema) and swelling
(oedema) and a scoring system based on severity of reaction
■ Used to provide a semi-objective classification of substances
as irritants

– Eye irritation tests

■ Substance applied inside lower eye lid
■ Severity of response judged by scoring redness and swelling
of conjunctiva and eyelids, opacity of the cornea and effects
on iris over a period of up to 21 days
 Sensitisation studies
■ Skin sensitisation studies usually performed on guinea pigs
– A series of exposures applied to a part of the skin

– After a break from this induction phase (inducing the immune system
to respond) another part of skin is exposed to a lower (non-irritant)
dose of the substance

– If the substance has sensitising properties this shows as a skin

reaction
■ Reaction scored for severity – substance may be designated a
skin sensitiser depending on proportion of test animals showing
specified levels of response

– Newer approaches have been developed using mice and require less
severe responses
 Sensitisation studies
■ Attempts have been made (usually using guinea pigs), to
develop tests for predicting respiratory sensitisation potential
– However, no single validated method has been recognised

■ For humans who show signs of skin sensitisation, ‘challenge’

tests can be performed under medical supervision to help
diagnose the condition and identify the causative agent

■ As for skin sensitisation, humans who show signs of

occupational asthma can undergo ‘challenge’ tests to determine
causative agents
 Repeated dose toxicity studies
■ Testing for effects of repeated exposure to a substance is most often
carried out on rats, over varying periods of time
– 28 days – sub-acute testing
– 90 days – sub-chronic testing
– One or two years – chronic testing

■ Exposure usually by oral route (less commonly by inhalation route)

■ Three dose levels usually used

– Highest dose chosen to induce clear toxic effects
– Middle dose chosen to induce less effects
– Lowest dose chosen to induce no effects
– (Shorter term tests can act as a guide to select doses for longer
term tests)
 Repeated dose toxicity studies
■ At end of exposure period a range of measurements
undertaken on the animals together with an examination
of body tissues and organs
– Range of tissues and organs examined increases with increasing
length of exposure period
– Number of animals used is generally larger in the longer
exposure period studies

■ Main aims of these studies are to produce information on:

– Type of toxicity
– Potential target organs
– Dose-response relationships
– If possible, the No Observed Adverse Effect Level
 Genotoxicity studies
■ Wide range of tests
– Strategic step-wise approach usually adopted

■ Initial in-vitro tests on bacterial cells to screen whether a

substance may be genotoxic (Ames test)
– Ames test based on assumption that if substance is genotoxic to
Salmonella bacteria used in test it may also be a carcinogen
– While some carcinogens do not give a positive Ames test (and vice-versa)
the ease and low cost of the test make it valuable as a screening test
– Some tests also conducted in presence of liver extract to investigate
possibility of genotoxic metabolites
– Other tests use yeast cells or fruit flies
– If all tests are negative, then usually the substance is not considered as
possessing genotoxic potential
 Genotoxicity studies
■ If initial in-vitro tests show genotoxic potential further studies can
be performed to determine if this potential could be expressed in
animals

– Generally performed in mice

– One or two doses of the substance administered and bone marrow

derived cells examined for evidence of genetic damage

– Positive results indicate potential to cause genetic damage and

substance is potentially a carcinogen

– If genetic damage found in normal (somatic) cells further tests on

germ cells (or offspring) undertaken to assess whether likely to
induce heritable genetic damage
 Reproductive and developmental
toxicity studies
■ Testing for a substance’s ability to cause toxicity to
reproduction usually carried out on mice or rats
– Groups of male and female animals dosed with a range of doses
– Allowed to breed in pairs and compared with a control group
– Some of the offspring of these tests may be dosed and allowed
to breed again (multi-generation studies)

– To investigate effects on developing offspring dosing may be

carried out during period of pregnancy
 Carcinogenicity studies
■ Testing for carcinogenic properties is an extension of the chronic
two-year repeated dose studies.
– Larger groups (50 male and female) of animals (usually mice or rats)
receive two years exposure

– Control group also observed

– Highest dose usually chosen to cause a small toxic effect but not enough
to cause excessive deaths

– At end of test (and whenever any animal dies) the animals are examined
for any tumours and their type determined

– If significant increase in number of tumours compared to control group

this may indicate the substance having carcinogenic properties in humans
 Allergy assessment methods in
humans
■ There are a number of different assessment methods that are
available for determining whether a person is allergic to a
particular substance including:
– Lung function tests (spirometry)

– Challenge tests

– Skin prick tests

– Patch tests

– Serological tests (Blood IgE testing)

 Lung function tests (spirometry)
■ Undertaken to evaluate how well the lungs are working
– Assess conditions such as asthma, pulmonary fibrosis and chronic
obstructive pulmonary diseases such as emphysema and chronic bronchitis

■ Uses a spirometer to measure the amount (volume) and speed (flow rate)
of air that can be inhaled or exhaled

■ Results given as actual values or as percentage of ‘predicted values’ for

given age, gender, height etc

■ Results within about 20% of predicted values considered ‘normal’

■ One limitation – highly dependent on patient co-operation and effort

Lung function tests (spirometry)
■ Common terms

– FVC – Forced vital capacity

– FEV1 – Forced expiratory volume in 1 second

– FEV1% - Ratio of FEV1 to FVC – in healthy adults this is

usually about 75 – 80 %

– PEF – Peak expiratory flow

 Challenge tests
■ Spirometry can also be used as part of a challenge test
– Undertaken to determine whether sudden contraction of the
bronchioles is as a result of exposure to a particular
substance

– Can confirm specific substance causing the sensitisation

– Starting with small dose, the challenge involves increasing

the dose of the substance in question

– Only performed under medical supervision

 Skin prick allergy tests

■ Usually first test recommended when allergy is

suspected
– Simple, quick, inexpensive
– Gives information on all types of allergy including
substances inhaled or ingested
– Suspected allergens mixed with liquid to form solution
– Up to 20 allergen solutions tested at a time by placing
drop on marked position on forearm
– Top surface of skin pricked beneath each drop
– Skin observed for reaction – usually within a few
minutes – reddening, itchy, swollen
 Patch testing
■ Skin prick testing introduces allergens into the body
– Used to test for allergies that do not necessarily occur on the skin

■ Patch testing places substances on the surface of the skin (usually

upper back) and aims to identify skin allergens
– Various test substances placed on the skin and covered for 48 hours
– At end of 48 hours, patch removed, skin examined and examined
again after further 48 hours
– Irritant reactions are most prominent immediately after patch
removed and fades over next day
– Allergic reactions take a few days to develop so is more prominent
on day 5 than when the patch is first removed
 Serological tests
■ Involve analysis of blood serum for presence and
quantity of specific IgE antibodies
– As quantitative measure of specific IgE in an individual
– For individuals taking antihistamine drugs
– For individuals with extensive skin disease such that prick
tests are difficult to undertake

– However, invasive, relatively costly and results not

available immediately
– Important that laboratories used take part in suitable
external proficiency testing schemes
 Dose response curves
■ Dose response relationships describe the effect on an
organism caused by differing levels of exposure (or dose)

■ Dose levels are usually expressed in mg/kg body weight of

the test animal for solids and mg/m3 or parts per million for
aerosols/vapours
– These levels can be plotted on a graph against the response

■ The dose response curve is a valuable tool to understand

the levels at which substances begin to exert adverse
effects and the degree of harm expected at various levels
 Dose response curves

■ Dose response curves can show a number of points

including:

– The ‘no effect level’ where no effect occurs or no effect is

detectable

– The threshold dose of the substance – the level at which the

effect starts to occur

– The levels at which the effect occurs in a set percentage or all

of the test animals
 The dose-response curve

threshold?
Response • It is possible to ‘overdose’
on large volumes of water
• A 10g dose of caffeine
causes convulsions and
vomiting (1 cup of coffee
~ 150 mg caffeine
• A fatal dose of salt is
estimated to be ~ 250g

Dose
 Dose response curves

■ Particular points on dose response curves include:

– LD50 (Lethal dose, 50%) the dose that kills 50% of the test population
– LC50 (Lethal concentration, 50%) the concentration that kills 50% of
the test population

– TD50 (Toxic dose, 50%) the dose that causes a particular effect in 50%
of the test population
– TC50 (Toxic concentration, 50%) the concentration that causes a
particular effect in 50% of the test population
 The dose-response curve
100% LD50 = 50% of species exposed
to dose die (Oral route)
measured in mg/Kg

threshold? LC50 = 50% of species exposed

% mortality

to concentration die (Inhalation

route)
50 measured in ppm or mg/m3
%

NOAEL – Highest dose at

which there is No Observed
Adverse Effect Level. Some
dose response curves may not
have a threshold, starting at
zero.

Dose
 No observed adverse effect level
(NOAEL)
■ Experimentally derived value – highest level/dose at which no
adverse effect observed in the test species

■ Lowest observed adverse effect level (LOAEL)

– Lowest dose/concentration that causes any observed effect

■ Other terms include:

– LD/LCLo (Lowest dose/concentration at which death occurs)
– TD/TCLo (Lowest dose/concentration at which particular effect occurs) –
effectively the same as LOAEL

■ As values usually established in species other than humans, various

safety or uncertainty factors are applied before this data is used in
the establishment of workplace exposure standards
 Threshold
■ Dividing line between no-effect and effect levels
of exposure

– It can be shown experimentally that thresholds exist, as

for a given population low levels of exposure do not
produce a detectable effect and as the dose increases the
effect appears

– Thresholds vary between species

– Thresholds vary within species due to natural variability

 Threshold
■ Dose response curves plotted from data from experimental
studies
– Curve drawn from a number of points, one from each exposure group
■ More exposure groups – more accurate the shape of the graph
■ Without infinite number of groups the precise shape of the curve
cannot be known accurately

■ For many substances the body can deal with the exposure
without an effect occurring
– At the threshold the defence mechanism is overwhelmed for more
susceptible individuals and the effect begins to appear
– Uncertainty on the shape of the curve is a particular concern at very
low doses
 Threshold
■ Uncertainty on the shape of the curve at very low doses
is a particular concern for carcinogens
– This has led to debate whether there is a threshold (no-effect)
level for carcinogenic effects
■ If no threshold the curve will go through the origin (zero dose)
■ If there is a threshold the curve will cross the axis at some
point greater than zero dose

– For carcinogens most experimental data has been derived at

high dose levels – so extrapolation of the curve to very low
levels is often difficult
– Low dose studies require too many animals to determine effects
 Variability
■ Within any group there are:
– Some susceptible individuals (hypersensitive) who are affected
at low concentrations
– Some highly resistant individuals (hyposensitive) who are not
affected at higher concentrations
– Vast majority of ‘average’ individuals

■ It is important to recognise that some hypersensitive individuals

may be in a work group and that they may suffer health effects at
exposures below the recognised exposure standard

■ Note: In animal testing studies the effects of variability are reduced

as much as possible by using groups of test animals that are bred to
be genetically very similar
 Slope of the curve

■ Another characteristic that can be examined is the slope of the

curve
– Slope of the curve can vary widely between different substances

– Some substances show a steep curve (rise rapidly from threshold

level to the ceiling level)
■ Particular care needs to be taken as exposures just above
threshold may produce effects in a large proportion of the group

– A relatively flat slope suggest the effect of an increase in dose is

generally minimal and that there is much greater variation in the
likelihood of the effect occurring in the whole exposed population
Slope of the curve