Toxicity Test

Farmakologi II dan
Toksikologi
 Dirangkum dari berbagai sumber :
Andersen and Krewski (2009). Toxicity Testing in the 21st Century: Bringing the Vision to Life. Tox. Sci., 107, 324-330.
FDA/CVM GFI No. 3, General Principles for Evaluating the Safety of Compounds Used in Food-Producing Animals (
http://www.fda.gov/downloads/AnimalVeterinary/GuidanceComplianceEnforcement/GuidanceforIndustry/UCM052180.pdf)
VICH Guidances (http://www.vichsec.org, VICH GL33 etc.)
Sumol Pavittranon. Toxicology and Risk analysis
Society of toxicology, USA

2 toxicity test 6/3/2019

 Toxicology is an applied science
with many areas of specialization

Images from Gray’s Anatomy

Images from webelements.com

Image from Molecular Cell Biology

3 toxicity test 6/3/2019

 What are major areas of
specialization in toxicology?

• Mechanistic toxicology (basic biology

and chemistry)
• Descriptive toxicology (testing)
• Regulatory toxicology (rule making
and compliance)
• Risk assessment (modeling)
• Translational and clinical (applying basic research
to patient care)

4 toxicity test 6/3/2019

 Descriptive Toxicology

• Typically involves toxicity testing

• Broad spectrum of responses reflects toxicity
– Functional effects, such as immunological responses
– Growth inhibition
– Reproductive impairment
– Increase in cancer incidence
– Mortality

5 toxicity test 6/3/2019

 Descriptive Toxicology
Toxicity Testing
• Assesses the concentration-
dependent hazard a chemical may
present
– Human health
– Natural populations
• Results typically applied to
– Approval of product use
– Regulating allowable concentrations in
6 the environment. toxicity test 6/3/2019
 Descriptive Toxicology
Types of toxicity testing

• In vitro (test tube)—useful in detecting potential biochemical and genetic effects

– Use model systems (bacteria, cultured animal cells, DNA interactions)
• In vivo (animal)—are essential for detecting health effects
– Acute, chronic, multi-generation
– Experimental animals may be treated with high doses over a lifetime to evaluate
potential to cause cancer

• In silico (computer-based)—biological
experiments conducted by computer
models; these depend on data previously
collected in other
Completion experiments
of all toxicity tests may take
7 five or six years and is very costly toxicity test 6/3/2019
Descriptive Toxicology
What private and public sectors invest in toxicity
testing that aims to protect human health?

• Chemical Manufacturers
• Pharmaceutical Industry
• US Federal Agencies and Programs
– National Toxicology Program (NTP)
– Environmental Protection Agency (EPA)
– National Institute of Environmental
Health Sciences (NIEHS)
– Food and Drug Administration (FDA)
• State and Local Governmental Bodies
 Translational science is the application of biomedical research
and drug development to efficiently use a promising drug in
the right patient circumstances and assess its efficacy in the
Translational human using appropriate indicators such as biomarkers.

• Scientists work in multidisciplinary teams

involving basic researchers, clinicians,
patient care providers, regulators, and ethics
boards.
• Basic scientists provide new tools for use in
patients and for assessment of their impact,
and clinical researchers make novel
observations about the nature and
progression of disease that can lead to
further basic research.
9 toxicity test 6/3/2019
 Toxicology

Paracelsus (1493-1541):
“All things are poison and nothing without poison; only the dose
makes that a thing is not a poison.”
Casarett & Doull’s Toxicology:
Toxicology is a study of the adverse effects of chemicals on
living organisms.

10 toxicity test 6/3/2019

 Toxicology Assessment

Hazard Identification
Chronic & acute effects
Toxicological endpoints
Hazard Characterization (Dose-Response)
Determine a No-Observed-Effect-Level (NOEL) and safe level of
exposure to humans

11 toxicity test 6/3/2019

 Toxicology Assessment
Toxicological Endpoints:
• Hepatic toxicity
• Renal/nephrotoxicity
• Reproductive toxicity
• Developmental toxicity
• Neurotoxicity (central or peripheral)
• Respiratory tract toxicity
• Immunotoxicity
• Dermal sensitization
• Dermal irritation

12 toxicity test 6/3/2019

 Toxicology Assessment

Safe Level of Exposure

Determine NOELs (or BMDLs- benchmark dose lower bound) from
dose-response curves obtained from toxicology studies
Determine the uncertainty factors to extrapolate the results from
animal studies to humans.

13 toxicity test 6/3/2019

 How Toxicity Is Assessed

Animal studies
Systemic toxicity studies (such as clinical signs and symptoms, clinical pathology,
histopathology)
Special functional tests (e.g., reproductive performance, immune system function,
neurological tests)
Human studies
Epidemiological studies
Human clinical studies
Case reports

14 toxicity test 6/3/2019

 Toxicology Assessment

Identify and characterize any potential adverse

health effects
Risk = Hazard X Exposure

Risk = Hazard X Exposure

15 toxicity test 6/3/2019

 Toxic Agents

•Chemicals
•Food additives
•Drugs
•Pesticides
•Metals
•Solvents
•Radiation
•Toxin
•Pollutants
16 toxicity test 6/3/2019
 Toxicity Testing

•Acutetoxicity
•Subacute toxicity
•Chronic toxicity
•Reproductive toxicity
•Genotoxicity
•Neurotoxicity
•Immunotoxicity

17 toxicity test 6/3/2019

 Acute toxicity testing

•Adverse effect within 24 hr

•Life threatening, accidental, overdose
•Define intrinsic toxicity of the chemical
•Oral, dermal, inhalation, skin, eye irritation
•Define LD50

•LD50 = Dose that cause 50 % mortality

18 toxicity test 6/3/2019

 Sub chronic testing

•Repeated dose for up to 6 months

•10 % life span
•2 spp, rodent and non-rodent
•reflect cumulative effect, latent period
•and reversibility
•non-lethal parameter
•target organ arranged
•Data for chronic study

19 toxicity test 6/3/2019

20 toxicity test 6/3/2019
 Chronic toxicity testing
•Life span of animal, 2 year in rat
•18 months in mice
•Similar metabolism in man
•Same route of administration
•Exposure duration similar to man
•3 treatment groups
•Maximum tolerated dose
•Pathology data, bl. chem, urianalysis
•Good Laboratory practice

21 •To define safety factor

toxicity test 6/3/2019
 Animals

Rodent and non-rodent

Avain
Fish
Aquatic invertibrate
Ferret
non primates
dog
22
rabbit
toxicity test 6/3/2019
 Reproductive toxicity testing

•Reproductive efficiency, 2 generation

•Fertility profile 70 days
•Semen analysis
•Pathology, gross and histo
•Oogenesis
•In vitro method
•Teratogenetic
•Biochemistry parameters

23 toxicity test 6/3/2019

 Genotoxicity testing
•Gene mutation assay (Ame’s assay)
•Chromosome effect
Sister chromatic exchange
Micronucleus test
•DNA interaction (DNA unscheduled synthesis)
•Neoplastic cell tranformation
BALB/3T3 cells

24 toxicity test 6/3/2019

 Neurotoxicity testing
•Condition behaviors
•Unconditioned behaviors
•Affective behaviors
•Social behavior
•Motor acts
•Learning & Memory
•Biochemical
NTE
cAMP, cGMP
GABA
Dopamines
25 toxicity test 6/3/2019
 Immunotoxicology testing

Immune disfunction

increase tumor susceptability

decrease host resistance

26 toxicity test 6/3/2019

 Recommended Testing Approach
by FDA
Toxicology
Testing

Additional
Basic Toxicology Special
Toxicology
Studies Studies
Studies

Two 90-day Subchronic Effects on human gut flora,

One 1-year Chronic Carcinogenicity
2-Gen Reproductive in rats Immunotoxicity Mode of action
One or 2 Developmental Neurotoxicity, pharmaco-
A battery of Genotox Studies logical effects

27 toxicity test 6/3/2019

 VICH Safety Guidelines Implemented as FDA/CVM Guidance for
Industry (GFI)

VICH GL# CVM GFI# Subject

GL33 GFI 149 General Approach to Testing

GL31 GFI 147 Repeat-Dose (90-day) Toxicity Testing

GL37 GFI 160 Repeat-Dose (Chronic) Toxicity Testing

GL22 GFI 115 Reproductive Toxicity Testing

GL32 GFI 148 Developmental Toxicity Testing

GL23 GFI 116 Genotoxicity Testing

GL28 GFI 141 Carcinogenicity Testing

28 toxicity test 6/3/2019

 PerKa BPOM NOMOR 7 TAHUN 2014
TENTANG
PEDOMAN UJI TOKSISITAS NONKLINIK SECARA INVIVO

NO TEST PRINSIP UJI LUARAN

1 Uji toksisitas oral dosis tunggal, atau dosis berulang yang diberikan LD50
dalam waktu 24 jam.

2 uji toksisitas subkronik dosis berulang yang (No Observed Adverse Effect Level
diberikan secara oral pada hewan uji selama sebagian umur / NOAEL
oral; hewan, tetapi hematologi, biokimia klinis dan
tidak lebih dari 10% seluruh umur hewan. histopatologi
3 uji toksisitas kronik uji secara berulang sampai (NOAEL
seluruh umur hewan / >= 12 bulan neurologi, fisiologi,
oral hematologi, biokimia klinis dan
histopatologi.

4 uji teratogenisitas; uji selama abnormalitas bagian luar fetus

masa pembentukan organ fetus (masa organogenesis) (morfologi), jaringan lunak serta
kerangka fetus
5 uji sensitisasi kulit; hewan uji diinduksi kemudian dilakukan uji tantang (challenge dinilai berdasarkan skala
test). Magnusson dan Kligman
29 toxicity test 6/3/2019
lanjutan
PerKa BPOM NOMOR 7 TAHUN 2014
TENTANG
PEDOMAN UJI TOKSISITAS NONKLINIK SECARA INVIVO

NO TEST PRINSIP UJI LUARAN

6 uji iritasi mata uji (kelinci albino) pada mata cedera pada konjungtiva, kornea,
dan iris pada interval waktu tertentu

7 uji iritasi akut dermal (kelinci albino) pemaparan sediaan uji pada Derajat iritasi
dermal selama 3 menit sampai 4 jam dicek pada 1, 24, 48 dan 72 setelah
pemaparan untuk melihat
reversibilitas, pengamatan
dilanjutkan sampai 14 hari.
8 uji iritasi mukosa vagina Bahan uji dalam larutan NaCl 0,9% atau minyak zaitun histopatologi
dipaparkan kedalam mukosa >=5 kali dgn selang waktu 24 jam

9 uji toksisitas akut dermal sekali pemberian melalui rute derma LD50

10 uji toksisitas subkronik selama hematologi, biokimia klinis,

sebagian umur hewan, tetapi tidak lebih dari 10% seluruh umur histopatologi.
dermal hewan.

30 toxicity test 6/3/2019

 Difficulties in Toxicological studies
 Baseline study required (control group)
 Response not necessarily numerical
 Specificity of individual response
– Allergy or immunity
– Statistical study required
– Organism specific response, not applicable to humans
– Dosage response
– Response time, latency, acute versus chronic
– Difficulty in measuring intended variable (lead in liver measured by lead in blood)

31 toxicity test 6/3/2019

 Dose versus Response

 Run test on “large” population

 Given same dose (usually in
dose/body mass)
 Determine the number
or fraction of individuals that have
a response

32 toxicity test 6/3/2019

 Dose versus Response (cont)
 Repeat tests using different doses
 Find average response to each
dose
 Plot Response versus logarithm of
dose
 Forms Sigmoid shaped curve

33 toxicity test 6/3/2019

 Dose Limit Values

 EDf – Effective dose for f percent

of population. Reversible
response
 TDf – Toxic dose for f percent of
population. Undesirable response
that is irreversible
 LDf – Lethal dose for f percent of
population.

34 toxicity test 6/3/2019

 Definitions
 Therapeutic Margin
– TM = LD50% - ED50%
 Margin of Safety
– MOS = LD5% - ED95%
 Safety Index
– SI = LD5%/ED95%
 Therapeutic Index
– TI = LD50%/ED50%

35 toxicity test 6/3/2019

 How about food
toxicity test?

36 toxicity test 6/3/2019

 Toxicology Assessment

The general approach is to

Establish a human Acceptable Daily Intake
(ADI) level for total drug residues in edible
tissues based on toxicology testing
Determine if the compound is a carcinogen
Calculate the safe concentration value for total
residues in each edible tissue

37 toxicity test 6/3/2019

 Toxicological ADI for Total Residues

NOEL
Toxicological ADI =
Safety Factor
Toxicological ADI= NOEL/ Safety Factor

 Safety factor is determined by the type of the study,

species examined and toxicity endpoint (usually 100 to
1000-fold).
 The Benchmark Dose Lower Bound (BMDL) approach
may also be used.
 Example: NOEL = 0.125 mg/kg bw/day
toxicological ADI = 0.125/100 = 0.00125 mg/kg bw/day
= 1.25 µg/kg bw/day
38 toxicity test 6/3/2019
 Allocation of ADI
 ADI represents the amount of drug residues that can safely be consumed per day over a
human’s lifetime without adverse effects
 For drugs used in dairy cows and/or laying hens, ADI is partitioned amongst the edible
tissues (muscle, liver, kidney, fat), milk and eggs
 Otherwise, allocate the full ADI to the edible tissues (muscle, liver, kidney, and fat)
 Example: ADI = 10 µg/kg bw/day; partition 60% for milk, 10% for eggs, and 20% for
tissues
allocated ADI: 6 µg/kg bw/day (milk); 1 µg/kg bw/day (egg), and
2 µg/kg bw/day (tissues)

39 toxicity test 6/3/2019

 Safe Concentration (SC)

ADI x Average Human BW (kg)

SC =
Food Consumption (g)
SC= [ADI x Average Human BW (kg)] / Food Consumption (g)

Provide total drug residues allowed in each edible tissue

Calculated using the ADI (or partitioned ADIs when
applicable) and distributed amongst the edible tissues
(muscle, liver, kidney and fat), milk and eggs using food
consumption values

40 toxicity test 6/3/2019

 Ractopamine (NADA 140-863)
Summary of Toxicology Studies Conducted to Establish the Toxicological ADI

Type of Toxicology Study Tested Dose (mg/kg/day) NOEL (mg/kg/day)

Species

90-day oral Mouse 0, 25, 175, 1250 25

90-day oral Rat 0, 1.3, 14.3, 154.8 1.3

Two-generation Reproduction Rat 0, 0.15, 1.4, 15, 160 15

14-day oral Dog 0, 0.05, 0.15, 1.5 0.05

1-year oral Dog 0, 0.112, 0.224, 5.68 NA

90-day gavage Monkey 0, 0.125 0.125

6-week gavage Monkey 0, 0.25, 0.5, 4.0 0.25

1-year oral Monkey 0, 0.125, 0.5, 4.0 0.125

2-year oral oncogenicity Mice 0, 35, 175, 1085 320

Single dose oral Man 5 - 40 mg total dose 0.1

41 toxicity test 6/3/2019

 Ractopamine (NADA 140-863)
- Toxicological ADI Determination

The 1-year oral study in the monkey was selected as the study
with the lowest appropriate NOEL for determining the toxicological
ADI.
Toxicological ADI = NOEL/Safety Factor = [(0.125mg/kg bw/day)/100] = 0.00125 mg/kg bw/day = 1.25µg/kg bw/day

NOEL 0.125 mg/kg bw/day

Toxicologi cal ADI  
Safety Factor 100
 0.00125 mg/kg bw/day  1.25 µg/kg bw/day

42 toxicity test 6/3/2019

 Ractopamine (NADA 140-863) – ADI Determination

Toxicological ADI = 1.25 µg/kg bw/day

An microbiological ADI is not needed.
Final ADI = toxicological ADI
= 1.25 µg/kg bw/day

43 toxicity test 6/3/2019

 Ractopamine (NADA 140-863) – Calculation of Safe
Concentrations

ADI  Human Weight

Safe Concentrat ion (SC) 
Food Consumptio n Value
1.25 µg/kg bw/day  60 kg

Food Consumptio n Value (g)

Edible Tissue Food Consumption (g) Calculated SC (ppm)

Muscle 300 0.25
Liver 100 0.75
Kidney 50 1.5
Fat 50 1.5

44 toxicity test 6/3/2019

 Will these methods last forever?

6/3/2019 toxicity test 45

 Increasing frustration with current approaches to
toxicity testing from many sectors…

Low throughput;
expensive
Questionable
relevance to actual
human risks
Conservative
extrapolation defaults
Traditional
approaches dating to
1930’s
Little use of modern
Question: What are the
biology, mode of action limitations/advantages of the
Reliance on animals current testing approach that
6/3/2019 toxicity test relies on animals? 46
 Design Criteria:
Toxicity Testing of Environmental
Agents

Broadest coverage
of chemicals, end
points, life stages

Fewest animals; Lowest cost;

least suffering for least time
those used

Detailed mode of action and

dose response information
for human health risk
assessment

47 toxicity test 6/3/2019

 Options for Future Toxicity Testing Strategies Table 2-1

Option I Option II Option III Option IV

In Vivo Tiered In Vivo In Vitro/In Vivo In vitro

Animal biology Animal biology Primarily human Primarily human

biology biology
High doses High doses Broad range of doses Broad range of doses
Low throughput Improved throughput High and medium High throughput
throughput
Expensive Less expensive Less expensive Less expensive
Time consuming Less time consuming Less time Less time
consuming consuming
Relative large Fewer animals Substantially fewer Virtually no animals
number of animals animals
Apical endpoints Apical endpoints Perturbations of Perturbations of
toxicity pathways toxicity pathways
Some in silico and In silico screens In silico screens
in vitro screens possible
48 toxicity test 6/3/2019
 Toxicity Testing

… a not-so-distant future where all routine toxicity

testing will be conducted in human cells or cell lines in
vitro by evaluating perturbations of cellular responses
in a suite of toxicity pathway assays.
Andersen and Krewski (2009). Toxicity Testing in the 21 st Century: Bringing the
Vision to Life. Tox. Sci., 107, 324-330.

Question: How long will it take

to implement this new toxicity
testing paradigm?
49 toxicity test 6/3/2019
 Dose-Response and Perturbation of
Toxicity Pathways
Exposure

Tissue Dose Low Dose

Higher Dose
Higher yet
Biologic Interaction

Perturbation

Normal
Biologic
Biologic
Inputs Function

Early Cellular
Changes

Adaptive Stress
Cell Morbidity
Responses Injury and
Mortality
Question: How do we
distinguish adaptive versus
6/3/2019 adverse (toxic) responses? toxicity test 50
Targeted Testing – toxicogenomics, etc.
Assess pathways,
integrate tissue
responses, and in
some cases evaluate
metabolites

Discuss use of new

technologies in
targeted testing
strategies
 In the new approach,
Toxicity pathways assays, better reflecting biological targets and modes of action
Increased speed and throughput for chemicals and decreased costs and animal
usage
Move away from extrapolating from high dose animal results to low doses in humans
and focus on results of perturbations of toxicity pathways in humans
Now extrapolations include in vitro - in vivo and across levels of biological
organization

6/3/2019 toxicity test 52

 Dose Response and in vitro to
in vivo extrapolations

Dose response modeling of perturbations of pathway

function would be organized around computational
systems biology models of the circuitry underlying
each toxicity pathway. In vitro to in vivo extrapolations
would rely on pharmacokinetic models – ideally
physiologically based pharmacokinetic models - that would
predict human blood and tissue concentrations under
specific exposure conditions.
Andersen and Krewski (2009). Toxicity Testing in the 21 st Century: Bringing the
Vision to Life. Tox. Sci., 107, 324-330.

53 toxicity test 6/3/2019

 Some advantages
Toxicity testing more focused on human biology; not an uncertain
reflection of high dose animal studies for what is expected at low doses
in humans
Creates detailed understanding of pathway targets, functional design of
pathway circuitry , more diverse dose response models for target and
integrated cellular responses for ties to possible outcomes

And promises
Human relevance
Dose relevance
Chemical coverage
Mixtures effects on toxicity pathways
Mechanistic focus: mode of action based
Cost effective
Fast
The 3 Rs: replacement, reduction, refinement
6/3/2019 toxicity test 54
 Terimakasih ^^

6/3/2019 toxicity test 55