Basic principles of toxicology for
PDE preparation
Adam Woolley
Toxicology:
basic principles
• Brief history and context
• Dose response
• Review of the main areas of concern for PDEs:
Pharmacology, ADME, Toxicology, genotoxicology,
carcinogenicity, reproductive and developmental
effects; other studies, especially sensitisation
• No toxicity without exposure
• Context
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Toxicology is multidiscplinary
Pathology Biochemistry Pharmacology
Molecular
Chemistry
biology
Toxicology Analytical
Medicine chemistry
Physiology Physics
Pharmaceutical
Epidemiology science
Toxicology:
basic principles
• Toxicology has a long history
• Ebers papyrus from Egypt
• Roman / Italian poisonings
• Digitalis / foxgloves
• Modern toxicology starting in 2nd half of 20th century.
• Thalidomide
• Vioxx
• Cisapride
Toxicology:
basic principles
• Paracelsus: the dose makes the poison – nothing is
“safe”
• Two main factors to remember:
– Dose
– Individual response
• PLUS the potency of the substance
• No toxicity without exposure
Expression of toxicity
Toxicity is determined by:
• Dose (usually mg/kg; or mg/m2 body surface)
• Frequency & duration of dosing
• Route of exposure
• Physical form or formulation
• Absorption, Distribution, Metabolism, Elimination (ADME)
• Species and/or phenotype
• Presence of receptors
• The potency of the substance
Expression of toxicity
Acute toxicity of different compounds
Compound LD50 (mg/kg)
Diazepam (mouse, oral) 50
Diazepam (women) 5 (TDLo)
Paracetamol (mice, oral) 340
Paracetamol (man, est oral) 250
Ethanol (man, estimated oral) 7000
Ethanol (rat oral) 10,000
Expression of toxicity
• Acute toxicity of dioxin (TCDD) in different species
Species LD50 (μg/kg)
Guinea pig 1
Male rat 22
Female rat 45
Mouse 114
Rabbit 115
Hamster 5000
Monkey 70
Expression of toxicity
Expression of toxicity – influenced by:
• Age
• Genetics
• Pregnancy
• Diet
• Co-medication
• Occupation
• Life style
• Disease
• Microbiota
Toxicity testing
objectives
• Dose response curve
• Presence of thresholds
• NOEL
• NOAEL
• Target organs
• Natural does not mean safe
• Man made is not necessarily toxic
PDE preparation:
guideline objective
EMA guideline states:
The objective of this guideline is to recommend an
approach to review and evaluate pharmacological
and toxicological data of individual active substances
and thus enable determination of threshold levels as
referred to in the GMP guideline.
Toxicity and PDE
preparation: hazards of concern
Four main hazard points in EMA guideline:
• Genotoxicant
• Reproductive developmental toxicant
• Carcinogen
• Highly sensitising potential
Toxicity and PDE
preparation: areas for review
• Pharmacology
• ADME (Absorption, Distribution, Metabolism &
Elimination)
• Toxicology – single and repeat dose
• Genotoxicology & carcinogenicity
• Reproductive and developmental effects
• Other studies, especially sensitisation
Toxicity and PDE
preparation: areas of concern
Pharmacology
• Primary vs secondary
• Safety pharmacology
• Animal receptors may show
• different binding and affinity from human equivalents
• different activities
• Effects at concentrations significantly higher than
human exposure may not be relevant
• Beware of differences in hormone action between
animals and humans (males and females)
Toxicity and PDE
preparation: areas of concern
ADME
• Determines exposure – extent and duration
• Parameters to watch:
• Half-life of elimination (t1/2)
• Time of maximum concentration (tmax)
• Maximum concentration (Cmax)
• AUC – area under the curve
• Clearance
Toxicity and PDE
preparation: areas of concern
ADME
• Absorption
• Route: dermal < oral < inhalation = or < injection
• Dependent on formulation
• Distribution
• Beware accumulation (e.g. in lipid or bone)
• Metabolism may be species specific; 1st pass
• Elimination (excretion) may be
• Hepatic bile faeces OR blood urine
• Renal urine
Toxicity and PDE
preparation: areas of concern
Toxicology
• Single dose
• Repeat dose
• Usually in two species
• Should indicate dose response curve
• Hazards and effects
• Target organs
• NOAEL or NOEL
• Reversibility
Toxicity and PDE
preparation: areas of concern
Toxicology
• Repeat dose investigations
• In life – clinical signs, food consumption, bodyweight,
eyes, neurological examination
• Clinical pathology
• Pathology
• Necropsy
• Organ weights
• Histopathology
Toxicity and PDE
preparation: areas of concern
Genotoxicology & carcinogenicity
• Genotoxicity
• Is a hazard of concern
• May not indicate carcinogenicity
• Carcinogenicity in animals may not equate to human
cancer hazard or risk
Toxicity and PDE
preparation: areas of concern
Genotoxicology
• Standard (gold standard) tests:
• Bacterial reversion (Ames) test
• Mammalian cells in culture
• Tests in vivo – usually rats or mice
• Some others that are not standard or very old
• Mammalian cell tests subject to false positives
• Weight of evidence is vital; one positive does not
necessarily mean genotoxicity
Toxicity and PDE
preparation: areas of concern
Carcinogenicity
• Gold standard studies
• Bioassays in rats and mice, usually 2-years
• BUT epidemiology may be important
• Mechanism
• Genotoxic versus non-genotoxic
• Non-genotoxic mechanism in animals usually not
relevant to humans
• Carcinogenicity in animals may not equate to human
cancer hazard or risk
Toxicity and PDE
preparation: areas of concern
Reproductive and developmental effects
• Studies may include:
• Fertility
• Embryo/foetal development
• Peri- and post-natal development
• Should give NOAEL or NOEL
• NOAEL for maternal effects
• NOAEL for malformations or foetal effects
• All should be related to AUC
Toxicity and PDE
preparation: areas of concern
Reproductive effects: Fertility
• Usually in rats
• Males treated 28 or 63 days; females treated 28 days
• Necropsy around day 15
• Reduced fertility is a warning for man
• Depends on mechanism
Toxicity and PDE
preparation: areas of concern
Developmental effects: Embryo/foetal toxicity
• Studies in rats & rabbits; perhaps mice or minipigs.
• Treatment from implantation to palate closure
• Rats gestation days 6 to 16; rabbits days 6 to 28
• Necropsy: rats day 20; rabbits day 29
• Malformations vs variants
• Developmental toxicity with maternal toxicity is
serious
• Delayed development may be seen as low pup
weight or delayed ossification
Toxicity and PDE
preparation: areas of concern
Reproductive effects: Peri- post-natal development
• Studies nearly always in rats
• Treatment from day 6 gestation to day 21 lactation
• Some flexibility in design
• Offspring (F1 generation) mated but not treated
• Investigations include:
• Maternal lactation, nursing etc and pup survival
• Physical development (ears, teeth, onset of puberty)
• Neurological development (eyes, hearing, balance, water
maze etc)
Toxicity and PDE
preparation: areas of concern
Other studies: local tolerance and sensitisation
• Dermal irritation – acute studies in rats or repeat
dose with topical products
• Ocular irritation – rabbits
• Sensitisation
• Local Lymph Node Assay in mice
• Buehler or Magnusson Kligman studies in guinea
pigs
• Beware false postives
Toxicity testing
references
• David Woolley and Adam Woolley: Practical Toxicology: evaluation,
prediction and risk (3rd edition, Taylor & Francis, 2017)
• ICH (International Conference on Harmonisation of Technical
Requirements for Registration of Pharmaceuticals for Human Use):
http://www.ich.org/
• OECD Test guidelines:
http://www.oecd.org/env/ehs/testing/oecdguidelinesforthetestingofchemical
s.htm
• OECD Good Laboratory Practices:
http://www.oecd.org/env/ehs/testing/goodlaboratorypracticeglp.htm
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