Toxicology Flashcards
Potential toxicities of new drugs are assessed after..
the pharmacological efficacy of the drug is proven
Requirement of the FDA to assess potential toxicities is due to..
how drugs can cause adverse effects in man
What is involved in tox testing?
acute tests that provide the basis for further study and chronic tox performed under GLP to determine long-term tox potential
Instance of tox of a drug not being found before being on market
1956-60 thalidomide was prescribed to pregnant women for morning sickness and resulted in malformed children
Thalidomide was not tested on the most sensitive species
In silico approaches
developed to characterise and predict toxic outcomes
methods already used for regulatory purposes, will be more prominent in future
Why are in silico tests important
large scale of chemicals studied, endpoints and pathways, exposure etc. all analysed and conditions considered simultaneously
(Kavlock et al, 2008)
Software for in silico
DEREK used to compare test drugs structures to highlight any tox
Single dose acute studies
tox that results from a single dose of compound
What does single dose acute studies give..
an early warning whether a drug is highly toxic
What can single dose acute studies establish
a non-lethal dose range and aids the choice of doses for repeat dose studies
provide information on target organs and limiting tox due to pharmacological effects
Single dose acute studies is carried out on..
2 different species
one rodent
one non-rodent
Investigational product in single dose studies is administered..
at different dose levels and observed for 14 days
Single does studies allow..
LD50 to be determined, 50% lethal dose
Stage 2 of acute dose studies
repeat of stage 1 in small groups (3-5 animals per sex)
span estimated NTEL
Observations made throughout and blood samples taken .. example
Irwin’s test for nervous system
Parasuraman 2011 example
Used Draize test
acute tox test to measure harmfulness of chemicals on rabbits and guinea pigs
(0.5g of substance and observe for 24 days)
Dermal and ophthalmic preparations tested
Look for redness, swelling, discharge etc
Dose ranging studies
Tox of repeated doses at dose levels ranging from ED50 to max non-lethal dose
Dose ranging studies help establish
Max tolerated dose (MTD)
MTD
highest dose that can be given to animals for the duration of the test that doesnt produce overt tox
Dose ranging studies are carried out in..
rodent and non-rodent
they are sacrificed and post mortem and histological analysis of major organs and tissues taken
One month repeated dose can be used to..
support clinical trials
One month repeated dose allow
administration of single or multiple doses in man for up to 6 months
One month repeated dose aid..
selection of doses for long-term investigations and help to identify possible target organs and tissues
Use what for one month repeated dose
proposed administration route
minimum of 3 dose levels
dosed for 29 days
Survivors sacrificed day 30
Detailed monitoring of what in one month repeated dose studies
food/ water intake body weight general behaviour blood/ urine chemistry haematology
Important feature of one month repeated dose studies
recovery group for both species
can be observed for up today 56
usually have 2nd satellite group of dosed rates so toxicokinetics can be assessed in this species
Greaves et al 2004
Showed if tox studies are shorter than 1 month, risk of certain organ tox being overlooked
single studies have capacity to detect many of most important tox
Six month repeat dose studies (chronic)
necessary for long-term clinical trials (Phase II and III) and for marketing approval
Six month repeat dose studies are similar to..
30-day study
treatments and free recovery period
assess reversibility of tox effects after 3 months
Acute short and long term studies are standard as..
performed for majority of candidate drugs
Specialist test later in development: oncogenicity and reproductive tox
Oncogenicity studies required when ..
compound is intended for long-term use in man (>6 months)
if drug has long half-life
where there is concern because of the compound pharmacologies or toxic effects
if mutagenicity tests are positive
Oncogenicity tests procedure
administered orally in 2 rodent species
18 months in mice and 2 years in rats
Terminal investigations of oncogenicity studies aim to..
determine total incidence of tumour bearing animals
total number of tumours
incidence of benign and malignant tumours
dose-response relationship
time to tumour recognition
oncogenicity studies serve as..
long-term tox test
Reproduction studies are used to determine..
Whether the compound has any adverse effects on reproductive function
male/ female fertility
foetal and post-natal development
Repro studies are done..
to support phase II/III trials and for marketing approval
Teratology studies are usually carried out if..
that women of child bearing age are going to be prescribed the drug
Dosing of teratology studies
during organogenesis
a period when embryonic tissues undergo rapid development
Development stage is a target of teratogen
chemicals that cause foetal malformation
Repro studies procedure
pregnant animales sacrificed 1 day before parturition
day 22 for rats
day 29 for rabbits
delivered by Caesarean section
Post-natal development tests
Usually use rats
investigate effect of compound on late uterine growth, parturition and post-natal development
may also reveal if agent affects lactation
Pups are assessed for
behaviour and physical development
some pups may be mated to assess their reproductive capacity
In vitro approaches
harmful effect of chemicals on DNA due to mutagenic quality of the drugs
rely on detection of DNA damage (or its immediate outcome) as an indicator of potential mutagenicity or carcinogenicity in man
expected to detect virtually all human mutagens
all test methods use some measure of tox and chemicals are generally assessed at levels below prescribed level of tox, in absence of tox and conc up to standard limit
set by regulatory authorities
Ames test
widely used method for detecting mutagenic chemicals that induce mutations in DNA (Zeiger and Mortelmans, 2001)
detect point mutations
Use S. typhimurium because ..
has a mutant gene so its incapable of synthesising histidine
strain cant grow in normal culture medium unless supplemented with amino acid
Affected S. typhimurium can mutate back to active form similar to WT then..
can grow in absence of amino acid
reverse mutation resulting in colonies that are revertants
His (-) strain undergoes spontaneous mutation to..
His (+)
Chemical mutagens increase frequency of..
back mutation
Ames is a sensitive test..
Large number of bacteria exposed to the chemical
Test organism divides in the presence of compound
Test bacteria carry mutations that increase permeability to foreign molecules and compromise bacteriums ability to repair any damage that DNA test substrate may cause
Ames test is
Cheap, fast (48 hour incubation period) and results are easy to analyse and interpret
Main disadvantage of Ames test - false negatives
Not all carcinogens are mutagens (e.g. asbestos)
target is small, single gene (can be overcome by using several test strains or different micro-organisms with different existing mutations
Mammalian cell mutation test
detect gene mutation and chromosome aberration
ICH recommend what for mammalian cell mutation test
L5178Y cells and TK locus
Mammalian cells have 2 copies of each gene but mouse lymphoma cell line only has..
1 functional copy of TK +/-
Tk is not an essential enzyme and
scavenges free thymidine which incorporates into cell DNA
Measure resistance to lethal nucleoside analogue TFT..
Interferes with cell metabolism and results in cell death
Lloyd and Kidd, 2002
Chromosome aberration test
Used to detect chromosome breakage
Chromosome aberration test procedure..
Treatment of cells with DNA-damaging agents can result in unrepairable lesions in both strands of DNA
Lead to chromosome breakage which can be seen in cell division
Chromosome aberration assay performed
with cultured human peripheral blood lymphocytes
various cultured cell lines, e.g. chinese hamster lung
Problem with cultured cell lines..
tend to lose and gain chromosome and parts of chromosomes spontaneously show high and unpredictable rate of chromosome aberrations
(Ishidate and Sofuni, 1985)
Cells assed for what in Chromosome aberration assay..
different types of chromosome aberrations
e.g. gaps, deletions and exchanges
Initial proportion of metaphase in test compound is compared to..
control range also treated in cell culture
Micronucleus test
to determine chromosome breakage and loss
Traditionally micronucleus test is..
performed in rats
Micronuclei form as a result of
chromosomal breakage or spindle damage
Fragments of whole chromosomes may not be included in nuclei of daughter cells following division they form..
single or multiple micronuclei (Howell-Joly bodies) in the cytoplasm of cells
Erythrocytes used for micronuclei test as
they no obstruction by main nucleus and easily detected
Micronuclei stain
Acrinide organe
assessed under fluorescent light
Bone marrow cell tox (or depression) indicated by
Decrease in proportion of immature erythrocytes
V large decrease in proportion indicative of cytostatic or cytotoxic effects
Comet assay
to detect DNA damage and chromosome breakage
Comet assay further developed by
Singh et al
Involving electrophoresis under alkaline pH
Single cell gel electrophoresis is
Simple, rapid and sensitive for analysing and quantifying DNA damage in individual cells
Comet assay can distinguish between
genotoxicity and cytotoxicity induced chromosomal damage
DNA damage induces relaxation in ..
supercoiled DNA coils or results in small fragments
Why is alkaline condition required
to ensure DNA is unwound especially if tox causes single-strand break (Tice et al., 2000)
Comet assay end result
Pulled to one side by electrophoresis
Extent of DNA liberated from head of comet is directly proportional to
DNA damage
Tail length in comet assay
measurement from point of greatest intensity within comet head to end of fluorescing tail
Tail movement
product of tail length and fraction of total DNA present in tail
