ALTERNATIVE METHODS IN TOXICOLOGY TESTING

STRENGTHS AND WEAKNESS – PROS AND CONS: ANIMAL TESTING AS A BASIS FOR RISK ASSESSMENT  Advantages  Considerable experience  Characterization of potency, target organs, type and time dependence of lesion development  Toxicokinetics covered (animals)  Disadvantages  Cost and labor intensive  Completion of toxicity “package” may take up to 5 years  Use of large number of rodentsnonrodents  Need for extrapolation from animals to humans  Exposure assessment often imprecise, worstcase used Fixed Dose Procedure (FDP) — OECD TG 420. This method does not use death as an endpoint, instead it uses the observation of clear signs of toxicity developed at one of a series of fixed dose levels to estimate the LD50. — Acute Toxic Class method (ATC) — OECD TG 423. This method does not use death as the only endpoint, it also uses signs of toxicity in its stepwise approach to estimating the LD50. — UpandDown Procedure (UDP) — OECD TG 425. This method does still use death as an endpoint, but doses animals one at a time to see if the dose needs to be put up or down to achieve an estimate of the LD50 therefore giving the minimum number of animals a lethal dose of the test substance.

ALTERNATIVE METHODS IN

TOXICOLOGY TESTING

Thursday 8 December 2011

Herman Autrup

STRENGTHS AND WEAKNESS – PROS AND CONS:

ANIMAL TESTING AS A BASIS FOR RISK ASSESSMENT

 Advantages

 Considerable experience

 Characterization of potency, target organs, type and

time-dependence of lesion development

 Toxicokinetics covered (animals)

 Disadvantages

 Cost- and labor intensive

 Completion of toxicity “package” may take up to 5 years

 Use of large number of rodents/non-rodents

 Need for extrapolation from animals to humans

 Exposure assessment often imprecise, worst-case used

Limit the use of animal experiments in toxicity

testing

Reduce Refine Replace

REACH regulation and EU cosmetic directives

Fixed Dose Procedure (FDP) — OECD TG 420 This method does not use death as an endpoint, instead it uses the observation of clear signs of toxicity developed at one

of a series of fixed dose levels to estimate the LD50.

— Acute Toxic Class method (ATC) — OECD TG 423 This method does not use death

as the only endpoint, it also uses signs of toxicity in its stepwise approach to

estimating the LD50.

— Up-and-Down Procedure (UDP) — OECD TG 425 This method does still use death

as an endpoint, but doses animals one at a time to see if the dose needs to be put

up or down to achieve an estimate of the LD50 therefore giving the minimum

number of animals a lethal dose of the test substance.

REDUCE – LD50

REFINE – GENOTOXICITY STRATEGY

Tier 1 In vitro test, bacterial mutation, and in vitro

micronucleus, depends on specific features of test

compound

If both negative – non genotoxic

If one or more positive - in vivo tests ( erythrocyte nicronuclei, transgenic rodents assay, comet)

if one is positive – in vivo genotoxin

EFSA Journal 2011 9: 2379

REFINE – SENSITIVE ANIMALS

Transgenic mouse models –Carcinogen identification

+ car – car -noncar + noncar accuracy

Trp 53 +/- 21 10 27 1 81% Tg/AC 17 6 29 10 74%

Ras H2 21 7 17 6 75% NTP rat + genotox

36 0 7 23 65%

 Pharmacokinetics & metabolism

 Repeated Dose/organ toxicity

 Reproductive development toxicity

 Skin Irritation/corrosion

Skin sensitization

NEW APPROACHES TO TOXICITY TESTING

 Toxicity testing in the 21 st century (US NAS, 2007)

 Advantages: attempt to comprehensively cover relevant molecular events,

high throughput prescreening, highly automated, expected to be rapid, exposure driven ?

 Disadvantages: What to do with false-positives/negatives, conflicting data ?

Do we know all relevant disease pathways ? Relation of pathway perturbation (yes/no, 10 or 100 %) to in vivo toxicity ? Toxicokinetics not integrated in initial approach !

 Testing based on toxicity end-points in cell culture and integration

of toxicokinetics (several EU-funded projects)

 Advantages: (attempted) integration of kinetics, biotransformation, effects

concentrations

 Disadvantages: Relevance of response in a single cell type for effects on the

tissue/organ level? What is the best test system and test strategy (combinations) ? Stability of cell culture systems ?

TOXICITY IN THE 21 CENTURY - CONCEPT

TOXICITY TESTING IN THE 21 ST CENTURY I

 Initial examination of the physicochemical properties and chemical and biological fate of chemical

 Toxicity testing which comprises two components: toxicity pathway assays in which initial perturbations of relevant biological systems are assessed followed by complementary targeted testing

 Dose-response relationships involving three elements of extrapolation:

 A quantitative mechanistic understanding of the relevant

mechanistic pathway

 Physiologically-based kinetik modelling

 Utilization of any suitable human data

 Exposure data based on bio-monitoring and human surveillance data

TOXICITY TESTING IN THE 21 ST CENTURY II

 A wide range of in vitro test systems derived from human

tissues The assumption here is that cells derived from humans must be more representative of humans than other in vitro

systems

 High throughput systems to detect biological changes caused by exposure to the chemical under investigation, e.g genomics and enable their interpretation in terms of mechanisms

 Rapid data processing systems and machine-learning systems to deal with the large amount of data generated

 Improvements in exposure assessment modeling using

information from a range of sources

 In vivo models selected to address specific questions witch will narrow uncertainties in the risk assessment, if needed

TESTING BASED ON TOXICITY END-POINTS IN CELL CULTURE AND INTEGRATION OF TOXICOKINETICS

Toxicity testing in cell culture

Concentration of drugs giving response for sensitive and relevant endpoint

MOS

Selection of drug candidates

Therapeutic dose level (animal and/or human)

Interindividual susceptibilities

Chemical Grouping in Toxicology

In SILICO TOXICOLOGY

OECD DEFINITION OF A CATEGORY

“ group of chemicals whose

physico-chemical and toxicological properties are

likely to be similar or follow a regular pattern

as a result of structural similarity ”

GROUPING COMPOUNDS TO FILL A DATA GAP

 Form a rational group of compounds

 Requires a transparent basis

 Often termed a category

 Obtain relevant toxicology, or other, data and

information for the group

 Interpolate activity within the group

 Read-across

 Qualitative or occasionally quantitative

 OECD (Q)SAR Application Toolbox

 Helps the user group chemicals together and

• Does not imply congeneric series: domains

defined by (mechanistic organic) chemistry

• Useful for mechanisms when we know there is

a “reactive mechanism of action”

• Maybe qualitative or quantitative

Mode of Action (Receptor Mediated) Analogues

O

H

OH

O H

OH

O H

METHODS TO GROUP COMPOUNDS

Mode of Action (Receptor Mediated) Analogues

O

H

OH

O H

OH

O H

METHODS TO GROUP COMPOUNDS

• Useful for receptor mediated mechanisms

• Requires knowledge and definition of receptor binding

domain

• May be only feasible solution to some chronic endpoints

• Qualitative identification of hazard

Calculations of Similarity

METHODS TO GROUP COMPOUNDS

N NH

O O

O

N

O O

O

N NH

O O

O

N NH

O O

O

N NH

O O

O

N NH

O O

O

N NH

O O

N NH

O O

mephobarbital 0.60

metharbital 0.68

IN SILICO TOXICITY – SKIN SENSITISATION

CRAMER RULES IN TOXTREE

The Cramer classification scheme is probably the best known approach for structuring chemicals in order to estimate a Threshold of Toxicological Concern.

Chemicals are divided into three structural classes based on a decision tree This comprises 33

structural rules and places evaluated compounds into one of three classes:

• Class I substances are simple chemical structures with efficient modes of metabolism suggesting a

low order of oral toxicity

• Class II are of intermediate toxicity

• Class III substances are those that permit no strong initial presumption of safety, or may even suggest significant toxicity or have reactive functional groups

Cramer GM, Ford RA & Hall RL (1978) Estimation of Toxic Hazard - A Decision Tree Approach J

Cosmet Toxicol., Vol.16, pp 255-276, Pergamon Press.

CRAMER RULES IN TOXTREE

HYDROLYSIS REACTIONS IN TOXTREE

A limited number of hydrolysis reactions (Cramer rules #15, #17,

#29, #30 and #31) are implemented, based on expert advice

2-(Benzoyloxy)propyl benzoateEXAMPLES OF QUERY

EXPOSURE DRIVEN APPROACHES:THRESHOLD OF

TOXICOLOGICAL CONCERN (TTC)

 The TTC is a concept that refers to the establishment of a

human exposure threshold value for all chemicals, below

which there would be no appreciable risk to health

 Tolerable exposures based on potency distribution for

chemicals in toxicity assays and statistical evaluation of

NOAELs

 Tolerable exposures defined by type of effects (carcinogen)

and chemical structure (Cramer classes)

 Requires reliable exposure assessment and confidence in

data evaluation

TTC CONCEPT

Can be used for compounds of known chemical

structure (Cramer classes) but lacking toxicity data Reliable exposure levels

TTC levels

Structural alerts for genotoxicity 0.15 ug/person/day Structural alert for neurotoxicity (PPP)

Concerns – Cramer class based upon oral exposures – validation for dermal and

inhalation exposures ongoing

ISSUES ON THE USE OF THE TTC CONCEPT

 Are the compound similar to the compound used to classify in Cramer classes

 Difference in metabolism between route of exposure (Cramer class based upon oral

exposure)

 Default adjustment factors for uptake

 Intermittent exposures

 Total (aggregate) exposure

 Simultaneous exposure to other agents