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 1 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 2 3R Limit the use of animal experiments in toxicity testing Reduce Refine Replace REACH regulation and EU cosmetic directives 3 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 4 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 5 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% 6 REPLACE In vitro toxicological models OECD- ECVAM In silico QSAR 7 IN VITRO TESTSYSTEMS Acute systemic toxicity Carcinogenicity Dermal Penetration Eye irritation/corrosion Genotoxicity Neurotoxicity Pharmacokinetics & metabolism Repeated Dose/organ toxicity Reproductive development toxicity Skin Irritation/corrosion Skin sensitization 8 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 ? 9 TOXICITY IN THE 21 CENTURY - CONCEPT 10 [...]... uncertainties in the risk assessment, if needed 12 TESTING BASED ON TOXICITY END-POINTS IN CELL CULTURE AND INTEGRATION OF TOXICOKINETICS Characterization of cytotoxicity of chemical in “relevant” cell culture model supported by “omics” and other molecular technics Toxicity testing in cell culture Concentration ofdrugs giving response for sensitive and relevant endpoint NOEC Toxicokinetic modelling...TOXICITY TESTING IN THE 21ST 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... transform concentration responses seen in vitro to dose-response in animals/humans Transformation ofin vitro concentrations to dose received by animal or humans usingPBPK-modelling Estimation of NOAEL Interindividual susceptibilities Therapeutic dose level (animal and/or human) MOS Selection of drug candidates 13 In SILICO TOXICOLOGY Chemical Grouping in Toxicology 14 OECD DEFINITION OF A CATEGORY “ group... 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... 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 11 TOXICITY TESTING IN THE 21ST CENTURY II A wide range of in vitro test systems derived from human tissues The assumption here is... of Action (Receptor Mediated) Analogues OH OH HO HO HO 19 METHODS TO GROUP COMPOUNDS Mode of Action (Receptor Mediated) Analogues OH OH HO HO HO •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 20 METHODS TO GROUP COMPOUNDS Calculations of Similarity O O... Sci 28: 696-708 21 IN SILICO TOXICITY – SKIN SENSITISATION 22 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:... ongoing 31 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 Used for unknown contaminants in food, flavouring... genotoxic carcinogens ( AFB, Azoxycompounds, nitrosamines) Potent non-genotoxic carcinogens (steroids) PBT compounds ( PCB, dioxines) Risk of allergenicity (Proteins) 28 29 30 TTC levels Structural alerts for genotoxicity 0.15 ug/person/day Structural alert for neurotoxicity (PPP) Non structural alert 1.5 ug/person/day Concerns – Cramer class based upon oral exposures – validation for dermal and inhalation... be similar or follow a regular pattern as a result of structural similarity ” 15 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 16 Free(ish) Tools . (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. uncertainties in the risk assessment, if needed 12 TESTING BASED ON TOXICITY END-POINTS IN CELL CULTURE AND INTEGRATION OF TOXICOKINETICS Characterization of cytotoxicity of chemical in “relevant”. 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