Rome, December 17 th , 2012 Serena Cinelli Improving test methods in the spirit of the 3Rs; the point of view of a contract research organization
Why a CRO? CRO is the bridge between companies and a compliant dossier CROs have more knowledge than average industrial companies for their: � involvement in the safety assessment of a wide range of products � tendency to deal with different industry segments (different needs, approaches, flexibility, drivers...)
Main functions of CROs � comply with regulatory directives and guidelines � ensure an updated knowledge of the new method status
The role of contract organization in toxicological development General trend of industry → contract out safety program Reasons: � small and mid-size companies (limited experience) � preference to rely on established techniques � importance of trained personnel � assign valuable resources on internal projects � rare occasions to carry out the methods
Importance of CRO in supporting the 3Rs philosophy CRO plays a central role to encourage the use of alternative methods as preliminary assays CRO can promote the use of an “intelligent testing strategy” with a tiered experimental approach using validated in vitro methods
Validation process Academia Research and Industry CRO Development Implementation Industry Pre-validation Regulatory acceptance Validation CRO Regulators Industry OECD
Validated in vitro methods Regulatory Species Mechanism Endpoints Suggested equivalent reference addressed information ER TA Endogenous Estrogen agonist OPPTS 890.1300 * human ER α ER α , ER β Effect on ovary/uterus OECD TG 455 dependent size, histology, transcriptional OECD TG 457 * male/female fertility human ER α , ER β Estrogen agonist, activation antagonist ER binding Uterine cytosol Estrogen agonist, ER binding Effect on ovary/uterus OPPTS 890.1250 from rats antagonist size, histology, male/female fertility AR binding Prostate cytosol Androgen agonist, AR binding Effect on testes size, OPPTS 890.1150 from rats antagonist histology, male/female fertility Steroidogenesis Human Steroid synthesis Testosterone, Effect on * OPPTS 890.1550 (H295R) (estrogen and estrogen hormone estrogen/testosterone OECD TG 456 testosterone) levels levels, sex organs, male/female fertility Human Aromatase 3 H 2 O released Effect on Aromatase OPPTS 890.1200 inhibition, the during the estrogen/testosterone * enzyme responsible conversion of levels, sex organs, for the conversion of androstenedione male/female fertility androgens to to estrone estrogens
Endocrine disruptor in vitro assay TG 455 Test system: stably transfected cells � ER α α α α -HeLa-9903 � � � Restrictions: Biological risk Technical challenges: difficulties to maintain stability and integrity of the cell line
Endocrine disruptor in vitro assay TG 457 BG1Luc Estrogen Receptor Transactivation Test system: stably transfected cells Test Method for � � � BG1Luc4E2 � Identifying Estrogen Receptor Agonists and Restrictions: Biological risk Antagonists Technical challenges: difficulties to maintain stability and integrity of the cell line
Endocrine disruptor in vitro assay Restrictions: Radioactive risk
Endocrine disruptor in vitro assay TG 456 Test system: H295R Restrictions: None
Endocrine alterations Hormonal systems are complicated and stand alone in vitro methods are not sufficient to predict in vivo effects •Results of in vivo studies in rodents – decrease of mating and reproductive efficiency – affect foetuses or newborn animals – toxic effects on parental endocrine organs • in vitro methods can help to – understand the product mode of action – limit unnecessary in vivo studies – support the toxicologist in data interpretation – define the most appropriate follow-up studies
OECD Conceptual framework Note: Document prepared by the Secretariat of the Test Guidelines Programme based on the agreement reached at the 6th Meeting of the EDTA Task Force OECD Conceptual Framework for the Testing and Assessment of Endocrine Disrupting Chemicals Level 1 - physical & chemical properties, e.g., MW, reactivity, volatility, biodegradability, Sorting & prioritization based - human & environmental exposure, e.g., production volume, release, use patterns - hazard, e.g., available toxicological data upon existing information Level 2 - ER, AR, TR receptor binding affinity -High Through Put Prescreens - Transcriptional activation - Thyroid function In vitro assays providing - Aromatase and steroidogenesis in vitro - Fish hepatocyte VTG assay - Aryl hydrocarbon receptor recognition/binding - Others (as appropriate) mechanistic data - QSARs Level 3 - Uterotrophic assay (estrogenic related) In vivo assays providing data - Hershberger assay (androgenic related) - Fish VTG (vitellogenin) assay - Non -receptor mediated hormone function (estrogenic related) about single endocrine - Others (e.g. thyroid) Mechanisms and effects Level 4 In vivo assays providing data - enhanced OECD 407 (endpoints based on endocrine mechanisms) - Fish gonadal histopathology assay about multiple endocrine - male and female pubertal assays - Frog metamorphosis assay Mechanisms and effects - adult intact male assay - 1-generation assay (TG415 enhanced) 1 Level 5 - 2-generation assay (TG416 enhanced) 1 - Partial and full life cycle assays In vivo assays providing data on - reproductive screening test (TG421 enhanced) 1 in fish, birds, amphibians & - combined 28 day/reproduction screening test invertebrates (developmental and effects from endocrine & (TG 422 enhanced) 1 reproduction) other mechanisms 1 Potential enhancements will be considered by VMG mamm
RTC experience on REACH Reach test programme does not include specific tests for endocrine disruption Annex VIII : products produced > 100 tonnes per year documentation available May 2013
RTC experience on REACH CRO clients : Companies with very different in size and experience � different approach to safety assessment program. Often consortia of companies producing the same chemical compound which may include both large- scale and small-scale producers
RTC experience on REACH The clients usually approaches the CRO with all the relevant information: • physical-chemical properties • human exposure • application
RTC experience on REACH • environmental exposure • available toxicological data Level 1 - physical & chemical properties, e.g., MW, reactivity, volatility, biodegradability, Sorting & prioritization based - human & environmental exposure, e.g., production volume, release, use patterns - hazard, e.g., available toxicological data upon existing information
RTC experience on REACH OECD (Q)SAR Application Toolbox Combination of toxicological knowledge, expert judgment, and statistically derived models Alerts Chemical structure Elaboration results
RTC experience on REACH Consortia including large companies usually have this information and also data on in vitro and in vivo mechanistic studies described in Level 2 and Level 3 of OECD framework Level 2 - ER, AR, TR receptor binding affinity -High Through Put Prescreens - Transcriptional activation - Thyroid function In vitro assays providing - Aromatase and steroidogenesis in vitro - Fish hepatocyte VTG assay - Aryl hydrocarbon receptor recognition/binding - Others (as appropriate) mechanistic data - QSARs Level 3 - Uterotrophic assay (estrogenic related) In vivo assays providing data - Hershberger assay (androgenic related) - Fish VTG (vitellogenin) assay - Non -receptor mediated hormone function (estrogenic related) about single endocrine - Others (e.g. thyroid) Mechanisms and effects Information from Levels 1, 2 and 3 make easier to decide on the next steps of safety program
RTC experience on REACH Consortia with only small companies : TG 421 TG 407 TG 422
Reduce and Refine with OECD in vivo studies
Importance of CRO in the supporting the 3Rs philosophy Importance of CRO in designing the safety program in the spirit of 3Rs: � include the right in vitro alternative methods � limit animal use in the in vivo studies � refine in vivo studies with extra toxicity end-points
Conclusion Small companies do not ask for in vitro tests (concern of time and budget) � screening in vivo studies are always necessary for safety classification Poor interest from Sponsors � scarce offer from CROs. Only few CROs have the complete panel of in vitro mechanistic studies
Conclusion Promotion of alternative tests may be challenging CRO plays a central role to encourage the use of new tests and overcome the natural cautious aptitude of industrial sponsors
Rome, December 17 th , 2012 Serena Cinelli Thank you
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