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

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1 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

2 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...)

3 Main functions of CROs comply with regulatory directives and guidelines ensure an updated knowledge of the new method status

4 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

5 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

6 Validation process Research and Development Academia Industry CRO Industry Implementation Pre-validation CRO Industry Validation Regulatory acceptance Regulators OECD

7 Validated in vitromethods * * * * Regulatory reference ER TA OPPTS OECD TG 455 OECD TG 457 ER binding OPPTS AR binding OPPTS Steroidogenesis OPPTS OECD TG 456 Aromatase OPPTS Species Endogenous human ERα human ERα, ERβ Uterine cytosol from rats Prostate cytosol from rats Human (H295R) Human Mechanism addressed Estrogen agonist Estrogen agonist, antagonist Estrogen agonist, antagonist Androgen agonist, antagonist Steroid synthesis (estrogen and testosterone) Aromatase inhibition, the enzyme responsible for the conversion of androgens to estrogens Endpoints ERα, ERβ dependent transcriptional activation ER binding AR binding Testosterone, estrogen hormone levels 3 H 2 O released during the conversion of androstenedione to estrone Suggested equivalent information Effect on ovary/uterus size, histology, male/female fertility Effect on ovary/uterus size, histology, male/female fertility Effect on testes size, histology, male/female fertility Effect on estrogen/testosterone levels, sex organs, male/female fertility Effect on estrogen/testosterone levels, sex organs, male/female fertility

8 Endocrine disruptor in vitro assay TG 455 Test system: stably transfected cells ERα-HeLa-9903 Restrictions: Biological risk Technical challenges: difficulties to maintain stability and integrity of the cell line

9 Endocrine disruptor in vitro assay TG 457 BG1Luc Estrogen Receptor Transactivation Test Method for Identifying Estrogen Receptor Agonists and Antagonists Test system: stably transfected cells BG1Luc4E2 Restrictions: Biological risk Technical challenges: difficulties to maintain stability and integrity of the cell line

10 Endocrine disruptor in vitro assay Restrictions: Radioactive risk

11 Endocrine disruptor in vitro assay TG 456 Test system: H295R Restrictions: None

12 Endocrine alterations Hormonal systems are complicated and stand alone in vitromethods are not sufficient to predict in vivoeffects 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

13 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 Sorting & prioritization based upon existing information - physical & chemical properties, e.g., MW, reactivity, volatility, biodegradability, - human & environmental exposure, e.g., production volume, release, use patterns - hazard, e.g., available toxicological data Level 2 In vitro assays providing mechanistic data - ER, AR, TR receptor binding affinity -High Through Put Prescreens - Transcriptional activation - Thyroid function - Aromatase and steroidogenesis in vitro - Fish hepatocyte VTG assay - Aryl hydrocarbon receptor recognition/binding - Others (as appropriate) - QSARs Level 3 In vivo assays providing data about single endocrine Mechanisms and effects Level 4 In vivo assays providing data about multiple endocrine Mechanisms and effects - Uterotrophic assay (estrogenic related) - Hershberger assay (androgenic related) - Non -receptor mediated hormone function - Others (e.g. thyroid) - enhanced OECD 407 (endpoints based on endocrine mechanisms) - male and female pubertal assays - adult intact male assay - Fish VTG (vitellogenin) assay (estrogenic related) - Fish gonadal histopathology assay - Frog metamorphosis assay Level 5 In vivo assays providing data on effects from endocrine & other mechanisms - 1-generation assay (TG415 enhanced) 1-2-generation assay (TG416 enhanced) 1 - reproductive screening test (TG421 enhanced) 1 - combined 28 day/reproduction screening test (TG 422 enhanced) 1 1 Potential enhancements will be considered by VMG mamm - Partial and full life cycle assays in fish, birds, amphibians & invertebrates (developmental and reproduction)

14 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

15 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 largescale and small-scale producers

16 RTC experience on REACH The clients usually approaches the CRO with all the relevant information: physical-chemical properties human exposure application

17 RTC experience on REACH environmental exposure available toxicological data Level 1 Sorting & prioritization based upon existing information - physical & chemical properties, e.g., MW, reactivity, volatility, biodegradability, - human & environmental exposure, e.g., production volume, release, use patterns - hazard, e.g., available toxicological data

18 RTC experience on REACH OECD (Q)SAR Application Toolbox Combination of toxicological knowledge, expert judgment, and statistically derived models Alerts Chemical structure Elaboration results

19 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 In vitro assays providing mechanistic data - ER, AR, TR receptor binding affinity -High Through Put Prescreens - Transcriptional activation - Thyroid function - Aromatase and steroidogenesis in vitro - Fish hepatocyte VTG assay - Aryl hydrocarbon receptor recognition/binding - Others (as appropriate) - QSARs Level 3 In vivo assays providing data about single endocrine Mechanisms and effects - Uterotrophic assay (estrogenic related) - Hershberger assay (androgenic related) - Non -receptor mediated hormone function - Others (e.g. thyroid) - Fish VTG (vitellogenin) assay (estrogenic related) Information from Levels 1, 2 and 3 make easier to decide on the next steps of safety program

20 RTC experience on REACH Consortia with only small companies: TG 407 TG 421 TG 422

21 Reduce and Refine with OECDin vivostudies

22 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

23 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

24 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

25 Thankyou Rome, December 17 th, 2012 Serena Cinelli