Improving test methods in the spirit of the 3Rs; the point of view - - PowerPoint PPT Presentation

Improving test methods in the spirit of the 3Rs; the point of view of a contract research

• rganization

Rome, December 17th, 2012

Serena Cinelli

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

Research and Development Pre-validation Validation Regulatory acceptance Implementation Academia Industry CRO Industry CRO Industry

Regulators OECD

Regulatory reference Species Mechanism addressed Endpoints Suggested equivalent information

ER TA OPPTS 890.1300 OECD TG 455 OECD TG 457

Endogenous human ERα human ERα, ERβ Estrogen agonist Estrogen agonist, antagonist ERα, ERβ dependent transcriptional activation Effect on ovary/uterus size, histology, male/female fertility

ER binding OPPTS 890.1250

Uterine cytosol from rats Estrogen agonist, antagonist ER binding Effect on ovary/uterus size, histology, male/female fertility

AR binding OPPTS 890.1150

Prostate cytosol from rats Androgen agonist, antagonist AR binding Effect on testes size, histology, male/female fertility

Steroidogenesis OPPTS 890.1550 OECD TG 456

Human (H295R) Steroid synthesis (estrogen and testosterone) Testosterone, estrogen hormone levels Effect on estrogen/testosterone levels, sex organs, male/female fertility

Aromatase OPPTS 890.1200

Human Aromatase inhibition, the enzyme responsible for the conversion of androgens to estrogens

3H2O released

during the conversion of androstenedione to estrone Effect on estrogen/testosterone levels, sex organs, male/female fertility

* * * *

Validated in vitro methods

Endocrine disruptor in vitro assay

TG 455 Test system: stably transfected cells

• ERα

α α α-HeLa-9903 Restrictions: Biological risk Technical challenges: difficulties to maintain stability and integrity of the cell line

TG 457 Test system: stably transfected cells

• BG1Luc4E2

Restrictions: Biological risk Technical challenges: difficulties to maintain stability and integrity of the cell line

BG1Luc Estrogen Receptor Transactivation Test Method for Identifying Estrogen Receptor Agonists and Antagonists

Endocrine disruptor in vitro assay

Restrictions: Radioactive risk

Endocrine disruptor in vitro assay

TG 456 Test system: H295R Restrictions: None

Endocrine disruptor in vitro assay

Endocrine alterations

• 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

Hormonal systems are complicated and stand alone in vitro methods are not sufficient to predict in vivo effects

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

Level 2

In vitro assays providing mechanistic data

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

Level 5

In vivo assays providing data on effects from endocrine &

• ther mechanisms
• 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
• 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
• Uterotrophic assay (estrogenic related)
• Hershberger assay (androgenic related)
• Non -receptor mediated hormone function
• Others (e.g. thyroid)
• Fish VTG (vitellogenin) assay

(estrogenic related)

• Fish gonadal histopathology assay
• Frog metamorphosis assay
• 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)

• enhanced OECD 407 (endpoints based on

endocrine mechanisms)

• male and female pubertal assays
• adult intact male assay

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

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

OECD (Q)SAR Application Toolbox Combination of toxicological knowledge, expert judgment, and statistically derived models

RTC experience on REACH

Chemical structure Alerts Elaboration results

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

RTC experience on REACH

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

• n the next steps of safety program

Consortia with only small companies:

RTC experience on REACH

TG 407 TG 421 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 17th, 2012

Serena Cinelli

Thank you