Application of a novel, pathway-based screen for Developmental And Reproductive Toxicity (DART)

Dr. Marjolein WildwaterProgram leader alternative test systems HAN BioCentre

Dr. Chantal SmuldersShell

Alternative in vivo 3R species for developmentaland reproduction (DART) hazard prediction

Unique features • Well known 3D systems

with tissues and organs

• Conserved molecular andcellular pathways

• Huge amount of availabletools/technologies

• High throughput

• Low cost

• Rapid generation time

• 3R

Challenges

• Not human

• Further requirement forvalidation and determinationof applicability domain

• Not yet accepted byregulators

A combinatorial approach to identify biomarkers and AOPs for developmental and reproductive

toxicity

D. discoideum, C. elegans and D. rerio as alternative test systems for developmental and

reproductive toxicity

You tube: by Tglab You tube: tony hyman lab You tube: Karlstrom and Kane

What makes these organisms predictable for effects in humans???

Shell International B.V. April 2016

C. elegans genome wide RNAi library

Easy AOP identification High throughput methodology

D. rerio robotic injection equipment

Craniofacial bone reporter Notch receptor

C. elegans

Fluorescent reporters

Available tools allow rapid and unique testing

Comparable organs, tissues and cell biology; conserved genetic responses/pathways!

http://www.sfu.ca/biology/faculty/hutter/hutterlab/research/Ce_nervous_system.html

• Muscles• Skin• Neurons• intestine C. elegans neurons visualised by GFP

D. rerio• Muscles• Skin• Neurons• Intestine• Heart• Eye• cartilage

C. elegans cell lineage is known and invariable

An example: Pax6, an evolutionary conservedgene for eye development

Washington et al PLoSBiol e1000247

Pax6 important in C. elegans for normal headdevelopment

Cinar and Chisholm., Genetics 168: 1307–1322

Ultimate goal: medium throughput tiered 3R-approach to predict DART potential in man

Phenotypic responses of 3 species to selected chemicals

Internal dose assessment exposure

Mapping of adverse outcome pathways by RNA-sequencing, RNAi and kinomics approach

Q-SAR

e.g. EST

Combinatorial testing pipe line for DART shows high predictivity to mammalian data

Standard operational protocols for exposure

Internal dose assessment after exposure

Omics: transcriptomic, and kinomic analysis and RNAi to identify mode of action

Dose-response curves

Phenotypic data alignment

Validation of the test system

Compound exposure

Shell International B.V.

APPLICATION OF A NOVEL, PATHWAY-BASED SCREEN FOR DEVELOPMENTAL AND REPRODUCTIVE TOXICITY

Chantal Smulders ,

Shell

April 2016

Shell International B.V.

INDUSTRY VALUE OF DART SCREEN & 3R BENEFITS

April 2016

Product development pipeline

early selection of promising substance

avoidance of animal testing multiple new substances

Support read-across strategies and categorization

maximize use of existing animal data

avoidance of additional animal testing

Part of a weight-of-evidence approach

AOP approach allows better prediction of potential human

outcome

Shell International B.V.

APPLICATION OF NOVEL DART SCREEN

April 2016

New process chemicals - 1 existing (Reprox Cat 2)

- 3 structural analogues (A,B,C)

Company name appears here Month 2010Footer

To demonstrate chemical safety, alternative testing and translational toxicology can provide insight in human safety

Alternative testing using high content assays (e.g. genomics) can provide insight in (absence) of potential effects and help elucidate new AOPS

Basis: key pathways for reproduction and development are preserved across species in evolution

Testing in more primitive, less sentient species can provide insight in potential perturbance of human pathways

Major reproductive and developmental toxicity phenotypes in mammals (humans) can be predicted

Monday, July 27, 2015

DEVELOPMENT OF A NEW SCREENING ASSAY

Shell International B.V. April 2016

ASSAYS DEVELOPED IN NC3RS PREDART CRACK-IT PROJECT

Collaborative project between Shell and Syngenta

Aim to assess biomarker development in a panel of in vitro and in vivo alternative test species:-

amoeba (Dictyostelium discoideum),

nematode (Caenorhabditis elegans)

zebrafish (Danio rerio)

and embryonic stem cells)

Assess if these can enable reliable prediction of potentially hazardous compounds taking advantage of evolutionary conserved molecular pathways across the test species

15

Shell International B.V.

INDUSTRIAL APPLICATION OF THE TEST SPECIES: EXPOSURE TO A DART CHEMICAL THAT SHOULD INDUCE

DART ONLY AT A HIGH DOSE: DEVELOPMENTAL DELAY

April 2016

Compound B

A.

B.

C. D.

4 dpf control

4 dpf 10mM PIP

Adult control10mM PIP

Existing

Existing

Shell International B.V.

ANALOGOUS COMPOUND ASSESSMENT INDICATESDEVELOPMENTAL DELAY MOST STRONGLY AT HIGHER

DOSE

April 2016

0102030405060708090

100

zebrafish

nematode

Trip-Hep (AVG)

A. B.

C. D.

Piperazine 1‐(2‐Hydroxyethyl Piperazine)

1,4‐Bis(2‐Hydroxyethyl)piperazine 

concentration

concentration concentration

concentration

% Develop

men

tal delay

% Develop

men

tal delay

% Develop

men

tal delay

% Develop

men

tal delay

0102030405060708090100

zebrafish

nematode0

102030405060708090100

Control (H2

O)

1µM

10µM

100µ

M

1mM

10mM

zebrafish

nematode

0102030405060708090100

zebrafish

nematode

Existing A

B C

Shell International B.V.

PERCENTAGE OF AFFECTED ZEBRAFISH LARVAE -TERATOGENIC EFFECTS

April 2016

ExistingABC

Number of fish

Shell International B.V.

C. ELEGANS: EFFECTS ON BROOD SIZE ONLY AT HIGH CONCENTRATIONS

Control 0.1µM 1µM 10µM 100µM 1mM 10mM

Existing - - - - *x *x *xA - - - - * * *xB - - - * * - *C - - - - - - -

April 2016

* statistically significant effects on brood size compared to controlx statistically significant effects on development compared to control

Shell International B.V.

SUMMARY RESULTS DART SCREEN

April 2016

Reproductive toxicity effects in nematodes and zebrafish only observed at high concentrations (100 µM and above)

These concentrations are unlikely to be reached in vivo in humans under conditions of use.

The existing substance - showing reproductive effects at high concentration only - was the most potent reproductive toxicant to nematodes and zebrafish.

These results are in alignment with the reported test data for rats and rabbits on this substance, where indications of reproductive effects were only observed at high test concentrations and are considered to be a consequence of maternal toxicity rather than a direct reproductive effect

The structural analogs tested are unlikely to be developmental toxicants in humans.

Shell International B.V.

CONCLUSIONS USABILITY DART SCREEN

April 2016

The results of the zebrafish and nematodes are in alignment with data obtained from mammalian toxicity studies, indicating that these have potential as developmental toxicity screens

Despite not replacing a reprotoxicity study in animals one-on-one, significant reduction of animal testing can be achieved

Duration and cost of the DART screen allows application early in the product development pipeline

Further mapping of developmental AOP across species is needed

In vitro – in vivo dose extrapolation is key for successful use of the screen in risk assessment

If care is taken regarding relevance of exposure pathways useful environmentally relevant (ecotoxicological) data can also be obtained

Acknowledgements

At what stage would you be willing to invest money when using the alternative test models for DART testing?

1. Now. They can already be used for pre-screening of large compound sets.

2. Not yet. More data/information is required before I trust these systems.

3. Only when they are officially accepted by the regulators.

4. Never

Now.

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31%

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Interactive question 1

Please contact wildwaterm@gmail.com for more information

In principle, would your company/institution be willing to join the effort to help us to further validate these organismal 3Rs models?

1. Yes, we could provide compounds

2. Yes, compounds with mammalian data

3. Yes, investment (time, money and/or resource) to enable testing in parallel to mammalian studies

4. Yes, investment (time, money and/or resource) to pre-screen chemicals to inform choices for further product development (e.g. prioritisation)

5. No

6. Unable to comment

Yes, 

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 c...

Yes, 

comp

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Yes, 

invest

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Yes, 

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Unab

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comm

ent

12% 12%

54%

7%9%6%

Interactive question 2