new approaches to using mechanistic toxicology data in

New approaches to using mechanistic toxicology

data in human health risk assessment

XXXVth EAPCCT Congress, 28 May 2015, St Julians, Malta

Prof Martin F Wilks

Director, SCAHT

Universität Basel

Klingelbergstrasse 61

4056 Basel

www.scaht.org

Outline

Toxicological disasters and early regulation

The changing face of risk assessment

Safety testing – from haruspicy to systems toxicology

2

Sulfanilamide (1937)

Sulfanilamide – antibacterial agent discovered in 1935 and

available in tablet and powder form

In 1937, S.E. Massengill company produced ‘elixir

sulfanilamide‘ using diethylene glycol as solvent

No toxicity testing had been perfomed, but the product had

passed testing for appearance, flavour and fragrance

Within 4 weeks, 353 patients had received treatment, 105

died (incl. 34 children), primarily from renal failure

The incident facilitated the passing of the 1938 Food, Drug

and Cosmetic Act which required companies to submit safety

testing information to the US FDA

Further mass poisoning incidents occurred in South Africa

(1969), India (1986), Nigeria (1990), Bangladesh (1990/92),

Haiti (1995/96), China, Panama (2006), Nigeria (2008)

In all cases medicines or personal care products had been

prepared with DEG as a substitute of, or contaminant in, other

solvents such as glycerine or propylene glycol

3

http://en.wikipedia.org/wiki/File:Elixir_Sulfanilamide.jpg

http://en.wikipedia.org/wiki/File:Sulfanilamide-skeletal.svg

http://en.wikipedia.org/wiki/File:Diethylene_glycol_structure.svg

Thalidomide (1958 – 1962)

Developed by Grünenthal and sold as

sedative since late 1957

Shown to be particularly effective against

morning sickness in pregnant women

Standard safety tests on non-pregnant rats

showed no appreciable toxicity even at high

doses

An estimated 10‘000 – 20‘000 children were

born with limb deformities caused by

thalidomide

Extensive investigations in the 1960s showed

a marked species difference in teratogenicity

with New Zealand White Rabbits being

particularly sensitive

Testing for developmental effects in two

species became a regulatory requirement in

the USA in 1966

4

http://toxsci.oxfordjournals.org/content/122/1/1/F1.large.jpg

Toxicology and regulation

Regulatory toxicology is the process whereby information

relevant to assessing the toxicity of agents, which may be

biological, chemical or physical in nature, is obtained and

evaluated by or on behalf of governmental or international

organizations.

The aim is to protect workers, consumers, the public generally

and the environment

Illing HPA & Marrs TC

General and Applied Toxicology, 2009

5

Toxicology and regulation

“Regulatory toxicology is to toxicology what military

music is to music“

Sir Colin Berry

Em. Professor of Morbid Anatomy and Histopathology

Queen Mary College

University of London

6

OECD Testing Guidelines

http://www.oecd-ilibrary.org/environment/oecd-guidelines-for-the-testing-of-chemicals-section-4-health-effects_20745788

7

OECD Guidelines for the Testing of Chemicals

Section 4: Health Effects

401: Acute Oral Toxicity

402: Acute Dermal Toxicity

403: Acute Inhalation Toxicity

404: Acute Dermal Irritation/Corrosion

405: Acute Eye Irritation/Corrosion

406: Skin Sensitisation

407: Repeated Dose 28-day Oral Toxicity Study in Rodents

408: Repeated Dose 90-Day Oral Toxicity Study in Rodents

409: Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents

410: Repeated Dose Dermal Toxicity: 21/28-day Study

411: Subchronic Dermal Toxicity: 90-day Study

412: Subacute Inhalation Toxicity: 28-Day Study

413: Subchronic Inhalation Toxicity: 90-day Study

414: Prenatal Development Toxicity Study

415: One-Generation Reproduction Toxicity Study

416: Two-Generation Reproduction Toxicity

417: Toxicokinetics

418: Delayed Neurotoxicity of Organophosphorus Substances Following Acute

Exposure

419: Delayed Neurotoxicity of Organophosphorus Substances: 28-day Repeated

Dose Study

420: Acute Oral Toxicity - Fixed Dose Procedure

421: Reproduction/Developmental Toxicity Screening Test

422: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental

Toxicity Screening Test

423: Acute Oral toxicity - Acute Toxic Class Method

424: Neurotoxicity Study in Rodents

425: Acute Oral Toxicity: Up-and-Down Procedure

426: Developmental Neurotoxicity Study

427: Skin Absorption: In Vivo Method

428: Skin Absorption: In Vitro Method


430: In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER)

431: In Vitro Skin Corrosion: Human Skin Model Test

432: In Vitro 3T3 NRU Phototoxicity Test

435: In Vitro Membrane Barrier Test Method for Skin Corrosion

436: Acute Inhalation Toxicity – Acute Toxic Class Method

437: Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and

Severe Irritants

438: Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants

439: In Vitro Skin Irritation

440: Uterotrophic Bioassay in Rodents

441: Hershberger Bioassay in Rats

442A: Skin Sensitization

442B: Skin Sensitization

451: Carcinogenicity Studies

452: Chronic Toxicity Studies

453: Combined Chronic Toxicity/Carcinogenicity Studies

455: The Stably Transfected Human Estrogen Receptor-alpha Transcriptional Activation Assay for

Detection of Estrogenic Agonist-Activity of Chemicals

471: Bacterial Reverse Mutation Test

473: In vitro Mammalian Chromosome Aberration Test

474: Mammalian Erythrocyte Micronucleus Test

475: Mammalian Bone Marrow Chromosome Aberration Test

476: In vitro Mammalian Cell Gene Mutation Test

477: Genetic Toxicology: Sex-Linked Recessive Lethal Test in Drosophila melanogaster

478: Genetic Toxicology: Rodent Dominant Lethal Test

479: Genetic Toxicology: In vitro Sister Chromatid Exchange Assay in Mammalian Cells

480: Genetic Toxicology: Saccharomyces cerevisiae, Gene Mutation Assay

481: Genetic Toxicology: Saacharomyces cerevisiae, Miotic Recombination Assay

482: Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells in

vitro

483: Mammalian Spermatogonial Chromosome Aberration Test

484: Genetic Toxicology: Mouse Spot Test

485: Genetic toxicology, Mouse Heritable Translocation Assay

486: Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo

487: In Vitro Mammalian Cell Micronucleus Test

(http://www.oecd.org) 8

Haruspicy

Haruspex = a person trained to practice a form of

divination called haruspicy (haruspicina) involving

the inspection of the entrails of sacrificed animals,

especially the livers.

Practised at least since Babylonian times, it was an

integral part of many ancient cultures.

After ritual scrifice the haruspex examined the size,

shape, color, markings etc. of certain internal

organs, usually the liver (hepatoscopy), but also the

gall, heart and lungs

Assyrian clay liver

models dated to the

19th or 18th century BC

Etruscan inscriptions on

the bronze sheep's liver

of Piacenza

9



















417: Toxicokinetics


Exposure


Dose Study


















Severe Irritants























vitro






(http://www.oecd.org)

OECD TG408: Repeated Dose 90-day Oral

Toxicity Study in Rodents

Gross necropsy

All animals in the study shall be subjected to a full, detailed

gross necropsy which includes careful examination of the

external surface of the body, all orifices, and the cranial,

thoracic and abdominal cavities and their contents. The

liver, kidneys, adrenals, testes, epididymides, uterus,

ovaries, thymus, spleen, brain and heart of all animals ...

should be trimmed ... and their wet weight taken.

10



















417: Toxicokinetics


Exposure


Dose Study


















Severe Irritants























vitro






(http://www.oecd.org) 11

A paradigm shift (NRC, 2007)

Where we are:

Complex array of studies

Evaluate observable outcomes

in whole animals

Time-consuming and resource-

intensive

Where we need to be:

Broad coverage of chemicals,

chemical mixtures, outcomes, and

life stages

Reduce the cost and time of testing

Use fewer animals

Robust scientific basis for assessing

health effects

12

A vision for toxicity testing (NRC 2007)

13

Toxicity Pathways

Toxicity Pathways

OECD 2011. Series on Testing and Assessment No. 138. Report of the Workshop on Using Mechanistic Information

in Forming Chemical Categories. ENV/JM/MONO(2011)8, 18-May-2011.

14

A hypothesized mode of action is considered to comprise a set of

critical key events from administration of the substance to a final

specific toxic outcome.

Mode of action is to be distinguished from mechanism of toxicity,

which is a detailed knowledge of the molecular interactions leading

to the toxic effect.

The mechanism of toxicity is fully elucidated for only a few

chemicals… but many more chemicals have a reasonably well

understood MOA, in that the key events are known, measurable,

necessary, and consistent.

Carmichael N et al. Crit Rev Toxicol. 2011 Mar;41(3):175-86.

What is a mode of action?

WHO/IPCS MOA framework

15

Mode of action example

Chloroform organ toxicity and tumor development

Key events

1. Absorption and

distribution of

chloroform to

target tissue

2. Generation of

cytotoxic metabolite

(phosgene) by P450

(CYP2E1)

3. Sustained cytotoxicity

4. Regeneration and

proliferation

Application of key events analysis to chemical carcinogens and noncarcinogens.

Boobis AR, Daston GP, Preston RJ, Olin SS. Crit Rev Food Sci Nutr. 2009 Sep;49(8):690-707.

WHO/IPCS MOA framework

16

OECD Adverse Outcome Pathways

OECD 2011. Series on Testing and Assessment No. 138. Report of the Workshop on Using Mechanistic Information

in Forming Chemical Categories. ENV/JM/MONO(2011)8, 18-May-2011.

17


OECD 2011. Series on Testing and Assessment No. 138. Report of the Workshop on Using Mechanistic Information in

Forming Chemical Categories. ENV/JM/MONO(2011)8, 18-May-2011.

http://www.oecd.org/officialdocuments/displaydocument/?cote=env/jm/mono(2011)8&doclanguage=en

AOPs can be used to address a number of decisions, including:

(1) preliminary priority setting for further testing

(2) hazard identification (e.g. with the OECD QSAR Toolbox)

(3) classification and labelling (possibly, not currently done)

(4) risk assessment, both preliminary and more complete

(i.e. with dose-response and ADME data)

Purpose

18

http://www.oecd.org/officialdocuments/displaydocument/?cote=env/jm/mono(2011)8&doclanguage=en


OECD 2012. Series on Testing and Assessment No.168. The Adverse Outcome Pathway for Skin Sensitization

Initiated by Covalent Binding to Proteins – Part 1

http://search.oecd.org/officialdocuments/displaydocumentpdf/?cote=env/jm/mono(2012)10/part1&doclanguage=en

19

http://search.oecd.org/officialdocuments/displaydocumentpdf/?cote=env/jm/mono(2012)10/part1&doclanguage=en

Pathway vs. network

Bensimon A, Heck AJR, Aebersold R. Annu. Rev. Biochem. 2012. 81:379–405

20

• Integrates classic toxicology approaches with

network models and quantitative measurements

of molecular and functional changes occurring

across multiple levels of biological organization.

• Evaluate interactions between potential hazards

and the components of a biological system.

• Aimed at developing a detailed mechanistic,

quantitative and dynamic understanding of

toxicological processes.

• Permits prediction and accurate simulation of

complex (emergent) adverse outcomes.

• Provides a basis for translation between model

systems (in vivo and in vitro) and study systems

(e.g., human, ecosystem).

• Extrapolates from early and highly sensitive

quantifiable molecular and cellular events to

medium- and long-term outcomes at the

organism level.

Artwork by Samantha J. Elmhurst (www.livingart.org.uk)

Published in: Shana J. Sturla; Alan R. Boobis; Rex E. FitzGerald; Julia Hoeng; Robert J. Kavlock; Kristin Schirmer; Maurice Whelan; Martin F.

Wilks; Manuel C. Peitsch; Chem. Res. Toxicol. 2014, 27, 314-329.

Copyright © 2014 American Chemical Society

Systems Toxicology

21

Summary

Safety testing is undergoing a paradigm shift from 20th century

‘organ-based’ to 21st century ‘mode-of-action’ based assessment

MOA and AOPs are frameworks for organizing complex biological

information into key events that are known, measurable, necessary

and consistent

Systems toxicology integrates classic toxicology approaches with

network models to create a basis for translation between model

systems (in vivo and in vitro) and human adverse outcomes

22

200

21st century B.C. 21st century A.D.?

23

new approaches to using mechanistic toxicology data in

Documents