R&D - SEAC Safety & Environmental Assurance Centre

Prototype risk assessment for

biopersistents using AOP approaches Bobbie Bradford, Paul Fowler, Annette Furniss, Andrew Homan, Penny Jones, Richard Stark and Andrew White

SEAC, Unilever, Colworth Science Park, Sharnbrook, Bedford, MK44 1LQ, UK

The traditional safety risk assessment of inhalable biopersistent materials relies on estimation of the consumer

lung exposure coupled with generation of in vivo inhalation toxicity data. Our aim is to replace the need for in vivo

studies using a combination of exposure-based waiving(1,2) and an understanding of the Adverse Outcome

Pathways (AOP) for key events in lung responses to fibrosis. The AOP framework includes exposure, transport to

the alveoli, and initiation of gene activation/protein production/altered signalling, inflammation/cell proliferation/

tissue remodelling and lung fibrosis and cancer. We are exploring the use of this AOP framework for risk

assessment using specific examples of inhalable biopersistent materials. By understanding the multistage

pathogenesis of well-studied human lung disorders induced by model biopersistents, early markers of pre-disease

in in vitro systems can be determined for use in consumer safety risk assessments for the use of novel

biopersistent materials.

(1) Carthew, P., Griffiths, H., Keech, S. and Hartop, P. (2002). Safety assessment for hairspray resins: risk assessment based on rodent

inhalation studies. Inhalation Toxicology 14, 401-416.

(2) Carthew, P., Clapp, C.. and Gutsell, S. (2009). Exposure based waiving: the application of the toxicological threshold of concern (TTC)

to inhalation exposure for aerosol ingredients in consumer products. Food Chem Toxicol. 47, 1287-95.

Adverse Outcome Pathway (AOP) for lung disease (fibrosis/cancer)

Exposure to

biopersistent

material

Bioavailable to

target cells - alveolar

macrophage,

epithelial cell,

fibroblasts

Gene activation

Protein production

Altered signalling

Cell death/proliferation,

Inflammation,

Remodelling

Lung

Pathology

Lung

Disease

Is there sufficient human exposure to cause

concern? Are the particles biopersistent? Can

they cause inflammation? What size are they?

Identification of the major gene/protein changes in human

disease progression informs the discovery of upstream

pre-disease markers i.e. TGFß. Also consider genetic

changes that could lead to fixed mutation.

Angiotensin

signaling

Fibrosis

FN1 EMT MAPK signaling

EDN1

TP53

signaling ATF2/JUN

signaling

DNA damage ER stress

ATF4 taof(XBP1)

ATF6 EIF2AK3

exp(CXCL12)

TLR/NFKB

signaling

JAK/STAT

signaling

taof(HIF1A)

Angiogenesis

E2F signaling

EGFR

signaling

Fibroblast proliferation

CDKN1A

CXCR4

Macrophage

activation

KEAP1 ROS

taof(NFE2L2)

Oxidative stress

MDM2

Hedgehog

signaling

Fibrocyte

recruitment

taof(FOXO1)

taof(FOXO3)

taof(RB1)

Collagen

RBL2

GSTP signaling

CCND1

taof(SRF)

MYOD1/

MEF2 activity

VEGFA

taof(PITX2)

IGFBP5

taof(SOX2)

taof(AHR)

exp(MUC5AC)

Epithelial cell

apoptosis

Mucus production

TGFB

signaling taof(CREB1)

VHL

Inflammation

Th1/Th2 cytokines

SOCS

kaof(KDR)

PDGFB

signaling

mTOR signaling

PI3K/AKT

signaling

WNT/b-catenin

signaling

FGF

signaling

Myofibroblast

differentiation

Differentiated myofibroblast Fibroblast

6 of the 12 BioMap® systems tested were responsive - Sensitive systems which detected all positive test materials:

• Mphg -Venular endothelial cells + macrophages

• MyoF - Lung fibroblasts (stimulated with TNFα and TGFβ)

• HDF3CGF – Fibroblasts (stimulated with TNFα, IL-1β,EGF, PDGF)

• KF3CT - Keratinocytes +fibroblasts (stimulated with TNFα, IL-1β , TGFβ)

• BE3C - Bronchial epithelial cells (stimulated with TNFα, IL-1β, IFNγ)

• BT-B cells + peripheral blood monocytes (stimulated with anti-IgM)

Cell systems were initially selected based on their observed

responses in levels of known biomarkers of pro-fibrotic effect

(i.e. collagen, osteopontin, αSMA, and specific genes).

Transcriptomic studies of in vitro cell lines and of relevant human

disease states in vivo have furthered our understanding of the

pathways involved. Sensitivity and specificity were tested using

previously characterised substances. Following this initial work,

relevant fibrosis BioMap systems (at BioSeek, DiscoveRX) were

used to identify the key events which are consistently activated

in response to known fibrosis-inducing materials.

SAFETY SCIENCE IN THE

21ST CENTURY

For more information visit

www.tt21c.org