Progress on alternative in vitro methods for
the safety assessment of chemicals
and their impact on human health:
international and Brazilian views
Skin corrosion and irritation: achievements of
Brazilian model
Silvya Stuchi Maria-Engler (FCF-USP)
SILVYA STUCHI MARIA-ENGLER
CLINICAL CHEMISTRY AND TOXICOLOGY DEPT
SCHOOL OF PHARMACEUTICAL SCIENCES
UNIVERSITY OF SÃO PAULO
UNDERSTAND ANIMAL TESTING
RISK ASSESSMENT
Determination of quantitative or qualitative value of risk related to a concrete
situation and a recognized threat (also called hazard).
Animal testing for
research and
development
80
90
Adopting of 3 Rs
- Reduction,
Refinement,
Replacement
(Russell & Burch,
1959).
ECVAM promotion
and validation of in
vitro assays
2000 -
REACH - EUROPEAN
Seventh
REGULATION, 2006
Amendment to the
Cosmetics.
Regulation on
imposes a ban on
Registration,
marketing cosmetics in
Evaluation,
Europe if the finished
Authorisation and
product or its
Restriction of
ingredients have been
Chemicals.
tested using animals.
Alternative Methods
Brazil
2008 - 2009
Publication of Law
AROUCA 11.794/08
Creation of the
National Council for
the Control of
Animal
Experimentation CONCEA
2012
Creation of BraCVAM Creation of RENAMA
- National Network
connected to the
of Alternative
National Institute of
Methods (MSTI)
Quality Control and
Health - INCQS
FIOCRUZ
2013
March - 2014
Law 15.316:
CONCEA:
After the validation of
the alternative
methods in Brazil,
companies will have
up to five years to
implement them
Ban the use of
animals in
cosmetics testing.
Government of
São Paulo
ALL MODELS HAVE LIMITATIONS
NO MODEL CAN POSSIBLY EXPLAIN EVERY DETAIL
OF A SCIENTIFIC PHENOMENA
THREE-DIMENSIONAL (3D) CELL CULTURE AND ORGANOTYPICAL CULTURES
•  Reproduces similar results
in vitro and in vivo:
•  Multicellular tumor spheroids
generated in vitro exhibit
characteristics of avascular tumors,
respect to tumor physiology and
microenvironment.
•  The interaction among cells in a
tridimensional culture environment
enhances cell differentiation and
leads to luminal formation in some
breast-derived cell cultures.
•  3D tissues retaining the original
functions of tissues/organs in vitro
•  is crucial for optimal tissue
engineering and regenerative
medicine.
WHY THIS IS IMPORTANT IN BRAZIL?
IN VITRO
DERMAL EQUIVALENTS/ SKIN RECONSTRUCTIONS
•  BRAZIL HAS LIMITATION TO IMPORT COMERCIAL KITS
•  BRAZIL AS A HUGE COSMETIC MARKET
•  IMPROVEMENT OF INNOVATION
•  NEW COMPOUNDS BASED ON BRAZILIAN FLORA/
PLANT EXTRATS
•  NEW SYNTHETIC MOLECULES/ COMPOUNDS
•  PHARMACOLOGICAL POTENTIAL AND CLINICAL
APPLICATIONS IN SKIN
•  ANTI-TUMORAL (melanoma, cervical cancer)
•  SKIN DISORDERS (psoriasis)
(3R) RECYCLING SKIN
Foreskin
fibroblasts
Foreskin
melanocytes
Separation and digestion
keratinocytes
+KeM
AIR LIQUID
INTERFACE
DERMAL
EQUIVALENT:
COLLAGEN+
FIBROBLASTS
Collagen
IV film
KERATINOCYTES AND
MELANOCYTES
DERMAL EQUIVALENT
2 WEEKS LATER
SKIN
RECONSTRUCTED
KCs$
1 DAY IN
MEDIUM
12 DAYS IN AIRLIQUID
INTERFACE
EPIDERMAL
EQUIVALENTS
RENAMA ACHIEVEMENTS
•  Epidermal Equivalent in vitro
VMR= validated methods references
Skin
USP (2014)
•  Draize Test
EpiSkin
L´Oreal
EpiDerm™ Tisue
Model
MatTek®
SkinEthic
L´Oreal
Human epidermis
Catarino et al, 2015
Pedrosa et al, 2015
BIOPRINTING))
)SKIN)ON)BIOPOLIMERIC)MEMBRANE)
Carolina Catarino
MSc. Student FAPESP
ELETROSPINING PRINTHEAD
PRINTED
LAYERS
ELETROSPUN
FIBERS
MB (PET + Collagen I)
A.P.S. Immich, P. C. Pennacchi, S. S. Maria-Engler, R. L. Boemo, L. H. Catalani“Artificial Graft for Eardrum Regeneration”. Termis – 2013 – Istambul, Turquia.
• 2/17 methods of Brazilian CONCEA
RN17, 2014
•  OECD TG 431 – Dermic corrosion test
•  OECD TG 439 – Skin Irritation test
1.  MORPHOLGY AND DIFFERANTIATION/
2.  BARRIER FUCNTION
3.  VIABILITY
HE
CK10
Ki67
Filagrinn
CK14
Involucrin
HE
CK10
3 min
1h
3 min
1h
Positive
Control
Acetic
Acid
Negative
Control
NaCl 0,9 %
Viab.: 100%
Viab.: 100%
Viab.: 0,99%
Viab.: 2,84%
Láctic
Acid
2-Bromo
etil
benzene
Viab.: 82%
Viab.: 80%
Benzil
acetone
Viab.: 8%
Viab.: 2%
Viab.:8,3%
Viab.: 2,3%
Octanóic
Acid
Viab.: 80%
Viab.: 97%
13
SKIN BIOLOGY LABSCHOOL OF PHARMACEUTICAL SCIENCES,
UNIVERSITY OF SÃO PAULO
Co-coordination
Silvia B. M. Barros
Collaborators:
Keiran Smalley,
Moffitt Cancer Center, USA
Márcia E. L. Consolaro, UEM,
PR
Enrique Bocardo, ICB-USP
Ana Campa, FCF-USP
Luis Catalani, IQ-USP
Lorena Cordeiro, USP RP
São Paulo School in advanced Science in Alternative methods: the science behind
FCF-USP 2015