How to develop safer cosmetics through in vitro science. This presentation was given to the Cosmetics Business Regulatory Summit 2018 by Dr Carol Treasure of XCellR8. It includes case studies of in vitro safety tests for skin sensitisation, advice on testing finished products and progress on a new model to predict mildness to skin.

1. Safer Cosmetics Through
In Vitro Science
Dr Carol Treasure
Founder & CEO, XCellR8 Ltd
carol.treasure@x-cellr8.com
@XCellR8_Labs
XCellR8; Dr Carol Treasure

2. About XCellR8
• XCellR8 provides 100% animal-free safety and
efficacy tests to the cosmetics and chemical industries
• Our mission: To accelerate the world’s transition to
100% animal-free testing through our scientifically
advanced and ethical approach
• Key clients include global cosmetic companies
including Lush and The Body Shop, ingredient
suppliers such as Croda and Innospec and SMEs
XCellR8 is GLP accredited by the MHRA for regulatory in vitro safety testing
Founded 2008 by
Bushra Sim and Dr Carol TreasureOur laboratory at Sci-Tech Daresbury Celebrating our 10th birthday

4. INTERNATIONAL
REGULATORY INFLUENCE
• Participation at OECD
Expert Working Groups
• OECD and ECHA
approval for animal-
product-free adaptations
of regulatory methods
GLOBAL THOUGHT
LEADERSHIP
• International conferences,
panels, debates, working groups
• Invited participant at the United
Nations General Assembly Sept
2018
INNOVATION / RESEARCH &
DEVELOPMENT
• Grants from Innovate UK
and the European Horizon
2020 programme to fund
the development of new in
vitro tests for acute toxicity
and mildness to skin
Our work beyond in vitro safety testing

5. The global
sustainability agenda
• 80% of countries still allow animal testing
for cosmetics
• This is now part of the United Nations’ 2030
Agenda: 17 Sustainability Development Goals
• SDG12: Sustainable Production and Consumption
• 12.8: “By 2030, ensure that people
everywhere have the relevant information and
awareness for sustainable development and
lifestyles in harmony with nature”.
• Watch our debate at the UN General
Assembly held on 27th Sept 2018
THE ROLE OF
100% ANIMAL-FREE TESTING

6. Are “alternatives” still alternative?!
Some “alternatives” are now
mainstream and adopted as the default
test methods for regulatory safety
testing eg REACH, CLP
• Extensive validation trials
(enhanced human safety)
• High level of confidence
internationally
• Large amount high quality of data

7. The “1R” journey
In
vivo
In
vitro
“3D” reconstructed
human skin cultures
(EpiDerm™)
“3D” human airway
epithelium,
(EpiAirway™)
Organ-on-a-chip
Genomic screening
(Senzagen
GARDSkin™)
Mechanistic tests
(anti-oxidant)
Animal-product-
free cell culture
Microbiome
3D bioprinting
Advanced in vitro:
Maximal human relevance
1R = Replacement
Huge progress over the last 20 years - uptake by industry is the key to the future!

8. Shifting the emphasis
From “No animal testing”
To “How do you test your
products and ingredients?”
Finished product testing is increasingly
important, helping companies to satisfy
consumer demands and go beyond the
“bare minimum” of a safety assessment
that relies on historical animal data for
ingredients
Instagram influencers visit the
XCellR8 lab to understand how
The Body Shop tests its ingredients
Well informed consumers
increasingly demand to know how
products are tested

9. What is truly animal-free testing?

10. Why animal-product-free (APF)?
Scientific advantages:
• Better model of human physiology
• Higher reproducibility (synthetic components)
Ethical advantages:
• Avoids animal welfare issues, eg Foetal Bovine Serum
(FBS) collection
• Helps companies meet consumer demands for
sustainable, ethical products with transparent supply chains
• Vegan compliant (and Halal?)
Uniquely, everything we do at XCellR8 is animal-product-free
Most in vitro methods still use animal-derived components
such as serum, tissue extracts and antibodies, so they still
require the sacrifice of animals and are not truly “animal-free”.
Uniquely, everything we do at XCellR8 is animal-product-free
(sometimes described as “vegan testing”)

11. Meeting consumer demands for ethical, safe
and sustainable cosmetics
Belot, N., Sim, B., Longmore, CL., Roscoe, L. and Treasure, C. (2017)
Adaptation of the KeratinoSens™ skin sensitisation test to
animal-product-free cell culture
http://www.altex.ch/resources/altex_2017_4_560_564_SC_Belot1.pdf
100% ANIMAL-FREE SKIN SENSITISATION TESTING
Edwards et al (2018)
Adaptation of the human Cell Line activation Test (h-CLAT)
to Animal-Product-Free Conditions
https://www.altex.org/publib/Edwards_of_180613_v2.pdf
Published in
ALTEX

12. Skin sensitisation adverse outcome pathway (AOP)
Regulatory guidance: “2 out of 3” approach
SENSITISER
T-CELL
1
2
KERATINOCYTES
CONTACT
Inflammatory Cytokine Release
3
4
LYMPHOCYTE PROLIFERATION
DENDRITIC
CELLS
MIGRATION TO LOCAL LYMPH
NODE
5
Key Events In Skin Sensitisation and
Related Tests
1. Contact
(Direct Peptide Reactivity Assay – DPRA)
2. Release of Pro-Inflammatory Cytokines by
Keratinocytes (KeratinoSensTM)
3. Dendritic Cell Activation/Maturation
(human Cell Line Activation Test – h-CLAT)
4. Migration
5. T-cell Proliferation
(Local Lymph Node Assay - LLNA)

13. Animal-product-free
(APF) adaptation of
KeratinoSens™
• Replaced foetal bovine serum (FBS) and porcine
culture reagents with human-based or synthetic
alternatives
• All 20 reference chemicals correctly classified in
line with Validated Reference Method (VRM)
• Data accepted by the OECD Expert Working
Group on Skin Sensitisation and WNT National
Co-Ordinators’ Committee
• Adapted method published as an Annex to the
VRM in the new version of OECD TG 442d 2018
• Therefore full acceptance as a regulatory
method

14. Animal-product-free
adaptation of h-CLAT
• Replaced foetal bovine serum (FBS), porcine
culture reagents and mouse-derived antibodies
with human-derived or synthetic alternatives
• All proficiency chemicals correctly classified –
equivalence with Validated Reference Method
(VRM)
• Data reviewed favourably by the OECD’s WNT
National Co-Ordinators’ Committee and adaptation
to TG442e will be discussed by Expert Working
Group in November 2018
• Data may be used in REACH submissions
supported by in-house validation results

15. How can the results be applied?

16. Skin sensitisation test results for a cosmetic ingredient
0
20
40
60
80
100
120
140
160
180
0.000
0.500
1.000
1.500
2.000
2.500
3.000
3.500
4.000
Percentageviabilityrelativetountreated
control(greyline)
FoldInduction(pinkbars)
Test item concentration (µg/ml)
KeratinoSens™ Test Results for a Cosmetic Ingredient
DPRA RESULTS FOR A COSMETIC INGREDIENT
Classification: Sensitiser
% cysteine depletion % lysine depletion Mean % peptide
depletion
Reactivity class Classification
32.3% 11.3% 21.8% Low Sensitiser
Overall “2 out of 3” classification:
Sensitiser
KERATINOSENS™ RESULTS
FOR A COSMETIC INGREDIENT
Classification: Sensitiser

17. Case study: Building in vitro data for
safer cosmetics
• Over the past 4 years, over 1,000 safety tests
on Lush ingredients & finished products
• Genotoxicity (450 ingredients)
• Skin sensitisation (including 140 finished products)
• Skin irritation
• Eye irritation

18. Building In Vitro data
for safer cosmetics;
the problem with safety data
sheets (SDS)
“One of the frustrations for us is that
whenever we receive a Material Safety
Data Sheet from an ingredient supplier it
always contains animal test data - usually
downloaded from the internet. We have to
do better than that.“
KARL BYGRAVE, DIRECTOR, LUSH
Read more >

19. Building in vitro data for safer cosmetics
• Advantages of an animal-free safety database for safety
assessment and product development
• Greater relevance to human physiology
• Higher confidence due to extensive validation of in vitro methods
• In vitro data provides a reliable, quick and often cost-effective
prelude to testing on human volunteers
• Learning more about the predictive capacity of the tests
• Encourage suppliers to test ingredients using animal-free
methods

20. Trend towards exposure-led safety assessment
High quality information for the safety assessment process
(includes in silico; in vitro; exposure-based scenarios using
parameters such as TTC* and DST* as endorsed by RIFM;
clinical testing)
*TTC – Threshold of Toxicological Concern; DST = Dermal Sensitisation Threshold

21. Testing finished products in vitro
FOR COMPLIANCE WITH THE
COSMETICS REGULATION 1223/2009

22. Can finished products be tested in vitro?
Method Regulatory Test Guideline Suitable for finished
products?
Eye Irritation (reconstructed
human cornea)
OECD TG 492 Yes
Skin Irritation (reconstructed
human skin)
OECD TG 439 Yes
Skin Sensitisation (DPRA) OECD TG 442C Yes (only for “mixtures of
known composition”)
Skin Sensitisation
(KeratinoSens)
OECD TG 442D Yes (limitations: solubility;
cytotoxicity)
Skin Sensitisation (h-CLAT) OECD TG 442E Yes (limitations: solubility;
cytotoxicity)

23. Skin sensitisation test results
for a bath bomb
To consider: classification threshold; compare with known human in vivo responses
Overall “2 out of 3” classification: Non-Sensitiser

24. Further reading
• Getting under the skin of
in vitro skin sensitisation
testing ebook
• Topics include potency
assessment and testing
finished products
Download your copy >
Available at x-cellr8.com/in-vitro-skin-sensitisation-testing/

25. Mildness testing
USING RECONSTRUCTED SKIN MODELS TO ASSESS
TODAY’S MILDER INGREDIENTS AND FORMULATIONS

26. Human in vitro / in vivo correlation of skin
mildness data for consumer products
• Innovate UK funded 2 year project
• Collaboration with Cutest, UK
• Optimise sensitive methods for assessing mild products
and ingredients
• Directly compare in vitro data with in vivo human patch
test data
• Includes funded industry case studies (PZ Cussons,
The Body Shop)
Contact us if you are interested in a funded case study for your company

27. In vitro skin mildness test
• 3D human tissue models, grown at the
air-liquid interface
• Suitable for testing ingredients and finished
products
• Applied directly to the tissue surface; good
model of “real life” exposure
• Classifies as Severe, Moderate, Mild or
Minimal / Non-Irritant
• “ET50” values allow rank order of mildness
to be determined in comparison with other
formulations / competitor and market leading
products
0.000
20.000
40.000
60.000
80.000
100.000
120.000
0 20 40 60 80 100 120
Percentageofviabilityrelativeto
NegativeControl
Time (minutes)
ET50 Calculation: SLS
Project will include additional markers such as cytokine release

28. Skin irritation
potential (mildness)
of surfactants
Skin Irritation Result ET50 Rank Position
Non Irritating >45 hrs 1
Non Irritating >45 hrs 1
Very Mild 12-18 hrs 2
Moderate to Mild 9.36 hrs 3
Moderate to Mild 7.46 hrs 4
Moderate to Mild 6.65 hrs 5
Moderate to Mild 5.33 hrs 6
Moderate 2.90 hrs 7
Moderate 2.29 hrs 8
Moderate 2.27 hrs 9
Moderate <2 hrs 10
Moderate <2 hrs 10
Moderate 1.38 hrs 11
Moderate 0.65 hrs 12
Moderate 0.56 hrs 13
Strong/Severe <0.5 hrs 14
Strong/Severe <0.5 hrs 14
Least irritating
(mildest)
Most irritating
Same methodology can be used to test eye mildness eg baby bath products

29. Future gazing
WHAT’S ON YOUR TESTING WISHLIST?

30. “Imagination is everything.
It is the preview of life’s
coming attractions”
ALBERT EINSTEIN

31. Genomic screening: the next generation
of skin sensitisation tests?
GARD measures the responses of >200 biomarkers and can predict potency of skin sensitisers

32. Microbiome – is it a skincare trend that’s
here to stay?
In vitro methods are available and undergoing further development
Recent Global Cosmetics News podcast: https://tinyurl.com/ybvjsdba

33. Personalised testing?
The technology is available for personalised
skin models – but it’s an expensive approach.
Will they ever be in real demand?

34. Thanks to the XCellR8 team!

35. Thank you!
Dr Carol Treasure
carol.treasure@x-cellr8.com
www.x-cellr8.com