In this presentation, first given at the SCS Science in a Bottle conference 2018, we share the results of our latest in vitro skin sensitisation tests, discuss whether finished products or mixtures can be tested in vitro, reveal progress towards a new model for predicting mildness to skin and how we used mechanistic data to assess antioxidant activity.

1. Future Gazing: Where Next for
In Vitro Testing?
Dr Carol Treasure
Founder & CEO, XCellR8 Ltd
carol.treasure@x-cellr8.com
@XCellR8_Labs

2. About XCellR8
• Founded 2008 by Dr Carol
Treasure and Bushra Sim
• Our Mission: To accelerate the
world’s transition to 100% animal-
free testing through our
scientifically advanced and ethical
approach

3. Are “Alternatives” Still Alternative?!
Some “alternatives” are now “mainstream” and adopted as the default
test methods for regulatory safety testing
• Extensive validation trials (enhanced human safety)
• High level of confidence internationally
• Large amount high quality of data

4. 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
Uptake by industry is the key to the future!

5. Shifting the emphasis:
From:
“No animal testing”
to:
“How do you test your products (and ingredients)?”

6. What is truly animal-free testing?

7. Why Animal-Product-Free (APF)?
• Scientific advantages:
• Better model of human physiology
• Ethical advantages:
• Avoids animal welfare issues, eg Foetal
Bovine Serum (FBS) collection
• Regulatory progress:
• APF adaptations of KeratinoSens™ and
h-CLAT sensitisation tests
• Driven by demand:
• Leading UK cosmetic companies
Everything we do at XCellR8 is animal-product-free

8. Skin Sensitisation – some latest work

9. Skin Sensitisation –
Background Biology
Regulatory guidance: “2 out of 3” approach

10. 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
%
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

11. How can the results be applied?

12. 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
https://uk.lush.com/article/fighting-animal-testing-thousand-times-over-lush-and-
xcellr8-continue-champion-safe-humane

13. Building In Vitro Data for Safer Cosmetics
• What is the problem with Safety Data Sheets (SDS)?
• Currently, almost all data on SDS is from historical animal studies
• Traceability: much data has been passed along through multiple
sources online
• The relevance to real-life human exposure is commonly questioned,
eg ECVAM strategy on acute systemic toxicity, Prieto et al 2014:
http://publications.jrc.ec.europa.eu/repository/bitstream/JRC90611/reqno_jrc9061
1_eurl%20ecvam%20strategy%20to%20replace%20reduce%20and%20refine%2
0the%20use%20of%20animals%20in%20the%20assessment%20of%20acute%2
0mamm.pdf
• Despite the risks, this data is still in widespread use as a critical part
of safety assessment for cosmetic ingredients.

14. 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

15. Building In Vitro Data for Safer Cosmetics
• Advantages of a fully in vitro safety database for safety
assessment and product development:
• Greater relevance to human physiology
• Higher confidence due to extensive validation of in vitro methods
• Supporting information for the safety assessment process
• Prelude to testing on human volunteers
• Learning more about the predictive capacity of the tests.
• Encourage suppliers to test ingredients using animal-free
methods

16. Testing finished products in vitro

17. 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)

18. Skin Sensitisation Test Results
for a Bath Bomb

19. eBook:
Getting under the skin of in vitro skin
sensitisation testing
Available at:
https://x-cellr8.com/in-vitro-skin-
sensitisation-testing/

20. Mildness testing:
Using reconstructed skin models to assess today’s
milder ingredients and formulations

21. Human in vitro / in vivo correlation of skin
mildness data for consumer products
• Innovate UK funded 2 year project
• Collaboration with Cutest
• 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)

22. 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

23. 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

24. Mechanistic data:
Case study: antioxidant testing

25. Antioxidant Activity
• Antioxidant activity = body’s natural defence against
reactive oxygen species (ROS) – by-products of
metabolism which can be harmful to cells.
• ROS levels in skin increased by many factors including
stress, ageing, air pollution.
• Anti-oxidant cosmetic ingredients may support a variety
of claims.
https://x-
cellr8.com/2017/12/08/focus-
one-cosmetic-claim-2018/
Case study
available
on our website as a
white paper

26. OxiSelect™ Antioxidant Test
• Measures antioxidant activity within cultured human cells - accounts for
pH, uptake, metabolism and efficacy of the test item
• Captures all major types of ROS (including hydroxyl, peroxyl,
superoxide, singlet oxygen)
Human epidermal
keratinocytes in
animal product-free
culture

27. In vitro assessment of the antioxidant activity of 5
natural extracts in the development of an anti-
ageing skincare formulation
• 5 natural extracts assessed alone and in combination to establish
any possible synergistic effects.
• 2 test concentrations (0.01% and 0.05%) – determined by
cytotoxicity pre-screen and discussion with client.
• 10 combinations placed into rank order of anti-oxidant activity in
human epidermal keratinocytes in animal-product-free culture.

28. Case Study: Antioxidant Activity

29. Case Study: AntiOxidant Activity
Test Item Anti-oxidant activity (% of negative control) Rank order of activity (1 =
highest)
Test concentration (% in cell culture medium)
0.01% 0.05%
A 81.93% 47.12%** 8
B 43.75%** 29.50%*** 2
C 69.88% 40.57%** 7
D 61.88%* 36.41%*** 6
E 91.91% 49.26%** 9
A+B 66.31%* 32.40%*** 3
A+C 84.82% 53.35%** 10
A+B+C 66.18%* 34.02%*** 4
A+B+D 55.90%** 34.10%*** 5
A+B+E 40.07%*** 22.58%*** 1
* p<0.05, ** p<0.01, *** p<0.001

30. Quick fire round! – more future gazing…..

31. Trend towards exposure-led safety
assessment

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

33. Microbiome

34. Personalised testing???

35. “Imagination is everything. It is the
preview of life’s coming attractions”
Albert Einstein

36. 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

37. Thanks to the XCellR8 team!

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