This presentation, originally shared at the Chemical Hazards Communication Society in 2018, discusses the regulatory status of in vitro safety assessments for REACH, CLP Regulation and Biocidal Products Regulation, gives an overview of current key methods for skin irritation, genotoxicity and acute toxicity, shows how finished mixtures or products can be tested, how to interpret results and barriers to progress towards an entirely animal-free testing strategy.

1. Advances in In Vitro Testing for Regulatory
Compliance in the Chemical Industry
CHCS 14th November 2018
Dr Carol Treasure
Founder & CEO
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

3. 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”)

4. • Context: regulatory status of in vitro methods
• Current key methods – overview
• Testing finished products
• Interpretation of results
• Barriers to progress
• A positive future!
What we’ll cover today

5. Context
REGULATORY STATUS OF IN VITRO METHODS

6. Where do in vitro tests fit into the safety
assessment process?
Literature review
In silico / read-across
Identify data gaps / formulate testing strategy
In vitro / in chemico tests Animal tests
If permissible and /
or required by
regulators eg ECHA

7. REACH – human health endpoints
• Mutagenicity (mammalian cells) or in
vitro micronucleus test
• Skin irritation*
• Eye irritation*
• Genotoxicity*
• Inhalation acute toxicity
• 28-day repeat dose toxicity
• Reproductive / developmental toxicity
*
Regulatory in vitro tests available / not available
Current status:
Read-across extensively used for “higher-tier” endpoints where in vitro tests not currently available
*In vivo tests permissible only if classification not obtained using in vitro methods
• Skin corrosion
• Skin irritation
• Eye irritation
• Skin sensitisation
• Mutagenicity (bacteria)
• Oral acute toxicity
Additional for Annex VIII
(10 -100 tonnes p/a)
Annex VII
(1-10 tonnes p/a)

8. • Adaptation of standard testing requirements
• In vitro tests:
• Can be non-regulatory
• Validation essential
• Adequate and reliable documentation required
• Scientifically robust – suitable for risk assessment, classification and labelling
• Weight-of-evidence
• Newly developed or equivalent test methods
REACH Annex XI
Adaptation of standard testing regimes

9. • Based on UN GHS system
• Testing requirements take lead from REACH
• Further guidance from IATA
• Non-animal approaches include:
• Chemical properties
• In silico tools (eg QSAR)
• Read-across
• Regulatory in vitro methods
• New (regulatory) in vitro tests including
genomics, with sufficient information and
justification
Other key regulations (Europe)
• Testing requirements take lead from REACH
• Further guidance from IATA
• In vitro tests should always be the starting
point
• Animal tests = last resort
*In vivo tests permissible only if classification not obtained using in vitro methods
CLP Regulation
Classification, Labelling and Packaging
Regulation EC 1272 / 2008
BPR Regulation
Biocidal Products Regulation
EC 528 / 2012

10. Options used by REACH registrants
Source: ECHA summary report, 2017 Source:
ECHA summary report, 2017

11. Overview
CURRENT KEY METHODS

12. Skin irritation: OECD TG 439 – EpiDerm™
• Chemical applied to the skin surface (x3): 30µl liquid or 25mg solid
• Negative control: phosphate buffered saline. Positive control: 5% SDS
• Incubation for 60 minutes at 37°C / 5% CO2
• Chemical / controls removed by washing
• Incubation for 42 hours (“recovery period”)
• Viability assessed by MTT conversion (healthy cells metabolise to purple formazan product,
detected by absorbance at 570nm)
• Absorbance readings expressed as % of negative control
• Viability < 50%: Skin Irritant (GHS Category 2 / R38)
• Viability >50%: Non-Irritant (No Category) (includes Optional Category 3 – Mild Irritant)
Additional testing requirement is rare in Europe. IATA published 2014

13. 0.0
20.0
40.0
60.0
80.0
100.0
120.0
140.0
NC1 NC2 NC3 PC1 PC2 PC3 TA1 TA2 TA3 TA4 TA5 TA6 TA7 TA8 TA9 TA10
Percentageofviabilityrelativetonegativecontrol
NC = negative control
PC = positive control
TA1 = naphthalene acetic acid
TA2 = isopropanol
TA3 = methyl stearate
TA4 = heptyl butyrate
TA5 = hexyl salicylate
TA6 = cyclamen aldehyde
TA7 = 1-bromohexane
TA8 = potassium hydroxide
(5% aqueous)
TA9 = 1-methyl-3-phenyl-1-
piperazine
TA10 = heptanal
Non-Irritant
Irritant
Skin irritation prediction of 10 proficiency chemicals
(OECD TG 439)

14. Skin corrosion: OECD TG 431 - EpiDerm™
• Chemical applied to the skin surface (x3): 30µl liquid or 25mg solid
• Negative control: phosphate buffered saline. Positive control: potassium hydroxide
• Incubation for 3 minutes and 60 minutes at 37°C / 5% CO2
• Chemical / controls removed by washing
• No recovery period
• Viability assessed by MTT conversion (healthy cells metabolise to purple formazan product, detected by
absorbance at 570nm)
• Absorbance readings expressed as % of negative control
• Viability < 50% after 3 minutes: Corrosive (GHS Category 1)
• Viability > 50% after 3 minutes but < 15% after 60 minutes: Corrosive (GHS Category 1)
• Viability >50% after 3 minutes and 60 minutes: Non-Corrosive (No Category)
DIFFERENCES FROM SKIN IRRITATION METHOD TG 439
Further testing may be required for sub-categorisation

15. Skin corrosion: OECD TG 431
STEP 2 FOR CHEMICALS IDENTIFIED AS CORROSIVE IN INITIAL TEST
EpiDerm™
• Viability < 25% after 3 minutes:
Optional Sub-Category 1A
• Viability > 25% after 3 minutes:
a combination of Optional Sub-
Categories 1B or 1C
Corrositex™
• Qualify test chemical (colour change) / check pH
• Add chemical to detection solution: warm to
70°C for 20 minutes
• Add to biomembrane discs
• Incubate overnight at 2-8°C
• Add to detection solution and categorize
• Add biomembrane to detection solution and
classify packing group
• Distinguish between sub-categories 1B and 1C
ECHA: no further testing required “if the results are adequate”

16. Eye irritation: OECD TG 492 - EpiOcular™
• Chemical applied to the skin surface (x3): 50µl liquid or 50mg solid
• Negative control: phosphate buffered saline. Positive control: methyl acetate
• Incubation for 30 minutes (liquids) or 60 hours (solids) at 37°C / 5% CO2
• Chemical / controls removed by washing
• No recovery period
• Viability assessed by MTT conversion (healthy cells metabolise to purple formazan product,
detected by absorbance at 570nm)
• Absorbance readings expressed as % of negative control
• Viability < 60%: Eye Irritant (No categorisation currently permitted)
• Viability > 60%: Non-Irritant (No Category)
DIFFERENCES FROM SKIN IRRITATION METHOD TG 439
EpiOcular™
For chemicals “not requiring classification and labelling for eye irritation or serious eye damage”

17. Eye irritation IATA*
* Integrated Approach on Testing and Assessment
BCOP = Bovine Corneal Opacity and
Permeability Test (TG 437)
ICE = Isolated Chicken Eye (TG 438)
STE = Short Time Exposure (TG 491)
FL = Fluorescein Leakage (TG 460)

18. Eye irritation IATA (Part 3) EpiOcular™
BCOP
ICE
STE
BCOP (TG 437)
ICE (TG 438)
STE (TG 491)
FL (TG 460)

19. CON4EI project
IMPROVED “IN VITRO”
STRATEGIES FOR PREDICTING
EYE IRRITATION POTENTIAL
• Aim: develop tiered testing strategy for the complete
replacement of OECD TG 405 (Draize test – rabbit eye)
• International consortium (Europe / US)
• 80 reference chemicals; 7 test methods
• Proposed 3 strategies:
• Stand-alone: EpiOcular™ ET50
• 2-tier (bottom-up): EpiOcular™ to BCOP LLBO*
• 3-tier (bottom-up): EpiOcular™ to BCOP “OP-KIT” SMI*
• Strategies successfully identified:
• 71.1% - 82.9% GHS Category 1 chemicals
• 64.2 – 68.5% Category 2 chemicals
• >80% No Category chemicals
• Promising for weight-of-evidence approaches and for
future regulatory acceptance of EpiOcular™ ET50
*BCOP LLBO = Bovine Corneal Opacity & Permeability Laser Light Based Opacitometer; SMI = Slug Mucosal Irritation
Note: stand-alone EpiOcular™
ET50 is the only truly in vitro /
animal-free approach

20. EpiOcular™ ET50 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 irritation 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

21. 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)

22. Direct Peptide Reactivity Assay
(DPRA): OECD TG 442c
• Models protein binding to the cell surface (Key Event 1)
• Test item incubated with cysteine or lysine peptides for
24 hours
• Peptide depletion measured by High Performance
Liquid Chromatography (HPLC)
%
Cysteine/Lysine
Depletion
Classification Reactivity Class
Non-
Sensitiser Minimal
Sensitiser Low
Sensitiser Moderate
Sensitiser High
0
100
• Validation data available using 15 Proficiency
Chemicals as per OECD TG 442c
• Validated for hazard identification but also
gives preliminary indication of potency

23. KeratinoSensTM test for skin sensitisation:
OECD TG 442d
Principle of the test
• Models Skin Cell Activation (Key Event 2)
• Sensitisers activate Anti-Oxidant Response
Element (ARE)
• Luciferase gene inserted into the cells,
linked to the ARE
• In the presence of a skin sensitiser, the ARE
is switched on and luciferase converts an
added substrate to produce emission of a
light signal
0
20
40
60
80
100
120
140
0.0
0.5
1.0
1.5
2.0
2.5
3.0
8 16 32 64 128
Viabilityrelativetountreatedcontrol
FoldInduction
Test item concentration (µM)
Skin sensitisation evaluation (KeratinoSens) of
positive control
Cinnamic Aldehyde
• EC1.5 = lowest concentration where the luciferase-linked gene was induced >1.5-fold (the
threshold for classification as a sensitiser)
• IMAX = maximum induction of the luciferase-linked gene. IMAX values can be used as a preliminary
comparison of the differences in skin sensitisation potential between products (potency)

24. Human Cell Line Activation
Test (h-CLAT) for skin
sensitisation: OECD TG 442e
THP-1 Cell
• Models Dendritic Cell Activation (Key Event 3)
• 24 hour exposure of the human monocytic leukaemia
cell line (THP-1)
• Detects changes in cell surface markers (CD54
and CD86)
• Measurement by flow cytometry following cell staining
with fluorochrome-tagged antibodies
• Upregulation above defined threshold results in
classification as a sensitiser

25. Skin sensitisation tests - regulatory status
• Regulatory expectation: 2 out of 3 approach to classify
• Skin Sensitiser (GHS Category 1)
• Non-Sensitiser (GHS No Category)
• Potency data may be needed to sub-divide sensitisers into Category 1A (high frequency
/ potency) or 1B (low to moderate frequency / potency)
• Non-animal tests must be used as a first tier (since 2016) but…
• Follow-up animal tests may be required if in vitro and in chemico tests don’t give
conclusive results
• Animal tests may be requested by ECHA for potency data
• In vitro tests under validation for potency:
• Existing tests
• New: GARD POTENCY
• Cosmetic ingredients: animal tests permissible when assessing occupational
exposure for manufacturing workers

26. 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/

27. 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

28. Genotoxicity testing – regulatory in vitro methods
• Current in vitro regulatory methods
• Bacterial reverse mutagenicity (Ames test): OECD TG 471
• Detects mutagens only but…
• Doesn’t reflect eukaryote-specific mutagens
• Micronucleus test: OECD TG 487
• Detects clastogens and aneugens only
• V79 cells don’t express p53
• Chromosome aberration test: OECD TG 473
• Detects clastogens only
• None detect all major classes of genotoxins*
• High incidence of false positives
*Mutagens: change DNA sequence. Clastogens: break / damage chromosomes. Aneugens: change number of chromosomes

29. Genotoxicity testing
Non-regulatory
weight-of-evidence
approaches
Source:
Criteria for the RIFM evaluation process for fragrance materials.
Api et al (2015). Food and Chemical toxicology 82: S1-S19

30. Genotoxicity testing
Non-regulatory weight-of-evidence approaches
BlueScreen™
• Human TK6 cell line: p53 competent: reduced
false positives (Fowler et al, 2014. Mutation
Research 767: 28-36)
• Mutagens, aneugens, clastogens
• Detects changes in a stress pathway (GADD45a)
that is activated in human cells in the presence of
genotoxins
• Cells contain a Luciferase gene that converts an
added substrate to a light-emitting product when
the stress pathway becomes activated
• Extensive validation across industry
(Etter et al, 2015. Toxicol. In Vitro 29: 1425-1435)
BlueScreenTM Cells (human white blood
cell line): growing in suspension in
animal-product-free conditions

31. BlueScreenTM Animal-free test: sample data
SAMPLE ID AND
RESULTS
CYTOTOXICITY GENOTOXICITY
2 Result LEC Result LEC
- -
mg/ml mg/ml
Ref. No.
Concentration 0.05 mg/ml (Units)
Methylparaben
NEGATIVE NEGATIVE
L1051
CONTROLS
CYTOTOXIC CONTROLS GENOTOXIC CONTROLS
CELL LINE CELL DENSITY RESULT CELL LINE GLUC INDUCTION RESULT
HIGH LOW HIGH LOW
GenM-Gluc GenM-Gluc
PASS29.2 74.5 PASS 10.88 2.44

32. Acute toxicity screen
• Key regulatory safety requirement for REACH
• Animal tests (variants of LD50) still in use – widely discredited both scientifically and ethically
• XCellR8’s animal-free screen now in use by cosmetic companies to build a weight-of-evidence
• Validated (intra-laboratory) for 20 cosmetic ingredients
• Prediction model correlates in vitro IC50 value with predicted in vivo LD50 value and GHS Class for
oral acute toxicity
• In use as a non-regulatory screen; provides supporting information for regulatory submissions
Day 1
Cell seeding
Day 2
Dosing: 8
concentrations
Day 3
Cell viability evaluation

33. 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)

34. Interpretation of results

35. Interpretation of results
• Challenge
• Tests originally developed for hazard identification (“yes/no answer”)
• Trend towards more integrated approach
• Systemic exposure (including cosmetics)
• Exposure-led safety assessment
• This is not a new problem!
• Animal tests modelled systemic exposure but…
• Significant differences to humans eg metabolism
• Extrapolation dilemmas have switched!

36. Examples of exposure-led safety assessment
2 LEADING PERSONAL CARE COMPANIES
• Tiered approach
• Tier 1: in silico and weight-of-evidence;
formulate a hypothesis; testing
• Tier 2: in vitro screening*
• Tier 3: complex systems eg 3D human
tissue models
• *Pharmaceutical style, DMPK
• Ex vivo human skin absorption testing to
understand dermal kinetics
• Determine metabolic pathways
• Extracellular models (human liver S9)
• Intracellular metabolism (cell cultures)
• “We have had to take a leap of faith away
from animal models and have given up
relating back to animal data – we always use
the best new science available”
• Tiered approach
• Tier 1: in silico and weight-of-evidence;
formulate a hypothesis; testing
• Tier 2: in vitro screening
• Tier 3: clinical studies
• If in doubt, assume 100% skin absorption
(“worst case scenario”)
• Use historical animal data to calculate
systemic exposure and Margin of Safety
• In vitro studies determine hazards and limit of
exposure eg for contact allergens (Dermal
Sensitisation Threshold, DST, similar to TTC)
• Follow up with clinical studies to confirm in
vitro hypothesis (approach minimises human
risk)
COMPANY A COMPANY B

37. Barriers to progress

38. Barriers to progress
Standardised prediction models to relate test results to real-life
exposure scenarios
• Large companies working in isolation to look at systemic exposure.
Margin of safety etc
• Lack of good in vitro models for skin absorption
• Need models for repeat dose / chronic exposure (industry initiatives)
• Flawed benchmarks for validation
• Animal data provides an unreliable “gold standard”
• Recognition and shift to human-based prediction models
• Industry uptake / confidence
• Increased understanding of in vitro science needed
• Validation and regulatory approval lags behind technology
• eg GARD POTENCY
• Comfort zone / acceptance of status quo can limit resources made available
• Cosmetic industry progress

39. 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

40. Future gazing
• Global markets:

41. 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 – improved human safety

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

43. Conclusions

44. 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

45. Thanks to the XCellR8 team!

46. Thank you!
Dr Carol Treasure
carol.treasure@x-cellr8.com
www.x-cellr8.com
@XCellR8_Labs Dr Carol Treasure