Kimberly Norman, Ph.D., DABT

October 21, 2015

Current Approaches for Assessing Eye and Skin Safety of Cosmetics Using Non-Animal Methods

Animal-free Safety Assessment for Cosmetics

• European legislation and US activities towards 21st century toxicology are setting the standard for animal-free toxicology

• Per EU Regulation 1223/2009: Animal testing of cosmetic ingredients and finished products is banned in the EU, as well as marketing of those which were tested on animals for cosmetic safety

• In US, “Toxicity testing in the 21st Century: A Vision and a Strategy”

Proposes a paradigm shift in toxicology based on an increased use of computational and in vitro systems to reduce animal use in safety testing and more accurately assess human health effects

Cosmetic Safety Assessment

• Since cosmetics do not require pre-market approval, companies may chose how to design their testing strategies

• Developing non-animal programs may be spurred by:

1. Changing regulatory landscape

2. Ethical considerations (company policy)

3. Consumer expectations

4. Need for quick/relatively inexpensive tools for screening

• In general, the non-animal methods have been developed by industry/industry consortiums

• Companies routinely use assays as:

1. Screening tool during product development

2. Prior to clinical testing

3. As stand alone test(s) for safety

Critical Endpoints for Cosmetics

• Cosmetics should be assessed for local tolerance/topical exposure damage including:

1. Eye Irritation

2. Skin Irritation

3. Skin sensitization

• Many tools may be used to build effective testing strategies for each endpoint and an overall program

Cell culture insert

Basal layer of dividing

keratinocytes

Granular layer

Stratum corneum

Courtesy of MatTek Corporation

Alternative Safety Assessment Tools

Organotypic Models 96-well cell-based assays

Reconstructed Human Tissue Models In Chemico Assays

Petsko et al.

Eye Irritation

Common Modes of Chemical Action in Eye Irritation

Cell Membrane Lysis - Surface active agents solubilize

membrane lipids

- Organic solvents extract lipids

Protein Coagulation/Denaturation - Acids and certain solvents

Saponification

- Alkali (often progressive)

Chemical Reactivity

- Reactive materials such as bleaches

and peroxides Histologic section of human cornea. 1- epithelium, 2- Bowman’s layer, 3 – Stroma; 4- Descemet’s membrane, 5- endothelium Image from eyepathology.blogspot.com

Test system: corneas isolated from bovine eyes obtained from abattoir animals

Endpoints measured: corneal opacity and permeability

Protocol: liquids (neat) and surfactants (10%) exposed for 10 min plus 2 hours post-exposure incubation; solids (20%) exposed for 4 hours without post-exposure incubation

Status: validated and regulatory accepted for identifying UN GHS Cat. 1 and No Cat., but not Cat. 2 (OECD TG 437), US EPA cat. I / II / III

Applicability and limitations: according to TG 437,

No Cat.: high FPs in general

Cat. 1: high FPs for alcohols and ketones

Cat. 1: high FNs for solids

Tutorial on the BCOP: http://ec.europa.eu/enterprise/epaa/international-activities-3rs/index_en.htm

Bovine Corneal Opacity and Permeability Assay

a) 1.5% SLS 10-minute exposure

Opacity = 1.7 OD490= 0.302

b) 5% SLS 30-minute exposure

Opacity = 7.7 OD490= 2.54

Control

Cornea

Histopathology may be used to obtain more information on the degree of damage

and depth of penetration

Cross Sections of Bovine Corneas tested in a BCOP assay

Histopathology on Corneal Tissues

Test system: confluent monolayer of SIRC cells

Endpoints measured: cytotoxicity (MTT assay)

Protocol: test chemicals exposed at 5% and 0.05% for 5 min

Status: validated and recommended for identifying UN GHS Cat. 1 and No Cat., but not Cat. 2; OECD TG 491

Applicability and limitations:

No Cat.: high FNs for highly volatile chemicals with vapour pressure > 6 kPa and non-surfactant solids

Cat. 1: high FNs in general

Not applicable to test chemicals that are not soluble or do not form stable suspension in solvent for ≥ 5 min

Short Time Exposure (STE) Test Method

Test system: non-keratinized multi-layered epithelium reconstructed from primary human epidermal keratinocytes

Endpoints measured: cytotoxicity (MTT assay)

Protocol: liquids (neat) exposed for 30 min followed by 2 h post-exposure incubation; solids (neat) exposed for 6 h followed by 18 h post-exposure incubation

Status: validated and recommended for identifying UN GHS No Cat., but not Cat. 2 nor Cat. 1; OECD TG 492

Applicability and limitations:

Applicable to all types of chemicals

Intensely coloured chemicals addressed with HPLC/UPLC-spectrophotometry

EpiOcularTM Eye Irritation Test (EIT)

Ocular Irritection®

Test system: macromolecular matrix composed of lipids, (glyco-)proteins, carbohydrates and low MW components that mimics the highly ordered structure of the cornea

Endpoints measured: turbidity at 405 nm ("opacity")

Protocol: 24 h exposure to 5 different amounts of chemical; different protocols for surfactants & non-surfactants

Status: has undergone external validation; currently under evaluation by EURL ECVAM for identifying UN GHS Cat. 1 and No Cat., but not Cat. 2

Consider the following to select the best method(s):

- physicochemical properties of the sample: liquid/solid, viscosity, pH

- solubility: some assays are only compatible with water soluble samples

- ingredient/formulation: may be modeling different exposure conditions

Explore availability of selected method(s), ensure assay performance

Ensure protocol adheres to appropriate guidance for selected method.

Alternatively, alternate protocols may be used to enhance resolution

amongst prototypes during product development.

Conduct the assay(s) with proper controls

- negative controls, positive controls, assay acceptance criteria

- concurrently tested benchmarks or references may be useful

Practical Considerations for Testing Strategy

Skin Irritation

Slides courtesy of Gertrude-Emilia Costin, Ph.D., MBA

Assessing Skin Damage

Basal layer of dividing

keratinocytes

Stratum granulosum

Stratum corneum

Native human skin

Stratum spinosum

• Vast physical barrier against

mechanical, chemical and microbial

factors

• Immune network

• Unique defense system against UV

irradiation

CORROSION

• Irreversible damage of the skin following

exposure to a test substance

• Visible necrosis through the epidermis and

into dermis - macroscopically typified by

ulcers, bleeding, etc.

http://www.survivingdisasters.info/emergency-first-aid/c/chemical-burn

IRRITATION SKIN

• Reversible damage of the skin following

exposure to a test substance

• Characterized macroscopically by erythema

(redness) and oedema

• Damage to keratinocytes and dermal

cells leads to inflammation

• Registration and labeling of chemicals

• Transport of chemicals

• Occupational safety

• Safety of cosmetics, toiletries and household products

RHE Test Method- Skin Irritation Test (SIT)

Brief overview and current regulatory status

• Test system: 3-D RHE models (EpiDermTM (EPI-200), EpiSkin™ (SM),

SkinEthic™ RHE, LabCyte EPI-MODEL24)

• Assay endpoint: tissue viability (%) - MTT

• Regulatory Status: OECD Test Guideline 439

• Applicability: The results can be used for regulatory purposes to determine the skin irritancy of test

substances either as a stand-alone replacement for in vivo skin irritation testing or as partial

replacement test within a tiered testing strategy.

• Limitation: The method does not allow the classification of test substances to the optional UN GHS

Category 3 (mild irritants).

SkinEthic™ RHE EpiDerm™ (EPI-200) EpiSkin™ (SM) LabCyte EPI-MODEL

Tissue Receipt

Upon receipt, tissues are

incubated first for 1 hr

and then over night

(with media change) in

standard culture

conditions (37+10C in a

humidified atmosphere

of 5+1% CO2 in air).

Triplicate tissues are

treated topically with

control and test

substances (30 µL dose

for liquids; 25 mg for

solids).

Tissue Treatment

Spectrophotometric

Quantification

MTT Reduction Isopropanol Extraction

After exposure, tissues are rinsed and then placed in the

incubator at standard culture conditions for an initial post-

treatment incubation of 24±1 hr. After the initial post-treatment

expression incubation, the tissues are transferred in fresh

medium and placed back in the incubator for the remainder of the

42±2 hr post-treatment incubation (to capture delayed effects of

test substances on the reconstructed tissues).

Tissue Rinsing

RHE- Irritation: Typical Protocol

Post-treatment

Expression Incubation

In vitro result In vivo

prediction

UN GHS

CATEGORY

Mean tissue viability ≤ 50% Irritant (I) Category 2

Mean tissue viability > 50% Non-irritant (NI) No Category

Time-to-toxicity assay (Non-regulatory)

Uses human 3-D RHE models

Frequently used to support the needs of Product Development, New

Technology, Innovation Groups

Several exposure times are tested, ranging usually from 1 hr to 24 hrs

Depending on the RHE model, tissues can be exposed from 10 to 100 µL

of test material

Exposure is terminated by removal of the test material in rinsing procedure

Tissue viability assessed with MTT

ET50 values are interpolated from exposure time-response curves end

evaluated (rank order of test formulations, comparison to reference

materials, in-house prediction models for specific range of products, etc.)

The correlation of the results of this

in vitro assay with expected in vivo

has not been firmly established.

However, MatTek suggests the

following as a guideline for assigning

verbal descriptors for expected in

vivo irritation based on the ET50

scores using the EpiDermTM

(EPI-200) model.

MatTek Corporation MTT Effective Time-50 (ET-50) Protocol For Use with Epiderm™ Skin Model (EPI-200) (09/06/05).

Chemical Expected in vivo irritation ET50 (hrs)

Concentrated nitric acid Strong/severe, possibly corrosive <0.5

1% Sodium Dodecyl Sulfate Moderate 0.5-4

1% Triton X-100 Moderate to mild 4-12

Baby shampoo Very mild 12-24

10% Tween 20 Non-irritating 24

CORRELATION

Time-to-toxicity assay DATA – EXAMPLE

0

20

40

60

80

100

120

0 4 8 12 16 20 24

Tis

su

e V

iab

ilit

y (

% o

f C

on

tro

l)

Exposure Time (Hours)

Sample A Sample B Sample C

Non-irritant

Moderate irritant

Severe irritant

Skin Sensitization

Chemical (hapten)

penetrates skin and reacts

with protein(s)

Mechanism of Skin Sensitization

EPIDERMIS

DERMIS

Inflammation

Increased number of chemical-specific T-

cells released into the systemic circulation

Lymph node

Mature LC presents

chemical to T cells

Chemical is recognised by

Langerhans cells which

then migrate from the skin

to the draining lymph node

This causes proliferation

of specific T cells

Subsequent skin contact with

chemical activates the T cells

and leads to clinical

manifestation

INDUCTION

ELICITATION

LLNA

D. Basketter

Skin Sensitization Endpoint Evolution

1970s 1990s <2010

In silico

In chemico

In vitro

Human patch testing

Guinea Pig

Test Methods

Local Lymph Node

Assay

ECVAM Non-animal Strategy

• Direct Peptide Reactivity Assay (DPRA)

• KeratinoSensTM Assay (ARE-Nrf2 Luciferase Test

Method)

One method at an advanced stage of pre-validation:

• Human cell line activation test (h-CLAT)

• Conclusion: Each assay considered adequately

reproducible to be considered for inclusion in an ITS for

hazard identification

• Development of AOP-based ITS for hazard

identification

OECD Test Guidelines or 2 methods (5 Feb 2015):

AOP for Skin Sensitization

Molecular Initiating

Event

Cellular

Response

Organism

Response

Organ

Response

Electrophilic

reactivity

Covalent

interaction

with proteins

Expression of

cell surface

markers and

cytokines

Proliferation

of T-cells in

lymph nodes

Dermal

inflammation

(after

challenge)

Penetration

into the viable

epidermis

Molecular

properties

Direct Peptide Reactivity Assay (DPRA)

• In chemico

• The correlation of skin protein reactivity and skin sensitization is well established

Nucleophilic-electrophilic interaction:

E

:Nu

Chemical Allergen

Protein

Protein Cysteine, Lysine

Chemical

allergen

DPRA

Un-reacted Peptide

Test Chemical

Reaction Mixture Peak Area of un-reacted peptide

is compared to peak area of

reacted peptide

• Test chemical reacted with peptide for 24 hours

• Peptide depletion monitored by HPLC

KeratinoSens Assay

Natsch et al., 2009

• A feature all skin sensitizers have in common is their intrinsic

electrophilicity, or potential to be transformed to electrophilic chemicals

• The Nrf-2-electrophile sensing pathway comprising the repressor protein

Keap1, the transcription factor Nrf2 and the antioxidant response

element (ARE) detects skin sensitizers

Keap I

H

S

Nrf2

H

S Luciferase

ARE-element

N+

O

-ON+

O

O-

Cl

KeratinoSens reporter cell line

• HaCaT (immortalized keratinocyte cell line)

• Contains a reporter construct with a copy of the ARE-

element of the human AKRIC2 gene upstream of a

luciferase gene

• Endpoint: induction of luciferase activity by allergens

Nrf2

Induction phase

Lymph node

T T

T T

allergens

Keratinocyte Activation

LC activation

T-cell proliferation

LC: Langerhans cells

Human Cell Line Activation Test (h-CLAT)

Skin Penetration

Protein Reactivity

Langerhans cells (LC) play a critical

role in skin sensitization.

Upon antigen capture, LC undergo

maturation and migrate to the draining

lymph nodes.

LC maturation is characterized by the

up-regulation of CD86 and CD54 (Aiba

and Katz, 1990; Ozawa et al., 1996).

Assay Procedure:

Cells (THP-1) cultured with 8 doses of

chemical for 24 hours, then cell

staining (CD86 and CD54), and cell

viability (PI), followed by analysis by

flow cytometry

Image from Jowsey et al.

Induction phase

Lymph node

T T

T T

allergens

Keratinocyte Activation

LC activation

T-cell proliferation

LC: Langerhans cells

In Vitro Test Strategy Along AOP

Protein Reactivity

Ch

emical

Pro

perties

Mo

lecular

Initiatin

g

Even

ts

Cellu

lar

Resp

on

ses

Org

an

Resp

on

se

DPRA

KeratinoSens

h-CLAT

Image from Jowsey et al.

Substance Human LLNA DPRA KeratinoSe

ns

LuSens mMUSST1 h-CLAT2

1-Chloro-2,4-dinitrobenzene + + + + + + +

2-Mercaptobenzothiazole + + + + + + -

4-Phenylendiamine + + + + + + +

α-Hexyl-cinnamic aldehyde + + - + - + +

Cinnamic alcohol + + + + + - +

Citral + + + + + + +

Cobalt chloride + + + + + + +

Ethylene glycol dimethacrylate + + + + + + +

Eugenol + + + + + + +

Imidazolidinyl urea + + + + + + -

Isoeugenol + + + + + - +

MCI/MI + + + + + + +

Methyl methacrylate + + + - - + +

Nickel chloride + - + - - - -

Phenyl benzoate + + + - - + +

DL-lactic acid - - - - - - -

Isopropanol - - - - - - -

Salicylic acid - - - - - - -

Sodium lauryl sulfate - + - - - - -

Xylene - + - - - - -

1 Modified MUSST 2h-CLAT using THP-1 cells obtained from DSZM

sensitizer nonsensitizer

BASF Approaches

Bauch et al., Tox in Vitro 2011

Combining the data

Assay

Accuracy 54

chemicals (Bauch

et al., 2012)

compared to

human data

Accuracy 54

chemicals (Bauch

et al., 2012)

compared to

LLNA data

Accuracy 145

chemicals

(Natsch et al.,

2013) compared

to LLNA data

Individual

assays

DPRA 87% 79% 82%

ARE reporter gene

assay; LuSens or

KeratinoSens 82% 81% 79%

U937/CD86 Test

(MUSST-like tests) 85% 74% 71%

2 of 3

DPRA, ARE-based

assay and

U937/CD86 Test 94% 83% 81%

Expanding the dataset

• Assays validated using only pure substances

• IIVS may perform research services and encourages sharing data

• Industrial int., Medical Devices, surfactants

Case Study: Botanical Cosmetic Ingredients

• Botanical extracts are common components in cosmetics

• Botanical ingredients may be complex mixtures

• Safety assessment is challenging as molar concentrations of certain components are unknown

• A cosmetic ingredient containing botanical extracts are formulated with solvents, preservatives, and generally less than 10% extract

• The botanical extract needs to be assessed as part of the complete “ingredient”

• Thus, an assay was needed to sensitively detect sensitizing components within a complex mixture

Study performed in collaboration with

Botanical Cosmetic Ingredients

• We sought to establish if the KeratinoSens assay could detect sensitizing chemicals in a botanical ingredient mixture

• We measured the activity of 3 known sensitizers neat and then “spiked” at 1%- 2% into 4 different botanical ingredients (each with a different excipient solvent system)

1. Gluteraldehyde (GA)- strong sensitizer

2. Dimethyl maleate (DM)- moderate sensitizer

3. Cinnamic aldehyde (CA) – moderate sensitizer

• No sensitizing botanical ingredients available

• Activity of the spiked samples was measured and compared to the neat sensitizer and the unspiked botanical ingredient

Study performed in collaboration with

Dose Response Curves

Botanical ingredient #2 w/o spike *No botanical ingredients showed sensitization potential w/o spike

Botanical Matrix 2 –Gluteraldehyde 2% (MTT)

Name Supplier MW EC 1.5

(µm)

Sensitization

Class

Mode of

Action

EC 1.5 Data

Source

Gluteraldehyde Sigma Aldrich

100 20 Strong Shiff Base Natsch 2008 [9]

Dimethyl

Maleate Aldrich 172 4.93 Moderate

Michael

Acceptor IIVS

Cinnamic

Aldehyde Aldrich 132 ~15 Moderate

Michael

Acceptor IIVS

Summary of Results

Study performed in collaboration with

Spike Spike Concentration

EC 1.5 µg/mL

(test material)*

IC50 µg/mL^

(MTT)

IC50 µg/mL^

(NRU)

EC 1.5 (µM)

Observed#

EC 1.5 (µM)

published

Experiment 1 Matrix 1

Gluteraldehyde 2% 308.70 >1000 >1000 61.7 20.3

Dimethyl Maleate 2% 38.00 >1000 >1000 4.4 4.93

Dimethyl Maleate 1% 120.10 >400 >400 7.0 4.93

Cinnamic Aldehyde 2% 19.24 >400 >400 7.5 10.2

Cinnamic Aldehyde 1% 120.40 >400 >400 9.1 10.2

Cinnamic Aldehyde (Pos. Control) 10.2 NA

Experiment 2

Matrix 2 2% Gluteraldehyde 356.9 >1000 >1000 71.4 20.3

Matrix 3 2% Gluteraldehyde >1000 >1000 >1000 >200 20.3

Matrix 4 2% Gluteraldehyde 501.5 >1000 >1000 100.3 20.3

Matrix 2 1% Dimethyl Maleate 133.6 >1000 >1000 7.8 4.93

Matrix 3 1% Dimethyl Maleate 467.6 >1000 >1000 27.2 4.93

Matrix 4 1% Dimethyl Maleate 203.5 >1000 >1000 11.8 4.93

Cinnamic Aldehyde (Pos. Control) 13.3 * The EC1.5 value is the concentration for gene induction above the threshold (1.5 fold) as compared to the DMSO solvent controls.

These values are the average of the values from the two definitive trials for each test material.

^ The IC50 value is the concentration at which cells are 50% viable as compared to the DMSO solvent control wells. Cell viability was

measured using two separate cytotoxicity assays (MTT and NRU).

# The EC1.5 value observed (in µM of the sensitizer) was determined by calculating the concentration of the sensitizer spike in the EC 1.5

of the test material and converting that value to µM.

Study Conclusions

• The sensitizer spike could be detected within the botanical ingredient (EC1.5 value).

• The detection of the sensitizer (EC1.5 value) was impacted by the botanical matrix in some cases.

• These initial data suggest that the KeratinoSens assay may be a useful tool for the pre-clinical safety assessment of the sensitization potential of botanical ingredients and other mixtures.

• Givaudan

• P&G

• Mary Kay

• The Clorox Company for funds for IIVS participation in KeratinoSens

ring trial

• Colgate-Palmolive Company for funds for IIVS participation in

KeratinoSens ring trial

• IIVS lab team

Acknowledgements