Skin Sensitisation Test: KeratinoSens™ (OECD Test Guideline 442d):
Adaptation to Animal-Free Culture Conditions
Nathalie Belota, Bushra Sima, Matthew Fielda, Carol Barker-Treasurea
a
XCellR8, Daresbury UK
Results
Introduction
Skin sensitisation is defined as an allergic response following skin contact with the
tested chemical, as defined by the United Nations Globally Harmonized System of
Classification and Labelling of Chemicals (UN GHS).
CONTACT
SENSITISER
1
KEY EVENTS IN SKIN SENSITISATION and RELATED TESTS:
1) As described in the guideline, a basic experiment for ensuring optimal luminescence
measurements in the KeratinoSensTMassay was performed to ensure reliable results
with the luminometer
Results obtained showed that all acceptance criteria were met, with results even better
when using Human Serum as compared to Foetal Calf Serum (variability was lower).
2) In accordance with the OECD Test Guideline 442D, ten specified proficiency chemicals
were then assessed for their skin sensitisation potential in animal-free adapted
conditions.
1) CONTACT (DPRA)
2
KERATINOCYTES
2) RELEASE OF PRO-INFLAMMATORY CYTOKINES BY
KERATINOCYTES (KERATINOSENSTM)
3
3) DENDRITIC CELL ACTIVATION/MATURATION (hCLAT)
INFLAMMATORY
CYTOKINES
LANGERHANS
CELLS
4) MIGRATION
5) T-CELL PROLIFERATION (LLNA)
4
MIGRATION TO LOCAL LYMPH NODE
5
LYMPHOCYTE
PROLIFERATION
T-CELL
Example of results obtained for 2 proficiency chemicals: Isopropanol, non-sensitiser on the left, no luciferase induction; EGDMA on the
right (weak sensitiser) showing a dose-dependent increase of luciferase induction, at non cytotoxic concentrations. Threshold of cytotoxicity is defined as 70% viability while threshold of sensitisation (dashed line) is defined as 1.5 of the negative control set to 1.
Results obtained after application of the 10 proficiency chemicals in 3 independent experiments. Results were calculated on the 9 values obtained
The KeratinoSensTM method addresses the second key event of the skin sensitization
adverse outcome pathway (AOP) which takes place in the keratinocytes. It induces an
inflammatory response as well as gene expression associated with specific cell
signalling pathways. The KeratinoSens™ cell line is a human keratinocyte cell line
(HaCat), containing a luciferase substrate gene that is under control of a constitutive
promoter fused with an Antioxidant Response Element (ARE) from a gene that is
known to be up-regulated by contact sensitisers. Therefore, the luciferase signal
reflects the activation by sensitisers of endogenous Nrf2 dependent genes.
The standard KeratinoSens™ test described in the guideline included the use of animalderived components in the test system, such as bovine serum in the cell culture
medium. XCellR8, aiming to provide a total animal-free test, has adapted the cell
culture to animal product-free conditions, within the parameters allowed under OECD
Test Guideline 442d.
REFERENCE COMPOUNDS
ISOPROPANOL
SALICYLIC ACID
LACTIC ACID
GLYCEROL
CINNAMYL ALCOHOL
2MERCAPTOBENZOTHIAZOLE
METHYLDIBROMO
GLUTARONITRILE
4METHYLAMINOPHENOL SULFATE
2,4-DINITROCHLOROBENZENE
EGDMA
Method
Proficiency chemicals described in the OECD guideline TG 442d were evaluated in
the KeratinoSensTM test adapted to animal-free conditions. The proficiency chemicals
covered a range of sensitisation potentials.
In brief, 12 test concentrations of each test item were evaluated in the assay, and
incubated at 37°C/5% CO2 for 48h. After 48 hours, Luminescence was assessed. For
each test item, 3 independent runs (n=3) of the test were performed. A range of
acceptance criteria must be satisfied in order for each of the 3 independent
experimental runs, and the overall test, to be considered valid. Cinnamic aldehyde
was used as the positive reference of the assay. To ensure that the prediction of
sensitisation potential is made at subcytotoxic concentrations, a cytotoxicity test
(MTT) is performed in parallel plates. Viability of the negative control was set to 100%.
Data analysis provide the following parameters:
The EC1.5 value is the lowest concentration of test item that causes a luciferase
induction greater than 1.5-fold above the negative control. If any of the test
concentrations exceed the 1.5 threshold, the test item is classified as Positive
(Sensitiser).
The IMAX value is the maximum induction achieved at any of the test concentrations.
KeratinoSensTM cell line in animalproduct free culture medium
Non sensitizer
Non sensitizer
Non sensitizer
Non sensitizer
Sensitizer
(weak)
XCELLR8
EC1.5
(µM)
XCELLR8 Prediction
IC50
XCellR8
>1000
>1000
1.324
>2000
N/A
N/A
N/A
Non sensitizer
>2000
>1000
>1000
1.723
>2000
N/A
N/A
N/A
Non sensitizer
>2000
>1000
>1000
1.705
>2000
N/A
N/A
N/A
Non sensitizer
>2000
>1000
>1000
1.650
>2000
N/A
N/A
N/A
Non sensitizer
>2000
25-175
>1000
12.25
62.5
yes
yes
yes
Sensitizer
895.48
>500
15.04
62.5
yes
yes
yes
Sensitizer
>2000
20-100
3.2
15.63
yes
yes
yes
Sensitizer
96.500
<12.5
20-100
24.3
2.74
yes
yes
yes
Sensitizer
89.333
<12.5
5-20
26.1
2.74
yes
yes
N/A
Sensitizer
15.394
5-125
>500
8.98
24.7
yes
yes
yes
Sensitizer
>2000
Sensitizer
(Moderate) 25-250
Sensitizer
(Strong)
<20
Sensitizer
(Strong)
Sensitizer
(Extreme)
Sensitizer
(weak)
Imax
from 6
-9 values
for each chemical, with a maximum of 3 outliers removed. Imax: Maximum induction value obtained, EC1.5 (µM): Minimum concentration at which
an induction of at least 1.5 is obtained; IC50: Concentration at which 50% viability is observed.
Acceptance Criteria as per OECD Test Guideline 442D:
Acceptance Criteria
TM
The KeratinoSens cell line was adapted to animal product free culture conditions
and an in-house cell bank created under the new culture conditions.
EC 1.5
IC50 reIn vivo clas- refeference
sification rence
range
range
EC1.5 value
below
At EC1.5
dose1000 µM in value, viresat least 2 ability is
ponse
of 3 repeti>70%
tions
Acceptance
Criteria 1
Acceptance
Criteria 2
Acceptance
Criteria 3
Cinnamic aldehyde
(positive control) induction above 1.5 in at least
one concentration
-Positive control EC1.5
between 7 and 30 µM
- At 64 µM, induction
value between 2 and 8
CV of Blanks <20%
Pass/Fail/See
Notes
Pass
See Note
Pass
Note
Acceptance Criteria 2: In this study, the FCS was replaced by Human Serum. Positive control showed a luciferase induction
around 1.5 at the highest tested concentration of 64 µM. To respect the acceptance criteria point 2, the top concentration was
adapted to 128 µM, i.e at 128 µM, the induction value of the positive control is above 2.
These deviations from the acceptance criteria were considered
not to have affected the integrity or outcome of the study, especially as no border-line classifications were obtained.
Conclusion
The correct classification of all 10 reference chemicals in Proficiency Testing exercise
demonstrated that the KeratinoSensTM test can be successfully performed in animal productfree conditions, providing a fully human replacement for animal tests for skin sensitisation. It
also confirms that the use of Human Serum in place of FCS does not adversely affect the
classification of any of the Proficiency Chemicals.
The KeratinoSensTM test is fully accepted at a regulatory level for the hazard identification of
skin sensitisers and non-sensitisers in accordance with the UN GHS (OECD TG 442d, February
2015). It is expected to be incorporated into REACH (Registration, Evaluation, Authorisation
and restriction of CHemicals) legislation by 2016.
XCellR8 now offers this test from its GLP accredited lab in the UK, as an ethical testing option
for compliance with a range of legislation. This work was performed with the kind support of
Lush Ltd.
References: Emter et al, Toxicol. in vitro,2013; Natsch et al, Toxicol. in vitro , 2011; Natsch et
al, Toxicol.Sci. 8676; Emter et al, Toxicol. Appl. Pharmacol., 8676