Research Fragrance Chemical TM 11-0078 

Research Fragrance Chemical TM 10-0210 

Research Fragrance Chemical TM 10-0367 

Research Fragrance Chemical TM 09-0536 

Research Fragrance Chemical TM 11-0230 

Research Fragrance Chemical TM 08-0338 

09-0035 05-0293 

Authors 

Kimberly Norman, Ph.D., DABT 

Monica Krcha, B.S. 

Study Completion Date 

26 June 2014 

Performing Laboratory 

Institute for In Vitro Sciences, Inc. 

30 W. Watkins Mill Road, Suite 100 

Gaithersburg, MD 20878 

Study Number 

13AL98-AM03, AN30-AN31.170001 

Laboratory Project Number 

7560 

Page 1 of 16 
DEPENDENT GENE ACTIVITY AND CYTOTOXICITY (USING MTT) IN THE 

KERATINOCYTE ARE-REPORTER CELL LINE KERATINOSENS 

INTRODUCTION ............................................................................................................... 6 

MATERIALS AND METHODS ........................................................................................ ? 

APPENDIX A 

SP170001 (PROTOCOL) .............................................................................................. 1-17 

PROTOCOL ATTACHMENT 1 ......................................................................................... 1 

APPENDIX B (DOSE-RESPONSE CURVES) ..................................................................... B1-B3 

Project 7560, Final Report 2 
Completion Date: 

Sponsor: 

Sponsor's Representative: 

Testing Facility: 

Archive Location: 

Study Director: 

Laboratory Manager: 

Laboratory Supervisor: 

Project 7560, Final Report 

26 June 2014 

International Flavors & Fragrances Inc. 

800 Rose Lane 

Union Beach, NJ 07735 

Paul Ribeiro, Ph.D., Joan Cheng Ph.D., DABT 

Institute for In Vitro Sciences, Inc. 

30 W. Watkins Mill Road, Suite 100 

Gaithersburg, MD 20878 

Institute for In Vitro Sciences, Inc. 

Gaithersburg, MD 20878 

Nathan R. Wilt, B.S. 

Allison Hilberer, M.S. 

3 

; Ultl.-lC,1 UJ!tf 

Date 
TEST ARTICLE RECEIPT 

IIVS Test Sponsor's Physical Storage Article Receipt Date Designation Description Conditions * Number 

13AL98 Research Fragrance clear colorless 25 Nov 2013 2°C- 8°C Chemical TM 11-0078 non-viscous liquid 

13AL99 Research Fragrance clear colorless 25 Nov 2013 2°C- 8°C Chemical TM 10-0210 non-viscous liquid 

13AMOO Research Fragrance clear colorless 25 Nov 2013 2°C- 8°C Chemical TM 10-0367 non-viscous liquid 

13AM01 Research Fragrance clear colorless 25 Nov 2013 2°C- 8°C Chemical TM 09-0536 non-viscous liquid 

13AM02 Research Fragrance white powder 25 Nov 2013 2°C- 8°C Chemical TM 11-0230 

13AM03 Research Fragrance cloudy white 25 Nov 2013 room Chemical TM 08-03 3 8 semi-viscous liquid temperature 

13AN30 09-0035 clear colorless 14 Jan 2014 room non-viscous liquid temperature 

13AN31 05-0293 clear colorless 14 Jan 2014 room non-viscous liquid temperature 

* - Protected from exposure to light 

Project 7560, Final Report 4 
INDUCTION OF ANTIOXIDANT -RESPONSE-ELEMENT 

DEPENDENT GENE ACTIVITY AND CYTOTOXICITY (USING MTT) IN THE 

KERATINOCYTE ARE-REPORTER CELL LINE KERATINOSENS 

Project 7560, Final Report 5 
transcription factor Nrf2, which binds to the antioxidant I electrophile response element (ARE I 

EpRE), was shown to be a valuable cellular endpoint to detect skin sensitizers in vitro1·2• The 

induction of luciferase directly indicates the activation of ARE-dependent genes. Additionally, the 

cytotoxicity of a test article was assessed using cell viability endpoints with MTT (3-[4,5 -

dimethylthiazol-2-yl] - 2,5 - diphenyltetrazolium bromide) and Neutral Red vital dyes. 

Cytotoxicity was determined by measuring the relative conversion of MTT and Neutral Red 

uptake in test article-treated cultures compared to the solvent control. 

The experimental design of this study consisted of three definitive assays to determine the 

maximal induction (lmax), the concentration for maximal gene induction (Cimax), the EC1.5 value 

(concentration for an induction of 50% above solvent controls), and a mean IC50 (concentration 

leading to 50% viability as compared to solvent controls) for the test articles. For each definitive 

assay, the KeratinoSens cells were cultured in quintuplicate plates for 24 hours, treated with the 

test articles for 48 hours, and assessed for luciferase induction (3 plates) and cytotoxicity (2 

plates). The procedures that were performed in this assay were a modification of the procedures 

previously described by Natsch, et al. (2008) and were performed similar to those procedures 

performed by the Institute for In Vitro Sciences, Inc. in the KeratinoSens ring-study3. 

The Induction of Antioxidant-Response-Element Dependent Gene Activity in the 

Keratinocyte ARE-Reporter Cell Line KeratinoSens Assay was performed to determine the skin 

sensitization potential of the test articles, supplied by International Flavors & Fragrances Inc. The 

laboratory phase of this study was conducted from 8 January 2014 to 31 January 2014 at the 

Institute for In Vitro Sciences, Inc. (IIVS). 

1 Natsch, A. and Emter, R. (2008) Skin sensitizers induce antioxidant response element dependent genes: Application 

to the in vitro testing of the sensitization potential of chemicals. Toxicological Sciences. 1 02( 1 ): p. 110-119. 

2 Natsch, A., Emter, R., and Ellis, G. (2009) Filling the concept with data: Integrating data from different in vitro and 

in silico assays on skin sensitizers to explore the battery approach for animal-free skin sensitization testing. 

Toxicological Sciences. 107(1): p. 106-121. 

3 Natsch, A., Bauch C., Foertsch, L., Gerberick, F., Norman, K., Hilberer, A., Inglis, H., Landsiedel, R., Onken, S., 

Reuter, H., Schepky, A., and Emter, R. (2011) The intra- and inter-laboratory reproducibility and predictivity of the 

KeratinoSens assay to predict skin sensitizers in vitro: Results of a ring-study in five laboratories. Toxicology in Vitro. 

25(3): 733-774 

Project 7560, Final Report 6 
9.1% FBS and 500 G418. Batches of 1% FBS DMEM were prepared by supplementing 

DMEM with 1% FBS. The Dulbecco's Phosphate Buffered Saline without calcium and magnesium 

(CMF-DPBS) (Gibco) and EDTA powder (Sigma) were stored at room temperature. Batches of 

CMF-DPBS containing 0.05% EDTA, used for cell passaging, were prepared by supplementing the 

CMF-DPBS with a 10% EDTA solution. The 0.05% Trypsin/EDT A solution (Gibcollnvitrogen), 

lOX stock solution of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) 

(Sigma), and Dimethylsulfoxide (DMSO) (Sigma) were stored at -20 ± 5°C. The lyophilized 

substrate of the ONE-GloTM assay kit (Promega) and the ONE-Glo assay bufferTM were stored at -

20 ± 5°C. The ONE-GloTM reagent was prepared by adding the appropriate volume of reconstitution 

buffer to the lyophilized substrate. The 3.3 mg/mL Neutral Red stock solution, prepared from Neutral 

Red powder (Sigma) at IIVS, was stored at 2-8°C, protected from light. The Dulbecco's Phosphate 

Buffered Saline (DPBS) (Quality Biological) containing calcium and magnesium, used for the 

Neutral Red endpoint, was stored at 2-8°C. Upon receipt of cryopreserved KeratinoSens cells 

(Givaudan), the vial of cells was stored in a liquid nitrogen freezer. The remaining reagents were 

stored at room temperature. 

Cell Thawing Procedure 

A cryovial of the cryopreserved KeratinoSens cells was thawed in a water bath at 

approximately 37°C (in a beaker containing 70% ethanol) for as brief a time as possible. Once 

thawed, the cryovial was immediately decontaminated with 70% ethanol and placed in a laminar 

flow hood. The cells were added to a sterile conical tube and diluted by slowly adding 10 mL 

pre-warmed Assay Medium. The cells were then centrifuged at approximately 200 x g for 

5 minutes at room temperature. The supernatant was aspirated, the pellet was resuspended in 

approximately 15 mL of fresh pre-warmed Assay Medium, and then transferred into a T75 tissueculture flask. The flask was then incubated at 37 ± 1 °C, 90 ± 10% humidity, and 5.0 ± 1% C02 in 

air (standard culture conditions), until the KeratinoSens cells reached -60% to 90% confluence. 

Routine Culturing of the KeratinoSens Cells 

When the cultures reached -60 to 90% confluence, they were removed from the flask by 

trypsinization. The medium was aspirated and the cell sheet rinsed twice with approximately 

10 mL of CMF-DPBS containing 0.05% EDTA. One mL of trypsin/EDT A was added to cover the 

cell sheet. The flask was then placed into the incubator and incubated at standard culture 

conditions for 6-8 minutes, or until the cells became dislodged. When more than 50% of the cells 

became dislodged, the flask was rapped sharply against the palm of the hand. Then approximately 

7 mL of Maintenance Medium was added to each 7 5 cm2 flask to obtain a single cell suspension 

and cells passaged at appropriate densities. The KeratinoSens cells were routinely passaged every 

2-4 days. 

Project 7560, Final Report 7 
Three white-walled and two transparent plates were seeded for the test article replicate set 

for each assay. The plates were incubated for approximately 24 hours at standard culture 

conditions. The plate cultures seeded into the clear plates were examined under a phase contrast 

microscope and evaluated for uniform seeding and confluence prior to treating the cells with the 

test article or positive control. 

Solubility Determination 

The Sponsor specified that the test articles were soluble in DMSO, and therefore DMSO 

was selected as the solvent to dissolve and dilute the test articles. For each test article, the lOOX 

top stock prepared in DMSO was described as a clear colorless non-viscous solution. 

MTT Direct Reduction Test 

The ability of the test articles to directly reduce MTT was assessed at the same time of test 

article treatment in the definitive assays. A 1.0 mg/mL MTT solution was prepared by dissolving a 

10 mg/mL stock solution of MTT into warm MTT Addition Medium. Approximately 100 ~L of the 

lOOX test article concentration in DMSO was added to 1 mL of the MTT solution and then incubated 

in the dark at 37°C for one to three hours. One hundred ~L of a negative control (e.g. DMSO) was 

tested concurrently. If the MTT solution color turned blue/purple, the test articles were presumed to 

have reduced the MTT. The test article, Research Fragrance Chemical TM 11-0230, was found to 

directly reduce MTT. The test articles, Research Fragrance Chemical TM 11-0078, Research 

Fragrance Chemical TM 10-0210, Research Fragrance Chemical TM 10-0367, Research Fragrance 

Chemical TM 09-0536, Research Fragrance Chemical TM 08-0338, 09-0035, and 05-0293, did not 

directly reduce MTT. 

Controls 

Each assay plate included a range of doses of the positive control, Cinnamic Aldehyde 

(Sigma). A 100X concentration of positive control was prepared by weighing approximately 

84.6 mg of Cinnamic Aldehyde into a prelabeled conical tube. Ten mL of DMSO were added to 

prepare a 64,000 ~M dilution. The 64,000 ~M dilution was further diluted 1:100 in DMSO to 

prepare a 6400 ~M 100X stock concentration. The final 1X concentrations of the positive control 

were 64, 32, 16, 8, and 4 ~M. The solvent control for the test articles and the positive control was 

DMSO (to a final concentration of 1%) in 1% DMEM. Each plate included a set of 6 DMSO 

solvent control wells. 

Project 7560, Final Report 8 
Definitive Assays 

The test articles were tested in three definitive assays. Each definitive assay included a set 

of 4 plates (3 for gene induction, 1 for cytotoxicity assessment). Each plate contained up to seven 

test articles with a range of 12 dosing concentrations for each test article. Each plate also included 

5 wells designated for the positive control (tested over a range of 5 dosing concentrations), 6 wells 

designated as the DMSO solvent control, and 1 well which was left blank (See Figure 1). 

Each definitive assay was performed independently: 1) The plates for each definitive assay 

were seeded using separate flasks of cells by different individuals; 2) A separate 4X Assay Master 

plate was prepared for each definitive assay by different individuals; 3) Three different individuals 

dosed each set (5 total plates) of definitive assay plates; and 4) Separate batches of MTT and 

neutral red were prepared for each definitive assay. 

After at least 22 hours of incubation of the cells, the Assay Medium was removed from the 

cells. The plates were decanted and gently blotted on sterile paper towels. One hundred and fifty 

microliters of fresh pre-warmed 1% DMEM were added to all wells, including the blank. The 

plates were returned to the incubator until the dosing was initiated. 

For each test article, twelve decreasing doses were selected for the assay (test articles were 

diluted up to a final concentration of 200,000 in DMSO). For the positive control, 5 decreasing 

doses were prepared. For each set of 7 test articles, the positive control (5 doses), and the solvent 

control, a 100X DMSO master plate was made, followed by a 4X Master Plate. When added to the 

150 of 1% DMEM already in each well, the addition of the 50 4X dose brought the final dose 

on the plates to 1X. The preparation of the 100X DMSO Master, 4X Assay Master, and 1X plates is 

outlined in Figure 2. 

Project 7560, Final Report 9 
F 

G 

H 

T6C12 T6Cll T6C10 T6C9 T6C8 T?C12 T7CII T7C10 T7C9 T7C8 

NC NC NC NC NC 

Tl-T7 -Test Articles 

T6C1 T6C6 T6C5 T6C4 T6C3 

T7C7 T7C6 T7C5 T7C4 T7C3 

NC PC PC PC PC (0.4) (0.8) (1.6) (3.2) 

C1_12 -Test article concentrations (12 =lowest concentration, 1 =highest) 

NC -Solvent Control (DMSO) 

PC -Positive Control (6.4- 0.4 mM) 

blnk -Blank (contains no cells) 

1 OOX Master Plate Preparation 

T6C2 T6C1 

T?C2 T7C1 

PC blnk (6.4) 

The 100X DMSO Master Plate was labelled with the appropriate test article and control 

concentrations according to the standard plate map (see Figure 1). The 100X top stock 

concentration (200,000 of the test articles was prepared and then serially diluted to make the 

11 remaining doses. The 100X top stock for the positive control (6400 was prepared and then 

serially diluted to make the 4 remaining doses. The wells designated for the solvent control received 

DMSO. The well designated as blank was left empty. 

4X Master Plate Preparation 

The corresponding 4X Master Plate was similarly labeled and the 96-well plate was pre-filled 

with 300 of 1% DMEM in each well. The prepared dilutions from the 100X DMSO Master Plate 

were further diluted 25-fold by adding 12.5 each 100X sample (test article concentration, 

positive control concentration, or solvent control) to the 4X Master Plate. 

Test Article Treatment 

The resulting 4X Master Plate was then used to dose the replicate assay plates (transparent or 

white-walled) containing cells. A final 1X concentration was achieved for each dose by removing 

50 from each well of the 4X Master Plate and adding the dose to the corresponding well in the 

1X plates (already containing 150 of 1% DMEM in each well) . The blank well received 50 

of 1% DMEM. When all of the plates were dosed, the plates were sealed with a plate sealer to avoid 

evaporation of volatile compounds and to avoid cross-contamination between wells. The plates 

were then incubated at standard culture conditions for 48±1 hours. 

Project 7560, Final Report 10 
1:25 dilution 12.5 

1 :4 dilution I 

.x.x.x.x.x.xx X .x.x.x.x.x.xx X .X .X .X X X Y Y X X 

I I I x..X.X.XI IIIIIIX..A.X.XI X X X X T 

.XIIXXIXIII XXXXXXXIII X XIII .I 

_I I x.X.XXI IIIIIX.XXXI X I I I T 

0.98 2000 0.98 2000 0.98 2000 

X X y X y .x.x.x.xxx xx.x.x. I .X. .X. MTT/ Cvtotox Plate (lX) 

Luciferase Plates (1X) 

Visual Observations 

After approximately 48 hours of post-treatment incubation, visual observations of the 

cultures were performed for at least one of the cytotoxicity plates and recorded. Visual 

observations were performed to confirm the results of the cytotoxicity end point. 

Treatment Termination & Luciferase Induction Determination 

After 48 ±1 hours of exposure, each white-walled culture plate was removed from the 

incubator and allowed to equilibrate to room temperature for at least 30 minutes. Once at room 

temperature, the treatment medium was decanted from each plate. The cultures were rinsed with 

250 of CMF-DPBS (room temperature), the CMF-DPBS rinsate was decanted from the wells, 

and the plates were gently blotted onto paper towels. 

Project 7560, Final Report 11 
A 0.59 mg/mL MTT solution was prepared in 1% DMEM and used within 2 hours. After 

48 ±1 hours, the clear 96-well plates designated for the MTT endpoint were decanted and gently 

blotted on paper towels. No rinsing was performed. Two hundred IlL of 1% DMEM containing 

0.59 mg/mL MTT was added to each well. The plate was incubated with a plate seal at standard 

culture conditions for approximately 4 hrs. 

After approximately 4 hours, the MTT solution was decanted, the plate was blotted, and 

200 IlL of 10% SLS was added to each well. The plate was covered with a plate seal and 

incubated at standard culture conditions overnight. 

After the overnight incubation, the plate was placed on a plate shaker and shaken for at 

least 20 minutes at room temperature. The absorbance at 570 nm (OD570) of each well was 

measured with a Molecular Devices Vmax plate reader. 

Data Analysis 

For each set of test articles, a copy of the standard data file, provided by Givaudan, was 

made (i.e. 3 trials, each with 4 plates per trial-3 plates for the luciferase induction and 1 plate for 

MTT or NRU cytotoxicity). Raw data from the luminometer was transferred directly from the 

luminometer software into the designated Excel® spreadsheet for luminescence analyses. Raw 

data from the Vmax was transferred into the designated Excel® spreadsheet for cytotoxicity 

analyses. 

The data file automatically calculated the gene induction and the wells with statistically 

significant induction over a given threshold (default value set to 1.5 = 50% enhanced gene 

activity). Furthermore the maximal induction Cimax), the concentration for maximal gene induction 

(Cimax) and the EC value (concentration for induction above threshold), both with linear and loglinear extrapolation, was calculated similar to the LLNA (Local Lymph Node Assay). Relative 

survival (viability) was obtained by comparing the amount of MTT conversion or neutral red 

uptake by test article treated groups compared to the associated solvent treated group on the same 

plate. An IC50 value was determined for the MTT and neutral red cytotoxicity plates. A summary 

of the results from the 3 definitive assays was calculated. 

The luciferase induction and cytotoxicity data were plotted on the graphs. For the 

luciferase induction, the fold gene induction (as compared to the solvent controls) was plotted over 

the test article concentration. For the cytotoxicity, the % viability (as compared to the solvent 

controls) was plotted over the test article concentration. 

Project 7560, Final Report 12 
A test article was predicted to have sensitization potential if: 

1) The EC1.5 value fell below 1000 at least 2 of 3 repetitions; 

2) At the lowest concentration with a gene induction above 1.5, cellular viability was 

greater than 70%; and 

3) There was apparent overall dose response which was similar between repetitions. 

4 Natsch, A., Bauch C., Foertsch, L., Gerberick, F., Norman, K., Hilberer, A., Inglis, H., Landsiedel, R., Onken, S., 

Reuter, H., Schepky, A., and Emter, R. (2011) The intra- and inter-laboratory reproducibility and predictivity of the 

KeratinoSens assay to predict skin sensitizers in vitro: Results of a ring-study in five laboratories. Toxicology in Vitro. 

25(3): 733-774 

Project 7560, Final Report 13 
which the maximal fold induction occurs), is presented in Table 2. A summary graph 

representing the luciferase fold induction and the cell viability over concentration for each test 

article is included in Appendix B. 

The Induction of Antioxidant-Response-Element Dependent Gene Activity in the 

Keratinocyte ARE- Reporter Cell Line KeratinoSens assay was used to assess the skin 

sensitization potential of the test articles. A test article was predicted to have sensitization 

potential if: 1) The EC 1.5 value fell below 1000 in at least 2 of 3 repetitions; 2) At the 

lowest concentration with a gene induction above 1.5, cellular viability was greater than 70%; 

and 3) There was an apparent overall dose response which was similar between the three 

definitive assays. 

According to the current prediction model, the test article, Research Fragrance Chemical 

TM 11-0230, was predicted to be a skin sensitizer. The test articles, Research Fragrance 

Chemical TM 11-0078, Research Fragrance Chemical TM 10-0210, Research Fragrance 

Chemical TM 10-0367, Research Fragrance Chemical TM 09-0536, Research Fragrance 

Chemical TM 08-0338, 09-0035, and 05-0293, were predicted to be non-sensitizers. 

Although the current prediction model does not include guidance on potency, early data 

indicate a correlation between the EC 1.5 value and potency; the lower the EC 1.5 value the more 

potent the sensitizer, the higher the EC1.5 value the less potent the sensitizer. Cell toxicity was 

measured using two viability assays in order to account for any direct interactions with viability 

dye reagent and the test article (such as direct MTT reduction by a test article). It is important to 

accurately measure cell viability because it is a criterion in the prediction model. When 

evaluating some strong irritants in the KeratinoSens assay, such as sodium laurel sulfate, it was 

found that they caused gene induction above the 1.5 threshold even though they were nonsensitizers; however at the concentration at which these strong irritants caused gene induction 

above the 1.5 threshold they had caused significant cell death (greater than 30% cell death) and 

likely this irritation/toxicity was resulting in activation of the gene response. 

Project 7560, Final Report 14 
TM 11

0078 

13AL99 Research Fragrance Chemical >2000 110.10 NO* TM 10-0210 

8 Jan 2014 13AMOO Research Fragrance Chemical >2000 100.73 NO* TM 10-0367 

13AM01 Research Fragrance Chemical >2000 32.82 NO* TM 09-0536 

Cinnamic Positive Control 7.83 >64 YES Aldehyde 

13AM02 Research Fragrance Chemical 35.33 579.4 YES TM 11-0230 

13AM03 Research Fragrance Chemical >2000 209.1 NO TM 08-0338 

29 Jan 2014 13AN30 09-0035 >2000 192.8 NO* 

13AN31 05-0293 >2000 153.2 NO 

Cinnamic Positive Control 7.03 >64 YES Aldehyde 

NOTE: When an EC 1.5 or IC50 value was not obtained, the results were presented as greater than the 

highest dose tested. 

"- The EC 1.5 value is the concentration for gene induction above the threshold ( 1.5 fold) as 

compared to the DMSO solvent controls 

+ - A test article is predicted to have sensitization potential if: 

1) The EC 1.5 value falls below 1000 jlg/mL in at least 2 out of 3 independent trials 

2) At the lowest concentration with a gene induction above 1.5, cellular viability was greater 

than 70% 

3) There was an apparent overall dose response similar between repetitions 

* These test articles are not sensitizers per the KeratinoSens prediction model. Although in some of the 

repetitions within each independent trial the induction value was greater than 1.5, the overall induction was not statistically significant. The induction above 1.5 was observed at doses which were cytotoxic (as 

can be observed in the graphs presented on the following pages). 

Project 7560, Final Report 15 
13AL99 Research Fragrance Chemical 1.04 ND TM 10-0210 

8 Jan 2014 

13AMOO Research Fragrance Chemical 1.16 ND TM 10-0367 

13AM01 Research Fragrance Chemical 1.08 ND TM 09-0536 

13AM02 Research Fragrance Chemical 2.40 125 TM 11-0230 

13AM03 Research Fragrance Chemical 1.11 ND 

29 Jan 2014 TM 08-0338 

13AN30 09-0035 1.24 ND 

13AN31 05-0293 1.18 ND 

1

- Luciferase maximal fold induction as compared to the DMSO solvent controls. An Imax value 

1.5 produced an EC 1.5 value. 

2 

- Concentration where maximal fold induction occurred. When the fold induction did not cross 

the 1.5 fold threshold, the Clmax was presented as ND. 

ND - Not determined since the maximal induction was < 1.5 fold 

Project 7560, Final Report 16 
APPENDIX A 
0 

2.0 

3.0 

measured by its induction of luciferase in the KeratinoSens cells, a keratinocyte reporter 

cell line which contains an antioxidant response element (ARE). Luciferase induction 

directly indicates activation of ARE-dependent genes. Measurement of the induction of 

this signalling pathway in a reporter cell line provides a high-throughput cell-based in 

vitro test to screen for the skin sensitization potential of novel chemicals. Luciferase 

induction will be determined through the bioluminescent activity of the conversion of 

ATP and a luciferin substrate into light due to the enzymatic activity of luciferase. 

Additionally, the cytotoxicity of each test article will be determined using cell viability 

endpoints with MTT (3-[4,5- dimethylthiazol-2-yl]- 2,5- diphenyltetrazolium bromide). 

Cytotoxicity will be determined by measuring the relative conversion of MTT in the test 

article treated cultures as compared to the solvent control. 

SPONSOR 

2.1 Name: International Flavors & Fragrances Inc. 

2.2 Address: 800 Rose Lane 

Union Beach, NJ 07735 

2.3 Representatives: Paul Ribeiro, Ph.D., Joan Cheng Ph.D., DABT 

IDENTIFICATION OF TEST SUBSTANCES AND ASSAY CONTROLS 

3.1 Test Article: See Protocol Attachment 1 

3.2 Assay Controls: Positive: Cinnamic Aldehyde 

Solvent: Assay Media containing 1% DMSO 

3.3 Determination of Strength, Purity, etc. 

3.3.1 For GLP studies only, the Institute for In Vitro Sciences, Inc. (IIVS) will 

attempt to secure documentation of the analytical purity and composition 

of the test article and the stability and strength of the dosing solutions from 

the Sponsor. If the Sponsor is unable to provide such information, IIVS 

will retain documentation supporting attempts to obtain this information 

with the study file and an exception will be noted in the Statement of 

Compliance in the Final Report. 

3.3.2 IIVS will be responsible for the documentation of the analytical purity and 

composition of the positive and solvent controls used for the stock or 
c 

4.3 Study Director: 

Gaithersburg, MD 20878 

Kimberly Norman, Ph.D., DABT 

5.0 TEST SCHEDULE 

6.0 

7.0 

5.1 Proposed Experimental Initiation Date: 9 December 2013 

5.2 Proposed Experimental Completion Date: 27 December 2013 

5.3 Proposed Report Date: 21 February 2014 

TEST SYSTEM 

The KeratinoSens cells (Givaudan, Switzerland) have been propagated as a reporter cell 

line. The KeratinoSens cells are transfected HaCaT keratinocytes that include a 56-basepair insertion containing the ARE sequence from the AKR1C2 gene, a SV40 promoter, 

the luciferase gene in the vector pGL4 from Promega, and a stable insertion allowing for 

cells to be selected in the presence of Geneticin (G418). The signalling pathway with the 

repressor protein Keap 1 and the transcription factor Nrf2, which binds to the antioxidant I 

electrophile response element (ARE I EpRE), was shown to be a valuable cellular 

endpoint to detect skin sensitizers in vitro (Natsch & Emter, 2008; Natsch et.al, 2009). 

The induction of luciferase directly indicates the activation of ARE-dependent genes. 

EXPERThffiNTAL DESIGN AND METHODOLOGY 

The experimental design of this study consists of three definitive assays to determine 

average maximal induction of gene activity (Imax), the concentration for average maximal 

induction of gene activity (Cimax), the average concentration inducing luciferase gene 

activity >50% above solvent control values (EC1.5 value), and the average concentration 

leading to 50% cytotoxicity (IC50 ) for each test article. The luciferase gene induction 

(using the ATP luminescence endpoint) will be used to calculate the Imax, Clmax, and the 

EC 1.5 value. The induction of luciferase will be evaluated to determine sensitization 

potential of the test article. The cytotoxicity of the test articles will be evaluated on the 

basis of the NAD(P)H-dependent microsomal enzyme reduction ofMTT (Berridge, et al., 

1996) by the KeratinoSens cells. The amount of reduced MTT will be measured 

spectrophotometrically. Cytotoxicity data will be presented in the form of relative 

survival (relative MTT reduction) versus test article concentration. 

For each definitive assay, the KeratinoSens cells will be cultured in at least quadruplicate 
7 .1.1 Assay Medium: Dulbecco's Modified Eagle Medium(DMEM) containing 

GlutaMAXTM I, 1000 mg/L D-Glucose, Sodium Pyruvate (Gibco) 

supplemented with: 9.1% fetal bovine serum (FBS) (Hyclone) 

7 .1.2 Maintenance Medium: Dulbecco's Modified Eagle Medium(DMEM) 

containing GlutaMAXTM I, 1000 mg/L D-Glucose, Sodium Pyruvate 

supplemented with: 9.1% FBS and 5.5 mL Geneticin (G418) (Invitrogen) 

(final concentration 500 J,tg/mL) 

7 .1.3 1% DMEM: Dulbecco's Modified Eagle Medium containing GlutaMAXTM 

I, 1000 mg/L D-Glucose, Sodium Pyruvate supplemented with: 1% FBS 

7.1.4 Dulbecco's Phosphate Buffered Saline without Calcium & Magnesium 

supplemented with 0.05% EDTA 

7 .1.5 0.05o/o Trypsin/0.02% EDTA Solution (Gibco/Invitrogen) 

7.1.6 Dulbecco's Phosphate Buffered Saline without Calcium & Magnesium 

(CMF-DPBS) (Gibco) 

7.1.7 Dimethyl Sulfoxide (DMSO) (Sigma) 

7.1.8 Promega ONE-GloTM Luciferase Assay System (kit contains Luciferase 

Assay Buffer and lyophilized Luciferase Assay Substrate) (Promega) 

7.1.9 MTT (3-[4,5- dimethylthiazol-2-yl]- 2,5- diphenyltetrazolium bromide) 

(Sigma) 

7.1.10 Extraction solvent: Isopropanol (Sigma) [MTT Assay] 
7.3 

air containing 5 ±I% C02). 

If test articles are mixtures, or have no defined molecular weight they may be 

diluted on a ,...gtmL (w/v) basis, rather than based upon molarity as specified 

in Protocol Attachment 1. Unless otherwise specified these test articles will 

be tested considering a pro forma molecular weight of 200 g/mol, therefore 

the lOOX stock solution will be prepared to a top concentration of 40 mg/mL 

in DMSO or other appropriate solvent and diluted the same as the 200 mM 

top concentration amongst the DMSO Master Plate, 4X, and lX plates. 

The IX concentrations for the test articles dilutions based on molarity will be 

2000, IOOO, 500, 250, I25, 62.5, 31.3, I5.6, 7.8I, 3.9I, 1.95, and 0.975 mM. The 

IX concentrations for the test article dilutions based on weight/volume (w/v) will 

be 400, 200, IOO, 50, 25, I2.5, 6.25, 3.I3, 1.56, 0.78I, 0.391, and O.I95 

The stability of the test article(s) under the storage conditions at the testing facility 

and under the actual experimental conditions will not be determined by Institute 

for In Vitro Sciences, Inc. (IIVS). 

Controls 

Positive control: Cinnamic aldehyde will be serially diluted in DMSO to 

concentrations IOOX of the final concentrations for the assay. The IOOX DMSO 

Master Plate stock solutions will be added to the 4X Master Plate containing 

I% DMEM to a 4X concentration. Addition of the 4X concentrations to the wells 

already containing I 50 I% DMEM will result in a final IX concentration. The 

final concentrations of the positive control will be 6.40, 3.20, I.60, 0.800, and 

0.400 mM. Each plate will contain a serial dilution of the positive control. 

Solvent Control: Each plate will include 6 vehicle control wells (cells exposed to 

I% DMSO only). Han intermediate solvent is used (e.g., water), the concentration 

of the intermediate solvent will be the same as used with the test article treatment. 

Each plate will have a blank well (HI2). 
c 

( 

to have reduced the MTT. Water insoluble test materials may show direct reduction 

(darkening) only at the interface between the test article and the medium. 

7.5 Solubility Compatibility Testing 

7.6 

Prior to testing in the assay, the solubility of the test article in DMSO (or water, if 

not soluble in DMSO) may be assessed as determined by the Study Director, 

especially if the solubility of the test article is unknown. The preferred solvent is 

DMSO. The test article will be diluted in DMSO at the highest 100X 

concentration to be tested in the definitive assays. Some test articles may cause 

precipitation in DMSO or water. If the test article is not soluble in DMSO, the 

test article may be tested in sterile deionized water. If the test article forms a 

homogenous working suspension in either DMSO or water, the Study Director 

may elect to proceed with testing. 

Note: Although some chemicals may not be fully soluble at the maximum dosage, 

the gene induction may still be measured in many cases (Natsch, 2009 SOP). 

Maintenance of KeratinoSens cell line 

Cryopreserved KeratinoSens cells (Givaudan), tested for the presence of 

mycoplasma contamination, will be stored in the liquid nitrogen freezer. The 

stock ampules will be thawed and seeded as needed. The cells can be maintained 

for up to 25 passages. 

The cryopreserved KeratinoSens cells will be thawed in a water bath at 

approximately 37°C. The cryovial will be gently swirled in a beaker containing 

70% ethanol. Once thawed, the cryovial will be immediately decontaminated 

with 70% ethanol and the contents will be transferred to a sterile centrifuge tube 

and diluted by slowly adding approximately 9.0 mL of KeratinoSens Assay 

Medium. The cells will be centrifuged at 200 x g for approximately 5 minutes to 

remove the DMSO. Following centrifugation, the supernatant will be removed 

and the cells will be resuspended in approximately 15 mL of Assay Medium. 

Cultures will be incubated at 37 ± 1 oc humidified air containing 5 ± 1% C02 

(standard culture conditions). Cells are initially maintained in Assay Medium. 

They are passaged every 2-4 days to maintain a cell density less than 80% of 

confluence. Cells will be maintained in Maintenance Medium (Assay Medium 

supplemented with G418) after the first passage upon thawing. These stock 

cultures will be used to seed the 96-well plates. 
c 

7.8 

5-10 minutes (or until they became dislodged). When more than 50% of the cells 

become dislodged, the flask will be rapped sharply against the palm of the hand. 

When more than 90% of the cells become dislodged, the cells will be each 

re-suspended in 7 mL Maintenance Media. An appropriate volume of the 

trypsinized cells in Maintenance Medium will be added to new flasks for 

passaging. The KeratinoSens cells will be routinely passaged every 2-5 days. 

96-well plate cell procedures: When the flask(s) are 60--90% confluent, the 

KeratinoSens cells will be prepared for seeding in the 96-well plates. The 

medium will be aspirated and the cell sheet will be rinsed twice with -10 mL of 

DPBS containing 0.05% EDTA for approximately 2 minutes. One mL of 

trypsin/EDT A will be added to each flask to cover the cell sheet. The flask( s) will 

be incubated at standard culture conditions for 5-10 minutes (or until they become 

dislodged). When more than 50% of the cells become dislodged, the flask(s) will 

be rapped sharply against the palm of the hand. When more than 90% of the cells 

become dislodged, the cells will be each re-suspended in 5 mL Assay Medium 

(without G418). The concentration of cells will be determined with a coulter 

counter or hemacytometer. Cells will be diluted to approximately 1.0 x 105 

cells/mL in Assay Medium. 

Subculturing KeratinoSens Cells Into 96-well Plates 

Cultured cells that will be used in seeding the 96-well plates should be fed fresh 

Growth Medium the day before subculturing to the plates. Each definitive assay 

will have 4 plates; 3 white-walled plates will be used for the luciferase endpoint, 

while 1 clear bottomed plate will be used for the cytotoxicity endpoint. These 

plates are designated as the 1X plates. Each 96-well plate will be uniquely 

numbered and labeled with the seeding density, cell type, and date of seeding. Cell 

suspensions of 1x105cells/mL in the Assay Medium will be prepared. One hundred 

JA,L of the cell suspension will be added to the designated wells on the 96-well plate 

(i.e. all wells except well H12, which will be left blank). For example, using 8 tips, 

one hundred ~L of the cell suspension will be placed into the first 11 columns of the 

96-well tissue culture microtiter plate; one tip will be removed and 100 ~L will be 

added to the remaining column of wells (A12-G12) (1x105 cells/mL = 1000 

cells/well). Using a pipette, 100 ~L of Assay Medium will be placed into the well 

designated as blank (H12). The cells will be incubated for 24±1 hours at standard 

culture conditions. 
c 

On the day following seeding, visual observations will be performed on the clear 

plate(s) for appropriate cell growth, uniform seeding, and confluence. Prior to 

treatment, the Assay Medium was removed from the culture plates by careful 

inversion and blotting of the plates onto sterile paper towels. The cells were refed with 150 ~L of pre-warmed (37°C) 1% DMEM (DMEM containing 1% 

FBS). After re-feeding, the plates were incubated at standard culture conditions 

until treatment. 

The test article(s) will be prepared and treated as described below in Section 7.9.1 

(See also Figure 2). Test articles will be generally diluted in DMSO. Chemicals 

not soluble in DMSO may be dissolved and diluted in sterile deionized water, and 

then DMSO will be added to bring the final concentration ofDMSO to 1%. A 

1 OOX DMSO master plate, followed by a 4X Master Plate containing 1% DMEM, 

will be prepared for each set of test articles, the positive control, and the solvent 

control. The preparation procedures for each plate are described below; and a 

graphic overview is outlined in Figure 2. 

7. 9.1 Preparation of 1 OOX Master Plate 

The 100X DMSO Master Plate(s) will be labelled with the appropriate test 

article and control concentrations according to the standard plate map (see 

Figure 1). The 100X top stock will be 200 mM for neat chemicals, 

40 mg/mL for mixtures, and 6.4 mM for the positive control. The serial 

dilutions of the test article(s) and the positive control will be prepared in 

the 96-well plate. The dilutions of the test articles and positive control 

will be prepared according to their dilution schemes and then added to the 

appropriate wells on the 96-well plate. The highest stock concentrations 

for the test articles and the positive control will then be serially diluted in 

the DMSO master plate to create the other 11 (or 4) dilutions. 

Note: The wells designated as solvent controls will already contain 

150 f.tL of DMSO; therefore no dilutions will be made. The well 

designated as blank will remain empty. 

7.9.2 Preparation of 4X Master Plate 

The corresponding 4X Master Plate(s) with 1% DMEM will be similarly 

labeled as the 100X DMSO Master Plate. 300 ~L of 1% DMEM (DMEM 

without G418 containing 1% FBS) will be added to all applicable test wells. 
c 

c 

the 100X DMSO master plate will be added to the corresponding wells of 

4X Master Plate containing 300 of 1% DMEM. The well designated as 

blank will contain 300 of 1% DMEM. 

7.9.3 Addition of Test Article to Cultures (Final1X) 

The resulting 4X Master Plate with 1% DMEM will then be used to 

distribute doses of 50 from each well to the replicate assay plates 

containing cells. Dosing dilutions will be prepared at 4X the desired final concentration so that when 50 added to the 150 of 1% DMEM 

already in the each well, a final 1X dose will be obtained. 

A plate seal will be carefully placed directly over the culture wells. The 

sealer will be assured to adhere to each culture well. The 96-well plate 

cover will be placed loosely over the sealed plate and will be incubated 

with the plate under standard culture conditions. The plate cover should 

be placed loosely atop the plate seal to avoid deforming the sealer and 

causing the sealer to detach from culture wells. A loose fit of the plate lid 

is acceptable. The plates will be incubated (with the plate seals) for 

48± 1 hours at standard culture conditions. 

The 96 well template (Figure 1) illustrates the plate setup for treatment of 

the positive control and test articles. The test article concentrations 1-12 

correspond with the Dose ID numbers found on the dilution scheme for the 

specific test article. 
c 

A 

B 

c 

D 

E 

F 

G 

H 

TIC12 T2C12 

T3Ct2 T4Ct2 

T5C12 

T6C12 T7C12 

NC 

TIC11 TIC10 TIC9 

T2C11 T2C10 T2C9 

T3C11 T3C10 T3C9 

T4C11 T4C10 T4C9 T5Cu T5C10 T5C9 T6C11 T6C10 T6C9 

T7Cu T7C10 T7C9 

NC NC NC 

T 1-T7 - Test Articles 

TICs TIC1 TIC6 

T2Cs T2C7 T2C6 

T3Cs T3C7 T3C6 

T4Cs T4C7 T4C6 

T5Cs T5C7 T5C6 

TQCs T6C7 T6C6 TICs T1C1 T7C6 

NC NC PC (4) 

TIC5 TIC4 TIC3 

T2C5 T2C4 T2C3 

T3C5 T3C4 T3C3 T4C5 T4C4 T4C3 

T5C5 T5C4 TSC3 

T6C5 T6C4 T6C3 

T7C5 T7C4 T7C3 

PC PC PC 

(8) (I6) (32) 

C1_12 -Test article concentrations (12 =lowest concentration, 1 =highest) 

NC -Solvent Control (DMSO) 

PC -Positive Control (64- 4 

blnk -Blank (contains no cells) 

TIC2 TIC1 

T2C2 T2C1 

T3C2 T3C1 T4C2 T4C1 

TSC2 TSC1 

T6C2 T6C1 T7C2 T7Ct 

PC blnk (64) 

Note: Plate map applicable to testing 7 test articles. When less than 7 test articles will be 

tested in an assay, the location of each test article on the plate will be designated in the study 

workbook. The test article concentrations on the plate will remain lowest to highest from the 

left to right side of the plate. 
0.098 mM -------------7 200 mM 

1 :25 dilution ' 12.5 

(<lLJ nzflt-'lulr') 

1 :4 dilution 

0.98 !lM 2000 !lM f 

0.98 !lM 2000 !lM q 0.98 !lM 2000 !lM 0.98 !lM 2000 !lM XXJ...J..J..J...J..XXX.X 

xxxxxxxxxxx xxxxxxxxxxx xxxxxxxxxxx MTT Cytotox Plate (1X) 

Luciferase Plates (1X) 
Note Code Note Text Note Code Note Text 

1 Normal Cell Morphology p Precipitate observed in well 

2 Low Level of Cell Toxicity M Media discolored in well 

3 Moderate Level of Cell Toxicity N No cells observed in well 

4 High Level of Cell Toxicity 0 Precipitate in wells observed as oily droplets 

7.9.3 Preparation ofPromega ONE-GloTM Reagent 

The ONE-Glo TM Reagent will be prepared by reconstituting the 

lyophilized Substrate with the appropriate volume of Assay Buffer. The 

prepared reagent will be allowed to equilibrate for at least 15 minutes at 

room temperature (optimal temperature for enzymatic activity) prior to 

use. 

7.9.4 Treatment Termination: Luciferase Endpoint Assay 

After approximately 48 hours of exposure, each white-walled culture plate 

will be removed from the incubator and allowed to cool to room 

temperature for at least 30 minutes. Once at room temperature, the 

treatment medium will be decanted from each plate. The cultures will be 

rinsed with 250 J!L of CMF-DPBS (room temperature), and the DPBS 

rinsate will be decanted from the wells. Excess DPBS will be removed by 

blotting the plates gently onto paper towels. 

Fifty microliters of CMF-DPBS will be added to each well. Fifty 

microliters of Promega ONE-Glo TM Reagent will be added to each well 

and the plates will be allowed to incubate at room temperature in the dark 

for approximately 5 minutes before being read by the luminometer. 

The plates will be kept away from sunlight or bright fluorescent light 

before reading. Bright light may cause plate phosphorescence resulting in 

higher background levels. Plate phosphorescence has a half-life of several 

minutes. The plates will be read within 45 minutes of addition of the 

OneGlo reagent. Each plate will be placed onto the plate tray in the 

Berthold Detection Systems luminometer, and the luminescence 

determination will be initiated from an ffiM-PC hosting the Windowsbased SimplicityTM software. The light intensity in each well will be 
c 

7.10 

within 2 hours. After 48 ±1 hours, the clear 96-well plates designated for 

the MTT endpoint will be removed from the incubator. The test article 

dilutions will be decanted from the plates. No rinsing will be performed. 

Two hundred J..LL of 1% DMEM containing 0.59 mg/mL MTT will be 

added to each well. The plate will be incubated with a plate seal at 

standard culture conditions for 4±0.3 hrs. 

After 4±0.3 hours, the MTT solution will be decanted, the plate will be 

blotted, and 200 J..LL of 10% SLS will be added to each well. The plate will 

be covered with a plate seal and incubated at standard culture conditions 

overnight. 

After the overnight incubation, the plate(s) will be placed on a plate shaker 

and shaken for at least 20 minutes at room temperature. The absorbance at 

570 nm (0Ds7o) of each well will be measured with a Molecular Devices 

Vmax plate reader. The plate reader will be turned on at least 15 minutes 

prior to use, and will be calibrated according to standard operating 

procedures. The AUTO MIX setting will be selected. The reading will be 

saved to a designated print file and a print screen will be made of the raw 

data. Relative survival will be obtained by comparing the amount of MTT 

reduction by test article treated groups to the MTT reduction by the solvent 

treated group on the same plate. An IC50 value will be determined for the 

MTT cytotoxicity plate. 

Data Analysis 

For each set of chemicals, a copy of the standard Excel® data analysis spreadsheet 

file, provided by Givaudan, will be made (i.e. 3 trials, each with 4 plates per trial3 plates for the luciferase induction and 1 plate for MTT cytotoxicity). 

In the designated Excel® spreadsheet standard file, the fields which need to be 

filled in will be marked yellow. On the 'Summary sheet' the compound 

identifiers and the plate identifier will be inserted. On the sheet 'rep 1' the plate 

readout of the triplicate analysis of the first definitive assay will be directly 

inserted in the yellow areas marked repetition 1.1, 1.2, and 1.3. The plate readout 

of the triplicate analysis of the second definitive assay and third definitive assay 

will be added to the sheets marked 'rep2' and "rep3" as described for the first for 

the first definitive assay. The cytotoxicity plate readout results from each 

repetition will be inserted into the sheets labeled 'Cytotoxicity (1)- (3), with the 
The corrected OD570,550 value of the DMSO solvent control, each individual test 

article concentration, and the positive control will be determined by subtracting 

the raw ODs1o1sso value of the blank well (H12) from their mean 0Ds7o/sso values. 

Corrected ODs?o!sso value = Raw data 0Ds7o/sso - raw 0Ds7o/sso of blank well 

The mean corrected solvent control 0Ds70,550 value will be determined from the 6 

solvent control wells (A12, B12, C12, D12, E12, and F12). For each well, the 

percent viability will be calculated as the ratio of the corrected ODs1o1sso of the 

test or control sample to the corrected ODs1o1sso of the appropriate solvent 

control. 

Corrected OD5101550 of test or control article 

cell viability = -------------- X 100 

Mean corrected OD570,550 of Solvent Control 

The Givaudan Excel file will calculate an IC50 value using two concentrations, 

one of which results in viability of <50% and one >50% of the solvent control. 

The summary IC50 which appears on the 'Summary Results' page is calculated by 

determining the mean of the IC50 values from each definitive assay . 

. · 

7.10.2 Luciferase Data Analysis 

Raw data from the luminometer will be transferred directly from the luminometer 

software into the designated Excel® spreadsheet (provided by Givaudan) for 

luminescence analyses. Each definitive assay will include a set of 3 luciferase 

plates. Since each plate contains a set of solvent control wells and a blank well, 

the corrected RLUs6s values and individual well fold induction for each 

concentration of the test article or positive control will be determined using its 

corresponding plate solvent control wells and blank well. 

The corrected RLU565 value of the DMSO solvent control, each individual test 

article concentration, and the positive control will be determined by subtracting 

the raw RLUs6s value of the blank well (H12) from their mean RLUs65· 

Corrected RLUs65 value= Raw data RLUs6s- raw RLUs6s of blank well 

The file will then automatically calculate the gene induction and the wells with 

statistically significant induction over a given threshold (default value set to 1.5 = 

50% enhanced gene activity). To calculate the fold gene induction (over the 

controls), the corrected RLU565 value of each well will be divided by the mean 

corrected RLU565 value of the solvent control wells (which equals 1). 
c 

g 

p

p

(

y)

g

fold gene induction at each concentration will be used to calculate the EC 1.5 

value (concentration for induction 1.5 fold above threshold) for each definitive 

assay. For each test article at each concentration the values obtained from 

repetitions 1, 2, and 3 are compared to the average solvent control values of each 

plate from repetitions 1, 2, and 3 using an unpaired two-tailed t-test. An average 

fold induction value of greater than 1.5 which is statistically significant 

(significance is defined by a p-value of <0.5) is used to calculate the EC1.5 value. 

The spreadsheet calculates the EC1.5 both with linear and log-linear 

extrapolation. The final EC1.5 value for each test article will be determined by 

averaging the mean EC1.5 of the 3 definitive assays. 

Furthermore, the maximal induction (lmax) will be determined. The Imax for 

each test article run in each definitive assay is simply the largest average fold 

induction value for that test article. An Imax value for each test article will be 

calculated in the 'Summary page' by simply taking the arithmetic mean of the 

Imax values obtained for each test article from each definitive assay. The 

concentration for maximal gene induction (Cimax) will be calculated for each 

definitive assay. The Clmax is simply the concentration at which the largest 

average fold induction value, which is statistically significant, is achieved. 

The results from the different repetitions (definitive assays) will be summarized in 

the 'Summary' file. The data, including the average fold induction and IC50 

values for each test article at each concentration will be automatically plotted in 

the graphs on the 'Summary' page and in the different repetition sheets. The 

Study Director may manually modify values in the spreadsheet as needed. All 

manual modifications will be documented in the final report. 

8.0 CRITERIA FOR DETERMINATION OF A VALID TEST 

Average luciferase gene induction by the positive control ( cinnamic aldehyde) must cross 

the 1.5 threshold in at least one dose in each definitive assay. Furthermore, each definitive 

assay will be accepted (considered valid) if the cinnamic aldehyde luciferase gene 

induction falls within 2 standard deviations of the historical mean (updated every three 

months). Further determination of the validity of an assay will be at the discretion of the 

Study Director. Each assay will be assessed (but not necessarily deemed a valid assay) 

using similar criteria as outlined in the Ring Trial (Natsch, et al. 2011): 1) The standard 

deviation of the viability of the solvent controls for the luciferase plates for all of the 

definitive assays (9 total plates) will be <20%; 2) The cinnamic aldehyde will be 
c 

( 

sensitization potential if: 1) The EC1.5 value falls below 1000 J..tM (or 200 J..tg/mL) in at 

least 2 of 3 repetitions; 2) At the lowest concentration with a gene induction above 1.5, 

cellular viability should be greater than 70%; and 3) An apparent overall dose response 

should be similar between repetitions. 

The prediction mod;el for the KeratinoSens assay was based on studies with neat 

chemicals, not extracts or mixtures. The prediction model presented above based on 

(wlv) concentrations is based on the prediction model established for neat chemicals, 

but may not accurately assess mixtures. 

10.0 REPORT 

11.0 

A report of the results of this study will be prepared by the Testing Laboratory and will 

accurately describe all methods used for generation and analysis of the data. A copy of 

the protocol used for the study, any amendments and any significant deviation(s) from the 

protocol will appear as a part of the final report. 

RECORDS AND ARCHIVES 

A separate working notebook will be used to record the materials and procedures used to 

perform this study. Upon completion of the final report, all raw data, reports and 

specimens will be retained in the archives for a period of either a) 5 years, b) the length 

of time specified in the contract terms and conditions, or c) as long as the quality of the 

preparation affords evaluation, whichever is applicable. 

12.0 PROTOCOL AMENDMENTS 

13.0 

When it becomes necessary to change the approved protocol for a specific study, the 

change and the reason for it should be put in writing and signed by the Study Director as 

soon as practical. When the change may impact the study design and/or execution, verbal 

agreement to make this change should be made between the Study Director and Sponsor. 

This document is then provided to the Sponsor and is attached to the protocol as an 

amendment. 

REFERENCES 

Natsch, A. and Emter, R. (2008) Skin sensitizers induce antioxidant response element dependent 

genes: Application to the in vitro testing of the sensitization potential of chemicals. 

Toxicological Sciences. 102(1): p. 110-119. 
( 

14.0 

Natsch, A., Bauch C., Foertsch, L., Gerberick, F., Norman, K., Hilberer, A., Inglis, H., 

Landsiedel, R., Onken, S., Reuter, H., Schepky, A., and Emter, R. (201 I) The intra- and 

inter-laboratory reproducibility and predictivity of the KeratinoSens· assay to predict skin 

sensitizers in vitro: Results of a ring-study in five laboratories. Toxicology in Vitro. 

25(3): 733-774 

SPONSOR REPRESENTATIVE DATE 

Paul Ribeiro, Ph.D., Joan Cheng, Ph.D .. 

(Print or Type Name) 

l 

f 

i 

I 

I· 

I 

I. 

I 

I 

I 
g

13AMOO Research Fragrance Chemical TM 10-0367 184.2 g/mol 

13AM01 Research Fragrance Chemical TM 09-0536 192.2 g/mol 

13AM02 Research Fragrance Chemical TM 11-0230 148.0 g/mol 

13AM03 Research Fragrance Chemical TM 08-0338 194.2 g/mol 

13AN30 09-0035 200.2 g/mol 

13AN31 05-0293 156.2 g/mol 

Additional Testing Information: : The test articles, 13AL98- AM03, AN30-AN31 are neat 

chemicals and the 1 OOX top stock solution will be prepared to a 

concentration of 200 mM for each test article. 

REGULATORY REQUIREMENTS: 

Will this study be conducted according to GLPs? [ ] YES or [X ] NO 

If YES, please indicate which agency(ies) guidelines are to be followed: 

[ ] FDA; [ ] EPA TSCA; [ ] EPA FIFRA; [ ] OECD; [ ] Other _____ _ 
APPENDIXB 
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1.00 

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0.00 

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1.00 

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cone (uM) 

10.00 

cone (uM) 

100.00 1000.00 

-----induction • 3AL98 

---

140 120 

100 

00 

60 

40 

20 

0 

100.00 1000.00 

lnOUCliOn 1 Y 

--B-- Viti hility 1 ::!AI QQ 

140 120 100 

80 

60 

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0.00 +-------..,..---------r--11.-..--fl!l----.. 0 

1.00 10.00 100.00 1000.00 

cone {UM) -lrduc:ion 13Afi.IOO 

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1.00 10.00 100.00 1000.00 

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13AI'.1101 

3 .00 

140 

120 

100 

.g 1.50 80 c 

"0 

:e 60 

40 

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0.00 +---------------r---------------.--------------+ 0 

1.000 10.000 100.000 1000.000 

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cone (uM) ----+-induction 13AM02 

--E!-viability 13AM02 

3.00 

140 120 

1: 100 

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u 80 ::3 1.50 'C :c 

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1.000 10.000 100.000 1000.000 

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1.000 10.000 100.000 "1 000.000 

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