Reaction mass of methyl 2-({(E)-[4-(4-hydroxy-4-methylpentyl)cyclohex-3-en-1-ylidene]methyl}amino)benzoate and methyl 2-({(E)-[3-(4-hydroxy-4-methylpentyl)cyclohex-3-en-1-ylidene]methyl}amino)benzoate and methyl 2-({(Z)-[4-(4-hydroxy-4-methylpentyl)cyclohex-3-en-1-ylidene]methyl}amino)benzoate and methyl 2-({(Z)-[3-(4-hydroxy-4-methylpentyl)cyclohex-3-en-1-ylidene]methyl}amino)benzoate

Endpoint summary

Description of key information

The skin sensitisation is assessed with three in vitro tests with the following results:

DPRA (OECD TG 442C): Positive

KeratinoSens (OECD TG 442D): Positive

h-CLAT (OECD TG 442E): Positive

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Remarks:

Direct Peptide Reactivity Assay (DPRA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 23 March 2017 and 08 Apr 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Deviations:

no

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

Test Article Preparation
The test article was prepared at a 100 mM concentration in an appropriate solvent. Calculations using the molecular weight and purity of the test article were performed to determine the appropriate amount of test article to weigh out in order to achieve approximately 3 mL of the 100 mM sample.

The test article was weighed into a prelabeled glass vial and stored at room temperature. The test article was not dissolved in the solvent until immediately before mixing with the peptides.

Test Article Solubility TestA solubility test was performed for the test article in order to determine an appropriate solvent that completely dissolved the test article at a 100 mM concentration. The test article, Lyrame, was found to be soluble in acetonitrile with vortexing for approximately 1 minute.

Peptide Preparation
Custom synthetic peptides of cysteine or lysine (containing phenylalanine to aid in detection) were used in this assay. The purity of each peptide was at least 90%. Peptide samples were newly prepared for each sample set, and a single preparation of the peptide was used throughout the sample set. The cysteine peptide was prepared by weighing an appropriate amount of the peptide to achieve a 0.667 mM concentration in pH 7.6 phosphate buffer. The lysine peptide was prepared by weighing an appropriate amount of the peptide to achieve a 0.667 mM concentration in pH 10.1 ammonium acetate buffer. The peptide solutions were gently mixed on the shaker.

Peptide Standards
A set of serially diluted standards were prepared for each peptide. These standards were prepared by diluting the peptide solutions in dilution buffer (20% acetonitrile in either phosphate or ammonium acetate buffer). Six standards were prepared at concentrations of 0.534- 0.0167 mM. A seventh standard was prepared containing only dilution buffer. Approximately 1 mL of each standard was pipetted into the appropriate prelabeled autosampler vials.

Controls
The positive control used in this assay was cinnamic aldehyde prepared at a concentration of 100 mM. The positive control was reacted with the peptides in the same fashion as the test article. There were three sets of reference controls of acetonitrile run at different points throughout the assay (reference controls A-C). Solvent controls were also prepared for the solvent used in the assay(if different than acetonitrile). These controls consist of the solvent (acetonitrile) reacted with the peptide in the absence of test article. A coelution control was also prepared for the test article. The coelution control consisted of the test article without the peptide. The purpose of the coelution control was to determine if the test article elution from the HPLC column overlapped with the peptide elution.

HPLC Set-up and Operation
The separation module used in this assay was a Waters 2690/5 HPLC system. This system consisted of a solvent management system for the mobile phases and a sample management system for the test article and controls. The HPLC system was coupled to a photodiode array detector set at 220 nm. The dimensions of the column used were 2.1 mm x 100 mm x 3.5 micron. The column was primed for at least two hours before the start of the assay. To prime the column, equal parts of mobile phase A (0.1% trifluoroacetic acid in HPLC grade water) and mobile phase B (0.08% trifluoroacetic acid in HPLC grade acetonitrile) were passed through the column.

Once the column was equilibrated and the samples were prepared, the autosampler vials were placed into the designated locations of the separations module carousels. The samples were incubated in the dark at room temperature for 24± 2 hours.

A gradient elution was used in this assay. The mobile phase changed from 10-25% acetonitrile over a 10 minute period to allow for optimal separation and gradually elute most of the sample from the column. This was followed by a rapid increase to 90% acetonitrile to remove anything remaining on the column. The column was allowed to equilibrate back to initial specs for 7 minutes between injections.

The Empower PDA software (Version 3) was used to convert the absorbance data from the UV detector into chromatograms of intensity versus retention time for each sample and control. At the end of the run, each chromatogram was integrated in order for the software to calculate the area under the peptide peak. Cysteine and lysine elute from the column at known times, so it was possible to determine which peaks in the chromatograms represented the peptides and use the areas under those peaks for the subsequent calculations.

Key result

Run / experiment:

other: Cysteine and Lysine

Parameter:

other: Mean Depletion (%)

Value:

13.55

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

Solubility Assessment
The solubility of Lilial-Me Anthranilate in acetonitrile at a nominal concentration of 100 mM was confirmed by visual inspection.

Reactivity Assessment
All analytical acceptance criteria for each analytical run were met.
Only the cysteine result is reported by the application of the cysteine 1:10 reactivity prediction model (co eluting peaks). Based on this model, reactivity is classed as “no to minimal”, hence the DPRA prediction is negative.

DPRA Results

 

IIVS Test Article Number	Sponsor’s Designation	% Mean Peptide Depletion		% Mean Peptide Depletion of Cysteine and Lysine	Reactivity (Cysteine and Lysine)	Potential Sensitizer?
		Cysteine	Lysine
17AA44	Lyrame	17.02	10.07	13.55	Low	Yes
Positive Control	Cinnamic Aldehyde	74.78	61.55

Interpretation of results:

other: “low reactivity” and thus is predicted to be positive by DPRA for skin sensitization potential.

Conclusions:

Solutions of Lyrame were successfully analyzed by the validated DPRA analytical method in the Cysteine or Lysine containing synthetic peptides with all analytical acceptance criteria of the test being met.

The Mean Peptide Depletion of Cysteine and Lysine in the presence of Lyrame was 13.55% and from this result a DPRA prediction can be made. The reactivity of Lyrame with Cysteine and Lysine is classed as “low reactivity” and thus is predicted to be positive.

Executive summary:

The purpose of this study (based on the OECD guideline for the testing of chemicals, In chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA), OECD/OCDE document TG 442C) was to assess the reactivity and sensitizing potential of Lyrame.

Solutions of Lyrame were successfully analyzed by the validated DPRA analytical method in the Cysteine or Lysine containing synthetic peptides with all analytical acceptance criteria of the test being met.

The Mean Peptide Depletion of Cysteine and Lysine in the presence of Lyrame was 13.55% and from this result a DPRA prediction can be made. The reactivity of Lyrame with Cysteine and Lysine is classed as “low reactivity” and thus is predicted to be positive.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 Feb, 2017 - 21 Nov, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin sensitization tests is the KeratinoSensTM assay, which is recommended in international guidelines (e.g. OECD).

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

February 2015

Deviations:

no

GLP compliance:

yes

Type of study:

activation of keratinocytes

Details on the study design:

Receipt of Skin Sensitization Assay Reagents

Reagents used for the skin sensitization bioassay were inspected upon receipt and stored according to the manufacturer’s instructions. The DMEM (Dulbecco's Modified Eagle Medium), liquid with GlutaMAX™ I, 1000 mg/L D-Glucose, Sodium Pyruvate (Gibco) and Geneticin (G418) (Gibco) were stored at 2-8ºC. The Fetal Bovine Serum (FBS) (Atlanta Biologicals) was stored at 20 ± 5ºC. Batches of Assay Medium were prepared by supplementing DMEM with 9.1% FBS. Batches of Maintenance Medium were prepared by supplementing DMEM with 9.1% FBS and 500 µg/mL 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/EDTA solution (Gibco/Invitrogen) and 10X stock solution of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) (Sigma) were stored at -20 ± 5ºC. Dimethylsulfoxide (DMSO) (Sigma) was stored at room temperature under liquid nitrogen. The lyophilized substrate of the ONE-Glo™ assay kit (Promega) and the ONE-Glo assay buffer™ were stored at –20 ± 5ºC. The ONE-Glo™ reagent was prepared by adding the appropriate volume of reconstitution buffer to the lyophilized substrate. 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 37ºC (in a beaker containing 70% ethanol). 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 9 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 tissue-culture flask. The flask was then incubated at 37 ± 1ºC, 90 ± 10% humidity, and 5.0 ± 1% CO2 in air (standard culture conditions), until the KeratinoSens cells reached approximately 60% to 90% confluence.

Routine Culturing of the KeratinoSens Cells

When the cultures reached approximately 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/EDTA 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 T75 flask to obtain a single cell suspension and cells passaged at appropriate densities. The KeratinoSens cells were routinely passaged every 2-4 days.

Subculture of KeratinoSens Cells into 96-Well Plates

The KeratinoSens cells were subcultured into transparent Costar 96-well plates or white walled Perkin Elmer plates when the flasks were approximately 60 to 90% confluent. The flasks were rinsed and trypsinized as previously described. The cells were resuspended in 5 mL of Assay Medium per flask. The concentration of cells in suspension was determined using a Coulter Counter. A cell suspension of 1.0 x 105 cells/mL in Assay Medium was prepared. One hundred µL of the cell suspension was added to all but one well designated as a blank well (H12) (See Figure 1). The stock cell suspension was mixed often to ensure a uniform distribution of cells into each well.

Three white-walled and one transparent plate 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 solubility of the test article was tested in DMSO on the day of the initial definitive assay (at the highest 100X concentration of 200,000 µM) which was described as a clear light yellow non-viscous solution.

MTT Direct Reduction Test

The ability of the test article 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 µL of the 100X (200,000 µM) test article concentration in DMSO was added to 1 mL of the MTT solution and then incubated in the dark at 37ºC for one to three hours. One hundred µL of a negative control (e.g. DMSO) was tested concurrently. If the MTT solution color turned blue/purple, the test article was presumed to have reduced the MTT. The test article was observed to 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 an appropriate amount of Cinnamic Aldehyde into a prelabeled conical tube and adding the necessary amount of DMSO to prepare a 64,000 µM dilution. The 64,000 µM dilution was further diluted 1:10 in DMSO to prepare a 6400 µM 100X stock concentration. The final 1X concentrations of the positive control were 64, 32, 16, 8, and 4 µM. The solvent control for the test article and the positive control was 1% DMSO in the dilution solvent (1% DMEM.) Each plate included a set of 6 solvent control wells.

Testing Concentrations

The test article with a molecular weight of 343.46 g/mol was diluted based on molarity. The 100X stock dilution was prepared to a top concentration of 200,000 µM. The final 1X tested concentrations were 2000, 1000, 500, 250, 125, 62.5, 31.3, 15.6, 7.81, 3.91, 1.95, and 0.977 µM.

Positive control results:

The positive control Cinnamic Aldehyde caused a dose related induction of the luciferase activity. The EC1.5 was 12.28 µM.

Key result

Run / experiment:

other: 3 definitive assays

Parameter:

other: Mean EC1.5, uM

Value:

9.95

Positive controls validity:

valid

Remarks on result:

other: the test article Lyrame was predicted to have sensitization potential

Other effects / acceptance of results:

Criteria for Determination of a Valid Definitive Assay

The KeratinoSens assay was accepted when the positive control (cinnamic aldehyde) caused an EC1.5 value that fell within two standard deviations of the historical mean. Additionally, the results of the three definitive trials for each plate are assessed using similar criteria outlined in the validation ring trial . Those acceptance criteria included: 1) variability in DMSO solvent control wells for each definitive assay was <20%; and 2) the positive control produced a statistically significant induction above 1.5 fold below 64 µM in each definitive assay.

The test article was tested in three definitive assays. Each definitive assay included a set of 4 plates (3 for gene induction, 1 for cytotoxicity assessment). The test article was tested at 12 concentrations ranging from 0.977 to 2000 µM. The positive control, Cinnamic Aldehyde, was tested at 5 concentrations ranging from 4 to 64 µM. A summary ofthe EC1.5 (concentration for a statistically significant induction of 50% above solvent controls) andIC₅₀(concentration leading to 50% viability as compared to solvent controls) results of the definitive assays are presented in Table 1. Additional luciferase induction information (which was not used for the current prediction model) that includes the I_max(the maximal fold induction) and the CI_max(the concentration at which the maximal fold induction occurs), is also presented in Table 1. A summary graph representing the luciferase fold induction and the cell viability for each tested concentration of the test article is included in Appendix B.

 

The Induction of Antioxidant-Response-Element Dependent Gene Activity in the Keratinocyte ARE- ReporterCell Line KeratinoSens assaywas used to assess the skin sensitization potential of the test article. A test article was predicted to have sensitization potential if: 1) The EC1.5 value fell below 1000 µM 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 was predicted to be a sensitizer.

 

Although the current prediction model does not include guidance on potency, early data indicate a correlation between the EC1.5 value and potency; the lower the EC1.5 value the more potent the sensitizer, the higher the EC1.5 value the less potent the sensitizer. 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 laureth sulfate, it was found that they caused gene induction above the 1.5 threshold even though they were non-sensitizers; however at the concentration at which these strong irritants caused gene induction above the 1.5 threshold they had caused significant cell death and likely this irritation/toxicity was resulting in activation of the gene response. 

 

Deviation

The test article was stored at the room temperature upon receipt on 23 January 2017 until 26 January 2017 when the test article was moved to the 2-8ºC storage condition. This is a deviation from the SOP which states that each test article is stored under the conditions specified by the Sponsor. The Sponsor’s storage instruction for the test article was 2-8ºC. The Sponsor was notified and according to the Sponsor, the test article was suitable for use. Therefore, the study was unaffected by this deviation.

 

Table 1

Mean EC1.5, IC₅₀, I_max, CI_max 

Assay Date	IIVS Test Article	Sponsor Designation	Mean EC 1.5 (µM)	Mean IC50(µM)	Mean Imax	Mean CImax (µM)	Potential Sensitizer?
21 February 2017	17AA44	Lyrame	9.95	25.06	2.46	15.6	Yes
	Cinnamic Aldehyde	Positive Control	12.28	>64	NA	NA	Yes

NA – Not Applicable

 

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 µM in all 3 repetitions (or at least 2/3)

2) At the lowest concentration with a gene induction above 1.5, cellular viability should be greater than 70%

3) There should be an apparent overall dose response which is similar between repetitions.

 

IC₅₀– the concentration leading to 50% relative viability compared to the DMSO solvent controls

I_max– Luciferase average maximal fold induction compared to the DMSO solvent control

CI_max– Concentration where I_maxoccurred

Interpretation of results:

other: KeratinoSens assay was positive

Conclusions:

In a GLP-compliant guideline study, the test substance caused a biologically relevant induction in luciferase activity in Keratinosens assay. Based on this, the test substance is considered to give a positive result under the experimental conditions in this assay.

Executive summary:

The GLP-compliant in vitro KeratinoSens (ARE-Nrf2 luciferase reporter) assay was performed in accordance with OECD guideline 442D to assess the skin sensitising potential of the test substance. Three independent experiments were performed.  The test article was tested at 12 concentrations ranging from 0.977 to 2000 µM. Cinnamic Aldehyde was used as a positive control. The acceptance criteria were met. The Mean EC1.5 value was determined to be 9.95 uM for Lyrame. The mean maximum luciferase activity induction (Imax) was 2.46. The test item is classified as possitive in the KeratinoSens assay.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between August 2017 and Feburary 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD 442E; In vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT), July 2016.

Deviations:

no

GLP compliance:

yes

Type of study:

activation of dendritic cells

Details on the study design:

Routine Culturing of THP-1 Cells

Cryopreserved THP-1 cells, tested for and cleared of mycoplasma contamination, were stored in liquid nitrogen. The stock ampules were thawed and slowly diluted in approximately 9 mL of culture medium kept at 2-8°C. To wash the cells of cryopreservative, the cells were collected by centrifugation (200-300g, for 5 minutes, in a centrifuge set to 4°C). The rinse was repeated with the same volume of medium and centrifuge settings. After the second rinse, the cells were resuspended in an appropriate volume of culture medium warmed to approximately 37°C for the culture vessel used (typically either T25 or T75 flasks without a growth surface). The cells were maintained at 37±1ºC in a humidified atmosphere of 5±1% CO2 in air (standard culture conditions) with at least one agitation per each day. Cells were typically refed every 2-3 days with culture medium warmed to approximately 37°C until the cells became confluent enough to be passaged or transferred to a larger culture vessel.

Cells were routinely passaged every 2 to 3 days and seeded at a density of 0.1×106 to 0.2×106 cells/mL. The cells were routinely maintained at densities ranging from 0.1 to 0.8×106 cells/mL. The cell density should not exceed 1.0×106 cells/mL. Cells were propagated up to two months after thawing but not in excess of 30 passages post thawing.

The medium used for cell culturing was the RPMI 1640 formulation with 25 mM HEPES buffer and 2 mM L-glutamine (Gibco). The medium was supplemented with 10% heat-inactivated fetal bovine serum (Atlanta Biological), and 0.1% 2-mercaptoethanol (Gibco). Since the cells were suspension cells, removal from a culture vessel consisted of removing the entire cell suspension and adding the appropriate volume of cell suspension to the new culture vessel containing fresh medium, pre-warmed to 37°C. The cells were maintained at static standard culture conditions.

Reactivity Check

At least two weeks after thawing, the cells underwent a reactivity check to ensure that the cells were expressing appropriate levels of reactivity to sensitizing and non-sensitizing chemicals. Only the cells which pass the reactivity check were used in subsequent studies. Routine cell culture activities and reactivity check assay were documented in the cell culture records and briefly summarized in the study report.

Prior to an assay, cells were seeded in culture flasks at densities of 0.1 or 0.2×106 cells/mL and pre-cultured for approximately 72 or 48 hours, respectively. The culture conditions and cell density defined for this pre-assay culture conditioning were maintained as consistently as possible to ensure optimal CD54 and CD86 induction and expression. On the day of testing, cells were harvested from the culture flasks and seeded into 24-well plates, as described in the dose range finding assay section.

Solubility Testing

Prior to the use, a solubility test was performed for the test article to determine the most appropriate solvent for use in the assay. The test article was initially prepared as 500 mg/mL dilutions in saline (0.9% NaCl) and was found to be insoluble. The test article was then prepared as 500 mg/mL in dimethyl sulfoxide (DMSO) (Sigma) and was determined to be soluble.

Assay Controls

The dose range finding assay and definitive assays included the positive control, DNCB, prepared at a top stock concentration of 2 mg/mL in DMSO. The positive control was further diluted 1:250 in culture medium and then 500 µL were dosed on the cells.

The dose range assay and definitive assays also included a solvent control for the solvent used, prepared as 4 µL/mL for DMSO and dosed 500 µL on the cells. In order to correctly perform the required calculations for determining the passing criteria of the assay, a medium control was required for correcting the solvent control calculations. The medium control consisted of 500 µL of culture medium dosed on the cells.

The definitive assays also required isotype controls for each antibody stained sample. The isotype control accounted for non-specific antibody binding on the cell surface and served as a background that was subtracted from the antibody stained cells during data analysis.

Dose Range Finding Assay

The test article was tested in a valid dose range finding assay to determine the highest doses to be used in the definitive assays, which were targeted to be 1.2-fold higher than the calculated CV75 concentration (i.e., the test article concentration resulting in 75% cell viability relative to the solvent control).

Preparation of Solutions

The test and control articles were prepared on the day of testing and applied to the test system within one hour of preparation to minimize potential for chemical degradation or breakdown. The test article was dissolved up to a maximum final concentration of 500 mg/mL in DMSO.

From the initial test article dilution, 2-fold serial dilutions were prepared using the same solvent to obtain eight serial stock solutions. These stock solutions were further diluted 250-fold (for test articles diluted in DMSO) in the culture medium (2X dosing solutions). These dosing dilutions were prepared at 2X the desired final concentration so that when 500 μL of each dosing dilution are added to 500 µL of cell suspension in the 24-well plate, a 1X final dose concentration is achieved.

The solvent control was DMSO in culture medium. A single concentration of the solvent control was diluted in the same manner as described for the test article dosing solutions so that the final concentration of DMSO was 0.2% on the cells.

Preparation of the Test System

On the day of dosing, cells were collected by centrifugation (200-300g, in a centrifuge set for 5 minutes at room temperature). The cells were resuspended in fresh culture medium to a density of 2.0×10e6 cells/mL, and 500 μL of the cell suspension were seeded into the appropriate wells of a 24-well plate (resulting in 1.0×10e6 cells/well). The plates were maintained at standard culture conditions.

Test System Exposure

The 2X dosing solutions were applied to the cells by pipetting 500 μL of each of the 2X dosing solutions directly to the appropriate wells containing 500 μL of cell suspension. The treated plates were sealed with plate sealers prior to incubation (to avoid evaporation or cross-contamination of volatile test articles), and were incubated for 24±0.5 hours at standard culture conditions with at least 1 agitation per each day.

Cytotoxicity Assessment - Propidium Iodide (PI) Staining

After 24±0.5 hours of exposure, the samples were removed from the 24-well plates and added to labeled micro-centrifuge tubes. The cells were collected by centrifugation (200-300g, in a centrifuge set to 5 minutes at 4ºC). The supernatants were carefully decanted into a waste container. The remaining cell pellets were resuspended with 1 mL of FACS buffer and centrifuged again using the above centrifuge settings and decanting the supernatant. The rinsing process was performed 2 additional times using 1 mL of FACS buffer. After the three rinses, each cell pellet was resuspended in 600 µL of FACS buffer and 200 µL were transferred to the appropriate wells of a 96-well round-bottom plate. Propidium Iodide was added to the appropriate samples of the 96-well plate to make a final concentration of 0.625 µg/mL of PI in the plate.

Cytotoxicity Measurement and Calculation of CV75

The PI uptake was analyzed using flow cytometry. Cells stained with PI represented the non-viable cell population and were gated out to identify the viable populations. Approximately 10,000 living (PI negative) cells were acquired. When the cell viability was low, up to approximately 30,000 cells including dead cells were acquired. Alternatively, the data acquisition can be finished one minute after the initiation. The cell viability was calculated (e.g. PI negative events versus total events).

The CV75 value, a concentration expected to result in 75% cell viability, was calculated using the following formula:

(75-C)Log(B) - (75-A)Log(D)
Log of CV75 = –––––––––––––––––––––––––
A-C

Where:
A is the minimum value of cell viability over 75% in testing groups
C is the maximum value of cell viability below 75% in testing groups
B and D is the concentration showing the value of cell viability A and C, respectively

The CV75 value was used to calculate the test article concentrations tested in the definitive assays.


Definitive Assays

The test article was tested in five definitive assays. The results of the third definitive assay (Trial 3, performed on 29 November 2017) and fourth definitive assay (Trial 4, performed on 6 December 2017) were considered invalid because the control results were unacceptable. Only the results of the first, second, and fifth definitive assays (Trial 1 on 24 August 2017, Trial 2 on 29 August 2017, and Trial 5 on 15 January 2018) with acceptable control results were considered valid and are presented in this report.

After Trial 1 and Trial 2, an additional assay was performed as instructed by the study director to verify the sensitization potential of the test article. Later, a calculation error was identified and corrected that affected the analyzed results of Trial 1 and Trial 2. Based on correct data analysis, all three valid assays showed sensitization potential of the test article. The cells were prepared and seeded into the 24-well dosing plates in the same manner as for the dose range assay.

Test Article Dose Selection

The highest test article dose in the first and second definitive assays was 1.2×CV75 value determined from the dose range finding assay (final concentration of 87.0 µg/mL). Seven serial doses using a dilution factor of 1.2 were prepared such that eight doses ranging from 0.335×CV75 to 1.2×CV75 were tested in Trial 1 and Trial 2. In Trials 3-5, the highest test article dose was increased to 125 µg/mL as instructed by the study director. Seven serial doses using a dilution factor of 1.5 were prepared.

Preparation of Stock and 2X Dosing Solutions

The same solvent used in the dose range finding assay was used to dissolve the test articles in the definitive assays. The test articles was prepared as stock concentrations corresponding to 500-fold (for DMSO) higher than the final dose concentrations. Seven serial dilutions using a dilution factor of 1.2 to 1.5 were made using the same solvent to obtain eight serial solutions. These solutions were then further diluted 250-fold (for test articles diluted in DMSO) in the culture medium (2X dosing solutions). These dosing dilutions were prepared at 2X the desired final concentration so that when 500 μL of each dosing dilution are added to 500 μL of cell suspension in the 24-well plate, a 1X final dose concentration is achieved. The test article dilutions were exposed to the cells within one hour of preparation.

The solvent control was DMSO in culture medium. A single concentration of the solvent control was diluted in the same manner as described for the test article dosing solutions so that the final concentration of DMSO was 0.2% on the cells.

Preparation of the Test System

The preparation procedure was identical to the procedure described in the dose range finding assay section.

Test System Exposure

The exposure procedure was identical to the procedure described in the dose range finding assay section.

Staining and Analysis

After 24±0.5 hours of exposure, the samples were placed into labeled micro-centrifuge tubes and the cells were collected by centrifugation as described in the dose range finding assay section. The supernatants were carefully decanted into a waste container. The remaining cell pellets were resuspended with 1 mL of FACS buffer and centrifuged. The rinsing process was performed 2 additional times using 1 mL of FACS buffer. Finally, cells were resuspended in 600 µL of 0.01% (w/v) blocking suspension (prepared in FACS buffer from a 1% (w/v) stock suspension immediately before use) and incubated at 2-8°C in the dark for 15±1 minutes.

After the 15±1 minute incubation, the samples were divided into 3 aliquots of 180 µL. These aliquots were placed into the designated wells of a 96-well round-bottom plate. The 96-well round bottom plate was then immediately centrifuged. After centrifugation to pellet the cells, the supernatants were aspirated without disturbing the cell pellet. A master mixture of each antibody (CD54, CD86 and mouse IgG isotype control) was prepared based on the number of samples needing to be stained with each antibody so that each sample receives 50 µL of the appropriate antibody dose. For each test article dilution or control there were three cell populations each treated with a different antibody mixture. There was a separate cell population treated with FITC anti-CD54, FITC anti-CD86, and FITC isotype control. The antibody mixtures were prepared in FACS buffer using the following ratios:

3 µL of CD54 to 50 µL total
6 µL of CD86 to 50 µL total
3 µL of isotype control to 50 µL total

Fifty microliters of each antibody mixture were added to the appropriate wells of the 96-well plate. The plate was gently agitated by hand to mix the reagents and then incubated in the dark at 2-8ºC for 30±1 minutes. Following the incubation, 150 µL of FACS buffer were added to each well and the plate was centrifuged as described previously. The supernatant was aspirated and the wash step was repeated twice with 200 µL of FACS buffer. Finally, cells were resuspended in 200 µL of FACS buffer. Concentrated PI (20X) at 12.5 µg/mL was added to the appropriate wells of the 96-well plate to make a final concentration of 0.625 µg/mL of PI in each well of the plate (10.5 µL of concentrated PI in each well containing 200 µL of cell suspension).

The expression of CD54, CD86, isotype control and PI uptake was analyzed using flow cytometry. Cells stained with PI were gated out to identify the viable populations. Approximately 10,000 living (PI negative) cells were acquired. When the cell viability was low, up to 30,000 cells including dead cells can be acquired. Alternatively, the data acquisition can be finished one minute after the initiation. The cell viability was calculated (e.g. PI negative events versus total events). In addition the MFI of the antibody stained cell populations was calculated. The MFI values were used to calculate the RFI values to determine skin sensitization predictions.

Definitive Assay Data Analysis

The following plots were prepared using the flow cytometry software:

Side Scatter (SSC) versus Forward Scatter (FSC)
FSC is a measure of cell size. SSC is a measure of cell granularity. This plot is created to confirm a single population of cells is present without excessive debris.

2 Histogram Plots (Cell Count versus PI) (Cell Count versus FITC)
These plots were used to determine the percentage of each cell population expressing PI (for cell viability) or FITC (for upregulation of CD54 and CD86).

A gate was placed between the peak of the PI negative fraction and the PI positive fraction on the histogram using the DNCB-treated isotype control cells. The PI negative fraction corresponds to living cells which were used for subsequent analysis. The MFI of the living populations of each cell sample was determined by the software and used in the following formula to determine the RFI values for each test article treated sample.

MFI of test article treated cells − MFI of test article treated isotype control cells
RFI = –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
MFI of solvent treated control cells − MFI of solvent treated isotype control cells

The isotype controls consisted of the same test article concentrations tested for the CD54 and CD86 staining, but these samples were treated with isotype control consisting of mouse IgG. Use of the isotype control allowed for the distinction between specific CD54 and CD86 antibody binding and non-specific background antibody binding.

Prediction Model

The test article was tested in three valid definitive assays to derive a single prediction (skin sensitizer or non-sensitizer). The definitive assays may be performed on the same day provided that for each assay: a) independently harvested cells were used (i.e. cells collected from different culture flasks), and b) independent fresh stock solutions of the 2X dosing solutions of the test articles and antibodies were prepared.

If the RFI of CD86 was equal to or greater than 150 at any tested dose with >50% cell viability in at least two independent assays and/or if the RFI of CD54 was equal to or greater than 200 at any tested dose with >50% cell viability in at least two independent assays, the prediction was considered as positive (sensitizer). Otherwise, the prediction was considered as negative.

For test articles considered to be sensitizers, two effective concentrations (EC) values, the EC150 for CD86 and EC200 for CD54 were calculated using the following formulas. These values were calculated from 2 consecutive doses starting from the lowest concentration. The EC values represented the calculated test article concentration at which an RFI of 150 or 200 is achieved.

EC150 (for CD86) = Bdose + [(150-BRFI)/(ARFI - BRFI)(Adose-Bdose)]

EC200 (for CD54) = Bdose + [(200-BRFI)/(ARFI - BRFI)(Adose-Bdose)]

Where:
Adose is the lowest concentration in µg/mL with RFI ≥150 (CD86) or 200 (CD54)
Bdose is the highest concentration in µg/mL with RFI <150 (CD86) or 200 (CD54)
ARFI is the RFI value associated with Adose
BRFI is the RFI value associated with Bdose

Criteria for Determination of a Valid Test

The assay results were accepted if the following criteria were met. The cell viability values of the solvent control(s) were > 90%. For the solvent control(s), RFI values of both CD86 and CD54 were less than the positive criteria (CD86 RFI <150 and CD54 RFI <200). For the positive control (DNCB), RFI values of both CD86 and CD54 were predicted to be positive (CD86 RFI ≥150 and CD54 RFI ≥200), and cell viability was >50%. For the medium and solvent controls, the MFI ratio of both CD86 and CD54 to isotype control were >105%. The cell viability of the test article-treated cultures were >50% in at least four doses.

Positive control results:

The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50%.

Key result

Run / experiment:

other: 3 independent runs

Parameter:

other: EC200 of CD54 RFI, ug/mL

Value:

33.9

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Interpretation of results:

other: the test article Lyrame was predicted to be a potential sensitizer

Conclusions:

Based on the results of the definitive assays, the test article, Lyrame, was classified as a potential sensitizer.

Executive summary:

The Human Cell Line Activation Test (h-CLAT) was used to determine the skin sensitization potential of the test article, Lyrame, by measuring the upregulation of cell surface markers, CD54 and CD86 on the surface of human monocytes (THP-1) after a 24 hour exposure to eight concentrations of each test article. The upregulation of the surface markers along with the cell viability was measured using flow cytometry after staining with fluorescently labeled antibodies for the surface markers and viability staining with PI.

Based on the results of the definitive assays, the test article, Lyrame, was classified as a potential sensitizer.

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

The skin sensitization potential is assessed with in silico and in vitro methods and is shortly summarized. Thereafter the executive summaries of the individual in vitro studies are presented.

Summary of information:

In silico: OECD Toolbox and Derek Nexus identify no structural alerts for skin sensitization.

In vitro: The substance tested was positive in the DPRA, positive in KeratinoSens assay, and positive in h-CLAT.

Overall conclusion: The test substance has a skin sensitization potential based on the key information of three in vitro skin sensitization tests, all of which were positive. Even though OECD Toolbox and Derek Nexus do not identify any alerts for skin sensitization, based on the in vitro testing results the substance is considered positive for skin sensitization.

DPRA:

The purpose of this study (based on the OECD guideline for the testing of chemicals, In chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA), OECD/OCDE document TG 442C) was to assess the reactivity and sensitizing potential of Lyrame.

Solutions of Lyrame were successfully analyzed by the validated DPRA analytical method in the Cysteine or Lysine containing synthetic peptides with all analytical acceptance criteria of the test being met.

The Mean Peptide Depletion of Cysteine and Lysine in the presence of Lyrame was 13.55% and from this result a DPRA prediction can be made. The reactivity of Lyrame with Cysteine and Lysine is classed as “low reactivity” and thus is predicted to be positive.

KeratinoSens:

The GLP-compliant in vitro KeratinoSens (ARE-Nrf2 luciferase reporter) assay was performed in accordance with OECD guideline 442D to assess the skin sensitising potential of the test substance. Three independent experiments were performed. The test article was tested at 12 concentrations ranging from 0.977 to 2000 µM.Cinnamic Aldehyde was used as a positive control. The acceptance criteria were met. The Mean EC1.5 value was determined to be 9.95 uM for Lyrame. The mean maximum luciferase activity induction (Imax) was 2.46. The test item is classified as positive in the KeratinoSens assay.

h-CLAT:

The Human Cell Line Activation Test (h-CLAT) was used to determine the skin sensitization potential of the test article, Lyrame, by measuring the upregulation of cell surface markers, CD54 and CD86 on the surface of human monocytes (THP-1) after a 24 hour exposure to eight concentrations of each test article. The upregulation of the surface markers along with the cell viability was measured using flow cytometry after staining with fluorescently labeled antibodies for the surface markers and viability staining with PI.

Based on the results of the definitive assays, the test article, Lyrame, was classified as a potential sensitizer.

Justification for classification or non-classification

Based on the presented information the substance needs to be classified as a skin sensitiser Category 1 according to EU CLP (EC 1272/2008 and its amendments)