Reaction mass of (E)-1-(3,6-dimethylcyclohex-3-en-1-yl)-2-methylpent-1-en-3-one and (E)-1-(4,6-dimethylcyclohex-3-en-1-yl)-2-methylpent-1-en-3-one

Endpoint summary

Description of key information

Skin sensitisation: DPRA (OECD TG 442C): Predicted non-sensitiser.

Skin sensitisation: ARE-Nrf2 Luciferase Test (KeratinoSens™) (OECD TG 442D): Predicted non-sensitiser.

Skin sensitisation: h-CLAT (OECD TG 442E): Predicted sensitiser.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

The study was conducted between 08 January 2019 and 11 January 2019.

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 (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

The DPRA test allows quantification of a chemical’s reactivity and is used to categorize a substance in one of four classes of reactivity to allow discriminating between skin sensitizing and non-sensitizing chemicals and thus assesses their sensitization potential.

Specific details on test material used for the study:

Identification PG-RAW-90-032
Appearance: Clear liquid
Storage conditions: Refrigerated temperature (2°C to 8°C)

Details on the study design:

Peptide and Positive Control
Synthetic peptide containing Cysteine
Alternative name: Ac-RFAACAA-OH
Batch number: 1857724
Stated purity: 96.2% (by HPLC)
Molecular Weight: 751 g/mol
Supplier: AnaSpec
Storage conditions: Frozen (-10°C to -30°C)

Synthetic peptide containing Lysine
Alternative name: Ac-RFAAKAA-OH
Batch number: 1658141
Stated purity: 96.8% (by HPLC).
Molecular Weight: 776 g/mol
Supplier: AnaSpec
Storage conditions: Frozen (-10°C to -30°C)

Cinnamic Aldehyde (Positive control)
Batch number: MKCB9907
Stated purity: 99.1%
Molecular Weight: 132 g/mol
Supplier: SAFC
Storage conditions: Room temperature (15°C to 25°C)
Expiry/retest date: November 2021

Apparatus
High performance liquid chromatograph (HPLC): Waters Alliance 2695 separation module and 2487 dual wavelength detector
Balances fitted with printers: Capability of weighing to 5 decimal places
General laboratory apparatus and glassware.

Analytical Procedure
Reagents
Acetonitrile (ACN): HPLC gradient grade
Trifluoroacetic acid (TFA): 99% Pure
Water: Deionised reverse osmosis
Ammonium Acetate: Analytical reagent
Sodium Phosphate, monobasic: Analytical reagent
Sodium Phosphate, dibasic: Analytical reagent
Ammonium Hydroxide: Analytical reagent
100 mM Phosphate buffer, pH 7.5: In house preparation
100 mM Ammonium Acetate buffer, pH 10.2: In house preparation
HPLC Mobile Phase A: 0.1% v/v TFA in Water, in house preparation
HPLC Mobile Phase B: 0.085% v/v TFA in ACN, in house preparation

Assessment of Test Item Solubility
The solubility of PG-RAW-90-032 was assessed in acetonitrile at a nominal concentration of 100 mM.

Preparation of Peptide Stock Solutions
Stock solutions of each peptide at concentrations of 0.667 mM were prepared by dissolution of pre-weighed aliquots of the appropriate peptide in ca 20 mL aliquots of the appropriate buffer solution (Cysteine in 100 mM phosphate buffer pH 7.5, Lysine in 100 mM Ammonium acetate buffer pH 10.2).

Preparation of Peptide Calibration Standards
Calibration standards of both peptides were prepared by diluting the requisite stock solution in the appropriate buffer and acetonitrile and contained each peptide at concentrations of 0.0167 mM, 0.0334 mM, 0.0667 mM, 0.133 mM, 0.267 mM and 0.534 mM. A buffer blank was also prepared.

Preparation of Stability Controls and Precision Control
Stability controls (Reference Control B), precision controls of both peptides were prepared at a concentration of 0.5 mM in acetonitrile/buffer.

Preparation of Positive Control Solution and Test Item Stock Solution
The positive control chemical (Cinnamic Aldehyde) was prepared at a concentration of 100 mM in acetonitrile. A 100 mM stock solution of PG-RAW-90-032 was also prepared in acetonitrile.

Preparation of Positive Control and Cysteine Peptide Depletion Samples and Co-elution Controls
Triplicate solutions each of the positive control and PG-RAW-90-032 stock solutions were diluted with the Cysteine peptide stock solution so as to prepare solutions containing 0.5 mM Cysteine and 5 mM of Cinnamic Aldehyde or 5 mM PG-RAW-90-032. For the co-elution control, buffer solution was used in place of the Cysteine stock solution.

Preparation of Positive Control and Lysine Peptide Depletion Samples and Co-elution Controls
Triplicate solutions each of the positive control and PG-RAW-90-032 stock solution were diluted with the Lysine peptide stock solution so as to prepare solutions containing 0.5 mM Lysine and 25 mM of Cinnamic Aldehyde or 25 mM PG-RAW-90-032. For the co-elution control, buffer solution was used in place of the Lysine stock solution.

Incubation
The appearance of the PG-RAW-90-032 and positive control samples in the HPLC vials was documented following preparation and then the vials placed into the autosampler of the HPLC set at 25°C for a minimum of 22 hours incubation prior to initiation of the analysis run. Prior to initiation of the run the appearance of the samples in the vials was assessed and documented again.

Analysis
The concentration of both the Cysteine and Lysine peptides in the presence of PG-RAW-90-032 and the associated positive controls was quantified by HPLC using UV detection as detailed in the chromatographic section.

Instrumentation Parameters
High performance liquid chromatograph (HPLC): Waters Alliance 2695 separation module and 2487 dual wavelength detector
Column: Agilent Zorbax SB C18, 3.5 µm, 100 x 2.1 mm
Guard column: Phenomenex AJO4286
Column temperature: 30 °C
Sample temperature: 25 °C
Mobile Phase (MP) A: 0.1% TFA in Water
Mobile Phase (MP) B: 0.085% TFA in ACN
Gradient: Time (minutes) MP A (%) MP B (%)
0 90 10
20 75 25
21 10 90
23 10 90
23.5 90 10
30 90 10
Flow rate: 0.35 mL/minute
Stroke volume 25 µL
Detector wavelength: UV, 220 nm
Injection volume: 2 µL (slow draw rate)
Run time: 30 minutes
Approximate retention time (Cysteine) 11 minutes
Approximate retention time (Lysine) 7 minutes

Run / experiment:

mean

Parameter:

other: % Overall mean peptide depletion (reactivity)

Value:

0.622

Positive controls validity:

valid

Solubility Assessment

Solubility of PG-RAW-90-032 was achieved at a nominal concentration of 100 mM in acetonitrile.

Reactivity Assessment

All analytical acceptance criteria for each peptide run were met:

		Peptide		Standard Linearity		Positive control depletion (%)		Reference controls		Test item
Acceptance criteria		Cysteine		r^2>0.99		60.8-100 (SD <14.9%)		0.45-0.55 mM (CV <15%)		SD <14.9%
		Lysine		r^2>0.99		40.2-69.0 (SD <11.6%)		0.45-0.55 mM (CV <15%)		SD <11.6%
Achieved results		Cysteine		r^2>0.999		71.6 (SD, 0.16%, n=3)		B: 0.500 mM (CV 0.48%, n=6)		SD 0.12% (n=3)
		Lysine		r^2>0.999		57.1 (SD, 0.68%, n=3)		B: 0.500 mM (CV 0.66%, n=6)		SD 0.67% (n=3)

CV         Coefficient of Variation

SD         Standard deviation

The depletion of peptide in the presence of PG-RAW-90-032 was:

Peptide	Reference Control	Mean peak area of peptide with test item(µV.sec)	Mean peptide depletion by PG-RAW-90-032 (%)
Cysteine	Control B: 924280 (n=6)	912780 (n=3)	1.24
Lysine	Control B: 797070 (n=6)	807570 (n=3)	-1.32

Applying the following reactivity prediction depletion model (below), reactivity of PG-RAW-90-032 is classed as “no or minimal” and the DPRA prediction is therefore negative and is therefore predicted not to be a potential skin sensitizer. 

Mean of cysteine and lysine% depletion	Reactivity Class	DPRA Prediction
0%≤ mean% depletion ≤6.38%	No or minimal reactivity	Negative
6.38%< mean% depletion ≤22.62%	Low reactivity	Positive
22.62%< mean% depletion ≤42.47%	Moderate reactivity
42.47%< mean% depletion ≤100%	High reactivity

There were no co-elution peaks in either the Cysteine or Lysine assay.   

Overall Achieved Depletion Values

Test item		Cysteine peptide depletion (%)		Lysine peptide depletion (%)		Overall mean depletion (%)		Reactivity class		DPRA prediction
PG-RAW-90-032		1.24		0.00^1		0.622		Minimal		Negative

¹          For calculation of overall mean depletion a negative value counts as zero

Individual Achieved Depletion Values

Cysteine Peptide Depletion

Sample	Peak area (µV.sec)	Peptide concentration^1(µg/mL)	Peptide Depletion^2(%)	Mean Depletion (%)	SD  (%)
Positive control	260938	106	71.8	71.6	0.16
	263778	107	71.5
	263076	107	71.5
PG-RAW-90-032	911464	370	1.39	1.24	0.12
	913600	371	1.16
	913262	371	1.19

SD      Standard Deviation

¹        Samples prepared at a nominal concentration of 376 µg/mL (0.5 mM)

²        Calculated against a mean Reference Control (B) area of 924280 µV.sec (n=6)

Lysine Peptide Depletion

Sample	Peak area (µV.sec)	Peptide concentration^1(µg/mL)	Peptide Depletion^2(%)	Mean Depletion (%)	SD  (%)
Positive control	336270	164	57.8	57.1	0.68
	341481	167	57.2
	347028	169	56.5
PG-RAW-90-032	808183	394	-1.39	-1.32	0.67
	812568	396	-1.94
	801959	391	-0.613

SD      Standard Deviation

¹         Samples prepared at a nominal concentration of 388 µg/mL (0.5 mM)

²         Calculated against a mean Reference Control (B) area of 797070 µV.sec (n=6)

Interpretation of results:

other: Predicted to be negative

Remarks:

Criteria used: EU CLP

Conclusions:

Solutions of PG-RAW-90-032 were successfully analyzed by the validated DPRA analytical method (Envigo analytical method FIA/M101/15) in both the Cysteine and Lysine containing synthetic peptides. With overall mean peptide depletion (reactivity) of 0.622% in the presence of the test item, PG-RAW-90-032 is only minimally reactive and is predicted by DPRA as negative and therefore unlikely to be a potential skin sensitizer based on this assay.

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 PG-RAW-90-032. 

Solutions of PG-RAW-90-032 were successfully analyzed by the validated DPRA analytical method (Envigo analytical method FIA/M101/15) in both the Cysteine and Lysine containing synthetic peptides. There was minimal reactivity of both peptides in the presence of PG-RAW-90-032. With an overall depletion of peptide of 0.622% the reactivity of PG-RAW-90-032 is henceforth classified as “no or minimal” therefore the DPRA prediction is negative. PG-RAW-90-032 is likely not to be a potential skin sensitizer based on this assay. 

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

The study was conducted between 10 December 2018 and 10 January 2019.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

The KeratinoSens™ in vitro skin sensitisation assay uses an immortalised adherent cell line derived from HaCaT human keratinocytes stably transfected with a selectable plasmid. The cell line contains the luciferase gene under the transcriptional control of a constitutive promoter fused with an ARE element from a gene that is known to be up-regulated by contact sensitisers. The luciferase signal reflects the activation by sensitisers of endogenous Nrf2 dependent genes, and the dependence of the luciferase signal in the recombinant cell line on Nrf2 has been demonstrated. This allows quantitative measurement (by luminescence detection) of luciferase gene induction, using well established light producing luciferase substrates, as an indicator of the activity of the Nrf2 transcription factor in cells following exposure to electrophilic test chemicals.

Specific details on test material used for the study:

Test item: PG-RAW-90-032
Storage conditions: Refrigerated (2 - 8˚C), in the dark
Appearance: Clear liquid

Details on the study design:

Cell Culture
The cells used in this assay were the transgenic cell line KeratinoSens™ with a stable insertion of the luciferase construct supplied by Givaudan (Dubendorf, Switzerland). The cells were routinely grown and subcultured in maintenance medium at 37°C ± 2°C in a humidified atmosphere containing 5% CO2 in air. Maintenance medium was 500 mL Dulbecco’s Modified Eagles Medium containing Glutamax (DMEM) (Gibco 21885), supplemented with 50 mL foetal bovine serum (FBS) (Gibco 10270) and 5.5 mL Geneticin (Gibco 10131).

Cell Culture from Frozen Stocks
Vials of KeratinoSens™ cells, stored frozen in cryotubes at -196°C under liquid nitrogen, in DMEM containing 10% dimethyl sulphoxide and 20% FBS, were thawed rapidly at 37°C in a water-bath. The cells were then resuspended in a total of 10 mL of pre-warmed maintenance medium without geneticin and pelleted by centrifugation at 125 g for 5 minutes. The cell pellet was resuspended in maintenance medium without geneticin in tissue culture flasks. The flasks were incubated until 80-90% confluent cell monolayers had been obtained. Geneticin-containing medium was used in subsequent passages. Establishing cell cultures from frozen stocks and subsequent passage was conducted prior to the start of this study.

Cell Passage
Actively growing cell stocks were maintained and expanded by subculturing (passage). When the cells had reached 80 – 90% confluence, the medium from each flask was removed, the cells washed twice with Dulbecco’s phosphate buffered saline (DPBS) (Gibco 14190) and harvested using trypsin-EDTA solution. Cultures were incubated at 37 ± 2°C in a humidified atmosphere containing 5% CO2 in air until complete detachment and disaggregation of the cell monolayer had occurred. The cells were then resuspended in medium to neutralise the trypsin (cells from several flasks were pooled at this point). The cells were resuspended and distributed into flasks containing fresh maintenance medium. This passage procedure was repeated to provide a sufficient number of cells for a test, and were passaged at least twice before using the cells in a test. The passages of KeratinoSens™ cells were limited to 25 passages from frozen stock.

Preparation of Test Cell Cultures
The cells from flasks of actively growing cultures were detached and disaggregated as described above. The number of viable cells in the prepared cell suspension were determined by counting a trypan blue-stained cell preparation using an Improved Neubauer Haemocytometer. The cell suspension was diluted with maintenance medium without geneticin to give 1 x 10^5 viable cells/mL and 100 µL volumes pipetted into all wells except one well of sterile 96-well flat-bottomed microtitre plates which received 100 µL maintenance medium without geneticin with no cells. On each occasion four plates were prepared in parallel: three white plates for measuring luminescence and one transparent plate for measuring cell viability using the MTT assay. The plates were incubated for 24 ± 2 hours at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air, to allow the cells to attach.

Preparation of the Positive Control
Cinnamic aldehyde (Sigma, 239968, lot: STBG0250V, expiry: July 2020) was prepared by weighing between 20 – 40 mg into a tared glass container and diluted to a final concentration of 200 mM in DMSO using the following formula:
V = 5 x (((p / 100) x w) / MW) - w / 1000
Where
V = volume of DMSO in mL to be added
p = purity of the chemical in %
MW = molecular weight of the chemical in g/mol
w = exact weight of the chemical added to the vial in mg
The 200 mM cinnamic aldehyde solution was further diluted to a final concentration of 6.4 mM by adding 32 µL of the 200 mM solution to 968 µL of DMSO.
Preparation of the positive control was shared with other studies performed in the same assay.

Test Item Solubility
Prior to commencing testing, the solubility of the test item, PG-RAW-90-032, in DMSO was assessed.
The test item, PG-RAW-90-032, was found to be soluble in DMSO at 200 mM, the highest concentration as recommended by the guideline this test follows.

Preparation of the Test Item
A stock solution of the test item, PG-RAW-90-032, was prepared by weighing the test item into a tared glass container and diluting to 200 mM in DMSO using the formula above.

Preparation of the Dilution Plate
The 100x solvent plate was replicated into a fresh 96 well plate by adding 240 µL of assay medium to each well and then 10 µL solution per well from the 100x solvent plate was added to equivalent wells on the dilution plate. Assay medium was 495 mL DMEM (Gibco 21885), supplemented with 5.0 mL FBS.

Treatment of Cultured Plates
Approximately 24 hours after the test cell culture plates were established, the medium was removed from the wells by careful inversion of the plates and blotting onto sterile paper towel. 150 µL of assay medium was added to every well of the 96 well plates. 50 µL from each well of the dilution plate was transferred to equivalent wells in the 96 well plates. Three white plates were dosed for measuring luminescence and one transparent plate for measuring cell viability using the MTT assay.
The plates were then covered with a plate seal and placed in the incubator at 37 ± 2°C, in a humidified atmosphere of 5% CO2 in air for 48 ± 2 hours.

Cell Viability Measurement
After incubation, the transparent plate was removed from the incubator and the plate seal discarded. The cell culture medium was removed by careful inversion of the plate and blotted onto sterile paper towel to remove residual culture medium. 100 µL fresh assay medium was added to each well. 10 µL of MTT solution (5 mg/mL in PBS) was added to each well of the 96-well plate. The plate was incubated at 37 ± 2C in a humidified atmosphere of 5% CO2 in air for 4 hours ± 10 minutes. The medium was then removed by careful inversion of the plate and blotted onto sterile paper towel to remove residual culture medium. 50 µL of DMSO was added to each well. The plate was then placed in the incubator at 37 ± 2°C, in a humidified atmosphere of 5% CO2 in air, protected from light, for at least 10 minutes. The absorbance value of each well was read using a plate reader with a 540 nm filter.

Luciferase Measurement
Luciferase was measured using the Steady Glo® Luciferase Assay system kit supplied by Promega (E2550). Steady-Glo® luciferase reagent was prepared by transferring the contents of one bottle of Steady-Glo® buffer to one bottle of Steady-Glo® substrate. The reagent was mixed by inversion until the substrate had dissolved. The reconstituted reagent was used on the same day it was prepared for test 2. Frozen reconstituted reagent was used for test 1 and was thawed to room temperature before use.
After incubation the medium was removed from the wells of the triplicate white plates by careful inversion of the plates and blotting on sterile absorbent paper. 100 µL of fresh assay medium was added to each well before 100 µL of Steady-Glo® luciferase reagent was added to each well of the plate. The plates were shaken on a plate shaker for at least 5 minutes until the cells had lysed. Luminescence (emitted light) was measured using a SpectraMax L luminometer. Each plate was read for total photon count with an integration time of 1 second. The plates were dark adapted for 1 minute prior to measurement.

Number of Tests Required
Two independent tests each containing three replicates (i.e. n=6) were required to make a conclusion. Each independent test was performed on a different day with fresh stock solutions of the chemicals and independently harvested cells.

Positive control results:

The luciferase activity induction obtained with the positive control, cinnamic aldehyde, was statistically significant above the threshold of 1.5 in at least one of the tested concentrations (4 to 64 μM) in both tests.
The EC1.5 values of the positive control, cinnamic aldehyde, were 13.26 μM and 8.36 μM for test 1 and 2, respectively, which lay within the historical control range for this laboratory. The average induction in the three replicates for cinnamic aldehyde at 64 µM were 4.64 and 6.36 for test 1 and 2, respectively, which met the acceptance criterion of between 2 and 8.

Run / experiment:

other: 1

Parameter:

other: Imax

Value:

1.25

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: predicted not skin sensitizing

Run / experiment:

other: 2

Parameter:

other: Imax

Value:

1.05

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: predicted not skin sensitizing

Run / experiment:

other: 1

Parameter:

other: IC50

Remarks:

in µM

Value:

43.08

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: predicted not skin sensitizing

Run / experiment:

other: 2

Parameter:

other: IC50

Remarks:

in µM

Value:

49.75

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: predicted not skin sensitizing

Other effects / acceptance of results:

The average coefficient of variation of the luminescence reading for the negative solvent control (DMSO) was 8.6% and 11.8% for test 1 and 2, respectively, which met the acceptance criterion of below 20%.

Results for PG-RAW-90-032 – Test 1

Test item conc. (µM)	0.98	1.95	3.91	7.81	15.63	31.25	62.5	125	250	500	1000	2000
Mean fold induction	1.01	1.07	1.07	1.08	1.12	1.25	0.01	0.00	0.00	0.00	0.00	0.05
Statistically significant	No	No	No	No	No	No	No	No	No	No	No	No
Viability (%)	125.15	117.33	105.72	106.80	110.24	110.72	3.17	3.31	3.37	2.90	3.10	2.63
Imax	1.25
EC1.5(µM)	N/A
IC30(µM)	43.08
IC50(µM)	48.89

 

Determination criteria for the skin sensitisation potential of the test item	Result
Is the Imax>1.5 fold and statistically significant	No
Is the cellular viability >70% at the lowest concentration at the EC1.5 determining concentration	N/A
Is the EC1.5 value <1000µM	N/A
Is there an apparent overall dose-response for luciferase induction	No
KeratinoSens™ prediction	Negative

 

Results for Cinnamic Aldehyde – Test 1

Positive control conc. (µM)	4	8	16	32	64
Mean fold induction	1.38	1.22	1.64	2.00	4.64
Statistically significant	No	No	Yes	Yes	Yes
Viability (%)	103.50	103.56	103.56	107.07	102.08
Imax	4.64
EC1.5 (µM)	13.26
IC30 (µM)	N/A
IC50 (µM)	N/A

 

Test Acceptance Criteria	Result
Luciferase activity induction obtained with the positive control statistically significant above the threshold of 1.5 in at least one of the test concentrations	Yes	Pass
Average induction of positive control at 64 µM between 2 – 8	Yes (4.64)	Pass
EC1.5 of positive control within two standard deviations of the historical mean (-5.71 to 38.79)	Yes (13.26)	Pass
CV% of blank values < 20%	Yes (8.6%)	Pass

Results for PG-RAW-90-032 – Test 2

Test item conc. (µM)	0.98	1.95	3.91	7.81	15.63	31.25	62.5	125	250	500	1000	2000
Mean fold induction	0.96	0.92	0.97	0.96	1.01	1.05	0.14	0.00	0.00	0.00	0.00	0.00
Statistically significant	No	No	No	No	No	No	No	No	No	No	No	No
Viability (%)	101.25	99.66	95.33	108.64	110.51	118.10	3.06	3.26	3.59	3.13	3.26	3.00
Imax	1.05
EC1.5 (µM)	N/A
IC30 (µM)	44.32
IC50 (µM)	49.75

 

Determination criteria for the skin sensitisation potential of the test item	Result
Is the Imax>1.5 fold and statistically significant	No
Is the cellular viability >70% at the lowest concentration at the EC1.5 determining concentration	N/A
Is the EC1.5 value <1000µM	N/A
Is there an apparent overall dose-response for luciferase induction	No
KeratinoSens™ prediction	Negative

Results for Cinnamic Aldehyde – Test 2

Positive control conc. (µM)	4	8	16	32	64
Mean fold induction	1.23	1.48	1.99	2.87	6.36
Statistically significant	No	No	Yes	Yes	Yes
Viability (%)	116.70	108.04	107.51	108.04	101.85
Imax	6.36
EC1.5 (µM)	8.36
IC30 (µM)	N/A
IC50 (µM)	N/A

 

Test Acceptance Criteria	Result
Luciferase activity induction obtained with the positive control statistically significant above the threshold of 1.5 in at least one of the test concentrations	Yes	Pass
Average induction of positive control at 64 µM between 2 – 8	Yes (6.36)	Pass
EC1.5of positive control within two standard deviations of the historical mean (-5.71 to 38.79)	Yes (8.36)	Pass
CV% of blank values < 20%	Yes (11.8%)	Pass

Interpretation of results:

other: predicted as not skin sensitizing

Remarks:

Criteria used: EU CLP

Conclusions:

It was concluded that PG-RAW-90-032 gave a negative response in the ARE-Nrf2 Luciferase Test (KeratinoSens™), supporting the prediction that PG-RAW-90-032 is not a skin sensitizer.

Executive summary:

The purpose of this study was to support a predictive, adverse-outcome-pathway evaluation of whether the test item, PG-RAW-90-032, is likely to be a skin sensitizer using the OECD TG 442D: ARE-Nrf2 Luciferase Test (KeratinoSens™).

The I_max for PG-RAW-90-032 was 1.25 in test 1 and 1.05 in test 2. The I_max for both tests was <1.5 fold compared to the DMSO control and therefore the EC_1.5 could not be calculated. The cellular viability fell below 70% in both tests. The IC₃₀value was 43.08 µM in test 1 and 44.32 µM in test 2 and the IC₅₀values were 48.89 µM and 49.75 µM in tests 1 and 2, respectively. Graphs for PG-RAW-90-032 showed no overall dose-response for luciferase induction.

All acceptance criteria for the positive control, cinnamic aldehyde,were met.

It was concluded that PG-RAW-90-032 gave a negative response in the ARE-Nrf2 Luciferase Test (KeratinoSens™), supporting the prediction that PG-RAW-90-032 is not a skin sensitizer.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

The study was conducted between 28 January 2019 and 15 March 2019.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for the Testing of Chemicals: OECD 442E: In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT), June 2018

Deviations:

yes

Remarks:

These deviations to the study plan, however, do not affect the validity of the study.

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Justification for non-LLNA method:

An alternative method has been developed for the evaluation of the skin sensitisation potential by measuring phenotypic changes, such as CD86 and CD54 expression on dendritic cells. The human leukemia cell line THP-1 is used as a surrogate for human myeloid dendritic cells, since these cells also show enhanced CD86 and/or CD54 expression when treated with sensitisers.

Specific details on test material used for the study:

Identification: PG-RAW-90-032
Partition coefficient (n-octanol/water): log Pow: 4.88 (EPISuite modeling)
Water solubility: 2.767 mg/L (EPISuite modeling)
Appearance: Colourless liquid
Storage Conditions: At +2 to +8 °C, light protected
Stability in Solvent: Stable in water (not quantified)

Details on the study design:

Controls for Cytotoxicity Test and h-CLAT
Concurrent controls were used for several Envigo CRS GmbH studies performed simultaneously.

Medium Control and Solvent Control for the Test Item
Name: Culture medium

Positive Control (h-CLAT)
Name: DNCB (2,4-dinitrochlorobenzene, CAS No.: 97-00-7) final concentration: 3 and 4 µg/mL, Purity ≥ 99%)
Solvent: DMSO

Solvent Control for the Positive Control (h-CLAT)
Name: DMSO (Dimethyl sulfoxide, CAS No. 67-68-5) in culture medium, final concentration 0.2%, Purity ≥ 99%

TEST ITEM PREPARATION
On the day of the experiment (prior to start) PG-RAW-90-032 was prepared in culture medium which formed a stable suspension/dispersion.
As tested by a solubility test, 5000 µg/mL in culture medium (the OECD 442E guideline recommended maximal to be tested test item concentration) was used as highest test item concentration in the cytotoxicity test.
For the cytotoxicity test (dose finding assay) eight concentrations of the test item were analysed. For this, dilutions were prepared by 1:2 serial dilutions.

TEST SYSTEM AND SUPPORTING INFORMATION
Reasons for the Choice of THP-1 Cells
THP-1 cells (Human monocytic leukemia cell line) were purchased from ATCC, #TIB-202. THP-1 cells are used as a surrogate for human myeloid dendritic cells, because the THP-1 cells also show enhanced CD86 and/or CD54 expression when treated with sensitisers.

THP-1 Cell Cultures
Stocks of the THP-1 cell line are stored in liquid nitrogen in the cell bank of Envigo CRS GmbH (aliquots of cells in freezing medium at 1 x 10^6 to 2 x 10^6 cells/mL) allowing the repeated use of the same cell culture batch in experiments. Therefore, the parameters of the experiments remain similar, because of the reproducible characteristics of the cells. Thawed stock cultures are propagated at 37 ± 1.5 °C in plastic flasks. The cells are sub-cultured twice weekly. The cell density should not exceed 1 x 10^6 cells/mL. The THP-1 cell suspension is incubated at 37 ± 1.5 °C and 5.0 ± 0.5 % carbon dioxide atmosphere. Cells can be used up to two months after thawing (passage number should not exceed 30).
The passage numbers of the used THP-1 cells were 15 and 16 in the cytotoxicity tests and 20, 22, 26, 28, 17 and 20 in the h CLAT for runs 1 to 6, respectively.

Culture Medium
RPMI 1640 Medium, GlutaMAXTM Supplement including 25 mM HEPES, supplemented with 10 % FBS (v/v), 0.05 mM 2 mercaptoethanol, 4.5 g/L glucose, 1% (v/v) sodium pyruvate and appropriate antibiotics (100 U/mL of penicillin and 100 µg/mL of streptomycin) is used to culture the cells during the assay. Medium with supplements has to be stored at 2 - 8 °C and used within one month. The culture medium has to be warmed to room temperature just before use.

Preparation and Seeding of THP-1 Cells
On the day of the cytotoxicity or main experiment (h-CLAT) directly before the treatment of the cells, a volume of 500 µL with a cell density of 1.8 - 2 x 10^6 THP-1 cells/mL was seeded in each corresponding well of a 24-well flat bottom plate.
 
Experimental Design and Procedures of the Cytotoxicity Test
Dose Finding Assay (Flow cytometer)
The test item concentrations investigated in the main experiment (h-CLAT) were determined with two cytotoxicity tests. The tests were performed on different days. The test item was prepared separately for each run.

Treatment of the Cells
The test item dilutions were prepared freshly before each experiment.
Each volume (500 µL) of the dilutions of the test item and culture medium was added to the cells.
The treated THP-1 cells were incubated for 24 ± 0.5 hours. All dose groups were tested in one replicate for each cytotoxicity test. At the end of the incubation period, the cell cultures were microscopically evaluated for morphological alterations.
Each concentration of the test item, culture medium and solvent control were prepared for the 7-AAD staining.

Staining of the Cells
Each test item-treated and not test item treated cells were collected in sample tubes centrifuged (approx. 250 x g, 5 min), washed twice (2 - 8 °C) with 2 mL FACS buffer (PBS with 0.1% (w/v) BSA) and re-suspended in a final volume of 2 mL/tube FACS buffer. At least 10 minutes before the flow cytometry acquisition, 5 µL of a 7-AAD solution were added in each sample tube.

Flow Cytometry Acquisition (Cytotoxicity Test)
Before using the flow cytometer (FACSCalibur, Becton Dickinson GmbH), the device was calibrated with appropriate beads in accordance with the manufacturer’s instructions.
The cytotoxicity was analysed by flow cytometry using the software Cellquest Pro 6.0. The 7-AAD acquisition channel (FL-3) was set for the optimal detection of DNA-bound 7-AAD fluorescence signal.
 
Preparation of the acquisition (Cytotoxicity Test)
The following acquisition plots were prepared:
• 2D plot consisting of FSC (Forward Scatter) versus SSC (Side Scatter)
• Histogram plot of the FL-3 channel
The voltage of FSC and SSC was set to appropriate levels. FSC and SSC are not needed for the analysis, but the FSC/SSC plot should be checked to make sure that a single population appears without contamination or excessive debris. The FL-3 voltage was set and compensate to appropriate position (FACSCalibur, Becton Dickinson GmbH, software FACSComp 6.0).
The cell viability was measured by gating-out dead cells stained with 7-AAD. A total of 10,000 living cells were analysed.
The maintenance of the flow cytometer was in accordance with the manufacturer’s instructions. The process of washing was conducted very carefully since insoluble chemicals could flow in the flow line.

Flow Cytometry Analysis (Cytotoxicity Test)
The cell viability is shown by the cytometry analysis program (% total) or is calculated according to the following equation:
Cell Viability (%) = (Number of living cells / Number of acquired cells) x 100

The CV75 value, a concentration showing 75% of THP-1 cell survival (25% cytotoxicity), is calculated by log-linear interpolation using the following equation:
Log CV75 = ((75 - c) x Log (b) - (75 - a) x Log (d)) / a - c
Where:
a is the minimum value of cell viability over 75%
c is the maximum value of cell viability below 75%
b and d are the concentrations showing the value of cell viability a and c respectively

Acceptability of the Cytotoxicity Assay
The cytotoxicity test is considered to be acceptable if it meets the following criteria:
• The cell viability of the medium control should be more than 90%.

Calculation of the Test Doses for the Main Experiment (h-CLAT)
The mean of two CV75 values was used to determine the dose-range for the main experiment (h-CLAT).
Eight final concentrations (µg/mL) were used for the test item in the main experiment (h CLAT). The highest concentration used was 1.2 × mean CV75 and seven further concentrations of the test item were prepared by serial 1:1.2 dilution.

Experimental Design and Procedures of h-CLAT
The test item was tested in six independent runs of which one was not valid. The tests were performed on different days. The test item was prepared separately for each run.

Treatment of the Cells
For the test item exposure the highest dose solution calculated from the cytotoxicity test was prepared corresponding to 1.2 × mean CV75. Further 7 dilutions were prepared by serial 1:1.2 dilution. The dilutions were prepared freshly before each experiment.
Each volume (500 µL) of the dilutions of the test item, medium control, positive and DMSO control was added to the cells. The treated THP-1 cells were incubated for 24 ± 0.5 hours. At the end of the incubation period, the cell cultures were microscopically evaluated for morphological alterations.
Each concentration of the test item, medium control, positive and DMSO control was prepared in triplicates for the different staining (with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1).

Staining of the Cells
The triplicates of each test item-treated and not test item-treated cells were pooled and equally distributed into three sample tubes, collected by centrifugation (approx. 250 x g, 5 min) and then washed twice with approx. 2 mL of FACS buffer (PBS with 0.1% (w/v) BSA). Thereafter, the cells were centrifuged, re-suspended and blocked with 600 µL of blocking solution at 2 8 °C (on ice) for approx. 15 min. After blocking, the cells were centrifuged and the cell pellets were re-suspended in 100 µL FACS buffer. The cells were stained with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1 (isotype control).
All solutions were kept light protected at 2 - 8 °C or on ice during the staining and analysis procedures.
The cells with the different antibodies or the IgG1 were mixed and incubated light protected for 30 ± 5 min. at 2 - 8 °C (on ice).

Sample Preparation for Measurement
After staining with the antibodies, the cells were washed twice (2 - 8 °C) with 2 mL FACS buffer and re-suspended in a final volume of 2 mL/tube FACS buffer. At least 10 minutes before the flow cytometry acquisition, 5 µL of a 7-AAD solution were added.

Flow Cytometry Acquisition
Before using the flow cytometer (FACSCalibur, Becton Dickinson GmbH), the device was calibrated with appropriate beads in accordance with the manufacturer’s instructions.
The expression of cell surface antigens (CD54, CD86) was analysed by flow cytometry using the software Cellquest Pro 6.0. The FITC acquisition channel (FL-1) was set for the optimal detection of the FITC fluorescence signal, and the 7-AAD acquisition channel (FL-3) was set for the optimal detection of DNA-bound 7 AAD fluorescence signal.
Preparation of the acquisition
The following acquisition plots were prepared:
• 2D plot consisting of FSC (Forward Scatter) versus SSC (Side Scatter)
• Histogram plot of each channel (FL-1 and FL-3, respectively)
The voltage of FSC and SSC was set with untreated cells to appropriate levels. FSC and SSC are not needed for the analysis, but the FSC/SSC plot was checked to make sure that a single population appears without contamination or excessive debris. The FL-1 and FL-3 voltage were set and compensate to appropriate position. The FL-1 voltage was set using the FITC labelled-mouse IgG1 medium-treated cells tube, as such the MFI of control cells was set in the range between 1.0 and 4.0 (Geo Mean) and in the range between 3.0 and 4.0 (Geo Mean) with the FITC labelled CD54 medium-treated cells (FACSCalibur, Becton Dickinson GmbH).
The cell viability was detected by setting an R1-gate (dead cells are gated-out by staining with 7-AAD). Therefore, the R1 gate was set approximately at the middle position between the peak of the negative fraction and the positive fraction in the FL-3 histogram using DNCB-treated cells. The negative fraction corresponds to the living cells and was kept for the subsequent analyses. For each control and all test item concentrations, the cell viability was recorded from the isotype control cell tube (stained with FITC labelled-mouse IgG1), the CD54 and CD86 cell tube, where only the isotype control cells were used for the cell viability evaluation.
The maintenance of the flow cytometer was in accordance with the manufacturer’s instructions. The process of washing was conducted very carefully since insoluble chemicals could flow into the flow line.

Acquisition
Dead cells were gated-out by staining with 7-AAD. Gating by FSC (forward scatter) and SSC (side scatter) was not done. A total of 10,000 living cells were analysed. Mean fluorescence intensity (MFI) of viable cells and viability for each sample were used for analysis. The other tubes were acquired without changing the settings of the cytometer. The MFI was recorded for each condition. The relative fluorescence intensity (RFI) was calculated, but excluded from the evaluation, if the cell viability was less than 50% (due to diffuse labelling of cytoplasmic structures that could be generated due to cell membrane destruction).

Data Analysis and Interpretation
Flow Cytometry Analysis
The RFI is used as an indicator of CD86 and CD54 expression, and is calculated as follows for each concentration of every chemical:
RFI [%] = ((MFI of test item treated cells) - (MFI of test item treated isotype control cells) / (MFI of solvent control cells) - (MFI of solvent isotype control cells)) x 100
MFI = Geometric Mean Fluorescence Intensity (GeoMean)

The cell viability from the isotype control cells, CD54 and CD86 cells is calculated according to the following equation:
Cell Viability (%) = (Number of living cells / Total number of acquired cells) x 100
where only the isotype control cells (which are stained with mouse IgG1 (isotype) antibodies) are used for the cell viability evaluation.

Run / experiment:

other: 1 and 2

Parameter:

other: % RFI of CD86

Value:

150

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: in at least one concentration

Run / experiment:

other: 3

Parameter:

other: % RFI of CD86

Value:

150

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: at any dose

Run / experiment:

other: 3

Parameter:

other: % RFI of CD54

Value:

200

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: at any dose

Run / experiment:

other: 4

Parameter:

other: % RFI of CD54

Value:

200

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: in the three highest concentrations

Run / experiment:

other: 5

Parameter:

other: % RFI of CD86

Value:

150

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: at any dose

Run / experiment:

other: 5

Parameter:

other: % RFI of CD54

Value:

200

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: at any dose

Run / experiment:

other: 6

Parameter:

other: % RFI of CD86

Value:

150

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: in all tested concentrations

Results of the first h-CLAT run for the Test Item PG-RAW-90-032

	Concentration (µg/mL)	RFI (%) CD54 Antibody	RFI (%) CD86 Antibody	Cell Viability (%)
Medium Control	-	100.0	100.0	96.22
DMSO Control	-	100.0	100.0	96.04
Positive Control (DNCB)	3.0	188.3^#	592.1*	80.69
	4.0	334.3*	464.2*	80.62
Test Item	24	55.7	166.1*	94.80
	29	103.1	113.9	95.84
	35	84.7	133.9	94.80
	42	77.9	49.3	93.64
	50	69.5	145.7	94.72
	60	84.0	67.1	93.84
	73	63.4	108.9	94.76
	87	79.4	105.7	93.10

*   RFI value of CD86 or CD54 fulfilled the positive criteria (CD86≥150% and CD54≥200%).

^#    CD54 RFI value of the positive control (3.0 µg/mL DNCB) did not fulfil the positive criterion (CD54 ≥ 200%).

 

Results of the second h-CLAT run for the Test Item PG-RAW-90-032

	Concentration (µg/mL)	RFI (%) CD54 Antibody	RFI (%) CD86 Antibody	Cell Viability (%)
Medium Control	-	100.0	100.0	95.87
DMSO Control	-	100.0	100.0	96.12
Positive Control (DNCB)	3.0	421.4*	370.6*	83.43
	4.0	534.3*	508.1*	75.61
Test Item	24	71.7	175.1*	94.94
	29	75.0	179.0*	92.69
	35	87.0	216.6*	93.58
	42	71.7	192.6*	93.66
	50	77.2	231.4*	94.36
	60	97.8	266.8*	92.82
	73	67.4	191.3*	93.10
	87	93.5	187.8*	92.98

*   RFI value of CD86 or CD54 exceeded the positive criteria (CD86≥150% and CD54≥200%).

 

Results of the third h-CLAT run for the Test Item PG-RAW-90-032

	Concentration (µg/mL)	RFI (%) CD54 Antibody	RFI (%) CD86 Antibody	Cell Viability (%)
Medium Control	-	100.0	100.0	93.91
DMSO Control	-	100.0	100.0	96.17
Positive Control (DNCB)	3.0	251.6*	609.2*	87.24
	4.0	338.5*	562.3*	85.59
Test Item	24	91.0	121.1	93.63
	29	96.4	134.1	93.43
	35	93.7	122.8	92.58
	42	110.8	127.6	88.53
	50	92.8	127.0	95.15
	60	96.4	124.8	95.56
	73	94.6	119.4	92.65
	87	91.0	109.3	89.13

*   RFI value of CD86 or CD54 exceeded the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).

 

Results of the fourth h-CLAT run for the Test Item PG-RAW-90-032

	Concentration (µg/mL)	RFI (%) CD54 Antibody	RFI (%) CD86 Antibody	Cell Viability (%)
Medium Control	-	100.0	100.0	95.14
DMSO Control	-	100.0	100.0	95.41
Positive Control (DNCB)	3.0	303.4*	386.8*	85.44
	4.0	499.5*	350.6*	82.85
Test Item	35	137.6	89.4	95.11
	42	160.1	92.0	95.33
	50	189.9	124.2	94.77
	60	199.4	110.9	94.47
	72	197.2	109.6	94.60
	87	254.5*	114.8	94.15
	104	264.0*	122.1	94.34
	125	285.4*	131.7	93.15

*   RFI value of CD86 or CD54 exceeded the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).

 

Results of the fifth h-CLAT run for the Test Item PG-RAW-90-032

	Concentration (µg/mL)	RFI (%) CD54 Antibody	RFI (%) CD86 Antibody	Cell Viability (%)
Medium Control	-	100.0	100.0	95.74
DMSO Control	-	100.0	100.0	95.60
Positive Control (DNCB)	3.0	468.5*	365.8*	84.16
	4.0	523.3*	464.5*	75.27
Test Item	35	119.7	120.6	95.38
	42	95.5	110.1	94.54
	50	124.2	131.7	94.52
	60	90.9	112.1	94.17
	72	116.7	114.1	94.09
	87	124.2	107.0	93.37
	104	153.0	140.2	91.70
	125	157.6	125.6	92.76

*   RFI value of CD86 or CD54 exceeded the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).

 

The fifth run was not valid, since the cell viability was not below 90 % at any tested concentration and the result was negative.

 

Results of the sixth h-CLAT run for the Test Item PG-RAW-90-032

	Concentration (µg/mL)	RFI (%) CD54 Antibody	RFI (%) CD86 Antibody	Cell Viability (%)
Medium Control	-	100.0	100.0	96.96
DMSO Control	-	100.0	100.0	96.87
Positive Control (DNCB)	3.0	364.9*	758.9*	83.56
	4.0	552.6*	1020.0*	80.84
Test Item	50	91.7	182.7*	95.83
	60	96.7	200.5*	95.72
	72	108.3	161.4*	95.69
	87	121.7	203.0*	94.27
	104	135.0	263.5*	91.37
	125	163.3	300.0*	90.91
	150	143.3	311.2*	90.36
	180	140.0	269.0*	92.49

*   RFI value of CD86 or CD54 exceeded the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).

Results of the h-CLAT Test

Results of the first h-CLAT run for the Test Item PG-RAW-90-032

	Concentration (µg/mL)	Antibody / ISO	MFIGeoMean(FITC)	MFI-ISO	RFI (%)	Cell Viability (%)	Cell Viability ISO (%)
Medium control	-	ISO	2.14			96.22	96.22
		CD54	3.45	1.31	100.0	95.79
		CD86	4.94	2.80	100.0	98.04
DMSO control	-	ISO	2.06			96.04	96.04
		CD54	3.43	1.37	100.0	96.04
		CD86	4.71	2.65	100.0	97.88
Positive control (DNCB)	3	ISO	3.49			80.69	80.69
		CD54	6.07	2.58	188.3^#	79.96
		CD86	19.18	15.69	592.1	89.32
	4	ISO	3.03			80.62	80.62
		CD54	7.61	4.58	334.3	82.13
		CD86	15.33	12.30	464.2	88.89
Test Item	24	ISO	2.18			94.80	94.80
		CD54	2.91	0.73	55.7	94.84
		CD86	6.83	4.65	166.1	97.57
	29	ISO	2.35			95.84	95.84
		CD54	3.70	1.35	103.1	96.01
		CD86	5.54	3.19	113.9	97.61
	35	ISO	2.32			94.80	94.80
		CD54	3.43	1.11	84.7	95.29
		CD86	6.07	3.75	133.9	96.95
	42	ISO	2.30			93.64	93.64
		CD54	3.32	1.02	77.9	94.19
		CD86	3.68	1.38	49.3	93.54
	50	ISO	2.26			94.72	94.72
		CD54	3.17	0.91	69.5	93.93
		CD86	6.34	4.08	145.7	97.06
	60	ISO	2.20			93.84	93.84
		CD54	3.30	1.10	84.0	94.58
		CD86	4.08	1.88	67.1	95.56
	73	ISO	2.24			94.76	94.76
		CD54	3.07	0.83	63.4	95.14
		CD86	5.29	3.05	108.9	97.90
	87	ISO	2.22			93.10	93.10
		CD54	3.26	1.04	79.4	93.30
		CD86	5.18	2.96	105.7	95.45

^#    CD54 RFI value of the positive control (3.0 µg/mL DNCB) did not fulfil the positive criterion (CD54 ≥ 200%).

 

Results of the second h-CLAT run for the Test Item PG-RAW-90-032

	Concentration (µg/mL)	Antibody / ISO	MFIGeoMean(FITC)	MFI-ISO	RFI (%)	Cell Viability (%)	Cell Viability ISO (%)
Medium control	-	ISO	2.35			95.87	95.87
		CD54	3.27	0.92	100.0	95.72
		CD86	4.64	2.29	100.0	98.53
DMSO control	-	ISO	2.27			96.12	96.12
		CD54	2.97	0.70	100.0	95.98
		CD86	4.62	2.35	100.0	98.57
Positive control (DNCB)	3	ISO	2.98			83.43	83.43
		CD54	5.93	2.95	421.4	84.42
		CD86	11.69	8.71	370.6	89.95
	4	ISO	3.60			75.61	75.61
		CD54	7.34	3.74	534.3	76.10
		CD86	15.54	11.94	508.1	85.76
Test Item	24	ISO	2.52			94.94	94.94
		CD54	3.18	0.66	71.7	94.84
		CD86	6.53	4.01	175.1	98.24
	29	ISO	3.42			92.69	92.69
		CD54	4.11	0.69	75.0	93.83
		CD86	7.52	4.10	179.0	97.27
	35	ISO	2.56			93.58	93.58
		CD54	3.36	0.80	87.0	93.59
		CD86	7.52	4.96	216.6	96.70
	42	ISO	2.70			93.66	93.66
		CD54	3.36	0.66	71.7	94.24
		CD86	7.11	4.41	192.6	97.31
	50	ISO	2.61			94.36	94.36
		CD54	3.32	0.71	77.2	94.28
		CD86	7.91	5.30	231.4	97.96
	60	ISO	2.65			92.82	92.82
		CD54	3.55	0.90	97.8	92.45
		CD86	8.76	6.11	266.8	96.55
	73	ISO	2.73			93.10	93.10
		CD54	3.35	0.62	67.4	93.26
		CD86	7.11	4.38	191.3	97.07
	87	ISO	2.65			92.98	92.98
		CD54	3.51	0.86	93.5	93.39
		CD86	6.95	4.30	187.8	97.27

 

Results of the third h-CLAT run for the Test Item PG-RAW-90-032

	Concentration (µg/mL)	Antibody / ISO	MFIGeoMean(FITC)	MFI-ISO	RFI (%)	Cell Viability (%)	Cell Viability ISO (%)
Medium control	-	ISO	2.20			93.91	93.91
		CD54	3.31	1.11	100.0	93.54
		CD86	5.75	3.55	100.0	95.98
DMSO control	-	ISO	2.07			96.17	96.17
		CD54	3.68	1.61	100.0	96.05
		CD86	5.78	3.71	100.0	95.77
Positive control (DNCB)	3	ISO	2.67			87.24	87.24
		CD54	6.72	4.05	251.6	86.24
		CD86	25.27	22.60	609.2	93.69
	4	ISO	2.73			85.59	85.59
		CD54	8.18	5.45	338.5	86.84
		CD86	23.59	20.86	562.3	92.55
Test Item	24	ISO	2.30			93.63	93.63
		CD54	3.31	1.01	91.0	94.33
		CD86	6.60	4.30	121.1	96.35
	29	ISO	2.35			93.43	93.43
		CD54	3.42	1.07	96.4	93.70
		CD86	7.11	4.76	134.1	95.78
	35	ISO	2.36			92.58	92.58
		CD54	3.40	1.04	93.7	93.22
		CD86	6.72	4.36	122.8	95.55
	42	ISO	2.38			88.53	88.53
		CD54	3.61	1.23	110.8	89.52
		CD86	6.91	4.53	127.6	92.08
	50	ISO	2.35			95.15	95.15
		CD54	3.38	1.03	92.8	95.65
		CD86	6.86	4.51	127.0	98.04
	60	ISO	2.38			95.56	95.56
		CD54	3.45	1.07	96.4	95.39
		CD86	6.81	4.43	124.8	97.74
	73	ISO	2.43			92.65	92.65
		CD54	3.48	1.05	94.6	92.44
		CD86	6.67	4.24	119.4	94.87
	87	ISO	2.43			89.13	89.13
		CD54	3.44	1.01	91.0	89.77
		CD86	6.31	3.88	109.3	92.99

Results of the fourth h-CLAT run for the Test Item PG-RAW-90-032

	Concentration (µg/mL)	Antibody / ISO	MFIGeoMean(FITC)	MFI-ISO	RFI (%)	Cell Viability (%)	Cell Viability ISO (%)
Medium control	-	ISO	2.10			95.14	95.14
		CD54	3.88	1.78	100.0	96.01
		CD86	8.25	6.15	100.0	98.05
DMSO control	-	ISO	2.13			95.41	95.41
		CD54	4.17	2.04	100.0	95.72
		CD86	8.44	6.31	100.0	98.07
Positive control (DNCB)	3	ISO	2.57			85.44	85.44
		CD54	8.76	6.19	303.4	86.43
		CD86	26.98	24.41	386.8	94.11
	4	ISO	2.71			82.85	82.85
		CD54	12.90	10.19	499.5	85.15
		CD86	24.83	22.12	350.6	93.34
Test Item	35	ISO	2.30			95.11	95.11
		CD54	4.75	2.45	137.6	95.54
		CD86	7.80	5.50	89.4	98.44
	42	ISO	2.30			95.33	95.33
		CD54	5.15	2.85	160.1	95.71
		CD86	7.96	5.66	92.0	98.31
	50	ISO	2.34			94.77	94.77
		CD54	5.72	3.38	189.9	95.00
		CD86	9.98	7.64	124.2	98.44
	60	ISO	2.34			94.47	94.47
		CD54	5.89	3.55	199.4	94.76
		CD86	9.16	6.82	110.9	97.78
	72	ISO	2.29			94.60	94.60
		CD54	5.80	3.51	197.2	95.24
		CD86	9.03	6.74	109.6	98.23
	87	ISO	2.31			94.15	94.15
		CD54	6.84	4.53	254.5	94.70
		CD86	9.37	7.06	114.8	97.99
	104	ISO	2.29			94.34	94.34
		CD54	6.99	4.70	264.0	95.00
		CD86	9.80	7.51	122.1	98.49
	125	ISO	2.35			93.15	93.15
		CD54	7.43	5.08	285.4	94.23
		CD86	10.45	8.10	131.7	97.62

 Results of the fifth h-CLAT run for the Test Item PG-RAW-90-032

	Concentration (µg/mL)	Antibody / ISO	MFIGeoMean(FITC)	MFI-ISO	RFI (%)	Cell Viability (%)	Cell Viability ISO (%)
Medium control	-	ISO	2.51			95.74	95.74
		CD54	3.17	0.66	100.0	95.45
		CD86	4.50	1.99	100.0	97.48
DMSO control	-	ISO	2.52			95.60	95.60
		CD54	3.25	0.73	100.0	95.47
		CD86	4.80	2.28	100.0	98.05
Positive control (DNCB)	3	ISO	3.35			84.16	84.16
		CD54	6.77	3.42	468.5	84.13
		CD86	11.69	8.34	365.8	91.38
	4	ISO	3.96			75.27	75.27
		CD54	7.78	3.82	523.3	77.28
		CD86	14.55	10.59	464.5	86.35
Test Item	35	ISO	2.68			95.38	95.38
		CD54	3.47	0.79	119.7	94.51
		CD86	5.08	2.40	120.6	96.96
	42	ISO	2.85			94.54	94.54
		CD54	3.48	0.63	95.5	94.93
		CD86	5.04	2.19	110.1	97.31
	50	ISO	2.80			94.52	94.52
		CD54	3.62	0.82	124.2	93.95
		CD86	5.42	2.62	131.7	96.81
	60	ISO	2.95			94.17	94.17
		CD54	3.55	0.60	90.9	94.17
		CD86	5.18	2.23	112.1	96.83
	72	ISO	2.66			94.09	94.09
		CD54	3.43	0.77	116.7	94.36
		CD86	4.93	2.27	114.1	96.47
	87	ISO	2.81			93.37	93.37
		CD54	3.63	0.82	124.2	93.83
		CD86	4.94	2.13	107.0	96.51
	104	ISO	2.74			91.70	91.70
		CD54	3.75	1.01	153.0	93.14
		CD86	5.53	2.79	140.2	95.74
	125	ISO	2.80			92.76	92.76
		CD54	3.84	1.04	157.6	93.30
		CD86	5.30	2.50	125.6	95.98

 

Results of the sixth h-CLAT run for the Test Item PG-RAW-90-032

	Concentration (µg/mL)	Antibody / ISO	MFIGeoMean(FITC)	MFI-ISO	RFI (%)	Cell Viability (%)	Cell Viability ISO (%)
Medium control	-	ISO	2.64			96.96	96.96
		CD54	3.24	0.60	100.0	96.50
		CD86	4.61	1.97	100.0	98.19
DMSO control	-	ISO	2.65			96.87	96.87
		CD54	3.22	0.57	100.0	96.64
		CD86	4.50	1.85	100.0	97.99
Positive control (DNCB)	3	ISO	3.89			83.56	83.56
		CD54	5.97	2.08	364.9	83.07
		CD86	17.93	14.04	758.9	94.64
	4	ISO	3.78			80.84	80.84
		CD54	6.93	3.15	552.6	81.08
		CD86	22.65	18.87	1020.0	92.73
Test Item	50	ISO	2.67			95.83	95.83
		CD54	3.22	0.55	91.7	95.71
		CD86	6.27	3.60	182.7	99.18
	60	ISO	2.71			95.72	95.72
		CD54	3.29	0.58	96.7	95.85
		CD86	6.66	3.95	200.5	99.17
	72	ISO	2.68			95.69	95.69
		CD54	3.33	0.65	108.3	94.90
		CD86	5.86	3.18	161.4	98.94
	87	ISO	2.69			94.27	94.27
		CD54	3.42	0.73	121.7	94.39
		CD86	6.69	4.00	203.0	98.60
	104	ISO	2.92			91.37	91.37
		CD54	3.73	0.81	135.0	91.13
		CD86	8.11	5.19	263.5	98.09
	125	ISO	2.99			90.91	90.91
		CD54	3.97	0.98	163.3	90.63
		CD86	8.90	5.91	300.0	98.15
	150	ISO	3.03			90.36	90.36
		CD54	3.89	0.86	143.3	91.03
		CD86	9.16	6.13	311.2	98.09
	180	ISO	2.85			92.49	92.49
		CD54	3.69	0.84	140.0	91.86
		CD86	8.15	5.30	269.0	98.35

 

Acceptance Criteria of the h-CLAT

Acceptance Criteria of the first h-CLAT run for the Test Item PG-RAW-90-032

Cell viability of medium control and DMSO control should be more than 90%.            Medium =       96.22%            DMSO =         96.04%

In the positive control (DNCB), RFI values of both CD54 and CD86 should exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability should be > 50%.            3 µg/mL DNCB (CD 54):       188.3%^#            3 µg/mL DNCB (CD 86):       592.1%            4 µg/mL DNCB (CD 54):       334.3%            4 µg/mL DNCB (CD 86):       464.2%

In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 should not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%).            CD54:   104.6%            CD86:    94.6%

For medium and DMSO controls, the MFI ratio of CD54 and CD86 to isotype control should be > 105%.            Medium CD 54:         161.2%            Medium CD 86:         230.8%            DMSO CD 54:           166.5%            DMSO CD 86:           228.6%

^#             CD54 RFI value of the positive control (3.0 µg/mL DNCB) did not fulfil the positive criterion (CD54 ≥ 200%).

Acceptance Criteria of the second h-CLAT run for the Test Item PG-RAW-90-032

Cell viability of medium control and DMSO control should be more than 90%.            Medium =       95.87%            DMSO =         96.12%

In the positive control (DNCB), RFI values of both CD54 and CD86 should exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability should be > 50%.            3 µg/mL DNCB (CD 54):       421.4%            3 µg/mL DNCB (CD 86):       370.6%            4 µg/mL DNCB (CD 54):       534.3%            4 µg/mL DNCB (CD 86):       508.1%

In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 should not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%).            CD54:    76.1%            CD86:   102.6%

For medium and DMSO controls, the MFI ratio of CD54 and CD86 to isotype control should be > 105%.            Medium CD 54:         139.1%            Medium CD 86:         197.4%            DMSO CD 54:           130.8%            DMSO CD 86:           203.5%

Acceptance Criteria of the third h-CLAT run for the Test Item PG-RAW-90-032

Cell viability of medium control and DMSO control should be more than 90%.            Medium =       93.91%            DMSO =         96.17%

In the positive control (DNCB), RFI values of both CD54 and CD86 should exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability should be > 50%.            3 µg/mL DNCB (CD 54):       251.6%            3 µg/mL DNCB (CD 86):       609.2%            4 µg/mL DNCB (CD 54):       338.5%            4 µg/mL DNCB (CD 86):       562.3%

In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 should not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%).            CD54:   145.0%            CD86:   104.5%

For medium and DMSO controls, the MFI ratio of CD54 and CD86 to isotype control should be > 105%.            Medium CD 54:         150.5%            Medium CD 86:         261.4%            DMSO CD 54:            177.8%            DMSO CD 86:            279.2%

Acceptance Criteria of the fourth h-CLAT run for the Test Item PG-RAW-90-032 

Cell viability of medium control and DMSO control should be more than 90%.            Medium =       95.14%            DMSO =         95.41%

In the positive control (DNCB), RFI values of both CD54 and CD86 should exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability should be > 50%.            3 µg/mL DNCB (CD 54):       303.4%            3 µg/mL DNCB (CD 86):       386.8%            4 µg/mL DNCB (CD 54):       499.5%            4 µg/mL DNCB (CD 86):       350.6%

In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 should not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%).            CD54:   114.6%            CD86:   102.6%

For medium and DMSO controls, the MFI ratio of CD54 and CD86 to isotype control should be > 105%.            Medium CD 54:         184.8%            Medium CD 86:         392.9%            DMSO CD 54:            195.8%            DMSO CD 86:            396.2%

Acceptance Criteria of the fifth h-CLAT run for the Test Item PG-RAW-90-032

Cell viability of medium control and DMSO control should be more than 90%.            Medium =       95.74%            DMSO =         95.60%

In the positive control (DNCB), RFI values of both CD54 and CD86 should exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability should be > 50%.            3 µg/mL DNCB (CD 54):       468.5%            3 µg/mL DNCB (CD 86):       365.8%            4 µg/mL DNCB (CD 54):       523.3%            4 µg/mL DNCB (CD 86):       464.5%

In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 should not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%).            CD54:   110.6%            CD86:   114.6%

For medium and DMSO controls, the MFI ratio of CD54 and CD86 to isotype control should be > 105%.            Medium CD 54:         126.3%            Medium CD 86:         179.3%            DMSO CD 54:            129.0%            DMSO CD 86:            190.5%

Acceptance Criteria of the sixth h-CLAT run for the Test Item PG-RAW-90-032

Cell viability of medium control and DMSO control should be more than 90%.            Medium =       96.96%            DMSO =         96.87%

In the positive control (DNCB), RFI values of both CD54 and CD86 should exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability should be > 50%.            3 µg/mL DNCB (CD 54):        364.9%            3 µg/mL DNCB (CD 86):        758.9%            4 µg/mL DNCB (CD 54):        552.6%            4 µg/mL DNCB (CD 86):       1020.0%

In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 should not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%).            CD54:   95.0%            CD86:   93.9%

For medium and DMSO controls, the MFI ratio of CD54 and CD86 to isotype control should be > 105%.            Medium CD 54:         122.7%            Medium CD 86:         174.6%            DMSO CD 54:            121.5%            DMSO CD 86:            169.8%

Interpretation of results:

other: Predicted positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP)

Remarks:

Criteria used: EU CLP

Conclusions:

The test item PG-RAW-90-032 with a log Pow of 4.88 activated THP-1 cells under the test conditions of this study. Therefore the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Executive summary:

This in vitro Human Cell Line Activation Test (h-CLAT) was performed to assess the dendritic cell activation potential (third key event of a skin sensitization AOP) of PG-RAW-90-032 which formed a stable suspension/dispersion in culture medium when administered to THP-1 cells for 24 ± 0.5 hours. The highest test item concentration for the main experiment (h-CLAT) of PG-RAW-90-032 was previously determined by two cytotoxicity tests.

Cytotoxic effects were observed following incubation with the test item starting with the concentration of 78.1 µg/mL up to the highest tested concentration (5000 µg/mL) in the first cytotoxicity test and starting with the concentration of 156 µg/mL up to the highest tested concentration (5000 µg/mL) in the second cytotoxicity test (threshold of cytotoxicity: < 75%). The mean CV75 value of both cytotoxicity tests was calculated as 72.32 µg/mL.

The following concentrations of the test item were tested in the main experiments (h-CLAT):

24, 29, 35, 42, 50, 60, 73, and 87 µg/mL in the first to third run,

35, 42, 50, 60, 72, 87, 104 and 125 µg/mL in the fourth and fifth run, and

50, 60, 72, 87, 104, 125, 150 and 180 µg/mL in the sixth h-CLAT run.

The test item with a log Pow of 4.88 (EPISuite modeling) was tested in 6 independent runs. The RFI of CD86 was greater than 150% in at least one concentration of the first and second run. However, since the CD86 was only greater than 150% in the lowest tested concentration of the first run, a confirmatory third run was conducted. The RFI of CD86 and CD54 was not equal or greater than 150% and 200%, respectively at any dose in the third independent run. Furthermore, the observed cell viability was only slightly below 90% in two tested concentrations of the third run. Therefore, a fourth h-CLAT run was conducted with increased test item concentrations. Since the RFI of CD54 was greater than 200% in the three highest concentrations of the fourth run, a fifth h-CLAT run was performed. The fifth run showed that the RFI of CD86 and CD54 was not equal or greater than 150% and 200%, respectively at any dose. However, the fifth run was not valid since the cell viability was not < 90% at any tested test item concentration. Therefore, a sixth h-CLAT run was conducted with further increased test item concentrations. For the sixth h-CLAT run, the RFI of CD86 was greater than 150% in all tested test item concentrations. Therefore, in conclusion to all tested and valid h-CLAT runs, the h-CLAT prediction is considered positive for the tested test item in this h-CLAT.

In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%). 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%, with one exception. The CD54 RFI value of the positive control (3.0 µg/mL DNCB) in the first h-CLAT run did not exceed the positive criterion (CD54 ≥ 200%). However, this one exception is considered to be acceptable since the CD54 RFI value of the positive control (4.0 µg/mL DNCB) in the first h CLAT run exceeded the positive criteria.

In conclusion, the test item PG-RAW-90-032 with a log Pow of 4.88 activated THP-1 cells under the test conditions of this study. Therefore the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

In chemico skin sensitisation: DPRA (OECD TG 442C)

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 PG-RAW-90-032. 

Solutions of PG-RAW-90-032 were successfully analyzed by the validated DPRA analytical method (Envigo analytical method FIA/M101/15) in both the Cysteine and Lysine containing synthetic peptides. There was minimal reactivity of both peptides in the presence of PG-RAW-90-032. With an overall depletion of peptide of 0.622% the reactivity of PG-RAW-90-032 is henceforth classified as “no or minimal” therefore the DPRA prediction is negative. PG-RAW-90-032 is likely not to be a potential skin sensitizer based on this assay.

In vitro skin sensitisation: h-CLAT (OECD TG 442E)

This in vitro Human Cell Line Activation Test (h-CLAT) was performed to assess the dendritic cell activation potential (third key event of a skin sensitization AOP) of PG-RAW-90-032 which formed a stable suspension/dispersion in culture medium when administered to THP-1 cells for 24 ± 0.5 hours. The highest test item concentration for the main experiment (h-CLAT) of PG-RAW-90-032 was previously determined by two cytotoxicity tests.

Cytotoxic effects were observed following incubation with the test item starting with the concentration of 78.1 µg/mL up to the highest tested concentration (5000 µg/mL) in the first cytotoxicity test and starting with the concentration of 156 µg/mL up to the highest tested concentration (5000 µg/mL) in the second cytotoxicity test (threshold of cytotoxicity: < 75%). The mean CV75 value of both cytotoxicity tests was calculated as 72.32 µg/mL.

The following concentrations of the test item were tested in the main experiments (h-CLAT):

24, 29, 35, 42, 50, 60, 73, and 87 µg/mL in the first to third run,

35, 42, 50, 60, 72, 87, 104 and 125 µg/mL in the fourth and fifth run, and

50, 60, 72, 87, 104, 125, 150 and 180 µg/mL in the sixth h-CLAT run.

The test item with a log Pow of 4.88 (EPISuite modeling) was tested in 6 independent runs. The RFI of CD86 was greater than 150% in at least one concentration of the first and second run. However, since the CD86 was only greater than 150% in the lowest tested concentration of the first run, a confirmatory third run was conducted. The RFI of CD86 and CD54 was not equal or greater than 150% and 200%, respectively at any dose in the third independent run. Furthermore, the observed cell viability was only slightly below 90% in two tested concentrations of the third run. Therefore, a fourth h-CLAT run was conducted with increased test item concentrations. Since the RFI of CD54 was greater than 200% in the three highest concentrations of the fourth run, a fifth h-CLAT run was performed. The fifth run showed that the RFI of CD86 and CD54 was not equal or greater than 150% and 200%, respectively at any dose. However, the fifth run was not valid since the cell viability was not < 90% at any tested test item concentration. Therefore, a sixth h-CLAT run was conducted with further increased test item concentrations. For the sixth h-CLAT run, the RFI of CD86 was greater than 150% in all tested test item concentrations. Therefore, in conclusion to all tested and valid h-CLAT runs, the h-CLAT prediction is considered positive for the tested test item in this h-CLAT.

In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%). 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%, with one exception. The CD54 RFI value of the positive control (3.0 µg/mL DNCB) in the first h-CLAT run did not exceed the positive criterion (CD54 ≥ 200%). However, this one exception is considered to be acceptable since the CD54 RFI value of the positive control (4.0 µg/mL DNCB) in the first h CLAT run exceeded the positive criteria.

In conclusion, the test item PG-RAW-90-032 with a log Pow of 4.88 activated THP-1 cells under the test conditions of this study. Therefore the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

In vitro Skin sensitisation: ARE-Nrf2 Luciferase Test (KeratinoSens™) (OECD TG 442D)

The purpose of this study was to support a predictive, adverse-outcome-pathway evaluation of whether the test item, PG-RAW-90-032, is likely to be a skin sensitizer using the ARE‑Nrf2 Luciferase Test (KeratinoSens™).

The I_max for PG-RAW-90-032 was 1.25 in test 1 and 1.05 in test 2. The I_max for both tests was <1.5 fold compared to the DMSO control and therefore the EC_1.5 could not be calculated. The cellular viability fell below 70% in both tests. The IC₃₀ value was 43.08 µM in test 1 and 44.32 µM in test 2 and the IC₅₀values were 48.89 µM and 49.75 µM in tests 1 and 2, respectively. Graphs for PG-RAW-90-032 showed no overall dose-response for luciferase induction.

All acceptance criteria for the positive control,cinnamic aldehyde,were met.

It was concluded that PG-RAW-90-032 gave a negative response in the ARE-Nrf2 Luciferase Test (KeratinoSens™), supporting the prediction that PG-RAW-90-032 is not a skin sensitizer.

Justification for classification or non-classification

The substance PG-RAW-90-032 is predicted negative for AOP key event 1 in the DPRA skin sensitisation study.

The substance PG-RAW-90-032 is predicted negative for AOP key event 2 in the KeratinoSens™skin sensitisation study.

The substance PG-RAW-90-032 is predicted positive for AOP key event 3 in the h-CLAT skin sensitisation study.

Two out of the three assays were negative. According to the '2 out of 3' approach described in OECD Guidance Document No. 256 and Annex I, overall prediction can be made that the substance is not a skin sensitizer and hazard classification is not needed.