Registration Dossier

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Administrative data

Description of key information

DPRA (OECD 442C): positive

KeratinoSens (OECD 442D): positive

hCLAT (OECD 442E): positive

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
The study was conducted between 11 December 2017 and 15 December 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 (incl. QA statement)
Type of study:
direct peptide reactivity assay (DPRA)
Specific details on test material used for the study:
Sponsor’s identification: PG-RAW-0004
Appearance: Clear colourless liquid
Storage conditions: Refrigerated temperature (2°C to 8°C) in the dark
Details on the study design:
Peptide and Positive Control
Synthetic peptide containing Cysteine
Alternative name: Ac-RFAACAA-OH
Batch number: 1658140
Stated purity: 97.8% (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: 1556172
Stated purity: 94.1% (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-0004 in acetonitrile was assessed at a 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 prepared as well.

Preparation of Stability Controls and Precision Control
Stability controls (Reference Control B) and precision controls of both peptides were prepared at a concentration of 0.5 mM. Reference Control B samples and the precision control sample contained acetonitrile.

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-0004 was also prepared.

Preparation of Positive Control and Cysteine Peptide Depletion Samples and Co-elution Controls
Triplicate solutions each of the positive control and PG-RAW-0004 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 PG-RAW-0004. 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-0004 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 PG-RAW-0004. For the co-elution control, buffer solution was used in place of the Lysine stock solution.

Incubation
The appearance of the PG-RAW-0004 and positive control samples in the HPLC vials was documented after 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-0004 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, 3.5µm, 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: Mean peptide depletion (%)
Value:
61.5
Positive controls validity:
valid
Remarks on result:
other: predicted to be a potential skin sensitizer

Solubility Assessment

The solubility of PG-RAW-0004 in acetonitrile at a nominal concentration of 100 mM was confirmed.

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

r2>0.99

60.8-100
(SD <14.9%)

0.45-0.55 mM (CV <15%)

SD <14.9%

Lysine

r2>0.99

40.2-69.0
(SD <11.6%)

0.45-0.55 mM (CV <15%)

SD <11.6%

Achieved results

Cysteine

r2>0.999

71.1
(SD, 0.38%, n=3)

B: 0.505 mM (CV 0.46%, n=6)

SD 0.21% (n=3)

Lysine

r2>0.999

58.8
(SD, 0.31%, n=3)

B: 0.504 mM (CV 0.31%, n=6)

SD 6.81% (n=3)

CV         Coefficient of Variation

SD         Standard deviation

The depletion of peptide in the presence of PG-RAW-0004 was:

Mean peak area of reference control(µV.sec)

Mean peak area of peptide with test item(µV.sec)

Mean peptide depletion by PG-RAW-0004 (%)

Cysteine

Control B: 821710 (n=6)

3312 (n=3)

99.6

Lysine

Control B: 761210 (n=6)

583010 (n=3)

23.4

Applying the following depletion model (below), reactivity is classed as “High” and the DPRA prediction is positive, hence PG-RAW-0004 is predicted as 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 of the Lysine or Cysteine assays.  

Overall Achieved Depletion Values

Test item

Cysteine peptide depletion (%)

Lysine peptide depletion (%)

Overall mean depletion (%)

Reactivity class

DPRA prediction

PG-RAW-0004

99.6

23.4

61.5

High

Positive

 

Individual Achieved Depletion Values

Cysteine Peptide Depletion

Sample

Peak area (µV.sec)

Peptide concentration1
(µg/mL)

Peptide Depletion2(%)

Mean Depletion (%)

SD
 (%)

Positive control

235619

108.483

71.3

71.1

0.38

240001

110.513

70.8

236026

108.673

71.3

PG-RAW-0004

3346

0.98571

99.6

99.6

0.21

1567

0.16275

99.8

5022

1.7617

99.4

SD      Standard Deviation

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

2                Calculated against a mean Reference Control B area of 821710 µV.sec(n=6)

3          Individual and mean concentrations of the positive control are outside of the range established during the validation study (Annex 1, Table B). There is however considered to be no impact on the data reporting as both each individual and the overall mean depletion values are within the stated acceptance criteria of60.8%-100%depletion.

Lysine Peptide Depletion

Sample

Peak area (µV.sec)

Peptide concentration1(µg/mL)

Peptide Depletion2(%)

Mean Depletion (%)

SD
 (%)

Positive control

311208

159.78

59.1

58.9

0.31

315814

162.15

58.5

312399

160.40

59.0

PG-RAW-0004

642860

330.54

15.5

23.4

6.81

554679

285.14

27.1

551490

283.50

27.6

SD      Standard Deviation

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

2                Calculated against a mean Reference Control B area of 761210 µV.sec(n=6)

Interpretation of results:
other: potential skin sensitizer
Conclusions:
Solutions of PG-RAW-0004 were successfully analysed by the validated DPRA analytical method (Envigo analytical method FIA/M101/15) in both Cysteine and Lysine containing synthetic peptides. The overall mean depletion result of 61.5% places PG-RAW-0004 in the reactivity class of “high” and hence it is predicted by DPRA to be a potential skin sensitizer.
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-0004. 

Solutions of PG-RAW-0004 were successfully analysed by the validated DPRA analytical method (Envigo analytical method FIA/M101/15) in both Cysteine and Lysine containing synthetic peptides. The overall result of 61.5% depletion places PG-RAW-0004in the reactivity class of “high reactivity” and therefore it is predicted by DPRA to be a potential skin sensitizer. 

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date: 17 November 2017 Experimental completion date: 21 December 2017
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:
yes
Remarks:
Please see any other information on materials and methods section
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
Skin sensitisers have been reported to induce genes that are regulated by the antioxidant response element (ARE). The KeratinoSens™ test is a method for which validation studies have been completed followed by an independent peer review conducted by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM). The KeratinoSens™ test method was considered scientifically valid to be used as part of an IATA (Integrated Approach to Testing and Assessment), to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling. The method cannot be used on its own, neither to sub-categorise skin sensitisers into subcategories lA and lB as defined by the UN GHS, for authourities implementing these two optional subcategories, nor to predict potency for safety assessment decisions. However, depending on the regulatory framework a positive result may be used on its own to classify a chemical int o UN GHS category 1.
Specific details on test material used for the study:
Test Item Name: PG-RAW-0004
Supplier Code: PG-RAW-0004
Supplier batch/lot number: RDRW004-3
CAS number: 2109705-93-1; 2111193-86-1
Purity: 95.5%
Expiry Date: 01 Sep 19
Physical state: Clear liquid
Storage Conditions: Refrigerated
Details on the study design:
Method of administration of test item:
Per plate, single application of 12 concentrations (2000, 1000, 500, 250, 125, 62.5, 31.25, 15.625, 7.813, 3.906, 1.953, 0.977 μM) of the test item was applied in cell culture medium (dilution factor of 2) with a final concentration of DMSO of 1%. The top concentration was previously determined by solubility testing.

Method of administration of reference items:
Per plate, single application of 5 concentrations of the positive control was applied in cell culture medium (dilution factor of 2) with a final concentration of DMSO of 1% and single application of culture medium with 1% DMSO was applied as the negative control (6 wells per plate).
One well per plate is left empty (no cells).

Exposure times of test items and reference items:
Cells were incubated with the test or reference item for 48 ± 2h before endpoints measurements.

Number of repetitions:
Three repetitions (runs) were performed. Each repetition consisted of 3 x 96-well plates for luminescence and 2 x 96-well plates for MTT. The validity of each repetition was assessed following acceptance criteria described.

Overview
Preliminary testing: Determination of the top concentration by solubility testing
Day 1: Seeding cells (3 x 96-well plates for Luminescence; 2 x 96-well plate for MTT); 10,000 cells per well, p14.
Day 2: 24h after seeding the test and control items were applied and plates were incubated at 37°C, 5% CO2, 2: ≥ 95% relative humidity for 48 ± 2h.
Day 4: Evaluation of luciferase activity by luminescence (3 plates) and cell viability by MTT testing (2plates)

Data Analysis
XCellR8 Forms F0056 A (version 01) and B (version 04): Data Analysis for KeratinoSensTM were used to analyse data. These forms are Microsoft Excel workbooks containing formulae to process the raw data as described in SOP L0057. The spreadsheets have been validated in-house (July 2017).
The following parameters were calculated in the KeratinoSensTM test method:
• the maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the test item and positive control;
• the EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5- fold threshold (i.e. 50% enhanced luciferase activity);
• For each concentration showing > 1.5-fold luciferase activity induction, statistical significance is calculated (e.g. by a two-tailed Student’s t-test), comparing the luminescence values for the three replicate samples with the luminescence values in the solvent (negative) control wells to determine whether the luciferase activity induction is statistically significant (p <0.05). The lowest concentration with > 1.5-fold luciferase activity induction is the value determining the EC1.5 value. It is checked in each case whether this value is below the IC30 value, indicating that there is less than 30% reduction in cellular viability at the EC1.5 determining concentration.
• The percentage of viability as compared to the Negative control
Positive control results:
Positive Control (PC) (Cinnamic aldehyde) induced ≥1.5-fold in at least one concentration. The average induction of cinnamic aldehyde was 1.91
Run / experiment:
mean
Parameter:
other: EC1.5
Value:
11.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Other effects / acceptance of results:
Solubility Assessment
The test concentrations of PG-RAW-0004 used in the KeratinoSensTM test were selected on the basis of solubility test carried out prior to the study:
Solubility of PG-RAW-0004 (MW: 154) in cell culture medium (confirmed up to 30.8mg/ml (200mM)); subsequent dilution in cell culture medium-1% DMSO giving a top concentration of 2000µM).


Discussion
The human skin sensitisation potential of PG-RAW-0004 was assessed using validated in vitro method: the KeratinoSensTM test to determine keratinocyte activation. The method was adapted to animal product-free conditions by XCellR8 and reference chemicals described in the guideline and in the performance standards were used to confirm the reliability, accuracy, sensitivity and specificity values. The adapted method showed full concordance with the Validated Reference Method (VRM) – the KeratinoSensTM standard protocol. We recently obtained clarification from the European Chemicals Agency (ECHA) that data using the adapted method may be used in REACH submissions, provided that the Performance Standards data, demonstrating equivalence with the VRM, is included in the dossier.

In this study, PG-RAW-0004 was classified as a sensitiser to human skin.

The sensitisation potential of PG-RAW-0004 was quantified by calculating 2 parameters known as the EC1.5 and the IMAX value. The meanings of these are as follows:

• The EC1.5 value means the Effective Concentration (EC) of test item that caused an induction of luciferase activity of greater than 1.5-fold over untreated controls. If at least one concentration induces luciferase activity to >1.5, then the product is classified as a skin sensitiser. (Note: this classification also requires the cell viability measured by MTT to be greater than 70%).

PG-RAW-0004 did cause luciferase induction >1.5 in all 3 repetitions. Therefore, PG-RAW-0004 was classified as a sensitiser.


• The IMax value is the maximum-fold induction observed within the concentration range tested. Although the KeratinoSensTM test is not validated to predict potency, the IMAX value can provide a useful tool for a very preliminary comparison of sensitisation potential between test items. As shown in Section 13.2, the maximum induction was observed at a test concentration of 2000µM, which showed an Imax value of 1904.107 in repetition 1; 35.506 at 2000µM in repetition 2; 2358.35 at 1000µM in repetition 3. For reference, during test validation, sensitising proficiency chemicals produced Imax values of up to 36-fold over untreated controls.

All of the formal acceptance criteria of the tests were met.

Determination of the skin sensitisation potential of PG-RAW-0004

Sensitisation Potential of the test item repetition 1

REP 1

Test item concentration (µM)

0.977

1.953

3.906

7.813

15.625

31.250

62.5

125

250

500

1000

2000

Mean of fold induction

0.941

0.902

1.156

1.274

1.434

1.478

2.186

3.061

5.489

14.129

101.988

1904.107

SD

0.039

0.045

0.049

0.085

0.150

0.273

0.091

0.219

0.945

0.920

9.893

104.463

Viability %

118.13

108.40

96.08

96.65

94.49

95.13

107.09

112.29

130.72

136.18

157.23

140.53

Imax

1904.107 at 2000µM

EC1.5

32.22µM

IC50

>2000µM as viability at 2000µM was 140.53%

IC30

>2000µM as viability at 2000µM was 140.53%

Sensitisation Potential of the test item repetition 2

REP 2

Test item concentration (µM)

0.977

1.953

3.906

7.813

15.625

31.250

62.5

125

250

500

1000

2000

Mean of fold induction

1.144

1.165

1.254

1.434

1.350

1.293

1.581

1.801

2.656

4.082

8.710

35.506

SD

0.064

0.125

0.212

0.183

0.191

0.127

0.165

0.316

0.283

0.442

0.879

2.429

Viability %

96.15

86.87

96.06

100.59

97.83

99.59

102.22

118.07

128.96

161.25

210.83

229.39

Imax

35.506 at 2000µM

EC1.5

53.74µM

IC50

>2000µM as viability at 2000µM was 229.39%

IC30

>2000µM as viability at 2000µM was 229.39%

Sensitisation Potential of the test item repetition 3

REP 3

Test item concentration (µM)

0.977

1.953

3.906

7.813

15.625

31.250

62.5

125

250

500

1000

2000

Mean of fold induction

0.836

1.107

1.048

1.223

1.472

1.908

3.404

6.756

26.094

338.892

2358.350

0.010

SD

0.040

0.202

0.073

0.081

0.115

0.098

0.303

0.713

1.935

35.363

123.870

0.003

Viability %

95.90

88.13

90.31

103.20

100.58

103.66

110.91

134.53

152.64

164.49

158.82

0.43

Imax

2358.35 at 1000µM

EC1.5

16.63µM

IC50

1687µM

IC30

1561µM

Determination criteria for the skin sensitisation potential ofPG-RAW-0004

 

REP1

REP2

REP3

Does at least one concentration ofTest Iteminduce luciferase activity>1.5-fold:

Yes

Yes

Yes

Does the first concentration inducing luciferase activity above 1.5, have a viability above 70%:

Yes

Yes

Yes

Does EC1.5value occur at a concentration <1000µM (or <200µg/ml)

Yes

Yes

Yes

Does the test item induce the luciferase in a dose-dependent manner

Yes

Yes

Yes

Classification

Sensitiser

Sensitiser

Sensitiser

Assay Acceptane Criteria (Mean of the 3 repetitions)

Criteria

Result

Pass or Fail

1-Positive Control (PC) (Cinnamic aldehyde) induction>1.5-fold in at least one concentration 

Yes

Pass

2-Average induction of PC at 32µM is [1.6-3.0]

Yes (2.11)

Pass

3-EC1.5value is [6-39µM]

Yes (11.50)

Pass

4-CV% of blank values < 20%

Yes (13.06%)

Pass

Interpretation of results:
study cannot be used for classification
Conclusions:
PG-RAW-0004 was classified as sensitiser
Executive summary:

The human skin sensitisation potential of PG-RAW-0004 was assessed using the validated in vitro method, the KeratinoSensTMassay,adapted to fully animal-free by XCellR8,and validated in-house to determine keratinocyte activation.

 

After 48h exposure of cells with 12 concentrations of PG-RAW-0004,Luciferase measurements andMTT viability testing were performed.

PG-RAW-0004 was classified as sensitiser

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date: 20 November 2017 Experimental completion date: 30 November 2017
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)
Version / remarks:
July 2016
Deviations:
yes
Remarks:
The cytotoxicity measurement and estimation of the CV75 value of the dose finding assay is performed by XTT test instead of flow cytometry.
GLP compliance:
yes (incl. QA statement)
Type of study:
other: In vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)
Specific details on test material used for the study:
Identification: PG-RAW-0004
Chemical name: Reaction mass of 2-methoxy-6-methylocta-1,5-diene and 2-methoxy-6-methylocta-2,5-diene
CAS Numbers: 2109705-93-1 / 2111193-86-1
Batch: RDRW004-3
Purity: 95.5%*
Expiry Date: 01 September 2019
Storage Conditions: In the refrigerator, light protected
* Dose calculation was not adjusted to purity.
Details on the study design:
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: 2 and 3 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 (immediately prior to start) PG-RAW-0004 was stable suspended in culture medium.
The maximum concentration of test item was 5000 μg/mL culture medium as tested by a solubility test.
For the XTT test (dose finding assay) eight concentrations of the test item were analysed. For this, dilutions were prepared by 1:2 serial dilution from 5000 μg/mL culture medium.

Experimental Design and Procedures of XTT
Dose Finding Assay (XTT Test)
The test item concentrations investigated in the main experiment (h-CLAT) were determined with two XTT tests.
The XTT test is based on the cleavage of the yellow tetrazolium salt XTT [= (sodium 3'-(1-phenylaminocarbonyl) - (3,4 - tetrazolium) – bis - (4 – methoxy – 6 - nitro) - benzenesulfonic acid hydrate)] to form an orange water soluble formazan dye by dehydrogenase activity in active mitochondria. This method was first described 1988 by SCUDIERO et al. and improved in subsequent years by several other investigators.
Two independent cytotoxicity experiments were performed with different cell cultures and on different days to obtain a reliable CV75. The mean of two CV75 values was used to determine the dose-range for the main experiment (h-CLAT).
XTT Labelling Mixture
The XTT labelling mixture consists of two components, a XTT buffer solution and the substrate solution. Both components were mixed right before application at a ratio of 1:100.
Treatment
After the cell seeding, 100 μL of the test item dilutions, the medium and solvent controls, respectively, were added to the cells. All dose groups were tested in 7 replicates for each XTT test. At the end of the incubation period of 24 ± 0.5 hours, the cell cultures were microscopically evaluated for morphological alterations.
XTT Labelling and Measurement
At the end of the incubation period, 50 μL of the XTT labelling mixture were added to each well. The cells were incubated and subsequently transferred to a microplate reader (Versamax® Molecular Devices). The absorbance was measured at 450 nm (reference wavelength 690 nm). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).

Experimental Design and Procedures of h-CLAT
The test item was tested in two independent runs.
Treatment of the Cells
For the test item exposure the highest dose solution calculated from the XTT assay was prepared corresponding to 1.2 × 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 × 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 that 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 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 determined 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. If the cell viability will be low, then up to 30,000 cells including dead cells should be acquired. Alternatively, the acquisition can be finished 1 minute after the acquisition start. 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 not calculated, if the cell viability was less than 50% (due to diffuse labelling of cytoplasmic structures that could be generated due to cell membrane destruction).
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%.
Run / experiment:
other: 2 independent runs
Parameter:
other: % relative fluorescence intensity (RFI) of CD86
Value:
150
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Run / experiment:
other: 2 independent runs
Parameter:
other: % relative fluorescence intensity (RFI) of CD54
Value:
200
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Other effects / acceptance of results:
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-0004 stable suspended 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-0004 was previously determined by two XTT tests.
Cytotoxic effects were observed following incubation with the test item starting with the concentration of 625 μg/mL up to the highest tested concentration (5000 μg/mL) in both XTT tests (threshold of cytotoxicity: < 75%). The mean CV75 value of both XTT tests was calculated as 478.45 μg/mL.
The following concentrations of the test item (dissolved in culture medium) were tested in the main experiment (h-CLAT):
160, 192, 231, 277, 332, 399, 478 and 574 μg/mL
The test item with an estimated log Pow of 3.68 – 3.76 was tested in 2 independent runs. The two highest test item concentrations in the second run were excluded from the evaluation, since the cell viability was below 50%. The RFI of CD86 and CD54 was greater than 150% and 200%, respectively, in at least one concentration of both runs. Therefore 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%. For details see Annex 2.
Further results of the testing battery (including e.g. DPRA, ARE-Nrf2 luciferase test method) based on the OECD adverse outcome pathway for the assessment of the skin sensitisation potential are not available. Therefore, consideration of the test method results within the context of an IATA (Integrated Approaches to Testing and Assessment) is not possible.

Results of the first XTT test for Test Item PG-RAW-0004

 

Microscopic Evaluation

Photometric Evaluation


Test Group

Concen-tration
[µg/mL]

Cytotoxicity

Mean Ab-sorbance*§

Standard-Deviation

Chem. Blank§

Mean Ab-sorbance – Chemical Blank

Absorbance in % of Solvent Control**

Medium Control

-

no

0.910

0.039

0.241

0.670

105.54

Solvent Control

-

no

0.877

0.071

0.243

0.634

100.00

Test Item

39.1

no

1.015

0.071

0.234

0.781

123.06

78.1

no

1.188

0.093

0.242

0.946

149.14

156.3

no

1.232

0.074

0.238

0.994

156.66

312.5

no

0.967

0.121

0.244

0.723

113.99

625

no

0.495

0.029

0.264

0.230

36.33

1250

no

0.401

0.023

0.286

0.115

18.17

2500

no

0.399

0.068

0.352

0.047

7.40

5000

yes

0.556

0.130

0.480

0.076

11.93

Shaded test groups: cytotoxic effects occurred in the photometric evaluation (< 75% cell viability)

§ Given absorbances are rounded values.

* mean absorbance (absolute) of 7 wells

** relative absorbance [rounded values]

The mean viability of the solvent control in comparison to the medium control was 94.75%.

The CV75 value of the first XTT test: 469.4 μg/mL

Results of the second XTT test for Test Item PG-RAW-0004

 

Microscopic Evaluation

Photometric Evaluation


Test Group

Concen-tration
[µg/mL]

Cytotoxicity

Mean Ab-sorbance*§

Standard-Deviation

Chem. Blank§

Mean Ab-sorbance – Chemical Blank

Absorbance in % of Solvent Control**

Medium Control

-

no

0.649

0.030

0.236

0.412

105.53

Solvent Control

-

no

0.633

0.028

0.242

0.391

100.00

Test Item

39.1

no

0.756

0.051

0.239

0.516

132.19

78.1

no

0.834

0.044

0.240

0.595

152.19

156.3

no

0.871

0.079

0.250

0.622

159.18

312.5

no

0.800

0.062

0.257

0.543

139.02

625

no

0.385

0.025

0.289

0.096

24.67

1250

yes

0.330

0.009

0.357

-0.027

-6.85

2500

yes

0.391

0.049

0.528

-0.137

-35.05

5000

yes

0.556

0.107

0.950

-0.394

-100.76

Shaded test groups: cytotoxic effects occurred in the photometric evaluation (< 75% cell viability)

§ Given absorbances are rounded values.

* mean absorbance (absolute) of 7 wells

** relative absorbance [rounded values]

The mean viability of the solvent control in comparison to the medium control was 94.76%.

The CV75 value of the second XTT test: 487.5 μg/mL

The mean CV75 value of both XTT tests: 478.45 μg/mL

Results of the first h-CLAT run for the Test Item PG-RAW-0004

 

Concentration (µg/mL)

RFI (%)
CD 54 Antibody

RFI (%)
CD 86 Antibody

Cell Viability (%)

 

Medium Control

-

100.0

100.0

100.0

DMSO Control

-

100.0

100.0

100.0

Positive Control (DNCB)

2.0

232.4*

542.7*

67.2

3.0

277.6*

534.8*

70.0

Test Item

160

151.7

189.9*

82.8

192

121.5

135.5

93.5

231

138.3

229.3*

82.0

277

201.3*

350.0*

79.2

332

232.2*

364.5*

69.8

399

359.7*

423.2*

61.5

478

312.8*

388.4*

58.9

574

224.2*

333.3*

53.1

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

Results of the second h-CLAT run for the Test Item PG-RAW-0004

 

Concentration (µg/mL)

RFI (%)
CD 54 Antibody

RFI (%)
CD 86 Antibody

Cell Viability (%)

 

Medium Control

-

100.0

100.0

100.0

DMSO Control

-

100.0

100.0

100.0

Positive Control (DNCB)

2.0

210.6*

531.2*

73.0

3.0

210.6*

490.2*

74.2

Test Item

160

295.6*

254.6*

84.3

192

362.9*

325.8*

72.5

231

471.7*

323.3*

63.0

277

522.4*

303.3*

78.0

332

441.0*

257.9*

80.3

399

162.9

292.0*

50.6

478

120.5

416.3*

23.7

574

49.8

340.1*

17.4

Shaded test groups: cell viability below 50%, are excluded from the evaluation

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

Interpretation of results:
study cannot be used for classification
Conclusions:
The test item PG-RAW-0004 with an estimated log Pow of 3.68 – 3.76 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-0004 stable suspended 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-0004 was previously determined by two XTT tests.

Cytotoxic effects were observed following incubation with the test item starting with the concentration of 625 μg/mL up to the highest tested concentration (5000 μg/mL) in both XTT tests (threshold of cytotoxicity: < 75%). The mean CV75 value of both XTT tests was calculated as 478.45 μg/mL.

The following concentrations of the test item (dissolved in culture medium) were tested in the main experiment (h-CLAT):

160, 192, 231, 277, 332, 399, 478 and 574 μg/mL

The test item with an estimated log Pow of 3.68 – 3.76 was tested in 2 independent runs. The two highest test item concentrations in the second run were excluded from the evaluation, since the cell viability was below 50%. The RFI of CD86 and CD54 was greater than 150% and 200%, respectively, in at least one concentration of both runs. Therefore 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%.

In conclusion, the test item PG-RAW-0004 with an estimated log Pow of 3.68 – 3.76 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:
adverse effect observed (sensitising)
Additional information:

DPRA (OECD 442C):The purpose of this study (based on the OECD guideline for the testing of chemicals,In chemicoSkin Sensitisation: Direct Peptide Reactivity Assay (DPRA), OECD/OCDE document TG 442C) was to assess the reactivity and sensitizing potential of PG-RAW-0004. 

Solutions of PG-RAW-0004 were successfully analysed by the validated DPRA analytical method (Envigo analytical method FIA/M101/15) in both Cysteine and Lysine containing synthetic peptides. The overall result of 61.5% depletion places PG-RAW-0004in the reactivity class of “high reactivity” and therefore it is predicted by DPRA to be a potential skin sensitizer. 

KeratinoSens (OECD 442D): The human skin sensitisation potential of PG-RAW-0004 was assessed using the validatedin vitromethod, the KeratinoSensTMassay,adapted to fully animal-free by XCellR8,and validated in-house to determine keratinocyte activation.

After 48h exposure of cells with 12 concentrations of PG-RAW-0004,Luciferase measurements andMTT viability testing were performed.

PG-RAW-0004 was classified as sensitiser.

h-CLAT(OECD 442E): The skin sensitisation potential of the test substance has been tested according to OECD TG 442E: in vitro Human Cell Line Activation Test (h-CLAT) method. Tested at 160, 192, 231, 277, 332, 399, 478 and 574 μg/mL. The test item with an estimated log Pow of 3.68 – 3.76 was tested in 2 independent runs. The two highest test item concentrations in the second run were excluded from the evaluation, since the cell viability was below 50%. The RFI of CD86 and CD54 was greater than 150% and 200%, respectively, in at least one concentration of both runs. Therefore the h-CLAT prediction is considered positive for the tested test item in this h-CLAT.


Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)

Justification for classification or non-classification

The substance has been precited to be skin sensitizing in all three in vitro assays, i.e., DPRA, KaretinoSens, and h-CLAT. The substance was tested in more than 100 human subjects in an HRIPT at a dermal dosage of 1030 ug/cm2 and did not produce any adverse effects. Therefore, the substance may be classified for dermal sensitization of sub-category 1B according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 and its amendments.