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Studies conducted to internationally recognised testing guidelines with GLP certification.

Key value for chemical safety assessment

Skin sensitisation

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Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 January 2018 - January 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
February 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
This is an in chemico test and the test material is used in cosmetic ingredients. Regulation 1223/2009 Article 18 restricts the use of in vivo studies on these types of raw materials.
Test material information:
Composition 1
Specific details on test material used for the study:
The test article, a clear yellow liquid, was identified as Dermol GTR and was received at Covance on 31 October 2017 as follows:
- CAS Number: 101-34-8
- Storage:15 to 25°C, protected from light
- Purity: 100%
Details on study design:
The study was conducted to investigate the potential of the test material to induce genes that are regulated by the antioxidant response element (ARE). The data may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.
The ARE-Nrf2 luciferase test method utilises an immortalised adherent cell line derived from HaCaT human keratinocytes. The cell line is stably transfected with a plasmid containing a luciferase gene under the transcriptional control of the SV40 promoter fused with the ARE from a gene 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 substances.

Specifications
KeratinoSens™ cell line supplied by Givaudan Schweiz, Zurich, Switzerland. Identification
The test system was appropriately labelled with the study number, assay type, experiment number and test/positive/negative control.
Preparation of Cultures
A fresh vial of cells was used for each experimental occasion and cultured using Dulbecco’s modified Eagle medium (DMEM) containing serum and Geneticin.
Treatment Plate Preparation
The cells were 80-90% confluent (see Section 9 for details of protocol deviations). On the day prior to treatment, cells were harvested and distributed into 96-well plates (10000 cells/well) and incubated at 37±1°C, 5% (v/v) CO2, for 24±1 hours.
For each repetition, three replicates were used for the luciferase activity measurements and one parallel replicate used for the cell viability assay.
Treatment
At the end of the 24-hour incubation period, the medium was removed and replaced with fresh culture medium (containing serum but without Geneticin) to which test article and control formulations were added.
One well per plate was left empty (no cells and no treatment) to assess background values.
Each plate was sealed and incubated at 37±1°C, 5% (v/v) CO2 in air, in a humidified environment for 48±1 hours.
For each test article and positive control, one experiment was needed to derive a prediction (positive or negative), consisting of at two independent repetitions each containing three replicates of each concentration.
The data for repetition 1 was obtained from a repeat experiment as the initial experiment did not meet the acceptance criteria for the positive or negative controls.
The data from the initial experiment has not been reported.
Discordant results were obtained between the two repetitions, therefore a third repetition containing three replicates was performed.
Each independent repetition was performed on a different day with fresh stock solutions of chemicals and independently harvested cells. The cells came from different passages.
Cytotoxicity Assessment
After the 48-hour exposure period, the medium was replaced with fresh medium containing MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide).The plate was sealed and incubated for 4 hours at 37±1°C, 5% (v/v) CO2.
The MTT medium was removed and SDS (at 10% w/v) added per well. The plate was sealed and placed into an incubator at 37±1°C, 5% (v/v) CO2 in air and left overnight.
After the overnight incubation, the plate was shaken to ensure homogeneity of the solution in the wells and then absorption read at 600 nm using a SpectraMax M2e.

Luciferase Activity Measurements
After the 48-hour exposure period, the cells were washed with phosphate buffered saline and lysis buffer for luminescence readings was added to each well. The plates were then incubated for 20 minutes at 25±2°C, loaded into the luminescence plate
reader and read using the following parameters: 100 µL injection (Luciferase assay substrate), 15 second delay, 7 second luminescence integration time.
Positive control results:
Luciferase activity induction obtained with the positive control was statistically significant above the threshold of 1.5 at concentrations of 16 to 64 µM in Experiment 1 and at concentrations of 32 to 64 µM in Experiment 2. The EC1.5 value for the positive control were 11.02 and 18.11 µM in Experiments 1 and 2, respectively. The average induction in the two replicates for the positive control at 64 µM were 10.56 and 3.08 in Experiments 1 and 2, respectively.
Parameter:
other: Mamimal fold increase
Remarks:
Imax
Run / experiment:
Experiment 1
Value:
0.78
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of skin sensitisation
Parameter:
other: Maximal fold increase
Remarks:
Imax
Run / experiment:
Experiment 2
Value:
1.05
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
- Viability:
The cell viability measurement was not applicable as there were no EC1.5 determining concentrations in Experiments 1 or 2.
The IC50 value could not be calculated for Experiment 1 as all viability results were below 50% or in Experiment 2 as the minimum viability did not drop below 50%.
The IC30 value was 958.72 µM in Experiment 2. An IC30 value could not be calculated for Experiment 1 as all viability results were below 70%.

- Assay Acceptance:
Luciferase activity induction obtained with the positive control was statistically significant above the threshold of 1.5 at concentrations of 16 to 64 µM in Experiment 1 and at concentrations of 32 to 64 µM in Experiment 2.
The EC1.5 value for the positive control were 11.02 and 18.11 µM in Experiments 1 and 2, respectively. The average induction in the two replicates for the positive control at 64 µM were 10.56 and 3.08 in Experiments 1 and 2, respectively.
The average coefficient of variation of the luminescence reading for the negative control (DMSO) was 9.87% and 6.63% in Experiments 1 and 2, respectively.
All acceptance criteria were met, with the exception that in Experiment 1, the EC1.5 for the positive control was above the range specified in the protocol at 10.56. However, as luciferase induction values showed a clear dose response, it was considered that this was a valid experiment.
The assay was considered valid as the results in Experiment 1 were replicated in Experiment 2.
Interpretation of results:
GHS criteria not met
Conclusions:
The Imax in both experiments was less than 1.5 and not statistically significant, therefore the test article, Dermol GTR, was considered to be negative in the ARE-Nrf2 Luciferase Test.
Executive summary:

The study was conducted to investigate the potential of Dermol GTR to induce genes that are regulated by the antioxidant response element (ARE). The data may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.

The test article was dissolved in dimethylformamide (DMF) to the final stock concentration (200 mM). Serial dilutions were then made using DMF to obtain 12 master concentrations of the test article (0.098, 0.196, 0.391, 0.781, 1.563, 3.125, 6.25, 12.5, 25, 50, 100 and 200 mM).

The master concentrations were then further diluted 25 fold into culture medium containing serum and finally used for treatment with a further 4 fold dilution factor so that the final concentrations ranged from 0.98 to 2000 µM.

Aliquots of 50 µL of each of the final concentrationswere transferred to each of three luciferase plates and a single viability plate. Each plate was sealed using a plate sealer and then incubated at 37±1°C, 5% (v/v) CO2 in air, in a humidified environmentfor 48±1 hours.

After the 48-hour exposure period, the medium in the luciferase plates was replaced with fresh medium containing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT).The plate was sealed and incubated for 4 hours at 37±1°C, 5% (v/v) CO2. The MTT medium was then removed and SDS (at 10% w/v) added per well. The plate was sealed and placed into an incubator at 37±1°C, 5% (v/v) CO2 in air and left overnight. After the overnight incubation, the plate was shaken to ensure homogeneity of the solution in the wells and then absorption read at 600 nm using a SpectraMax M2e.

After the 48-hour exposure period, the cells in the viability plate were washed with phosphate buffered saline and lysis buffer for luminescence readings was added to each well. The plates were then incubated for 20 minutes at 25±2°C, loaded into the luminescence plate reader and read.

The results are summarised as follows:

Criteria

Experiment 1

Experiment 2

Imax

0.78

1.05

Cell Viability

N/A

N/A

EC1.5

>2000 µM

>2000 µM

Dose Response

No

No

 

All acceptance criteria were met, with the exception that in Experiment 1, the EC1.5 for the positive control was above the range specified in the protocol at 10.56. However, luciferase induction values showed a clear dose response, therefore it was considered that this was a valid experiment.

The test article, Dermol GTR, was considered to be negative in the ARE-Nrf2 Luciferase Test.

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 November 2017 to 18 January 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
05 Feb 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
direct peptide binding assay
Justification for non-LLNA method:
This is an in chemico test and the test material is used in cosmetic ingredients. Regulation 1223/2009 Article 18 restricts the use of in vivo studies on these types of raw materials.
Test material information:
Composition 1
Specific details on test material used for the study:
The test article, a clear, yellow liquid, was identified as chemical name Glyceryl Triacetyl Ricinoleate and was received at Covance on 31 October 2017 as follows:
CAS Number 101-34-8
Storage 15 to 25°C, protected from light
Purity 100%

The test article was dissolved in isopropanol. This was the first of the listed vehicles that produced a visually clear solution at a concentration of 100 mM.
The positive control was dissolved in acetonitrile at a concentration of 100 mM.
A stock solution containing cysteine at approximately 0.667 mM was prepared in 100 mM Phosphate Buffer pH 7.5 and a stock solution containing lysine at approximately 0.667 mM was prepared in 100 mM ammonium acetate buffer pH 10.2.
Formulations were prepared shortly before testing.

A certificate of analysis for the test article was provided by the Sponsor and is presented in the Certificate of Analysis.Cinnamaldehyde (CAS No. 104-55-2, batch number MKBT8955V, purity 99.1%, expiry 29 February 2020) was used as the positive control.
The peptides, cysteine (lot number P170608-LC180433, purity 96.07%) and lysine (lot number P170608-LC107617, purity 96.32%) were obtained from RS Synthesis, Louisville, Kentucky, USA.

Details on study design:
Objectives
The study was conducted to quantify the reactivity of the test article towards model synthetic peptides containing either lysine or cysteine. The data is used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling. The DPRA is an in chemico method which quantifies the remaining concentration of cysteine- or lysine-containing peptides following incubation with the test article. Relative peptide concentration was measured by high performance liquid chromatography (HPLC) with UV detection. Cysteine and lysine peptide percent depletion (PPD) values were then calculated and used in a prediction model which allows assigning the test article to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.

Test Article Incubation
Each test solution was prepared at ratios of 1:10 and 1:50 with the cysteine and lysine stock solutions, respectively. The preparations were placed in an incubator set at 25°C or 24±2 hours. At the end of the incubation period the samples were visually inspected for precipitate formation.

Analytical Method
The following HPLC conditions were applied:
Column: Agilent Zorbax SB-C18 2.1 mm x 100 mm, 3.5 µm or equivalent
Wavelength: 220 nm
Guard column: Phenomenex Security Guard c18 4 mm x 2 mm
Flow rate: 0.35 mL/min
Oven temperature: 30°C
Sample temperature: 25°C
Injection volume: 5 µL

Mobile Phase:
Phase A: 0.1% (v/v) of trifluoroacetic acid in MilliQ water
Phase B: 0.085% (v/v) of trifluoroacetic acid in acetonitrile

Gradient: Time (min) Phase A Phase B
0 90 10
10 75 25
11 10 90
13 10 90
13.5 90 10
20 90 10

Reference and Co-elution Controls

Reference controls were prepared for each peptide.
Reference Control A and B for each peptide were prepared by adding 750 µL of peptide stock solution to 250 µL of acetonitrile.
Reference Control C for cysteine was prepared by adding 750 µL of peptide stock solution to 200 µL of acetonitrile and 50 µL vehicle.
Reference Control C for lysine was prepared by adding 750 µL of peptide stock solution to 250 µL vehicle.
Reference Control A (in triplicate) was used to verify the HPLC system suitability prior to the analysis. Reference Control B (six replicates) was used to verify the stability of the reference controls over time and Reference Control C (in triplicate) was used to verify that acetonitrile did not impact the percent peptide depletion.

Co-elution controls were prepared to detect possible co-elution of the test article with the peptides. A mixture of 750 µL of 100 mM Phosphate Buffer pH 7.5, 200 µL of acetonitrile and 50 µL of test article solution was used to detect possible co-elution of the test article with cysteine. A mixture of 750 µL of 100 mM ammonium acetate buffer pH 10.2 and 250 µL of test article solution was used to detect possible co-elution of the test article with lysine.

Calibration Curves for Peptides
Calibration curves were prepared for each peptide using a range of concentrations from approximately 0.534 mM to 0.0167 mM (Standards 1 to 6).
Standard 1 for cysteine was prepared at approximatively 0.534 mM by dilution of 1600 µL of the peptide stock solution (0.667 mM) with 400 µL of acetonitrile.
Standards 2 to 6 for cysteine were prepared by serial dilution using dilution buffer (20% acetonitrile in 100 mM Phosphate Buffer pH 7.5).
Standard 1 for lysine was prepared at approximatively 0.534 mM by dilution of 800 µL of the peptide stock solution (0.667 mM) with 200 µL of acetonitrile.
Standards 2 to 6 for lysine were prepared by serial dilution using dilution buffer (20% acetonitrile in 100 mM ammonium acetate buffer pH 10.2).
Samples of dilution buffer alone were also prepared.

Sample Analysis Sequence
The analysis sequence for each peptide was as follows:
System suitability Standard 1 Dilution buffer
Calibration standards and reference controls Standard 1
Standard 2
Standard 3
Standard 4
Standard 5
Standard 6
Dilution Buffer
Reference Control A, rep 1
Reference Control A, rep 2
Reference Control A, rep 3
Co-elution controls Co-elution control for test article
Reference controls Reference Control B, rep 1
Reference Control B, rep 3
First set of replicates Reference Control C, rep 1
Positive Control, rep 1
Test sample, rep 1
Second set of replicates Reference Control C, rep 2
Positive Control, rep 2
Test sample, rep 2
Third set of replicates Reference Control C, rep3
Positive Control, rep 3
Test sample, rep 3
Reference controls Reference Control B, rep 4
Reference Control B, rep 5
Reference Control B, rep 6

Positive control results:
See tables in "Any other information" section
Key result
Parameter:
other: %
Run / experiment:
Cysteine depletion
Value:
ca. 2.72
Vehicle controls valid:
not examined
Negative controls valid:
not examined
Positive controls valid:
yes
Key result
Parameter:
other: %
Run / experiment:
Lysine depletion
Value:
ca. 0
Vehicle controls valid:
not examined
Negative controls valid:
not examined
Positive controls valid:
yes
Key result
Parameter:
other: %
Run / experiment:
Mean of the cysteine and lysine depletion
Value:
ca. 1.36
Vehicle controls valid:
not applicable
Negative controls valid:
not applicable
Positive controls valid:
not applicable

Protocol Deviations

Injection Volume

An injection volume of 5 uL was used and not 7 uL as specified in the protocol. This deviation from protocol did not affect the integrity or outcome of the study as all assay acceptance criteria were met for both the positive and negative controls.

Interpretation of results:
GHS criteria not met
Conclusions:
All acceptance criteria were met in both experiments.
The test article, Dermol GTR, was considered to be negative with no or minimal reactivity in the Direct Peptide Reactivity Assay.
Executive summary:

The study was conducted to quantify the reactivity of Dermol GTR towards model synthetic peptides containing either lysine or cysteine. The data may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.

The test article was dissolved in isopropanol at a concentration of 100 mM.

The test solutions were incubated at 1:10 and 1:50 ratios with the cysteine and lysine peptides, respectively, for 24±2 hours in glass autosampler vials, protected from light and set at 25°C.

The remaining concentration of cysteine- or lysine-containing peptides following the 24 hour incubation period was measured by high performance liquid chromatography (HPLC) with gradient elution and UV detection at 220 nm.

The cysteine depletion value was 2.72%, the lysine depletion value was 0.00% and the mean of the cysteine and lysine depletion values was 1.36%.

The test article, Dermol GTR, was considered to be negative with no or minimal reactivity in the Direct Peptide Reactivity Assay.

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 November 2017 - 10 April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
other: OECD 442E The human cell line activation test (h-CLAT)
Version / remarks:
29 July 2016
Deviations:
no
Principles of method if other than guideline:
The study was conducted to investigate the potential of the test material to activate monocytes and dendritic cells in the human monocytic leukemia cell line THP-1, by quantifying changes in the expression of cell surface markers (CD86 and CD54).
The human Cell Line Activation Test (h-CLAT) has been evaluated in a European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM)-coordinated validation study and subsequent independent peer review by the EURL ECVAMScientific Advisory Committee (ESAC) and has been recommended to be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between sensitisers and non-sensitisers for the purpose of hazard
classification and labelling.
GLP compliance:
yes (incl. certificate)
Type of study:
activation of dentritic cells
Justification for non-LLNA method:
This study is an in vitro alternative used to avoid the requirement to use in vivo studies to investigate this end point
Test material information:
Composition 1
Specific details on test material used for the study:
The test article, a clear yellow liquid, was identified as chemical name Glyceryl Triacetyl Ricinoleate and was received at Covance on 31 October 2017 as follows:
CAS Number: 101-34-8
Purity: 100%


Details on study design:
Test System

Specifications
Human monocytic leukemia cell line, THP-1 (an immortalised human monocyticleukemia cell line, used as a surrogate for dendritic cells), supplied by American Type Culture Collection (ATCC), Manassas, VA 20110 USA.

Identification
The test system was suitably labelled to clearly identify the study number, test article, test article concentration, positive and vehicle controls.

Cell Culture Maintenance
THP-1 cells were cultured, at 37ºC under 5% CO2 and humidified atmosphere, in RPMI0-1640 medium supplemented with 10% heat inactivated (HI) foetal bovine serum (FBS), 0.05 mM 2-mercaptoethanol, 100 units/mL penicillin and 100 µg/mL streptomycin
The cells were passaged every 2-3 days at a density of 0.1 to 0.2 x 10^6 cells/mL and maintained at a density from 0.1 x 10^6 to 0.8 x 10^6 cells/mL. Cell density did not exceed 1 x 10^6 cells/mL.

Reactivity Check
This was performed using 2,4-dinitrochlorobenzene (DNCB, CAS no. 97-00-7, ≥99% purity), nickel sulphate (CAS no. 10101-97-0, 99% purity) and lactic acid (CAS no. 50-21-5, 85% purity) two weeks after thawing. DNCB and nickel sulphate should produce a positive response of both CD86 and CD54 and lactic acid should produce a negative response of both CD86 and CD54.
Only cells which passed the reactivity check were used for the assay.

Plate Preparation
THP-1 cells were pre-cultured in culture flasks either at a density of 0.2 x 10^6 cells/mL for 48 hours or at a density of 0.1 x 10^6 cells/ml for 72 hours. On the days of testing, cells were harvested from the flasks and were resuspended with
fresh culture medium at 2 x 10^6 cells/mL. The cells were then distributed into a 24-well flat-bottomed plate (80 µL/1.6 x 10^5 cells per well).
Study Design

Dose Finding Assay
The test article working solutions or solvent controls were mixed 1:1 (v/v) with the cell suspensions in the 96-well plates. The plates were sealed and then incubated for 24 hours at 37°C, 5% CO2.
After the 24-hour incubation period, all cells from a 96-well flat-bottomed plate were transferred into a 96-well round-bottomed plate. The cells were washed at least twice in 200 µL of phosphate buffered saline containing 0.1% bovine serum albumin
(FACS buffer) and re-suspended in 190 µL of FACS buffer. 10 µL of propidium iodide solution (PI) was added just before FACS analysis (final concentration of PI = 0.625 µg/mL).

PI uptake was analysed using flow cytometry with the acquisition channel FL-3. A total of 10,000 viable cells were acquired.
Cell viability was calculated using the following equation:
Cell Viability =number of living cells/total number of acquired cells x 100
The CV75 value, i.e. a concentration showing 75% of THP-1 cell survival (25%
cytotoxicity), was 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 was the minimum value of cell viability over 75% in testing groups
c was the maximum value of cell viability below 75% in testing groups
b and d were the concentrations showing the value of cell viability a and c respectively

CD86/CD54 Expression Measurement
The prediction was derived from three valid, independent runs. Each independent run was performed on a different day. On the days of testing, cells harvested from the flasks were resuspended with fresh culture medium at 2 x 10^6 cells/mL. The cells were then distributed into a 24-well plate (500 µL/1 x 10^6 cells per well). The test article working solution or solvent control was mixed 1:1 (v/v) with the cell suspensions in the 24-well plates. The plates were sealed and then incubated for 24 hours at 37°C, 5% CO2. After the 24-hour incubation period, the cells were transferred into sample tubes, collected by centrifugation (approximately 250 g, 5 minutes) and washed twice with 1 mL of FACS buffer. After washing, the cells were blocked with 600 µL of blocking solution (FACS buffer containing 0.01% (w/v) globulin) at 4ºC for 15 minutes. After blocking, the cells were split into three aliquots of 180 µL into a 96-well plate and centrifuged (approximately 250 g, 3 minutes). After centrifugation, the cells were stained with 50 µL of FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 antibodies at 4ºC for 30 minutes. The stained cells were washed three times with an excess of FACS buffer, re-suspended in FACS buffer and 12.5 µg/mL PI solution was added (to give a final PI concentration of 0.625 µg/mL). The expression levels of CD86 and CD54 and cell viability were analysed using flow cytometry.

Test Article Formulation

Dose Finding Assay
The test article was dissolved at 100 mg/mL in DMF then eight stock solutions were prepared by 2-fold serial dilutions using the corresponding solvent.
The stock solutions were then further diluted 50-fold in culture medium (working solutions).

CD86/CD54 Expression Measurement
The test article was dissolved at 100 mg/mL in DMF then eight stock solutions were prepared by 1.2-fold serial dilutions using the corresponding solvent.
The stock solutions were then further diluted 250-fold into the culture medium (working solutions).

Data Evaluation

Analysis of Results
Based on the geometric mean fluorescence intensity (MFI), the relative fluorescence intensity (RFI) of CD86 and CD54 for the positive control cells and test article-treated cells were calculated according to the following equation:
RFI = (MFI of test article-treated cells – MFI of test article-treated isotype control cells)/(MFI of solvent-treated cells – MFI of solvent-treated isotype control cells) x100

The cell viability from the isotype control cells (stained with mouse IgG1 antibodies)
was calculated using the following equation:
Cell Viability = number of living cells/ total number of acquired cells x100

Prediction Model
An h-CLAT prediction is considered POSITIVE if at least one of the following conditions is met in 2 of 2 or in at least 2 of 3 independent runs, otherwise the h-CLAT prediction is considered NEGATIVE:
• The RFI of CD86 is ≥150% at any tested concentration (with cell viability ≥50%)
• The RFI of CD54 is ≥200% at any tested concentration (with cell viability ≥50%)

Calculation of Effective Concentration (EC) Values
For test articles predicted as POSITIVE, two EC values, the EC150 for CD86 and the EC200 for CD54, are calculated as follows:

EC150 (for CD86) = Bconcentration + [(150 – BRFI) / (ARFI – BRFI) x (Adose – Bdose)]
EC200 (for CD54) = Bconcentration + [(200 – BRFI) / (ARFI – BRFI) x (Adose – Bdose)]

Where:
Aconcentration is the lowest concentration in µg/mL with RFI >150 (CD86) or 200
(CD54)
Bconcentration is the highest concentration in µg/mL with RFI <150 (CD86) or 200
(CD54)
ARFI is the RFI at the lowest concentration with RFI >150 (CD86) or 200 (CD54)
BRFI is the RFI at the highest concentration with RFI <150 (CD86) or 200 (CD54).

Assay Acceptance Criteria
• The cell viabilities of medium and solvent control should be higher than 90%.
• In the solvent control, RFI values of both CD86 and CD54 should not exceed the positive criteria (CD86 RFI ≥150% and CD54 RFI ≥200%).
• For both medium and solvent controls, the MFI ratio of both CD86 and CD54 to isotype control should be >105%.
• In the positive control (DNCB), RFI values of both CD86 and CD54 should meet the positive criteria (CD86 RFI ≥150% and CD54 RFI ≥200%) and cell viability should be more than 50%.
• For the test article, the cell viability should be more than 50% in at least four tested doses in each run.

Negative Results
When 5000 µg/mL in saline (or medium), 1000 µg/mL in DMSO or the highest soluble concentration is used as the maximal test concentration of the test article, a negative result is acceptable even if the cell viability is above 90%.
Positive control results:
For the positive control, RFI values were ≥150% for CD86 and ≥200% for CD54 in all each independent runs. Cell viability was >50% in each independent run.
Parameter:
other: RFI (CD86)
Run / experiment:
17.44 µg/mL / Exp 1
Value:
91
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of skin sensitisation
Parameter:
other: RFI (CD86)
Run / experiment:
20.93 µg/mL / Exp 1
Value:
92
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of skin sensitisation
Parameter:
other: RFI (CD86)
Run / experiment:
25.12 µg/mL / Exp 1
Value:
91
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of skin sensitisation
Parameter:
other: RFI (CD86)
Run / experiment:
30.14 µg/mL / Exp 1
Value:
84
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of skin sensitisation
Parameter:
other: RFI (CD86)
Run / experiment:
36.17 µg/mL / Exp 1
Value:
88
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of skin sensitisation
Parameter:
other: RFI (CD86)
Run / experiment:
43.40 µg/mL / Exp 1
Value:
84
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of skin sensitisation
Parameter:
other: RFI (CD86)
Run / experiment:
52.08 µg/mL / Exp 1
Value:
84
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of skin sensitisation
Parameter:
other: RFI (CD86)
Run / experiment:
62.50 µg/mL / Exp 1
Value:
77
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of skin sensitisation
Parameter:
other: RFI (CD86)
Run / experiment:
17.44 µg/mL / Exp 2
Value:
89
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of skin sensitisation
Parameter:
other: RFI (CD86)
Run / experiment:
20.93 µg/mL / Exp 2
Value:
105
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of skin sensitisation
Parameter:
other: RFI (CD86)
Run / experiment:
25.12 µg/mL / Exp 2
Value:
93
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of skin sensitisation
Parameter:
other: RFI (CD86)
Run / experiment:
30.14 µg/mL / Exp 2
Value:
93
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of skin sensitisation
Parameter:
other: RFI (CD86)
Run / experiment:
36.17 µg/mL / Exp 2
Value:
87
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of skin sensitisation
Parameter:
other: RFI (CD86)
Run / experiment:
43.40 µg/mL / Exp 2
Value:
109
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of skin sensitisation
Parameter:
other: RFI (CD86)
Run / experiment:
52.08 µg/mL / Exp 2
Value:
121
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of skin sensitisation
Parameter:
other: RFI (CD86)
Run / experiment:
62.50 µg/mL / Exp 2
Value:
116
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of skin sensitisation
Parameter:
other: RFI (CD54)
Run / experiment:
17.44 µg/mL / Exp 1
Value:
84
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of skin sensitisation
Parameter:
other: RFI (CD54)
Run / experiment:
20.93 µg/mL / Exp 1
Value:
77
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of skin sensitisation
Parameter:
other: RFI (CD54)
Run / experiment:
25.12 µg/mL / Exp 1
Value:
76
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of skin sensitisation
Parameter:
other: RFI (CD54)
Run / experiment:
30.14 µg/mL / Exp 1
Value:
78
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of skin sensitisation
Parameter:
other: RFI (CD54)
Run / experiment:
36.17 µg/mL / Exp 1
Value:
84
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of skin sensitisation
Parameter:
other: RFI (CD54)
Run / experiment:
43.40 µg/mL / Exp 1
Value:
74
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of skin sensitisation
Parameter:
other: RFI (CD54)
Run / experiment:
52.08 µg/mL / Exp 1
Value:
71
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of skin sensitisation
Parameter:
other: RFI (CD54)
Run / experiment:
62.50 µg/mL / Exp 1
Value:
69
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of skin sensitisation
Parameter:
other: RFI (CD54)
Run / experiment:
17.44 µg/mL / Exp 2
Value:
95
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of skin sensitisation
Parameter:
other: RFI (CD54)
Run / experiment:
20.93 µg/mL / Exp 2
Value:
99
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of skin sensitisation
Parameter:
other: RFI (CD54)
Run / experiment:
25.12 µg/mL / Exp 2
Value:
96
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of skin sensitisation
Parameter:
other: RFI (CD54)
Run / experiment:
30.14 µg/mL / Exp 2
Value:
124
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of skin sensitisation
Parameter:
other: RFI (CD54)
Run / experiment:
36.17 µg/mL / Exp 2
Value:
99
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of skin sensitisation
Parameter:
other: RFI (CD54)
Run / experiment:
43.40 µg/mL / Exp 2
Value:
101
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of skin sensitisation
Parameter:
other: RFI (CD54)
Run / experiment:
52.08 µg/mL / Exp 2
Value:
101
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of skin sensitisation
Parameter:
other: RFI (CD54)
Run / experiment:
62.50 µg/mL / Exp 2
Value:
104
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
- Dose Finding Assay:
No CV75 value was calculated, as there was no effect on viability.
An oily precipitate was observed during the dilution steps at concentrations of 62.5 to 1000 µg/mL, therefore the maximum attainable concentration for the CD86/CD54 expression measurements was 62.5 µg/mL.

- Assay Acceptance Criteria Results:
All assay acceptance criteria were met.
The cell viabilities of medium and solvent control were higher than 90% in each independent run.
In the solvent control, RFI values of both CD86 and CD54 did not exceed the positive criteria (CD86 RFI ≥150% and CD54 RFI ≥200%).
For both medium and solvent controls, the MFI ratio of both CD86 and CD54 to isotype control was >105% on all occasions.
For the positive control, RFI values were ≥150% for CD86 and ≥200% for CD54 in all each independent runs. Cell viability was >50% in each independent run.
For the test article, the cell viability was more than 50% in all tested concentrations in each independent run.
Interpretation of results:
GHS criteria not met
Conclusions:
The test article was considered to be negative in the human Cell Line Activation Test, under the conditions of this study.
Executive summary:

The study was conducted to investigate the potential of EC 202 -935 -1 to activate monocytes and dendritic cells in the human monocytic leukemia cell line THP-1, by quantifying changes in the expression of cell surface markers (CD86 and CD54). The data may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.

The test article was dissolved in isopropanol and a dose finding assay was conducted to determine a concentration showing 75% THP-1 cell survival (CV75). No CV75 value was calculated as there was no effect on viability at the maximum attainable concentration (62.50 µg/mL).

Eight stock solutions were prepared by 1.2-fold serial dilutions using isopropanol to give eight doses ranging from 8.72 to 31.25 mg/mL. These stock solutions were then diluted 250‑fold into the culture medium (working solutions).

Aliquots of 500 µL of each of the working solutionswere mixed 1:1 (v/v) with cell suspensions at 1 x 106 cells per well. Each plate was sealed using a plate sealer and then incubated at 37±1°C, 5% CO2 in air, in a humidified environmentfor 24 hours.

After the 24-hour incubation period, the cells were transferred into sample tubes, collected by centrifugation, washed with FACS buffer and then blocked with 600 µL of blocking solution (FACS buffer containing 0.01% (w/v) globulin) at 4°C for 15 minutes.

After blocking, the cells were split into three aliquots of 180 µL into a 96-well plate, centrifuged and then stained with FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 antibodies at 4°C for 30 minutes.

The stained cells were washed with FACS buffer and re-suspended in FACS buffer and propidium iodide solution was added. The expression levels of CD86 and CD54 and cell viability were analysed using flow cytometry.

The relative fluorescence intensity (RFI) values for the test article were calculated as follows:

Concentration (µg/mL) RFI (CD86) RFI (CD54)
Exp 1 Exp 2 Exp 1 Exp 2
17.44 91 89 84 95
20.93 92 105 77 99
25.12 91 93 76 96
30.14 84 93 78 124
36.17 88 87 84 99
43.4 84 109 74 101
52.08 84 121 71 101
62.5 77 116 69 104
Negative control 134 126 110 149
Positive control 286 221 284 187

The test article,Dermol GTR, was considered to be negative in the human Cell Line Activation Test, under the conditions of this study.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The registered substance failed to induce sensitisation reactions in any of the in vitro and in chemico tests performed. The registered substance is therefore considered not to fulfill the criteria for classification as a skin sensitiser under the Classification, Labelling, and Packaging (CLP) regulation (1272/2008).