Dioctyl maleate, branched

Endpoint summary

Description of key information

The key studies were conducted to internationaly recognised testing guidelines and with GLP certification.

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:

key study

Study period:

31 October 2017- 23 November 2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Results were very weakly positive with low reactivity

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

Regulation 1223/2009, Article 18, restricts the use of in vivo studies on cosmetic raw materials (the sole use of this substance), therefore a recognised in chemico test was conducted on the test material.

Specific details on test material used for the study:

Test Article Bernel Ester DCM
CAS Number 85566-63-8
Storage 15 to 25˚C, protected from light
Purity 92.78%*

* Assumed 100% for testing

Details on the study design:

Test Article Formulation
The test article was dissolved in acetonitrile. 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.

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 for 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: 7 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 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).
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 2
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

DATA EVALUATION
Data Analysis and Calculations
The chromatographic data were collected and processed using Chromeleon, a validated data capture system.
A calibration curve was generated based on the concentration of standards and the peak area. All the peaks of the samples were integrated (standards, samples and controls) “valley to valley” (when possible) and Cysteine and Lysine peptides concentrations were determined from absorbance at 220 nm using the respective calibration curves. Areas were determined for all the samples.
The PPD of each peptide was determined in each sample with the following formula:

PPD = (1-(Peptide peak area in replicate injection/Mean peptide peak area in reference controls C) x 100

The mean peptide peak areas for the nine Reference Controls B and C, Standard Deviation (SD) and Coefficient of Variation (CV) were calculated.
The mean peptide peak areas and the mean peptide concentrations (mM) for the three Reference Controls C were calculated.
The chromatograms of Co-elution Controls were compared to the chromatograms of Reference Controls C.
For the positive control and for the test article, the PPD in each replicate was calculated from the peptide area of the replicate injection and the mean peptide peak for Reference Control C. The PPD of every injected positive control and test chemical replicate was calculated. The mean PPD of the three replicate determinations and SD were calculated.
The mean percent cysteine and percent lysine depletion values were calculated for each test chemical (negative depletion is considered as “0” when calculating the mean).

Assay Acceptance Criteria
The following criteria should be met for a run to be considered valid:
• The standard calibration curve should have a r2>0.99.
• The mean peptide concentration for reference controls A should be 0.50±0.05 mM and the coefficient of variation (CV) of peptide peak areas for the nine reference controls B and C should be <15.0%.
• The mean PPD value of the three replicates for the positive control and maximum standard deviation (SD) must fall within the ranges in the following table:

Peptide Mean PPD Values (%)
Lower Bound Upper Bound SD
Cysteine 60.8 100 <14.9
Lysine 40.2 69.0 <11.6

The following criteria should be met for the test article’s result to be considered valid:
• The maximum standard deviation for the test article replicates should be <14.9 for the percent cysteine depletion and <11.6 for the percent lysine depletion.
• The mean peptide concentration of the three reference controls C in the appropriate should be 0.50±0.05 mM.

#Prediction Model
The mean percent cysteine and percent lysine depletion value was calculated. By using the cysteine 1:10/lysine 1:50 prediction model below, the threshold of 6.38% average peptide depletion was used to support the discrimination between skin sensitisers and non-sensitisers in the framework of an IATA.

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
22.62% < mean % depletion ≤42.47% Moderate reactivity Positive
42.47% < mean % depletion ≤100% High reactivity

The mean percent depletion fell in the range of 3% to 10% for the cysteine 1:10/lysine 1:50 prediction model, therefore a second run was required.

Positive control results:

Cinnamic aldehyde (CAS No. 104-55-2, batch number MKBT8955V purity 99.1%, expiry 29 February 2020) was used as the positive control.
The mean percentage peptide depletion (PPD) values for the positive control were:
- 62.06for lysine
- 51.04 for cysteine

Run / experiment:

other: 1-3

Parameter:

other: % peptide depletion (PPD) lysine

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: 1-3

Parameter:

other: % peptide depletion (PPD) cysteine

Value:

6.29

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

All acceptance criteria were met in both Experiments, with the exception that in Experiment 2, the mean peptide concentration for Reference Control A was just below the range specified, at 0.43 mM and 0.44 mM for cysteine and lysine, respectively. As the routine system suitability test prior to sample analysis was satisfactory, the results were considered valid.

Lysine Depletion, Experiment 1

The percentage peptide depletion values were as follows:

Substance	Replicate Peptide Peak Areas	Reference Control C Mean Peptide Peak Area	PPD	Mean PPD	SD
Test Article	33.86	38.04	10.99	8.02	2.68
	35.84		5.78
	35.27		7.28
Positive Control	13.16	38.04	65.4	62.06	3.41
	15.75		58.6
	14.39		62.17

The r² value for the standard calibration curve was 0.99995.

The peptide concentrations for the Reference Controls A and C were as follows:

Reference Control	Peptide Concentration (mM)
	Replicate 1	Replicate 2	Replicate 3	Mean
A	0.46	0.47	0.48	0.47
C	0.47	0.48	0.48	0.48

The peak area results for Reference Controls B and C were as follows:

Reference Control	Replicate	Peptide Peak Area
B	1	37.49
	2	36.67
	3	40.15
	4	38.58
	5	39.19
	6	39.42
C	1	37.28
	2	38.43
	3	38.41
Mean		38.4
SD		1.11
CV		2.89

Lysine Depletion, Experiment 2

The percentage peptide depletion values were as follows:

Substance	Replicate Peptide Peak Areas	Reference Control C Mean Peptide Peak Area	PPD	Mean PPD	SD
Test Article	33.85	28.84	0	1.24	1.25
	28.12		2.5
	28.49		1.21
Positive Control	13.75	28.84	52.32	51.04	9.89
	17.14		40.57
	11.47		60.23

The r² value for the standard calibration curve was 0.99994.

The peptide concentrations for the Reference Controls A and C were as follows:

Reference Control	Peptide Concentration (mM)
	Replicate 1	Replicate 2	Replicate 3	Mean
A	0.44	0.43	0.44	0.44
C	0.45	0.45	0.46	0.45

The peak area results for Reference Controls B and C were as follows:

Reference Control	Replicate	Peptide Peak Area
B	1	33.42
	2	27.95
	3	27.71
	4	28.5
	5	31.77
	6	28.03
C	1	28.72
	2	28.37
	3	29.45
Mean		29.32
SD		1.97
CV		6.7

Cysteine Depletion, Experiment 1

The percentage peptide depletion values were as follows:

Substance	Replicate Peptide Peak Areas	Reference Control C Mean Peptide Peak Area	PPD	Mean PPD	SD
Test Article	24.75	26.41	6.29	7.03	0.65
	24.45		7.42
	24.46		7.38
Positive Control	6.34	26.41	75.99	76.47	1.46
	6.52		75.31
	5.78		78.11

The r² value for the standard calibration curve was 0.99865.

The peptide concentrations for the Reference Controls A and C were as follows:

Reference Control	Peptide Concentration (mM)
	Replicate 1	Replicate 2	Replicate 3	Mean
A	0.44	0.49	0.5	0.48
C	0.49	0.48	0.49	0.49

The peak area results for Reference Controls B and C were as follows:

Reference Control	Replicate	Peptide Peak Area
B	1	26.53
	2	26.61
	3	27.12
	4	25.89
	5	25.58
	6	25.94
C	1	26.86
	2	25.87
	3	26.48
Mean		26.32
SD		0.52
CV		1.98

Cysteine Depletion, Experiment 2

The percentage peptide depletion values were as follows:

Substance	Replicate Peptide Peak Areas	Reference Control C Mean Peptide Peak Area	PPD	Mean PPD	SD
Test Article	15.46	17.2	10.12	12.29	7.17
	13.71		20.29
	16.09		6.45
Positive Control	6.11	17.2	64.48	65.72	4.66
	6.57		61.8
	5.01		70.87

The r² value for the standard calibration curve was 0.99874.

The peptide concentrations for the Reference Controls A and C were as follows:

Reference Control	Peptide Concentration (mM)
	Replicate 1	Replicate 2	Replicate 3	Mean
A	0.41	0.44	0.42	0.43
C	0.44	0.46	0.44	0.45

The peak area results for Reference Controls B and C were as follows:

Reference Control	Replicate	Peptide Peak Area
B	1	16.55
	2	16.9
	3	15.05
	4	14.04
	5	14.16
	6	14.89
C	1	17.13
	2	17.7
	3	16.78
Mean		15.91
SD		1.38
CV		8.66

Interpretation of results:

study cannot be used for classification

Conclusions:

The test article, Bernel Ester DCM, was considered to be weakly positive (low reactivity) in the Direct Peptide Reactivity Assay.

Executive summary:

The study was conducted to quantify the reactivity of Bernel Ester DCM 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 acetonitrileat 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.

In Experiment 1, the cysteine depletion value was 7.03%, the lysine depletion value was 8.02% and the mean of the cysteine and lysine depletion values was 7.52%. As the results fell within the range of 3% to 10% for the cysteine 1:10/lysine 1:50 prediction model, a further run was conducted.

In Experiment 2, the cysteine depletion value was 12.29%, the lysine depletion value was 1.24% and the mean of the cysteine and lysine depletion values was 6.76%. These results were concordant with those of Experiment 1.

All acceptance criteria were met in both Experiments, with the exception that in Experiment 2, the mean peptide concentration for Reference Control A was just below the range specified, at 0.43 mM and 0.44 mM for cysteine and lysine, respectively. As the routine system suitability test prior to sample analysis was satisfactory, the results were considered valid.

The test article, Bernel Ester DCM, was considered to be weakly positive with low reactivity in the Direct Peptide Reactivity Assay.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 October 2017 to 18 December 2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Weakly positive results

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

The study was conducted to meet the known requirements of OECD Guidelines for Testing of Chemicals Method 442D (adopted February 2015).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Specific details on test material used for the study:

The test article, a clear colourless liquid, was identified as Bernel Ester DCM and was received at Covance as follows:

Test Article Bernel Ester DCM
CAS Number 85566-63-8
Storage 15 to 25˚C, protected from light.
Purity 92.78% (assumed 100% for testing)

A Certificate of Analysis for the test article was provided by the Sponsor.

Details on the study design:

Objectives
The study was conducted to investigate the potential of Bernel Ester DCM 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.

TEST SYSTEM

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/untreated 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.

METHODS

Treatment Plate Preparation
The cells were 80-90% confluent. 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.
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.
Untreated controls were included, since the chosen solvent is not specified in the OECD test guideline.

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.

CONTROLS
Dimethyl sulfoxide (DMSO) supplied by Sigma Aldrich Chemical Co. Ltd. was used as the negative control.
Cinnamic aldehyde (CAS No. 14371-10-9), supplied by Sigma Aldrich Chemical Co. Ltd. was used as the positive control.

Test Article Formulation
Test formulations were prepared using dimethylformamide (DMF). This was used as the test article was insoluble in the vehicles listed in the protocol. A visually clear solution was produced at a concentration of 200 mM in DMF.
Serial dilutions were made (from the 200 mM stock) using the vehicle to obtain 12 master concentrations (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 range from 0.98 to 2000 µM.
Formulations were prepared shortly before testing.

Negative and Positive Control Article Formulation
The negative control was diluted into culture medium containing serum so that the final concentration was 1%.
The positive control was prepared at a concentration of 6.4 mM in DMSO. Five master concentrations ranging from 0.4 to 6.4 mM were prepared in DMSO (from a 6.4 mM stock solution). 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 range from 4 to 64 µM.

DATA EVALUATION

Analysis of Results
The following parameters were calculated:
• The maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the test article 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) was obtained; and
• The IC50 and IC30 concentration values for 50% and 30% reduction of cellular viability.

Imax
Fold luciferase activity induction at each concentration of test article and positive control was calculated as follows:
Fold induction = (Lsample - Lblank) / (Lsolvent - Lblank)

where
• Lsample is the luminescence reading in the test article well,
• Lblank is the luminescence reading in the blank well containing no cells and no treatment,
• Lsolvent is the average luminescence reading in the wells containing cells and solvent (negative control).
The overall maximal average fold induction (Imax) was calculated as the average of the individual repetitions.

EC1.5
EC1.5 was calculated by linear interpolation as follows:
EC1.5 = (Cb – Ca) x (1.5 – Ia) / (Ib – Ib) + Ca

where
• Ca is the lowest concentration in µM with >1.5-fold induction
• Cb is the highest concentration in µM with <1.5-fold induction
• Ia is the fold induction measured at the lowest concentration with >1.5-fold induction (mean of three replicate wells)
• Ib is the fold induction at the highest concentration with <1.5-fold induction (mean of three replicate wells).
The overall EC1.5 was calculated as the geometric mean of the individual repetitions.

Viability
Viability was calculated as follows:
Viability = (Vsample - Vblank) / (Vsolvent - Vblank) x 100

where
• Vsample is the MTT-absorbance reading in the test article well
• Vblank is the MTT-absorbance reading in the blank well containing no cells and no treatment
• Vsolvent is the MTT-absorbance reading in the wells containing cells and solvent (negative control).

IC50 / IC30
IC50 and IC30 were calculated by linear interpolation as follows:
ICx = (Cb – Ca) x ( (100 – x) –Va ) / (Vb - Va) + Ca

where
• x is the % reduction at the concentration to be calculated (50 and 30 for IC50 and IC30)
• Ca is the lowest concentration in µM with >x% reduction in viability
• Cb is the highest concentration in µM with • Va is the % viability at the lowest concentration with >x% reduction in viability
• Vb is the % viability at the highest concentration with The overall IC50 and IC30 were calculated as the geometric mean of the individual repetitions.

Assay Acceptance Criteria
The following acceptance criteria should be met:
• The luciferase activity induction obtained with the positive control should be statistically significant above the threshold of 1.5 in at least one of the tested concentrations (from 4 to 64 µM).
• The EC1.5 value for the positive control should be within two standard deviations of the historical mean (e.g. between 7 and 30 µM based on the validation dataset) and the average induction in the three replicates for the positive control at 64 µM should be between 2 and 8. If the latter criterion is not fulfilled, the dose-response of cinnamic aldehyde will be evaluated to determine acceptability.
• The average coefficient of variation of the luminescence reading for the negative control (DMSO) should be below 20% in each repetition which consists of 6 wells in triplicate.

Prediction Model
A KeratinoSens™ prediction is considered positive if the following 4 conditions are all met in 2 of 2 or in the same 2 of 3 repetitions, otherwise the KeratinoSens™ prediction is considered negative:
1. The Imax is >1.5-fold and statistically significantly different when compared to the solvent (negative) control (se determined by a two-tailed, unpaired Student’s T-test);
2. The cell viability is >70% at the lowest concentration with induction of luciferase activity above 1.5-fold (i.e. at the EC1.5 determining concentration);
3. The EC1.5 value is <1000 µM (or <200 µg/mL for test articles with no defined MW);
4. There is an apparent overall dose response for luciferase induction or if the dose response curve is biphasic.

Positive control results:

Luciferase activity induction obtained with the positive control was statistically significant above the threshold of 1.5 at concentrations of 32 to 64 µM in Experiment 1 and Experiment 2.
The EC1.5 values for the positive control were 16.81 and 17.13 µM in Experiments 1 and 2, respectively. The average inductions in the two replicates for the positive control at 64 µM were 5.15 and 2.99 in Experiments 1 and 2, respectively.

Run / experiment:

other: Experiment 1

Parameter:

other: Imax

Value:

1.54

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: Experiment 2

Parameter:

other: Imax

Value:

2.5

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: Experiment 1

Parameter:

other: EC1.5

Value:

29.75

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: Experiment 2

Parameter:

other: EC1.5

Value:

18.93

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

Calculation of Imax and EC1.5 Values
Luminescence readings and fold increases are given in Table 10.1 and Table 9.2 (attached).
The maximal fold increases (Imax) were 1.54 and 2.50 in Experiments 1 and 2, respectively.
The EC1.5 values were 29.75 and 18.93 in Experiments 1 and 2, respectively.

Viability
MTT-absorbance readings are given in Table 10.3 (attached).
Cell viability at the EC1.5 determining concentration was 71.13% and 162.84% in Experiments 1 and 2, respectively.
The overall IC50 (the geometric mean of the individual repetitions) was >2000 and 248.22 in Experiments 1 and 2, respectively.
The overall IC30 (the geometric mean of the individual repetitions) was 1970.36 and 59.95 in Experiments 1 and 2, respectively.

ACCEPTANCE
Luciferase activity induction obtained with the positive control was statistically significant above the threshold of 1.5 at concentrations of 32 to 64 µM in Experiment 1 and Experiment 2.
The EC1.5 values for the positive control were 16.81 and 17.13 µM in Experiments 1 and 2, respectively. The average inductions in the two replicates for the positive control at 64 µM were 5.15 and 2.99 in Experiments 1 and 2, respectively.
The average coefficient of variation of the luminescence reading for the negative control (DMSO) was 26.83% and 12.21% in Experiments 1 and 2, respectively. The assay was considered valid as the results in Experiment 1 were replicated in Experiment 2.

Interpretation of results:

study cannot be used for classification

Conclusions:

All four conditions required for a positive prediction were met in both experiments, therefore the test article, Bernel Ester DCM, was considered to be positive in the ARE-Nrf2 Luciferase Test. However, the results were very weakly positive.

Executive summary:

Summary

The study was conducted to investigate the potential of Bernel Ester DCM 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 at37±1°C, 5% (v/v) CO₂in 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) CO₂in 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	1.54	2.50
Cell Viability	71%	162%
EC1.5	29.75 μM	18.93 μM
Dose Response	Yes	Yes

All acceptance criteria were met in Experiment 1, with the exception that the coefficient of variation of the luminescence readings for the negative control was 26.83%. All acceptance criteria were met in Experiment 2. The assay was considered valid as the results in Experiment 1 were replicated in Experiment 2.

All four conditions required for a positive prediction were met in both experiments, therefore the test article,Bernel Ester DCM, was considered to be weakly positive in the ARE-Nrf2 Luciferase Test.

Reference 3

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5 March 2018 to 15 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD 442E The human cell line activation test (h-Clat)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes

Type of study:

activation of dendritic 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

Specific details on test material used for the study:

Test Article: Bernel Ester DCM
Cas Number 85566-63-8
Storage 15 - 25 °C, protected from light#
Purity 92.78%

Details on the study design:

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 106 cells/mL and maintained at a density from 0.1 x 106 to 0.8 x 106 cells/mL. Cell density did not exceed 1 x 106 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 106 cells/mL for 48 hours or at a density of 0.1 x 106 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 106 cells/mL. The cells were then distributed into a 24-well flat-bottomed plate (80 µL/1.6 x 105 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 106 cells/mL.Thecellswere then distributed into a 24-well plate (500 µL/1 x 106 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.

Positive control results:

For the positive control, RFI values were ≥150% for CD86 and ≥200% for CD54 in all 3 independent runs. Cell viability was >50% in each independent run.

Run / experiment:

other: Experiment 1& 2

Parameter:

other: CD86 Expression Result

Remarks on result:

other: See results table below

Run / experiment:

other: Experiment 1 & 2

Parameter:

other: CD54 Expression Result

Remarks on result:

other: See results table below

CD86/CD54 expression results

Concentration (µg/mL)	RFI (CD86)		RFI (CD54)
	Exp 1	Exp 2	Exp 1	Exp 2
69.77	125	113	56	53
83.73	125	105	64	63
100.47	124	109	61	58
120.57	118	110	63	60
144.68	112	110	58	56
173.62	121	103	50	66
208.34	119	101	50	74
250.01	121	104	60	78

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 Bernel Ester DCM 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 (250 µg/mL).

Eight stock solutions were prepared by 1.2-fold serial dilutions using isopropanol to give eight doses ranging from 34.88 to 125 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 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% (v/v) CO₂ in air, in a humidified environmentfor 24±0.5 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 (or sample tubes), 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
69.77	125	113	56	53
83.73	125	105	64	63
100.47	124	109	61	58
120.57	118	110	63	60
144.68	112	110	58	56
173.62	121	103	50	66
208.34	119	101	50	74
250.01	121	104	60	78

The test article, Bernel Ester DCM, was considered to be negativein the human Cell Line Activation Test.

Endpoint conclusion

Endpoint conclusion:

no study available (further information necessary)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The test substance was negative in the hCLAT test and weakly positive (low reactivity) in the DPRA and Keratosens assays. Further testing will be needed to confirm whether the test substance is conclusively a skin sensitiser or not.