Registration Dossier

Administrative data

Description of key information

Effects on skin sensitisation:Not sufficient for classification under GHS

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:
key study
Study period:
12/9/2017 to 30/11/2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD Guideline 442E (human Cell Line Activation Test (h-CLAT)
Version / remarks:
Adopted 29 July 2016.
GLP compliance:
yes
Type of study:
activation of dendritic cells
Specific details on test material used for the study:
The test article, a clear liquid, was identified as Bernel Ester TCC and was received at Covance on 30 June 2017 as follows:
Test Article: Bernel Ester TCC
CAS Number: 16502-99-1
Storage: "15 to 25°C protected from light
Batch Number ESTS185/17, P7803
Retest Date 23-Jan-19
Purity 94%
A certificate of analysis for the test article was provided by the Sponsor. The solvent control was DMSO (at a concentration of 0.4% in culture medium). DMSO was supplied by Sigma Aldrich, Gillingham.
Details on study design:
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

Negative results are acceptable only for test articles exhibiting a cell viability of less than 90% at 1.2 x CV75 (highest concentration). If the cell viability at 1.2 x CV75 is equal to or greater than 90% the negative results should be discarded and the dose selection should be refined by repeating the CV75 determination.
When 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%.
Parameter:
other: Relative Fluorescence Intensity - RFI (%)
Run / experiment:
Expression levels of CD86 and cell viability were analysed using flow cytometry. Cell viability >= 50 %
Value:
< 200
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
not specified
Remarks on result:
no indication of skin sensitisation
Parameter:
other: Relative Fluorescence Intensity - RFI (%)
Run / experiment:
Expression levels of CD54 and cell viability were analysed using flow cytometry. Cellviability >= 50 %
Value:
< 200
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
not specified
Remarks on result:
no indication of skin sensitisation

RESULTS

Dose Finding Assay

The cell viability results are given in the table below:

  Viability (%) at Concentration (µg/mL) 
Run 0.781 1.562 3.125 6.25 12.5 25 50 100
1 99.1 98.6 98.8 99.0 98.9 99.0 99.3 99.0
2 99.1 98.8 99.2 99.3 99.0 99.2 99.0 98.8

No effect on viability was noted, therefore no CV75 value could be calculated.

CD86/CD54 Expression Results

Geometric mean fluorescence intensity (MFI) and viability results are given in the table below:

Experiment 1


  MFI (Geo Mean) Corrected MFI Viability
Concentration (ug/mL) CD86 CD54 Isotype CD86 CD54 CD86 CD54
27.9 1108 736 596 512 140 97.5 97.9
33.48 976 714 596 380 118 98.7 98.6
40.18 969 705 573 396 132 98.6 98.9
48.22 948 720 583 365 137 98.7 98.8
57.87 947 742 588 359 154 98.7 98.8
69.44 951 730 587 364 143 98.6 98.8
83.33 911 713 582 329 131 98.5 98.5
100 937 725 572 365 153 98.5 98.6
Solvent 1063 719 595 468 124 98.5 98.9
Positive control 2182 1141 717 1465 424 89.7 90.7

Experiment 2

  MFI (Geo Mean) Corrected MFI Viability
Concentration (ug/mL) CD86 CD54 Isotype CD86 CD54 CD86 CD54
27.9 1301 663 538 763 125 98.3 98.3
33.48 1276 661 536 740 125 98.4 98.8
40.18 1267 641 528 739 113 98.3 98.6
48.22 1322 658 539 783 119 98.3 98.3
57.87 1385 653 552 833 101 97.8 97.9
69.44 1336 662 557 779 105 98.4 98.7
83.33 1293 656 551 742 105 98.5 98.5
100 1392 672 550 842 122 98.3 98.8
Solvent 1282 675 571 711 104 98.7 99
Positive control 3097 2276 747 2350 1529 82.5 82.2

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

Concentration (ug/mL) RFI (CD86) RFI (CD54)
Exp Exp 1 Exp 2 Exp 1 Exp 2
27.9 109 107 113 120
33.48 81 104 95 120
40.18 85 104 106 109
48.22 78 110 110 114
57.87 77 117 124 97
69.44 78 110 115 101
83.33 70 104 106 101
100 78 118 123 117
Interpretation of results:
other: The test article, Bernel Ester TCC, was considered to be negative in the human Cell Line Activation Test.
Conclusions:
CONCLUSION
The RFI of CD86 was <150% at all tested concentrations (with cell viability ≥50%) and the RFI of CD54 was <200% at all tested concentrations (with cell viability ≥50%) in both independent runs.
The test article, Bernel Ester TCC, was therefore considered to be negative in the human Cell Line Activation Test.
Executive summary:

The study was conducted to investigate the potential of Bernel Ester TCC 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 anhydrous analytical grade dimethyl sulphoxide (DMSO) and a dose finding assay was conducted to determine a concentration showing 75% THP-1 cell survival (CV75).

Eight stock solutions were prepared by 1.2-fold serial dilutions using DMSO to give eight doses ranging from 0.335 x CV75 to 1.2 x CV75. These stock solutions were then diluted 250-fold (for DMSO) into the culture medium (working solutions).

Aliquots of 500 µL of each of the working solutions were 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 environment for 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.

No effect on viability was noted during the dose finding assay, therefore no CV75 value could be calculated.

The RFI of CD86 was <150% at all tested concentrations (with cell viability ≥50%) and the RFI of CD54 was <200% at all tested concentrations (with cell viability ≥50%) in both independent runs.

The test article, Bernel Ester TCC, was therefore considered to be negative in the human Cell Line Activation Test.

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25/7/2017 to 24/11/2017
Reliability:
1 (reliable without restriction)
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:
other: Direct Pepditde reactivity Assay - DPRA
Justification for non-LLNA method:
This is a Non in vivo 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.
Specific details on test material used for the study:
The test article, a clear liquid, was identified as Bernel Ester TCC and was received at
Covance as follows:

Test Article Bernel Ester TCC
CAS Number 16502-99-1
Storage 15 to 30˚C, protected form light
Batch Number ESTS185/17, P7803
Retest Date 23 January 2019
Purity 94%

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.

A certificate of analysis for the test article was provided by the Sponsor and is presented in the Certificates 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 P161108-LC180433, purity 95.92%) and lysine (lot number P160825-LC107617, purity 99.26%) were obtained from RS Synthesis, Louisville, Kentucky, USA.
Details on study design:
Objectives
The study was conducted to quantify the reactivity of Isodecyl 3,5,5-trimethylhexanoate 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 all test article and co-elution
samples contained small particles, therefore all samples were centrifuged at 400 g for 5 minutes.

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 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 on results incl. tables".
Parameter:
other: Percent Peptide Depletion (PPD) - Lysine
Run / experiment:
1-3
Value:
> -4.38 - < -0.23
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid
Remarks on result:
other: The max PPD value across the 3 runs was -0.23, with a mean PPD of 0.00 (negative value) for the 3 runs
Parameter:
other: Percent Peptide Depletion (PPD) - Cysteine
Run / experiment:
1-3
Value:
> 2.31 - <= 3.69
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid
Remarks on result:
other: The max PPD value across 3 runs was 3.69, with a mean PPD of 3.05 for the 3 runs.

Lysine depletion

The percentage peptide depletion values were as follows:

Substance

Replicate Peptide Reference Control C

Peak Areas         Mean Peptide Peak Area

PPD

Mean

PPD

SD

Test Article

40.73

39.79

 

39.70

-2.59#

-0.23#

 

0.00

 

2.1

 

41.44

 

-4.38#

 

 

Positive Control

18.17

17.83

 

39.70

54.23

55.09

 

54.63

 

0.4

 

18.03

 

54.58

 

 

# Negative value was considered as “0” when calculating the mean

The r2value for the standard calibration curve was 0.99989.

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

0.487                     0.488

0.488

0.49

C

0.488                     0.487

0.494

0.49

 

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

Reference Control

Replicate

Peptide Peak Area

B

1

2

3

4

40.708 40.565 39.904

39.599

 

5

39.551

 

6

39.657

C

1

2

39.555 39.449

 

3

40.086

Mean

 

39.90

SD

 

0.46

CV

 

1.16

 

Cysteine Depletion

The percentage peptide depletion values were as follows:

Substance

Replicate Peptide Reference Control C

Peak Areas         Mean Peptide Peak Area

PPD

Mean

PPD

SD

Test Article

27.91                      

28.07                    28.98

3.69

3.14

 

3.05

 

0.7

 

28.31                      

2.31

 

 

Positive Control

8.10                        

7.98                       28.98

72.05

72.46

 

72.48

 

0.4

 

7.85                        

72.91

 

 

The r2value for the standard calibration curve was 0.995196.

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

0.492                     0.498

0.489

0.49

C

0.469                     0.485

0.463

0.47

 

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

Reference Control

Replicate

Peptide Peak Area

B

1

2

3

4

29.944 29.227 28.740

27.960

 

5

29.727

 

6

27.718

C

1

2

28.772

29.815

 

3

28.364

Mean

 

28.92

SD

 

0.82

CV

 

2.82

 

Conclusions:
The test article, Bernel Ester TCC, was considered to be negative in the Direct Peptide Reactivity Assay.
Executive summary:

The study was conducted to meet the known requirements of OECD Guidelines for Testing of Chemicals Method 442C (adopted 04 February 2015).

The test article, Bernel Ester TCC, was considered to be negative in the Direct Peptide Reactivity Assay.

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31/07/2017 to 30/10/2017
Reliability:
1 (reliable without restriction)
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 a Non in vivo 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.
Specific details on test material used for the study:
The test article, a clear liquid, was identified as Bernel Ester TCC, and was received at Covance on 30 June 2017 as follows:
Test Article Bernel Ester TCC
CAS Number 16502-99-1
Storage 15 to 25°C, protected from light
Batch Number ESTS185/17 P7803
Retest Date 23 January 2019
Purity 94%

A Certificate of Analysis for the test article was provided by the Sponsor.Dimethyl sulfoxide (DMSO) supplied by Sigma-Aldrich Chemical Company, Gillingham, UK was used as the negative control.
Cinnamic aldehyde (CAS No. 14371-10-9), supplied by Sigma-Aldrich Chemical Company, Gillingham, UK was used as the positive control.

Test Article Formulation
Test formulations were prepared using DMSO.
A master stock solution at a concentration of 50 mg/mL (94.5 mM) was prepared. This was the maximum attainable concentration in any of the listed vehicles.
Serial dilutions were made (from the 94.5 mM stock) using the vehicle to obtain 12 master concentrations (0.05, 0.09, 0.19, 0.37, 0.75, 1.5, 3.0, 5.9, 11.8, 23.5, 47 and 94 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 0.46 to 940 µM (see Section 9 for details of protocol deviations).
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.
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.
Parameter:
other: maximal fold increases (Imax)
Run / experiment:
1-3
Value:
> 1.39 - <= 2.72
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
other: The EC1.5values for the positive control were 8.13, 6.47 and 17.92 µM in experiments 1, 2 and 3, respectively.
Remarks on result:
other: The maximal fold increases (Imax) were 1.39, 2.72 and 1.44 for experiments 1, 2 and 3 respectively.
Parameter:
other: EC1.5 values
Run / experiment:
1-3
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
other: The EC1.5 values for the positive control were 8.13, 6.47 and 17.92 µM in Experiments 1, 2 and 3, respectively.
Remarks on result:
other: The EC1.5 value for Experiment 2 was 19.42µg/mL. There were no EC1.5 values for experiments 1 and 3 as there were no statistically significant increases in induction.
Other effects / acceptance of results:
Protocol Deviations

Procedure:
Concentrations for Testing

Protocol Deviation:
The maximum attainable concentration was 50 mg/mL (94.5 mM) in DMSO, therefore the concentration range specified in the Protocol could not be achieved. This deviation from Protocol did not impact on the integrity or outcome of the study as the study was considered acceptable at the maximum attainable concentration.

Calculation of Imaxand EC1.5Values

Luminescence readings and fold increases are given in Table 10.1, Table 10.2 and Table 10.3.

The maximal fold increases (Imax) were 1.39, 2.72 and 1.44 for Experiments 1, 2 and 3, respectively.

The EC1.5value for Experiment 2 was 19.42µg/mL. There were no EC1.5values for

Experiments 1 and 3 as there were no statistically significant increases in induction.

7.2    Viability

MTT-absorbance readings are given in Table 10.4.

Cell viability was 108% at the EC1.5determining concentration in Experiment 2. This measurement was not applicable for Experiments 1 and 3 as there were no EC1.5determining concentrations in these experiments.

In Experiment 2 there was an overall dose response for lucipherase.

In Experiments 1 and 3, there was no apparent overall dose response for luciferase and the dose response curves were not biphasic.

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, at concentrations of 8 to 64 µM in Experiment 2 and at concentrations of 32 to 64 µM in Experiment 3.

The EC1.5values for the positive control were 8.13, 6.47 and 17.92 µM in

Experiments 1, 2 and 3, respectively. The average induction in the three replicates for the positive control at 64 µM was 12.04, 8.67 and 2.82 in Experiments 1, 2 and 3, respectively.

The average coefficient of variation of the luminescence reading for the negative control (DMSO) was 14.36%, 7.49% and 17.91% in Experiments 1, 2 and 3, respectively.

Conclusions:
The test article, Bernel Ester TCC, was considered to be negative in the
ARE-Nrf2 Luciferase Test at the maximum attainable concentration achieved under the conditions of this study.
Executive summary:

The study was carried out in accordance with OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method).

The test article, Bernel Ester TCC, was considered to be negative in the ARE-Nrf2 Luciferase Test at the maximum attainable concentration achieved under the conditions of this study.

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

Justification for classification or non-classification