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Diss Factsheets

Administrative data

Description of key information

Key information was conducted to internationally recognised study guidelines and with GLP certification.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 October 2017 to 18 December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD Guidelines for Testing of Chemicals Method 442E
Version / remarks:
Adopted 29 July 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
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 2,3 propanetricarboxylic acid, 2-hydroxy-, 1,2,3-tris (2-octyldodecyl) ester and trioctyldodecyl citrate, was received at Covance on 27 July 2017 as follows:
CAS Number Storage Purity
126121-35-5 15 to 25°C, protected from light 99.39%
A certificate of analysis for the test article was provided by the Sponsor, see Certificate of Analysis.
Details on the study design:
Objectives:
The study was conducted to investigate the potential of Citmol 320 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 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 ECVAM Scientific 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.

Test System:

Specifications:
Human monocytic leukemia cell line, THP-1 (an immortalised human monocytic leukemia 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 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 96 well flat-bottomed plate (80 µL/1.6 x 105 cells per well) for the dose finding assay or into a 24-well plate (500 µL/1 x 106 cells per well) for the expression measurements.

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.
The final CV75 value was the mean of the results from two assays.

CD86/CD54 Expression Measurement:

One experiment (consisting of two independent runs) was needed to drive a prediction. The independent runs were performed on different days.
On the days of testing, cells harvested from the flasks were resuspended with fresh culture medium at 2 x 106 cells/mL. The cells were 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±0.5 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 500 mg/mL in isopropanol then eight stock solutions were prepared by 2-fold serial dilutions using the corresponding solvent.
The stock solutions were then further diluted 250-fold in culture medium (working solutions).
The top three concentrations following dilution in culture medium (250, 500 and 1000 µg/mL produced oily emulsions and were not treated. The maximum attainable concentration was therefore 125 µg/mL.

CD86/CD54 Expression Measurement
The test article was dissolved at 62.5 mg/mL in isopropanol then eight stock solutions of each test article were prepared by 1.2-fold serial dilutions using the corresponding solvent to give a range from 17.44 to 62.5.
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:
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%.



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.
Run / experiment:
other: 34.9 µg/mL / 1
Parameter:
other: RFI (CD86)
Value:
98
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Run / experiment:
other: 34.9 µg/mL / 2
Parameter:
other: RFI (CD86)
Value:
95
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Run / experiment:
other: 41.9 µg/mL / 1
Parameter:
other: RFI (CD86)
Value:
109
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Run / experiment:
other: 41.9 µg/mL / 2
Parameter:
other: RFI (CD86)
Value:
92
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Run / experiment:
other: 50.2 µg/mL / 2
Parameter:
other: RFI (CD86)
Value:
111
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Run / experiment:
other: 50.2 µg/mL / 2
Parameter:
other: RFI (CD86)
Value:
97
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Run / experiment:
other: 60.3 µg/mL / 1
Parameter:
other: RFI (CD86)
Value:
124
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Run / experiment:
other: 60.3 µg/mL / 2
Parameter:
other: RFI (CD86)
Value:
95
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Run / experiment:
other: 72.3 µg/mL / 1
Parameter:
other: RFI (CD86)
Value:
125
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Run / experiment:
other: 72.3 µg/mL / 2
Parameter:
other: RFI (CD86)
Value:
91
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Run / experiment:
other: 86.8 µg/mL / 1
Parameter:
other: RFI (CD86)
Value:
131
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Run / experiment:
other: 86.8 µg/mL / 2
Parameter:
other: RFI (CD86)
Value:
94
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Run / experiment:
other: 104.2 µg/mL / 1
Parameter:
other: RFI (CD86)
Value:
134
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Run / experiment:
other: 104.2 µg/mL / 2
Parameter:
other: RFI (CD86)
Value:
105
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Run / experiment:
other: 125. µg/mL / 1
Parameter:
other: RFI (CD86)
Value:
131
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Run / experiment:
other: 125 µg/mL / 2
Parameter:
other: RFI (CD86)
Value:
90
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Run / experiment:
other: 34.9 µg/mL / 1
Parameter:
other: RFI (CD54)
Value:
102
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Run / experiment:
other: 34.9 µg/mL / 2
Parameter:
other: RFI (CD54)
Value:
78
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Run / experiment:
other: 41.9 µg/mL / 1
Parameter:
other: RFI (CD54)
Value:
92
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Run / experiment:
other: 41.9 µg/mL / 2
Parameter:
other: RFI (CD54)
Value:
82
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Run / experiment:
other: 50.2 µg/mL / 1
Parameter:
other: RFI (CD54)
Value:
92
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Run / experiment:
other: 50.2 µg/mL / 2
Parameter:
other: RFI (CD54)
Value:
77
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Run / experiment:
other: 60.3 µg/mL / 1
Parameter:
other: RFI (CD54)
Value:
90
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Run / experiment:
other: 60.3 µg/mL / 2
Parameter:
other: RFI (CD54)
Value:
76
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Run / experiment:
other: 72.3 µg/mL / 1
Parameter:
other: RFI (CD54)
Value:
92
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Run / experiment:
other: 72.3 µg/mL / 2
Parameter:
other: RFI (CD54)
Value:
71
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Run / experiment:
other: 86.8 µg/mL / 1
Parameter:
other: RFI (CD54)
Value:
98
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Run / experiment:
other: 86.8 µg/mL / 2
Parameter:
other: RFI (CD54)
Value:
79
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Run / experiment:
other: 104.2 µg/mL / 1
Parameter:
other: RFI (CD54)
Value:
91
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Run / experiment:
other: 104.2 µg/mL / 2
Parameter:
other: RFI (CD54)
Value:
94
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Run / experiment:
other: 125 µg/mL / 1
Parameter:
other: RFI (CD54)
Value:
91
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Run / experiment:
other: 125 µg/mL / 2
Parameter:
other: RFI (CD54)
Value:
107
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
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 test article, the cell viability was more than 50% in all tested concentrations in each independent run.

Dose Finding Assay

The cell viability results are given in the table below:

Dose Finding Assay: Cell Viability

Run Viability (%) at Concentration (µg/mL)
7.8 15.6 31.3 62.5 125
1 99.2 99.4 99.1 98.8 98.4
2 99.3 99.3 99.2 99.3 99.2

An oily emulsion was observed during the dilution steps at concentrations of 250, 500 and 1000 µg/mL, therefore the maximum attainable concentration was 125 µg/mL.

No CV75 value was calculated, as there was no effect on viability.

CD86/CD54 Expression Results

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

Experiment 1

Concentration (µg/mL) MFI (Geo Mean) Corrected MFI Viability
CD86 CD54 Isotype CD86 CD54 IgG CD86 CD54
34.9 1219 656 546 673 110 98.7 98.4 98.8
41.9 1292 643 544 748 99 98.8 98.3 98.5
50.2 1308 644 545 763 99 98.8 98 98.5
60.3 1396 640 543 853 97 98.6 98.1 98.1
72.3 1399 643 544 855 99 98.6 98.3 98.3
86.8 1437 643 537 900 106 98.4 98.2 98.2
104.2 1461 641 543 918 98 98.7 98.3 98.3
125 1435 637 539 896 98 98.3 97.9 98
Medium 1271 665 539 732 126 98.8 98.6 98.8
Solvent 1226 648 540 686 108 98.9 98.7 98.7
Positive control 3479 1252 630 2849 622 90.4 84.8 86.9

Experiment 2

Concentration (µg/mL) MFI (Geo Mean) Corrected MFI Viability
CD86 CD54 Isotype CD86 CD54 IgG CD86 CD54
34.9 1301 635 552 749 83 98.7 97.7 98
41.9 1278 634 546 732 88 98.6 98 98
50.2 1316 632 550 766 82 98.7 97.7 98
60.3 1301 632 551 750 81 98.7 97.7 98.1
72.3 1278 631 555 723 76 97.9 97.4 97.5
86.8 1287 625 541 746 84 98.3 97.4 97.5
104.2 1372 639 538 834 101 98.6 96.6 97.6
125 1243 648 533 710 115 98.5 97.8 98
Medium 1354 653 548 806 105 98.4 98.4 98.1
Solvent 1334 649 542 792 107 98.6 98.1 97.8
Positive control 3716 1623 646 3070 977 79.3 74.9 76.2

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
34.9 98 95 102 78
41.9 109 92 92 82
50.2 111 97 92 77
60.3 124 95 90 76
72.3 125 91 92 71
86.8 131 94 98 79
104.2 134 105 91 94
125 131 90 91 107
Solvent control 94 98 86 102
Positive control 342 303 438 782

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.
Executive summary:

The study was conducted to investigate the potential ofCitmol 320to 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 (125 µg/mL).

Eight stock solutions were prepared by 1.2-fold serial dilutions using isopropanol to give a range from 17.44 to 62.5 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 106cells per well. Each plate was sealed using a plate sealer and then incubated at37±1°C, 5% (v/v) CO2in 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
34.9 98 95 102 78
41.9 109 92 92 82
50.2 111 97 92 77
60.3 124 95 90 76
72.3 125 91 92 71
86.8 131 94 98 79
104.2 134 105 91 94
125 131 90 91 107

The test article,Citmol 320, was considered to be negativein the human Cell Line Activation Test.

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 September 2017 - 19 December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
05 Feb 2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
direct peptide 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 and received at Covance as follows:
CAS Number 126121-35-5
Storage 15 to 25°C, protected from light
Expiration Date 31 December 2018

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

Test Article Formulation
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.
Run / experiment:
other: 1 / 1
Parameter:
other: Lysine Peptide depletion (PPD)
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Run / experiment:
other: 2 / 1
Parameter:
other: Lysine Peptide depletion (PPD)
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Run / experiment:
other: 3 / 1
Parameter:
other: Lysine Peptide depletion (PPD)
Value:
0.97
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Run / experiment:
mean
Parameter:
other: Lysine Peptide depletion (PPD)
Value:
0.32
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Run / experiment:
other: 1 / 1
Parameter:
other: Cysteine peptide depletion (PPD)
Value:
30.3
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Run / experiment:
other: 2 / 1
Parameter:
other: Cysteine peptide depletion (PPD)
Value:
33.51
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Run / experiment:
other: 3 / 1
Parameter:
other: Cysteine peptide depletion (PPD)
Value:
38.27
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Run / experiment:
mean
Parameter:
other: Cysteine peptide depletion (PPD)
Value:
34.02
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation

It should be noted that the data for both peptides were obtained in repeat experiments. The reference controls were outside of the Protocol specified range in the initial experiment, no data were obtained in the first repeat experiment due to an equipment malfunction and in the second repeat experiment, the reference controls for cysteine were outside of the Protocol specified range.

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

29.11

28.08

23.65

0.00

0.00

0.32

0.56

 

23.42

 

0.97

 

 

Positive Control

13.70

14.33

31.24

56.15

54.13

55.85

1.59

 

13.35

 

57.27

 

 

The r2 value for the standard calibration curve was 0.9999.

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

0.47                       0.48

0.48

0.48

C (Positive Control)

0.48                       0.48

0.50

0.49

C (Test Article)

0.36                       0.34

0.41

0.37

The peak area results for reference controls B and C (acetonitrile) were as follows:

Reference Control

Replicate

Peptide Peak Area

B

1

2

3

4

32.13 32.34 32.18

30.76

 

5

31.89

 

6

32.16

C (Positive Control)

1

2

30.96

30.82

 

3

31.93

Mean

 

31.69

SD

 

0.64

CV

 

2.03

The peak area results for reference controls B and C (isopropanol) were as follows:

Reference Control

Replicate

Peptide Peak Area

B

1

2

3

4

32.13 32.34 32.18

30.76

 

5

31.89

 

6

32.16

C (Test Article)

1

2

23.10

21.57

 

3

26.27

Mean

 

29.16

SD

 

4.33

CV

 

14.84

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

13.46

12.84                    19.31

30.30

33.51

34.02

4.01

 

11.92

38.27

 

 

Positive Control

2.82

2.75                       19.02

85.17

85.54

85.47

0.27

 

2.72

85.70

 

 

The r2value for the standard calibration curve was 0.99974.

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

0.49                       0.50

0.50

0.49

C (Positive Control)

0.49                       0.48

0.51

0.49

C (Test Article)

0.51                       0.49

0.50

0.50

The peak area results for reference controls B and C (acetonitrile) were as follows:

Reference Control

Replicate

Peptide Peak Area

B

1

2

3

4

19.42 19.27 19.48

18.80

 

5

19.29

 

6

19.98

C (Positive Control)

1

2

18.86

18.56

 

3

19.64

Mean

 

19.26

SD

 

0.45

CV

 

2.32

The peak area results for reference controls B and C (isopropanol) were as follows:

Reference Control

Replicate

Peptide Peak Area

B

1

2

3

4

19.42 19.27 19.48

18.80

 

5

19.29

 

6

19.98

C (Test Article)

1

2

19.78

18.89

 

3

19.27

Mean

 

19.35

SD

 

0.38

CV

 

1.95

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The test article, Citmol 320, was considered to be positive with low reactivity 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, Citmol 320, was considered to be positive with low reactivity in the Direct Peptide Reactivity Assay.

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 September 2017 - 30 September 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
February 2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
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 Citmol 320, also known as 2,3 propanetricarboxylic acid, 2-hydroxy-, 1,2,3-tris (2-octyldodecyl) ester and trioctyldodecyl citrate, was received at Covance on 27 July 2017 as follows:
CAS Number Storage Purity
126121-35-5 15 to 25°C, protected from light 99.39%

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.
Details on the 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 8 to 64 µM in Experiment 1 and at concentrations of 16 to 64 µM in Experiment 2.
The EC1.5 values for the positive control were 5.91 and 12.83 µM in Experiments 1 and 2, respectively. The average induction in the two replicates for the positive control at 64 µM was 4.8.
Run / experiment:
other: 1
Parameter:
other: maximal average fold increases (Imax)
Value:
1.67
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Run / experiment:
other: 2
Parameter:
other: maximal average fold increases (Imax)
Value:
1.38
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Run / experiment:
other: 1
Parameter:
other: EC1.5 (µM)
Value:
61.25
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Run / experiment:
other: 2
Parameter:
other: EC1.5 (µM)
Value:
2 000
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
MTT-absorbance readings are given below.
Cell viability at the EC1.5 determining concentration was 41.33% in Experiment 1. No EC1.5 determining concentration was obtained for Experiment 2.

The average coefficient of variation of the luminescence reading for the negative control (DMSO) was 10.56% and 13.99% in Experiments 1 and 2, respectively.

Table 1 Luminescence Readings for Experiment 1

Substance Concentration (µM)
0.98 1.95 3.91 7.81 15.63 31.25 62.5 125 250 500 1000 2000
Test Article Plate 1 1543168 1716888 1331594 1315789 1622793 1513157 1552492 1800022 1965316 1785354 1771369 1980176
Plate 2 1260152 1431601 1325672 1132655 1417618 1141759 1626698 1630547 1552627 1436187 1430972 1583847
Plate 3 1567568 1525120 1969936 1222825 1176571 1300467 1626504 1182489 1798114 1530318 1255817 1643321
Mean Fold Induction 1.37 1.47 1.47 1.15 1.32 1.24 1.51 1.44 1.67 1.49 1.39 1.63

Substance Individual Values
Negative Control Plate 1 996389 1165954 1325068 1200351 1007688 1028106
Plate 2 1072441 1102455 1125619 1075240 1112966 983413
Plate 3 910765 1182607 1022956 988821 996411 852643

Substance Individual Values
Untreated Control Plate 1 231109 324801 263910 277165 271943 227283
Plate 2 222678 230236 226502 277985 213420 236246
Plate 3 221073 213204 243438 234992 266381 255785
Mean Fold Induction 0.21 0.24 0.23 0.25 0.24 0.23

Substance Concentration (µM)
4 8 16 32 64
Positive Control Plate 1 1431035 1835723 2978093 3069113 5669837
Plate 2 1395302 1633747 1916742 3046923 5175213
Plate 3 1495939 1795741 2005825 2753672 5707566
Mean Fold Induction 1.36 1.65 2.15 2.78 5.2

Table 2 Luminescence Readings for Experiment 2

Substance Concentration (µM)
0.98 1.95 3.91 7.81 15.63 31.25 62.5 125 250 500 1000 2000
Test Article Plate 1 1533217 1831023 2024406 1700206 1693024 1586190 1944731 1740189 1851405 2102393 1910927 2027599
Plate 2 1700734 1837135 2002173 2083845 2535908 2237423 2009566 2132399 2615251 2544230 2297559 2012024
Plate 3 1690890 1656271 1588240 1490599 1417681 1501171 1529517 1775703 1534145 1611129 1612342 1835399
Mean Fold Induction 1.1 1.19 1.25 1.17 1.23 1.17 1.22 1.26 1.31 1.38 1.29 1.32

Substance Individual Values
Negative Control Plate 1 1343544 1402264 1471825 1433334 1397110 1513574
Plate 2 1739936 1650344 1558418 1802835 1889729 1822263
Plate 3 1071972 1425564 1417094 1402845 1246272 1463716

Substance Individual Values
Untreated Control Plate 1 236583 243472 231726 240778 243859 255048
Plate 2 340410 319892 318889 310664 341686 313092
Plate 3 272027 239692 243633 236943 267273 267926
Mean Fold Induction 0.19 0.18 0.18 0.17 0.19 0.19

Substance Concentration (µM)
4 8 16 32 64
Positive Control Plate 1 1706855 1923384 2154105 3172802 6613567
Plate 2 2430683 2669507 3235224 3850181 7882148
Plate 3 1626853 1789618 2176803 4068620 5133834
Mean Fold Induction 1.27 1.41 1.66 2.49 4.33

Table 3 MTT-Absorbance Readings

Substance Concentration (µM)
0.98 1.95 3.91 7.81 15.63 31.25 62.5 125 250 500 1000 2000
Test Article Experiment 1 0.385 0.474 0.44 0.471 0.417 0.364 0.414 0.329 0.432 0.598 0.656 0.802
Experiment 2 0.488 0.523 0.514 0.517 0.402 0.487 0.437 0.457 0.56 0.67 0.721 0.836
Mean Viability (%) 39.73 45.65 43.56 45.24 37.73 38.62 39.03 35.65 45.09 57.97 63.05 75.18

Substance Individual Values
Negative Control Experiment 1 1.005 1.035 0.987 1.022 0.966 0.991
Experiment 2 1.316 1.176 1.28 1.156 1.089 1.125

Substance Concentration (µM)
4 8 16 32 64
Positive Control Experiment 1 1.038 0.974 1.063 1.019 1.051
Experiment 2 1.147 1.124 1.135 1.219 1.089
Mean Viability (%) 100.02 95.84 100.79 102.07 98.25
Interpretation of results:
GHS criteria not met
Conclusions:
The test article did not meet all 4 conditions for a positive prediction in either repetition and was therefore considered to be negative in the ARE-Nrf2 Luciferase Test.
Executive summary:

The study was conducted to investigate the potential of Citmol 320 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 isopropanol to the final concentration (200 mM). Serial dilutions were then made using isopropanol to obtain 12 master concentrations of the test article (0.098, 0.195, 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) CO2in air, in a humidified environment for 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:

Condition

Rep 1

Rep 2

IMAX (fold increase)

1.67

1.38

Cell Viability at the EC1.5 (%)

41.33

N/A

EC1.5 (mg/mL)

61.25

>2000

Dose response for luciferase Induction

No

No

For each repetition the acceptance criteria were met, with the exception that in Repetition 1 the EC1.5 for the positive control was 5.91 and was therefore just below the range specified in the protocol.

The test article, Citmol 320, did not meet all 4 conditions for a positive prediction in either repetition and was therefore considered to be negativein the ARE-Nrf2 Luciferase Test.

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

Three in chemico / in vitro tests were conducted on the registered substance. The human cell line assay and the keratinosens study produced definitive negative results, while the direct peptide binding assay produced a low positive result for Cysteine peptide depletion only. Lyseine peptide depletion in the same test produced no reaction.

Based upon all the information available it is considered that the registered substance does not meet the criteria for classification according to the Classification, Labelling, and Packaging (CLP) regulation.