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Registration Dossier
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EC number: 224-931-9 | CAS number: 4560-68-3
Skin sensitisation
Administrative data
- 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
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 017
- Report date:
- 2017
Materials and methods
Test guideline
- 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
Test material
- Reference substance name:
- tris(2-octyldodecyl) citrate
- EC Number:
- 603-121-2
- Cas Number:
- 126121-35-5
- Molecular formula:
- C66H128O7
- IUPAC Name:
- tris(2-octyldodecyl) citrate
- Test material form:
- liquid
- Details on test material:
- Tris(octyldodecyl) citrate 99.87 %
Bis(octyldodecyl) citrate 0.13 %
Constituent 1
- 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.
In vitro test system
- 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%.
Results and discussion
- 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.
In vitro / in chemico
Resultsopen allclose all
- 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.
Any other information on results incl. tables
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.
Applicant's summary and conclusion
- 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.
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