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Diss Factsheets

Administrative data

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From July 24th to September 12th, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD Guideline for the Testing of Chemicals, Part 442E: In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT).
Version / remarks:
July 29th, 2016
Deviations:
yes
Remarks:
In the pre-test and experiment I and II the stop criterion during measurement was 10,000 events instead of 10,000 viable cells. For this reason experiment I and II were declared invalid.
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of dendritic cells

Test material

Constituent 1
Reference substance name:
Reaction mass of dodecane-1-thiol and tridodecyl trithiophosphite
Molecular formula:
Not applicable - Multiconstituent substance
IUPAC Name:
Reaction mass of dodecane-1-thiol and tridodecyl trithiophosphite
Test material form:
liquid

In vitro test system

Details on the study design:
PRELIMINARY SOLUBILITY TEST
The solubility of the test item was determined in a non-GLP pre-test in culture base medium RPMI 1640 and in DMSO. The best result was obtained in DMSO. Therefore, DMSO was used as solvent of the test item.

PRELIMINARY AUTOFLUORESCENCE TEST
A possible autofluorescence of the test item was determined using a 2475 Multi-λ Fluorescence Detector and an Excitation wavelength of 488 ± 2 nm. The autofluorescence of the test item has no influence on the result of the assay.

PREPARATION OF STOCK SOLUTION FOR PRE-TEST DOSE RANGE FINDING TEST AND FOR MAIN TESTS
First, a stock solution containing 500 mg/ml was prepared and used to prepare a geometric series of solutions (factor 2 for pre-test; factor 1.2 for experiments). Afterwards all concentrations were further diluted (1:250 fold) in complete culture medium (working solutions). Another 1:2 dilution was achieved by adding 1 part of each test item concentration to 1 part of the cell suspension in the treatment plate. In the end, the total dilution factor was 1:500. The stock solution as well as the dilutions were freshly prepared on the day of treatment.

CONTROLS
The controls were tested at only one concentration.
- Solvent control: Dimethyl sulfoxide (DMSO). Final concentration: 0.2 %
- Positive control: 2,4-dinitrochlorobenzene (DNCB). Final concentration: 4 µg/ml.

TEST SYSTEM
- Cell cultures:
THP-1 cells are stored in liquid nitrogen in the cell bank of LAUS GmbH to allow a continuous stock of cells, which guarantees similar parameters of the experiment and reproducible characteristics of the cells. For the pre-test cells of passage 11 were used. For the main experiments cells of passage 18, 6, 15 and 17 were used. After thawing the cells were cultivated in RPMI 1640 complete culture medium in cell culture flasks at 37 ± 1 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2.

- Reactivity Check:
Two weeks after thawing, a reactivity check of the cells was performed. For that, the two positive controls 2,4-dinitrochlorobenzene (DNCB) (CAS n. 97-00-7, ≥ 99 % purity, test concentration: 4 µg/ml) and nickel sulfate (NiSO4) (CAS n. 10101-97-0, ≥ 99% purity, test concentration: 200 µg/ml) as well as the negative control, lactic acid (LA) (CAS n. 50-215, ≥ 85 % purity, test concentration: 1000 µg/ml) were used. These substances as well as all additional information are given by the OECD 442E. The experimental procedure was identical to those of the main experiments.
The two positive controls produced a clearly positive result of the two surface markers CD86 and CD54. The negative control produced a clearly negative result of both surface markers. Therefore the cells were found to be suitable for the experiments. For the pre-test as well as the experiments only cells which have successfully passed the reactivity check were used.

MEDIUM: culture base medium RPMI 1640
TEST VESSEL: the test was performed on 24-well plate or on 96-well plates.

PRE-TEST (DOSE RANGE FINDING)
A preliminary cytotoxicity test was performed to determine the concentrations to be used for the CD86/CD54 expression measurement in the main experiments. The following 8 concentrations of the test item were tested with an incubation time of 24 h in the pre-test: 1000 µg/ml, 500 µg/ml, 250 µg/ml, 125 µg/ml, 62.5 µg/ml, 31.3 µg/ml, 15.6 µg/ml and 7.8 µg/ml. For that 1.6 * 10^5 cells / well of a 96-well plate were exposed to each concentration of the test item for 24 h at 37.0 ± 1.0 °C and 5.0 ± 0.5 % CO2. Following treatment, the cells were washed three times with staining buffer (centrifugation: 250 * g for 5 min). After that, the cells were resuspended in staining buffer and PI solution was added to achieve a final concentration of 0.595 µg/ml. The plate was incubated for 15 min on ice and 5 minutes at room temperature in the dark. Afterwards, the PI uptake was analysed by flow cytometry with the acquisition channel PerCP (corresponds to the FL-3 channel of the predecessor model). The cell viability was automatically calculated by the flow cytometer.

MAIN TESTS:
Since no CV75 could be determined (no cytotoxic effect) in the pre-test, and the solvent of the test item is DMSO, the maximal test item concentration in the experiments was again 1000 µg/ml. To achieve the highest test item concentration, a 500 x stock solution in DMSO was prepared and diluted. The performance of experiment III and IV was identical. 8 test item concentrations were tested in each experiment. 1 * 10^6 cells / well of a 24-well plate were exposed to each concentration of the test item for 24 h at 37.0 ± 1.0 °C and 5.0 ± 0.5 % CO2. During treatment the plates were closed with a sealing tape to avoid cross-contamination. Following treatment, the solutions were transferred into sample tubes and washed twice with 1 ml staining buffer (centrifugation: 250 * g for 5 min). After that, the cells were resuspended in 600 µl blocking solution and incubated on ice for 15 min. Then, 180 µl of the cell suspension were added in three wells of a 96-well round button plate respectively. The plates were centrifuged at 250 * g for 5 minutes and the supernatant was discarded. After that, 50 µl of the three FITC-labelled antibody solutions were added in one of the three wells respectively and the plate was incubated on ice for 30 min in the dark. Before use, the antibodies were diluted 3:25 v/v for CD86, 3:50 v/v, for CD54 and IgG1 with staining buffer. After the incubation on ice the cells were washed 3 times with 200 µl staining buffer (centrifugation: 250 * g for 5 min) before 150 µl staining buffer as well as 7.5 µL PI working solution were added to each well. Afterwards the plate was incubated for 15 min on ice and 5 min at room temperature in the dark before measurement at the flow cytometer. In total 10,000 viable cells (PI negative) were acquired and analysed.

DATA ANALYSIS
The cytotoxicity/viability was directly measured and calculated by the flow cytometer and is given as % values. For the evaluation of the CD54/CD86 expression, the “mean fluorescence intensity (MFI)” was also analysed by the software of the flow cytometer. The EC150 for CD86 as well as the EC200 for CD54 could not be calculated. For calculation of the end results, a validated Microsoft Excel® spreadsheet file was used
RFI= (MFI chemical-treated cell - MFI chemical-treated isotype control cell) * 100/ (MFI solvent-treated control cells- MFI solvent-treated isotype control cells).

EVALUATION CRITERIA
For CD86/CD54 expression measurement, each test item is tested in at least two independent runs to derive a single prediction. 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 ≥ than 150 % at any tested concentration with cell viability ≥ 50 %
− The RFI of CD54 is ≥ than 200 % at any tested concentration with cell viability ≥ 50 %
If the first two runs are concordant for both markers, a third experiment is not necessary. Then, the study is completed. If the first two runs are not concordant for at least one of the markers (CD54 or CD86), a third run is needed and the final prediction will be based on the majority result of the three individual runs (i.e. 2 out of 3). In this respect, it should be noted that if two independent runs are conducted and one is only positive for CD86 and the other is only positive for CD54, a third run is required. If this third run is negative for both markers, the h-CLAT prediction is considered negative. On the other hand, if the third run is positive for either marker or for both markers, the h-CLAT prediction is considered positive.
In case of a negative result, special care should be taken if the test item
- has a Log Kow > 3.5. Those results should not be considered. However, positive results obtained with those test chemicals can be used for analysis.
- is a pro-hapten or a pre-hapten
- has an autofluorescence and is emitting at the same wavelength as FITC or as PI.

ACCEPTABILITY
The assay is considered acceptable if it meets the following criteria:
- The cell viabilities of medium and solvent/vehicle controls are higher than 90 %.
- In the solvent/vehicle control, RFI values of both CD86 and CD54 do 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 iso-type control should be > 105 %.
- In the positive control, 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 item, the cell viability should be more than 50% in at least four tested concentrations in each run.
If any of these criteria is not met, the experiment is considered not valid and needs to be repeated.

Results and discussion

In vitro / in chemico

Resultsopen allclose all
Key result
Run / experiment:
other: CD86 ≥ 150 for all test item non cytotoxic concentrations in experiment III and in experiment IV.A calculation of the EC150 and EC200 was not possible since all RFI values for CD86 were above 150 and none of the RFI values for CD54 were above 200.
Parameter:
other: CD86
Value:
150 %
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Run / experiment:
other: for 2 test item non cytotoxic concentrations in experiment IV (482.3 μg/ml and 833.3 μg/ml ). These concentrations were not consecutive and no calculation of EC200 was possible.
Parameter:
other: CD54
Value:
200 %
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
RESULTS OF MAIN STUDY:
Two valid experiments with a treatment period of 24 hours were performed. In the experiments, the highest nominal applied concentration (1000 µg/ml) was chosen based on the results obtained in the pre-test. A geometric series (factor 1.2) of 7 dilutions thereof was prepared. As solvent control for the test item, DMSO was used in a final concentration of 0.2 % in culture medium. As positive control, 2,4-dinitrochlorobenzene (DNCB, CAS n. 97-00-7, ≥ 99 % purity) was used.
In experiment III all of the tested concentrations showed viability values above 50 %. In experiment IV the highest test item concentration induced a cytotoxic effect (viability below 50 %) and the results could therefore not be used for the final evaluation of the CD86 und CD54 expression.
In both experiments (experiment III and IV) the RFI values of CD86 were > 150 % in all tested concentrations. Concerning the CD54 expression, none of the values exceeded the threshold in experiment III. However in experiment IV the RFI values of CD54 exceeded the threshold of 200 % in two test item concentrations (833.3 µg/ml and 482.3 µg/ml) with cell viability ≥ 50 %. Therefore, in accordance to the classification criteria the result of this study is positive. In conclusion, it can be stated that under the experimental conditions of this study, the test item was positive in the h-CLAT and is therefore considered to have the potential to activate dendritic cells and to be a potential skin sensitiser.

REACTIVITY CHECK:
Prior to the study, the cells, that were used in the experiments were checked in a reactivity check and were found to be suitable for the experiments.

PRELIMINARY TESTS AND DOSE RANGE FINDING
In the pre-test and experiment I and II the stop criterion during measurement was 10,000 events instead of 10,000 viable cells. Therefore, the size of the population that was used for the evaluation of the results was smaller than indicated by the OECD 442E. An independent analysis with a proficiency chemical (2,4-dinitrochlorobenzene (DNCB, CAS n. 97-00-7, ≥ 99 % purity)) revealed that the difference in viability due to this deviation is only minimal. Therefore, the pre-test is still valid but the main experiments were declared as invalid and were repeated.
In dose-range finding test no considerable reduction of the viability was detected till concentration of 1000 μg/ml. Therefore a CV75 value was not determined and the highest tested concentration chosen for the main test was 1000 μg/ml.


DEMONSTRATION OF PROFICIENCY
Prior to routine use, the validity of the h-CLAT test at laboratory was demonstrated in a proficiency study. In this study, 10 proficiency chemicals (indicated by the OECD 442E guideline) were tested. As prescribed by the guideline, more than 7 of the results were correctly categorized. Therefore, the proficiency of the test system was demonstrated.

ACCEPTANCE CRITERIA
All validity criteria were fulfilled:
- The cell viabilities of medium and solvent/vehicle controls were higher than 90%.
- In the solvent/vehicle control, RFI values of both CD86 and CD54 do not exceed the positive criteria (CD86 RFI ≥ 150 % and CD54 RFI ≥ 200 %)
exp III:DMSO: RFI of CD86= 96, 90 and RFI of CD54= 106, 107; exp IV:DMSO: RFI of CD86= 92, 93 and RFI of CD54= 102, 96.
- For both medium and solvent controls, the MFI ratio of both CD86 and CD54 to isotype control should be > 105%.
exp III:Medium: MFI RATIO to isotype for CD86= 176 and for CD54= 130;DMSO: MFI RATIO to isotype for CD86= 171, 168 and for CD54= 131, 131;
exp IV: Medium: MFI RATIO to isotype for CD86= 187 and for CD54= 135; MFI RATIO to isotype for CD86= 177, 174 and for CD54= 135, 131.
- In the positive control, 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 %.
exp III:DNCB viability > 87.24 %: RFI of CD86= 753 and RFI of CD54= 370; exp IV:DNCB viability > 80.36 %: RFI of CD86= 935 and RFI of CD54= 431.
- For the test item, the cell viability should be more than 50% in at least four tested concentrations in each run.
If any of these criteria is not met, the experiment is considered not valid and needs to be repeated.
All validity criteria were met. Therefore, the study was considered as valid.

Any other information on results incl. tables

Results of experiment III

  Concentration [µg/ml] Viability Antibodies MFI  MFI ratioto Isotype % RFI value
Medium  - 97.06% CD86 1379 176  
97.48% CD54 1013 130  
97.13% ISO 782    
DMSO - 97.30% CD86 1373 171 96
97.80% CD54 1047 131 106
97.61% ISO 801    
DMSO - 97.60% CD86 1326 168 90
97.45% CD54 1038 131 107
97.27% ISO 790    
DNCB 4 87.24% CD86 4973   753
87.50% CD54 1857   370
87.86% ISO 939    
Test Item 279.1 85.08% CD86 2452   153
85.38% CD54 1877   123
85.96% ISO 1575    
Test Item 334.9 86.10% CD86 2367   161
86.15% CD54 1804   145
85.19% ISO 1447    
Test Item 401.9 81.71% CD86 2616   190
82.92% CD54 1902   150
82.24% ISO 1532    
Test Item 482.3 82.84% CD86 2664   195
82.64% CD54 1922   152
82.53% ISO 1548    
Test Item 578.7 82.01% CD86 2538   166
82.54% CD54 1946   146
82.02% ISO 1586    
Test Item 694.4 70.48% CD86 2826   177
71.01% CD54 2242   175
70.10% ISO 1812    
Test Item 833.3 69.79% CD86 2831   186
69.59% CD54 2200   175
71.54% ISO 1769    
Test Item 1000 72.46% CD86 2780   186
75.00% CD54 2123   165
74.84% ISO 1718    

Results of experiment IV

  Concentration [µg/ml] Viability Antibodies MFI  MFI ratioto Isotype % RFI value
Medium  - 97.30% CD86 1318 187  
97.56% CD54 953 135  
97.67% ISO 704    
DMSO - 97.67% CD86 1297 177 92
97.21% CD54 985 135 102
97.19% ISO 731    
DMSO - 97.85% CD86 1340 174 93
97.89% CD54 1009 131 96
97.55% ISO 771    
DNCB 4 82.26% CD86 6244   935
83.28% CD54 1951   431
80.36% ISO 925    
Test Item 279.1 82.44% CD86 3015   245
81.74% CD54 2120   194
82.66% ISO 1628    
Test Item 334.9 81.78% CD86 3008   240
81.35% CD54 2083   170
79.77% ISO 1651    
Test Item 401.9 73.41% CD86 3154   237
72.83% CD54 2269   180
72.39% ISO 1812    
Test Item 482.3 72.09% CD86 3183   237
73.80% CD54 2359   204
70.40% ISO 1842    
Test Item 578.7 73.28% CD86 3148   223
73.55% CD54 2379   193
73.77% ISO 1888    
Test Item 694.4 78.45% CD86 3099   210
78.62% CD54 2313   157
77.88% ISO 1913    
Test Item 833.3 54.24% CD86 3660   238
55.21% CD54 2859   215
55.32% ISO 2312    
Test Item 1000 43.10% CD86 3254   233
42.62% CD54 2652   283
44.55% ISO 1934    

Preliminary test results

Substance Concentration Viability %
Medium  - 98.09
Solvent control test item (DMSO) - 97.78
Solvent control positive control (DMSO) - 97.65
Positive control DNCB 4.0 84.34
Test item 7.8 96.92
Test item  15.6 97.38
Test item 31.3 97.2
Test item  62.5 96.08
Test item 125 94.93
Test item  250 95.14
Test item 500 93.68
Test item  1000 91.83

Applicant's summary and conclusion

Interpretation of results:
other: classified as skin sensitiser according to the CLP Regulation (EC n.1272/2008).
Conclusions:
The test item resulted to be positive (sensitiser) in h-CLAT test.
Executive summary:

The substance was tested to assess its sensitising potential by quantifying changes in the expression level of the two cell surface markers CD86 and CD54 in the THP-1 cells, according to the h-CLAT test protocol, OECD TG 442E.

In total one pre-test and four experiments (experiment I - IV) with a treatment period of 24 hours were performed whereby experiment I and II were invalid and had to be repeated. Therefore, in total two valid experiments (experiment III and IV) were performed. The results and data of the invalid experiments are not reported here.

In the experiments, the highest nominal applied concentration (1000 µg/ml) was chosen based on the results obtained in the pre-test. A geometric series (factor 1.2) of 7 dilutions thereof was prepared.

As solvent control for the test item, DMSO was used in a final concentration of 0.2 % in culture medium.

As positive control 2,4-dinitrochlorobenzene (DNCB, CAS n. 97-00-7, ~ 99% purity) was used.

In both experiments the RFI of CD86 was > 150 % in all tested non-cytotoxic (viability ~ 50 %) test item concentrations. In experiment IV also the RFI of CD54 was > 200 % at two non-cytotoxic test item concentrations.

Conclusion:

Under the experimental conditions of this study, the test item was positive in the h-CLAT and is therefore considered to have the potential to activate dendritic cells and therefore to be a potential skin sensitiser.