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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 24th July to 21st September, 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
Chemical structure
Reference substance name:
Tris(ethyl acetoacetato-O1',O3)aluminium
EC Number:
239-343-8
EC Name:
Tris(ethyl acetoacetato-O1',O3)aluminium
Cas Number:
15306-17-9
Molecular formula:
C18H27AlO9
IUPAC Name:
tris(ethyl acetoacetato-O1',O3)aluminium
Test material form:
solid

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 the culture base medium RPMI 1640 and in DMSO. The test item is insoluble in all solvents at room temperature. The best result was obtained in DMSO after sonication for 30 min at approximately 37 °C. However, even then the solubility seems to be a critical factor since the results regarding the cytotoxicity were not concordant.

PRELIMINARY AUTOFLUORESCENCE TEST
A possible autofluorescence of the test item was determined using a 2475 Multi-λ Fluorescence Detector and an Excitation wavelength of 480 ± 5nm. 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 in pre-warmed (37 °C) DMSO was prepared, sonicated 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 solutions 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:
The OECD 442E Assays address the Key Event on activation of dendritic cells on the Adverse Outcome pathway for Skin Sensitisation including h-CLAT based on the human monocytic leukemia cell line, THP-1. The cells were purchased by CLS (Eppelheim, Germany). 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 valid pre-test cells of passage 12 were used. For the main experiments cells of passage 18, 6 and 14 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. 50-21-5, ≥ 85 % purity, test concentration: 1000 µg/ml) were used. 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 dose range finding 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 96- and 24-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: 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, 3.9 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 transferred into sample tubes and centrifuged for 5 min. Afterwards the supernatant was removed and the pellet was suspended in 600 µl staining buffer before 200 µl respectively were transferred into one well of a 96-well plate. After that, the cells were washed three times with staining buffer. After that, the cells were resuspended in 150 µl staining buffer and 7.5 µl Propidium iodide (PI) solution (12.5 µg/ml) were 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 I and II was again 500 µg/ml. But since strong cytotoxic effects were observed in both experiments 347.2 µg/ml were used as maximum test item concentration in experiment III, IV and V. In every experiment, a 500 x stock solution in DMSO was prepared and diluted. The performance of all experiments 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. 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 bottom plate respectively. The plates were centrifuged 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 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. Except experiment I and II, 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.
Log CV75= [(75 - c)*Log(b) - (75 - a)+Log(d)] / (a - c)
where:
a= the minimum value of cell viability over 75 %
c= the maximum value of cell viability below 75 %
b and d are the concentrations showing the value of cell viability a and c respectively.
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: Experiment V
Parameter:
other: EC150 for CD86
Value:
149.73
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: Experiment V
Parameter:
other: EC200 for CD54
Value:
280.6
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
RESULTS OF MAIN STUDY:
In total three valid experiments (experiment III, IV and V) were performed. In the experiments, the highest nominal applied concentration (347.2 µg/ml) was chosen based on the solubility and toxicity of the test item. A geometric series (factor 1.2) of 7 dilutions thereof was prepared. As solvent control for the test item, pre-warmed (37 °C) 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. 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. All acceptance criteria were met and therefore, the study was considered valid. In experiment III all tested concentrations were analysable (viability > 50 %). The RFI of CD54 was not ≥ 200 in any tested concentration but the RFI of CD86 was > 150 % in two test item concentrations (116.3 µg/ml and 347.2 µg/ml). The result of the experiment is therefore “positive”. In experiment IV again all tested concentrations were analysable (viability > 50 %). The RFI of CD54 and CD86 was not ≥ 200 % or ≥ 150 %, respectively, in any tested concentration. The result of experiment IV is therefore “negative”. According to the OECD 442E a third experiment should be performed in case of equivocal result in two valid experiments. Therefore, a third repetition (experiment V) was performed. In experiment V all tested concentrations except the concentration 289.3 µg/ml were analysable (viability > 50 %). At this concentration the viability was reduced to 12 - 13 %. This effect is declared as an outlier. The RFI of CD54 was > 200 % only in the non-cytotoxic  test item concentration 347.2 µg/ml. In addition the RFI of CD86 was > 150 % in four non-cytotoxic test item concentrations (347.2 µg/ml, 241.1 µg/ml, 200.9 µg/ml, 167.4 µg/ml). The EC150 value for CD86 was calculated and is 149.73. Also the EC200 value for CD54 was calculated and is 280.60. Due to the cytotoxic effect in concentration 289.3 µg/ml, this value was calculated on the basis of 347.2 µg/ml and 241.1 µg/ml. The result of experiment V is therefore “positive”. Since the majority result of the three individual runs is positive, the end result of this h-CLAT is considered as “positive”.
 
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 total two pre-tests and five experiments (experiment I - V) with a treatment period of 24 hours were performed. In the first pre-test the stock solution became solid after sonication. The deviation was considered as critical and the pre-test was terminated and repeated heating up the stock solution till 37 °C to optimize the solubility of the test item. Experiment I and II were invalid due to an error of the definition of the stop criterion. In the second 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 TG 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 is only minimal. Therefore, the second pre-test and the results of the viability of experiment I and II is still valid but the RFI values in experiments I and II were declared as invalid. Therefore the experiments I and II were repeated. The results of the cytotoxicity measurement were used anyway. Since strong cytotoxic effects were detected, the concentrations for experiment III - V were adjusted. However, due to the very low solubility of the test item, the cytotoxicity values fluctuate between the experiments.
 
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= 89, 100 and RFI of CD54= 86, 93;exp IV:DMSO: RFI of CD86= 98, 99 and RFI of CD54= 80, 86; exp V: DMSO: RFI of CD86= 73,79 and RFI of CD54= 104, 98.
- 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= 174 and for CD54= 137;DMSO: MFI RATIO to isotype for CD86= 176, 181 and for CD54= 137, 137;
exp IV:Medium:MFI RATIO to isotype for CD86= 188 and for CD54= 148;DMSO: MFI RATIO to isotype for CD86= 185, 185 and for CD54= 138, 140;
exp V:Medium:MFI RATIO to isotype for CD86= 196 and for CD54= 136;DMSO: MFI RATIO to isotype for CD86= 168, 172 and for CD54= 137, 134.
- 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 > 85.96 %: RFI of CD86= 919 and RFI of CD54= 406; exp IV:DNCB viability > 85.51 %: RFI of CD86= 890 and RFI of CD54= 372; exp. V:DNCB viability > 73.64 %: RFI of CD86= 809 and RFI of CD54= 356.
- 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.
 
EVALUATION OF THE RESULTS
Since the majority result of the three individual runs is positive, the end result of this h-CLAT is considered as “positive”.

Any other information on results incl. tables

Results of experiment III

  Concentration [µg/ml] Viability Antibodies MFI 

MFI

ratio

to Isotype %

RFI value
Medium  - 93.86% CD86 1406 174  
94.69% CD54 1105 137  
95.15% ISO 809    
DMSO - 97.77% CD86 1231 176 89
97.58% CD54 956 137 86
97.25% ISO 700    
DMSO - 97.04% CD86 1332 181 100
97.13% CD54 1010 137 93
97.24% ISO 736    
DNCB 4 85.96% CD86 6299   919
86.03% CD54 1936   406
86.15% ISO 823    

Test

Item

96.9 96.69% CD86 1328   105
96.94% CD54 1024   99
96.08% ISO 771    

Test

Item 

116.3 87.54% CD86 1946   208
90.17% CD54 1127   113
90.03% ISO 839    

Test

Item

139.5 96.55% CD86 1333   107
96.81% CD54 1029   103
96.65% ISO 766    

Test

Item 

167.4 95.58% CD86 1463   135
95.42% CD54 1010   102
95.21% ISO 748    

Test

Item

200.9 94.78% CD86 1240   92
95.51% CD54 1038   112
96.05% ISO 751    

Test

Item 

241.1 96.26% CD86 1282   94
96.06% CD54 1089   119
96.19% ISO 785    

Test

Item

289.3 94.61% CD86 1347   106
94.38% CD54 1103   125
94.92% ISO 783    

Test

Item 

347.2 79.73% CD86 2141   249
80.90% CD54 1256   171
82.63% ISO 817    

Results of experiment IV

  Concentration [µg/ml] Viability Antibodies MFI  MFI ratioto Isotype % RFI value
Medium  - 96.46% CD86 1372 188  
97.12% CD54 1081 148  
97.35% ISO 729    
DMSO - 97.54% CD86 1370 185 98
97.66% CD54 1020 138 80
97.68% ISO 739    
DMSO - 97.07% CD86 1384 185 99
97.83% CD54 1049 140 86
97.20% ISO 747    
DNCB 4 85.51% CD86 6522   890
85.97% CD54 1976   372
85.90% ISO 853    

Test

Item

96.9 97.26% CD86 1313   89
97.08% CD54 1046   106
96.57% ISO 749    

Test

Item 

116.3 95.76% CD86 1392   104
96.37% CD54 1038   108
95.51% ISO 735    

Test

Item

139.5 95.96% CD86 1284   90
96.15% CD54 1010   104
96.61% ISO 719    

Test

Item 

167.4 96.57% CD86 1358   98
96.23% CD54 1030   104
96.08% ISO 738    

Test

Item

200.9 95.65% CD86 1326   89
95.86% CD54 1088   115
96.08% ISO 766    

Test

Item 

241.1 95.69% CD86 1244   77
96.15% CD54 1062   109
95.74% ISO 756    
Test Item 289.3 94.85% CD86 1346   93
94.99% CD54 1068   109
94.68% ISO 761    
Test Item  347.2 90.73% CD86 1397   96
90.44% CD54 1152   128
90.74% ISO 791    

Results of experiment V

  Concentration [µg/ml] Viability Antibodies MFI  MFI ratioto Isotype % RFI value
Medium  - 97.23% CD86 1371 196  
97.66% CD54 954 136  
97.60% ISO 701    
DMSO - 97.96% CD86 1203 168 73
97.50% CD54 978 137 104
97.58% ISO 714    
DMSO - 98.10% CD86 1260 172 79
98.03% CD54 980 134 98
97.76% ISO 731    
DNCB 4 78.57% CD86 5159   809
77.46% CD54 1768   356
73.64% ISO 881    
Test Item 96.9 97.07% CD86 1350   123
97.15% CD54 1029   107
96.77% ISO 747    
Test Item  116.3 96.70% CD86 1305   116
96.40% CD54 1015   105
95.62% ISO 737    
Test Item 139.5 95.84% CD86 1426   128
95.80% CD54 1096   112
95.65% ISO 801    
Test Item  167.4 95.69% CD86 1663   188
94.73% CD54 1065   121
95.80% ISO 746    
Test Item 200.9 92.81% CD86 1716   191
91.88% CD54 1132   132
92.12% ISO 783    
Test Item  241.1 89.44% CD86 1930   240
88.61% CD54 1148   149
87.86% ISO 754    
Test Item 289.3 13.25% CD86 1948   192
13.25% CD54 1591   220
12.39% ISO 1009    
Test Item  347 .2 77.83% CD86 3617   455
78.77% CD54 2148   286
84.54% ISO 1392    

Preliminary test results

Substance Concentration Viability
Medium  - 97.7
Solvent control test item (DMSO) - 98.5

Solvent control positive control

(DMSO)

- 98.33
Positive control DNCB 4 88.34
Test item 3.9 98.49
Test item  7.8 97.75
Test item 15.6 98.22
Test item  31.3 98.22
Test item 62.5 97.98
Test item  125 98.09
Test item 250 97.81
Test item  500 92.54

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:

Tris(ethyl acetoacetato-O1',O3)aluminium 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. This method is associated with the process of activation of monocytes and dendritic cells representing the KE3 in the AOP to produce a skin sensitiser effect.

Two pre-tests and five experiments (experiment I - V) with a treatment period of 24 hours were performed. The first pre-test was terminated due to the low solubility of the test item and repeated by heating up the stock solution till 37 °C to optimize the solubility of the test item. Experiment I and II were invalid due to an error of the definition of the stop criterion (the stop criterion during measurement was 10,000 events instead of 10,000 viable cells).

In total three valid experiments (experiment III, IV and V) were performed. In the experiments, the highest nominal applied concentration (347.2 µg/ml) was chosen based on the solubility of the test item. A geometric series (factor 1.2) of 7 dilutions thereof was prepared. As solvent control for the test item, pre-warmed (37 °C) 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 the RFI of CD54 was not ≥ 200 % in any tested concentration but the RFI of CD86 was > 150 % in two test item concentrations (116.3 µg/ml and 347.2 µg/ml). The result of the experiment is “positive”. In experiment IV the RFI of CD54 and CD86 was not ≥ 200 % or ≥ 150 % in any tested concentration. The result of experiment IV is “negative”. Since the results of experiment III and IV were equivocal a third repetition (experiment V) was performed. In experiment V the RFI of CD54 was > 200 % only in the non-cytotoxic test item concentration 347.2 µg/ml. In addition the RFI of CD86 was > 150 % in four non-cytotoxic test item concentrations (347.2 µg/ml, 241.1 µg/ml, 200.9 µg/ml, 167.4 µg/ml). The result of experiment V is “positive”.

Since the majority result of the three individual runs is positive, the end result of this h-CLAT is considered as “positive”.