Tris(ethyl acetoacetato-O1',O3)aluminium

Skin sensitisation

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

Test material information

Constituent 1

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

Results

Results 1

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

Results 2

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”.