Zinc bis(benzenesulphinate)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From April 6 to May 4, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD guideline 442E

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Test material

Test material information

Constituent 1

Reference substance name:

Zinc bis(benzenesulphinate)

EC Number:

246-148-1

EC Name:

Zinc bis(benzenesulphinate)

Cas Number:

24308-84-7

Molecular formula:

C12H10O4S2Zn

IUPAC Name:

zinc bis(benzenesulphinate)

In vitro test system

Details on the study design:

TEST SYSTEM
Cells
The THP-1 is an immortalized human monocytic leukemia cell line derived from an acute monocytic leukemia patient.The THP-1 cells are stored in a cryoprotective medium in a liquid nitrogen container. Cells were grown using general culture procedures. They were cultured in cRPMI medium and maintained in a humidified incubator set at 37 °C, 5 % CO2 and were not allowed to exceed a cell density of 1.0E+06 cells/ml or more than 30 passages.
The culture medium (cRPMI) was composed of RPMI 1640 with 10 % FBS, 0.05 mM 2-mercaptoethanol and with penicillin and streptomycin.
During cell culturing, cell viability was checked using trypan blue.

Cell culture for testing
For testing, THP-1 cells were seeded at a density between 0.1E+06 cells/ml and 0.2E+06 cells/ml, and pre-cultured in culture flasks for 48 hours to 72 hours, respectively. Cells did not exceed density of 1.0E+06 cells/ml. On the day of testing, cells harvested from culture flasks were resuspended with fresh culture medium at 2.0E+06 cells/ml. Then, 500 μl of cells suspension were distributed into a 24-well flat-bottom plate (i.e. 1.0E+06 cells/well).

Reactivity check
2 weeks after thawing, a reactivity check was performed to qualify the cells of each working cell bank before testing. A reactivity check assay was performed by testing cell response after contact with lactic acid, DNCB and NiSO4.

STUDY DESIGN
Dose-Range Finding assay (DRF)
DRF was performed to assess test item toxicity and, determine the CV75 i.e. the test item concentration that result in 75 % cell viability compared to the vehicle control. Secondly, based on cytotoxicity data obtained from the DRF, a concentration series was tested in successive runs in the main tests.
The DRF consisted in two separated assays.
Treatments of DRF assays were performed at the following concentrations: 1.95, 3.91, 7.81, 15.63, 31.25, 62.50, 125 and 250 μg/ml.
Test item stock solutions were prepared at 8 different concentrations by 2-fold dilutions using the selected vehicle. These stock formulations were then diluted 250-fold (as DMSO is the selected vehicle) into cRPMI to obtain working solutions.
The working solutions were finally used for exposure by adding 500 μl of working solutions to the volume of THP-1 cell suspension in the plate (500 μl) to achieve a further 2-fold dilution. In order to avoid evaporation of volatile chemicals and cross-contamination between wells, a sealer was placed on each 24-well plate just after treatment, before putting the plastic lids back on each plate.
The treated plates were then incubated for 24 hours ± 30 minutes in a humidified incubator set at 37 °C and 5 % CO2.
At the end of the treatment phase, cells were transferred into sample tubes and collected by centrifugation. The supernatants were discarded and the remaining cells were re-suspended with 600 μl of FACS buffer. Finally, cells were resuspended in 200 μl FACS buffer and the plate was positioned into the plate-reader of the flow cytometer. A volume of 10 μl of Propidium Iodide (PI) solution at 12.5 μg/ml was added automatically by the flow cytometer before acquisition of a sample to obtain a final PI concentration of 0.625 μg/ml per well.

Main test
The main test consisted in two separated runs. Test item stock solutions were prepared at 8 different concentrations by 1.2-fold dilutions using the selected vehicle. The highest concentration corresponded to 1.2-fold the mean CV75. The maximum concentration in the plates was 250 μg/ml. All stock formulations were then 250-fold diluted into cRPMI to obtain working solutions.
In parallel, the working solutions of positive controls DNCB and NiSO4 and vehicle control were prepared.
All working solutions were finally used for exposure by adding 500 μl of working solutions to the volume of THP-1 cell suspension in the plate (500 μl) to achieve a further 2-fold dilution. In order to avoid evaporation of volatile chemicals and cross-contamination between wells, a sealer was placed on each 24-well plate just after treatment, before putting the plastic lids back on each plate.
The treated plates were then incubated for 24 hours ± 30 minutes in a humidified incubator set at 37 °C and 5 % CO2.
During the main test, treatments were performed at the following final concentrations: 69.77, 83.72, 100.47, 120.56, 144.68, 173.61, 208.33 and 250 μg/ml.
At the end of the treatment phase, cells were transferred into sample tubes and collected by centrifugation, washed twice with 1 ml FACS buffer and blocked with 600 μl of blocking solution and incubated at 4 °C for 15 minutes (± 1 minute). After blocking, cells were split in three aliquots of 180 μl into a 96-well round bottom plate and centrifuged before staining with antibodies. A volume of 50 μl of FITC-labeled anti-CD86, anti-CD54 or mouse IgG1 (isotype) antibodies prepared in FACS buffer was added to each aliquot before incubation for 30 minutes (± 2 minutes) at 4 °C.
Finally, cells were washed with 150 μl FACS buffer 2 times and re-suspended in 200 μl FACS buffer. The plate was then positioned into the plate-reader of the flow cytometer. A volume of 10 μl of PI solution at 12.5 μg/ml was added automatically by the flow cytometer before acquisition of a sample to obtain a final PI concentration of 0.625 μg/ml per well.

FLOW CYTOMETRY ANALYSIS
DRF assays
The PI uptake is analyzed using flow cytometry with the acquisition channel B3. A total of 10000 living cells (PI negative) are acquired. In case of low viability which does not allow obtaining 10000 living cells, a total of 30000 events is acquired. Alternatively, cells were acquired for a maximum of 1 minute after the initiation of the acquisition.

Main test
The non-specific binding of IgG1 and the expression CD86 and CD54 was analyzed by flow cytometry with the acquisition channel B1 in order to obtain the Mean Fluorescence Intensity (MFI); whereas the viability (PI uptake) was analyzed with the acquisition channel B3. A total of 10000 living cells (PI negative) were acquired. When the viability was low and did not allow obtaining 10000 living cells, a total of 30000 events was acquired. Alternatively, cells were acquired for a maximum of 1 minute after the initiation of the acquisition.
In case cell viability is less than 50 %, no MFI is presented in the study report and the corresponding test item concentration are considered too high for interpretation because of the diffuse labeling cytoplasmic structures that are generated following cell membrane destruction.

CALCULATIONS
Estimation of the CV75 value
The percentage of living cells (PI negative cells) is used as the value for cell viability.
The CV75 value is derived from the dose-response curve (75 % of cell viability, lying between a and c, at concentrations of b and d). CV75 is defined as the estimated concentration that is required to elicit 75 % cell viability. The CV75 value is calculated by log-linear interpolation according to:
log CV75 = [(75 - c) × log b - (75 - a) × log d] / (a - c)

Main test
Based on the Mean Fluorescence Intensity (MFI), the Relative Fluorescence Intensity (RFI) of CD86 and CD54 were calculated according to:
RFI = 100 × ( MFI of test item treated (CD86 or CD54) - MFI of test item treated IgG1 ) / ( MFI of control-treated (CD86 or CD54) - MFI of control-treated IgG1 )
RFI = Relative Fluorescence Intensity
MFI = Mean Fluorescence Intensity

Acceptance criteria
DRF
- viability of control cells treated with cRPMI should be ≥ 90 %,
- viability of control cells treated with 0.2 % DMSO should be ≥ 90 %.

Main test
The following applies for each run.
Controls acceptance criteria
- viability of cells treated with cRPMI and DMSO controls should be ≥ 90 %,
- in cRPMI and DMSO control wells, MFI ratio of both CD86 and CD54 to isotype control should be > 105 %,
- in the DMSO control, RFI values of both CD86 and CD54 should not exceed the positive criteria (CD86 RFI > 150 % and CD54 RFI ≥ 200 %),
- in the positive controls (DNCB and NiSO4), RFI values of both CD86 and CD54 should meet positive criteria (CD86 RFI ≥ 150 and CD54 RFI ≥ 200) and cell viability should be more than 50 %.

Test item acceptance criteria
- for a test item noted as cytotoxic in the DRF phase, and resulting in a negative outcome in the main test, cell viability at 1.2 × CV75 should be < 90 % in each run,
- cell viability of at least four out of eight concentrations should be > 50 %.

Individual run interpretation
A run conclusion is positive if at least one of the conditions below is met:
- RFI of CD86 is ≥ 150 at any concentration leading to ≥ 50 % viability,
- RFI of CD54 is ≥ 200 at any concentration leading to ≥ 50 % viability.
In other circumstances, the run is considered as negative.

Prediction model
Based on the individual run conclusions, a final prediction is made as follows:
- if the first two runs are both positive for CD86 and/or are both positive for CD54, the h-CLAT prediction is considered POSITIVE and a third run does not need to be conducted,
- if the first two runs are negative for both markers, the h-CLAT prediction is considered NEGATIVE (with due consideration of the highest-tested dose conditions) without the need for a third run,
- if however, 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. two out of three). In this respect, it should be noted that if two independent runs are conducted and one is only positive for CD86 (hereinafter referred to as P1) and the other is only positive for CD54 (hereinafter referred to as P2), a third run is required. If this third run is negative for both markers (hereinafter referred to as N), the h-CLAT prediction is considered NEGATIVE. On the other hand, if the third run is positive for either marker (P1 or P2) or for both markers (hereinafter referred to as P12), the h-CLAT prediction is considered POSITIVE.

Results and discussion

Positive control results:

Run A - RFI
DNCB: 370 (CD86), 1065 (CD54)
NiSO4: 355 (CD86), 6507 (CD54)

Run B
DNCB: 213 (CD86), 549 (CD54)
NiSO4: 255 (CD86), 1989 (CD54)

In vitro / in chemico

Results

Results 1

Run / experiment:

other: exp. A

Parameter:

other: % RFI for CD86

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Results 2

Run / experiment:

other: exp. B

Parameter:

other: % RFI for CD86

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 3

Run / experiment:

other: exp. A and B

Parameter:

other: % RFI for CD54

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

Results obtained from the solubility assay are summarized in the table below:
vehicle concentration (mg/ml) aspect retained vehicle and maximum stock concentration?
0.9% NaCl 100 heterogenous white suspension no
DMSO 500 heterogenous white suspension no
DMSO 250 heterogenous white suspension no
DMSO 125 white heterogenous suspension yes
with particles, becoming a white
homogeneous suspension after
5-minute sonication

Therefore, DMSO was selected as vehicle, and the following test item concentrations were tested in the DRF phase: 1.95, 3.91, 7.81, 15.63, 31.25, 62.50, 125 and 250 μg/ml.

DRF RESULTS
During both DRF phases, the stock formulations were white homogeneous suspensions. The following results were obtained in the DRF 1:
- at post-treatment observation, no abnormalities such as precipitate/emulsion at any tested concentrations,
- flow cytometry measurement after Propidium Iodide staining revealed a cell viability decrease below 75 % at the concentration of 250 μg/ml only. The corresponding CV75 value was 244.32 μg/ml.
The following results were obtained in DRF 2:
- at post-treatment observation, or cell morphology modification was observed at any tested concentrations,
- flow cytometry measurement after Propidium Iodide staining revealed a cell viability decrease below 75 % at the concentration of 250 μg/ml only. The corresponding CV75 value was 184.58 μg/ml. Based on the results from both DRF runs, the mean CV75 was 214.45 μg/ml. As 1.2-fold the mean CV75 was equal to 257.34 μg/ml, i.e. above the highest tested concentration due to solubility limit, the highest concentration used in the main test was 250 μg/ml (i.e. highest homogenous achievable concentration).

MAIN TEST: individual run results
All acceptance criteria were reached in each run.
During both main test runs, the stock formulations were white homogeneous suspensions.
Run A:
- post-treatment observations: no precipitate/emulsion was noted in treated wells,
- run outcome: RFI CD86 exceeded the positivity threshold at the concentrations of ≥ 208.33 μg/ml and at all concentrations for RFI CD54. The run was therefore considered positive.
Run B:
- post-treatment observations: no precipitate/emulsion was noted in treated wells,
- run outcome: RFI CD86 did not exceed the positivity thresholds at any tested concentration but RFI CD54 exceeded the positivity threshold at all concentrations. The run was therefore considered positive.

Any other information on results incl. tables

conc. µg/ml	RFI for CD86		RFI for CD54		viability %		run conclusion		general conclusion
	A	B	A	B	A	B	A	B
69.77	113	113	1559	532	94.0	91.9	positive for CD86 and CD54	positive for CD54	positive
83.72	107	137	1418	464	92.9	92.7
100.47	109	148	1953	636	92.1	90.5
120.56	116	144	3429	1036	89.5	86.1
144.68	118	134	4012	900	87.4	88.9
173.61	84	131	6347	1400	80.2	80.9
208.33	182	127	4171	1525	79.2	76.8
250.00	157	116	4071	1559	72.6	71.9

Applicant's summary and conclusion

Interpretation of results:

other: the study is part of a global assessment on skin sensitisation

Conclusions:

Positive in human Cell Line Activation Test due to:
RFI (CD86) > 150 % in exp. A
RFI (CD86) < 150 % in exp. B
RFI (CD54) > 200 % in exp. A and B

Executive summary:

Method

The human Cell Line Activation Test (hCLAT) was conducted according to OECD guideline 442E.

Based on a maximum attainable concentration of test substance in DMSO of 125 mg/ml, a maximum test concentration of 250 μg/ml was used in DRF.

The calculated mean CV75 value determined in 2 DRF runs was 214.45 µg/ml. As 1.2 -fold the mean CV75 was 257.34 µg/ml, the highest concentration used in the main experiment was limited to 250 μg/ml.

The skin sensitizing potential of the test item was then tested in the main test in two successive runs at concentrations up to 250 µg/ml.

In each run, the test item formulations were applied to THP-1 cells and cultured in a 24-well plate for 24 h ± 30 minutes at 37 °C, 5 % CO₂ in a humidified incubator. A set of control wells was also added in each plate to guarantee the validity of each run. At the end of the incubation period, cells from each well were distributed to three wells of 96-well plate: the first well was labeled with IgG1-FITC antibodies, the second one was labeled with CD86-FITC antibodies and the third one was labeled with CD54-FITC antibodies. Then, just before flow cytometry analysis of CD86 and CD54 expression, all cells were dyed with Propidium Iodide for viability discrimination.

For each run, the Mean Fluorescence Intensity (MFI) obtained for each test sample was corrected by the isotype control IgG1 MFI value to obtain the corrected MFI. Corrected MFI value from the corresponding vehicle control was set to 100 % CD54 and CD86 expression by default. Then, corrected MFI values from each test sample were compared to the corresponding vehicle control to obtain the Relative Fluorescence Index for CD86 and CD54 expression for each tested concentration (RFI CD86 and RFI CD54).

Results

In run A, RFI values > 150 % for CD86 and > 200 % for CD54 were found, i.e. both positive.

In run B, RFI values < 150 % for CD86, but > 200 % for CD54 were found, i.e. only CD54 was positive.

Based on the prediction model for hCLAT test method, the substance was considered as positive in the hCLAT.