Diammonium [[N,N'-ethylenebis[N-(carboxymethyl)glycinato]](4-)-N,N',O,O',ON,ON']cobaltate(2-)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From November 05 to 20, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 442E

Version / remarks:

June 25, 2018

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Test material

Test material information

Constituent 1

Reference substance name:

Cobalt diammonium EDTA

IUPAC Name:

Cobalt diammonium EDTA

Test material form:

solid

In vitro test system

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:

Pre-tests
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 first 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 * 105 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 at 250 * g. Afterwards the super-natant was removed and the pellet was suspended in 200 µL staining buffer before 200 µL were transferred into one well of a 96-well plate. After that, the cells were washed three times with staining buffer (centrifugation: 250 * g for 5 min). After that, the cells were re-suspended in 150 µL staining buffer and 7.5 µL 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). In total 10 000 (± 10 %) viable cells (PI negative) were acquired. The cell viability was automatically calculated by the flow cytometer. Based on these results, the concentrations for the main experiments were defined.
Results
None of the controls induced a cytotoxic effect. Since the cell viabilities of medium and solvents controls were higher than 90 % and the viability of all test item concentrations were >50 %, the pre-test was valid.
Since none of the tested concentration induced a cytotoxic effect with RPMI 1640 as sol-vent and the test item was soluble at the concentration 500 mg/mL a second pre-test using the following concentrations was performed:
5000 µg/mL, 2500 µg/mL, 1250 µg/mL, 625 µg/mL, 312.5 µg/mL, 156.3 µg/mL, 78.1 µg/mL and 39.1 µg/mL.
Exposure Concentrations and Dose Selection
Since no CV75 could be determined (no reduction of viability below 75 %) in the pre-tests, and the solvent of the test item is RPMI 1640, the maximal test item concentration in the experiments was 5000 µg/mL.
To achieve the highest test item concentration, a 100x stock solution in RPMI 1640 was prepared and diluted.
Experimental Procedure
The performance of experiment I and II was identical. Eight test item concentrations were tested in each experiment 1395.4 µg/mL, 1674.5 µg/mL, 2009.4 µg/mL, 2411.3 µg/mL, 2893.5 µg/mL, 3472.2 µg/mL, 4166.7 µg/mL, 5000.0 µg/mL.
1 * 106 cells / well of a 24-well plate were exposed to each concentration of the test item for 24 h ± 0.5 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 re-suspended 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 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 150 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 (BD FACS LyricTM). FITC-Detection: (Excitation: 488 nm / Detection filter 527/32; corresponding to the FL-1 channel). In total 10,000 viable cells (PI negative) were acquired and analysed.

Results and discussion

In vitro / in chemico

Results

Results 1

Run / experiment:

other: Experiment I and II

Parameter:

other: RFI of CD54

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Run / experiment:

other: Experiment I

Parameter:

other: RFI of CD86

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Parameter:

other: RFI of CD86

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Interpretation of results:

other: Skin sensitising

Remarks:

according to the criteria listed in the OECD guideline 442E

Conclusions:

Skin sensitising

Executive summary:

Method

This in vitro study was performed to assess the sensitising potential of the test item by quantifying changes in the expression level of the two cell surface markers CD86 and CD54, which are associated with the process of activation of monocytes and dendritic cells, according to the OECD guideline 442E.

Two valid experiments with a treatment period of 24 hours were performed.

In the experiments, the highest nominal applied concentration (5000 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 and tested. Precipitation of the test item was not visible in any of the experiments.

As solvent control for the test item, RPMI 1640 was used in a final con-centration of 1 % in culture medium. As positive control, 2,4-dinitrochlorobenzene was used. Prior to the study, the cells 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.

Results

In both experiments (I and II) no cytotoxicity occurred at any tested concentration.

In both experiments (I and II) the RFI of CD54 was ≥ 200 % at any tested concentration with cell viability ≥ 50 %.

In experiment I and the RFI of CD86 was ≥ 150 % in the following concentrations (1395.4, 1674.5, 2009.4 and 2411.3 µg/mL). The EC 200 for CD54 is 329.25 µg/mL.

In experiment II the RFI of CD86 was not ≥ 150 % in any tested concentration.

Conclusion

In accordance with the classification criteria, the result of this study is positive and the substance is considered to be skin sensitising.