Manganese Violet

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

25 August 2021 - 01 October 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 442E (In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT))

Version / remarks:

2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

human Cell Line Activation Test (h-CLAT)

Test material

Test material information

Constituent 1

Reference substance name:

Ammonium manganese diphosphate, monoclinic and triclinic

Molecular formula:

MnKx (NH4)1-x P2O7 (0 =< x =< 0.5)

IUPAC Name:

Ammonium manganese diphosphate, monoclinic and triclinic

Test material form:

solid: particulate/powder

In vitro test system

Details of test system:

THP-1 cell line [442E]

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical stock solution: 40 μg/mL was the highest achievable concentration.
- Preparation of the test chemical serial dilutions: serial 1:1.2 dilution.
- Preparation of the positive controls: DNCB (2,4-dinitrochlorobenzene, CAS No. 97-00-7, purity ≥ 99%) was prepared in DMSO (dimethyl sulfoxide, CAS No. 67-68-5, purity ≥ 99%) at concentrations of 3 and 4 μg/mL.
- Preparation of the solvent control: DMSO in culture medium at 0.2% final concentration.
- Preparation of negative control: culture medium.
- Stable dispersion obtained: yes
- Log Kow of the test chemical: KOWWIN v1.68 estimate = -6.9.
- Other: water solubility: <0.01g/L

DOSE RANGE FINDING ASSAY:
- Highest concentration used: In the first dose finding assay 5000 μg/mL was used as top dose as recommended by the OECD 442E. Due to observed precipitates in the first dose finding assay, the highest tested test item concentration was 40 μg/mL the second and third dose finding assay. The first dose finding assay was invalid due to precipitates in all used concentrations. Therefore, only the results of the second and third dose finding assay were reported with the original numbering.
- Solubility assessment: The solubility of the test item was assessed visually for each preparation (particles, drops, cloudiness, non-miscible phases, etc) and recorded.
- Solubility in incubation medium: up to 5.00 µg/ml.
- Results of selecting appropriate concentration and determination of cytotoxicity e.g. CV75: Cytotoxic effects (threshold of cytotoxicity: <75%) were not observed following incubation with the test item up to the highest tested and manageable concentration (40 μg/mL). Due to the lack of cytotoxicity, a CV75 value could not be calculated. Therefore, the lowest concentration with precipitates (5.00 μg/mL) was used as top dose in three main experiments.
- Final concentration range selected on basis of: solubility (precipitation)

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates: 1
- Number of repetitions: 3
- Test chemical concentrations: 1.40, 1.67, 2.01, 2.41, 2.89, 3.47, 4.17, and 5.00 µg/ml.
- Application procedure: The test item dilutions were prepared freshly before each experiment.
Each dilution solution of the dilutions of the test item, medium control, positive and DMSO control was added to the cells. The treated THP-1 cells were incubated for 24 ± 0.5 h. At the end of the incubation period, the cell cultures were microscopically evaluated for morphological alterations. Each concentration of the test item, medium control, positive and DMSO control was prepared in triplicates for the different staining (with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1).
- Exposure time: 24 ± 0.5 hours
- Study evaluation and decision criteria used: As per OECD 442E. For CD86/CD54 expression measurement, each test item is tested in at least two independent runs to derive a single prediction (POSITIVE or NEGATIVE). A 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:
- RFI of CD86 is ≥ 150% at any concentration leading to ≥ 50% viability,
- RFI of CD54 is ≥ 200% at any concentration leading to ≥ 50% viability.
Otherwise, the h-CLAT prediction is considered NEGATIVE.
Based on the individual run conclusions, a final prediction is made. 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. Similarly, if the first two runs are negative for both markers, the h-CLAT prediction is considered NEGATIVE 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. 2 out of 3). 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.
- Description on study acceptance criteria: As per OECD 442E. For controls:
- Cell viability of medium and DMSO controls should be ≥ 90%,
- For both medium and DMSO control wells, MFI ratio of 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 control (DNCB), RFI values of both CD86 and CD54 should meet positive criteria (CD86 RFI ≥ 150 and CD54 RFI ≥ 200) and cell viability > 50% in at least one concentration of the two tested controls.
For results: Negative results are acceptable only for test items exhibiting a cell viability of <90% at the highest concentration tested (i.e. 1.2 × CV75).

SEEDING AND INCUBATION
- Seeding conditions (passage number and seeding density): On the day of the cytotoxicity or main experiment directly before the treatment of the cells, a volume of 500 μL with a cell density of 1.82*E06 THP-1 cells/mL was seeded in each corresponding well of a 24-well flat bottom plate (passage number should not exceed 30).
- Incubation conditions: 37 ± 1.5ºC and 5.0 ± 0.5 % CO2 atmosphere
- Washing conditions: washed twice (2-8ºC) with 2 mL FACS buffer (PBS with 0.1% (w/v) BSA)

MEASUREMENT OF CELL SURFACE EXPRESSION/LUCIFERASE ACTIVITY
- Flow cytometry used: FACSCalibur, Becton Dickinson GmbH, software FACSComp 6.0
- Plate used: 24-well flat bottom plate
- staining/cytotoxicity measurements: 2 cytotoxicity tests were performed with independent cell cultures, and 8 test item concentrations each. Each volume (500 μL) of the test item dilutions were added to the cells and incubated for 24 ± 0.5 h. At the end of the incubation period, cultures were microscopically evaluated for morphological alterations. Each well of test item-treated and untreated cells was collected in sample tubes, centrifuged, washed twice (2-8 °C) with 2 mL FACS buffer (PBS with 0.1% (w/v) BSA) and re-suspended in a final volume of 2 mL/tube FACS buffer. At least 10 min before the flow cytometry acquisition, 5 μL of a 7-AAD solution will be added in each sample tube.
- Preparation for CD54 and/or CD86 expression measurements/cell staining: The triplicates of each test item-treated and not test item-treated cells were pooled and equally distributed into three sample tubes, collected by centrifugation (approx. 250 x g, 5 min) and then washed twice with approx. 2 mL of FACS buffer (PBS with 0.1% (w/v) BSA). Thereafter, the cells were centrifuged, re-suspended and blocked with 600 μL of blocking solution at 2-8 °C (on ice) for approx. 15 min. After blocking, the cells were centrifuged, and the cell pellets were re-suspended in 100 μL FACS buffer. The cells were stained with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1 (isotype control).
All solutions were kept light protected at 2-8 °C or on ice during the staining and analysis procedures. The cells with the different antibodies or the IgG1 were mixed and incubated light protected for 30 ± 5 min at 2-8 °C (on ice). After staining with the antibodies, the cells were washed twice (2-8 °C) with 2 mL FACS buffer and re-suspended in a final volume of 2 mL/tube FACS buffer. At least 10 min before the flow cytometry acquisition, 5 μL of a 7-AAD solution were added.

DATA EVALUATION
- Cytotoxicity assessment: flow cytometry
- Prediction model used: The expression of cell surface antigens (CD54, CD86) was analysed by flow cytometry using the software Cellquest Pro 6.0. The FITC acquisition channel (FL-1) was set for the optimal detection of the FITC fluorescence signal, and the 7-AAD acquisition channel (FL-3) was set for the optimal detection of DNA-bound 7-AAD fluorescence signal.

Vehicle / solvent control:

DMSO

Negative control:

not applicable

Positive control:

dinitrochlorobenzene (DNCB) [442E]

Results and discussion

Positive control results:

- DNCB: The RFI (CD86) was higher than 150 and the RFI (CD54) was higher than 200 in both runs, the result was considered POSITIVE.

In vitro / in chemico

Results

Results 1

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

RFI CD86>200 [442E]

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

RFI > 150

Results 2

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

other: RFI (CD86) and RFI (CD54)

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 3

Group:

test chemical

Run / experiment:

run/experiment 3

Parameter:

other: RFI (CD86) and RFI (CD54)

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Outcome of the prediction model:

negative [in vitro/in chemico]

Other effects / acceptance of results:

Based on the individual run results, the outcome of the study is NEGATIVE.
- First run (main test): The CD86 marker showed in 3 concentrations a CD86-RFI above 150%, indicating a positive outcome of main experiment 1. No cell viability was below 50%.
- Second run (main test): Neither RFI of CD54 nor RFI of CD86 showed values higher than 200% (CD54) or 150% (CD86), indicating a negative outcome of main experiment 2. No cell viability was below 50%.
- Third run (main test): Neither RFI of CD54 nor RFI of CD86 showed values higher than 200% (CD54) or 150% (CD86), indicating a negative outcome of main experiment 3. No cell viability was below 50%.

OTHER EFFECTS:
- Visible damage on test system: no
- Precipitation: In all main test runs, precipitation was observed at the highest tested concentration.

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS: All acceptance criteria were met.
- Acceptance criteria met for negative control: The cell viability of the medium and DMSO control was >90%. For both medium and solvent control, the MFI ratio of CD86 and CD54 to isotype control was >105%. In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 ≥200% and CD86 ≥150%).
- Acceptance criteria met for positive control: The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥200% and CD86 ≥150%) and the cell viability was >50%.
- Acceptance criteria met for the test item: the cell viability was more than 50% in at least four tested concentrations in each run.
- Range of historical values if different from the ones specified in the test guideline: see table in 'Any other information on results incl. tables'

Any other information on results incl. tables

Main Test Results:

Table 3. Main test individual results. Run 1.

	Concentration [µg/mL]	RFI CD54 antibody [%]	RFI CD86 antibody [%]	Cell viability [%]
Medium control	-	100.0	100.0	98.23
DMSO control	-	100.0	100.0	97.97
Positive control (DNCB)	3	295.5*	404.7*	92.62
	4	487.5*	365.1*	72.65
Test item	1.40	133.9	129.5	97.30
	1.67	116.1	147.1	98.08
	2.01	121.0	152.5*	97.85
	2.41	121.0	150.2*	97.68
	2.89	119.4	146.8	96.67
	3.47	140.3	160.0*	97.12
	4.17	132.3	154.2*	97.02
	5.00^P	137.1	125.1	97.10

Table 4. Main test individual results. Run 2.

	Concentration [µg/mL]	RFI CD54 antibody [%]	RFI CD86 antibody [%]	Cell viability [%]
Medium control	-	100.0	100.0	97.35
DMSO control	-	100.0	100.0	97.33
Positive control (DNCB)	3	417.5*	408.7*	93.01
	4	639.7*	436.7*	87.90
Test item	1.40	144.4	94.7	96.71
	1.67	153.3	119.1	96.79
	2.01	157.8	125.8	95.93
	2.41	153.3	121.1	96.17
	2.89	162.2	125.4	95.65
	3.47	166.7	134.0	95.71
	4.17	164.4	92.8	96.20
	5.00^P	173.3	126.8	96.58

Table 5. Main test individual results. Run 3.

	Concentration [µg/mL]	RFI CD54 antibody [%]	RFI CD86 antibody [%]	Cell viability [%]
Medium control	-	100.0	100.0	95.51
DMSO control	-	100.0	100.0	96.18
Positive control (DNCB)	3	357.5*	423.0*	90.13
	4	545.2*	394.2*	85.86
Test item	1.40	100.0	100.9	96.00
	1.67	101.3	100.9	95.91
	2.01	98.7	100.9	95.18
	2.41	105.1	92.8	95.05
	2.89	98.7	92.8	95.61
	3.47	126.9	106.7	94.45
	4.17	117.9	113.9	94.59
	5.00^P	119.2	95.1	94.87

^P Precipitation, are excluded from the evaluation; * RFI value of CD86 or CD54 fulfilled the positive criteria (CD86 ≥150% and CD54 ≥200%).

 

Acceptance criteria:

Table 6. Acceptance for the first run.

Cell viability of medium control and DMSO control should be more than 90%.             Medium:         98.23%             DMSO:           97.97%

In the positive control (DNCB), RFI values of both CD54 and CD86 should exceed the positive criteria (CD54 ≥200% and CD86 ≥150%) and the cell viability should be >50%.             3 µg/mL DNCB (CD 54):        295.5%             3 µg/mL DNCB (CD 86):        404.7%             4 µg/mL DNCB (CD 54):        487.5%             4 µg/mL DNCB (CD 86):        365.1%

In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 should not exceed the positive criteria (CD54 ≥200% and CD86 ≥150%).             CD54:    141.9%             CD86:      93.2%

For medium and DMSO controls, the MFI ratio of CD54 and CD86 to isotype control should be >105%.             Medium CD 54:          124.4%             Medium CD 86:          216.1%             DMSO CD 54:             136.4%             DMSO CD 86:             213.6%

Table 7. Acceptance for the second run.

Cell viability of medium control and DMSO control should be more than 90%.             Medium:         97.35%             DMSO:           97.33%

In the positive control (DNCB), RFI values of both CD54 and CD86 should exceed the positive criteria (CD54 ≥200% and CD86 ≥150%) and the cell viability should be >50%.             3 µg/mL DNCB (CD 54):        417.5%             3 µg/mL DNCB (CD 86):        408.7%             4 µg/mL DNCB (CD 54):        639.7%             4 µg/mL DNCB (CD 86):        436.7%

In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 should not exceed the positive criteria (CD54 ≥200% and CD86 ≥150%).             CD54:    140.0%             CD86:    109.6%

For medium and DMSO controls, the MFI ratio of CD54 and CD86 to isotype control should be >105%.             Medium CD 54:          115.7%             Medium CD 86:          173.1%             DMSO CD 54:            122.1%             DMSO CD 86:            180.4%

Table 8.Acceptance for the third run.

Cell viability of medium control and DMSO control should be more than 90%.             Medium:         95.51%             DMSO:           96.18%

In the positive control (DNCB), RFI values of both CD54 and CD86 should exceed the positive criteria (CD54 ≥200% and CD86 ≥150%) and the cell viability should be >50%.             3 µg/mL DNCB (CD 54):        357.5%             3 µg/mL DNCB (CD 86):        423.0%             4 µg/mL DNCB (CD 54):        545.2%             4 µg/mL DNCB (CD 86):        394.2%

In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 should not exceed the positive criteria (CD54 ≥200% and CD86 ≥150%).             CD54:      93.6%             CD86:    101.1%

For medium and DMSO controls, the MFI ratio of CD54 and CD86 to isotype control should be >105%.             Medium CD 54:          131.2%             Medium CD 86:          189.2%             DMSO CD 54:            129.1%             DMSO CD 86:            190.0%

 

Historical control data:

Table 9. Historical control data for 2020.

	Positive control				(MFI - MFI(ISO) DMSO)/ (MFI - MFI(ISO) Medium)
	DNCB (3 µg/mL)		DNCB (4 µg/mL)
Antibody	CD 54	CD 86	CD 54	CD 86	CD 54	CD 86
Value should be	≥200%	≥150%	≥200%	≥150%	<200%	< 50%
Mean value [%]	360.6	483.5	497.5	533.9	114.1	105.9
Standard deviation	127.7	141.9	153.5	180.7	17.2	13.7
Max value [%]	742.1	816.9	962.7	1027.9	172.5	136.8
Low value [%]	209.7	209.9	311.1	204.1	74.6	76.5
Total number of runs	35

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was found to be negative based on the individual results of three independent runs. Therefore, the test item may be classified as not skin sensitizer.

Executive summary:

An in vitro cell line activation test (h-CLAT) has been performed as part of an integrated approach to support the identification of the sensitization potential of the test item in accordance with the OECD Guideline 442E, under GLP conditions.

The h-CLAT method is based on changes in the quantification of cell surface marker expression (i.e. CD86 and CD54) on a human monocytic leukemia cell line, THP-1 cells. A solubility assay with the test item was performed, where DMSO was chosen as vehicle. Then, two Dose-Range Finding assays were performed with the test item. Based on the results of both DRF assays and the solubility test, the maximum concentration in the main test was 5 μg/mL. For the main test, two validated successive test runs were performed. In each run, the test item formulations (1.40, 1.67, 2.01, 2.41, 2.89, 3.47, 4.17, and 5.00 µg/ml) were applied to THP-1 cells and cultured for 24 hours ± 30 minutes at 37ºC, 5% CO₂ in a humidified incubator. Negative and positive controls were run in parallel. After incubation, all cells were dyed for viability discrimination and then CD86 and CD54 expression was measured by flow cytometry analysis. The Mean Fluorescence Intensity (MFI) was obtained for each test sample, corrected by the isotype control IgG1 and compared to the corresponding vehicle control to obtain the Relative Fluorescence Index (RFI) for CD86 and CD54 expression. All validity criteria were met. In two out of three runs, the results of RFI(CD86) and RFI(CD54) were less than 150 and 200 respectively in all concentrations tested. Therefore, the test item was found to be negative in the h-CLAT test.