Manganese Violet

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 August 2021 - 02 September 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:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

2021

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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

Test system:

human skin model

Remarks:

SkinEthic RHE®

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: not specified

Justification for test system used:

The SkinEthic RHE® model has been validated for irritation testing (Validation study based on the original ECVAM Performance Standards (21) in 2008) and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439); therefore, it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic RHE®
- Tissue batch number(s): 21-RHE-138 (living) and 21-RHE-104 (killed)
- Shipping date: 31 August 2021 and 22 June 2021
- Delivery date: 31 August 2021 and 01 September 2021 (defreezing).
- Expiration date: 6 September 2021
- Date of initiation of testing: 01 September 2021

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 25 x 1 mL of DPBS (Dutscher - Batch No. 7380521)
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 300 μL of a MTT solution at 1.0 mg/mL
- Incubation time: 3 hours and 02 minutes at 37°C, 5% CO2
- Spectrophotometer: ELx800 absorbance microplate reader (BioTek)
- Wavelength: 570 nm
- Filter: no
- Linear OD range of spectrophotometer: not specified.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD = 1.3 (CV = 6.4%), specification OD > 0.7
- Barrier function: > 10.0h (pecification 4.0 ≤ ET50 ≤ 10.0h); ET50 value was out of specification but declared satisfactory by dispensation. Quality of the RHE batch is good according to 2 further QC tests (normal values), especially histology showing good morphology.
- Morphology: 6.5 cell layers (specification ≥ 4); multi-layered, highly differentiated epidermis consisting of organized basal, spinous and granular layers, and a multilayered stratum corneum.
- Contamination: no, on the blood of the donors and the cells from the donors, absence of contamination was verified.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- 2 killed tissues
- Procedure used to prepare the killed tissues (if applicable): 2 killed Human skin model surfaces were treated in the same manner as the living tissues in order to generate non-specific MTT reduction.
- N. of replicates: 2
- Method of calculation used: True viability % = [(living tissues OD exposed to test item - killed tissues OD exposed to test item)/living tissues OD exposed to negative control] x 100

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin (or corrosive) if the viability after 42 minutes exposure and 42 hours of post-treatment incubation is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability after 42 minutes exposure and 42 hours of post-treatment incubation is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 mg (32 mg/cm2) (+10 μL distilled water)
- Vehicle: no

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16 μL of distilled water (ADL Prochilab, Batch No. 201117)

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 16 μL of 5% SDS.
- Concentration (if solution): 5% SDS solution was prepared by weighing 0.5000 g of SDS (SIGMA Batch No. STBJ3028) in a 10 mL volumetric flask q.s. 10 mL of distilled water

Duration of treatment / exposure:

42 min

Duration of post-treatment incubation (if applicable):

41 hours

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean (42 min exposure)

Value:

94.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100% viability (distilled water)

Positive controls validity:

valid

Remarks:

1.8% viability (5% SDS)

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: yes (relevant control added)
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes. A full demonstration of proficiency was
performed for the EpiSkin-SM model, plus a reduced validation with the SkinEthic RHE model.
Adequate results were obtained for the evaluated chemicals.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes. Mean OD of the tissue replicates treated with
the negative control was in the range ≥ 0.8 and ≤ 3.0.
- Acceptance criteria met for positive control: yes. Mean viability of the tissue replicates treated
with the positive control (SDS 5%), expressed as % of the negative control was < 40%
- Acceptance criteria met for variability between replicate measurements: yes. Standard deviation
between tissue replicates was SD ≤ 18%

Any other information on results incl. tables

Table 1. Summary of results.

	Well ID	OD	Mean OD /        disc (#)	Mean OD / product	Viability %	Mean viability %	SD viability	Conclusion
Negative control	SPL 1	0.922 0.941 0.926	0.929	0.902	103.0 100.7	100.0	3.4
	SPL 2	0.851 0.873 0.884	0.869
	SPL 3	0.870 0.934 0.920	0.908
Positive control	SPL 4	0.018 0.016 0.018	0.017	0.016	1.9 1.8	1.8	0.1	Irritant
	SPL 5	0.016 0.015 0.015	0.015
	SPL 6	0.016 0.016 0.016	0.016
Test item PH- 21/0781	SPL 7	0.969 0.996 0.917	0.960	0.862	106.4 87.8	95.5	9.7
	SPL 8	0.861 0.836 0.803	0.833
	SPL 9	0.824 0.773 0.781	0.792
Test item PH- 21/0781 NSMTT	SPL 10	0.007 0.008 0.008	0.007	0.007	0.8 0.7	0.7	0.1
	SPL 11	0.006 0.007 0.007	0.006
Test item PH- 21/0781 Corrected						94.8		Non irritant

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Non-irritant to skin, no category (CLP Regulation EC no. 1272/2008)

Conclusions:

Under test conditions, the mean percent viability of the treated tissues was 94.8% versus 1.8% with the positive control item (5% SDS). Therefore, the test item is not irritant to the skin.

Executive summary:

An in vitro skin irritation test was performed with the test item in a reconstructed human epidermis SkinEthic RHE® model, according to OECD TG 439, under GLP conditions.

Three epidermis units were treated with 16 mg test item for 42 minutes at room temperature. Exposure to the test item was terminated by rinsing with 25 x 1 mL of DPBS. The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO₂. The viability of each disk was assessed by incubating the tissues with MTT, extracting the precipitated formazan crystals using isopropanol for 2 hours under gentle agitation in the dark, and measuring the concentration of formazan by determining the OD at 570 nm, just after dilution of the extracts 1:2 in isopropanol. Distilled water was used as a negative control and 5% SDS solution as positive control; two killed control tissue models were added to the study which underwent the entire testing procedure to generate a non-specific MTT reduction control.

Under test conditions, the mean percent viability of the treated tissues was 94.8% versus 1.8% with the positive control item (5% SDS). Therefore, the test item must be considered as non-irritant to skin.