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Toxicological information

Skin irritation / corrosion

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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

Constituent 1
Chemical structure
Reference substance name:
Ammonium manganese(3+) diphosphate
EC Number:
233-257-4
EC Name:
Ammonium manganese(3+) diphosphate
Cas Number:
10101-66-3
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.