OligoActiv EDDHA-Mn

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 Feb 2021 to ... July 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 26. Jun. 2020

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

adopted 06. Jul. 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt Rheinland-Pfalz 15 May 2018

Test material

Test material information

Constituent 1

Reference substance name:

Acetic acid, oxo-, sodium salt, reaction products with ethylenediamine and phenol, manganese sodium salts

Molecular formula:

not applicable: UVCB

IUPAC Name:

Acetic acid, oxo-, sodium salt, reaction products with ethylenediamine and phenol, manganese sodium salts

Test material form:

solid: particulate/powder

In vitro test system

Test system:

human skin model

Remarks:

commercially available EpiDermTM-Kit, procured by MatTek

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis.

Details on animal used as source of test system:

not applicable

Justification for test system used:

This in vitro study was performed in order to evaluate the potential of "manganese-EDDHA sodium salts" to evoke skin irritation in a Reconstructed human Epidermis (RhE) test method.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RhE) TISSUE
- Model used:
EpiDermTM-Kit, procured by MatTek
- Designation of the kit:
1) EPI-200-SIT
2) EPI-218_SIT
- Tissue batch number(s):
1) 34134
2) 34162
- Delivery date:
1) 2021 March 16
2) 2021 May 25
- Date of initiation of testing:
2021 March
- Date of experimantal completion
2021 March

TEMPERATURE USED FOR TEST SYSTEM

- Temperature used during treatment / exposure: 35 minutes at 37 ± 1°C and 5.0 ± 1% CO2 and ≥ 95% relative humidity and 25 min at room temperature
- Temperature of post-treatment incubation (if applicable): 37 ± 1 °C and 5.0 ± 1% CO2 and ≥ 95% relative humidity

REMOVAL OF TEST MATERIAL AND CONTROLS
1 hour after the first application, the inserts were removed from the plates using sterile forceps and rinsed immediately in 1-minute-intervals.
After rinsing thoroughly with DPBS, each tissue was blotted with sterile cellulose tissue and then transferred into a new 6-well-plate with fresh assay medium (0.9 mL). The surface of the inserts was then carefully dried with a sterile cotton tipped swab.
Then, the tissues were set in the incubator for 24 hours at 37 ± 1°C and 5.0 ± 1% CO2 and ≥ 95% relative humidity.
After incubation the tissues were removed from the incubator and shaken for 5 minutes (120 rpm). 0.9 mL assay medium were filled in the lower row of the 6-well-plate. Then the inserts were transferred into the lower row of the 6-well-plate and set into the incubator for 19 hours and 40 minutes in the main test and 18 hours in the additional test for post-incubation at 37 ± 1°C and 5.0 ± 1% CO2 and ≥ 95% relative humidity.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: Microtiter plate photometer Anthos Reader 2010 Flexi (Anthos Microsysteme GmbH)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
Batch 34134
- Viability: MTT QC assay, 4 hours, n=3 -> OD=1.624 ± 0.135 (range 1 - 3 -> passed)
- Barrier function: ET50 assay (1 % Triton) -> ET50=4.84 h (range 4.77 - 8.72 h -> passed)
- Contamination: no contamination

Batch 34162
- Viability: MTT QC assay, 4 hours, n=3 -> OD=1.57 ± 0.047 (range 1 - 3 -> passed)
- Barrier function: ET50 assay (1 % Triton) -> ET50=6.28 h (range 4.77 - 8.72 h -> passed)
- Contamination: no contamination

NUMBER OF REPLICATE TISSUES:
3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues
- N. of replicates : 2
- Method of calculation used: Viability treated tissue (corrected) = Viability treated tissue (main test) – Viability treated tissue (additional test, no MTT assay)

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 irritating to skin if the viability after 60 minutes exposure is less than or equal to 50%
- The test substance is considered to be non-irritating to skin if the viability after 60 minutes exposure is greater than 50%

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
main test 26.3; 25.9; 26.3 mg
additional test 26.1; 25.9 mg

VEHICLE
no vehicle used

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL [“Dulbecco’s Phosphate Buffered Saline” (DPBS buffer without CaCl2 and without MgCl2)]
- Concentration (if solution): 100 %

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL [SDS-solution]
- Concentration (if solution): 5 %

Duration of treatment / exposure:

60 min

Duration of post-treatment incubation (if applicable):

43 hours and 40 minutes in the main test and 42 hours in the additional test

Number of replicates:

3 (main test), 2 (additional test)

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

91.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

90.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3

Value:

80.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 4

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

87.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
All validity criteria were met.
The values for negative control and for positive control were within the range of historical data of the test facility

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item Manganese-EDDHA sodium salts is considered as not irritant to skin. After the treatment, the mean value of relative tissue viability was 87.3 %. This value is well above the threshold for skin irritation (50%). The optical density of the negative control was well within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8. The positive control has met the acceptance criterion too, for thus ensuring the validity of the test system. The variation within replicates was within the accepted range for negative control, positive control and test item (required: ≤ 18%). For these reasons, the result of the test is considered valid.

Executive summary:

One valid experiment was performed according to OECD Guideline 439 following GLP with three tissues and two independent MTT measurements. Three tissues of the human skin model EpiDerm^TM were treated with the test item for 60 minutes. The test item was applied directly to each tissue and spread to match the tissue size (0.63 cm²; as indicated by the supplier). DPBS-buffer was used as negative control and 5% SDS solution was used as positive control. After treatment with the negative control, the mean absorbance value was within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8, OD was 1.653. The positive control showed clear irritating effects. The mean value of relative tissue viability was reduced to 2.8% (required: ≤ 20%). The variation within the tissue replicates of negative control, positive control and test item was acceptable (required: ≤ 18%). After the treatment with the test item, the mean value of relative tissue viability was 87.3%. This value is well above the threshold for skin irritation potential (50%). Thus, the test item Manganese-EDDHA sodium salts is considered as not irritant to skin. As the test item showed intense coloring in the pre-test, there was the risk to influence the photometric measurement and create a false negative result. Therefore, an additional test for intensely coloured test items was performed.The result of the valid additional test showed, that the test item colour did not critically influence the result of the study and no data correction is necessary.