Zinc EDDHA

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 Feb 2021 to 02 June 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 AUG 2021 - 10 NOV 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 435 (In Vitro Membrane Barrier Test Method for Skin Corrosion)

Version / remarks:

adopted 28. Jul. 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

artificial membrane barrier model

Cell source:

other: synthetic biobarrier (Corrositex®) produced by InVitro International, USA, 17751 Sky Park East, Suite G, Irvine, CA 92614 and procured by Romer Labs Deutschland GmbH.

Details on animal used as source of test system:

Commercially available Corrositex®-Kit was used.
The test system consists of two components: a proteinaceous macromolecular gel (=synthetic biobarrier) on a permeable membrane and a fluid, the Chemical Detection System (CDS).

Justification for test system used:

This test system was chosen as a suitable follow up test of the available RhE Test to clarify the C&L of the substance.

Vehicle:

unchanged (no vehicle)

Details on test system:

SOURCE AND COMPOSITION OF MEMBRANE BARRIER USED
- Was the Corrositex® test kit used: yes
- Components: a proteinaceous macromolecular gel (=synthetic biobarrier) on a permeable membrane and a fluid, the Chemical Detection System (CDS).
- Apparatus and preparation procedures: The test system consists of two components: a proteinaceous macromolecular gel (=synthetic biobarrier) on a permeable membrane and a fluid, the Chemical Detection System (CDS). The biobarrier was produced one day prior to the assay was performed. The membrane discs with the biobarrier were stored in the fridge (2-8 °C) overnight before use.

Corrositex®-Kit was produced by InVitro International, USA, 17751 Sky Park East, Suite G, Irvine, CA 92614 and procured by Romer Labs Deutschland GmbH.
Day of delivery: 15. Jul. 2021
Batch Corrositex®-Kit: CT051021

WAS THE TIMESCALE CATEGORY TEST PERFORMED: yes. The categorisation screen was used to choose the appropriate scoring scale. As the test item has a strong colour itself, the test item colour covered the colour change in the test tubes. Therefore, a dilution of 10% of the test item in demin. water was prepared and the pH value was determined. A pH value of 8 was determined.
Based on the pH value, 150 µL of the 10% test item solution were added in test tube B and the content was mixed. Then the pH value was measured again; the value was pH 6.
Because this value was ≤ 9, the test item was classified as category 2. Since the test item was classified as a category 2 substance, the vials had to be observed for 60 minutes (test item of category 2).

METHOD OF DETECTION
The evaluation was performed visually: break-through causes a change in the colour of the solution; the stop watch was operated accordingly.

METHOD OF APPLICATION: pipetting

NUMBER OF REPLICATES: 4

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if ≤ 60 min elapsed between application of the test substance to the membrane barrier and barrier penetration.
- The test substance is considered to be non-corrosive to skin if > 60 min elapsed between application of the test substance to the membrane barrier and barrier penetration.
- Justification for the selection of the cut-off point(s): prediction model for Category 2 test chemicals (test chemicals with high acid/alkaline reserve) according to OECD TG 435

Control samples:

yes, concurrent negative control

yes, concurrent no treatment

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
Vial 1: 497.8 mg
Vial 2: 490.2 mg
Vial 3: 493.7 mg
Vial 4: 494.4 mg
(used as solid)

NEGATIVE CONTROL
500 µL 10% citric acid solution

POSITIVE CONTROL
111.9 mg of sodium hydroxide (used as solid)

Duration of treatment / exposure:

Blank and negative control test item : 61 minutes
Positive control : 12 minutes

Number of replicates:

4

Irritation / corrosion parameter:

penetration time (in minutes)

Run / experiment:

Vial 1-4

Value:

> 60

Vehicle controls validity:

valid

Remarks:

Blank for CDS

Negative controls validity:

valid

Remarks:

10% citric acid solution

Positive controls validity:

valid

Remarks:

sodium hydroxide (used as solid)

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: No break-through

DEMONSTRATION OF TECHNICAL PROFICIENCY: The demonstration of proficiency was performed using proficiency chemicals under non-GLP conditions, but within the GLP-environment at LAUS GmbH. All of the listed proficiency chemicals except one met the right classification according to OECD 435. Selenic acid gave discrepant classification compared to the OECD guideline 435. This result can be seen as outlier and therefore as uncritical.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, negative control showed a clear negative result; no break-through of the negative control within 60 minutes
- Acceptance criteria met for positive control: the break-through time of the positive control falls within ± 2 standard deviations of the current historical mean value
- Acceptance criteria met for variability between replicate measurements: No break-through of the test item was observed in any replicate.

Interpretation of results:

other: Category 2 (irritant) based on EU GHS criteria

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item possesses no skin corrosion potential and is considered as a non-corrosive substance. Therefore, Zinc-EDDHA sodium salts is considered as non-corrosive to skin in the Corrositex® - Test. The test item is therefore not to be classified for corrosivity to skin according to GHS. The negative control showed no break-through and therefore no corrosive effects. The measured time of the positive control was 12 minutes and thus ensuring the validity of the test system. For these reasons, the result of the test is considered valid.

Executive summary:

The study was performed according to OECD Guideline 435 under GLP compliance using Corrositex® kit. A blank and 10% citric acid solution served as negative control and sodium hydroxide was the positive control. The test item was applied as a solid (fine powder). As the test item was classified as a category 2 substance, the test item vials were observed for 60 minutes. No break-through of the test item was observed within 61 minutes. Thus, it can be stated that in this study and under the experimental conditions reported, the test item possesses no skin corrosion potential and is considered as a non-corrosive substance. The negative control showed no break-through and therefore no corrosive effects. The measured time of the positive control was 12 minutes and thus ensuring the validity of the test system. For these reasons, the result of the test is considered valid.

Therefore, Zinc-EDDHA sodium salts is considered as non-corrosive to skin in the Corrositex® - Test. The test item is therefore not classified for corrosivity to skin according to GHS.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

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

1

Reference substance name:

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

Molecular formula:

non specified (UVCB substance)

IUPAC Name:

Acetic acid, oxo-, sodium salt, reaction products with ethylenediamine and phenol, zinc 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 "zinc-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:
EPI-200-SIT
- Tissue batch number(s):
34134
- Delivery date:
2021 March 16
- Date of initiation of testing:
2021 March 15 (pre-tests)
- Date of experimantal completion
2021 March 19

TEMPERATURE USED FOR TEST SYSTEM
Tissues were dosed in 1-minute-intervals. After dosing the last tissue, all plates were transferred into the incubator for 35 minutes at 37 ± 1°C and 5.0 ± 1% CO2 and ≥ 95% rel-ative 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 post-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 18 hours and 30 minutes 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

NUMBER OF REPLICATE TISSUES:
3

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- 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 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):
25.9; 26.4; 26.6 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):

42.5 h

Number of replicates:

3

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

37.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

30.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3

Value:

30.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 4

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

32.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive 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:

Category 2 (irritant) based on GHS criteria

Conclusions:

The test item Zinc-EDDHA sodium salts is considered as at least irritant to skin. After the treatment, the mean value of relative tissue viability was reduced to 32.6. This value is below 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 reduced to 32.6%. This value is below the threshold for skin irritation potential (50%). Test items that induce values below the threshold of 50% are considered at least irritant to skin. The OECD guideline 439 addresses the human health endpoint skin irritation. Therefore, further testing on skin corrosion potential with another suitable in vitro study (e.g. OECD 431) may be required. Therefore, the test item Zinc-EDDHA sodium salts is considered at least irritant to skin in the Reconstructed human Epidermis (RhE) Test Method.