Reaction mass of potassium;sodium;2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetate;manganese, potassium;sodium;2-[bis[2-[bis(carboxymethyl)amino]ethyl]amino]acetate;manganese and potassium;sodium;2-[2-[bis(carboxymethyl)amino]ethyl-(2-hydroxyethyl)amino]acetate;manganese

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

REPORTING FORMAT FOR THE CATEGORY APPROACH

1. HYPOTHESIS FOR THE CATEGORY APPROACH (ENDPOINT LEVEL)
See the documentation in support for the category approach.

2. CATEGORY APPROACH JUSTIFICATION (ENDPOINT LEVEL
See the documentation in support for the category approach.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

29 July 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU Method B.40bis

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of ammonium;potassium;sodium;2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetate;magnesium, ammonium;potassium;sodium;2-[bis[2-[bis(carboxymethyl)amino]ethyl]amino]acetate;magnesium and ammonium;potassium;sodium;2-[2-[bis(carboxymethyl)amino]ethyl-(2-hydroxyethyl)amino]acetate;magnesium

EC Number:

902-533-2

IUPAC Name:

Reaction mass of ammonium;potassium;sodium;2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetate;magnesium, ammonium;potassium;sodium;2-[bis[2-[bis(carboxymethyl)amino]ethyl]amino]acetate;magnesium and ammonium;potassium;sodium;2-[2-[bis(carboxymethyl)amino]ethyl-(2-hydroxyethyl)amino]acetate;magnesium

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Source strain:

not specified

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: epiCS, Cell Systems
- Tissue batch number(s): Batch No.100-AG1428-1)
- Production date: 02 October 2017
- Shipping date: 02 October 2017
- Delivery date: 03 October 2017
- Date of initiation of testing: 04 October 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 minutes at room temperature and during 1 hour at 37°C ± 1°C, 5% ± 1% CO2,
- Temperature of post-treatment incubation (if applicable): for 2 hours 55 minutes between 36.5°C and 38°C, 5% CO2..

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 3 minutes and 55 minutes after the test item application, the human epidermis was washed 20 times with 1 mL of DPBS (DPBS – Dutscher, Batch No. 1310817).
- Observable damage in the tissue due to washing:
The rinsed tissues were checked for any coloration and noted to be whitish, comparable coloration to that of the negative control tissues.
- Modifications to validated SOP: not specified

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time:
The skin samples were placed in 300 μL of a MTT solution at 1 mg/mL for 2 hours and 55 minutes between 36.5°C and 38.0°C, 5% CO2.
- Spectrophotometer: not specified
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability:
- Barrier function: 55.00%
- Morphology: sufficient cornified (5) and vital (4) cell layers
- Contamination: no indication of HIV1, HBV, HCV, bacteria, yeast or mycoplasma found.
- Reproducibility: not specified

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
no interference

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 corrosive to skin if Viability measured after exposure time points (t=3 and 60 minutes) < 50% after 3 min exposure
- The test substance is considered to be corrosive to skin if Viability measured after exposure time points (t=3 and 60 minutes) ≥ 50% after 3 min exposure AND < 15% after 60 min exposure
- The test substance is considered to be non-corrosive to skin if Viability measured after exposure time points (t=3 and 60 minutes) ≥ 50% after 3 min exposure AND ≥ 15% after 60 min exposure
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439: not applicable

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg

CONTROLS
In the same experimental conditions, a positive control (50 μL of 8N KOH – Sigma, Batch No. SLBD3295V) and a negative control (50 μL of distilled water– Prochilab, Batch No. 1007123) were carried out.

Duration of treatment / exposure:

The test item was applied, at the dose of 25 mg, during 3 minutes at room temperature and during 1 hour at 37°C ± 1°C, 5% ± 1% CO2., to the epidermal surface of 2 living human skin models previously moistened with 25 μL of distilled water.

Duration of post-treatment incubation (if applicable):

The skin samples were placed in 300 μL of a MTT solution at 1 mg/mL for 2 hours and 55 minutes between 36.5°C and 38.0°C, 5% CO2.

Number of replicates:

2

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Replicate 1

Value:

93.1

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Replicate 2

Value:

87.12

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

3 minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with the test item were 93.10% and 87.12%, versus 57.77% and 0.62%, respectively, with the positive control item (potassium hydroxide 8N).

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In accordance with the Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions enable to conclude that the test item Reaction mixture of MgEDTA, MgDTPA and MgHEEDTA does not have to be classified in Category 1 “Corrosive”.
The hazard statement “H314: Causes severe skin burns and eye damage” with the signal word “Danger” are not required.

Executive summary:

The aim of the study was to evaluate the possible corrosive effects of the test item after topical administration onin vitrohuman reconstituted epidermis (epiCS®, CellSystems®).

The test item Reaction mixture of MgEDTA, MgDTPA and MgHEEDTA was applied as supplied, at the dose of 25 mg, to 2 living Human skin model surfaces (epiCS®, CellSystems®) during 3 minutes and 55 minutes. The application was followed by a rinse with 20 mL of DPBS. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues. The experimental protocol was established in accordance with the O.E.C.D. Test Guideline No. 431 dated 28 July 2015 and the method B.40bis of the Council regulation No. 440/2008.

3 minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with the test item were 93.10% and 87.12%, versus 57.77% and 0.62%, respectively, with the positive control item (potassium hydroxide 8N).

In accordance with the Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions enable to conclude that the test item Reaction mixture of MgEDTA, MgDTPA andMgHEEDTA does not have to be classified in Category 1 “Corrosive”.
The hazard statement “H314: Causes severe skin burns and eye damage” with the signal word “Danger” are not required.