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

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

dicopper(2+) 2-({2-[bis(carboxymethyl)amino]ethyl}(2-hydroxyethyl)amino)acetate 2-[bis({2-[bis(carboxymethyl)amino]ethyl})amino]acetate

EC Number:

915-008-8

IUPAC Name:

dicopper(2+) 2-({2-[bis(carboxymethyl)amino]ethyl}(2-hydroxyethyl)amino)acetate 2-[bis({2-[bis(carboxymethyl)amino]ethyl})amino]acetate

Test material form:

solid

Remarks:

solid with lumps

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: 07 August 2017
- Shipping date: 07 August 2017
- Delivery date: 08 August 2017
- Date of initiation of testing: 08 August 201

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 3 hours between 36.2°C and 37.8°C, 5% CO2..

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 3 minutes and 1 hour after the test item application, the human epidermis was washed 20 times with 1 mL of DPBS (DPBS – Dutscher, Batch No. 7530417).
- Observable damage in the tissue due to washing:
The rinsed tissues were checked for any coloration. The tissues applied for 3 minutes and 1 hour were noted to be slightly blue.
- 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 sample was placed in MTT solution of 1 mg/mL concentration, except the eight tissues for the non-specific control which were placed in MTT assay medium (Cell Systems Batch No. 303-AG1218), for 3 hours between 36.2°C and 37.8°C, 5% CO2..
- Spectrophotometer: not specified
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability:
- Barrier function: 55.09%
- 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
- Fresh tissues / killed tissues
As the test item was identified as potentially producing direct MTT reduction and causing colour interference, four additional killed control tissues were added to the study (two by exposition time). These four control tissues were incubated with culture medium instead of MTT solution during the MTT incubation step to generate a non-specific colour control (NSC killed).
- Procedure used to prepare the killed tissues (if applicable): freezing
- N. of replicates : 4
- Method of calculation used:
As the test item is identified as producing both direct MTT reduction and colour interference:
True viability % =
[(OD of living tissues exposed to test item - OD of killed tissues exposed to test item - OD of living tissues exposed to test item incubated with medium without MTT + OD of killed tissues exposed to test item incubated with medium without MTT) / OD of living tissues 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 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 (8N KOH – Sigma, Batch No. SLBD3295V) and a negative control (50 μL of distilled water– Prochilab, Batch No. 20160608) were carried out.

Duration of treatment / exposure:

The test item was applied, as supplied, after being applied on a nylon mesh, at the dose of 25 mg, during 3 minutes at room temperature and during 1 hour at 37°C ± 1°C, 5% ± 1% CO2,

Duration of post-treatment incubation (if applicable):

The skin sample was placed in MTT solution of 1 mg/mL concentration, except the eight tissues for the non-specific control which were placed in MTT assay medium (Cell Systems Batch No. 303-AG1218), for 3 hours between 36.2°C and 37.8°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:

97.68

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:

100.57

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 corrected percent viability of the epidermis skins treated with the test item were 97.68% and 100.57% (considered as 100%), versus 17.98% and 0.33%, 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 CuDTPA and CuHEEDTA 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 on in vitro human reconstituted epidermis (epiCS®, CellSystems®).

The test item Reaction mixture of CuDTPA and CuHEEDTA was applied as supplied on a nylon mesh at the dose of 25 mg then to 2 living and 4 killed Human skin model surfaces (epiCS®, CellSystems®) previously moistened with 10 μL of distilled water during 3 minutes and 1 hour. 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.

Additionally, 2 living and 4 killed Human skin model surfaces (epiCS®, CellSystems®) were treated in the same manner during both 3 minutes and 1 hour, but they were incubated in in MTT assay medium instead of MTT solution in order to generate non-specific living and killed colour controls.
The experimental protocol was established in accordance with the O.E.C.D. Test Guideline No. 431 dated 29 July 2016 and the method B.40bis of the Council regulation No. 440/2008.

3 minutes and 1 hour after the test item application, the mean corrected percent viability of the epidermis skins treated with the test item were 97.68% and 100.57% (considered as 100%), versus 17.98% and 0.33%, 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 CuDTPA and CuHEEDTAdoes 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.