Diammonium [[N,N'-ethylenebis[N-(carboxymethyl)glycinato]](4-)-N,N',O,O',ON,ON']cuprate(2-)

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August-September 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Diammonium [[N,N'-ethylenebis[N-(carboxymethyl)glycinato]](4-)-N,N',O,O',ON,ON']cuprate(2-)

EC Number:

268-018-3

EC Name:

Diammonium [[N,N'-ethylenebis[N-(carboxymethyl)glycinato]](4-)-N,N',O,O',ON,ON']cuprate(2-)

Cas Number:

67989-88-2

Molecular formula:

C10H12CuN2O8.(NH4)2

IUPAC Name:

copper(2+) ion 2-({2-[(carboxylatomethyl)(carboxylmethyl)amino]ethyl}(carboxymethyl)amino)acetate diamine

Test material form:

other: solution

Details on test material:

Description: clear blue liquid
Batch: CFC-10635 (555H1011)
Purity/Composition: ~55%
Test substance storage: at room temperature in the dark
Stability under storage conditions: stable
Expiry date: 1 September 2013
General information: avoid contact with aluminium, nickel, zinc, copper and copper alloys. Store in PVC, PP, PE, Bituminized tank

No correction was made for the purity/composition of the test compound.

Test animals

Species:

other: in vitro

Details on test animals or test system and environmental conditions:

Test system: EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 12-EKIN-031).
This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Rationale: In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Source: SkinEthic Laboratories, Lyon, France.

Test system

Type of coverage:

other: not applicable

Preparation of test site:

other: not applicable

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

The liquid test substance was applied undiluted (25 µl) directly on top of the tissue.

Duration of treatment / exposure:

See below

Observation period:

Not applicable

Number of animals:

Not applicable

Details on study design:

See below

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 min

Value:

11

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

EDTA-Cu(NH4)2 was checked for possible direct MTT reduction by adding the test substance to MTT medium. Because no colour change was observed it was concluded that EDTA-Cu(NH4)2 did not interact with MTT.
The mean absorption at 570 nm measured after treatment with EDTA-Cu(NH4)2 and controls are presented in Table 1.
Table 2 shows the mean tissue viability obtained after 15 minutes treatment with EDTA-Cu(NH4)2 compared to the negative control tissues. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with EDTA-Cu(NH4)2 compared to the negative control tissues was 11%. Since the mean relative tissue viability for EDTA-Cu(NH4)2 was below 50% it is considered to be irritant.

The positive control had a mean cell viability after 15 minutes exposure of 4%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 8%, indicating that the test system functioned properly.

Any other information on results incl. tables

Table1            Mean absorption in the in vitro skin irritation test with EDTA-Cu(NH4)2

	A (OD570)	B (OD570)	C (OD570)	Mean (OD570)		SD
Negative control	0.883	1.018	0.969	0.957	±	0.069
EDTA-Cu(NH4)2	0.061	0.123	0.144	0.109	±	0.043
Positive control	0.029	0.035	0.044	0.036	±	0.007

OD = optical density

SD = Standard deviation

Triplicate exposures are indicated by A, B and C.

In this table the values are corrected for background absorption (0.042). Isopropanol was used to measure the background absorption.

 

Table2            Mean tissue viability in thein vitroskin irritation test with EDTA-Cu(NH4)2

	Mean tissue viability (percentage of control)
Negative control	100
EDTA-Cu(NH4)2	11
Positive control	4

 

Applicant's summary and conclusion

Interpretation of results:

other: irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test substance is irritating in an in vitro test (OECD 439).

Executive summary:

This report describes the ability of EDTA-Cu(NH4)2 to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small Model (EPISKIN-SM^TM)). The possible skin irritation potential of EDTA-Cu(NH4)2 was tested through topical application for 15 minutes.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

Batch CFC-10635 (555H1011) of EDTA-Cu(NH4)2 was a clear blue liquid with a purity of ~55%. EDTA-Cu(NH4)2 was applied undiluted (25 µl) directly on top of the skin tissue for 15 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with EDTA-Cu(NH4)2 compared to the negative control tissues was 11%. Since the mean relative tissue viability for EDTA-Cu(NH4)2 was below 50% after 15 minutes treatment it is considered to be irritant.

The positive control had a mean cell viability of 4% after 15 minutes exposure. The absolute mean OD₅₇₀(optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 8%, indicating that the test system functioned properly.

Finally, it is concluded that this test is valid and that EDTA-Cu(NH4)2 is irritant in the in vitro skin irritation test under the experimental conditions described in this report.