Bis[N-(2-hydroxyethyl)-N-methylglycinato-N,O,ON]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:

2016-12-126 to 2017-01-19

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: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

29 July, 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

May 30, 2008

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Bis[N-(2-hydroxyethyl)-N-methylglycinato-N,O,ON]copper

EC Number:

300-491-4

EC Name:

Bis[N-(2-hydroxyethyl)-N-methylglycinato-N,O,ON]copper

Cas Number:

93940-93-3

Molecular formula:

C10H20CuN2O6

IUPAC Name:

bis[N-(2-hydroxyethyl)-N-methylglycinato-N,O,ON]copper

Test material form:

solid: particulate/powder

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: human-derived epidermal keratinocytes

Source strain:

other: not applicable

Vehicle:

unchanged (no vehicle)

Remarks:

To ensure good contact with the skin the test item was moistened with 25 µL H2O.

Details on test system:

- Source: MatTek Corporation
- The EpiDerm™ tissue: normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis
- Surface: 0.63 cm2
- Pre-incubation: 60 minutes in the incubator (37 ± 1 °C, 5% CO2, 95% RH) in 6-well plates

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 mg of the test item

Duration of treatment / exposure:

3 min and 60 min

Number of replicates:

2

Test system

Details on study design:

Details of the test procedure used:
- EpiDerm™ tissue of human-derived epidermal keratinocytes was used (MatTek, EPI-200-SCT)
- Conditions of exposure: 37 ± 1 °C, 5% CO2, 95% RH
- Washing: inserts gently rinsed with PBS about 20 times (3 min and 60 min exposure time)
- The test was performed on a total of 4 tissues per dose group, 2 replicates for each treatment period (3 min and 60 min exposure time).
- MTT assay: incubation with 0.3 mL of MTT solution for 60 minutes, 3 minutes and 60 minutes at 37 ± 1 °C, 5% CO2

- Data evaluation:
The following was calculated: Corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after 3 min and 60 min treatment compared to the negative control tissues concurrently treated with Aqua dest (= 100%)

- Description of evaluation criteria:
In step 1
The mean tissue viability (% negative control)
< 50% after 3 min exposure: predicted as corrosive
≥ 50% after 3 min exposure AND < 15% after 60 min exposure: predicted as corrosive (a combination of optional sub-categories 1B and 1C)
≥ 50% after 3 min exposure AND ≥ 15% after 60 min exposure: predicted as Non-Corrosive
In step 2:
The mean tissue viability (% negative control)
< 25% after 3 min exposure: optional Sub-category 1A
≥ 25% after 3 min exposure: a combination of optional Sub-categories 1B and 1C

- Historical data positive control: Relative Tissue Viability [%] (60 min experiment): 6.1%; Rel. Standard Deviation: 1.99%
- Historical data negative control: Mean Absorption (60 min experiment): 1.867; Rel. Standard Deviation: 0.261

- Acceptability of the Assay:
- mean OD570 nm of the two negative control tissues of the 3 min and 60 min treatment period is between 0.8 and 2.8,
- mean relative tissue viability of the two positive control tissues of the 60 min treatment period is < 15%,
- coefficient of variation (CV) (in the range of 20 – 100% viability) between two tissues treated identically is <= 30%..

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

for the 3 min treatment

Run / experiment:

4 tissues per dose group, 2 replicates

Value:

>= 84.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no corrosive effect

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

for the 60 min treatment

Run / experiment:

4 tissues per dose group, 2 replicates

Value:

>= 90.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

no corrosive effect

Other effects / acceptance of results:

The acceptance criteria were met

Any other information on results incl. tables

Pre-experiments:

The MTT-reducing capacity was tested. The mixture turned blue/purple. Therefore the part of absorption due to non-specific reductin of MTT (NSMTT) was determined:

3 min: NSMTT = 1.3 %

60 min: NSMTT = -1.5 %

This means that the test item was washed away almost completely before the addition of MTT solution.

The non-specific colour (NSC living) was determined using MTT-free assay medium:

3 min: NSC = 0.1 %

60 min: NSC = 0.1 %

Since the outcome is < 5 % no correction of the results was necessary.

Main experiment

Summary results after 3 min and 60 min exposure:

		Negative control		Test item		Positive control
Exposure		1	2	1	2	1	2
3 min	Mean OD 570 (blank corrected)	1.984	1.706	1.809	1.256	0.055	0.141
	Total mean OD570 (blank corrected)	1.845		1.533		0.098
	Mean relative tissue viability (%)	100		83.1		5.3
	NSMTT-corrected tissue viability (%)	-		81.8		-
	CV (%)	10.7		25.5		62.2
60 min	Mean OD 570 (blank corrected)	1.997	2.241	1.946	1.817	0.101	0.1
	Total mean OD570 (blank corrected)	2.119		1.882		0.1
	Mean relative tissue viability (%)	100		88.8		4.7
	NSMTT-corrected tissue viability (%)	-		90.3		-
	CV (%)	8.1		4.9		0.7

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was found to be not corrosive to skin.

Executive summary:

This in vitro study was performed to assess the corrosivity potential of the test item by means of the Human Skin Model Test. The test was according to OECD guideline 431 and in compliance to GLP.

The test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 3 min and 60 min exposure period and compared to those of the concurrent negative controls.

The test item showed MTT-reducing capability. Since the substance was classified as “non-corrosive” in the main experiment, NSMTT (non specific reduction of MTT) was determined and calculated to 1.3% after 3 min treatment, and – 1.5% after 60 min treatment. Moreover, the test item showed colouring potential after mixture with water and isopropanol in the relevant range of 570 ± 30 nm. Therefore, NSC_living  (non-specific colour of additional viable tissues) was determined and calculated to 0.1% for both treatment periods. Since NSC_livingwas ≤ 5% relative to the negative control of living epidermis, no correction of the results and no determination of NSC_killed was necessary.

The corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after 3 min and 60 min treatment compared to the negative control tissues concurrently treated with Aqua dest (= 100%). The mean relative tissue viability (% negative control) was 84.4 % (NSMTT corrected), which is ≥ 50% after 3 min treatment and was 90.1 % (90.1%, NSMTT-corrected) ≥ 15 % after 60 min. Therefore it is concluded that the substance shows no corrosive effects.