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Diss Factsheets

Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-12-09 to 2020-03-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
adopted 18th June 2019
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Bis(glycinato)copper
EC Number:
236-783-2
EC Name:
Bis(glycinato)copper
Cas Number:
13479-54-4
Molecular formula:
C4H8CuN2O4
IUPAC Name:
copper;2-aminoacetate
Test material form:
solid: granular
Details on test material:
Appearance Grey to bluish powder/granule
Homogeneity homogeneous
Storage room temperature (20 ± 5 °C)
Stability stable under storage conditions
CAS No. 13479-54-4
EC-No. 236-783-2
Chemical Class organometallic chelate

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: not specified
Justification for test system used:
This in vitro study was performed in order to evaluate the potential of Copper bisglycinate to evoke skin irritation in a reconstructed human epidermis (RhE) test method. The test method can diskriminate between non-classification and classification in Cat 1 or Cat 2 according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS).
Vehicle:
unchanged (no vehicle)
Remarks:
Tissues wetted with DPBS buffer
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: The test system is a commercially available EpiDerm™-Kit. The EpiDerm™ tissue consists of human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers representing main lipid classes analogous to those found in vivo.
- Tissue batch number(s): Kit: 30851
- Delivery date: 2020-03-10
- Date of initiation of testing: 2020-03-11

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 1 hour after the first application, the inserts were removed from the plates using sterile forceps and rinsed immediately in 1-minute-intervals.
- Observable damage in the tissue due to washing: not observed

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Anthos Reader 2010 Flexi (Anthos Microsysteme GmbH)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: MTT QC assay, 4 hours, n=3: OD (540-570 nm)[1.0 - 3.0], Result: 1.771 ± 0.07
- Barrier function: ET-50 assay, 100 µL 1% Triton-X-100, 4 time-points, n=3, MTT assay; ET-50 [4.77 - 8.72 h];, Results: 5.13 h
- Contamination: Long term antibiotic and antimycotic free culture; no contamination, Result Sterile

NUMBER OF REPLICATE TISSUES: three tissues were used for each treatment

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
-The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after 1h exposure and post-treatment incubation is less than or equal (≤) to 50%.
- The test chemical may be considered as non-irritant to skin in accordance with UN GHS No Category if the tissue viability after 1h exposure and post-treatment incubation is more than (>) 50%.
Control samples:
yes, concurrent negative control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): Tissue 1: 26.4 mg, Tissue 2: 26.1 mg, Tissue 3: 25.1 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL DPBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL SDS solution
- Concentration (if solution): 5%
Duration of treatment / exposure:
1 h
Duration of post-treatment incubation (if applicable):
42 h
Number of replicates:
3

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
tissue 1
Value:
34
Vehicle controls validity:
not examined
Negative controls validity:
valid
Remarks:
OD between 0.8 and 2.8
Positive controls validity:
valid
Remarks:
< 20% of negative control
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
tissue 2
Value:
30.5
Vehicle controls validity:
not examined
Negative controls validity:
valid
Remarks:
OD between 0.8 and 2.8
Positive controls validity:
valid
Remarks:
< 20% of negative control
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
tissue 3
Value:
35.6
Vehicle controls validity:
not examined
Negative controls validity:
valid
Remarks:
OD between 0.8 and 2.8
Positive controls validity:
valid
Remarks:
< 20% of negative control
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: Not reported
- Direct-MTT reduction: not observed
- Colour interference with MTT: not observed

DEMONSTRATION OF TECHNICAL PROFICIENCY: The validity of the skin irritation study at LAUS GmbH was demonstrated in a proficiency study. For this purpose, 10 proficiency chemicals (indicated by the OECD 439 guideline) were tested.
All of the 10 proficiency chemicals were correctly categorized. Therefore, the proficiency of the skin irritation study was demonstrated.


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, OD should be between 0.8 and 2.8, mean OD for the negative control was 1.411
- Acceptance criteria met for positive control: yes, The viability should be < 20% of the negative control, the mean viability was 3.3%
- Acceptance criteria met for variability between replicate measurements: yes, the variability should be < 18%, the variability were 3.0% for the negative control, 0.1% for the positive control and 10.2% for the test item
- Range of historical values if different from the ones specified in the test guideline: No

Any other information on results incl. tables

Historical data

Parameter

Negative Control
(OD)

Positive Control
(% OD compared to

Negative Control)

Substance

DPBS buffer

Sodium Dodecyl Sulphate

Solution 5%

Mean

1.779

4.2%

Standard deviation

0.310

2.9%

Range

0.476 – 2.471

1.7% - 17.1%

Study

1.411

3.3%

Applicant's summary and conclusion

Interpretation of results:
other: based on the conditions used in the present test the substance is at least classified as Category 2, irritant to skin
Conclusions:
In the present test the skin irritation potential of bis(glycinato)copper was evaluated according to OECD guideline 439 (2019). Human keratinocytes, 3 tissues, were exposed to 25 mg test item for 1h. After a postincubation time of 42 h the viability was determined using the MTT assay. The mean viability of three tissues was < 50% (33.4%), thus the test item is considered to be at least irritating to the skin accoding to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System of Classifcation and Labelling of Chemicals (GHS).
Executive summary:

In a dermal irritation study performed in accordance with OECD Guideline 439 (In Vitro Skin Irritation) (2019), bis(glycinato)copper (100% a.i.) was applied to the three-dimensional human epidermis model tissue for an exposure period of 1 h in triplicates. 25 μL of DPBS buffer were topically applied to the epidermal surface in order to improve further contact between the solid and the epidermis. Each approximately 25 mg of the test item were applied to the wetted tissues. The test item was spread to match the surface of the tissue.


After dosing the last tissue the plates were transferred to an incubator for 35 minutes exposure at 37±1°C and 5.0±1% CO2, the tissues were then washed with phosphate buffered saline to remove any residual test material after 60 minutes after the first application. Subsequently the tissue constructs were incubated for 42 h at 37°C. Cytotoxicity (irritancy) was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.


The positive (5% SDS) and negative (DPBS buffer) control gave responses that were within the acceptance criteria and as such demonstrated the validity of the study.


The relative mean tissue viability obtained after 60 minutes treatment with bis(glycinato)copper compared to the negative control tissues was 33.4%. Since the mean relative tissue viability for the test substance was below 50%, Bis(glycinato)copper is identified to be at least irritating to the skin under the described test conditions.


Thus, based on OECD guideline, the test item is considered to be at least irritating to the skin according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System of Classification and Labelling of Chemicals (GHS).