Copper glycinate sulfate

Endpoint summary

Description of key information

Skin irritation: according a read-across approach, copper monoglycinate sulfate is expected to be classified as 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).

Eye irritation: according to a read-across approach, copper monoglycinate sulfate is not expected to be irritating to the eye according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System of Classification and Labelling of Chemicals (GHS).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

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

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 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

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

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%

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).

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

(Q)SAR

Adequacy of study:

supporting study

Study period:

2020

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a (Q)SAR model, with limited documentation / justification, but validity of model and reliability of prediction considered adequate based on a generally acknowledged source

Justification for type of information:

1. SOFTWARE
OECD QSAR Toolbox 4.4.1
2. MODEL (incl. version number)
Version 4.4.1
3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
Molecular Structure of chemical formula: C4H8CuN2O4
4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
For detailed information please refer to the attached justification section
5. APPLICABILITY DOMAIN
For detailed information please refer to the attached justification section
6. ADEQUACY OF THE RESULT
For detailed information please refer to the attached justification section

Qualifier:

no guideline followed

Principles of method if other than guideline:

Skin irritation/corrosion alerts gathered from QSAR Toolbox

GLP compliance:

no

Irritation / corrosion parameter:

other: QSAR prediction

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

other: QSAR prediction: BfR exclusion rules: Based on physico-chemical properties of the substance the manganese monoglycinate sulfate is not considered to be corrosive to the skin.

Interpretation of results:

other: QSAR prediction

Conclusions:

Using the molecular structure of bis(glycinato)copper in the QSAR prediction profiler 'BfR exclusion rules' revealed that the substance is not considered to be corrosive to the skin based on its physico-chemical properties.

Executive summary:

There are no data about the corrosivity to the skin of bis(glycinato)copper. Hence, a QSAR prediction was performed using the OECD QSAR Toolbox and the profiler 'BfR exclusion rules'. Of the rules available, three were met for bis(glycinato)copper, namely a low LogKow (-3.21), a predicted high melting point (decomp. > 200°C) and a low lipid solubility < 0.01 g/kg.

Thus, the substance is not considered to be corrosive.

Reference 3

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

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

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across hypothesis is based on transformation of the target and source substances to common compounds (scenario 1 of the RAAF). The target substance copper monoglycinate sulfate and the source substances copper sulfate and copper bisglycinate consist of the Cu2+ cation and the respective anion. The amino acid glycine is constituent of both the target substance copper monoglycinate sulfate and the source substance copper bisglycinate.
It is generally accepted that the Cu2+ cation (as measure for dissolved copper species) is the determining factor for toxicity and ecotoxicity, but not sulfate or glycine.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target substance copper monoglycinate sulfate is a chelate-complex which consists of the divalent copper ion as centre-ion and glycine as ligand. The remaining sulfate group stabilizes the center ion within the complex.
Copper monoglycinate sulfate and the source substance copper sulfate are ionic and consist of the Cu2+ cation and the respective anions. It is generally accepted that the copper cation is the determining factor for toxicity and ecotoxicity. Therefore, this read-across approach is based on the assumption that the metal cation of both the target and the source substance, copper, is the relevant component for assessment of toxicity and ecotoxicity.
The anion of the target substance is the essential amino acid glycine and the sulfate anion. In the source substance, it is the sulfate anion. These anions are not considered as (eco)toxicologically relevant at the given concentrations.
Please refer to the justification for read-across analogue approach in Chapter 13.2 for more detailed information.
3. ANALOGUE APPROACH JUSTIFICATION
Please refer to the justification for read-across analogue approach in Chapter 13.2 for more detailed information.
4. DATA MATRIX
Please refer to the justification for read-across analogue approach in Chapter 13.2 for more detailed information.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

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 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

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

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%

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

The target substance copper monoglycinate sulfate is classified as 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).

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. 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).

There are no data about the corrosivity to the skin of bis(glycinato)copper. Hence, a QSAR prediction was performed using the OECD QSAR Toolbox and the profiler 'BfR exclusion rules'. Of the rules available, three were met for bis(glycinato)copper, namely a low LogKow (-3.21), a predicted high melting point (decomp. > 200°C) and a low lipid solubility < 0.01 g/kg. Thus, the substance is not considered to be corrosive.

The target substance copper monoglycinate sulfate is therefore classified as 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).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-12-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

October 2017

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

February 2017

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for the Testing of Chemicals, Series on Testing and Assessment No. 160

Version / remarks:

“GUIDANCE DOCUMENT ON “THE BOVINE CORNEAL OPACITY AND PERMEABILITY (BCOP) AND ISOLATED CHICKEN EYE (ICE) TEST METHODS: COLLECTION OF TISSUES FOR HISTOLOGICAL EVALUATION AND COLLECTION OF DATA ON NON-SEVERE IRRITANTS”; 25. Oct. 2011

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: slaughterhouse Müller Fleisch GmbH, Industriestraße 42, 75217 Birkenfeld, Germany
- Number of animals: not specified
- Characteristics of donor animals (e.g. age, sex, weight): between 12 and 60 months old
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): The eyes were transported to the test facility in Hanks’ Balanced Salt Solution with 1% Penicillin-Streptomycin solution (Penicillin 100 U/mL, Streptomycin 100 µg/mL) in a suitable cooled container within 1 hour and 10 minutes.
- Time interval prior to initiating testing: 1 h
- indication of any existing defects or lesions in ocular tissue samples: only corneas which were free from damages were used
- Indication of any antibiotics used: no

Vehicle:

Hank's balanced salt solution

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): 20 % (w/v)
VEHICLE
- Amount(s) applied (volume or weight with unit): 750 µL

Duration of treatment / exposure:

4 h

Duration of post- treatment incubation (in vitro):

90 min

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS: After the arrival of the corneas, they were examined and only corneas which were free from damages were used. The corneas were excised with a scalpel and cut from the globe with a 2-3 mm ring of sclera around the outside. Each cornea was transferred to a cornea holder in which pre-warmed cMEM (32 ± 1 °C) without phenol red was filled. The holders were then incubated for 1 hour in the incubation chamber at 32 ± 1 °C. After the initial incubation, the medium was completely changed and the baseline opacity for each cornea was recorded.
QUALITY CHECK OF THE ISOLATED CORNEAS: None of the corneas showed tissue damage; therefore, all corneas were used.
NUMBER OF REPLICATES: 3
NEGATIVE CONTROL USED: yes, concurrent vehicle
POSITIVE CONTROL USED: yes, Imidazole, 20 % solution in HBSS
APPLICATION DOSE AND EXPOSURE TIME: 750 µL each for 4 h
TREATMENT METHOD: closed chamber for controls. Open chamber for test item suspension.
POST-INCUBATION PERIOD: yes, 90 min
REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: thorough rinsing
METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD492)
SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
DECISION CRITERIA: as indicated in the TG.

Irritation parameter:

in vitro irritation score

Run / experiment:

1-3

Value:

-0.04

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: not reported
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Opacity and Permeability Values

The illuminance (unit: LUX) values which were measured before and after exposure are given in the following table:

Table 1: Illuminance Values. Rep. = Replicate

Parameter	Negative Control			Test Item			Positive Control
	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.
(I) Measured values before exposure	1051	1056	1057	1027	1032	1039	1017	1027	1028
(I) Measured values after exposure	1039	966	1026	987	987	985	350	389	366

The values in the following present the calculated opacity values, according to evaluation.

Table 2: Opacity Values Negative Control. Rep. = Replicate

Parameter	Negative Control
	1. Rep.	2. Rep.	3. Rep.
Opacity before exposure	2.10	1.90	1.86
Opacity after exposure	2.58	5.75	3.11
Opacity Difference	0.48	3.85	1.25
Mean Opacity Difference	1.86

Table 3: Opacity Values Test Item and Positive Control. Rep. = Replicate.

Parameter	Test Item			Positive Control
	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.
Opacity before exposure	3.07	2.86	2.58	3.49	3.07	3.03
Opacity after exposure	4.79	4.79	4.88	85.25	72.75	79.80
Opacity Difference	1.72	1.93	2.30	81.76	69.68	76.77
Opacity Difference corrected	-0.14	0.07	0.44	79.90	67.82	74.91
Mean Opacity Difference corrected	0.13			74.21

For the permeability measurement, three replicates for each treatment group were measured three times. cMEM without phenol red was measured as blank value as well. The optical density values at 492 nm are given in the following tables:

Table 4: Optical density at 492 nm of Blank.

Parameter	cMEM without phenol red
1. Measurement	0.037
2. Measurement	0.037
3. Measurement	0.034
Mean	0.036

Table 5: Optical density at 492 nm of Negative Control, Test Item and Positive Control. Rep. = Replicate. * Note: Two replicates for the positive control were obtained by measurement of a fivefold diluted solution and multiplication of the absorbances with factor 5.

Parameter	Negative Control			Test Item			Positive Control
	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.
1.Measure-ment	0.052	0.053	0.047	0.037	0.040	0.039	1.771	0.638	0.599
2.Measure-ment	0.052	0.055	0.046	0.036	0.045	0.042	1.746	0.690	0.589
3.Measure-ment	0.052	0.054	0.048	0.035	0.045	0.042	1.769	0.685	0.060
1.Measure-ment – blank	0.0160	0.0170	0.0110	0.0010	0.0040	0.0030	1.7350	0.6020	0.5630
2.Measure-ment – blank	0.0160	0.0190	0.0100	0.0000	0.0090	0.0060	1.7100	0.6540	0.5530
3.Measure-ment – blank	0.0160	0.0180	0.0120	-0.0010	0.0090	0.0060	1.7330	0.6490	0.0236
Mean of each replicate	0.0160	0.0180	0.0110	0.0000	0.0073	0.0050	1.7260	0.6350	0.3799
Mean of the 3 replicates	0.0150			--			--
Corrected	--	--	--	-0.0150	-0.0077	-0.0100	1.7110	3.1600*	1.8843*
Corrected mean of the 3 replicates	--			-0.0109			2.2518

IVIS Values

The calculated IVIS for each replicate and the corresponding means are presented in the following table:

Table 6: IVIS

Test Group	IVIS	Mean IVIS	Relative Standard Deviation IVIS
Negative Control HBSS	0.72	2.08	86.23%
	4.12
	1.41
Test Item	-0.36	-0.04	858.13%
	-0.05
	0.29
Positive Control 20% imidazole solution	105.57	107.99	5.91%
	115.22
	103.18

Note: the high relative standard deviations of the IVIS of the negative control and test item are due to mathematical reasons, as the respective means are very small.

Validity

According to the guideline, the test is considered as valid if the positive control causes an IVIS that falls within two standard deviations of the current historical mean.

The mean IVIS of the negative control has to show an IVIS ≤ 3.

The validity criteria and findings are given in the following table:

Table 7: Validity

Parameter	Criterion	Found	Assessment
Mean IVIS of negative control HBSS	≤ 3	2.08	ok
Mean IVIS of positive control 20% imidazole solution	75.63 – 146.38	107.99	ok

Values for negative and positive controls were within the range of historical data of the test facility. Therefore, the test system was acceptable.

Assessment

According to OECD Guideline no. 437 (Oct. 2017), a substance with an IVIS ≤ 3 requires no classification for eye irritation or serious eye damage.

Table 8: Classification Scheme

IVIS	UN GHS
≤ 3	No category
> 3 and ≤ 55	No prediction can be made
> 55	Eye damage Category I

In the negative control, no signs of eye irritation were observed.

The positive control induced serious eye damage, which would be classified as GHS category I.

The test item showed no effects on the cornea of the bovine eye. The calculated mean IVIS (In Vitro Irritancy Score) was -0.04. The experiment is considered as sufficient for the classification of the test item, because all three replicates of the test item lead to the same assessment for the test item.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of this ex vivo study (BCOP), a 20 % suspension of the test item in HBSS did not induce an increase of the corneal opacity and permeability as compared to negative control. The calculated mean in vitro score was -0.04, which corresponds to "no category" according to UN GHS.

Executive summary:

In this ex vivo study according to OECD guideline no. 437 (Oct. 2017), the corneal damage potential of Bis(glycinato)copper was assessed by quantitative measurements of changes in opacity and permeability in a bovine cornea.

Bovine corneas were collected from slaughtered cattle that were between 12 and 60 months old.

The test item was applied onto the cornea of a bovine eye which had been previously incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been measured.

The test item was incubated on the cornea for 4 hours at 32 ± 1 °C. After removal of the test item, opacity and permeability values were measured.

Hank’s Balanced Salt Solution (HBSS) was used as negative control. The negative control showed no irritating effect on the cornea and the calculated mean IVIS (In Vitro Irritancy Score) was 2.08.

20 % imidazole solution was used as positive control. The positive control induced serious eye damage on the cornea and was within two standard deviations of the current historical mean. The calculated mean IVIS was 107.99.

Under the conditions of this study, the test item Bis(glycinato)copper showed no effects on the cornea of the bovine eye. The calculated mean IVIS was -0.04.

According to OECD guideline no. 437 (Oct. 2017), a substance with an IVIS ≤ 3 requires no classification for eye irritation or serious eye damage.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2019-12-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across hypothesis is based on transformation of the target and source substances to common compounds (scenario 1 of the RAAF). The target substance copper monoglycinate sulfate and the source substances copper sulfate and copper bisglycinate consist of the Cu2+ cation and the respective anion. The amino acid glycine is constituent of both the target substance copper monoglycinate sulfate and the source substance copper bisglycinate.
It is generally accepted that the Cu2+ cation (as measure for dissolved copper species) is the determining factor for toxicity and ecotoxicity, but not sulfate or glycine.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target substance copper monoglycinate sulfate is a chelate-complex which consists of the divalent copper ion as centre-ion and glycine as ligand. The remaining sulfate group stabilizes the center ion within the complex.
Copper monoglycinate sulfate and the source substance copper sulfate are ionic and consist of the Cu2+ cation and the respective anions. It is generally accepted that the copper cation is the determining factor for toxicity and ecotoxicity. Therefore, this read-across approach is based on the assumption that the metal cation of both the target and the source substance, copper, is the relevant component for assessment of toxicity and ecotoxicity.
The anion of the target substance is the essential amino acid glycine and the sulfate anion. In the source substance, it is the sulfate anion. These anions are not considered as (eco)toxicologically relevant at the given concentrations.
Please refer to the justification for read-across analogue approach in Chapter 13.2 for more detailed information.
3. ANALOGUE APPROACH JUSTIFICATION
Please refer to the justification for read-across analogue approach in Chapter 13.2 for more detailed information.
4. DATA MATRIX
Please refer to the justification for read-across analogue approach in Chapter 13.2 for more detailed information.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

October 2017

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

February 2017

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for the Testing of Chemicals, Series on Testing and Assessment No. 160

Version / remarks:

“GUIDANCE DOCUMENT ON “THE BOVINE CORNEAL OPACITY AND PERMEABILITY (BCOP) AND ISOLATED CHICKEN EYE (ICE) TEST METHODS: COLLECTION OF TISSUES FOR HISTOLOGICAL EVALUATION AND COLLECTION OF DATA ON NON-SEVERE IRRITANTS”; 25. Oct. 2011

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: slaughterhouse Müller Fleisch GmbH, Industriestraße 42, 75217 Birkenfeld, Germany
- Number of animals: not specified
- Characteristics of donor animals (e.g. age, sex, weight): between 12 and 60 months old
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): The eyes were transported to the test facility in Hanks’ Balanced Salt Solution with 1% Penicillin-Streptomycin solution (Penicillin 100 U/mL, Streptomycin 100 µg/mL) in a suitable cooled container within 1 hour and 10 minutes.
- Time interval prior to initiating testing: 1 h
- indication of any existing defects or lesions in ocular tissue samples: only corneas which were free from damages were used
- Indication of any antibiotics used: no

Vehicle:

Hank's balanced salt solution

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): 20 % (w/v)
VEHICLE
- Amount(s) applied (volume or weight with unit): 750 µL

Duration of treatment / exposure:

4 h

Duration of post- treatment incubation (in vitro):

90 min

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS: After the arrival of the corneas, they were examined and only corneas which were free from damages were used. The corneas were excised with a scalpel and cut from the globe with a 2-3 mm ring of sclera around the outside. Each cornea was transferred to a cornea holder in which pre-warmed cMEM (32 ± 1 °C) without phenol red was filled. The holders were then incubated for 1 hour in the incubation chamber at 32 ± 1 °C. After the initial incubation, the medium was completely changed and the baseline opacity for each cornea was recorded.
QUALITY CHECK OF THE ISOLATED CORNEAS: None of the corneas showed tissue damage; therefore, all corneas were used.
NUMBER OF REPLICATES: 3
NEGATIVE CONTROL USED: yes, concurrent vehicle
POSITIVE CONTROL USED: yes, Imidazole, 20 % solution in HBSS
APPLICATION DOSE AND EXPOSURE TIME: 750 µL each for 4 h
TREATMENT METHOD: closed chamber for controls. Open chamber for test item suspension.
POST-INCUBATION PERIOD: yes, 90 min
REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: thorough rinsing
METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD492)
SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
DECISION CRITERIA: as indicated in the TG.

Irritation parameter:

in vitro irritation score

Run / experiment:

1-3

Value:

-0.04

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: not reported
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Opacity and Permeability Values

The illuminance (unit: LUX) values which were measured before and after exposure are given in the following table:

Table 1: Illuminance Values. Rep. = Replicate

Parameter	Negative Control			Test Item			Positive Control
	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.
(I) Measured values before exposure	1051	1056	1057	1027	1032	1039	1017	1027	1028
(I) Measured values after exposure	1039	966	1026	987	987	985	350	389	366

The values in the following present the calculated opacity values, according to evaluation.

Table 2: Opacity Values Negative Control. Rep. = Replicate

Parameter	Negative Control
	1. Rep.	2. Rep.	3. Rep.
Opacity before exposure	2.10	1.90	1.86
Opacity after exposure	2.58	5.75	3.11
Opacity Difference	0.48	3.85	1.25
Mean Opacity Difference	1.86

Table 3: Opacity Values Test Item and Positive Control. Rep. = Replicate.

Parameter	Test Item			Positive Control
	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.
Opacity before exposure	3.07	2.86	2.58	3.49	3.07	3.03
Opacity after exposure	4.79	4.79	4.88	85.25	72.75	79.80
Opacity Difference	1.72	1.93	2.30	81.76	69.68	76.77
Opacity Difference corrected	-0.14	0.07	0.44	79.90	67.82	74.91
Mean Opacity Difference corrected	0.13			74.21

For the permeability measurement, three replicates for each treatment group were measured three times. cMEM without phenol red was measured as blank value as well. The optical density values at 492 nm are given in the following tables:

Table 4: Optical density at 492 nm of Blank.

Parameter	cMEM without phenol red
1. Measurement	0.037
2. Measurement	0.037
3. Measurement	0.034
Mean	0.036

Table 5: Optical density at 492 nm of Negative Control, Test Item and Positive Control. Rep. = Replicate. * Note: Two replicates for the positive control were obtained by measurement of a fivefold diluted solution and multiplication of the absorbances with factor 5.

Parameter	Negative Control			Test Item			Positive Control
	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.
1.Measure-ment	0.052	0.053	0.047	0.037	0.040	0.039	1.771	0.638	0.599
2.Measure-ment	0.052	0.055	0.046	0.036	0.045	0.042	1.746	0.690	0.589
3.Measure-ment	0.052	0.054	0.048	0.035	0.045	0.042	1.769	0.685	0.060
1.Measure-ment – blank	0.0160	0.0170	0.0110	0.0010	0.0040	0.0030	1.7350	0.6020	0.5630
2.Measure-ment – blank	0.0160	0.0190	0.0100	0.0000	0.0090	0.0060	1.7100	0.6540	0.5530
3.Measure-ment – blank	0.0160	0.0180	0.0120	-0.0010	0.0090	0.0060	1.7330	0.6490	0.0236
Mean of each replicate	0.0160	0.0180	0.0110	0.0000	0.0073	0.0050	1.7260	0.6350	0.3799
Mean of the 3 replicates	0.0150			--			--
Corrected	--	--	--	-0.0150	-0.0077	-0.0100	1.7110	3.1600*	1.8843*
Corrected mean of the 3 replicates	--			-0.0109			2.2518

IVIS Values

The calculated IVIS for each replicate and the corresponding means are presented in the following table:

Table 6: IVIS

Test Group	IVIS	Mean IVIS	Relative Standard Deviation IVIS
Negative Control HBSS	0.72	2.08	86.23%
	4.12
	1.41
Test Item	-0.36	-0.04	858.13%
	-0.05
	0.29
Positive Control 20% imidazole solution	105.57	107.99	5.91%
	115.22
	103.18

Note: the high relative standard deviations of the IVIS of the negative control and test item are due to mathematical reasons, as the respective means are very small.

Validity

According to the guideline, the test is considered as valid if the positive control causes an IVIS that falls within two standard deviations of the current historical mean.

The mean IVIS of the negative control has to show an IVIS ≤ 3.

The validity criteria and findings are given in the following table:

Table 7: Validity

Parameter	Criterion	Found	Assessment
Mean IVIS of negative control HBSS	≤ 3	2.08	ok
Mean IVIS of positive control 20% imidazole solution	75.63 – 146.38	107.99	ok

Values for negative and positive controls were within the range of historical data of the test facility. Therefore, the test system was acceptable.

Assessment

According to OECD Guideline no. 437 (Oct. 2017), a substance with an IVIS ≤ 3 requires no classification for eye irritation or serious eye damage.

Table 8: Classification Scheme

IVIS	UN GHS
≤ 3	No category
> 3 and ≤ 55	No prediction can be made
> 55	Eye damage Category I

In the negative control, no signs of eye irritation were observed.

The positive control induced serious eye damage, which would be classified as GHS category I.

The test item showed no effects on the cornea of the bovine eye. The calculated mean IVIS (In Vitro Irritancy Score) was -0.04. The experiment is considered as sufficient for the classification of the test item, because all three replicates of the test item lead to the same assessment for the test item.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of this ex vivo study (BCOP), a 20 % suspension of the test item in HBSS did not induce an increase of the corneal opacity and permeability as compared to negative control. The calculated mean in vitro score was -0.04, which corresponds to "no category" according to UN GHS.

Executive summary:

In an ex vivo study according to OECD guideline no. 437 (Oct. 2017), the corneal damage potential of Bis(glycinato)copper was assessed by quantitative measurements of changes in opacity and permeability in a bovine cornea. Bovine corneas were collected from slaughtered cattle that were between 12 and 60 months old. The test item was applied onto the cornea of a bovine eye which had been previously incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been measured. The test item was incubated on the cornea for 4 hours at 32 ± 1 °C. After removal of the test item, opacity and permeability values were measured. Hank’s Balanced Salt Solution (HBSS) was used as negative control. The negative control showed no irritating effect on the cornea and the calculated mean IVIS (In Vitro Irritancy Score) was 2.08. 20 % imidazole solution was used as positive control. The positive control induced serious eye damage on the cornea and was within two standard deviations of the current historical mean. The calculated mean IVIS was 107.99. Under the conditions of this study, the test item Bis(glycinato)copper showed no effects on the cornea of the bovine eye. The calculated mean IVIS was -0.04. According to OECD guideline no. 437 (Oct. 2017), a substance with an IVIS ≤ 3 requires no classification for eye irritation or serious eye damage.

The target substance copper monoglycinate sulfate is therefore not expected to be irritating to the eye according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System of Classification and Labelling of Chemicals (GHS).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Skin irritation

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).

There are no data about the corrosivity to the skin of bis(glycinato)copper. Hence, a QSAR prediction was performed using the OECD QSAR Toolbox and the profiler 'BfR exclusion rules'. Of the rules available, three were met for bis(glycinato)copper, namely a low LogKow (-3.21), a predicted high melting point (decomp. > 200°C) and a low lipid solubility < 0.01 g/kg.

The target substance copper monoglycinate sulfate is therefore expected to be classified as 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).

 

Eye irritation

In an ex vivo study according to OECD guideline no. 437 (Oct. 2017), the corneal damage potential of Bis(glycinato)copper was assessed by quantitative measurements of changes in opacity and permeability in a bovine cornea. Bovine corneas were collected from slaughtered cattle that were between 12 and 60 months old. The test item was applied onto the cornea of a bovine eye which had been previously incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been measured. The test item was incubated on the cornea for 4 hours at 32 ± 1 °C. After removal of the test item, opacity and permeability values were measured. Hank’s Balanced Salt Solution (HBSS) was used as negative control. The negative control showed no irritating effect on the cornea and the calculated mean IVIS (In Vitro Irritancy Score) was 2.08. 20 % imidazole solution was used as positive control. The positive control induced serious eye damage on the cornea and was within two standard deviations of the current historical mean. The calculated mean IVIS was 107.99. Under the conditions of this study, the test item Bis(glycinato)copper showed no effects on the cornea of the bovine eye. The calculated mean IVIS was -0.04. According to OECD guideline no. 437 (Oct. 2017), a substance with an IVIS ≤ 3 requires no classification for eye irritation or serious eye damage.

The target substance copper monoglycinate sulfate is therefore not expected to be irritating to the eye according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System of Classification and Labelling of Chemicals (GHS).