Copper glycinate sulfate

Eye irritation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

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

Test guideline 2

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

Test guideline 3

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)

Test material

Test material information

Constituent 1

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

Test animals / tissue source

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

Test system

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.

Results and discussion

In vitro

Results

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

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.