Copper diformate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-10-08

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

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:

2017

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)

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Guidance Document on ‘The Collection of Tissues for Historical Evaluation and Collection of Data’. Series on Testing and Assessment, No. 160.

Version / remarks:

2018

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Copper diformate

EC Number:

208-865-8

EC Name:

Copper diformate

Cas Number:

544-19-4

Molecular formula:

CH2O2.1/2Cu

IUPAC Name:

copper diformate

Test material form:

solid: particulate/powder

Details on test material:

blue in color

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

- Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals.
- Eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL).
- They were transported to the test facility over ice packs on the same day of slaughter.
- The corneas were prepared immediately on arrival.
- All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): Approximately 0.8783 g of the solid test item was found to adequately cover the corneal surface.
- Concentration (if solution):

VEHICLE
- Amount(s) applied (volume or weight with unit): 0.75 ml
- Concentration (if solution): used as supplied


POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 0.75 ml
- Concentration: Imidazole, was used as a 20% w/v solution in sodium chloride 0.9% w/v


Treatment of corneas
- The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea

Duration of treatment / exposure:

240 min at 32 ± 1 ºC

Duration of post- treatment incubation (in vitro):

90 minutes at 32 ± 1 ºC (sodium fluorescein)

At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed 3 times with fresh complete EMEM (Eagle’s Minimum Essential Medium) containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed.
Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.

Number of animals or in vitro replicates:

3 corneas for the test item
3 corneas as negative controls treated with sodium chloride 0.9%
3 corneas as positive controls treated with imidazole 20% in sodium chloride 0.9%

Details on study design:

Preparation of Corneas:
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 77 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

Selection of Corneas and Opacity Reading:
The medium from both chambers of each holder was replaced with fresh complete EMEM.
A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer.
Three corneas were randomly allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

Treatment of Corneas:
The EMEM was removed from the anterior chamber of the BCOP holder and the test item or control items were applied to the cornea. Approximately 0.8783 g of the solid test item was found to adequately cover the corneal surface. 0.75ml of each control item was applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 240 minutes.
At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed 3 times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed.

Application of Sodium Fluorescein:
Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.

Permeability Determinations:
After incubation the medium in the posterior chamber of each holder was decanted and retained.
360 µL of media representing each cornea was dispensed into the appropriate wells of a pre-labeled 96-well plate. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader.

Histopathology:
The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.

Data Evaluation:
Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.

Opacity Measurement
The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.

Permeability Measurement:
The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.

In Vitro Irritancy Score
The following formula was used to determine the In Vitro Irritancy Score:
In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value)
Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced a response through only one of the two endpoints.

Visual Observation:
The condition of the cornea was visually assessed post treatment.

SCORING SYSTEM:
In Vitro Irritancy Score (IVIS). This was detailed in the study report as follows:
<= 3 = No UN GHS Category
> 3; <= 55 = No UN GHS Prediction can be made
> 55 = Category 1.
An identification of test substances that should be classified as irritating to eyes (UN GHS Category 2 or Category 2A) or test substances that should be classified as mildly irritating to eyes (UN GHS Category 2B) cannot be made.

Major Computerized Systems:
The following computerized systems were used in the study:
Delta Building Monitoring System Version 3.4.0.
Labtech LT-4500 microplate reader and LT-com software Version 7.0.

Criteria for an Acceptable Test:
For an acceptable test the following positive control criterion should be achieved:
20% w/v Imidazole was used for positive control purposes. The test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the historical mean collated during the previous 12 months for this testing facility.
For an acceptable test the following negative control criteria should be achieved:
Sodium chloride 0.9% w/v was used for negative control purposes. The test was acceptable if the negative control produced an In Vitro Irritancy Score which is less than or equal to the upper limit for background opacity and permeability values calculated from the previous 12 months data for this testing facility.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

71

Negative controls validity:

valid

Remarks:

IVIS: 0.7

Positive controls validity:

valid

Remarks:

IVIS: 109.8

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

Corneal Epithelium Condition:
- The corneas treated with the test item were cloudy with opaque blue patches post treatment.
- The corneas treated with the negative control item were clear post treatment.
- The corneas treated with the positive control item were cloudy post treatment.

The positive control In Vitro Irritancy Score was within the acceptance range. The positive control acceptance criterion was therefore satisfied.
The negative control gave opacity and permeability values below the established upper limits. The negative control acceptance criteria were therefore satisfied.

Any other information on results incl. tables

Table 1: Individual and mean corneal opacity and permeabiltiy measurements

Treatment	Cornea Number	Opacity				Permeability (492) OD		In Vitro Irritancy Score	Observation post treatment
		Pre-Treatment	Post-Treatment	Post-Treatment-Pre-Treatment	Corrected Value		Corrected Value
Negative Control	2	3	3	0		0.001			clear
	3	3	4	1		0.001			clear
	6	3	4	1		0.001			clear
				0.7*		0.001*		0.7
Positive Control	9	5	92	87	86.3	1.655	1.654		cloudy
	10	5	78	73	72.3	2.490	2.489		cloudy
	11	2	81	79	78.3	2.010	2.009		cloudy
					79.0*		2.051*	109.8
Test Item	12	4	69	65	64.3	0.062	0.061		Cloudy with opaque blue patches
	14	3	97	94	93.3	0.007	0.006		Cloudy with opaque blue patches
	15	4	59	55	54.3	0.003	0.002		Cloudy with opaque blue patches
					70.7*		0.023*	71.0

* = mean value

Applicant's summary and conclusion

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

The test item causes serious eye damage accordingly to category 1. CLP.