Copper dichromium tetraoxide

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental work was both started and completed on 25 July 2017.

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 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)

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)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Copper dichromium tetraoxide

EC Number:

234-634-6

EC Name:

Copper dichromium tetraoxide

Cas Number:

12018-10-9

Molecular formula:

Cr2CuO4

IUPAC Name:

dichromium(3+) copper(2+) tetraoxidandiide

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Identification: Copper dichromium tetraoxide
CAS Number: 12018-10-9
Sample: CuCr2O4
Batch: EX.14502.6
Purity: 88.5%
Physical state/Appearance: Dark blue powder
Expiry Date: 01 January 2023
Storage Conditions: Room temperature in the dark

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

- Source: Local abattoir (by-product from freshly slaughtered animals)
- Characteristics of donor animals: Adult cattle (typically 12 to 60 months old)
- Storage and transport conditions of ocular tissue: The eyes 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.
- Indication of any existing defects or lesions in ocular tissue samples: All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

- Amount applied: 0.75 mL
- Concentration: 20% w/v in sodium chloride 0.9% w/v

Duration of treatment / exposure:

240 minutes

Details on study design:

- Selection and 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 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.
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. The average opacity for all corneas was calculated. Three corneas with opacity values close to the median value of all corneas were allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

- Number of replicates: 3 negative control, 3 positive control, 3 test item.

- Negative control used: Aqueous sodium chloride 0.9% w/v (see 'Any other information on materials and methods' for further details)

- Positive control used: Imidazole (see 'Any other information on materials and methods' for further details)

- Application dose and exposure time: The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item preparation or control items were 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.

- Removal of test substance: At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed three 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.


- Scoring system: In Vitro Irritancy Score (IVIS)

- 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 OD 492 was calculated by subtracting the mean OD 492 of the negative control corneas from the OD 492 value of each treated cornea. The OD 492 value of each treatment group was calculated by averaging the corrected OD 492 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.

- Data interpretation:
The test item was classified according to the following prediction model:

IVIS: CLASSIFICATION
≤ 3:  No category. Not requiring classification to UN GHS or EU CLP
> 3; ≤55: No prediction of eye irritation can be made
>55: Category 1. UN GHS or EU CLP Causes serious eye damage

- 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 during 2015 for this testing facility.  Therefore the In Vitro Irritancy Score should fall within the range of 50.8 to 100.4.

For an acceptable test the following negative control criteria should be achieved:
Aqueous 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 during 2015 for bovine corneas treated with the respective negative control.  When testing solids the negative control limit for opacity should be ≤5.4 and for permeability ≤0.070.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

Test item

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- Corneal epithelium condition: The corneas treated with the test item or negative control item were clear post treatment. The corneas treated with the positive control item were cloudy post treatment.
- Acceptance criteria met for negative control: The negative control gave opacity of ≤5.4 and permeability ≤0.070. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The positive control In Vitro Irritancy Score was not within the range of 50.8 to 100.4. The positive control acceptance criterion was therefore not satisfied and has been reported as a deviation (see 'Any other information on materials and methods').

Any other information on results incl. tables

Corneal Opacity and Permeability Measurement

Individual and mean corneal opacity measurements and individual and mean corneal permeability measurements are given in Table 1.

Table 1:  Individual and Mean Corneal Opacity and Permeability Measurements

Treatment	Cornea Number	Opacity				Permeability (OD)		In Vitro Irritancy Score
		Pre-Treatment	Post-Treatment	Post-Treatment - Pre-Treatment	Corrected Value		Corrected Value
Negative Control	7	3	5	2		0.004
	8	3	4	1		0.039
	9	3	5	2		0.008
				1.7*		0.017 		1.9
Positive Control	10	2	83	81	79.3	2.520°	2.503
	11	3	81	78	76.3	2.100°	2.083
	12	3	98	95	93.3	3.065°	3.048
					83.0**		2.545**	121.2
Test Item	4	3	4	1	0.0	0.018	0.001
	5	5	4	-1	0.0	0.096	0.079
	6	4	7	3	1.3	0.044	0.027
					0.4**		0.036**	1.0

OD = Optical density    * = Mean of the post-treatment - pre-treatment values   = Mean permeability

° = 1 in 5 dilution performed  ** = Mean corrected value    

Corneal Epithelium Condition

The condition of each cornea is given in Table 2.

Table 2:  Corneal Epithelium Condition Post Treatment

Treatment	Cornea Number	Observation Post Treatment
Negative Control	7	Clear
	8	Clear
	9	Clear
Positive Control	10	Cloudy
	11	Cloudy
	12	Cloudy
Test Item	4	Clear
	5	Clear
	6	Clear

In Vitro Irritancy Score

The In Vitro irritancy scores are summarized as follows:

Treatment	In Vitro Irritancy Score
Test Item	1.0
Negative Control	1.9
Positive Control	121.2

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

No category. Not requiring classification to UN GHS or EU CLP.

Executive summary:

The study was conducted to GLP and according to OECD Guideline for the Testing of Chemicals No. 437 (updated 26 July 2013) “Bovine Corneal Opacity and Permeability Assay” and Method B.47 of Commission Regulation (EC) No. 440/2008. The test item is not classified according to GHS criteria. Items not classified for eye irritation or serious eye damage are defined as those that do not meet the requirements for classification as UN GHS/EU CLP Category 1 or 2 (2A or 2B).