Cobalt molybdate

Eye irritation

Administrative data

Endpoint:

eye irritation

Remarks:

other: in-vitro assay

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Comparable to GLP-guideline study with acceptable restrictions: limited documentation; no scientific justification is given why the test substance was tested neat by direct application. The conclusion "A mean IVIS of -0.3690 was calculated, corresponding to a classification as not eye irritant." is not correct according to regulatory acceptance in the EU. The BCOP test is an in-vitro test method that can be used to classifiy substances as "ocular corrosives and severe irritants". For identifying non-severe irritants and non-irritants, in-vivo testing needs to take into account.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 437 dated Sept. 2009

Deviations:

yes

Remarks:

- limited documentation

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Kaiser-Friedrich-Straße 7, 55113 Mainz, Germany

Test material

Test material information

Constituent 1

Reference substance name:

Cobalt molybdate

EC Number:

237-358-4

EC Name:

Cobalt molybdate

Cas Number:

13762-14-6

Molecular formula:

CoMoO4

IUPAC Name:

λ²-cobalt(2+) dioxomolybdenumbis(olate)

Details on test material:

- Name of test material (as cited in study report): cobalt molybdenum oxide
- Physical state: dark blue powder
- Analytical purity: 99%
- Batch No.: EH 110005/1
- Expiration date of the batch: June 2013
- Storage condition of test material: room temperature

Test animals / tissue source

Species:

other: cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

TEST METHOD
The bovine corneal opacity and permeability (BCOP) test is an in-vitro test method used to classify substances as “ocular corrosives and severe irritants”. The potential of a test substance to cause ocular corrosivity or severe irritancy is measured by its ability to induce opacity and increased permeability in an isolated bovine cornea. The opacity and permeability assessments of the cornea are combined to derive an in-vitro irritancy score (IVIS), which is used to classify the irritancy level of the test substance.

IDENTIFICATION OF THE SOURCE OF THE EYES, STORAGE AND TRANSPORT CONDITIONS
- Source: slaughterhouse Müller Fleisch GmbH (Enzstraße 2.4, 75217 Birkenfeld, Germany)
- Donor animals: between 12 and 60 months old
- Date and time of eye collection: 13 Apr 2011
- Transport medium and temperature conditions: Hanks´ Balanced Salt Solution (HBSS)

PREPARATION OF THE EYES (BEFORE EXPOSURE)
- Eyes free of defects (scratches, neovascularisation): yes
- Dissection of the eyes and treatment: Corneas were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in cornea holders.
- Type of cornea holder used: no data
- Description of the cornea holder: The cornea holders consist of an anterior and a posterior compartment, which interface with the epithelial and endothelial sides of the cornea.
- Test medium and temperature conditions used in the cornea holder: pre-warmed (32 °C) Eagle´s Minimum Essential Medium (MEM; supplemented with sodium bicarbonate, L-glutamine and 1% fetal calf serum.) without phenol red
- Equilibration time: 1 h at 32 ± 1 °C
- Quality check of the equilibrated corneas: The medium was changed and the baseline opacity for each cornea was recorded. The baseline opacity was measured by placing the holder with the cornea in a spectral photometer and recording the absorption at 570 nm. None of the corneas showed tissue damage.

DETERMINATION OF THE INITIAL OPACITY
- Method: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: number of eyes for the negative control: 3; number of eyes for the positive control: 3

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied in the test: 1039.0 mg, 1091.6 mg and 1068.1 mg

NEGATIVE CONTROL
- Substance: physiol. saline
- Concentration (if solution): 0.9% NaCl solution in deionised water
- Amount(s) applied in the test: 750 µL

POSITIVE SUBSTANCE
- Substance: imidazole
- Concentration: 20% imidazole solution in physiol. saline
- Amount(s) applied in the test: 750 µL

Duration of treatment / exposure:

4 h at 32 °C

Observation period (in vivo):

not applicable

Number of animals or in vitro replicates:

number of eyes for the test substance: 3

Details on study design:

TEST CONDITIONS
- Short description of the method used: open-chamber method: The glass window from the anterior chamber was removed prior to treatment. The controls or test substance were applied directly to the epithelial surface of the cornea. After dosing, the glass window was replaced on the anterior chamber to recreate a closed system.

POST-EXPOSURE TREATMENT
- Removal of the test substance: The test substance was removed from the anterior chamber and the epithelium washed with medium
- Medium for washing the corneas: MEM containing phenol red
- Medium for final rinsing: MEM without phenol red

DETERMINATION OF THE FINAL OPACITY
- Method: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer.
- Time of determination: Directly after refilling fresh MEM without phenol red in the anterior chamber, the final opacity was measured.

DETERMINATION OF THE CORNEAL PERMEABILITY:
- Method: Sodium fluorescein solution was added to the anterior chamber of the cornea holder while the posterior chamber was filled with fresh medium. The amount of sodium fluorescein that crosses into the posterior chamber was quantitatively measured via UV/VIS spectrophotometry at 490 nm recorded as optical density (OD490).
- Amount and concentration of the dye: 1 mL sodium fluorescein solution (5 mg/mL; dissolved in PBS)
- Incubation time: x min at y °C: 90 ± 5 min at 32 ± 1 °C

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

other: opacity

Basis:

mean

Remarks:

out of all 3 eyes

Time point:

other: 4 h

Score:

0.205

Reversibility:

other: not applicable

Remarks on result:

other: test substance

Results 2

Irritation parameter:

other: permeability

Basis:

mean

Remarks:

out of all 3 eyes

Time point:

other: 4 h

Score:

-0.004

Reversibility:

other: not applicable

Remarks on result:

other: test substance

Results 3

Irritation parameter:

other: IVIS

Basis:

mean

Remarks:

out of all 3 eyes

Time point:

other: 4 h

Score:

-0.369

Reversibility:

other: not applicable

Remarks on result:

other: test substance

Results 4

Irritation parameter:

other: opacity

Basis:

mean

Remarks:

out of all 3 eyes

Time point:

other: 4 h

Score:

0.333

Reversibility:

other: not applicable

Remarks on result:

other: negative control

Results 5

Irritation parameter:

other: permeability

Basis:

mean

Remarks:

out of all 3 eyes

Time point:

other: 4 h

Score:

0.066

Reversibility:

other: not applicable

Remarks on result:

other: negative control

Results 6

Irritation parameter:

other: IVIS

Basis:

mean

Remarks:

out of all 3 eyes

Time point:

other: 4 h

Score:

1.323

Reversibility:

other: not applicable

Remarks on result:

other: negative control

Results 7

Irritation parameter:

other: opacity

Basis:

mean

Remarks:

out of all 3 eyes

Time point:

other: 4 h

Score:

49.255

Reversibility:

other: not applicable

Remarks on result:

other: positive control

Results 8

Irritation parameter:

other: permeability

Basis:

mean

Remarks:

out of all 3 eyes

Time point:

other: 4 h

Score:

2.983

Reversibility:

other: not applicable

Remarks on result:

other: positive control

Results 9

Irritation parameter:

other: IVIS

Basis:

mean

Remarks:

out of all 3 eyes

Time point:

other: 4 h

Score:

93.671

Reversibility:

other: not applicable

Remarks on result:

other: positive control

Any other information on results incl. tables

Data evaluation

Calculation of opacity value: Opacity was calculated from the measured absorption at 570 nm.

O=1/10^(-A)

O= Opacity

A= Absorption at 570 nm

Correction of measured absorption at 490 nm: As cuvettes with a pathlength of 0.2 cm were used in the measurement of the Fluorescein-Na solution in the spectral photometer, the pathlength must be corrected to 1 cm. Coefficient: 1/0.2=5

All absorptions were multiplied with this coefficient.

Calculation of IVIS (in-vitro irritancy score)

For the negative control: IVIS= mean opacity value + (15x mean corrected OD490 value)

For the test substance and the positive control: IVIS= (opacity - opacity (negative control) + [15x (corr. OD490 - mean corr. OD490 negative control).

Table 1: Absorption and opacity values 

Parameter	Negative control			Test substance			Positive control
Absorption before exposition	0.1399	0.1525	0.1113	0.1509	0.1801	0.1504	0.1842	0.1646	0.2069
Absorption after exposition	0.2200	0.2820	0.1816	0.1851	0.2362	0.2346	1.5811	1.8850	1.5742
Opacity before exposition	1.3799	1.4208	1.2922	1.4154	1.5139	1.4256	1.5282	1.4607	1.6101
Opacity after exposition	1.6594	1.9144	1.5192	1.5313	1.7228	1.7163	38.1132	76.7361	37.5155
Opacity difference	0.2795	0.4936	0.2270	0.1159	0.2089	0.2907	36.5850	75.2754	35.9054

Mean opacity difference of the negative control is 0.3334.

 

Table 2: Optical density at 490 nm

	Negative control			Test substance			Positive control
Meas.	0.0129	0.0140	0.0128	0.0126	0.0145	0.0028	0.4596	0.6091	0.7609
Corr	0.0643	0.0698	0.0639	0.0632	0.0724	0.0142	2.2979	3.0456	3.8044
Mean	0.0660			-
Corr. – Mean negative control				-0.0028	0.0064	-0.0518	2.2319	2.9796	3.7384

 

In order to correct the path length, a factor of 5 was taken into account when calculating the IVIS.

 

Table: 3: IVIS values

IVIS was calculated using the values in tables 1 and 2 and the equation stated before:

	IVIS	Mean IVIS
Negative control	1.2440	1.3230
	1.5406
	1.1855
Test substance	-0.2595	-0.3690
	-0.0285
	-0.08197
Positive control	69.7301	93.6710
	119.6360
	91.6480

 

- Evaluation criteria:

Test substance with an IVIS ≥ 55.1 was regarded as severe irritant/corrosive and labelled Category 1/R41.

 

- Acceptance criteria:

OECD 437: “A test is considered acceptable if the positive control gives an IVIS that falls within two standard deviations of the current historical mean, which is to be updated at least every three months, or each time an acceptable test is conducted in laboratories where tests are conducted infrequently (i.e., less than once a month). The negative or solvent/vehicle control responses should result in opacity and permeability value that less than the establisher upper limits for background opacity and permeability values for bovine corneas treated with the respective negative or solvent/vehicle control.”

Applicant's summary and conclusion

Interpretation of results:

other: not corrosive

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

There is regulatory acceptance in the EU that a substance can be considered as severe eye irritant (Serious eye damage Category 1/R41) based on a positive result in one of the available in-vitro test methods e.g. the Bovine Corneal Opacity and Permeability (BCOP) test. Negative in-vitro corrosivity responses are not conclusive with respect to non-classification or classification as irritant (Category 2/R36) and shall therefore be subject to further evaluation.