Ashes (residues), vanadium-contg.

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 437 "Bovine Corneal Opacity and Permeability Test Method for Identifiying Ocular Corrosives and Severe Irritants"

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Ashes (residues), vanadium-contg.

EC Number:

282-208-3

EC Name:

Ashes (residues), vanadium-contg.

Cas Number:

84144-87-6

Molecular formula:

Not available

Constituent 2

Reference substance name:

Vanadium concentrate

IUPAC Name:

Vanadium concentrate

Details on test material:

- Name of test material (as cited in study report): vanadium concentrate
- Physical state: white powder
- Lot/batch No.: 14-45810
- Storage condition of test material: room temperature, protected from light

Test animals / tissue source

Details on test animals or tissues and environmental conditions:

PREPERATION OF CORNEAS
The assay uses isolated corneas obtained as a by-product from an abattoir from freshly slaughtered animals
The eyes will be carefully examined for defects and any defective eyes is discarded.
The tissue surrounding the eyeball is carefully pulled away and the cornea is stored in a petri dish containing HBSS. Corneas are then mounted in corneal holders (MC2, Clermont, France) with the endothelial side against the O-ring of the posterior chamber. The anterior chamber is then positioned on top of the cornea and tightened with screws. The chamber of the corneal holder is then filled with RPMI (without phenol red) containing 1 % FBS and 2 mM L-glutamine (complete RPMI). The posterior chamber is always filled first. The corneas are incubated for 1 h at 32 +-1 °C in a water bath.

Test system

Vehicle:

physiological saline

Remarks:

0.8 g of the test item was diluted with 0.9 % NaCl ad 4 ml, to gain a 20 % concentration

Controls:

yes

Amount / concentration applied:

750 µl

Duration of treatment / exposure:

4 h +- 5 min

Observation period (in vivo):

none

Number of animals or in vitro replicates:

- 3 corneas for the test item
- 3 corneas as negative control treated with physiological saline 0.9 % NaCl
- 3 corneas as positive control treated with 20 % imidazole in physiological saline 0.9 % NaCl
The BCOP assay is considered to be valid if the in vitro score obtained with the positive control falls within the two standard deviations of the current historical mean.

Details on study design:

TREATMENT OF THE CORNEAS
After the incubation period, the medium is removed from both chambers and replaced with fresh complete RPMI. An initial opacity measurement is performed on each of the corneas using an opacitometer (MC2, Clermont, France). Three corneas with opacity readings appproximately equivalent to the median opacity of all corneas are selected as negative-control corneas. The opacity of each cornea are read against an air-filled chamber and recorded. Corneas that have an initial opacity reading that is 7 or more units greater or lower then the average opacity from all used corneas are not dosed. The medium is removed from the anterior chamber and replaced with the test item or control.
750 µl of the test item preparation or the control substance are introduced into the anterior chamber. After 4 h +- 5 min incubation at 32 +- 1 °C either the test substance or control substance is removed and the epithelium is washed.

REMOVAL OF TEST SUBSTANCE
- Washing: at least 3x with MEM (containing phenol red). Once the medium is free of test substance, the cornea is finally rinsed with complete RPMI (without phenol red).
- Time after start of exposure: 4h +- 5 min

TREATMENT AFTER REMOVAL OF TEST SUBSTANCE
The anterior chamber is refilled with complete RPMI and an opacity measurement is performed. After the measurement, the medium is removed from both chambers of the holder. The posterior chamber is refilled with fresh complete RPMI. 1 ml of a 5 mg/ml sodium fluorescein solution is added to the anterior chamber and the corneas are incubated for 90 min at 32 +- 1 °C. Then the medium from the posterior chamber is removed and its optical density (OD490) is determined, using a spectrophotometer.

EVALUATION OF RESULTS
The change in opacity for each cornea is calculated by subtracting the initial opacity reading from the final opacity reading. These values are corrected by subtracting from each the average change in opacity observed for the negative-control corneas. The mean opacity value for each treatment is calculated by averaging the corrected opacity values of each cornea for a given treatment.
The mean OD490 for the blank wells is calculated. The mean blank OD490 is subtracted from the OD490 of each well (corrected OD490). Any dilutions that are made to bring the OD490 values into the linear range of the spectrophotometer (OD490 should be less than 1500), are taken into account by multiplying the OD490 value of the dilution by the dilution factor. The final-corrected OD490 of the test artivle and the positive control is calculated by subtracting the average-corrected OD490 of the negative control corneas from the corrected OD490 value of each treated cornea:
Final-corrected OD490 = (OD490 - mean blank OD490) - average-corrected negative control OD490
The mean OD490 value of each treatment group is calculated by averaging the final-corrected OD490 values of the treated corneas for that treatment condition.
The following formula is used to determine the in vitro score:
In vitro score = mean opacity value + (15x mean OD490 value)
The results are evaluated according to Table 1.

SCORING SYSTEM:
see Table 1.

Results and discussion

In vivo

Results

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

in vitro

Time point:

other: 4 h

Score:

9.048

Reversibility:

not specified

Irritant / corrosive response data:

The mean in vitro score was calculated to be 9.048. Therefore the test item was classified as mild irritant. The in vitro score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid.

Any other information on results incl. tables

Table 1: Opacity

Cornea-No.	Test item	Opacity blank value	Opacity post dose	Change of opacity values	Corrected opacity values
1.000	Negative Control	5	3	-2
2.000		3	3	0
3.000		6	4	-2
MV		4.667	3.333	-1.333
4.000	Positive Control Imidazole	6	170	164	165.333
5.000		6	185	179	180.333
6.000		6	153	147	148.333
MV		6.000	169.333	163.333	164.667
7.000	Test item Vanadium concentrate	3	9	6	7.333
8.000		4	14	10	11.333
9.000		3	11	8	9.333
MV		3.333	11.333	8.000	9.333

Table 2: Permeability

Cornea-No.	Test item	OD490	Corrected OD490 values
1.000	Negative Control	0.012
2.000		0.017
3.000		0.033
MV		0.021
4.000	Positive Control Imidazole	2.107	2.086
5.000		2.115	2.094
6.000		2.110	2.089
MV		2.111	2.090
7.000	Test item Vanadium concentrate	0.001	-0.020
8.000		0.008	-0.013
9.000		-0.004	-0.025
MV		0.002	-0.019

Table 3: in vitro Score

Cornea-No.	Test item	Corrected opacity value	Corrected OD490 value	in vitroscore
1.000	Negative Control	-2.000	0.012
2.000		0.000	0.017
3.000		-2.000	0.033
MV		-1.333	0.021
4.000	Positive Control Imidazole	165.333	2.086
5.000		180.333	2.094
6.000		148.333	2.089
MV		164.667	2.090	196.017
7.000	Test item Vanadium concentrate	7.333	-0.020
8.000		11.333	-0.013
9.000		9.333	-0.025
MV		9.333	-0.019	9.048

Applicant's summary and conclusion

Interpretation of results:

irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

According to the evaluation criteria the test item Vanadium concentrate is classified as mild eye irritant.

Executive summary:

In a Bovine Corneal Opacity and Permeabiliy Assay (BCOP) 750 µl of 20% Vanadium concentrate was exposed in vitro to 3 corneas for 4 h. The mean in vitro score was calculated to be 9.048. Therefore, Vanadium concentrate is mildly irritating to the eye.