Cobalt wolframate

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: Fully Guideline- and GLP-compliant

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: Commission Directive 2001/59/EC (6 August 2001)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD-Guideline 437, "Bovine Corneal Opacity and Permeability Test Method (BCOP) ", 07 September 2009

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EPA (1996) EPA737-B-96-001

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: UN (2007) GHS

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

4-amino-2-methylpyrimidine-5-methylamine

EC Number:

202-384-7

EC Name:

4-amino-2-methylpyrimidine-5-methylamine

Cas Number:

95-02-3

IUPAC Name:

5-(aminomethyl)-2-methylpyrimidin-4-amine

Details on test material:

Name of test material (as cited in study report): Cobalt wolframate
Chemical name: Cobalt wolframate
Molecular formula: CoWO4
Batch No.: 750702_20100329
CAS No.: 10101-58-3
Appearance: Darb blue powder
Solubility: Not soluble in water.
pH: -
Conditions of storage: At room temperature.
Stability at conditions of storage: Stable.
Date of expiry: 31 May 2015

Test animals / tissue source

Species:

other: invitro

Details on test animals or tissues and environmental conditions:

Eyes were collected by slaughterhouse employees and were enucleated as soon as possible after death. To prevent exposure of the eyes to potentially irritant substances, the slaughterhouse employees did not use detergent when rinsing the head of the animal.
Eyes were immersed completely in cold HBSS+ in a container. To minimize the risk of contamination 100 IU/ml penicillin and streptomycin was added to the HBSS and for transport the container was kept in a cold box with cooling packs.
Collection of the eyes and use of corneas occured on the same day. The time interval between collection and use was kept as short as possible and recorded. All eyes used in the assay were from the same group of animals of one slaughterhouse.
After arrival at the laboratory, the eyes were carefully visually examined for defects including increased opacity, scratches and neovascularization. Only corneas from eyes free of such defects were used. All corneas were dissected with a 2 to 3 mm rim of sclera with care to avoid damage to the corneal epithelium and endothelium. Isolated corneas were mounted in cornea holders that consist of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea. Both chambers (posterior chamber first) were filled with pre-warmed cEMEM. The device was then equilibrated at 32 °C +/-1 °C for at least one hour in an air incubator to achieve normal metabolic activity.

Test system

Vehicle:

water

Amount / concentration applied:

The test substance was administered at a concentration of 20 % (w/v) in deionised water (sterile). The test substance preparation was a suspension.

Duration of treatment / exposure:

Bovine Corneal Opacity and Permeability Test Method:
• 1 hour equilibration
• 4 hours test substance incubation
• 1.5 hours fluorescein incubation

Observation period (in vivo):

n.a

Number of animals or in vitro replicates:

Three cornas were used for each treatment (test substance, negative and positive control). As negative control deionised sterile water and as positive control 20% Imidazole were used.

Details on study design:

Bovine corneas were isolated and mounted in cornea holders and equilibrated for one hour (at 31.1 °C – 33.0 °C) to achieve normal metabolic activity. After exclusion of corneas which did not achieve quality criteria, the corneas were distributed into groups (3 per group) and exposed to 750 µl test substance preparation (suspension), the negative control and the positive control for 4 hours. Incubation temperature was 31.1 °C – 33.0 °C. After the exposure period substances were removed from the anterior chamber and the epithelium was washed eight times with EMEM+ to determine the effectiveness of rinsing acidic or alkaline materials and to remove substance residues. cEMEM was used as a final rinse to ensure removal of the phenol red from the anterior chamber prior to the opacity measurement. Both chambers were then refilled with fresh cEMEM. Opacity was determined by the amount of light transmission through the cornea. Corneal opacity was measured quantitatively in Lux with the aid of an opacitometer-kit BASF-OP2.0, which was calibrated with a standard filter set before the corneas were measured. 1 ml sodium fluorescein solution was added to the anterior chamber of the cornea holder and then incubated in a horizontal position for 90 minutes. Incubation temperature was 31.1 °C – 33.0 °C. Permeability was determined by the amount of sodium fluorescein dye that penetrated all corneal cell layers. The amount of sodium fluorescein that crossed into the posterior chamber was quantitatively measured with the aid of a Bio-Tek EL800 microtiter plate reader at 490 nm. For measurement 360 µl of cEMEM from the posterior chamber were transferred into the wells of a 96-well microtiter plate (triplicates). Data were recorded as OD490 values which were equivalent to the OD490 values based upon a visible light spectrophotometer using a standard 1 cm path length. To proof that the measurement was performed in the linear range wells containing 8 concentrations (ranging from 12.5 µg/ml to 0.78 µg/ml) of fluorescein solutions were additionally prepared.

Results and discussion

In vivo

Results

Irritation parameter:

other: Mean IVIS

Basis:

mean

Time point:

other: Endpoint

Score:

17

Reversibility:

other: n.a

Remarks on result:

other: not an ocular corrosive or severe irritant

Irritant / corrosive response data:

The mean IVIS of the three substance treated bovine corneas was 17.0.
The mean IVIS of the three negative control treated bovine corneas was 3.3.
The mean IVIS of the three positive control treated bovine corneas was 105.8.

Other effects:

Assay acceptance criteria according to the OECD Guideline 437:
• The mean IVIS of the three negative control treated bovine corneas was 3.3. This study met the acceptance critera because the mean opacity and the mean permeability of the corneas, treated with deionised water (NC) were 3.3 and 0.000 respectively, are less than the established upper limits for background opacity and permeability values of the historical data of the negative controls.
• The mean IVIS of the three positive control treated bovine corneas was 105.8. This study met the acceptance criteria because the mean IVIS of the corneas, treated with 20 % imidazole (PC) was 105.8, is within the range of the historical data.
The validity of the opacitometer is given since the measurements of the control filters were within the range of the historical data. The validity of the cornea holder is given because the cornea holder control value Io was within the range of the historical data. The correlation coefficient of the fluorescein dilutions was 0.9999. Since the correlation coefficient was > 0.99 the linearity was given.

Applicant's summary and conclusion

Interpretation of results:

other: not an ocular corrosive or severe irritant

Remarks:

Criteria used for interpretation of results: other: OECD Guideline 437

Conclusions:

According to the results of this study the test substance
"Cobalt wolframate" is considered to be not an ocular corrosive or severe irritant.
According to the results of this study and the Directive 2001/59/EC for classification, the test substance "Cobalt wolframate" requires further testing as outlined in the OECD guideline 405.

Executive summary:

Aim and Method

The study was performed to evaluate possible ocular corrosion and severe irritation of"Cobalt wolframate"according tothe OECD Guideline 437 for the testing of chemicals “Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants”,. The BCOP Testhas sufficient accuracy and reliability for the prediction of ocular corrosion or irritancy of test substances.

Bovine corneas are isolated and mounted in cornea holders and equilibrated for at least one hour to achieve normal metabolic activity. After exclusion of corneas which do not achieve certain quality, 3 corneas / substance are distributed into groups and exposed to the test substance, the negative control and the positive control for 4 hours. Then the substances are removed and the corneas are accurately washed. Afterwards the opacity and permeability of each cornea are recorded. Opacity is measured quantitatively with the aid of an opacitometer. Permeability is determined by the amount of sodium fluorescein dye that penetrates all cornea layers. For this purpose fluorescein solution is filled into anterior chamber followed by an incubation for 90 minutes. The amount of sodium fluorescein that crosses into the posterior chamber is quantitatively measured with a spectrophotometer at OD₄₉₀. Using opacity and permeability data an in vitro irritancy score (IVIS) is calculated. If the IVIS of the controls do not meet the acceptance criteria the study is invalid and will be repeated.

• Investigations performed were in conformance with

OECD-Guideline 437, Bovine Corneal Opacity and Permeability Test Method (BCOP), 7^thof September 2009.

“The OECD-Principles of Good Laboratory Practice”, OECD Environment Health and Safety Publications, Series on Principles of Good Laboratory Practice and Compliance Monitoring No. 1,1998.

 U.S.EPA (1996). Label Review Manual: 2^ndEdition. EPA737-B-96-001.,:Environmental Protection Agency.

 EU (2001). Commission Directive 2001/59/EC ofadapting to technical progress for the 28^thtime Council Directive 67/548/EEC on the approximation of the laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances. Official Journal of the European Communities

L255:1-333.

UN (2007). Globally Harmonized System of Classification and Labelling of Chemicals (GHS).&: United Nations Publications.

Results

The validity of the opacitometer is given since the measurements of the control filters were within the range of the historical data.

The validity of the cornea holder is given because the cornea holder control value I_owas within the range of the historical data.

The mean opacity and the mean permeability of the corneas, treated with deionised water (NC) were 3.3 and 0.000 respectively, which are less than the established upper limits for background opacity and permeability values of the historical data of the negative controls.

The mean IVIS of the corneas, treated with 20 % imidazole (PC) was 105.8, which is within the range of the historical data.

The control substances were in the required ranges, thus demonstrating the validity of the experiment.

Thecorrelation coefficientof the fluorescein dilutions was0.9999. Since the correlation coefficient was > 0.99 the linearity was given.

CONCLUSION

The IVIS of"Cobalt wolframate"was 17.1. Furthermore, no important increase in opacity and no increase in permeability was observed.