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Description of key information

Vanadium, oxalate complexes is corrosive to the eye and the pH of a saturated solution is acidic (pH 1.9). Vanadium, oxalate complexes is only manufactured and marked as aqueous solution, and inhalation of the solution can be considered negligible. Hence, vanadium, oxalate complexes solution cannot possess an irritation potential in the respiratory tract.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-11-29 to 2012-11-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study reliable without restrictions
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 435 (In Vitro Membrane Barrier Test Method for Skin Corrosion)
Version / remarks:
adopted 2006-07-19
Deviations:
no
Qualifier:
according to
Guideline:
other: ICCVAM Minimum Performance Standards: In Vitro Membrane Barrier Test Systems for Skin Corrosion, June 23, 2003
Deviations:
no
Qualifier:
according to
Guideline:
other: ICCVAM Recommended Performance Standards for in vitro Test Methods for Skin Corrosion (May 2004)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2009-03-30
Test material information:
Composition 1
Details on test animals and environmental conditions:
Not applicable - Since this is an in vitro study there is no information on test animals.
Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): approx. 500 mg of the test item were dispensed directly atop the bio-barrier.
Duration of treatment / exposure:
60 minutes
Observation period:
not applicable
Number of animals:
not applicable
Details on study design:
TEST SYSTEM
- Test kit:
The test kit (Name: Corrositex™; Supplier: Transia GmbH, 61239 Ober-Mörlen, Germany; Lot No.: CT052112) consists of a bio-barrier (collagen gel) and a chemical detection system separated by a cellulose membrane.
- Preparation of the bio-barrier:
One day prior to testing the bio-barrier matrix was prepared. The bio-barrier powder was solved in the bio-barrier diluent and heated for 20 ± 2 minutes at 68 – 70 °C in a water bath under continuous stirring. The temperature did not exceed 70 °C. The mixture was allowed to cool in the non-heated water bath for another 10 minutes. The mixture was then filled into the membrane holders (200 µL per membrane holder). Air bubbles were avoided. The filled membrane holders were sealed with parafilm and were stored at 2 – 8 °C until further use.

EXPERIMENTAL PERFORMEANCE
- Qualification Test: to test whether the test system is suitable for the test item, i.e. the test substance is detectable by the CDS, approx. 100 mg of the test item were transferred into the “Qualification Test Vial”. The vial was shaken until the solution appeared homogenous, and incubated for at least 1 minute. The colour change was recorded. Since a change in colour was visible in the “Qualification Test Vial”, the test item was considered to be suitable for the next step.
- Categorisation Test: qualified test substance is classified into categories (Category 1: strong acids or bases; Category 2: less strong acids and bases) by a screening test. The category, that a test substance is assigned to, will determine how the breakthrough time (if one occurs) will be interpreted. A test substance is assigned to a category based on its ability to induce a pH change in one of two defined buffers. One buffer is designed for detecting acids, the other for detecting bases.
The test item was categorised according to the pH value method to distinguish between weak and strong acids or bases as described in the Corrositex™ Instruction Manual (1995)*. The pH of the solution containing the test item in a “Category A Vial” and a “Category B Vial” was measured using pH sticks, where the Category A vial contained the acid buffer and Category B contained the base buffer. Test items with a solution pH that is ≤ 5 in the “Category A Vial” and ≥ 9 in the “Category B Vial”, i.e. strong acids or bases, were assigned to Category 1. Test items with a solution pH that is ≥ 5 in the “Category A Vial” and ≤ 9 in the “Category B Vial”, i.e. less strong acids and bases, were assigned to Category 2.
- Classification Test: 7 vials containing the CDS were acclimated at room temperature. A total of 4 vials was used for quadruplicate measurements of the test item, one vial was used as positive control (single measurement), and one vial was used as negative control (single measurement). One vial was used as colour reference for the CDS.
The prepared bio-barriers were placed on top of the CDS vials (not longer than 2 minutes prior to an application) and approx. 500 mg of the test item or 500 µL of the respective controls (positive control: sulfuric acid 95-97% (lot no. K40281631 929, Merck, 64295 Darmstadt, Germany); negative control: citric acid (lot no. 140986235008107, Sigma, 89555 Steinheim, Germany, 10% (w/v) solution in deionised water)) were applied per bio-barrier for 1 hour or 4 hours, depending on the results of the categorisation test. The time of the first colour change or precipitation in the CDS solution, i.e. bio-barrier penetration, was recorded.

INTERPRETATION OF THE RESULTS
Each vial was monitored for 60 minutes or 240 minutes depending on the results of the classification test, and the time of the first observable change in the CDS solution was recorded. The elapsed time between application and penetration of the bio-barrier was determined, and the mean of the quadruplicate measurements was calculated. The test item was categorised according to the criteria in table 1, which can be seen in the field "Any other information on materials and methods incl. tables" below.

ACCEPTABILITY OF THE ASSAY
The results were considered valid if the following acceptance criteria were met:
The test item induces a physical change (colour or precipitation) in the CDS solution in the qualification test, and the test substance is detectable by the CDS.
The negative control does not induce a change (i.e. colour or precipitation) in the CDS solution over 60 minutes in the classification test, and the functional integrity of the bio-barrier is demonstrated.
The positive control induces a change (i.e. colour or precipitation) in the CDS solution in the classification test after 0 – 3 minutes, and the breakthrough time of the positive control is within the acceptable range. The Corrositex™ assay will be accepted if the positive control time falls within ± two standard deviations of the positive control historical mean breakthrough time.

* Reference
- Corrositex™ instruction manual (1995). In Vitro International, 16632 Millikan Avenue, Irvine, CA 92606, USA.
Irritation / corrosion parameter:
other:
Value:
0
Remarks on result:
other:
Remarks:
Basis: other: minutes (mean of 4 replicates). Time point: after 60 minutes of treatment. Remarks: A change of colour of CDS reagent after treatment of bio-barriers with test item was not observed during the test, i.e. for 60 minutes. . (migrated information)
Irritant / corrosive response data:
A change of colour of the CDS reagent after treatment of the bio-barriers with the test item was not observed for 60 minutes in any of the four replicates indicating that it did not break through the bio-barrier. The test item is not corrosive.

HISTORICAL DATA:

Table: Historical range of breakthrough times for the positive control

Historical mean time of first colour change of positive control [minutes]

1.31

Standard deviation [minutes]

0.45

Data of 46 studies performed at Harlan CCR from 2006 until 2012.

QUALIFICATION TEST:

The test item induced a change in colour in the qualification test after 1 minute of incubation. Since a change in colour was visible in the “Qualification Test Vial”, the test item was thus detected by the CDS and considered suitable for the next step.

CATEGORISATION TEST:

Since the pH value of the test item was 5 in the “Category A Vial” and 9 in the “Category B Vial”, the test item does not appear to be a strong acid or base and was assigned to category 2. It was concluded that the required duration of the classification test was 60 minutes.

CLASSIFICATION TEST:

The negative control did not penetrate the bio-barrier, and thus the bio-barrier was considered functionally integer.

The positive control, i.e. a colour change, was detected by the CDS after 1 minute, and thus the breakthrough time of the positive control fell within ± two standard deviations of the positive control historical mean breakthrough time.

A change in the CDS was not observed for 60 minutes in any of the four replicates of the test item indicating that it did not break through the bio-barrier.


Test Group

Time Interval of
Colour Change

UN Packaging Group

R-Sentence

GHSand Regulation (EC) No 1272/2008 (CLP)

Negative Control

Colour change was not observed for 60 minutes

-

-

-

Positive Control

1 minute

I

R35

1A

Test Item

Colour change was not observed for 60 minutes

Non-corrosive

-

-

All validity criteria of the test were met. No other effects were observed in any of the treatments.

 
Interpretation of results:
other: the test item is not corrosive.
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Vanadium, oxalate complexes is not corrosive to skin according to the EC-Commission directive 67/548/EEC and EC-Regulation 1272/2008 and subsequent regulations.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-02-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study reliable without restrictions
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
adopted 2009-09-07
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
2010
Deviations:
no
Qualifier:
according to
Guideline:
other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2009-03-30
Test material information:
Composition 1
Details on test animals or tissues and environmental conditions:
Not applicable - Since this is an in vitro study there is no information on test animals.
Vehicle:
other: 0.9% (w/v) NaCl in deionised water
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
A 20% (w/v) suspension of the test item in the vehicle was prepared using ultrasonic technique.
Duration of treatment / exposure:
240 ± 15 minutes
Observation period (in vivo):
not applicable
Number of animals or in vitro replicates:
not applicable
Details on study design:
COLLECTION OF BOVINE EYES
Freshly isolated bovine eyes from at least 9 month old donor cattle were collected from the abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were transported to the laboratory in Hank’s Ballanced Salt Solution (HBSS) supplemented with streptomycin / penicillin at ambient temperature. The corneae were isolated on the same day after delivery of the eyes, inserted in pre-cooled preservation medium composed of Medium 199 (© Biochrom) supplemented with L-glutamine, Na-bicarbonate and Taurine, and stored in the refrigerator at 2 – 8 °C until the following day. Shortly before use, Dextran was added to the medium.

PREPARATION OF CORNEAE
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. The cornea was carefully removed from the eye using scalpel and rounded scissors.
Each isolated cornea was mounted in a specially designed cornea holder as described in OECD guideline 437, annex III, that consists of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. After the anterior part of the holder was positioned on top of the cornea and fixed in place with screws, both compartments of the holder were filled with complete medium. The posterior compartment was filled first to return the cornea to its natural convex position. Care was taken to assure that no air bubbles were present within the compartments.
For equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath.
At the end of the incubation period, the basal opacity was determined (t0). Each cornea with a value of the basal opacity > 7 was discarded and not used in the test.

OUTLINE OF STUDY
Complete medium was completely removed from the anterior compartment and replaced by the test item, positive control (10% (w/v) Benzalkonium chloride (Sigma, 89555 Steinheim, Germany, lot no. 036K0208) in 0.9% (w/v) NaCl solution in deionised water (saline, produced in-house, lot no. 180113)) or negative control (0.9% (w/v) NaCl in deionised water (produced in-house, lot no. 180113)).
The anterior compartment received the test item suspension or negative or positive control at a volume of 0.75 mL each on the surface of the corneae. Since the test item could not be suspended homogeneously, each 0.75 mL of the so prepared stock was distributed to each cornea. Thereby it was taken care of that the corneae were evenly covered with the test item.
The test item, positive control and negative control were tested in triplicate.
The anterior compartment was then plugged again. The corneae were turned into a horizontal position and slightly rotated to ensure uniform covering of the corneae with the test or control items and were incubated in a water-bath in horizontal position at 32 ± 1 °C for 240 minutes.
After the incubation, the test item or control items, respectively, were rinsed off from the application side with 0.9% (w/v) NaCl in deionised water at least three times or until no visual evidence of the test substance was observed. Fresh cMEM was added into the anterior compartment and opacity was measured (t240).
In the second step of the assay, permeability of the cornea was determined. 1 mL of a Na-fluorescein solution, 0.5 % (w/v) dissolved in HBSS (Hank’s buffered salt solution), was placed in the anterior compartment, replacing the cMEM. Corneae were incubated again in a horizontal position for an additional 90 minutes at 32 ± 1 °C in the water-bath. The optical density of an aliquot of the mixed complete medium from the posterior chamber was measured spectrophotometrically at 490 nm (OD490).

CRITERIA FOR DETERMINATION OF A VALID TEST
According to the OECD 437 protocol, the test its considered acceptable if the positive control gives an In Vitro Irritation Score (IVIS) that falls within two standard deviations of the current historical mean. The negative or solvent/vehicle control responses should result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneas treated with the respective negative or solvent/vehicle control. Accordingly and based on historical control data, the test was acceptable if the in vitro irritation score of the positive control was ≥ 30 and the in vitro irritation score of the negative control was ≤ 3.

EVALUATION OF RESULTS
- Opacity: the change of opacity value of each treated cornea or positive and negative control corneae was calculated by subtracting the initial basal opacity from the post treatment opacity reading (t240 – t0), for each individual cornea.
The average change in opacity of the negative control corneae was calculated and this value was subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.
- Permeability: the corrected OD490 value of each cornea treated with positive control and test item was calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.

IN VITRO IRRITATION SCORE CALCULATION
The following formula was used to determine the in vitro irritation score of the negative control:
In vitro Irritation Score = opacity value + (15 x OD490 value)
The following formula was used to determine the in vitro irritation score of the positive control and the test item:
In vitro Irritation Score = (opacity value – opacity value mean negative control) + (15 x corrected OD490 value)
The in vitro irritation score was calculated for each individual treatment and positive control cornea. The mean in vitro irritation score irritation value of each treated group was calculated from the individual in vitro irritation score values.
Depending on the score obtained, the test item was classified into the following category according to OECD guideline 437 (table 1 in the field "Any other information on materials and methods incl. tables" below).
Irritation parameter:
other: In vitro irritation score
Basis:
mean
Time point:
other: 240 minutes
Score:
69.31
Irritant / corrosive response data:
Relative to the negative control, exposure of the test item vanadium, oxalate complexes to the corneae did not induce an increase of the corneal permeability, but a clear increase of the opacity values occurred. The calculated mean in vitro irritation score was 69.31 (threshold for corrosivity / severe irritancy: ≥ 55.1). According to OECD guideline 437, the test item is classified as corrosive / severe irritant to the eye.

Table 1: Results after 240 Minutes Incubation Time


Test Group

Opacity value = Difference (t240-t0) of Opacity

Permeability at 490 nm (OD490)

In vitro IrritationScore 

Mean in vitro irritation score± Standard Deviation

Proposed in vitro Irritation Scale

 

 

Mean

 

Mean

 

 

 

Negative Control

5

2.00

0.052

0.055

5.78

2.83 ± 2.60

Non corrosive / non severe irritant

1

0.055

1.83

0

0.059

0.89

Positive Control

146.00*

0.001*

146.01*

213.03 ± 90.54

Corrosive / severe irritant

316.00*

0.002*

316.03*

177.00*

0.003*

177.04*

Vanadium, oxalate complexes

85.00*

0.044*

85.66*

69.31 ± 14.32

Corrosive / severe irritant

63.00*

0.018*

63.27*

59.00*

- 0.010*/** 

59.00*

 * Corrected values (subtraction of mean opacity/permeability of negative control from measured opacity/permeability of each replicate, respectively)

** Value was set to “0” for the calculation of the irritation score

- With the negative control (0.9% (w/v) NaCl in deionised water) neither an increase of opacity nor permeability of the corneae could be observed (mean in vitro irritation score 2.83). The score is well within the concurrent negative control range, i.e. within two standard deviations of the current historical mean.

- The positive control (10% (w/v) Benzalkonium chloride in 0.9% (w/v) NaCl in deionised water) induced clear opacity of the corneae (mean in vitro irritation score 213.03) corresponding to a classification as corrosive/severe irritant to the eye (CLP/EPA/GHS (Cat 1)). The score is well within the concurrent positive control range, i.e. within two standard deviations of the current historical mean.

- Thus, based on historical positive and negative control data, this test meets the acceptability criteria in accordance with OECD 437.

Table 2: Historical data

 

Positive Control

Negative Control

Mean in vitro Irritation Score

176.71

1.78

Standard Deviation

42.65

0.75

Range of in vitro Irritation Scores

99.4 - 292.3

0.41 - 2.99

Values of 138 studies with solid test items performed between February 2007 and November 2012

Interpretation of results:
Category 1 (irreversible effects on the eye)
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Vanadium, oxalate complexes is corrosive / severely irritating to the eye and should be classified and labelled as severe eye irritant according to Regulation (EC) No. 1272/2008 (Category 1).
Endpoint conclusion
Endpoint conclusion:
no study available

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for selection of skin irritation / corrosion endpoint:
A reliable in vitro GLP guideline study (OECD 435) indicates that vanadium, oxalate complexes is not corrosive to skin. Based on a worst-case assumption and weight-of-evidence, vanadium, oxalate complexes is considered to be irritating to skin since its saturated aqueous solution has a pH of 1.9.

Justification for selection of eye irritation endpoint:
A reliable in vitro GLP guideline study (OECD 437) indicates that vanadium, oxalate complexes is corrosive to the eye.

Effects on skin irritation/corrosion: irritating

Effects on eye irritation: corrosive

Justification for classification or non-classification

Skin irritation:

Vanadium, oxalate complexes does not possess a potential for skin corrosion, but requires classification in Skin irritation category 2 (H315: Causes skin irritation) according to Regulation (EC) 1272/2008. According to Directive 67/548/EEC and subsequent adaptations, vanadium, oxalate complexes is corrosive (Xi; R38 Irritating to skin).

Eye irritation:

Vanadium, oxalate complexes possesses a corrosive potential and requires classification for irreversible effects on the eye (Category 1; H318: Causes serious eye damage) according to Regulation (EC) 1272/2008. According to Directive 67/548/EEC and subsequent adaptations, vanadium, oxalate complexes is corrosive (Xi; R41 Risk of serious damage to eyes).

Respiratory irritation:

Vanadium, oxalate complexes is only manufactured and marked as aqueous solution, and inhalation of vanadium, oxalate complexes (solution) can be considered negligible. Hence, vanadium, oxalate complexes (solution) cannot possess an irritation potential in the respiratory tract and therefore does not require classification as respiratory irritant according to Directive 67/548/EEC and Regulation (EC) 1272/2008.