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Description of key information

An in vitro skin irritation study (EpiSkinTM) was performed with divanadium trioxide (Heppenheimer, 2010), and results indicate that it is not irritant to skin.

An acute eye irritation / corrosion test according to OECD 405 (Leuschner, 2010) was performed with divanadium trioxide, and results indicate that it is irritant to eyes according to Regulation (EC) 1272/2008 (mild reversible effects).

No respiratory irritation was observed in acute and subacute inhalation toxicity tests.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-03-03 to 2010-03-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: Commission Regulation (EC) No. 440/2008 B.46 (draft)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ECVAM international validation study on in vitro tests for acute skin irritation (Altern Lab Anim. 2007 Dec; 35 (6):559-601)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD Guideline for the Testing of Chemicals; Draft proposal for a new guideline, in vitro skin irritation: Reconstructed Human Epidermis (RhE) Test method, 11 December 2009, Vers. 4.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2009-03-30
Details on test animals or test system and environmental conditions:
Not applicable - This was a in vitro study without any test animals.
Vehicle:
water
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): Approx. 15 mg of the test item were applied to each of triplicate tissues.
No further information on the amount/concentration applied was stated.
Duration of treatment / exposure:
15+/- 1 min
Observation period:
not applicable
Number of animals:
not applicable
Details on study design:
CELL CULTURE
EpiSkin TM kits (Lot No.: 10-EKIN-007) were purchased from Skinethic Laboratories (06000 Nice, France). The EpiSkin TM tissue consists of normal, human-derived epidermal keratinocytes that have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiSkin TM tissues (surface 0.38 cm^2) are cultured on specially prepared cell culture inserts.
EpiSkin TM tissues were shipped with ice packs on medium-supplemented agarose gels in a 12-well plate. On day of experiment EpiSkin TM tissues were transferred to 12-well plates with maintenance medium.

TREATMENT:
The negative control (deionised water (lot no. 1.3.10, produced in-house; 15 µL were applied to each of triplicate tissues) and positive control (5% sodium lauryl sulphate (lot no. 1353471 51508322; Fluka, Sigma-Aldrich, 89555 Steinheim, Germany) solution in deionised water, prepared freshly prior to the performance of the experiment; 15 µL were applied to each of triplicate tissues), and the test item were added into the insert atop the concerning EpiSkin TM triplicate tissues. Additionally, the test item tissues were wetted with 15 µl of deionised water. The plates were placed into the incubator for 15+/- 1 min at 37 +/- 1.5 °C, 5 +/- 0.5 % CO2.
After the end of the treatment interval the inserts were removed immediately from the plate. Using a wash bottle the tissues were gently rinsed with PBS to remove any residual test material. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper. The inserts were placed in the plates with 2 mL maintenance medium. The tissues were incubated for 42 +/- 1 hour at 37 +/- 1.5 °C, 5 +/- 0.5 % CO2.

MTT ASSAY:
Cell viability is measured by dehydrogenase conversion of MTT [(3-4,5-dimethyl thiazole 2-yl) 2,5-diphenyl-tetrazoliumbromide], present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues (Faller, C., Bracher, M., Dami, N., Roguet, R., 2002. Predictive ability of reconstructed human epidermis equivalents for assessment of skin irritation of cosmetics. Toxicology in vitro 16 (5), 557-552.).The percent reduction of cell viablity in comparison of untreated negative controls is used to predict skin irritation potential.
After the treatment procedure (42 hours) was completed for all tissues of each time point cell culture inserts were transferred from the holding plates to plates containing 2 mL assay medium containing 0.3 mg/mL MTT per well. After a 3 hour incubation period (37 +/- 1.5 °C, 5 +/- 0.5 % CO2) MTT solution was aspirated from the wells and the wells were rinsed three times with PBS. Tissues samples were cut out of the inserts with a biopsy punch and transferred into plastic vials. The tissue samples were immersed into extractant solution by gently pipetting 0.5 mL extractant solution (isopropanol) into each vial. The tissue samples were completely covered by isopropanol. The vials were sealed to inhibit isopropanol evaporation. The formazan salt was extracted for approx. 72 hours in the refrigerator.
Per each tissue sample 2 X 200 µL aliquots of the formazan blue solution were transferred into a 96-well flat bottom microtiter plate. OD was read in a microplate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany) at 570 nm without reference filter. Mean values were calculated from the 2 wells per tissue sample.

EVALUATION OF RESULTS:
The mean OD of the three negative control tissues was calculated. This value corresponds to 100% tissue viability in the current test. For each individual tissue treated with the test item or the positive control the individual relative tissue viability is calculated according to the following formula:
Relative viability (%) = [OD test item/ OD negative control] * 100
For the test item and the positive control the mean relative viability +/- standard deviation of the three individual tissues are calculated and used for classification according to the following prediction model:
For the current test, an irritation potential of a test item according to EU classification R38 is predicted if the mean relative tissue viablity of three individual tissues is reduced below 50 % of the negative control.

ACCEPTABILITY OF THE ASSAY:
The absolute OD 570 nm of the negative control tissues in the MTT test is an indicator of tissue viability obtained after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is meeting the acceptance criterion if the mean OD of the three tissues is ≥0.6 till ≤ 1.5.
An assay is meeting the acceptance criterion if mean relative tissue viability of the positive control is ≤ 40%.
The standard deviations in between tissues of the same treatment group should be ≤ 18%.

The data of the quality control (determined by Skinethic Laboratories, 06000 Nice, France) of the respecitve EpiSkin TM lot is mentioned below under "Attached background material" (the acceptance limit of the IC50 should be in the between 1.0 and 3.0 mg/L after 18 hours treatment with SLS).

TEST FOR DIRECT MTT REDUCTION.
For correct interpretation of results it was necessary to assess the ability of the test item to directly reduce MTT. To test for this ability approximately 15 mg of the test item were added to 1 mL of MTT solution and the mixture was incubated in the dark at room temperature for 60 minutes. Untreated MTT medium was used as control. If the MTT solution colour turned blue/purple, the test item was presumed to have reduced the MTT.
A colour change was not observed.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
86.2
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
After treatment with divanadium trioxide, the relative absorbance values decreased to 86.2%. This value is well above the threshold for irritancy of ≤ 50 %. Therefore, the test item is not considered to possess an irritant potential.

Results after treatment with divanadium trioxide

 

Dose group

Treatment Interval

Absorbance 570 nm
Tissue 1

Absor-bance 570 nm
Tissue 2

Absorbance 570 nm
Tissue 3

Mean Absorbance of 3 Tissues

Absorbance [%] Tissue 1, 2 + 3

Rel. Standard Deviation

Rel. Absorbance

[% of Negative Control]

Negative Control

15 min

0.878

0.819

0.873

0.857

102.5
95.6
101.9

3.8

100.0

Positive Control

15 min

0.293

0.209

0.203

0.235

34.2
24.4
23.7

5.9

27.4

Divana-dium trioxide

15 min

0.790

0.712

0.714

0.739

92.1
83.1
83.4

5.1

86.2

Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.

Historical data:

Positive Control

Negative Control

Number of Studies

73

Number of Studies

73

Period

July 2007 - March 2010

Period

July 2007 - March 2010

Mean Viability

16.5 %

Mean OD

1.081

Standard Deviation

11.0%

Standard Deviation

0.262

Range of Viabilities

3% - 36%

Range of ODs

0.7 – 1.6*

* The upper OD value is outside of the range of 0.6 - 1.5 recommended by the OECD guideline. Nevertheless since the OD value is only slightly above the required range, the historical data can still be considered as valid.

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
After treatment with divanadium trioxide, the relative absorbance values decreased to 86.2%. This value is well above the threshold for irritancy of ≤ 50 %. Therefore, the test item is not considered to possess an irritant potential. The test item is not classified as skin irritant according to Directive 67/548/EEC and according to regulation (EC) No.: 1272/2008.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-05-18 to 2010-06-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
, 2002-04-24
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2009-11-12
Species:
rabbit
Strain:
Himalayan
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: LPT Laboratory of Pharmacology and Toxicology GmbH & Co. KG, Branch Löhndorf, 24601 Löhndorf/Post Wankendorf, Germany
- Age at study initiation: Approx. 3 - 4 months
- Weight at study initiation: Animal 1: 2.2 kg; Animal 2: 2.3 kg; Animal 3: 2.0 kg
- Housing: For 8 hours following test item application, the animals were kept singly in restrainers which allowed free movement of the head but prevented a complete body turn, wiping of the eyes with the paws and excluded irritation of the eye by excrements and urine. During the acclimatisation period and after the 8-hour period in restrainers, the animals were kept singly in cages with dimensions of 380 mm x 425 mm x 600 mm (manufacturer: Dipl.Ing. W. EHRET GmbH, 16352 Schönwalde, Germany).
- Diet (ad libitum): Commercial diet, ssniff® K-H V2333 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water (ad libitum before and after the exposure period): Tap water
- Acclimation period: At least 20 adaptation days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 °C +/- 3 °C (maximum range)
- Relative humidity: 30% - 70% (maximum range; aim was 50% - 60%)
- Photoperiod (hrs dark / hrs light): 12/12
No further information on the test animals was stated.
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 100 mg of the test item were administered into one eye each of three animals. The test item was placed into the conjunctival sac of the right eye of each animal after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second in order to prevent loss of the material. The left eye, which remained untreated, served as a control.
No further information on the amount/concentration applied was stated.
Duration of treatment / exposure:
1 hour (One hour after application the eyes were rinsed.)
Observation period (in vivo):
1, 24, 48 and 72 hours and 4 to 8 days after the administration
Number of animals or in vitro replicates:
3 male rabbits
Details on study design:
It is explicitly noted and in accordance with the guideline that the test was performed initially using one animal. As no corrosive or severe irritant effects were observed in this animal, 2 further animals were employed 24 hours after start of the initial test.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): The eyes were rinsed with portions of 20 mL 0.9% aqueous NaCl solution, each.
- Time after start of exposure: 1 hour after administration

SCORING SYSTEM: Draize scoring system

TOOL USED TO ASSESS SCORE: The eyes were examined ophthalmoscopically with a slit lamp prior to the administration and 1, 24, 48 and 72 hours and 4 to 8 days after the administration. The eye reactions were observed and registered.
24 hours and 7 days after administration, fluorescein (Fluorescein SE Thilo drops (ALCON PHARMA GmbH, 79108 Freiburg, Germany)) was applied to the eyes before being examined to aid evaluation of the cornea for possible lesions.

OTHER OBSERVATIONS: Any further lesions not covered by the scoring system were recorded. Body weight of all animals was measured at the beginning of the study and at the end of the study. Behaviour and food consumption were monitored.
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
animal #1
Time point:
24/48/72 h
Score:
1
Max. score:
1
Reversibility:
fully reversible within: 4 days
Irritation parameter:
iris score
Basis:
mean
Remarks:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
0
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
animal #1
Time point:
24/48/72 h
Score:
2
Max. score:
2
Reversibility:
fully reversible within: 7 days
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
animal #1
Time point:
24/48/72 h
Score:
1.67
Max. score:
2
Reversibility:
fully reversible within: 4 days
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
animal #2
Time point:
24/48/72 h
Score:
0.67
Max. score:
1
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
iris score
Basis:
mean
Remarks:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
0
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
animal #2
Time point:
24/48/72 h
Score:
2
Max. score:
2
Reversibility:
fully reversible within: 8 days
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
animal #2
Time point:
24/48/72 h
Score:
1.67
Max. score:
2
Reversibility:
fully reversible within: 4 days
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
animal #3
Time point:
24/48/72 h
Score:
1
Max. score:
1
Reversibility:
fully reversible within: 6 days
Irritation parameter:
iris score
Basis:
mean
Remarks:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
0
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
animal #3
Time point:
24/48/72 h
Score:
2
Max. score:
2
Reversibility:
fully reversible within: 8 days
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
animal #3
Time point:
24/48/72 h
Score:
1.67
Max. score:
2
Reversibility:
fully reversible within: 4 days
Other effects:
There were no systemic intolerance reactions.

Corneal opacity (grade 1: Scattered or diffuse areas of opacity (other than slight dulling of normal lustre), details of iris clearly visible) was observed in all animals 24 and 48 hours, in animal no. 1 until 72 hours and in animal no. 3 until 5 days after instillation. The mean score per animal, following grading at 24, 48 and 72 h after instillation of the test material was calculated ≤ 1. The irises were not affected by instillation of the test item (grade 0 in all three animals). Conjunctival redness, i.e. grade 1 (some blood vessels hyperaemic (injected)) or grade 2 (diffuse, crimson colour; individual vessels not easily discernible)) was observed in all animals 60 minutes up to 6 days, in animal nos. 2 and 3 until 7 days after instillation. The mean score per animal, following grading at 24, 48 and 72 h after instillation of the test material was calculated 2. Chemosis was observed in all animals 24 and 48 hours, i.e. grade 2 (Obvious swelling with partial eversion of lid) and 72 hours grade 1 (Some swelling above normal) after instillation. The mean score per animal, following grading at 24, 48 and 72 h after instillation of the test material was calculated < 2.

In addition, secretion was observed in all animals 24 to 6 days, in animal nos. 2 and 3 from 60 minutes after instillation.

The fluorescein test performed 24 hours after instillation revealed corneal staining in all animals (up to 1/4 of the surface). The fluorescein test performed 7 days after instillation did not reveal any pathological findings.

The effects observerd were fully reversible within 21 days of instillation (8 days). In accordance with EEC 67/548, the overall irritation results can be summarized as follows:

Opacity (cornea and iris), 1h after application: mean score=0.0

Opacity (iris), 24, 48 and 72h after application: mean score=0.0

Opacity (cornea), 24, and 48 h after application: mean score=1.0

Opacity (cornea), 72 h after application: mean score=0.66

Opacity (cornea), 4d after application: mean score=0.33

Opacity (cornea), 6d after application: mean score=0.0

 

Conjunctival redness, 1h after application: mean score=1.0

Conjunctival redness, 24, 48 and 72h after application: mean score=2.0

Conjunctival redness, 4d after application: mean score=1.33

Conjunctival redness, 7d after application: mean score=0.66

Conjunctival redness, 8d after application: mean score=0.0

 

Conjunctival chemosis, 1h after application: mean score=0.0

Conjunctival chemosis, 24 and 48h after application: mean score=2.0

Conjunctival chemosis, 72h after application: mean score=1.0

Conjunctival chemosis, 4d after application: mean score=0.0

Interpretation of results:
other: Category 2 (irritating to eyes)
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
According to the EC-Commission directive 67/548/EEC and the results obtained under the present test conditions, Divanadium trioxide is non-irritating to eyes, hence, no labelling is required.

According to EC Regulation No. 1272/2008 and the mean scores in at least in 2 of 3 tested animals for corneal opacity (≥ 1) and conjunctival redness (≥ 2), divanadium trioxide produced reversible effects and needs to be classified for eye irritation Category 2 (H319: Causes serious eye irritation; if appropriately: Precautionary Statement Prevention: P264 + P280; Precautionary Statement Response P305 + P351 + P338, P337 + P313).
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Additional information

Justification for classification or non-classification

Skin irritation:

Divanadium trioxide does not possess a skin irritation potential and does not require classification as skin irritant according to Regulation (EC) 1272/2008.

 

Eye irritation:

Divanadium trioxide meets classification criteria as irritating to eyes (Category 2) according to Regulation (EC) 1272/2008.

Respiratory irritation:

The observed histopathological changes in the acute inhalation toxicity test with V2O3 (Leuschner, 1991) were regarded as unspecific effects, which usually occur after an inhalative exposure to a dust. Irritating effects in the respiratory tract, specific to the exposure of V2O3, were not observed.

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