Divanadium pentaoxide

Endpoint summary

Description of key information

Key studies demonstrate an eye and respiratory irritation potential:

An in vitro skin irritation study (EpiSkinTM) was performed with divanadium pentaoxide (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 pentaoxide, and results indicate that it is considered to be damaging to the eyes.

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 - Since this is an in vitro study there is no information on 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) are purchased from Skinethic Laboratories (06000 Nice, France). The EpiSkin TM tissue consists of normal, human-derived epidermal keratinocytes which 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 could not be observed.
No further information on the study design was stated.

Irritation / corrosion parameter:

% tissue viability

Remarks:

(mean)

Value:

88.2

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

After treatment with the test item divanadium pentaoxide the relative absorbance values decreased to 88.2%. 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 pentaoxide

 

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 penta-oxide	15 min	0.716	0.779	0.772	0.756	83.6 90.9 90.0	4.0	88.2

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:

GHS criteria not met

Conclusions:

The test item is not irritating to the skin.
According to the Regulation (EC) No 1272/2008 and subsequent regulations, the test item is not irritating to the skin.

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 2010-05-31

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. 4 - 5 months
- Weight at study initiation: Animal 1: 2.1 kg; Animal 2: 2.3 kg; Animal 3: 2.5 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

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.

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 and 5 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 used, 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 and 5 days after the administration. The eye reactions were observed and registered.
24 hours 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:

2

Max. score:

4

Reversibility:

not reversible

Remarks on result:

other: Due to character and severity of the damages in the eyes of the animal the study was discontinued for animal 1 on test day 6.

Irritation parameter:

iris score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

1.33

Max. score:

2

Reversibility:

not reversible

Remarks on result:

other: Due to character and severity of the damages in the eyes of the animal the study was discontinued for animal 1 on test day 6.

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

2.33

Max. score:

3

Reversibility:

not reversible

Remarks on result:

other: Day 4 and 5: Eye swollen, could not be opened. Due to character and severity of the damages in the eyes of the animal the study was discontinued for animal 1 on test day 6.

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not reversible

Remarks on result:

other: Due to character and severity of the damages in the eyes of the animal the study was discontinued for animal 1 on test day 6.

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

2.67

Max. score:

4

Reversibility:

not reversible

Remarks on result:

other: Due to character and severity of the damages in the eyes of the animal the study was discontinued for animal no. 2 on test day 5.

Irritation parameter:

iris score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

2

Reversibility:

not reversible

Remarks on result:

other: Due to character and severity of the damages in the eyes of the animal the study was discontinued for animal no. 2 on test day 5.

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

3

Max. score:

3

Reversibility:

not reversible

Remarks on result:

other: Day 4: Eye swollen, could not be opened. Due to character and severity of the damages in the eyes of the animal the study was discontinued for animal no. 2 on test day 5.

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not reversible

Remarks on result:

other: Due to character and severity of the damages in the eyes of the animal the study was discontinued for animal no. 2 on test day 5.

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

animal #3

Time point:

24/48/72 h

Score:

2.67

Max. score:

4

Reversibility:

not reversible

Remarks on result:

other: Due to character and severity of the damages in the eyes of the animal the study was discontinued for animal no. 3 on test day 5.

Irritation parameter:

iris score

Basis:

mean

Remarks:

Animal #3

Time point:

24/48/72 h

Score:

1.67

Max. score:

2

Reversibility:

not reversible

Remarks on result:

other: Due to character and severity of the damages in the eyes of the animal the study was discontinued for animal no. 3 on test day 5.

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

animal #3

Time point:

24/48/72 h

Score:

3

Max. score:

3

Reversibility:

not reversible

Remarks on result:

other: Day 4: Eye swollen, could not be opened. Due to character and severity of the damages in the eyes of the animal the study was discontinued for animal no. 3 on test day 5.

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

animal #3

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not reversible

Remarks on result:

other: Due to character and severity of the damages in the eyes of the animal the study was discontinued for animal no. 3 on test day 5.

Irritant / corrosive response data:

Corneal opacity was observed in all animals:
- animal no. 1: 24 hours (grade 1), 48 hours (grade 2) and 72 hours (grade 3) after instillation;
- animal nos. 2 and 3: 24 hours (grade 2) and 48 and 72 hours (grade 3) after instillation.
The mean score per animal, following 24, 48 and 72 h after instillation of the test material was calculated ≤ 3.

The fluorescein test performed 24 hours after instillation revealed corneal staining in all animals (1/4 to 1/2 of the surface).

Irritation of the iris was observed in all animals:
- animal no. 1: 24 and 48 hours (grade 1) and 72 hours (grade 2) after instillation;
- animal no. 2: 24 to 72 hours (grade 2) after instillation;
- animal no. 3: 24 hours (grade 1) and 48 and 72 hours (grade 2) after instillation.
The mean score per animal, following grading at 24, 48 and 72 h after instillation of the test material was calculated ≤ 1.5 in one animal; in two of these three animals ≥ 1.5.

Conjunctival redness was observed in all animals:
- animal no. 1: 60 minutes (grade 1), 24 and 48 hours (grade 2) and 72 hours (grade 3) after instillation;
- animal no. 2: 60 minutes (grade 2) and 24 to 72 hours (grade 3) after instillation;
- animal no. 3: 60 minutes (grade 1) and 24 to 72 hours (grade 3) after instillation.
The mean score in two of three animals, following grading at 24, 48 and 72 h after instillation of the test material was calculated to be 3.

Chemosis was observed in all animals:
- animal nos. 1 and 3: 24 to 72 hours (grade 2) after instillation;
- animal no. 2: 60 minutes (grade 1) and 24 to 72 hours (grade 2) after instillation.
The mean score in all three animals, following grading at 24, 48 and 72 h after instillation of the test material was calculated to be 2.

In addition, secretion was observed in animal no. one 24 hours to 72 hours, in animal nos. two and three 60 minutes to 72 hours after instillation.

Due to character and severity of the damages in the eyes of the animal the study was discontinued for animal 1 on test day 6 and animal nos. 2 and 3 on test day 5.

Other effects:

There were no systemic intolerance reactions.

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

The test item is severly irritating to the eyes.
According to the Regulation (EC) No 1272/2008 and subsequent regulations, the test item is severly irritating to the eyes (Category 1).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Additional information

Skin irritation

An in vitro skin irritation study (EpiSkin^TM) was performed with the substance (Heppenheimer, 2010), and results indicate that it is not irritant to skin.

Eye irritation

An acute eye irritation / corrosion test according to OECD 405 (Leuschner, 2010) was performed with the substance,and results indicate that it is damaging to eyes.

Respiratory irritation:

There are several epidemiological studies linking upper respiratory symptoms to vanadium pentoxide exposure (Kiviluoto, 1980; Kiviluoto et al., 1979a; Lewis, 1959, Zenz and Berg, 1967 Zenz et al. 1962). Effects were observed even after single exposure (e.g. Zenz & Berg, 1967). Long-term chronic exposure data of workers in the vanadium industry are reported in several publications by Kiviluoto et al. In a factory manufacturing vanadium pentaoxide, 63 workers exposed toV₂O₅at concentrations of 0.1 to 3.9 mg V/m³measured as total dust for 11 years (average 0.2-0.5 mg V/m³) did not have an increased prevalence of upper respiratory symptoms in the case study by Kiviluoto et al (1979a,b, 1980, 1981a,b). However, other epidemiological data support that respiratory symptoms are observed at exposure concentrations of V₂O₅that are above 0.1 mg/ V/m³.

Justification for classification or non-classification

Skin irritation:

The study by Heppenheimer (2010) is an in vitro skin irritation study, is considered the key study for skin irritation, and will be used for classification. After treatment with divanadium pentaoxide, the relative absorbance values decreased to 88.2%. 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 EC Regulation No. 1272/2008.

Eye irritation:

Divanadium pentaoxide is considered to be damaging to eyes, due to the fact that the effects observed (e.g. iritis) could be regarded as non reversible within a time period of 21 days. According to the EC Regulation 1272/2008, divanadium pentaxoxide is classified Category 1: H318: Causes serious eye damage.

Respiratory irritation:

Based on human data, exposure to divanadium pentaoxide may result in respiratory irritation. Thus, criteria for classification in STOT-SE Category 3 - respiratory tract irritation are met. According to the EC Regulation 1272/2008, divanadium pentaxoxide is classified STOT SE Category 3: H335: May cause respiratory irritation.