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Description of key information

An in vitro skin irritation study (Epi-200 SIT) was performed with sodium metavanadate (Heppenheimer, 2012), and results indicate that it is not irritant to skin. An in vivo eye irritation / corrosion test according to OECD 405 (Hansen, 2013) was performed with sodium metavanadate, and results indicate that it is irritant to eyes (Cat. 2). Beforehand, an in vitro eye corrosion test had been conducted according to OECD 437 (Heppenheimer, 2013) to assure that sodium metavanadate is not corrosive to the eye.

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:
2012-11-07 to 2012-11-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2010-07-22
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2009
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: In vitro EpiDerm TM Skin irritation Test (EPI-200-SIT) for use with MatTek Corporation's Reconstructed Human Epidermal Model EpiDerm (EPI-200), Rev. 1/19/2010
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2009-03-30
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature (further detailed: Harlan CCR SOP SUBST.doc)
Test system:
human skin model
Source species:
human
Cell type:
other: normal, human-derived epidermal keratinocytes
Cell source:
other: not specified
Source strain:
not specified
Details on animal used as source of test system:
not applicable
Justification for test system used:
In a prevalidation study performed by ECVAM, the in vitro skin irritation test using the human skin model EpiDerm™ and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for skin irritancy potential.
Vehicle:
other: Dulbecco's Phosphate Buffered Saline (DPBS)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm TM tissues (purchased from MatTek Corporation)
- Tissue lot number: 16852
- Delivery date: 2012-11-06
- Date of initiation of testing: 2012-11-07

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C (incubator; duration: 35 minutes) followed by placing the plates into a sterile hood (duration: 25 minutes)(total exposure duration: 60 minutes)
- Temperature of post-treatment incubation: 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
At the end of the treatment interval the inserts were removed from the plate and tissues were rinsed with DPBS in order to remove any residual test material. After the rinsing, the inserts were submerged in DPBS. Afterwards the inserts were again rinsed with DPBS. The tissues were then transferred into plates with fresh assay medium. The surface of the tissues was dried using sterile cotton tipped swaps. Tissues were incubated for about 23 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2.
New plates (or lower row of the same plates) were filled with fresh assay medium, and the inserts were transferred onto the new plates. The wells were incubated for nearly 18 hours post-incubation at 37 ± 1.5 °C, 5 ± 0.5 % CO2. The complete incubation period was about 41 hours.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 300 µL
- Incubation time with MTT: 3 hours
- Extraction of formazan: after the incubation period, the MTT solution was aspirated from the wells, and the wells were rinsed with DPBS. Inserts were transferred onto new plates. The inserts were immersed into extractant solution by pipetting isopropanol in each insert. The tissues were completely covered from both sides. The plate was sealed to inhibit the isopropanol evaporation. The formazan salt was extracted for about 69 hours at room temperature.
After the extraction period was completed, the inserts were pierced to allow the extract to run into the well from which the insert was taken and the insert was discarded. The plates were shaken for 15 minutes until the solution was homogeneous in colour.
Per each tissue, 3 × 200 μL aliquots of the blue formazan solution were transferred into a 96-well flat bottom microtiter plate from the 15 minutes exposure. The optical density was determined with a spectrophotometer. Mean values were calculated from the 3 wells per tissue.
- Spectrophotometer: Versamax® Molecular Devices
- Wavelength: 570 nm
- Filter bandwidth: 1 nm

TEST FOR DIRECT MTT REDUCTION
To test for the ability of the test item to directly reduce MTT approx. 25 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 colour of the MTT would turn blue/purple, the test item is assumed to have reduced MTT.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Barrier function: tissues pass analysis for tissue functionality
- Contamination: absence of bacteria, yeast, and other fungi (long term antibiotic, antimycotic free culture) as absence of HIV1- virus, Hepatitis B virus, Hepatitis C virus
Please also refer to the field "Attached background material" below.

PREDICTION MODEL / DECISION CRITERIA
The mean optical density 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 was calculated according to the following formula: relative viability(%) = (OD test item/ OD mean of negative control) x 100
For the test item and the positive control the mean relative viability ± relative standard deviation of the three individual tissues was calculated and used for classification according to the following prediction model: if the mean relative tissue viability of three individual tissues is less than or equal to 50% of the negative control, the test item needs to be classified and labeled for its skin irritation potential: Category 2 – irritant, H315 according to Regulation (EC) No 1272/2008.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): approximately 25 mg of the test item were applied to each tissue replicate, wetted with the vehicle

VEHICLE
- Amount(s) applied (volume or weight with unit): 25 μL of DPBS

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
Duration of treatment / exposure:
60 minutes
Duration of post-treatment incubation (if applicable):
about 41 hours
Number of replicates:
Test item: triplicates
Negative control: triplicates
Positive control: triplicates
Irritation / corrosion parameter:
% tissue viability
Value:
72.2
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: the optical evaluation of the MTT-reducing capacity of the test item after 1-hour incubation with the MTT-reagent indicated that it did not turn blue. Therefore, the test item did not reduce MTT directly and a functional test with freeze-killed tissue was not deemed necessary.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: after treatment with the negative control, the absorbance values (2.117, 2.423, and 2.175, respectively) were well above the required acceptability criterion of mean OD ≥ 1.0 and ≤ 2.5 for the 60 minutes treatment interval.
- Acceptance criteria met for positive control: treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 3.8% (acceptability criterion: positive control is ≤ 20 %).
- Acceptance criteria met for variability between replicate measurements: the relative standard deviations of readings for tissue replicates of the test item, positive and negative controls were all below 18%, respectively (threshold of the "OECD Guideline for the Testing of Chemicals 439: In vitro Skin Irritation: Reconstructed Human Epidermis Test Method”: 18%).

HISTORICAL DATA

Positive Control

Negative Control

Number of Studies

67

Number of Studies

67

Period

May 2010 – November 2012

Period

May 2010 – November 2012

Mean Viability

6.6%

Mean Absorption

1.717

Standard Deviation

2.1%

Standard Deviation

0.274

Range of Viabilities

4% - 9.4%

Range of Vabilities

1.423 – 2.651

Table 1: Results after treatment with sodium metavanadate and the controls

Dose group

Treatment interval

Absorbance
570 nm
Tissue 1*

 

Absorbance
570 nm
Tissue 2*

 

Absorbance
570 nm
Tissue 3*

 

Mean Absor-bance
of 3 Tissues

 

Mean Rel. Absorbance

[% of Negative Control]**

 

Negative control

60 min

2.117

2.423

2.175

2.239

100.0

Positive control

60 min

0.091

0.088

0.077

0.085

3.8***

Test item

60 min

1.674

1.529

1.647

1.617

72.2

* Mean of three replicate wells after blank correction

** relative absorbance [rounded values]: 100 x (absorbancetest item)/(absorbance negative control)

*** The viability of the positive control is below the historical limit of 4.0%. Nevertheless, since the positive control induced a clear positive effect, and is only slightly below the historical limit, it 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 adaptations, 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:
2013-02-25 to 2013-03-12
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:
adopted 2012-10-02
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: approximately 7 months
- Weight at study initiation: 2.50 - 2.85 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; before and after the exposure period): commercial diet, ssniff® K-H V2333 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water (ad libitum; before and after 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)
- 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:
One hour
Observation period (in vivo):
Prior to the administration and 1, 24, 48, 72 hours and 4 to 6 days after the administration
Number of animals or in vitro replicates:
3 female rabbits
Details on study design:
USE OF TOPICAL ANAESTHETICS AND SYSTEMIC ANALGESICS
Sixty minutes prior to test item administration, 0.01 mg Buprenovet®/kg b.w. were administered by subcutaneous injection to all animals to provide a therapeutic level of systemic analgesia to avoid or minimize pain and distress.
Five minutes prior to the test item administration, one or two drops of Ophtocain®, a topical anaesthetic, was applied to each eye of all animals, to the right eye, in which the test item was to be applied, and to the left eye, which served as control.
In addition, 8 hours after administration of the test item all animals were treated with 0.01 mg Buprenovet®/kg b.w. in conjunction with 0.5 mg Metacam®/kg b.w., subcutaneously.

INITIAL TEST AND CONFIRMATORY TEST
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: each eye was rinsed with 20 mL of 0.9% aqueous NaCl solution.
- Time after start of exposure: one hour after instillation

SCORING SYSTEM: according to the Draize scale
Any further lesions are listed.

TOOL USED TO ASSESS SCORE: the eyes were examined ophthalmoscopically with a slit lamp prior to the administration and 1, 24, 48, 72 hours and 4 to 6 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.

OBSERVATIONS:
General criteria: body weight of all animals was measured at the beginning 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:
3.33
Max. score:
4
Reversibility:
fully reversible within: 6 days
Remarks on result:
other: Corneal staining (3/4 to whole surface) was observed during the 24 hours fluorescein test.
Irritation parameter:
iris score
Basis:
mean
Remarks:
animal #1
Time point:
24/48/72 h
Score:
0.67
Max. score:
2
Reversibility:
fully reversible within: 4 days
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
animal #2
Time point:
24/48/72 h
Score:
3
Max. score:
3
Reversibility:
fully reversible within: 6 days
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
animal #1
Time point:
24/48/72 h
Score:
4
Max. score:
4
Reversibility:
fully reversible within: 6 days
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
animal #2
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 5 days
Remarks on result:
other: Corneal staining (1/2 to 3/4 of the surface) was observed during the 24 hours fluorescein test.
Irritation parameter:
iris score
Basis:
mean
Remarks:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
animal #2
Time point:
24/48/72 h
Score:
2.67
Max. score:
3
Reversibility:
fully reversible within: 5 days
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
animal #2
Time point:
24/48/72 h
Score:
3
Max. score:
4
Reversibility:
fully reversible within: 6 days
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
animal #3
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 5 days
Remarks on result:
other: Corneal staining (3/4 to whole surface) was observed during the 24 hours fluorescein test.
Irritation parameter:
iris score
Basis:
mean
Remarks:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
animal #3
Time point:
24/48/72 h
Score:
2
Max. score:
3
Reversibility:
fully reversible within: 5 days
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
animal #3
Time point:
24/48/72 h
Score:
2
Max. score:
4
Reversibility:
fully reversible within: 4 days
Irritant / corrosive response data:
A single instillation of 100 mg Sodium metavanadate per animal into the conjunctival sac of the right eye of three rabbits caused the following changes:
Cornea opacity was observed in all animals:
- one animal: 24 and 72 hours (grade 3), 48 hours (grade 4), 4 and 5 days (grade 1) after instillation;
- second and third animal: 24 hours to 4 days (grade 1) after instillation.

The irises (grade 1) were observed in animal no. one 48 and 72 hours after instillation.

Conjunctivae redness was observed in all animals:
- one animal: 60 minutes and 4 to 5 days (grade 1), 24 to 72 hours (grade 3) after instillation;
- second animal: 60 minutes and 4 days (grade 1), 24 and 48 hours (grade 3), 72 hours (grade 2) after instillation;
- third animal: 60 minutes and 72 hours to 4 days (grade 1), 24 hours (grade 3), 48 hours (grade 2) after instillation.

Chemosis was observed in all animals:
- one animal: 60 minutes and 4 to 5 days (grade 1), 24 to 72 hours (grade 4) after instillation;
- second animal: 60 minutes and 4 days (grade 1), 24 hours (grade 4), 48 hours (grade 3), 72 hours (grade 2) after instillation;
- third animal: 24 hours (grade 3), 48 hours (grade 2), 72 hours (grade 1) after instillation.
Other effects:
There were not any systemic intolerance reactions concerning behaviour, body weight and food consumption.
Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Conclusions:
Sodium metavanadate is irritating to the eyes.
According to the EC-Regulation 1272/2008 and subsequent adaptations, sodium metavanadate is classified as rritating to the eyes (Category 2).
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:

Sodium metavanadate does not possess an irritation potential and does not require classification as skin irritant according to Regulation (EC) 1272/2008.

Eye irritation:

Sodium metavanadate possesses an irritation potential and requires classification as eye irritant (Category 2) according to Regulation (EC) 1272/2008.

Respiratory irritation:

Sodium metavanadate is neither irritating/corrosive to skin nor corrosive to eyes. Only mild, reversible effects were observed in the in vivo eye irritation test (Hansen, 2013). Furthermore, local reversible or irreversible adverse health effects were not observed below lethal levels (i.e. no pathological findings) in the acute inhalation toxicity test (Leuschner, 1992). Hence, sodium metavanadate does not possess an irritation potential in the respiratory tract and does not require classification as respiratory irritant according to Regulation (EC) 1272/2008.