Divanadium tris(sulphate)

Endpoint summary

Description of key information

Trivalent (V2O3), tetravalent (VOSO4), and pentavalent (NaVO3) vanadium substances failed to elicit any skin sensitising response in GLP-compliant skin sensitisation studies according to Magnusson and Kligman (OECD 406).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-05-18 to 2010-09-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

, 1992-07-17

Deviations:

yes

Remarks:

, guinea pigs were housed at a temperature of 22°C +/- 3°C instead at a temperature of 20 +/- 3°C

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2009-11-12

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

see attached justification document in the endpoint summary for skin sensitisation

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services Germany GmbH, Stolzenseeweg 32 – 36, 88353 Kißlegg, Germany
- Age at study initiation: 32 days
- Weight at study initiation: 325 - 361 g (excluding positive control group); Positive control group: 312 - 381 g
- Housing: The animals were kept in pairs in MAKROLON cages (MZK 80/25). Granulated textured wood (Granulat A2, J. BRANDENBURG, 49424 Goldenstedt, Germany) was used as bedding material in the cages.
- Diet (ad libitum): Commercial diet, ssniff® Ms-H V2233 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water (ad libitum): Tap water
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C ± 3°C (maximum range)
- Relative humidity (%): 55% ± 15% (maximum range)
- Photoperiod (hrs dark / hrs light): 12/12
No further information on the test animals was stated.

Route:

intradermal and epicutaneous

Vehicle:

water

Remarks:

Aqua ad iniectabilia (Batch nos. 911728 and 002152; Delta Select GmbH, 63303 Dreieich, Germany)

Concentration / amount:

Intracutaneous (Induction): 0.01% solution of sodium metavanadate in aqua ad iniectabilia
Topical (induction): 1% solution of sodium metavanadate in aqua ad iniectabilia
Topical (challenge): 0.05% solution of sodium metavanadate in aqua ad iniectabilia

Route:

epicutaneous, occlusive

Vehicle:

water

Remarks:

Aqua ad iniectabilia (Batch nos. 911728 and 002152; Delta Select GmbH, 63303 Dreieich, Germany)

Concentration / amount:

Intracutaneous (Induction): 0.01% solution of sodium metavanadate in aqua ad iniectabilia
Topical (induction): 1% solution of sodium metavanadate in aqua ad iniectabilia
Topical (challenge): 0.05% solution of sodium metavanadate in aqua ad iniectabilia

No. of animals per dose:

Preliminary test: 16 animals
Main test: 10 animals (treatment group) plus 5 animals (vehicle control group)
Positive control group: 20 animals

Details on study design:

RANGE FINDING TESTS:
The aim of the preliminary study was to determine the appropriate dose level of the test item following intracutaneous and topical administration. The concentration used in the main study following the induction procedure should produce mild to moderate (grade 1 or grade 2) skin reactions following topical application and be adjusted to the highest level that can be well tolerated locally and generally following intracutaneous injection. For the challenge exposure a subirritating concentration was used which produced no evidence of skin irritation in non-sensitised animals.
The shoulder and the flank region of the animals were shaved or shaved and depilated (approx. 5 cm x 5 cm).
(a) intracutaneous: 0.1 mL of the prepared test item was administered intracutaneously (shoulder region).
Three concentrations of the test item were injected intradermally into four animals, respectively. In all, twelve concentrations were tested intracutaneously.
(b) topical: The test area of 12 animals was shaved (6 animals) or shaved and depilated (6 animals). 2 mL of the test preparation was spread over a filter paper (2 cm x 4 cm) and applied to the test area and held in contact by an occlusive dressing.
Two concentrations each were applied to the shaved or shaved and depilated flanks.
The occlusive dressing and the filter paper containing the test item were removed after 24 or 48 hours and the application sites were assessed immediately, 24 and 48 (depilated) or immediately and 24 hours (non-depilated) after removal of the filter paper for erythema and oedema using the grading of MAGNUSSON/KLIGMAN.
Results:
Twelve concentrations of sodium metavanadate were tested by intracutaneous injection: 0.00001, 0.00005, 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.1, 0.5, 1, 5 or 10% solutions in aqua ad iniectabilia.
Concentrations up to 0.005% did not reveal any skin reaction. A concentration of 0.01% revealed a moderate and confluent erythema 24, 48 and 72 hours after administration. A concentration of 0.1% revealed a moderate and confluent erythema 24 hours and an intense erythema and swelling 48 and 72 hours after administration. A concentration of 0.5% revealed an intense erythema and swelling 24, 48 and 72 hours after administration. The animal employed with the concentrations of 1, 5 and 10% died prematurely within 24 hours after administration.
Twelve concentrations of sodium metavanadate were tested by topical application: 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1 0.5, 1, 5, 10, 25 and 50% concentrations in aqua ad iniectabilia.
Non-depilated skin: Concentrations up to 0.05% did not reveal any skin reactions. Concentrations of 0.1 or 0.5% revealed a discrete or patchy erythema, a concentrations of or 5% revealed a moderate and confluent erythema, concentrations of 10% and above revealed an intense erythema and swelling 48 and 72 hours after start of administration, respectively.
Depilated skin: Concentrations up to of 0.05% did not reveal any skin reactions. A concentration of 0.1% revealed a discrete or patchy erythema 24, 48 and 72 hours after start of administration. A concentration of 0.5% revealed a moderate and confluent erythema 24 hours and an intense erythema 48 and 72 hours after start of administration. Concentrations of 1% and above revealed an intense erythema and swelling 24, 48 and 72 hours after start of administration.
It was decided to use a 0.01% solution for the 1st (intracutaneous) induction stage, a concentration of 1% for the 2nd (topical) induction stage and a 0.05% concentration for the challenge.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 (intradermal injection and topical administration)

- Test groups:
Stage 1:
Day 0
Three pairs of intradermal injections of 0.1 mL were given in the shoulder region which was cleared of hair so that one of each pair lay on each side of midline.
(1) Freund's complete adjuvant (Batch no. 049K8700; SIGMA-ALDRICH Chemie GmbH, 82024 Taufkirchen, Germany)(diluted 1 : 1 with 0.9% NaCl (Batch no. 005632; Delta Select GmbH, 63303 Dreieich, Germany))
(2) the test item (sodium metavanadate) (0.01 % in aqua ad iniectabilia)
(3) the test item in a 1:1 mixture (v/v) FCA/physiological saline
In injection 3, the final concentration of the test item was equal to that in injection 2.
Stage 2
Day 7
7 days after the intracutaneous injection, the shoulder region of the same animals was shaved again and treated topically using the patch-test technique (as described above under "Range finding tests)(exposure time: 48 hours). The patch was removed after 48 hours. No cleaning of the skin was necessary.

- Control group:
The vehicle control animals were treated in the same way as the animals of the test group, but received aqua ad iniectabilia instead of the test item.

Skin observations and scoring: The skin reaction results of the first induction exposure were evaluated at 24 and 48 hours, of the second induction at 48 and 72 hours after start of exposure. The skin reactions were graded following the grading scale of MAGNUSSON/KLIGMAN.

B. CHALLENGE EXPOSURE
- No. of exposures:

- Test groups:
Two weeks (Day 21) after the topical application (corresponds to a monitoring period of 21 days) the flanks of the same animals were shaved and depilated for a further topical application using the patch-test technique (as described above under "Range finding tests). The filter paper containing the test item was applied to the left flank, the filter paper with the vehicle to the right flank of the animal (exposure time: 24 hours). 21 hours after the filter paper had been removed the treated skin was cleaned and fur removed, if necessary.

- Control group:
The left flank was treated with the test item, the right flank with the vehicle i.e. in the same way as the test group.

- Evaluation (hr after challenge): 21 hours after removing the filter paper the challenge area was cleaned and cleared of hair if necessary. Three hours later (at 48 hours from the start of challenge application) the skin reaction was observed and recorded. 24 hours after this observation a second observation (72 hours) was made and recorded. The skin reactions were graded following the grading scale of MAGNUSSON/KLIGMAN.

OTHER OBSERVATIONS:
Mortality: daily during the observation period
Clinical signs: daily during the observation period
Body weight: at start of study and at study termination
Pathology: No necropsy was performed. No animal was found dead in the main study or was sacrificed in extremis.

POSITIVE CONTROL GROUP:
The animals of the positive control group (same origin (strain) as those used in the study) were treated with a 2% (w/v) benzocaine solution intracutaneously in stage 1 , a 5% (w/v) benzocaine solution topically in stage 2 and in stage 3. The positive control data are obtained from the historical background of the laboratory that conducted this study. The study was performed during March 2010.
No further information on the study design was stated.

The body weight was analysed statistically using STUDENT's t-test (p ≤ 0.01).

Challenge controls:

Vehicle control group: aqua ad iniectabilia
5 males were used for the vehicle control group.

Positive control substance(s):

yes

Remarks:

Benzocaine (dissolved in 40% ethanolic 0.9% NaCl solution)

Positive control results:

Animals of this strain treated with benzocaine in 40% ethanolic 0.9% NaCl solution exhibited a sensitising reaction in all animals in form of a moderate and confluent erythema or an intense erythema and swelling.

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

0.05% solution of sodium metavanadate in aqua ad iniectabilia

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No skin irritation was observed.

Remarks on result:

no indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

0.05% solution of sodium metavanadate in aqua ad iniectabilia

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No skin irritation was observed.

Remarks on result:

no indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

aqua ad iniectabilia

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

The vehicle control revealed no skin reactions.

Remarks on result:

no indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

aqua ad iniectabilia

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

The vehicle control revealed no skin reactions.

Remarks on result:

no indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

5% (w/v) benzocaine solution (benzocaine was dissolved in 40% ethanolic 0.9% NaCl solution)

No. with + reactions:

20

Total no. in group:

20

Clinical observations:

Animals exhibited a sensitising reaction in form of a moderate and confluent erythema or an intense erythema and swelling.

Remarks on result:

positive indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

5% (w/v) benzocaine solution (benzocaine was dissolved in 40% ethanolic 0.9% NaCl solution)

No. with + reactions:

20

Total no. in group:

20

Clinical observations:

Animals exhibited a sensitising reaction in form of a moderate and confluent erythema or an intense erythema and swelling.

Remarks on result:

positive indication of skin sensitisation

A 0.01% solution of sodium metavanadate in aqua ad iniectabilia chosen for the 1st (intracutaneous) induction stage revealed in all 10 animals a moderate and confluent erythema 24 hours and a discrete or patchy erythema 48 hours after administration.

2 mL of a 1% concentration of sodium metavanadate in aqua ad iniectabilia per animal chosen for the 2nd (topical) induction stage revealed in all 10 animals a discrete or patchy erythema 48 and 72 hours after start of exposure.

The challenge with 2 mL of a 0.05% solution of sodium metavanadate in aqua ad iniectabilia per animal revealed no skin irritation in any animal and, thus, the test item had no sensitising properties.

The body weight gain of the animals treated with sodium metavanadate was within the range of the vehicle control during the experiment.

Behaviour remained unchanged.

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the present test conditions, sodium metavanadate revealed no sensitising properties in guinea pigs in a test model according to MAGNUSSON and KLIGMAN.
Therefore, the test item must not be classified and labeled according to regulation (EC) No.: 1272/2008 and subsequent regulations.
Also, the test item must not be classified and labeled according to 67/548/EC and subsequent regulations.

Reference 2

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-05-18 to 2010-07-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

, 1992-07-17

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2009-11-12

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

see attached justification document in the endpoint summary for skin sensitisation

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services Germany GmbH, Stolzenseeweg 32 – 36, 88353 Kißlegg, Germany
- Age at study initiation: 32 days
- Weight at study initiation: 296 - 364 g (excluding positive control group); Positive control group: 312 - 381 g
- Housing: The animals were kept in pairs in MAKROLON cages (MZK 80/25). Granulated textured wood (Granulat A2, J. BRANDENBURG, 49424 Goldenstedt, Germany) was used as bedding material in the cages.
- Diet (ad libitum): Commercial diet, ssniff® Ms-H V2233 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water (ad libitum): Tap water
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20°C ± 3°C (maximum range)
- Relative humidity: 55% ± 15% (maximum range)
- Photoperiod (hrs dark / hrs light): 12/12
No further information on the test animals was stated.

Route:

intradermal and epicutaneous

Vehicle:

water

Remarks:

aqua ad iniectabilia (Batch no. 911728; Delta Select GmbH, 63303 Dreieich, Germany)

Concentration / amount:

Intracutaneous (Induction): 0.01% suspension of divanadium trioxide in aqua ad iniectabilia
Topical (induction): 50% suspension of divanadium trioxide in aqua ad iniectabilia
Topical (challenge): 25% suspension of divanadium trioxide in aqua ad iniectabilia

Route:

epicutaneous, occlusive

Vehicle:

water

Remarks:

aqua ad iniectabilia (Batch no. 911728; Delta Select GmbH, 63303 Dreieich, Germany)

Concentration / amount:

Intracutaneous (Induction): 0.01% suspension of divanadium trioxide in aqua ad iniectabilia
Topical (induction): 50% suspension of divanadium trioxide in aqua ad iniectabilia
Topical (challenge): 25% suspension of divanadium trioxide in aqua ad iniectabilia

No. of animals per dose:

Preliminary test: 8 animals
Main test: 10 animals (treatment group) plus 5 animals (vehicle control group)
Positive control group: 20 animals

Details on study design:

RANGE FINDING TESTS:
The aim of the preliminary study was to determine the appropriate dose level of the test item following intracutaneous and topical administration. The concentration used in the main study following the induction procedure should produce mild to moderate (grade 1 or grade 2) skin reactions following topical application and be adjusted to the highest level that can be well tolerated locally and generally following intracutaneous injection. For the challenge exposure a subirritating concentration was used which produced no evidence of skin irritation in non-sensitised animals.
The shoulder and the flank region of the animals were shaved or shaved and depilated (approx. 5 cm x 5 cm).
(a) intracutaneous: 0.1 mL of the prepared test item was administered intracutaneously (shoulder region).
Three concentrations of the test item were injected intradermally into one animal, three further concentrations into a second animal.
(b) topical: The test area of 6 animals was shaved (3 animals) or shaved and depilated (3 animals). 2 mL of the test preparation was spread over a filter paper (2 cm x 4 cm) and applied to the test area and held in contact by an occlusive dressing.
Two concentrations each were applied to the shaved or shaved and depilated flanks of 6 animals each.
The occlusive dressing and the filter paper containing the test item were removed after 24 or 48 hours and the application sites were assessed immediately, 24 and 48 (depilated) or immediately and 24 hours (non-depilated) after removal of the filter paper for erythema and oedema using the grading scale of MAGNUSSON/KLIGMAN.
Results:
Six concentrations of divanadium trioxide were tested by intracutaneous injection: 0.01, 0.1, 0.5, 1, 5 or 10% suspensions in aqua ad iniectabilia.
Concentrations of 0.01 or 0.1% revealed a discrete or patchy erythema, concentrations of 0.5 or 1% a moderate and confluent erythema and concentrations of 5 or 10% an intense erythema and swelling 24 to 72 hours after administration.
Six concentrations of divanadium trioxide were tested by topical application: 0.5, 1, 5, 10, 25 and 50% suspensions in aqua ad iniectabilia.
Divanadium trioxide is a black powder. A 50% concentration in aqua ad iniectabilia was the maximum concentration that could be prepared in order to produce a homogeneous test item-vehicle suspension. No higher concentrated suitable suspensions could be prepared.
Non-depilated skin: No skin reactions were observed at any concentration.
Depilated skin: No skin reaction was observed up to the concentration of 25%. A concentration of 50% revealed a discrete or patchy erythema 24 to 72 hours after administration.
It was decided to use a 0.01% suspension for the 1st (intracutaneous) induction stage, a concentration of 50% for the 2nd (topical) induction stage and a 25% concentration for the challenge.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 (intradermal injection and topical administration)

- Test groups:
Stage 1.
Day 0
Three pairs of intradermal injections of 0.1 mL were given in the shoulder region which was cleared of hair so that one of each pair lay on each side of midline.
(1) Freund's complete adjuvant (Batch no. 129K8700; SIGMA-ALDRICH Chemie GmbH, 82024 Taufkirchen, Germany) (diluted 1 : 1 with 0.9% NaCl (Batch no. 001033; Delta Select GmbH, 63303 Dreieich, Germany))
(2) the test item (divanadium trioxide) (0.01 % in aqua ad iniectabilia))
(3) the test item in a 1:1 mixture (v/v) FCA/physiological saline
In injection 3, the final concentration of the test item was equal to that in injection 2.
Day 6
As a 50% suspension of divanadium trioxide in aqua ad iniectabilia was not irritating to the non-depilated skin of the test animals in the preliminary experiment, the fur was shaved from the application area and the exposed skin was coated with 0.5 mL sodium laurylsulfate 10% in vaseline in order to induce a local irritation.
Stage 2:
Day 7
7 days after the intracutaneous injection, the shoulder region of the same animals was shaved again but not depilated and treated topically using the patch-test technique (as described above under "Range finding tests") (exposure time: 48 hours). The patch was removed after 48 hours. No cleaning of the skin was necessary..

- Control group:
The vehicle control animals were treated in the same way as the animals of the test group, but received aqua ad iniectabilia instead of the test item.

- Skin observations and scoring: The skin reaction results of the first induction exposure were evaluated at 24 and 48 hours, of the second induction at 48 and 72 hours after start of exposure. The skin reactions were graded following the grading scale of MAGNUSSON/KLIGMAN.

B. CHALLENGE EXPOSURE

- No. of exposures: 1 (topical administration)

- Test groups:
Stage 3:
Two weeks (Day 21) after the topical application (corresponds to a monitoring period of 21 days) the flanks of the same animals were shaved and depilated for a further topical application using the patch-test technique. The filter paper containing the test item was applied to the left flank, the filter paper with the vehicle to the right flank of the animal (exposure time: 24 hours). 21 hours after the filter paper had been removed the treated skin was cleaned and fur removed, if necessary .

- Control group:
The left flank was treated with the test item, the right flank with the vehicle i.e. in the same way as the test group

- Evaluation (hr after challenge): 21 hours after removing the filter paper the challenge area was cleaned and cleared of hair if necessary. Three hours later (at 48 hours from the start of challenge application) the skin reaction was observed and recorded. 24 hours after this observation a second observation (72 hours) was made and recorded. The skin reactions were graded following the grading scale of MAGNUSSON/KLIGMAN.

OTHER OBSERVATION:
Mortality: daily during the observation period
Clinical signs: daily during the observation period
Body weight: at start of study and at study termination
Pathology: No necropsy was performed. No animal was found dead in the main study or was sacrificed in extremis.

POSITIVE CONTROL GROUP:
The animals of the positive control group (same origin (strain) as those used in the study) were treated with a 2% (w/v) benzocaine solution intracutaneously in stage 1 , a 5% (w/v) benzocaine solution topically in stage 2 and in stage 3. The positive control data are obtained from the historical background of the laboratory that conducted this study. The study was performed during March 2010.

The body weight was analysed statistically using STUDENT's t-test (p ≤ 0.01).

Challenge controls:

Vehicle control group: aqua ad iniectabilia
5 males were used for the vehicle control group.

Positive control substance(s):

yes

Remarks:

Benzocaine (dissolved in 40% ethanolic 0.9% NaCl solution)

Positive control results:

Animals of this strain treated with benzocaine in 40% ethanolic 0.9% NaCl solution exhibited a sensitising reaction in all animals in form of a moderate and confluent erythema or an intense erythema and swelling.

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

25% suspension of divanadium trioxide in aqua ad iniectabilia

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No skin irritation was observed.

Remarks on result:

no indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

25% suspension of divanadium trioxide in aqua ad iniectabilia

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No skin irritation was observed.

Remarks on result:

no indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

aqua ad iniectabilia

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

The vehicle control revealed no skin reactions.

Remarks on result:

no indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

aqua ad iniectabilia

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

The vehicle control revealed no skin reactions.

Remarks on result:

no indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

5% (w/v) benzocaine solution (Benzocaine was dissolved in 40% ethanolic 0.9% NaCl solution)

No. with + reactions:

20

Total no. in group:

20

Clinical observations:

Animals exhibited a sensitising reaction in form of a moderate and confluent erythema or an intense erythema and swelling.

Remarks on result:

positive indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

5% (w/v) benzocaine solution (Benzocaine was dissolved in 40% ethanolic 0.9% NaCl solution)

No. with + reactions:

20

Total no. in group:

20

Clinical observations:

Animals exhibited a sensitising reaction in form of a moderate and confluent erythema or an intense erythema and swelling.

Remarks on result:

positive indication of skin sensitisation

A 0.01% suspension of divanadium trioxide in aqua ad iniectabilia chosen for the 1st (intracutaneous) induction stage revealed a discrete or patchy erythema in all 10 animals 24 hours after administration.

2 mL of a 50% concentration of divanadium trioxide in aqua ad iniectabilia/animal chosen for the 2nd (topical) induction stage were non-irritating to the shaved skin in the preliminary experiment. Hence, in the main study the skin was coated with sodium laurylsulfate on the day before stage 2 induction in order to induce a local irritation.

The challenge with 2 mL of a 25% suspension of divanadium trioxide in aqua ad iniectabilia/animal revealed no skin irritation in any animal and, thus, the test item had no sensitising properties.

The body weight gain of the animals treated with divanadium trioxide was within the range of the vehicle control during the experiment.

Behaviour remained unchanged.

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the present test conditions, divanadium trioxide revealed no sensitising properties in guinea pigs in a test model according to MAGNUSSON and KLIGMAN.
Therefore,divanadium trioxide must not be classified and labeled according to regulation (EC) No.: 1272/2008 and subsequent regulations.
Also, divanadium trioxide must not be classified and labeled according to 67/548/EC and subsequent regulations.

Reference 3

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Remarks:

in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-05-18 to 2010-09-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

, 1992-07-17

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2009-11-12

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

see attached justification document in the endpoint summary for skin sensitisation

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services Germany GmbH, Stolzenseeweg 32 – 36, 88353 Kißlegg, Germany
- Age at study initiation: 32 days
- Weight at study initiation: 323 - 360 g (excluding positive control group); Positive control group: 312 - 381 g
- Housing: The animals were kept in pairs in MAKROLON cages (MZK 80/25). Granulated textured wood (Granulat A2, J. BRANDENBURG, 49424 Goldenstedt, Germany) was used as bedding material in the cages.
- Diet (ad libitum): Commercial diet, ssniff® Ms-H V2233 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water (ad libitum): Tap water
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20°C ± 3°C (maximum range)
- Relative humidity: 55% ± 15% (maximum range)
- Photoperiod (hrs dark / hrs light): 12/12
No further information on the test animals was stated.

Route:

intradermal

Concentration / amount:

0.01 % of Freund's complete adjuvant (diluted 1:1 with 0.9 % NaCl)

Day(s)/duration:

Day 0

Route:

intradermal

Vehicle:

other: aqua ad iniectabilia

Concentration / amount:

0.1 mL of 0.01 % vanadium oxide sulphate pentahydrate

Day(s)/duration:

Day 0

Route:

intradermal

Vehicle:

other: aqua ad iniectabilia

Concentration / amount:

0.1 mL of vanadium oxide suphate pentahydrate in a 1:1 mixture (v/v) FCA/physiological saline

Day(s)/duration:

Day 0

Route:

epicutaneous, occlusive

Vehicle:

other: aqua ad iniectabilia

Concentration / amount:

10 % solution of vanadium oxide sulphate pentahydrate

Day(s)/duration:

Day 7 (exposure: 48 hours)

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

other: aqua ad iniectabilia

Concentration / amount:

0.5 % solution of vanadium oxide sulphate pentahydrate

Day(s)/duration:

Day 21 (exposure period: 24 hours)

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

Preliminary test: 10 animals
Main study: 10 animals (treatment group) plus 5 animals (vehicle control group)
Positive control group: 20

Details on study design:

RANGE FINDING TESTS:
The aim of the preliminary study was to determine the appropriate dose level of the test item following intracutaneous and topical administration. The concentration used in the main study following the induction procedure should produce mild to moderate (grade 1 or grade 2) skin reactions following topical application and be adjusted to the highest level that can be well tolerated locally and generally following intracutaneous injection. For the challenge exposure a subirritating concentration was used which produced no evidence of skin irritation in non-sensitised animals.
The shoulder and the flank region of the animals were shaved or shaved and depilated (approx. 5 cm x 5 cm).
(a) intracutaneous: 0.1 mL of the prepared test item was administered intracutaneously (shoulder region).
Three concentrations of the test item were injected intradermally into four animals, respectively. In all, twelve concentrations were tested intracutaneously.
(b) topical: The test area of 6 animals was shaved (3 animals) or shaved and depilated (3 animals). 2 mL of the test preparation was spread over a filter paper (2 cm x 4 cm) and applied to the test area and held in contact by an occlusive dressing.
Two concentrations each were applied to the shaved or shaved and depilated flanks of 6 animals each.
The occlusive dressing and the filter paper containing the test item were removed after 24 or 48 hours and the application sites were assessed immediately, 24 and 48 (depilated) or immediately and 24 hours (non-depilated) after removal of the filter paper for erythema and oedema using the grading scale of MAGNUSSON/KLIGMAN..
Results:
Twelve concentrations of vanadium oxide sulphate pentahydrate were tested by intracutaneous injection: 0.00001, 0.00005, 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.1, 0.5, 1, 5 or 10% solutions in aqua ad iniectabilia.
Concentrations up to 0.005% did not reveal any skin reaction. A concentration of 0.01% revealed a moderate and confluent erythema 24 hours and an intense erythema and swelling 48 and 72 hours after administration. Concentrations of 0.1% and above revealed an intense erythema and swelling 24 to 72 hours after administration.
Six concentrations of vanadium oxide sulphate pentahydrate were tested by topical application: 0.5, 1, 5, 10, 25 and 50% concentrations in aqua ad iniectabilia.
- Non-depilated skin: A concentration of 0.5% did not reveal any skin reactions. Concentrations of 1 or 5% revealed a discrete or patchy erythema, concentrations of 10% and above a moderate and confluent erythema 48 and 72 hours after start of administration.
- Depilated skin: A concentration of 0.5% did not reveal any skin reactions. A concentration of 1% revealed a discrete or patchy erythema 24 hours after administration. A concentration of 5% revealed an intense erythema and swelling 24 hours and a moderate and confluent erythema 48 and 72 hours after start of administration. Concentrations of 10% and above revealed an intense erythema and swelling 24, 48 and 72 hours after start of administration.
It was decided to use a 0.01% solution for the 1st (intracutaneous) induction stage, a concentration of 10% for the 2nd (topical) induction stage and a 0.5% concentration for the challenge.

MAIN STUDY
A. INDUCTION EXPOSURE

- No. of exposures: 2 (intradermal injection and topical administration)

- Test groups:
Stage 1:
Day 0
Three pairs of intradermal injections of 0.1 mL were given in the shoulder region which was cleared of hair so that one of each pair lay on each side of midline.
(1) Freund's complete adjuvant (Batch no. 049K8700; SIGMA-ALDRICH Chemie GmbH, 82024 Taufkirchen, Germany)(diluted 1 : 1 with 0.9% NaCl (Batch no. 005632; Delta Select GmbH, 63303 Dreieich, Germany))
(2) the test item (vanadium oxide sulphate pentahydrate) (0.01 % in aqua ad injectabilia)
(3) the test item in a 1:1 mixture (v/v) FCA/physiological saline
In injection 3, the final concentration of the test item was equal to that in injection 2.
Stage 2:
Day 7
7 days after the intracutaneous injection, the shoulder region of the same animals was shaved again but not depilated and treated topically using the patch-test technique (as described above under "Range finding tests") (exposure time: 48 hours). The patch was removed after 48 hours. No cleaning of the skin was necessary.

- Control group:
The vehicle control animals were treated in the same way as the animals of the test group, but received aqua ad iniectabilia instead of the test item.

- Skin observations and scoring: The skin reaction results of the first induction exposure (intradermal injection) were evaluated at 24 and 48 hours, of the second induction (topical administration) at 48 and 72 hours after start of exposure.

B. CHALLENGE EXPOSURE

- No. of exposures: 1 (topical administration)

- Test groups:
Stage 3:
Two weeks (Day 21) after the topical application (corresponds to a monitoring period of 21 days) the flanks of the same animals were shaved and depilated for a further topical application using the patch-test technique (as described above under "Range finding tests"). The filter paper containing the test item was applied to the left flank, the filter paper with the vehicle to the right flank of the animal (exposure time: 24 hours). 21 hours after the filter paper had been removed the treated skin was cleaned and fur removed, if necessary.

- Control group:
The left flank was treated with the test item, the right flank with the vehicle i.e. in the same way as the test group.

- Evaluation (hr after challenge):
21 hours after removing the filter paper the challenge area was cleaned and cleared of hair if necessary. Three hours later (at 48 hours from the start of challenge application) the skin reaction was observed and recorded. 24 hours after this observation a second observation (72 hours) was made and recorded. The skin reactions were graded following the grading scale of MAGNUSSON/KLIGMAN.

OTHER OBSERVATIONS:
Mortality: daily during the observation period
Clinical signs: daily during the observation period
Body weight: at start of study and at study termination
Pathology: No necropsy was performed. No animal was found dead in the main study or was sacrificed in extremis.

POSITIVE CONTROL GROUP:
The animals of the positive control group (same origin (strain) as those used in the study) were treated with a 2% (w/v) benzocaine solution intracutaneously in stage 1, a 5% (w/v) benzocaine solution topically in stage 2 and in stage 3.
The positive control data are obtained from the historical background of the laboratory that conducted this test. The study was performed during March 2010.
No further information on the study design was stated.

Challenge controls:

Vehicle control group: Aqua ad iniectabilia
5 males were used for the vehicle control group.

Positive control substance(s):

yes

Remarks:

Benzocaine (dissolved in 40% ethanolic 0.9% NaCl solution)

Positive control results:

Animals of this strain treated with benzocaine in 40% ethanolic 0.9% NaCl solution exhibited a sensitising reaction in all animals in form of a moderate and confluent erythema or an intense erythema and swelling.

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

0.5% solution of vanadium oxide sulphate pentahydrate in aqua ad iniectabilia

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No skin irritation was observed.

Remarks on result:

no indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

0.5% solution of vanadium oxide sulphate pentahydrate in aqua ad iniectabilia

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No skin reaction was observed.

Remarks on result:

no indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

aqua ad iniectabilia

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

The vehicle control revealed no skin reactions.

Remarks on result:

no indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

aqua ad iniectabilia

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

The vehicle control revealed no skin reactions.

Remarks on result:

no indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

5% (w/v) benzocaine solution (Benzocaine was dissolved in 40% ethanolic 0.9% NaCl solution)

No. with + reactions:

20

Total no. in group:

20

Clinical observations:

Animals exhibited a sensitising reaction in form of a moderate and confluent erythema or an intense erythema and swelling.

Remarks on result:

positive indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

5% (w/v) benzocaine solution (Benzocaine was dissolved in 40% ethanolic 0.9% NaCl solution)

No. with + reactions:

20

Total no. in group:

20

Clinical observations:

Animals exhibited a sensitising reaction in form of a moderate and confluent erythema or an intense erythema and swelling.

Remarks on result:

positive indication of skin sensitisation

A 0.01% solution of vanadium oxide sulphate pentahydrate in aqua ad iniectabilia chosen for the 1st (intracutaneous) induction stage revealed in all 10 animals a moderate and confluent erythema 24 hours and a discrete or patchy erythema 48 hours after administration.

2 mL of a 10% concentration of vanadium oxide sulphate pentahydrate in aqua ad iniectabilia/ animal chosen for the 2nd (topical) induction stage revealed in all 10 animals a discrete or patchy erythema 48 and 72 hours after start of exposure.

The challenge with 2 mL of a 0.5% solution of vanadium oxide sulphate pentahydrate inaqua ad iniectabilia/ animal revealed no skin irritation in any animal and, thus, the test item had no sensitising properties.

The body weight gain of the animals treated with vanadium oxide sulphate pentahydrate was within the range of the vehicle control during the experiment.

Behaviour remained unchanged.

Interpretation of results:

GHS criteria not met

Conclusions:

According to the Regulation (EC) 1272/2008 and subsequent adaptations, the substance is not sensitising to the skin.

Reference 4

Endpoint:

skin sensitisation: in vitro

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro skin sensitisation study does not need to be conducted because the available in vitro test methods are not applicable for the substance and therefore an in vivo skin sensitisation study was conducted

an in vitro skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The endpoint skin sensitisation of the vanadium category substances is not addressed by substance-specific information but by read-across of in vivo data available for representative tri-, tetra- and pentavalent vanadium substances. This is based on the assumption that once inorganic vanadium compounds become bioavailable within the skin (the localisation of the Langerhans cells, relevant for the immune response), this will be in the ionic form. In bioaccessibility tests of tri-, tetra- and pentavalent vanadium substances as well as “zero valency” vanadium metal shows that solely tetra- and pentavalent vanadium forms dissolve in artificial sweat. Pentavalent vanadium substances are released and retained as pentavalent forms, whereas the tetravalent forms transform to the pentavalent form. Thus, it can be assumed that vanadium speciation in body fluids is controlled by the conditions of the respective medium but not by the vanadium source. Thus, read-across of the available skin sensitisation data, covering tri-, tetra- and pentavalent vanadium substances to other vanadium category substances is justified. Further details on the principles of read-across of the vanadium category substances is given in the read-cross justification reports in section 13 of the technical dossier.

The studies by Haferkorn (2010a,b,c) are considered key studies on skin sensitisation and are used for classification. Representative trivalent (V2O3), tetravalent (VOSO4), and pentavalent (NaVO3) vanadium substances failed to elicit any skin sensitising response in sensitisation studies according to Magnusson and Kligman (OECD 406). A detailed evaluation of the relevance, reliability and adequacy of each study is presented in the individual study records. The "Rationale for conducting skin sensitisation testing according to the GPMT protocol (OECD 406)" is attached.

In addition, positive human experience with any vanadium substance have not been reported. Based on available evidence and considering guideline-conform in vivo studies conducted under GLP, vanadium substances, should be considered void of skin sensitisation. It is therefore concluded that none of the vanadium category substances requires classification and labelling as skin sensitising according to regulation (EC) 1272/2008.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Vanadium exposure has not been reported to induce immune responses in vanadium industry workers, and occupational asthma or reduced lung function have not been diagnosed in vanadium producing facilities.

Justification for classification or non-classification

According to negative results of sensitisation studies with inorganic tri-, tetra- and pentavalent vanadium forms according to Magnusson and Kligman, it can be concluded that inorganic tri-, tetra- and pentavalent vanadium substances do not have a sensitisation potential and therefore, must not be classified and labelled according to Regulation (EC) 1272/2008.