Cobalt wolframate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 August 2010 - 2 December 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

yes

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

yes

Principles of method if other than guideline:

The "maximisation test" of B. Magnusson and A. M. Kligman, modified according to Maurer & Hess was performed to reveal a possible sensitising potential of Cobalt wolframate. The use of this test is justified by the fact, that an LLNA (Local Lymph Node Assay) cannot be performed, as the test substance is poorly soluble in the solvents, commonly used for the LLNA. The same cause is the reason for choosing the modified test (Maurer and Hess, Fd Chem. Toxic. 1989, Vol. 27, No. 12, p. 807-811) instead of the original GPMT.
Deviations from the Guidelines: In step 1 of the experiment, the individual body weights of the animals were not determined at the end of the test period. This event did not adversely affect the outcome of the study.

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Test material

Test material information

Constituent 1

Reference substance name:

Cobalt wolframate

EC Number:

233-254-8

EC Name:

Cobalt wolframate

Cas Number:

10101-58-3

Molecular formula:

CoO4W

IUPAC Name:

cobalt wolframate

Details on test material:

Description by the sponsor
Name: COBALT WOLFRAMATE
Trade name: Cobalt wolframate
Chemical name: Cobalt wolframate
Supplier: Sponsor
Molecular formula: CoWO4
Batch No.: 750702_20100329
CAS No.: 10101-58-3
Appearance: Fine grained dark blue powder
Purity: Purity: > 98%
Impurity: WO3 < 2 %
Solubility in water: Not soluble
Conditions of storage: Room temperature
Stability at conditions of storage: Stable
Expiry date: 31 May 2015.

In vivo test system

Test animals

Species:

guinea pig

Strain:

Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
Species, strain: Guinea pigs, Hartley, Crl:Ha
Supplier: Charles River Laboratories, Germany
Sex, specification: Female healthy young adult and non pregnant animals
Age of the animals: Approx. 5 - 7 weeks at the first application
Weight range of the animals at the first application: Step 1: 293 g - 335 g. Step 2: 313 g - 338 g
Number of the animals in the main study: 10 + 10 animals for the test substance group, 5 + 5 animals for the control group
Spare animals: One additional animal per group was kept and administered under the same conditions as the other animals of the respective group Findings on the spare animals were only to be incorporated into this report if other animals of the test substance group or of the control group would have died spontaneously. Otherwise, the skin reactions of these animals were not used for the interpretation of the results.
ANIMAL MAINTENANCE
Hygiene: Optimal hygienic conditions
Room temperature / relative humidity: The animals were housed in fully air-conditioned rooms. Central air-conditioning guaranteed a range of 20 –
24°C for temperature and of 30 – 70% for relative humidity. There were no deviations from these ranges, which influenced the results of the study.
Air exchange: Approx. 12/h
Light: Only artificial light from 6.00 a.m. to 6.00 p.m
Cages:Group caging in plastic containers (48 cm x 115 cm x 36 cm), partly shaded, 6 (control group) or 11 (test substance group) animals per container
Feed: Ssniff Ms-H (Guinea Pig Maintenance Diet V2233), including ascorbic acid (2400 mg/kg), ad libitum, offered in stainless steel containers. Analysis of the feed for ingredients and contaminants are performed randomly by ssniff Spezialdiäten GmbH, Ferdinand-Gabriel-Weg 16, 59494 Soest, Germany.
Bedding material: Wood chips (aspen) from Fa. ABEDD®, LAB & VET Service GmbH, Hasnerstraße 84/6, 1160 Wien. Reduction of microorganisms by autoclaving
Water: Tap water offered in Makrolon bottles with stainless steel canules ad libitum. Random samples of the water are analysed by the "AGES", A-1226 Vienna, to assure that the water fulfils the requirements for drinking water for humans.
Identification of the animals: Numbers tattooed in the pinna of the right ear
Acclimatisation: 7 days

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

epicutaneous, occlusive

Vehicle:

petrolatum

Induction 2

Concentration / amount:

50 % in white petrolatum

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

petrolatum

Challenge 2

Concentration / amount:

50 % in white petrolatum

No. of animals per dose:

The study was performed in two consecutive steps: In the first step 5 control and 10 test substance animals were used and as after the challenge exposure negative results were obtained, additional 5 control animals and 10 test substance animals were exposed. This procedure is in accordance with the guidelines.

Details on study design:

First induction exposure: Commenced on Day 0.
Four separate intradermal injections of FCA, emulsified with isotonic saline (to enhance a possible sensitisation) were given at an area of approx. 2 x 4 cm in the interscapular region.
The injections were followed immediately afterwards by an epicutaneous application of the test substance, incorporated in white petrolatum (test substance groups) or plain white petrolatum (negative control groups) to the sites of the intradermal injections. Test patches (filter papers), 2 cm x 4 cm, with the test substance preparation (test substance group) or with the vehicle (negative control group), were applied. They were fixed with a strip of "Fixomull* stretch" (self adhesive non woven fabric, hypoallergenic, made by Beiersdorf AG, 20245 Hamburg, Germany).
The treated sites were covered occlusively with a Teflon ® - foil and kept in place and fixed with Guinea-Pig Jackets (Hugo Sachs Elektronik- Harvard Apparatus GmbH, 79232 March-Hugstetten, Germany).
24 h afterwards (Day 1) the jackets and the patches were removed.
Effects of the first induction exposure were checked by a skin examination 24 h after the end of the exposure period (Day 2).
On day 6, 24 h prior to the second induction exposure, the exposed skin sites were covered in all animals with a preparation of Na-dodecylsulfate, (E. Merck, 64271 Darmstadt, Germany, item No. 13760; approx. 0.5 g, 10 %, w/w, in white petrolatum), to produce a local hyperaemia.

Second induction exposure: Commenced on Day 7.
At the site of the preceding injections of the first induction exposure, an epicutaneous application of the test substance, incorporated in white petrolatum (test substance groups) or plain white petrolatum (negative control groups) to the sites of the intradermal injections analogously to the first induction exposure.
48 h afterwards (Day 9) the jackets and the patches were removed.
Effects of the second induction exposure were checked by a skin examination 48 h after the end of the exposure period (Day 11) in step 1 and 24 h after the end of the exposure period (Day 10 ) in step 2.

Challenge exposure: Commenced on Day 21 (step 1) and Day 20 (step 2).
In accordance with the test guidelines, the challenge exposure may be performed either on Day 20, 21 or 22.
The challenge exposure consisted of two separate epicutaneous applications, identically given to test substance and negative control group animals: One with a test substance preparation to the left flanks and one with the vehicle (white petrolatum) to the right flanks of all animals. Both exposed sites were apart from the exposure sites of the two induction exposures.
Test patches (now only 2 cm x 2 cm) and coverings were the same as in both induction exposures.
24 h afterwards (Day 22 in step 1 and Day 21 in step 2) the jackets and the patches were removed.
Effects of the challenge exposure were checked by a skin examination 24 h after the end of the exposure period (Day 23 in step 1 and Day 22 in step 2) and a second skin examination further 24 h later (Day 24 in step 1 and Day 23 in step 2).
Positive skin reactions of the test substance treated sites after the challenge exposure indicate a sensitising effect of the test substance, if the scores are higher than those of the vehicle treated sites and if the rate of those - positively reacting - animals is higher than the corresponding percentage of animals in the negative control group.

Challenge controls:

The challenge exposure consisted of two seperate applications, identically given to test substance and negative control animals.

Positive control substance(s):

yes

Results and discussion

Positive control results:

The net rate of animals with a positive skin reaction, regarded as sensitised by hexyl cinnamic aldehyde was 50 %.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

no

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 0. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no.

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

0

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

no

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 0. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no.

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

50 %

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

no

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 50 %. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: no.

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

50 %

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

no

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50 %. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: no.

Any other information on results incl. tables

All animals survived till the end of the study.

No effects of the test substance on body weights was derived from the data.

Immediately after the beginning of all epicutaneous exposures (inductions, challenge) the motor activities of all animals were decreased. This is due to the dressings which restrict the freedom of movement. Soon afterwards the behaviour was regular again. Except of the observations described above no abnormal behaviour or clinical signs were detected during the experiment in the animals.

Skin reactions after the intradermal FCA administration:

Cranial and caudal injection sites: Local irritations were observed in all animals beginning on the day after the injections. The irritations started with local erythema, which became more severe and led to ulcerations. Lesions did not heal until the end of the study. These local alterations are known effects of Freund's adjuvant.

Skin reactions after the first and the second induction exposure:

All animals of both groups had severe erythema and oedema in the interscapular region (score "3"), which were attributed to the effects of the adjuvant.

Skin reactions after the challenge exposure

No positive skin reaction in any animal at any reading time.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

The results of both steps were combined for the final conclusion.
The control sites of all animals of both groups were normal at each reading time.
After the challenge exposures, no animal of the test substance group had positive skin reactions at the test substance treated sites 24 hours and/or 48 hours after the end of the exposure. No adverse skin reactions were observed in the control animals. Therefore no animal of the test substance group was regarded as sensitised.
According to the guidelines, the results of this study and to the Directive 2001/59/EC for classification, the test substance "COBALT WOLFRAMATE" needs not to be labelled with "R43 May cause sensitisation by skin contact". "COBALT WOLFRAMATE" does not need to be classified according to Regulation (EC) 1272/2008 (CLP) with H317: May cause an allergic skin reaction.

Executive summary:

The "maximisation test" of B. Magnusson and A. M. Kligman modified according to Maurer & Hess was performed to reveal a possible sensitising potential of Cobalt wolframate.

Method

Investigations performed were in conformance with the Regulation (EC) 440/2008,Part B: Methods for the determination of toxicity and other health effects: Skin Sensitization; Official Journal of the European Union, No. L 142 and the OECD-guideline 406, "Skin Sensitisation".

The study was performed in two consecutive steps. In each step a test substance group with 10 female guinea pigs and a negative control group with 5 female guinea pigs was used.

The administered test substance concentrations were derived from the results of a preliminary test.

Two induction exposures were performed with a one week interval.

First induction exposure: Intracutaneous injection of Freund´s complete adjuvant emulsified with isotonic saline (1:1) and epicutaneous administration of the test substance (50 % in white petrolatum).

Second induction exposure: Preceded by a topical application of 10 % Na-dodecylsulfate in petrolatum for erythema induction 24 h before the epicutaneous administration of the test substance (50 % in white petrolatum).

Control animals were given plain white petrolatum at both inductions.

Challenge exposure: Two weeks after the second induction exposure. Epicutaneous administration of the test substance (50 % in white petrolatum) and the vehicle (white petrolatum), identical for the test substance group and the negative control group.

For the epicutaneous exposures occlusive dressings were used.

Results

The results of both steps were combined for the final conclusion.
All animals survived till the end of the study.
Intradermal injections of Freund's adjuvant caused severe local reactions in all animals, a known effect of the adjuvant. No other adverse effects were noted.

Skin reactions after the challenge exposure

The results of these skin examinations were decisive for the grading of the potential of sensitisation.

The control sites of all animals of both groups were normal at each reading time.

After the challenge exposures, no animal of the test substance group had positive skin reactions at the test substance treated sites 24 hours and/or 48 hours after the end of the exposure. No adverse skin reactions were observed in the control animals. Therefore no animal of the test substance group was regarded as sensitised.