Ashes (residues), vanadium-contg.

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18.08.2010-03.09.2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline 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:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EC No. 440/2008, L 142, Annex Part B, 30 May 2008 [4]

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Ashes (residues), vanadium-contg.

EC Number:

282-208-3

EC Name:

Ashes (residues), vanadium-contg.

Cas Number:

84144-87-6

Molecular formula:

Not available

Constituent 2

Reference substance name:

Vanadium Concentrate

IUPAC Name:

Vanadium Concentrate

Details on test material:

- Name of test material (as cited in study report): Vanadium concentrate
- Physical state: white powder
- Lot/batch No.: 14-45810
- Storage condition of test material: at room temperature, protected from light

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann, 33178 Borchen, Germany. Animals were derived from a controlled full-barrier maintained breeding system (SPF). According to Art. 9.2, No. 7 of the German Act on Animal Welfare[7] the animals are bred for experimental purposes.
- Age at study initiation: 8-9 w
- Weight at study initiation: weight was determined but no data are reported
- Housing: in groups of 5 in IVC cages, type II L, polysulphone cages on Altromin saw fibre bedding (lot no. 190110)
- Diet (e.g. ad libitum): free access to Altromin 1324 maintenance diet for rats and mice (lot no. 1020)
- Water (e.g. ad libitum): free access to tap water, sulphur acidified to a pH value of approx. 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Acclimation period: adequate, at least 5 days
- Other: Mice were healthy, nulliparous and non-pregnant. Cetrificates of food, water and bedding are filed at BSL Bioservice.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+-3 °C
- Humidity (%): 55+-10 %
- Air changes (per hr): at least 10/h
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

Based on the results observed in a preliminary test the following test item concentrations were selected for the main study:
6.25 %, 12.5 % and 25 % (diluted suspended in AOO).
The preparations were made immediately prior to each dosing.

No. of animals per dose:

5 mice/test group
3 mice/preliminary test

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: animal 1&2 were treated with a test item concentration of 25 % ; animal 3 served as negative control (treated with 100 % AOO)
- Irritation: none detected in any animal, neither signs of systemic toxicity
- Lymph node proliferation response: none

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: Annex I of Regulation (EC) 1272/2008 [6]

TREATMENT PREPARATION AND ADMINISTRATION:
The above mentioned concentrations were prepared and animals were selected randomly for treatment.
Topical application:
- Thickness of both ears of all animals was measured before start of administration.
- Mice were treated by topical application of 25 µl of the selected solution to the entire dorsal surface of each ear.
- Ear thickness was measured again approximately 48 h after first application.
- Topical applications were performed once daily over three consecutive days.
Administration of 3H-Methyl Thymidine:
- Five days after the first topical application all mice were dosed with 20 µCi 3H-Methyl thymidine by intravenous injection (tail vein) of 250 µl of 3H-Methyl thymidine, diluted to a working concentration of 80 µCi/ml.
Preparation of Cell Suspension:
- Thickness of ears was measured for the third time.
- Approximately 5 h after the injection of 3H-methyl thymidine all mice were sacrificed by cervical dislocation.
- The draining "auricular lymph nodes" were excised, individually pooled for each animal (2 lymph nodes/animal) and collected in phosphate buffered saline (PBS). A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through polyamide gauze (200 mesh size). After washing the gauze with PBS the cell suspension was pelleted in a centrifuge. The supernatant was discarded and the pellets were resuspended with PBS. This washing procedure was repeated.
- After the final wash each pellet was resuspended in approx. 1 ml 5 % TCA at approx. 4 °C for approx. 18 h for precipitation of macromolecules. Each precipitate was once washed again, resuspended in 1 ml 5 % TCA and 7 ml scintillation fluid was added. Then this solution was transferred into scintillation vials and stored at room temperature overnight.
Determination of incorporated 3H- Methyl thymidine:
The 3H-methyl thymidine-incorporation was measured in a beta-counter and expressed as the number of disintegrations per minute (DPM). Similarly, background 3H-methyl thymidine levels were also measured (5 % TCA). Determination of radioactivity was performed individually for each animal.

Positive control substance(s):

other: on 3 consecutive days concentrations of 1 % of P-Phenylenediamine (CAS 106-50-3, Sigma, purity <98 %; Lot 128K0093), AOO served as vehicle (4:1 (v/v) acetone/olive oil)

Statistics:

EC3 values, calculated concentrations which induce stimulation indices of three, are determined by linear interpolation {EC30c+[(3-d)/(b-d)]x(a-c)}, between two points of the stimulation indices axis, one above (a, b) and one below (c, d) the stimulation index of three. If all measured points are above or below the stimulation index of three, no EC3 value can be stated.

Results and discussion

Positive control results:

For results see "positive control" at "Attached Background Material".

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

None of the three tested concentrations of the test item reached the stimulation index of 3. The stimulation index at a concentration of 6.25 % was 1.4 The stimulation index at a concentration of 12.5 % was 1.4 The stimulation index at a concentration of 25 % was 2.2

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

Mean value for DPM in negative control AOO: 661.8 Mean value for DPM in Vanadium concentrate in AOO (Conc is 6.25 %): 912.8 Mean value for DPM in Vanadium concentrate in AOO (Conc is 12.5 %): 943.0 Mean value for DPM in Vanadium concentrate in AOO (Conc is 25 %): 1406.5 Mean value for DPM in background Szinti and TCA: 20.6

Any other information on results incl. tables

Table 1: Mean ear thickness

	Mean ear thickness [in mm] on
Test group	Day 1	Day 2	Day 3
6.25 %	0.20	0.20	0.21
12.5 %	0.20	0.20	0.21
25 %	0.19	0.20	0.21
negative control	0.19	0.20	0.21

The EC3 value (derived by linear interpolation) could not be calculated as the stimulation indices of all concentrations were below 3.

The results of radioactivity determination were supported by the means of the ear thickness per group, which showed no significant difference compared to the negative control.

Table 2: Radioactive Determination of the Test Substance Groups

POS	CPM	Test Item	Conc. [%]	Animal Number	DPM	DPM- mean background	DPM/Node	Stimulation Index
18	687*	Negative control AOO		16	1376*	n. d.	n. d.
19	311.0			17	661.0	640.4	320.2
20	372.0			18	745.0	724.4	362.2
21	219.0			19	439.0	418.4	209.2
22	399.0			20	802.0	781.4	390.7
MV	330.3			MV	661.8	641.2	320.6	1.0
SD	68.6			SD	138.0	138.0	69.0
42	241.0	Vanadium Concentrate in AOO	6,25	1	484.0	463.4	231.7	0.7
43	399.0			2	803.0	782.4	391.2	1.2
44	367.0			3	756.0	735.4	367.7	1.1
45	688.0			4	1390.0	1369.4	684.7	2.1
46	562.0			5	1131.0	1110.4	555.2	1.7
MV	451.4			MV	912.8	892.2	446.1	1.4
SD	156.4			SD	314.9	314.9	157.5	0.5
49	235.0	Vanadium Concentrate in AOO	12.5	6	473.0	452.4	226.2	0.7
50	798.0			7	1654.0	1633.4	816.7	2.5
51	496.0			8	1004.0	983.4	491.7	1.5
52	251.0			9	507.0	486.4	243.2	0.8
53	531.0			10	1077.0	1056.4	528.2	1.6
MV	462.2			MV	943.0	922.4	461.2	1.4
SD	207.3			SD	433.2	433.2	216.6	0.7
54	757.0	Vanadium Concentrate in AOO	25	11	1529.0	1508.4	754.2	2.4
55	253**			12	511**	n. d.	n. d.	n. d.
56	631.0			13	1272.0	1251.4	625.7	2.0
57	741.0			14	1507.0	1486.4	743.2	2.3
58	652.0			15	1318.0	1297.4	648.7	2.0
MV	695.3			MV	1406.5	1385.9	693.0	2.2
SD	54.6			SD	112.9	112.9	56.5	0.2
97	8.0	Background Szinti and TCA			16.0
98	7.0				14.0
99	8.0				16.0
100	19.0				38.0
101	9.0				19.0
MV	10.2			MV	20.6	0.0	0.0	0.0
SD	4.4			SD	8.8

n. d.   not determined

*       outlier, failed in Nalimov

**     outlier, failed in Grubbs, Nalimov, Dixon

If not noted individually, the results include both lymph nodes of an animal.

POS        position in counter

CPM       counts per minute

Conc.      concentration

DPM      disintegrations per minute

SD          standard deviation

MV        mean value

Szinti scintillation fluid

TCA       trichloroacetic acid

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

Vanadium concentrate as described in this report is expected to have no sensitising properties and therefore, should not be regarded as dermal sensitiser.

Executive summary:

In a dermal sensitization study with Vanadium Concentrate in AOO (acetone:olive oil 4:1), 20 eight to nine weeks old female CBA/CaOlaHsd mice were tested in an local lymph node assay (LLNA). Based on the results of the preliminary test the test item was assessed for sensitising properties at concentrations of 6.25 %, 12.5 % and 25 % (w/v). 20 animals were separated in four test groups, three treatment groups and the fourth group served as control animals which received the vehicle only. Clinical observations and weight changes were recorded daily. Positive control was P-Phenylenediamine 1 %, administered daily to the animals.

None of the three tested concentrations reached the stimulation index of 3, therefore the EC3 value (derived by linear interpolation) could not be calculated. The results of radioactivity determination were supported by the means of the ear thickness per group, which showed no significant difference compared to the negative control.

Consequently, according to OECD 429 and the criteria given in Annex I of Regulation (EC) 1272/2008, in this study, the test item Vanadium Concentrate is expected to have no sensitising properties and therefore, should not be regarded as dermal sensitizer.