Aluminum magnesium vanadium oxide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 Feb - 06 Mar 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

divanadium(5+) dialuminium(3+) magnesium(2+) nonaoxidandiide

EC Number:

692-448-4

Cas Number:

170621-28-0

Molecular formula:

Mg2xAl2xVyOz

IUPAC Name:

divanadium(5+) dialuminium(3+) magnesium(2+) nonaoxidandiide

Test material form:

solid: particulate/powder

Details on test material:

light yellow

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories B.V., AD Horst, The Netherlands
- Age at study initiation: 10-11 weeks (pre-test); 8-9 weeks (main study)
- Weight at study initiation: 20.7 g (animals of the range-finding test) and 20.2 g (animals of the main study)
- Housing: animals were housed in Makrolon Type II (pre-test)/III (main study) with wire mesh top (EHRET GmbH, Emmendingen, Germany) and granulated soft wood bedding (Rettenmaier & Söhne GmbH + Co. KG, Rosenberg, Germany).
- Diet: pelleted standard diet (Harlan Laboratories B.V., AD Horst, Netherlands), ad libitum
- Water: tap water (Gemeindewerke, Rossdorf, Germany), ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 32-65 (acclimation period); 45-65 (pre-test and main study)
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

5, 10, and 25% (w/v)

No. of animals per dose:

Range-finding test: 1 (in test groups)
Main study: 4 (controls), 4 (in test groups)

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: the compound solubility was determined according to the recommendations given in OECD guideline 429. The highest test item concentration, which could be technically used, was a 25% suspension in propylene glycol. At higher concentrations, an applicable formulation of the test item was not achieved, neither by the use of other vehicles nor by using additional methods to formulate the test item (e.g. sonication, warming to 37 °C).
- Irritation: to determine the highest non-irritant test concentration that at the same time did not induce signs of systemic toxicity, two mice were treated once daily on three consecutive days with concentrations of 10% and 25% by topical (epidermal) application to the dorsal surface of each ear. Clinical signs were recorded at least once daily. Signs of local skin irritation were documented and a score was used to grade a possible erythema of the ear skin. Prior to the first application of the test item (on Day 1), on Day 3 and before sacrifice (on Day 6) the ear thickness was determined using a micrometer (S0247 Kroeplin, Schlüchtern, Germany). Ear irritation was considered to be excessive if an erythema of the ear skin of a score value ≥ 3 was observed at any observation time and/or if an increase in ear thickness of ≥ 25% was recorded on Day 3 or Day 6. At the tested concentrations, the animals did not show any signs of local skin irritation or systemic toxicity. Based on these results, concentrations selected for the main study were 5, 10 and 25% (w/w).

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: ³H-methyl thymidine (³HTdR) incorporation determined by β-scintillation
- Criteria used to consider a positive response: the test substance was considered a skin sensitiser in the LLNA if the following criteria were met:
(1) the exposure to at least one concentration of the test substance resulted in an incorporation of ³HTdR at least 3-fold greater than in controls (as indicated by the stimulation index) and
(2) the data were compatible with a conventional dose response, although allowance had to be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION: each animal was treated by topical application of 25 μL of the different test substance concentrations and the respective vehicle (propylene glycol as vehicle control) on the entire dorsal surface of each ear. The application was repeated on Days 2 and 3. On Day 6, each animal was treated with 250 μL of 80.4 μCi/mL ³HTdR (corresponds to 20.1 µCi ³HTdR per mouse) in phosphate buffered saline (PBS) by intravenous injection via the tail vein. After five hours, mice were euthanised by intraperitoneal injection of sodium pentobarbital and the draining auricular lymph nodes were rapidly excised. The nodes of each animal were pooled (8 nodes per group) and single cell suspensions (SCS) in PBS were prepared by gentle mechanical disaggregation of each pooled lymph node cells (LNCs) through stainless steel gauze (200 µm mesh size). LNCs were pelleted by centrifugation, and washed twice with PBS. After removal of the washing solution, pellets were resuspended in 5% trichloroacetic acid and incubated at 4 °C for approximately 18 h for precipitation of macromolecules.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Mean values and standard deviations of body weights and DPM values were calculated. Statistical analysis of the mean DPM values was performed between treated and control groups to assess a dose-response relationship.

Results and discussion

Positive control results:

A significant increase in the stimulation indices (SI ≥ 3) was noted for the positive control substance hexyl cinnamic aldehyde in acetone:olive oil (4+1) at a concentration of 25% (SI = 5.9)

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.03

Test group / Remarks:

5% test substance

Results 2

Key result

Parameter:

SI

Value:

0.58

Test group / Remarks:

10% test substance

Results 3

Key result

Parameter:

SI

Value:

0.68

Test group / Remarks:

25% test substance

Results 4

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

The lymph nodes of each group were pooled and group DPM values were measured from the pooled lymph node cell suspensions. The measured group DPM values were corrected by subtracting the background DPM value (average of the two measured DPM values of 5% (w/v) trichloroacetic acid solutions). Treatment with 5, 10 and 25% (w/v) test substance in propylene glycol resulted in group DPM/lymph node of 250.1, 139.9 and 165.4, respectively (see Table 1 under "Any other information on results incl. table"). The DPM/lymph node value for the control group was 241.8.

Any other information on results incl. tables

No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

Table 1. Calculation of group DPM and SI values.

Test item concentration % (w/v)	Group	Measurement DPM	Calculation			Result
			DPM-BG*	Number of lymph nodes	DPM per lymph node**	SI
-	BG I	19	-	-	-	-
-	BG II	20	-	-	-	-
0	1	1954	1935	8	241.8	1.00
5	2	2020	2001	8	250.1	1.03
10	3	1139	1120	8	139.9	0.58
25	4	1343	1324	8	165.4	0.68

BG = background (1 mL 5% trichloroacetic acid)

1 = control group; 2-4: test groups

SI = stimulation index relative to the mean of the control group (group 1)

* values corrected for mean background value (BG I and BG II)

** Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

CLP: not classified