Niobium oxide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-08-31 to 2009-10-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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:

Diniobium pentaoxide

EC Number:

215-213-6

EC Name:

Diniobium pentaoxide

Cas Number:

1313-96-8

Molecular formula:

Nb2O5

IUPAC Name:

diniobium pentaoxide

Specific details on test material used for the study:

- Name: Diniobium Pentaoxide
- CAS: 1313-96-8
- Batch No.: AD/4199
- Density: ca. 4.5 g/cm³
- Active components: >98.5%
- Colour: pale yellow
- Physical state: solid
- Storage: at room temperature
- Stability: stable

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 Winkelmann, D-33178 Borchen, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: The animals were derived from a controlled full-barrier maintained breeding system (SPF).
- Age at study initiation: 8 - 9 weeks
- Weight at study initiation:
- Housing: The animals were kept in groups of 5 animals in IVC cages, type II L, polysulphone cages on Altromin saw fibre bedding (lot no. 04.05.09, main test; lot no. 050109, preliminary test)
- Diet (e.g. ad libitum): Free access to Altromin 1324 maintenance diet for rats and mice (lot no.0654)
- 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, microbiol. controlled at regular intervals)
- Acclimation period: at least 5 days
- Indication of any skin lesions: none

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): at least I 0 x I hour
- Photoperiod (hrs dark / hrs light): 12 / 12
- IN-LIFE DATES: From: 2009-09-14 To: 2009-10-01

Study design: in vivo (LLNA)

Vehicle:

other: acetone olive oil (AOO 3+1(v/v)

Concentration:

12.5%, 25% and 50% (suspended in AOO 3+1)

No. of animals per dose:

3 (2+1 control) animals for the pretest
5 animals/ test group in the main study

Details on study design:

PRE-SCREEN TESTS:
To determine the highest tolerated and non-irritant test concentration a preliminary test was performed. For this purpose, two animals were treated by topical application with the test item on three consecutive days at a concentration of 50% (suspended in AOO) to the entire dorsal surface of each ear. One further animal was treated with 100% AOO and served as negative control.
From day 1 to day 4 the ear thickness of each animal was measured. During this period also all clinical signs were recorded. Cageside observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge).
For details on results please refer to the results section "Any other information on results incl. tables ".

MAIN STUDY
- Topical Application
Each mouse was treated by topical application of 25 μL of the selected solution to the entire dorsal surface of each ear. 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 250μL of 3H-methyl thymidine, diluted to a working concentration of 80μCi/mL.
- Preparation of cell suspension
Approximately 5 hours after 3H-methyl thymidine-injection all mice were sacrificed by cervical dislocation. The draining “auricular lymph nodes” were excised, individually pooled for each animal (2 lymph nodes per 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 approximately 18 hours 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 ß-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.

OTHER:
Reliability checks with a positive control substance was performed periodically in the performing laboratory.

Positive control substance(s):

other: P-Phenylenediamine (CAS 106-50-3)

Results and discussion

Positive control results:

Reliability checks with a positive control substance was performed periodically in the performing laboratory. The mean SI-value of the latest reliability check was 13.8 (see table 2 in box "Any other information on results incl. tables).

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

0.7

Variability:

SD = 0.3

Test group / Remarks:

12% Diniobium pentaoxide

Results 2

Parameter:

SI

Value:

1.3

Variability:

SD = 0.4

Test group / Remarks:

25 % Diniobium pentaoxide

Results 3

Parameter:

SI

Value:

0.9

Variability:

SD = 0.4

Test group / Remarks:

50 % Diniobium pentaoxide

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION
For detailed results please refer to table 3 presented in box "Any other information on results incl. tables".

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

CLINICAL OBSERVATIONS:
All animals survived throughout the test period without showing any clinical signs.

BODY WEIGHTS
All animals showed the expected weight development, which includes a weight loss of up to 2 g throughout the study.

Any other information on results incl. tables

Results of the preliminary test:

Table 1: Ear Thickness - Preliminary test

Group	Animal No.	Day 1 [mm]	Day 2 [mm]	Day 3 [mm]	Day 4 [mm]
50% Diniobium Pentaoxide	1	0.18	0.17	0.17	0.18
	2	0.17	0.17	0.17	0.18
100 % AOO (3+1) Negative control	3	0.17	0.17	0.18	0.17

Neither signs of systemic toxicity nor signs of irritation at the application site could be detected in the animals.

Results of the Reliability Check (Positive Control)

Table 2: Stimulation indices of the Positive-Control Group of the Recent Study

Test Item	Concentration [%]	Animal Number	Stimulation Index
AOO (3+ I (v/v) Acetone/Olive Oil)	100	16
		17
		18
		19
		20
		MV	1.0
		SD
Phenylenediamine	1	81	10.2
		82	11.0
		83	14.0
		84	17.1
		85	16.8
		MV	13.8
		SD	2.9

Results of the Main Study

Table 3: Stimulation Indices obtained in the main study

Test Item	Concentration [%]	Animal Number	Stimulation Index
AOO (3+ I (v/v) Acetone/Olive Oil)	100	16
		17
		18
		19
		20
		MV	1.0
		SD
Diniobium Pentaoxide	12.5	1	1.0
		2	1.0
		3	0.4
		4	n.d.*
		5	0.5
		MV	0.7
		SD	0.3
Diniobium Pentaoxide	25	6	1.0
		7	1.0
		8	1.7
		9	0.7
		10	1.8
		MV	1.3
		SD	0.4
Diniobium Pentaoxide	50	11	0.3
		12	0.9
		13	1.3
		14	1.0
		15	n.d.*
		MV	0.9
		SD	0.4

* = outlier, failed Grubbs, Nalimov and Dixon; n.d. = not determined

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

SD = standard deviation; MV = mean value

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Diniobium pentaoxide is not sensitizing under the conditions of this LLNA study (OECD 429).

Executive summary:

In a dermal sensitization study conducted according to OECD 429 with Diniobium pentaoxide (> 98.5 % purity) suspended in AOO (3 +1 v/v acetone/olive oil), young adult female CBA/OlaHsd mice (5 per dose group) were tested at concentrations of 12.5% (w/v), 25 % (w/v) and 50 % (w/v) in a local lymph node assay (LLNA). Due to animal welfare reasons a periodically performed positive control (Phenylenediamine in AOO (3 +1 v/v) was used. There was no mortality nor clinical observations nor effects on body weights observed. None of the tested concentrations of the test substance reached the stimulation index threshold of 3. Therefore no EC3 value could be determined. In this study, Diniobium pentaoxide is not a dermal sensitizer.