Niobium hydride

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 May 2019 to 17 July 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

1992

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

2003

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: JMAFF guidelines

Version / remarks:

2000 (including the most recent revisions)

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The guinea pig Maximization test was selected since the test material could not be formulated homogenously in the vehicles accepted for the Local Lymph Node Assay as preferred alternative.
Attempts were made to conduct a LLNA on the substance. The testing laboratory performed trial preparations in an attempt to find a suitable vehicle for use in the study. As the substance consists of relatively big chunks the laboratory first ground some of these chunks using a mechanic grinder. The achieved powder was tested in the standard vehicles for LLNA studies: Acetone/Olive oil (4:1 v/v), N,N-dimethylformamide, methylethylketone, propylene glycol, dimethylsulfoxide and 1 % Pluronic© L92 in Elix water. In all these vehicles 50 and 20 % concentrations were tried but the substance settled out at the bottom of the container for each formulation. As such a suitable vehicle could not be found and it was not possible to continue with the LLNA study.

Test material

Test material information

1

Reference substance name:

Niobium hydride

EC Number:

237-769-9

EC Name:

Niobium hydride

Cas Number:

13981-86-7

Molecular formula:

HNb

IUPAC Name:

niobium(1+) hydride

Test material form:

solid

Details on test material:

Appearance: Silver grey solid

Specific details on test material used for the study:

PREPARATION OF TEST MATERIAL
Test material dosing formulations (w/w) were homogenised to visually acceptable levels at appropriate concentrations to meet dose level requirements.
The dosing formulations were kept at room temperature until dosing. The dosing formulations and vehicle were stirred until and during dosing.
No adjustment was made for specific gravity of the vehicle. No correction was made for the purity/composition of the test material.
Any residual volumes were discarded.

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Females (if applicable) nulliparous and non-pregnant: Yes
- Microbiological status of animals, when known: SPF-Quality
- Age at study initiation: Young adult animals (approximately 5 weeks old) were selected at the start of dosing.
- Weight at study initiation: 344 to 392 g at the start of dosing. Animals were assigned to the study according to body weights, with all animals within ± 20 % of the mean.
- Housing: On arrival and following assignment to the study, animals were group housed (up to 5 animals of the same sex and same dosing group together) in labelled cages (74 x 54 x 25 cm height) containing sterilised sawdust as bedding material equipped with water bottles. For psychological/environmental enrichment, animals were provided with shelters (90 x 5 x 125 mm).
- Diet: Complete maintenance diet for guinea pigs was provided ad libitum. In addition, hay was provided at least twice a week.
- Water: Municipal tap-water was freely available to each animal via water bottles.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 to 23 °C
- Humidity (relative): 42 to 71 %
- Air changes: Ten or greater air changes per hour with 100 % fresh air (no air recirculation) were maintained in the animal rooms
- Photoperiod: A 12-hour light/12-hour dark cycle

IN-LIFE DATES
The in-life phase of the study was completed on 12 July 2019

Study design: in vivo (non-LLNA)

Induction

Route:

intradermal and epicutaneous

Vehicle:

corn oil

Concentration / amount:

Guinea pigs were intradermally injected with a 5 % concentration and epidermally exposed to a 50 % concentration

Day(s)/duration:

Intradermal injections were made on day 1 and epidermal exposure took place on day 8

Adequacy of induction:

non-irritant substance, but skin pre-treated with 10% SDS

Challenge

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

corn oil

Concentration / amount:

50 %

Day(s)/duration:

Challenge took place on day 21

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

- Experimental group: 10 females
- Control group: 5 females

Details on study design:

PRELIMINARY IRRITATION STUDY
A preliminary irritation study was conducted in order to select test material concentrations to be used in the main study. The selection of concentrations was based on the following criteria:
- The concentrations are well-tolerated systemically by the animals.
- For the induction exposures: the highest possible concentration that produced mild to moderate irritation (grades 2 - 3).
- For challenge exposure: the maximum non-irritant concentration.
Series of test material concentrations were tested. Practical feasibility of administration determined the highest starting-concentration for each route. The starting and subsequent concentrations were taken from the series: 100 (undiluted), 50, 20, 10, 5, 2 and 1 %.
The test system and procedures were identical to those used during the main study, unless otherwise specified. The four animals selected were between 4 and 9 weeks old. No body weights were determined.

> Intradermal injections
A series of four test material concentrations was tested, the highest concentration being the maximum concentration that could technically be injected. Two animals received two different concentrations in duplicate (0.1 mL/site) in the clipped scapular region. The injection sites were assessed for irritation 24 and 48 hours after treatment.

> Epidermal application
A series of four test material concentrations was tested, the highest concentration being the maximum concentration that could technically be applied. Two different concentrations were applied (0.5 mL each) per animal to the clipped flank, using patches (2 x 3 cm) mounted on medical tape which were held in place with micropore tape and subsequently elastic bandage.
The animals receiving intradermal injections were treated with the lowest concentrations and two other animals with the highest concentrations. After 24 hours, the dressing was removed and the skin cleaned of residual test material using water. The treated skin areas were assessed for irritation 24 and 48 hours after removal of the dressings.

MAIN STUDY
The concentrations and induction method were selected based on the results of the preliminary irritation study.

> Induction: Experimental animals
- Day 1: The scapular region was clipped and three pairs of intradermal injections (0.1 mL/site) were made in this area as follows:
A) A 1:1 w/w mixture of Freund’s Complete Adjuvant with water for injection.
B) The test material at a 5 % concentration.
C) A 1:1 w/w mixture of the test material, at twice the concentration used in (B) and Freund’s Complete Adjuvant.
- Day 3: The dermal reactions caused by the intradermal injections were assessed for irritation.
- Day 7: The scapular area between the injection sites was clipped and subsequently rubbed with 10 % sodium-dodecyl-sulfate in Vaseline using a spatula. This concentration of SDS provokes a mild inflammatory reaction.
- Day 8: The 10 % SDS treated area between the injection sites was treated with 0.5 mL of a 50 % test material concentration using a patch (2 x 3 cm) mounted on medical tape, which was held in place with micropore tape and subsequently elastic bandage.
The dressing was removed after 48 hours exposure, the skin cleaned of residual test material using water and the dermal reactions caused by the epidermal exposure were assessed for irritation.

> Induction - Control animals
The control animals were treated as described for the experimental animals except that, instead of the test material, the vehicle was administered.

> Challenge - All animals
- Day 21: One flank of each animal was clipped and treated by epidermal application of a 50 % test material concentration and the vehicle (0.1 mL each, using patch test plasters). The patches were held in place with micropore tape and subsequently elastic bandage.
The dressing was removed after 24 hours exposure and the skin cleaned of residual test material and vehicle using water. The treated sites were assessed for challenge reactions 24 and 48 hours after removal of the dressing.
After termination, animals were sacrificed using isoflurane and an intracardial injection of Euthasol® 20 %.

> In-life Procedures, Observations, and Measurements
- Mortality/Moribundity Checks: Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from their cage during observation, unless necessary for identification or confirmation of possible findings.
- Toxicity: Observations for toxicity were performed once daily throughout the study.
- Body Weights: Animals were weighed individually on Day 1 (pre-dose) and after the challenge.
- Irritation: Irritation observations were performed according to the following numerical scoring system:

Grading of Irritation Reactions (intradermal reactions will be scored for erythema only or, if necrosis is present, the diameter of necrosis):
Erythema and eschar formation:
No erythema: 0
Slight erythema (barely perceptible): 1
Well-defined erythema: 2
Moderate erythema: 3
Severe erythema (beet redness) to slight eschar formation (injuries in depth): 4

Oedema formation:
No oedema: 0
Slight oedema (barely perceptible): 1
Well-defined oedema (edges of area well-defined by definite raising): 2
Moderate oedema (raised approximately 1 mm): 3
Severe oedema (raised more than 1 mm and extending beyond the area of exposure): 4

Grading of Challenge Reactions:
No visible change: 0
Discrete or patchy erythema: 1
Moderate and confluent erythema: 2
Moderate erythema and swelling: 3
Intense erythema and swelling: 4

- Terminal Procedures: Necropsy for gross macroscopic examination was not performed.

Challenge controls:

Two weeks after the epidermal application all animals were epidermally challenged with a 50 % test material concentration and the vehicle.

Positive control substance(s):

yes

Remarks:

ALPHA-HEXYLCINNAMALDEHYDE

Results and discussion

Positive control results:

Positive control concentrations selected for this study were:
- Intradermal induction: a 20 % solution in Corn oil.
- Epidermal induction: a 50 % solution in Corn oil.
- Challenge: a 50 % solution in Corn oil.

The skin reactions observed in all experimental animals in response to the 20 % concentration in the challenge phase were considered indicative of sensitisation, based on the absence of any response in the control animals. These results lead to a sensitisation rate of 100 % to the 50 % concentration.
From these results, it was concluded that the female guinea pig of the Dunkin Hartley strain is an appropriate animal model for the performance of studies designed to evaluate the sensitising potential of a test material in a Maximization type of test.

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

50 % concentration

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

None

Remarks on result:

no indication of skin sensitisation

Results 2

Key result

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

50 % concentration

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

None

Remarks on result:

no indication of skin sensitisation

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

50 % concentration

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

None

Remarks on result:

no indication of skin sensitisation

Results 4

Reading:

1st reading

Hours after challenge:

48

Group:

negative control

Dose level:

50 % concentration

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

None

Remarks on result:

no indication of skin sensitisation

Results 5

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

20 %

No. with + reactions:

5

Total no. in group:

10

Any other information on results incl. tables

Preliminary Irritation Study

Based on the results, the test material concentrations selected for the main study were a 5 % concentration for the intradermal induction and a 50 % concentration for the epidermal induction exposure.

No signs of irritation to the highest test material concentration epidermally tested were observed. Therefore, the test site of all animals of the main study were treated with 10 % SDS approximately 24 hours before the epidermal induction, to provoke a mild inflammatory reaction.

A 50 % test material concentration was selected for the challenge phase.

 

Main study

> Induction Phase: The reactions noted in the experimental and control animals after the epidermal induction exposure were considered to be enhanced by the SDS treatment.

> Challenge Phase: No skin reactions were evident after the challenge exposure in the experimental and control animals.

> Toxicity / Mortality: No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

> Body Weights: Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

 

Table 1: Induction Readings for Control Animals

Animal Number	Intradermal injections (readings Day 3)						Epidermal exposure (readings Day 10)
	1:1 Mixture of FCA and water for injection		Vehicle		1:1 Mixture of FCA and vehicle		Vehicle
	Erythema	Signs of necrosis (Ø mm)	Erythema	Signs of necrosis (Ø mm)	Erythema	Signs of necrosis (Ø mm)	Erythema	Oedema
21	2		0		1		0	0
22		3	0			2	0	0
23	2		1		1		0	0
24		2	1		2		0	0
25		2	1			2	0	0

 

Table 2: Induction Readings for Experimental Animals

Animal Number	Intradermal injections (readings Day 3)						Epidermal exposure (readings Day 10)
	1:1 Mixture of FCA and water for injection		5 % Test Material Concentration		1:1 Mixture of FCA and a 10 % Concentration		50 % Test Material Concentration
	Erythema	Signs of necrosis (Ø mm)	Erythema	Signs of necrosis (Ø mm)	Erythema	Signs of necrosis (Ø mm)	Erythema	Oedema
26		2	1		1		0t	0
27		1	1		2		0t	0
28		1	1			1	0t	0
29	2		2		2		0t	0
30	2		1		2		0t	0
31	1		1		2		0t	0
32		2	2			2	0t	0
33		1	1		1		0t	0
34	2		1			1	0t	0
35		1	1			1	0t	0

t: Grey staining of the skin, caused by staining properties of the test material

 

Table 3: Challenge Readings

Animal number	Day 23		Day 24		Comments
	Test material concentration 50 %	Vehicle	Test material concentration 50 %	Vehicle
Control
21	0	0	0	0	Not sensitised
22	0	0	0	0	Not sensitised
23	0	0	0	0	Not sensitised
24	0	0	0	0	Not sensitised
25	0	0	0	0	Not sensitised
Experimental
26	0	0	0	0	Not sensitised
27	0	0	0	0	Not sensitised
28	0	0	0	0	Not sensitised
29	0	0	0	0	Not sensitised
30	0	0	0	0	Not sensitised
31	0	0	0	0	Not sensitised
32	0	0	0	0	Not sensitised
33	0	0	0	0	Not sensitised
34	0	0	0	0	Not sensitised
35	0	0	0	0	Not sensitised

 

Applicant's summary and conclusion

Interpretation of results:

other: Not classified in accordance with EU criteria

Conclusions:

Under the conditions of this study, the test material was determined not to be a sensitiser in the guinea pig maximisation test.

Executive summary:

The purpose of this study was to evaluate whether the test material induces contact hypersensitivity in guinea pigs after intradermal and epidermal exposure in accordance with the standardised guidelines OECD 406, EU Method B.6, US EPA OPPTS 870.2600 and JMAFF Guidelines (2000). The study was conducted under GLP conditions.

Test material concentrations selected for the main study were based on the results of a preliminary study. In the main study, ten female Dunkin Hartley guinea pigs were intradermally injected with a 5 % concentration and epidermally exposed to a 50 % concentration. Five control animals were similarly treated, but with vehicle alone (corn oil). Approximately 24 hours before the epidermal induction exposure all animals were treated with 10 % SDS. Two weeks after the epidermal application all animals were epidermally challenged with a 50 % test material concentration and the vehicle.

There was no evidence that the test material had caused skin hypersensitivity in the guinea pig, since no responses were observed in the experimental animals in response to a 50 % test material concentration in the challenge phase. This result indicates a sensitisation rate of 0 per cent.

Under the conditions of this study, the test material was determined not to be a sensitiser in the guinea pig maximisation test.