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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2016-05-17 to 2016-06-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Due to the chemical nature of the test item, the Sponsor decided that the LLNA was not appropriate hence a study according to OECD Guideline 406 was required.

Test material

Constituent 1
Chemical structure
Reference substance name:
Zirconium diboride
EC Number:
234-963-5
EC Name:
Zirconium diboride
Cas Number:
12045-64-6
Molecular formula:
ZrB2
IUPAC Name:
zirconium diboride
Test material form:
solid: particulate/powder
Details on test material:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: ZrB2-3.2/14
- Expiration date of the lot/batch: 04 March 2017
- Purity: 99%
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25 ºC, below 70% relative humidity), protected from light and humidity
OTHER SPECIFICS:
Description: Grey powder (the colour was determined by visual inspection upon arrival at test facility)
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personal health and safety.
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Solubility and stability of the test substance in the solvent/vehicle: The selection of the vehicle was based on trial formulations with the test item. It is preferred to use aqueous vehicles whenever possible, therefore physiological saline solution was tried first (as distilled water is not compatible with intradermal treatments). This caused a noticeably rapidly settling formulation, therefore it was considered as not suitable for treatments. Consequently, 1% methyl cellulose was also tried as a vehicle, which resulted in an acceptably stable suspension, suitable for treatments.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The formulations were freshly formulated at appropriate concentrations in the vehicle on the day of administration. The stability and concentration in vehicle were not determined.
However, to limit the impact, the formulations were used within 4 hours after adding the vehicle to the test item and the test item preparation was performed with approved procedures and documented in detail. The formulations were homogenised to visually acceptable levels and stirred up to finishing the treatment to ensure sufficient homogeneity.

- No correction was made for the purity/composition of the test item. The dose levels and the vehicle selection for the main study were based on results of a Preliminary Dose Range Finding Study. The test item was weighed and formulations were prepared daily on a weight:volume basis (as % (w/v)) in the Pharmacy of CiToxLAB Hungary Ltd.

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: albinos, LAL/HA/BR
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: LAB-ÁLL Bt., Budapest, 1174 Hunyadi u. 7., Hungary
- Age at study initiation: around 6 weeks old (young adult)
- Weight at study initiation: 351-395 g
- Housing: Macrolon cages size IV, with 5 animals/cage to allow socialisation.
- Bedding: “Lignocel® 3/4-S Hygienic Animal Bedding” produced by J. Rettenmaier & Söhne GmbH & CO.KG (D-73494 Rosenberg, Germany) was available to animals during the study.
- Diet: Cunigra Diet for Rabbits (produced by Bonafarm-Bábolna Takarmány Ltd., Hungary - batch n° NN16201146, NN16200770, NN16201463), ad libitum.
- Water: Tap water from municipal supply as for human consumption, containing 50 mg/100 mL ascorbic acid, ad libitum. The drinking water is routinely analysed and is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
- Acclimation period: 13 days before start of treatment under laboratory conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.4 - 23.7°C
- Humidity (%): 24 - 77%
- Air changes (per hr): 15-20
- Photoperiod: 12 hours light daily, from 6.00 a.m. to 6.00 p.m.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
other: 1% methyl cellulose
Concentration / amount:
concentration: 5% (w/v)
volume: 0.1 mL per injection
Day(s)/duration:
day 1 of treatment
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
amount: 0.5 g
concentration: 100% (undiluted, dampened with saline)
Day(s)/duration:
day 8 of treatment; 48 hours of exposure
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
Challengeopen allclose all
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
amount: 0.5 g
concentration: 100% (undiluted, dampened with saline)
Day(s)/duration:
day 22 of treatment; 24 hours of exposure
Adequacy of challenge:
highest non-irritant concentration
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: 1% methyl cellulose in saline
Concentration / amount:
volume applied: 0.5 mL
concentration: 50% (w/v)
Day(s)/duration:
day 22 of treatment; 24 hours of exposure
Adequacy of challenge:
other: safeguard dose
No. of animals per dose:
Preliminary test: 8 animals (2 animals per concentration for each type of application)
Main test: 10 animals for the test group and 5 animals for the control group
Details on study design:
PRELIMINARY DOSE RANGE FINDING STUDY
- A day prior to the test, the hair was removed from the right and left sides of the animals (approximately 5x5 cm). The hair removal was performed carefully to ensure animals are closely shaven.
- A series of test item concentrations was tested to identify the primary irritation following intradermal injection and dermal application: 0.05, 0.1, 0.5, 1, 2.5 and 5% (w/v) concentrations were used for intradermal injection and 25, 50, 75% (w/v) and 100% (as supplied, dampened with saline) for dermal application. Local effects were examined and scored 1, 24, 48 and 72 hours after the treatment or after patch removal. Skin effects were scored for erythema and oedema, any other observations of changes to the skin were recorded.
- For the intradermal application, 0.1 mL per concentration was injected intradermally into the hair free skin of the animals. Two concentrations were injected on the right side and another two concentrations on the left side of the animals. The highest concentration (5%) was also tested in a 1:1 mixture (v/v) of Freund's Complete Adjuvant and physiological saline solution. Each concentration was injected in duplicate. Two animals were used per concentration. The highest concentration (5%) caused no more than mild-to-moderate erythema (score 1 or 2) during the observation period, therefore this concentration can be used in the main study.
- For the dermal application, the volume of the concentrations was 0.5 mL. For the 100% treatment, 0.5 g test item was dampened with saline, and then applied to the skin. A closed patch exposure was performed by means of an occlusive bandage using similar treatment procedures as for the main study. The time of exposure for the dermal application was 48 hours. One concentration was used on the right side and another concentration on the left side of animals. Two animals per concentrations were used.
- It was found that all the dermal and intradermal treatments formulated in the vehicle at the tested concentrations or applied without formulation produced no reaction on the skin of guinea pigs. Only the highest intradermal concentration (5%) formulated in a 1:1 mixture (v/v) of Freund's Complete Adjuvant and physiological saline solution caused scores above zero.

MAIN STUDY
The dose levels for the main study were selected based on the results of the preliminary test.
Control animals were treated similarly to test animals, except that during the induction phase, the test item was omitted.

MAIN STUDY - INTRADERMAL INDUCTION EXPOSURE
On the day before treatment, an area approximately 5x5 cm on the scapular region of the animals was clipped free of hair and was carefully shaved.
Three pairs of intradermal injections (0.1 mL/site) were made in the clipped region as follows (the first listed nearest the head):
Test group:
• 2 injections of Freund's Complete Adjuvant and physiological saline solution in a 1:1 (v/v) mixture,
• 2 injections of 5% test item in 1% methyl cellulose,
• 2 injections of 5% test item (in 1% methyl cellulose), formulated in a 1:1 mixture (v/v) of Freund's Complete Adjuvant and physiological saline solution.
Control group:
• 2 injections of Freund's Complete Adjuvant and physiological saline solution in a 1:1 (v/v) mixture,
• 2 injections of 1% methyl cellulose,
• 2 injections of 1% methyl cellulose, formulated in a 1:1 mixture (v/v) of Freund's Complete Adjuvant and physiological saline solution.

MAIN STUDY - DERMAL INDUCTION EXPOSURE
The same scapular region which received the intradermal injections, was used for dermal induction exposure.
Since the undiluted test item was not skin irritant in the dermal dose range-finding study, the test area was painted with 0.5 mL of 10% sodium dodecyl sulphate in Vaseline 24 hours prior to the topical induction application, in order to create a local irritation.
Seven days after the intradermal injections, the same hair-free scapular area was treated. 0.5 g of the test item was placed on a 2.5x2.5 cm sterile gauze patch (4 layers of porous gauze pads) and dampened with saline, then applied over the injection sites. The control group was treated with 1% methyl cellulose only.
The gauze patches were kept in contact with the skin by a patch with a surrounding adhesive hypoallergenic plaster. The treated areas were covered for 48 hours with a fully occlusive foil (Closed Patch Test). After the patch removal any remaining test item was removed with an ointment and gauze swab.
Following the dermal induction treatment, the animals were left untreated for 14 days prior to challenge applications.

MAIN STUDY - CHALLENGE EXPOSURE
Two weeks after the topical induction application, the animals were exposed to a dermal challenge dose. Approximately 24 hours before the treatment, the hair was removed from an area of approximately 5x5 cm on the left and right sides of each animal. 0.5 g of the test item was dampened with saline on a 5x5 cm patch of sterile gauze patch, then applied to the left side of all animals (both the test and the control).
Treatment was performed as in the dermal induction exposure (Closed Patch Test). The time of exposure was 24 hours. After the patch removal any remaining test item was removed with a wet gauze swab.

Terminal procedure:
Terminally animals were sacrificed under pentobarbital anaesthesia.

OBSERVATION AND SCORING
Detailed clinical observations were made on all animals outside the home cage in a standard arena before the first treatment (on the day of randomisation) and at least weekly thereafter. The dermal irritation scores (in cases of dermal induction exposures) were evaluated according to the scoring system by Draize (1959):
- Erythema and eschar formation:
0 = No Erythema
1 = Very slight Erythema (barely perceptible)
2 = Well defined Erythema
3 = Moderate to severe Erythema
4 = Severe Erythema (beef redness) to slight eschar formation (injuries in depth)

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

After the challenge exposure, each animal was examined and scored 24 and 48 hours after the end of the exposure period.
Grading was performed according to the following system:
0 = no visible change
1 = discrete or patchy erythema
2 = moderate and confluent erythema
3 = intense erythema and swelling
Challenge controls:
The right shaved side of all animals was treated with 50% dilution of the test item in 1% methyl cellulose.
Positive control substance(s):
yes
Remarks:
2-mercaptobenzothiazole

Results and discussion

Positive control results:
Challenge with reference item 2-mercaptobenzothiazole resulted in a positive response in test animals previously sensitised. The net response values at the 24 and 48 hours observations represented an incidence rate of 90% and 80% and net score values of 0.90 and 0.80 respectively. In the control animals no visible changes were found either at the 24 or 48 hours examinations following challenge with the reference item.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
100% (undiluted, dampened with saline)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no visible changes
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
100% (undiluted, dampened with saline)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no visible changes
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
100% (undiluted, dampened with saline)
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no visible changes
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
100% (undiluted, dampened with saline)
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no visible changes
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
50% w/v 2-mercaptobenzothiazole
No. with + reactions:
9
Total no. in group:
10
Clinical observations:
discrete erythema (score 1) on the skin of the animals
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
50% w/v 2-mercaptobenzothiazole
No. with + reactions:
8
Total no. in group:
10
Clinical observations:
discrete erythema (score 1) on the skin of the animals
Remarks on result:
positive indication of skin sensitisation

Any other information on results incl. tables

Skin effects after the challenge exposure:

The right shaved side of all animals was treated with a test item concentration of 50% (w/v) as a safeguard dose and no reaction was noted (test group and control group).

Clinical observations/mortality:

No signs of systemic or local toxicity were observed in any animal. No mortality was observed during the study.

Body weight:

There were no notable differences between the test animal group and the control group.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the assay, the test item zirconium diboride was shown to have no skin sensitisation potential and is consequently classified as a non-sensitizer, according to current EU-regulations.