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Toxicological information

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:
17 May 2016 to 1 July 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
other: read-across target
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted on read-across material
Justification for type of information:
Read-across to structurally similar substance erbium, gadolinium, yttrium and zirconium oxide (EC 946-001-8). The only difference between the substance being registered and the read-across substance is that the substance to be registered is missing the gadolinium oxide content (about 1.4 %). The absence of this compound at such a low % is considered highly unlikely to affect its properties.
Reason / purpose:
read-across source
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
75% (w/v) in sesame oil
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no visible changes
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
75% (w/v) in sesame oil
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no visible changes
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
75% (w/v) in sesame oil
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no visible changes
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
75% (w/v) in sesame oil
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no visible changes
Remarks on result:
no indication of skin sensitisation
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 sensitised guinea pigs
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
50% (w/v) mercaptobenzothiazole
No. with + reactions:
8
Total no. in group:
10
Clinical observations:
discrete erythema (score 1) on the skin of sensitised guinea pigs
Remarks on result:
positive indication of skin sensitisation

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:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
2003
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Due to the chemical nature of the test material, the Sponsor decided that the LLNA was not appropriate hence a study according to OECD Guideline 406 was required.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: PEGYZ-23
- Expiration date of the lot/batch: 31 March 2021
- Purity: 100%

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.

OTHER SPECIFICS
Description: Pink 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. Possibly irritative.
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Solubility and stability of the test material in the solvent/vehicle: The selection of the vehicle was based on trial formulations with the test material. Physiological saline, 1 % methyl cellulose and 1 % carboxymethylcellulose solutions caused noticeably rapidly settling formulations, therefore were considered as not suitable for treatments. Consequently, sesame oil was also tried as a vehicle, which resulted in an acceptably stable suspension, suitable for treatments. The stability and concentration in vehicle were not determined.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
For the intradermal induction treatment, the test material was ground with a Mixer Mill MM 400 ball mill (produced by Retsch GmbH, Germany) to a finer powder. The grinding was performed as follows:
Period of time: 3 minutes
Frequency: 30 1/sec

The formulation was homogenised to visually acceptable levels and stirred until animal treatment was completed, to ensure sufficient homogeneity.

The dose levels and the vehicle selection for the main study were based on results of a Preliminary Dose Range Finding Study. The test material was weighed and formulations were prepared daily on a weight:volume basis (as % (w/v)).

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 7 weeks old (young adult).
- Weight at study initiation: 410 - 445 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, NN16201718), 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: 20 days before start of treatment under laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.4 - 23.7 °C
- Humidity (%): 23 - 80 %
- 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: sesame oil
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)
Remarks:
dampened with saline
Concentration / amount:
amount: 0.5 g
Day(s)/duration:
day 8 of treatment; 48 hours of exposure
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Challengeopen allclose all
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: sesame oil
Concentration / amount:
concentration: 75% (w/v)
volume applied: 0.5 mL
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: sesame oil
Concentration / amount:
concentration: 37.5 % (w/v)
volume applied: 0.5 mL
Day(s)/duration:
day 22 of treatment; 24 hours of exposure
Adequacy of challenge:
other: safeguard dose
No. of animals per dose:
Preliminary test: 9 animals
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 5 x 5 cm). The hair removal was performed carefully to ensure animals are closely shaven.
- A series of test material concentrations was tested to identify the primary irritation following intradermal injection and dermal application: 0.01, 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, and 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 0 or 1) during the observation period, therefore this concentration can be used in the main study.
First, the intradermal application was tried with the test material in the form as it was supplied by the Sponsor, but the treatments could not be performed (the test material blocked the needle of the syringe). Because of this, the test material was ground with a ball mill, and the intradermal preliminary study was repeated. During the first intradermal experiment, one animal was used before it became clear that the test material is not suitable for the treatment.
- For the dermal application, the volume of the concentrations was 0.5 mL. For the 100 % treatment, 0.5 g test material was applied, and then dampened with saline. 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. Slight erythema was observed in one animal at a concentration of 100 % (as supplied, dampened with saline) at 1 and 24 hours. Besides this, the other dermal treatments at the tested concentrations produced no reaction on the skin of guinea pigs. The concentration used for the challenge exposure should be the highest non-irritant dose. Therefore, 75 % test material formulated in sesame oil was used for the challenge treatment.

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 material was omitted.

MAIN STUDY - INTRADERMAL INDUCTION EXPOSURE
On the day before treatment, an area approximately 5 x 5 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 material in sesame oil,
• 2 injections of 5 % test material (in sesame oil), 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 sesame oil,
• 2 injections of sesame oil, 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.
Seven days after the intradermal injections, the same hair-free scapular area was treated. 0.5 g of the test material was placed on a 2.5 x 2.5 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 sesame oil 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 material was removed with a wet 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 5 x 5 cm on the left and right sides of each animal. A 5 x 5 cm patch of sterile gauze patch was saturated with 75 % (w/v) dilution of test material and applied to the left side of all animals (both the test and the control). The right shaved side of all animals was treated with 50 % dilution of the maximum dermal challenge dose as a safeguard dose (37.5 % (w/v)).
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 material was removed with a wet gauze swab.

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 of classification of skin sensitisation:
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 maximum dermal challenge dose as a safeguard dose (37.5 % (w/v)).
Positive control substance(s):
yes
Remarks:
2-mercaptobenzothiazole

Results and discussion

Positive control results:
Challenge with reference material 2-mercaptobenzothiazole at 50 % (w/v) in 1 % methyl cellulose 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 material. The dermal scores represented discrete erythema (score 1) developed on the skin of sensitised guinea pigs. On the basis of the results of the relability check study, the reference material 2-mercaptobenzothiazole was classified as a skin sensitiser. This demonstrated that the experimental procedures and the test system were appropriate.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
75% (w/v) in sesame oil
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 group
Dose level:
75% (w/v) in sesame oil
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:
75% (w/v) in sesame oil
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:
75% (w/v) in sesame oil
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 sensitised guinea pigs
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) mercaptobenzothiazole
No. with + reactions:
8
Total no. in group:
10
Clinical observations:
discrete erythema (score 1) on the skin of sensitised guinea pigs
Remarks on result:
positive indication of skin sensitisation

Any other information on results incl. tables

Skin Effects after the Challenge Exposure

Test group: After the challenge with the test material at a concentration of 75 % (w/v), no positive response was observed in the treated animals. The mean of the scores was 0.00 according to the 24 and 48-hours results. The right shaved side of all animals was treated with a test material concentration of 37.5 % (w/v) as a safeguard dose and no reaction was noted.

 

Control group: After the challenge with the test material at a concentration of 75 % (w/v), no visible changes were found at the 24 and 48 hours examinations. The right shaved side of control animals was treated with a test material concentration of 37.5 % (w/v) as a safeguard dose and no reaction was noted.

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:
other: Not classified according to EU criteria
Conclusions:
Under the conditions of the assay, the test material was shown to have no sensitisation potential and is consequently classified as a non-sensitiser, according to current EU-regulations.
Executive summary:

The skin sensitisation potential of the test material was investigated in accordance with the standardised guidelines OECD 406, EU Method B.6 and EPA OPPTS 870.2600, under GLP conditions, following the Magnusson and Kligman method, using a maximisation method.

Based on the results of a preliminary test, ten test animals were subjected to sensitisation procedures in a two-stage process, named induction phase: i.e. an intradermal treatment and a 48-hour topical application (dermal treatment under an occlusive dressing). The test material was used at a concentration of 5 % (w/v) for intradermal injections and at a concentration of 100 % (undiluted, dampened with saline) for topical sensitisation treatment. Five control guinea pigs were simultaneously exposed to sesame oil only during the sensitisation phase.

Two weeks after the last induction exposure, a challenge dose at a concentration of 75 % (w/v) was administered on the left side of all animals. The right side of the animals was treated with 50 % dilution of the maximum dermal challenge dose as a safeguard dose (37.5 % (w/v)). Challenge was performed by dermal application of the test material. Skin reactions were measured 24 and 48 hours after patch removal.

Under the conditions of the study no signs of systemic toxicity were observed in any animal. Furthermore, no signs of contact sensitisation were detected in guinea pigs previously exposed to the test material during the experiment. In the control and treated animals the mean of the scores was 0.00 according to the 24 and 48-hour results.

The test material was therefore concluded to have no sensitisation potential and is considered a non-sensitizer, according to current EU-regulations.