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Diss Factsheets

Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
May 30th to June 30th, 2016
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Justification for type of information:
Justification for Read Across is given in section 13 of IUCLID.

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:
other: ISO 10993-10:2010 (Biological evaluation of medical devices - Part 10: Tests for irritation and skin sensitisation)
Qualifier:
according to guideline
Guideline:
other: ISO 10993-2:2006 (Biological evaluation of medical devices - Part 2: Animal welfare requirements)
GLP compliance:
no
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
A LLNA study has not been conducted because adequate data from guinea pig Maximisation test study are already available.

Test material

Constituent 1
Reference substance name:
not applicable
IUPAC Name:
not applicable

In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River- Calco (LC) - Italy
- Age at study initiation:
- Weight at study initiation: 300-500 g
- Housing: caged in groups of five (control group) and in group of ten (treated group) in NORYL cages (dimensions 74.3x54.3x25 cm). The cages and the housing room were cleaned before animal accomodation, then periodically disinfected.
- Diet: standard pellet complete diet supplied by the authorised breeder.
- Water: purified water ad libitum.
- Acclimation period: 5 days in which the animals were daily observed. at the end of the quarantine week the animals were carefully examined in order to evaluate their suitability for the study.

ENVIRONMENTAL CONDITIONS
- Photoperiod: 12hrs dark /12hrs light.
- Other: the housing room was lighted with fluorescent lamps. Room temperature and humidity were regulated by a conditioning plant and were daily monitored.

Study design: in vivo (non-LLNA)

Induction
Route:
intradermal and epicutaneous
Vehicle:
unchanged (no vehicle)
Concentration / amount:
neat (100%)
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Challengeopen allclose all
No.:
#1
Route:
epicutaneous, semiocclusive
Vehicle:
unchanged (no vehicle)
Day(s)/duration:
24 hours duration
Adequacy of challenge:
highest non-irritant concentration
No.:
#2
Route:
epicutaneous, semiocclusive
Vehicle:
unchanged (no vehicle)
Day(s)/duration:
24 hours duration
Adequacy of challenge:
other: re-challenge phase performed because of the positive results obtained during the challenge phase in order to confirm the results from first challenge
No. of animals per dose:
10 animals (group 1), 5 animals (control-group 2)
Details on study design:
Reliability check: the sensitivity and reliability of the experimental technique are assessed every six months according to OECD 406 and ISO 10993-10. A reliability check has been performed utilizing 2-mercaptobenzothiazole (CAS 149-30-4) in a concentation equal to 1.5 % in cottonseed oil for the intradermic injection phase, 30 % in Vaseline for the topic application of the induction phase and 27.5 % in vaseline for the challenge phase. In challenge phase 2 treated animals showed a skin reaction while nothing abnormal was detected in the control animals. Re-challenge phase confirmed results of challenge phase and two other animals resulted sensitised. 50 % of the treated animal have been shown a skin reaction while the control animals have been shown no signs of skin reaction.

RANGE FINDING TESTS: a preliminary study was intended to determine the concentration of the test sample to be used in the main test. For the topical induction phase, the highest concentration that causes mild to moderate erythema but does not otherwise adversely affect the animal was selected. For the challenge phase, the highest concentration that produces no erythema was selected. To select the sample dilution, four occlusive patches with 0.5 ml of the undiluted sample and diluted sample (90 %, 80 % and 70 % in sodium chloride injection) were applied to the dorsum of three additional animals. The dressings were left in place for 24 hours. After 24 hours of bandage removal, no erythema was observed in any treated sites.

MAIN STUDY
- Skin preparation: 24 hours before testing, fur was removed by shaving a 50 cm2 wide area on the back of the animals.
- Administration: the test consisted of an induction and a challenge phase

A. INDUCTION EXPOSURE
Intradermal injections:
group 1 - test group: 1- Stable emulsion of Freud's complete adjuvant (FCA) in sodium chloride injection 50:50(v/v), 2- test sample, 3- test sample diluted 50:50 (v/v) with stable emulsion of FCA and sodium chloride injection (50 %)
group 2-control group: 1- Stable emulsion of Freud's complete adjuvant (FCA) in sodium chloride injection 50:50(v/v), 2-sodium chloride injection, 3- sodium chloride diluted 50:50 (v/v) with stable emulsion of FCA and sodium chloride injection (50 %)
Day 0 - treated and control groups: three pairs of 0.1 intradermal injections were made in the interscapular region of each animal, on each side of the midline.
Dat 6 ± 1 - treated group and control group: after 6 days a topical application was made on all animals, with slight massage of 1 ml of sodium lauryl sulfate 10 % in vaseline.
Day 7±1- treated and control group: seven days after the intradermal injections 0.5 ml of the test sample was applied to each animal and held in place with an occlusive patch. The application was made so as to cover the intradermal injection sites. The dressing was left in place for 48 houts. The same treatment was performed on the control group, using sodium chloride injection instead of the test substance.

B. CHALLENGE EXPOSURE
Day 21±1- treated and control groups: an occlusive patch with 0.5 ml of the assay sample was applied to the flank of all 15 guinea pigs, while sodium chloride injection was applied on the left side. The dressing was left in place for 24 hours.

C. RE-CHALLANGE EXPOSURE
Day 28±1 - treated and control group: an occlusive patch with 0.5 ml of the assay sample was applied to the flank of all 15 guinea pigs, while sodium chloride injection was applied on the left side. The dressing was left in place for 24 hours.

OBSERVATIONS: 24±2 and 48±2 hours after removal of the patches, all treated and control animals were evaluated for a skin reaction. The intensity of erythema and/or edema were evaluated according to the following scale:
No visible change - 0
Discrete or patchy erythema -1
Moderate and confluent erythema - 2
Intense erythema and swelling - 3

INTERPRETATION OF RESULTS: Magnusson and Kligman grades of 1 or greater in the test group generally indicate sensitisation, provided grades of less than 1 are seen in control animals. If grades of 1 or greater are noted in control animals, then the reactions of test animals which exceed the most severe reaction in control animals are presumed to be due to sensitisation. If the response is equivocal, rechallenge is recommended to confirm the results from the first challenge. The outcome of the test is presented as the frequency of positive challenge results in the test and control animals.
Positive control substance(s):
no

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
100 %
No. with + reactions:
7
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
100 %
No. with + reactions:
7
Total no. in group:
10
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
0 %
No. with + reactions:
4
Total no. in group:
5
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
0 %
No. with + reactions:
4
Total no. in group:
5
Reading:
rechallenge
Hours after challenge:
72
Group:
test chemical
Dose level:
100 %
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no abnormalities were observed
Reading:
rechallenge
Hours after challenge:
72
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no abnormalities were observed

Applicant's summary and conclusion

Interpretation of results:
other: not classified for skin sensitisation according to the CLP Regulation (EC) No.1272/2008
Conclusions:
Not skin sensitiser
Executive summary:

The skin sensitisation of the test material was evaluated in the Guinea Pig Maximisation test according to the ISO 10993 -10. For this reason, 15 guinea pigs were used, 10 of which were treated with the test item and 5 were used as a control group and 3 for the range-finding test. During the preliminary test the concentration of the test sample to be used in the main test was determined.

During the induction phase, guinea pigs (test and control group) were treated with 3 pairs of intradermal injections. After six days, a local application of SLS at 10 % vaseline was performed. Topical application with the test sample and sodium chloride was performed for the treated and control group respectively 7 days after the injections. 21 days after the beginning of the treatment, both treated and control animals were subjected to a challenge phase, during which the test sample and sodium chloride was applied on the right and left side of the back respectively. The bandages were left for 24 hours. After 48 and 72 hours after the challenge, the reactions of both treated and control animals were evaluated: a slight erythema was observed in 7/10 animals in the treated group and 4/5 animals in the control group. Due to the positive results obtained in the control group the animals were subjected to a re-challenge. No abnormalities were observed in treated and control animals.

The test material is considered as non sensitiser.