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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:
2010-04-22 to 2010-07-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
Buehler test
Justification for non-LLNA method:
An LLNA study was not performed because the test method is not considered to be suitable for substances that contain silicon. Please refer to the attached document for further details.

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
Test System:
Animals: Albino Dunkin Hartley Guinea Pig, HsdPoc: DH, SPF
Rationale: Skin reactions in the guinea pig are classically used for determining the potential of test items to induce delayed contact hypersensitivity. No valid non-animal model (in-vitro) is available at present for the test of contact sensitization.
Breeder: Harlan Laboratories B.V., Kreuzelweg 53, 5961 NM / The Netherlands
Number of Animals for Main Study/Irritation Screening: 30 males / 6 males (Challenge: 20 test animals, 10 control animals; Irritation Screens: 6 animals)
Age at Delivery/Acclimatization Start: 4 to 5 weeks
Body Weight at Acclimatization Start: Test and control animals: 250 to 374 g, Animals used for irritation screens: 311 to 368 g
Identification: By unique cage number and corresponding individual animal number.
Randomization: Randomly selected by hand at time of delivery. No computer randomization.
Acclimatization: Seventeen days under laboratory conditions after health examination. Only animals without any visible signs of illness were used for the study. One to 3 days for the animals used in the irritation screening for induction and challenge. Only animals without any visible signs of illness were used for the study.

Environmental Conditions:
Conditions: Standard Laboratory Conditions: Air-conditioned with ranges for room temperature 22 ± 3 °C, relative humidity 30-70% and approximately 10-15 air changes per hour. Room temperature and humidity were monitored continuously. These data are retained at Harlan Laboratories. The animals were provided with an automatically controlled light cycle of 12 hours light and 12 hours dark. Music was played during the daytime light period.
Accommodation: Individually in Makrolon type-4 cages with standard softwood bedding (‘Lignocel’ J. Rettenmaier&Söhne GmbH&CoKG, 73494 Rosenberg/Germany, imported by Provimi Kliba AG, 4303 Kaiseraugst/Switzerland).
Diet: Pelleted standard Provimi Kliba 3418, batch no. 05/10 guinea pig breeding/maintenance diet, containing Vitamin C (Provimi Kliba AG, 4303
Kaiseraugst/Switzerland), ad libitum. Results of analyses for contaminants are archived at Harlan Laboratories Ltd.
Water: Community tap water from Füllinsdorf, ad libitum. Results of bacteriological, chemical and contaminant analyses are archived at Harlan Laboratories Ltd.

Study design: in vivo (non-LLNA)

Induction
Route:
epicutaneous, occlusive
Vehicle:
other: Acetone
Concentration / amount:
Concentration for 3-week induction: 100% (neat)
Concentration for challenge: 15% (w/w) in acetone
Challenge
Route:
epicutaneous, occlusive
Vehicle:
other: Acetone
Concentration / amount:
Concentration for 3-week induction: 100% (neat)
Concentration for challenge: 15% (w/w) in acetone
No. of animals per dose:
1 Irritation Screen for Induction and Challenge I: 3 (nos. 1 - 3)
2 Control Group: 10 (nos. 4 - 13)
3 Test Group: 20 (nos. 14 - 33)
4 Irritation Screen for Induction and Challenge II: 3 (nos. 34 - 36)
Details on study design:
Preparation of Dose Formulation:
The test item and vehicle were placed into a glass beaker on a tared Mettler balance and a weight/weight dilution was prepared. A magnetic stirrer was used to ensure homogeneous distribution of the test item preparation. The preparations were made immediately prior to each dosing. Homogeneity of the test item in the vehicle was maintained during administration using a magnetic stirrer. The test item formulations (in acetone) were applied between 15 and 45 minutes following their preparation. Dose levels are described in terms of material as supplied unless otherwise stated by the Sponsor.

Selection of Test Item Concentration for Main Study:
A number of factors contributed to the selection of the concentrations of test item including irritancy, slope of dose response curve and experience with similar test items. Irritation screenings were conducted to determine the minimal irritating concentration for the induction period and the highest non-irritating concentration for the challenge and re-challenge periods. The results of these screenings are described below.

Epidermal Induction:
For the epidermal induction phase, the concentration selected should produce some irritation but not adversely affect the animals. The selection of the concentration used for the main induction was done after the first irritation screen has been performed. During the first irritation screen, mild to moderate skin reactions were observed with the neat test item and the test item formulated at 75%, 50% and 25% in acetone, all applied topically and occlusively. The severity decreased at the 48-hour reading and all animals showed a slight irritation (grade 1) or no skin reactions anymore. Based on the first irritation screen results, the test item at 100% (neat) was used for the 3-week induction in the test group while the control group was treated with acetone alone. The test item concentration was agreed upon between the Study Director and Sponsor Representative.

Epidermal Challenge:
For the challenge phase, the concentration selected should be the maximum tested non-irritant concentration. As a non-irritant concentration could not be selected in the first irritation screen, a second irritation screen was performed with lower concentrations of 15%, 10%, 5% and 1% in acetone as well as Acetone alone. No local skin reaction was noted in the second irritation screen on the test sites treated with the test item at 15%, 10%, 5% and 1% in acetone as well as with Acetone alone. The non-irritating test item concentration of 15% in acetone required for the challenge was agreed upon between the Study Director and Sponsor Representative.

Rationale:
Dermal administration has historically been used as the route of choice for determining delayed contact hypersensitivity.

Observations:
Viability / Mortality: Daily from delivery of the animals to the termination of the test.
Clinical Signs / Grading of Skin Response: Daily from delivery of the animals to the termination of test. Skin responses were graded during the
irritation screening, induction and challenge periods.
Body Weights: At delivery/acclimatization start, at the end of the irritation screening, at test day 1 (day of treatment) and at the termination of the study.

Necropsy:
No necropsies were performed on the animals of the control and test group sacrificed at termination of their observation period or on the animals of the two irritation screenings sacrificed a few days after their skin assessment was performed. The animals were euthanized by intraperitoneal injection of pentobarbitone at a dose of at least 2.0 mL/kg body weight (equivalent to 324 mg sodium pentobarbitone/kg body weight) and discarded.

Treatment Method:
Patching method: The animal's fur was shaved with a fine clipper blade or equivalent just prior to the exposure. Closed patches were applied to the animals as follows: 0.5 mL of the test item or a freshly prepared test item dilution in a 25 mm Hill Top Chamber (approximately 4.9 cm2 skin exposure). The 25 mm Hill Top Chamber (skin application area of approximately 4.9 cm2) was firmly secured by an elastic plaster wrapped around the trunk of the animal and secured with impervious adhesive tape. The occlusive dressing was left in place for six hours (± 15 minutes). An identical patching method was used for the irritation screen, induction, challenge and rechallenge phases.

Observation and Scoring:
In order to evaluate the skin reactions following the irritation screenings, challenge and rechallenge, the trunk skin of each animal where the testing patches had been applied was depilated approximately 21 hours after the patches had been removed, using an approved depilatory cream (VEET Cream, Reckitt & Colman AG, 4123 Allschwil / Switzerland). The depilation was performed to clean the stratum corneum to facilitate the reading of the possible skin reaction. The depilatory cream was placed on the patch sites and surrounding areas, and left on for up to 3-5 minutes. It was then thoroughly washed off with a stream of warm, running water. The animals were then dried with a disposable towel, and returned to their cages. The grading method used for the irritation screenings, induction and challenges was identical. It was performed 24 ± 2 hours after removal of the patches for the irritation screening, induction and challenge and repeated 24 ± 2 hours later (48-hour grades) for the irritation screening and
the challenge.

The scoring was performed by visual assessment of erythema and oedema. They were assessed as follows:
0 = no visible change
1 = discrete or patchy erythema
2 = moderate and confluent erythema
3 = intense erythema and swelling
If observed, any other gross lesions not covered by this scoring system were described.

Grading of all animals was done by positioning each animal under true-light (Philips Master TLS HE 28W/840). For evaluation, two parameters were used: the incidence index and the severity index, for both test and control animals. The incidence index is an expression of the number of animals showing a response of grade 1 or greater at the 24- or 48-hour readings out of the total animals in the group. The severity index, designed to describe the intensity of the reaction, is calculated from the total sum of 24- and 48 hour response readings divided by the number of animals exposed.

Statistical Analysis:
Descriptive statistics (means and standard deviations) were calculated for body weights. No inferential statistics were used.

Data Compilation:
The following data were recorded on data sheets and transcribed for compilation and analysis: clinical signs (local/systemic), mortality/viability and skin reactions. The following data were recorded on-line: body weights. The RCC Tox Computer System (RCC-Tox-Lims) has been validated with respect to data collection, storage and retrievability.
Challenge controls:
10 naive control animals were challenged at the same test item concentration like the 10 test group animals.
Positive control substance(s):
yes
Remarks:
ALPHA-HEXYLCINNAMALDEHYDE (Harlan Laboratories Study C67807)

Results and discussion

Positive control results:
General:
The purpose of this skin sensitizing study was to confirm the allergenic potential of ALPHAHEXYLCINNAMALDEHYDE when administered topically to albino Dunkin Hartley guinea pigs. For this purpose the “Buehler Test” modified by Ritz, H.L. and Buehler, E.V. (1980) was used. Twenty male animals of the test group were treated topically with ALPHAHEXYLCINNAMALDEHYDE at 50% in PEG 300 once a week for a 3-week induction phase. Two weeks after the final induction application the animals were challenged with the same test item concentration of 5% in PEG 300 as used for induction. The ten animals of the control group were not treated during the induction. They were treated once at challenge with ALPHA-HEXYLCINNAMALDEHYDE at 5% in PEG 300.

Results:
None of the control animals were observed with skin reactions after the challenge treatment with the highest tested non-irritating concentration of ALPHA-HEXYLCINNAMALDEHYDE at 5% in PEG 300.
Nine and six of twenty test animals were observed with discrete/patchy erythema at the 24- and 48-hour readings, respectively, after the challenge treatment with the highest tested non-irritating concentration of ALPHA-HEXYLCINNAMALDEHYDE at 5% in PEG 300. No skin effect was observed in the control group.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
15% (w/w) in acetone
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
15% (w/w) in acetone
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
15% (w/w) in acetone
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
15% (w/w) in acetone
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
ALPHA-HEXYLCINNAMALDEHYDE at 5% in PEG 300
No. with + reactions:
9
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
ALPHA-HEXYLCINNAMALDEHYDE at 5% in PEG 300
No. with + reactions:
6
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation

Any other information on results incl. tables

RESULTS AND DISCUSSION

Observations

Viability / Mortality / Macroscopic Findings There were no deaths during the course of the study and no necropsies were performed.

Clinical Signs No signs of systemic toxicity were observed in any of the animals.

Body Weights (See attached Summary Tables and Individual Tables) The body weight of the animals was within the range commonly recorded for animals of this strain and age.

Skin Reactions in the Induction (See Individual Tables on p. ) No skin effect (grade 0) was observed in the control group after treatment with acetone alone during the three weeks of induction. Discrete/patchy erythema (grade 1) was observed in all (100%) animals in the first and third induction weeks and in 12 out of 20 (60%) animals in the second induction week after treatment with the test item at 100%.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the above mentioned findings in a non-adjuvant sensitization test in guinea pigs and in accordance to Regulation (EC) No 1272/2008, 2,4,6,8,10-Pentamethylcyclopentasiloxane does not have to be classified and labelled as a skin sensitizer.
Executive summary:

General

The purpose of this skin sensitizing study was to assess the ability of the test item 2,4,6,8,10-Pentamethylcyclopentasiloxane, to induce delayed contact hypersensitivity when applied topically to albino guinea pigs. A modified Buehler method (Ritz and Buehler, 1980) was used. The study was conducted according to OECD Guidelines for Testing of Chemicals, Number 406 "Skin Sensitization”.

Twenty male animals of the test group were treated topically with 2,4,6,8,10-Pentamethylcyclopentasiloxane at 100% (undiluted) once a week for a 3-week induction phase. Ten animals in the control group were treated in the same way as the test animals, but with the vehicle (acetone) only. Two weeks after the final induction application the control and test animals were challenged with the test item at 15% in acetone and acetone alone.

Results:

There was no skin reaction in the control and test group after the challenge procedure performed with 2,4,6,8,10-Pentamethylcyclopentasiloxane at 15% in acetone. The acetone, itself, did not show any local skin reactions and was, therefore, considered to be suitable for this type of study.

Based on the findings observed in a non-adjuvant sensitization test in guinea pigs, 2,4,6,8,10-Pentamethylcyclopentasiloxane does not have to be classified and labelled as a skin sensitizer.