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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:
weight of evidence
Study period:
2008-07-24 to 2008-09-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study reliable without restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
1992-07-17
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2007-04-20
Type of study:
guinea pig maximisation test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- CAS name: Cyclohexanol, 4-(1,1-dimethylethyl)-, trans-
- Molecular formula: C10H20O
- Molecular weight: 156.3 g/mol
- Physical state: solid

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland GmbH, Stolzenseeweg 32 - 36, 88353 Kißlegg, Germany
- Age at start of administration: 32 days
- Weight at start of administration: Vehicle control group: 300 - 321 g; test group: 294 - 332 g; positive control group: 257 - 347 g
- Housing: the animals were kept in pairs in MAKROLON cages (type III plus). granulated textured wood (Granulat A2, J. BRANDENBURG 49424 Goldenstedt, Germany) was used as bedding material in the cages. The cages were changed and cleaned twice a week.
- Diet (ad libitum): commercial diet, ssniff® Ms-H V2233 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water (ad libitum): tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C ± 3°C (maximum range)
- relative humidity: 55% ± 15% (maximum range)
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
other: sesame oil
Concentration / amount:
Main study:
- intracutaneous (induction): 10% suspension of BAS102 in sesame oil
- topical (induction): 50% suspension of BAS102 in sesame oil
- topical (challenge): 25% suspension of BAS102 in sesame oil
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: sesame oil
Concentration / amount:
Main study:
- intracutaneous (induction): 10% suspension of BAS102 in sesame oil
- topical (induction): 50% suspension of BAS102 in sesame oil
- topical (challenge): 25% suspension of BAS102 in sesame oil
No. of animals per dose:
Preliminary study: 8 animals
Main study: 10 animals (test group); 5 animals (control group)
Positive control group (historical background group): 20 animals
Details on study design:
PREPARATION OF THE TEST ITEM
The test item was not soluble in water. Therefore, the test item was suspended in sesame oil (batch no. 84260, henry Lamotte GmbH, 28197 Bremen, Germany) for all 3 stages. Sesame oil is known for not altering the existing response or the integrity of the epidermis and it is well applicable through a syringe.

RANGE FINDING TESTS:
The aim of the preliminary study was to determine the appropriate dose level of the test item following intracutaneous and topical administration. The concentration used in the main study following the induction procedure should produce mild to moderate skin reactions following topical application and be adjusted to the highest level that can be well tolerated locally and generally following intracutaneous injection. For the challenge exposure a subirritating concentration was used which produced no evidence of skin irritation in non-sensitised animals.
Eight animals (male) were used for the preliminary test: 6 animals for the topical administration and 2 animals for the intracutaneous administration.
The allocation of different test sites of the animals was alternated in order to minimize site-to-site variations in response.
The shoulder and the flank region of the animals were shaved or shaved and depilated4 (approx. 5 x 5 cm). Animals, even if only slightly injured, were replaced. The vehicle used during the preliminary test was sesame oil.
(a) intracutaneous
0.1 mL of the prepared test item was administered intracutaneously (shoulder region).
Three concentrations of the test item were injected intradermally into one, 3 further concentrations into a second animal. The concentration used were as follows: 0.01, 0.1, 0.5, 1, 5 and 10 % concentration of the test item.
(b) topical
The test area of 3 animals each was shaved or depilated. 2 mL of the test preparation was spread over a filter paper (2 x 4 cm) and applied to the test area and held in contact by an occlusive dressing (Urgoplast® (FOURNIER Pharma GmbH, 66280 Sulzbach, Germany).
Two concentrations each were applied to the shaved or shaved and depilated flanks of 3 animals each. The concentrations used were as follows: 0.5, 1, 5, 10, 25 and 50% concentration of the test item.
The occlusive dressing and the filter paper containing the test item were removed after 24 or 48 hours and the application sites were assessed immediately, 24 and 48 (depilated) or immediately and 24 hours (non-depilated) after removal of the filter paper for erythema and oedema using the Magnusson/Kligman grading scale.

Results:
Six concentrations of BAS102 were tested by intracutaneous injection: 0.01, 0.1, 0.5, 1, 5 or 10 % suspensions in sesame oil: no skin reactions were observed up to the concentration of 1%. A 5% concentration revealed a discrete or patchy erythema 24 hours, a concentration of 10% revealed a discrete or patchy erythema 24 to 72 hours after start of administration.
Six concentrations of BAS102 were tested by topical application: 0.5 1, 5, 10, 25 and 50% suspensions in sesame oil.
Undepilated skin: no skin reactions were observed at the concentration of 0.5%. Concentrations of 1 to 25% revealed a discrete or patchy erythema 48 hours, a concentration of 50% revealed a discrete or patchy erythema 48 and 72 hours after start of administration.
Depilated skin: concentrations of 0.5 to 25% did not reveal any skin reaction. A concentration of 50% revealed a discrete or patchy erythema 48 and 72 hours after administration. Hence, it was decided to use a 10% concentration for the 1st (intracutaneous) induction stage, a 50% concentration for the 2nd (topical) induction stage and a 25% concentration for the challenge.

MAIN STUDY
A 10% (w/v) suspension of BAS102 in sesame oil was the maximum technically feasible concentration that could be administered by intradermal injection, since higher concentrations blocked the syringe and did not allow proper intracutaneous administration.

A. INDUCTION EXPOSURE
1) TEST GROUP:
Day 0
Three pairs of intradermal injections of 0.1 mL were given in the shoulder region which was cleared of hair so that one of each pair lay on each side of midline.
(1) Freund's complete adjuvant (batch no. 058 K8711; SIGMA-ALDRICH Chemie GmbH, 82024 Taufkirchen, Germany) (diluted 1 : 1 with 0.9% NaCl (batch no. 05712201; Delta Select GmbH, 63303 Dreieich, Germany))
(2) the test item (BAS102) (10% concentration)
(3) the test item in a 1:1 mixture (v/v) FCA/physiological saline
In injection 3, the final concentration of the test item was equal to that in injection 2.
Injections (1) and (2) were given close to each other and nearest the head, while (3) was given towards the caudal part of the test area.

Day 6
As BAS102 was only slightly irritating to the non-depilated skin of the test animals in the preliminary experiment, the fur was shaved from the application area and the exposed skin was coated with 0.5 mL sodium laurylsulfate 10% (batch no. 31 K 0089; SIGMA Chemie GmbH, 89420 Höchstädt, Germany) in vaseline in order to induce a local irritation.

Day 7
7 days after the intracutaneous injection, the shoulder region of the same animals was shaved again and treated topically using the patch-test technique (exposure time: 48 hours). No cleaning of the skin was necessary.

2) VEHICLE CONTROL GROUP:
The vehicle control animals were treated in the same way as the animals of the test group, but received sesame oil instead of the test item.

B. CHALLENGE EXPOSURE
1) TEST GROUP:
Day 21
Two weeks after the topical application (corresponds to a monitoring period of 21 days) the flanks of the same animals were shaved and depilated for a further topical application using the patch-test technique. The filter paper containing the test item was applied to the left flank, the filter paper with the vehicle to the right flank of the animal (exposure time: 24 hours). 21 hours after the filter paper had been removed, no cleaning of the treated skin was necessary.

2) VEHICLE CONTROL GROUP
The left flank was treated with the test item, the right flank with the vehicle i.e. in the same way as the test group.

SKIN OBSERVATIONS AND SCORING:
- Induction: the skin reaction results of the first induction exposure (intradermally) were evaluated at 24 and 48 hours, of the second induction (topically) at 48 and 72 hours after beginning of exposure.
- Challenge (days 23 and 24): 21 hours after removing the filter paper the challenge area was cleaned and cleared of hair if necessary. Three hours later the skin reaction was observed and recorded. 24 hours after this observation a second observation was made and recorded.

The skin reactions were graded according to the Magnusson/Kligman grading scale.

OTHER EXAMINATIONS:
- mortality: daily during the observation period
- clinical signs: daily during the observation period
- body weight: at start of study and at study termination
- pathology: no necropsy was performed.
Challenge controls:
Vehicle control group: 5 male animals
Challenge dose: 25% suspension of BAS102 in sesame oil
Positive control substance(s):
yes
Remarks:
benzocaine (Dunkin-Hartley guinea pigs were treated with a 2% (w/v) benzocaine solution intracutaneously (induction) and with a 5% (w/v) benzocaine solution topically (induction & challenge)(benzocaine was dissolved in 40% ethanolic 0.9% NaCl solution)

Study design: in vivo (LLNA)

Statistics:
The body weight was analysed statistically using STUDENT's t-test (p ≤ 0.01).

Results and discussion

Positive control results:
Animals treated with benzocaine in 40% ethanolic 0.9% NaCl solution exhibited a sensitising reaction in all animals in form of a discrete or patchy erythema or a moderate and confluent erythema (grade 1 or 2).

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
25% suspension of BAS102
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No skin irritation in any animal.
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 25% suspension of BAS102. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No skin irritation in any animal..
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
25% suspension of BAS102
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No skin irritation in any animal.
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 25% suspension of BAS102. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No skin irritation in any animal..
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
25% suspension of BAS102
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
The vehicle control revealed no skin reactions.
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 25% suspension of BAS102. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: The vehicle control revealed no skin reactions..
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
25% suspension of BAS102
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
The vehicle control revealed no skin reactions.
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 25% suspension of BAS102. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: The vehicle control revealed no skin reactions..
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
5% (w/v) benzocaine solution
No. with + reactions:
20
Total no. in group:
20
Clinical observations:
Animals treated with benzocaine in 40% ethanolic 0.9% NaCl solution exhibited a sensitising reaction in all animals in form of a discrete or patchy erythema or a moderate and confluent erythema (grade 1 or 2).
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: 5% (w/v) benzocaine solution. No with. + reactions: 20.0. Total no. in groups: 20.0. Clinical observations: Animals treated with benzocaine in 40% ethanolic 0.9% NaCl solution exhibited a sensitising reaction in all animals in form of a discrete or patchy erythema or a moderate and confluent erythema (grade 1 or 2)..
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
5% (w/v) benzocaine solution
No. with + reactions:
20
Total no. in group:
20
Clinical observations:
Animals treated with benzocaine in 40% ethanolic 0.9% NaCl solution exhibited a sensitising reaction in all animals in form of a discrete or patchy erythema or a moderate and confluent erythema (grade 1 or 2).
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: positive control. Dose level: 5% (w/v) benzocaine solution. No with. + reactions: 20.0. Total no. in groups: 20.0. Clinical observations: Animals treated with benzocaine in 40% ethanolic 0.9% NaCl solution exhibited a sensitising reaction in all animals in form of a discrete or patchy erythema or a moderate and confluent erythema (grade 1 or 2)..

Any other information on results incl. tables

INDUCTION (skin observations):

A 10% suspension of BAS102 in sesame oil chosen for the 1st (intracutaneous) induction stage revealed a discrete or patchy erythema (grade 1) 24 hours after administration.

EXAMINATIONS:

- no animal was found dead or was sacrificed in extremis.

- the body weight gain of the animals treated with BAS102 was within the range of the vehicle control during the experiment.

- behaviour of the animals remained unchanged.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the present test conditions the test substance revealed no sensitising properties in guinea pigs in a test model according to MAGNUSSON and KLIGMAN.
According to the EC-Commission directive 67/548/EEC and its subsequent amendments, the test substance is not a skin sensitizer.
According to the EC-Regulation 1272/2008 and subsequent regulations, the test item is not a skin sensitizer.
Executive summary:

The GPMT appears well conducted, and the use of high test concentrations in combination with a complete absence of response in the test guinea pigs at a challenge concentration of 25% is consistent with an absence of skin sensitisation potential. However, this is undermined significantly by a gap in the positive control data for benzocaine. This substance is an OECD recommended positive control whose purpose is to demonstrate that the assay has been conducted to a sufficient quality and sensitivity, the latter being critical for the correct identification of weak skin sensitisers (Basketter et al. (1993) Results with OECD recommended positive control sensitizers in the maximisation, Buehler and local lymph node assays. Food and Chemical Toxicology 31, 63 - 67). In the present case, the positive control study shows positive reactions in the test animal, but no untreated control data are provided, meaning that one cannot judge whether the reactions seen are allergy or irritation. Nevertheless, it is reasonable to conclude that, on balance, this GPMT presents good evidence thatthe test item is not a skin sensitizer.