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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:
2008-02-18 to 2008-04-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study reliable without restrictions

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
, 1992-07-17
Deviations:
yes
Remarks:
, deviation to the guideline: 10 test and 5 control animals were used, which had been established as a common procedure for animal welfare reason.
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2007-04-20
Type of study:
guinea pig maximisation test

Test material

Constituent 1
Chemical structure
Reference substance name:
700-380-4
EC Number:
700-380-4
Molecular formula:
A: C25H50O2 B: C27H54O2
IUPAC Name:
700-380-4
Details on test material:
- Name of test material (as cited in study report): SymMollient S
- Molecular formula: A: C25H50O2; B: C27H54O2
- Molecular weight: A: 382.68 g/mol; B: 410.73 g/mol
- Physical state: Colourless liquid to solid
- Storage condition of test material: Ambient temperature (10 - 30 °C), dark, dry, in original container
No further information on the test material was stated.

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 study initiation: 32 days
- Weight at study initiation: 300 -335 g (excluding positive control group); positive control group: 289 - 349 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.
- 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
No further information on the test animals was stated.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
other: sesame oil
Concentration / amount:
The following concentrations were employed in the main study (determined in the preliminary test):
Intracutaneous (induction): 10 % solution of cetearyl nonanoate in sesame oil
Topical (induction): 50 % solution of cetearyl nonanoate in sesame oil
Topical (challenge): 10 % solution of cetearyl nonanoate in sesame oil
A 10 % (w/w) suspension of cetearyl nonanoate 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.
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: sesame oil
Concentration / amount:
The following concentrations were employed in the main study (determined in the preliminary test):
Intracutaneous (induction): 10 % solution of cetearyl nonanoate in sesame oil
Topical (induction): 50 % solution of cetearyl nonanoate in sesame oil
Topical (challenge): 10 % solution of cetearyl nonanoate in sesame oil
A 10 % (w/w) suspension of cetearyl nonanoate 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.
No. of animals per dose:
Main study:
- Vehicle control group: 5 animals
- Test group: 10 animals
The positive control group included 20 animals.
Details on study design:
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 reaction 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 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 depilated (approx. 5 X 5 cm). Animals, even if only slightly injured, were replaced.
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.
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 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 grading system of MAGNUSSON/KLIGMAN.

Results preliminary test:
Six concentrations of cetearyl nonanoate were tested by intracutaneous injection: 0.01, 0.1, 0.5, 1, 5 or 10 % solutions in sesame oil: No skin reactions were observed up to the top concentration of 10 %. 10% was the maximum technically feasible concentration that could be admiistered by intradermal injection, since higher concentrations blocked the syringe and did not allow proper i.c. administration.
Six concentrations of cetearyl nonanoate were tested by topical application: 0.5, 1, 5, 10, 25 and 50 % solutions in sesame oil.
Undepilated skin: No skin reactions were observed at any concentration
Depilated skin: Concentrations of 0.5 to 10 % revealed a discrete or patchy erythema 24 hours after administration. Concentrations of 25 and 50 % revealed a discrete or patchy erythema 24 and 48 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 10 % concentration for the challenge.

MAIN STUDY
A. INDUCTION EXPOSURE
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. 067 K8614; SIGMA-ALDRICH Chemie GmbH, 82024 Taufkirchen, Germany) (diluted 1:1 with 0.9 % NaCl (Batch no. 05712201; Delta Select, 63303 Dreieich, Germany))
2). 10 % solution of cetearyl nonanoate in sesame oil (Batch no. 4230101. Henry Lamotte GmbH, 28197 Bremen, Germany)
3).Cetearyl nonanoate in a 1:1 mixtue (v/v) FCA/physiological saline (The final concentration of the test item was equal to that in injection 2.)

Day 6:
As cetearyl nonanoate was non-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 was necessary.

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
Day 21:
Two weeks after the topical application 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.

Vehicle control group: The left flank was treated with the test item, the right flank with the vehicle i.e. in the same was as the test group.

Skin observations and scoring:
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 (at 48 hours from the start of challenge application) the skin reaction was observed and recorded
- 24 hours after this observation a second observation (72 hours) was made and recorded.
The skin reactions were graded following the grading scale of MAGNUSSON/KLIGMAN.

OTHER OBSERVATIONS:
- 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.
No further information on the study design was stated.
Challenge controls:
Vehicle control group: sesame oil
5 males were used in the vehicle group.
Positive control substance(s):
yes
Remarks:
Benzocaine (Benzocaine was dissolved in 40 % ethanolic 0.9 % NaCl solution); The animals were treated with a 2 % (w/v) benzocaine solution intracutaneously (induction) and a 5 % (w/v) benzocaine solution topically (induction and challenge).

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 ( for results see "Attached background material" below).

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
10 % solution of cetearyl nonanoate in sesame oil
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No skin reaction was observed in any animal.
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 10 % solution of cetearyl nonanoate in sesame oil. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No skin reaction was observed in any animal..
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
10 % solution of cetearyl nonanoate in sesame oil
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No skin reaction was observed in any animal.
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 10 % solution of cetearyl nonanoate in sesame oil. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No skin reaction was observed in any animal..
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
10 % solution of cetearyl nonanoate in sesame oil
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
The vehicle control revealed no skin reactions.
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 10 % solution of cetearyl nonanoate in sesame oil. 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:
10 % solution of cetearyl nonanoate in sesame oil
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
The vehicle control revealed no skin reactions.
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 10 % solution of cetearyl nonanoate in sesame oil. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: The vehicle control revealed no skin reactions..

Any other information on results incl. tables

The body weight gain of the animals treated with cetearyl nonanoate 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
Conclusions:
Under the present test conditions cetearyl nonanoate 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 classified as a skin sensitiser.
According to the EC-Regulation 1272/2008 and subsequent regulations, the test item is not classified as a skin sensitiser.

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