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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:
The study was performed between 28 January 2008 and 28 February 2008.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Inspection date: 11 January 2007. Date of decision: 15 February 2007
Type of study:
guinea pig maximisation test

Test material

Constituent 1
Reference substance name:
-
EC Number:
482-220-0
EC Name:
-
Cas Number:
848301-69-9
IUPAC Name:
1-(2-methylpentyl)-3-(6-methylundecyl)cyclopentane; 1-(3,7-dimethyldecyl)-3-(2-methylbutyl)cyclohexane; 1-(3-methyldecyl)-3-(2-methylpentyl)cyclohexane; 4,8,11-trimethylpentadecane; 6,9-dimethylhexadecane; 6-methylheptadecane

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: Four weeks old.
- Weight at study initiation: 229 to 265 g (mean: 248.9 g).
- Housing: The animals were housed in groups of two or three in makrolon cages furnished with woodflakes.
- Diet (e.g. ad libitum): Free access to food was allowed throughout the study.
- Water (e.g. ad libitum): Free access to mains tap water was allowed throughout the study.
- Acclimation period: At least five days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Temperature set to achieve limits of 19 to 22°C
- Humidity (%): Relative humidity was set to achieve limits of 45 to 70%
- Air changes (per hr): At least ten changes per hour
- Photoperiod (hrs dark / hrs light): Lighting was controlled by a time switch to give twelve hours continuous light and twelve hours darkness.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
other: The test material was freshly prepared in olive oil and liquid paraffin.
Concentration / amount:
Intradermal induction: 100%
Topical induction: 100%
Topical challenge: 100% and 50% in liquid paraffin.
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: The test material was freshly prepared in olive oil and liquid paraffin.
Concentration / amount:
Intradermal induction: 100%
Topical induction: 100%
Topical challenge: 100% and 50% in liquid paraffin.
No. of animals per dose:
Number of animals in test group: 10
Number of animals in control group: 5
Details on study design:
RANGE FINDING TESTS:
The concentrations of test material to be used at each stage of the main study were determined by 'sighting tests' in which groups of guinea pigs were treated with various concentrations of test material. The procedures were as follows:

Selection of Concentration for Intradermal Induction:
Intradermal injections (0.1 ml/injection site) were made on the clipped shoulder of two guinea pigs, at the concentrations of 100%, 50%, 25%, 12.5%, 6.25% and 3.125% by successive dilution (v/v) in olive oil. A macroscopic evaluation of the injection site was conducted approximately 24 hours after injection to determine whether these concentrations caused necrosis. Any evidence of systemic toxicity was also recorded. The highest concentration which did not cause necrosis was selected for the intradermal induction stage of the main study.

Selection of concentration for Topical Induction:
Two guinea pigs were treated with preparations of the test material at 100% and diluted at 50%, 25% and 12.5% by successive dilution (v/v) in liquid paraffin. Applications were made to the clipped flanks under occlusive dressings for an exposure period of 24 hours. The degree of erythema and oedema was evaluated approximately 24 hours after dressing removal. The highest concentration producing only moderate dermal irritation was selected for the topical induction stage of the main study.

Selection of Concentration for Topical Challenge:
Four preparations of the test material at 100% and diluted at 50%, 25% and 12.5% by successive dilution (v/v) in liquid paraffin were applied to the clipped flanks of three guinea pigs under occlusive dressings for an exposure period of 24 hours. These guinea pigs did not form part of the main study but had been treated identically to the control animals of the main study, up to Day 14.
The degree of erythema and oedema was evaluated approximately 24 hours after dressing removal. The highest non-irritant concentration of the test material and one lower concentration were selected for the topical challenge stage of the main study.


MAIN STUDY
A group of fifteen guinea pigs was used for the main studym ten tests and five controls. The bodyweight of each animal was recorded at the start and the end of the study.
Two phases were involved in the main study; (a) an induction of a response and (b) a challenge of that response.

A. INDUCTION EXPOSURE
Induction of the Test Animals: Shortly before treatment on Day 0 the hair was removed from an area approximately 40 mm x 60 mm on the shoulder region of each animal with veterinary clippers. A row of three injections (0.1 ml each) was made on each side of the spine. The injections were:
a) Freund's Complete Adjuvant plus isotonic sodium chloride in the ratio 1:1
b) 100% of the test material.
c) a 50% formulation of the test material in a 1:1 preparation of Freund's Complete Adjuvant plus isotonic sodium chloride.

On Day 6, the scapular region of all test and control animals was shaved and was brushed with a solution of sodium lauryl sulfate at 10% in thick vaseline, in order to create a local irritation.
On Day 7 the same area on the shoulder region used previously for intradermal injections was clipped again and treated with a topical application of the test material fonnulation. A topical application of the test material at 100% under occlusive dressing was made. The occlusive dressing was kept in place for 48 hours.

The degree of erythema and oedema was evaluated approximately 24 hours after dressing removal.


B. CHALLENGE EXPOSURE
The flanks of each animal were previously clipped free ofhair with veterinary clippers.

The test material formulation at the maximum non-irritant concentration (100%) was applied to one side of the shorn flank of each animal under an occlusive dressing. To ensure that the maximum non-irritant concentration was used at challenge, the test material at a concentration of 50% (v/v) in liquid paraffin was similarly applied under an occlusive dressing to the opposite skin site on the shorn flank.

After 24 hours, the dressing was carefully removed and discarded. The topical challenge sites were cleaned if required.

Prior to the 24-hour observation the flanks were clipped to remove regrown hair.

Approximately 24 and 48 hours after challenge dressing removal, the degree of erythema and oedema was quantified.

Any other reactions were also recorded.
Challenge controls:
Induction of the Control Animals: On Day 0, the intradermal induction was performed using an identical procedure to that used for the test animals except that the test material was omitted from the intradermal injections. Injection b) was therefore the vehicle alone, injection c) was a 50%
formulation of the vehicle in a 1:1 preparation of Freund's Complete Adjuvant plus isotonic sodium chloride. Similarly, on Day 7, the topical induction procedure was identical to that used for the test animals except that the test material was omitted.


The degree of erythema and oedema was evaluated approximately 24 hours after dressing removal.
Positive control substance(s):
yes
Remarks:
alpha-Hexylcinnamaldehyde

Results and discussion

Positive control results:
Based on the results, under the conditions of the test, the Positive control material, alpha-Hexylcinnamaldehyde, was classified as a sensitiser
according to EU labelling regulations Commission Directive 2001/59/EC

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
100 %
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 100 %. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
50 %
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 50 %. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
100 %
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 100 %. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
50 %
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50 %. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
100 %
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 100 %. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
50 %
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 50 %. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
100 %
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 100 %. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
50 %
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 50 %. No with. + reactions: 0.0. Total no. in groups: 5.0.

Any other information on results incl. tables

Results
Preliminary Sighting Tests

Skin Reactions Observed After Intradermal Induction

No skin reactions were noted at the intradermal induction sites of the preliminary sighting test animals following treatment with the test material at the concentrations of 100%, 50%, 25%, 12.5%,6.25% and 3.125%.

The concentration chosen for use in the Intradermal Induction phase of the main test was 100% (maximal non necrotising concentration (MNNC)).

Skin Reactions Observed After Topical Induction

No skin reactions were noted at the topical induction sites of the preliminary sighting test animals following treatment with the test material at concentrations of 100%,50%,25% and 12.5%.

The concentration chosen for use in the 2nd topical Induction of the main test was 100% and the

1st concentration chosen for the MNIC (maximal non irritant concentration) determination was 100%.

Skin Reactions Observed After Topical Challenge

No skin reactions were noted at the topical challenge sites of the preliminary sighting test animals following treatment with the test material at concentrations of 100%,50%,25% and 12.5%.

The concentrations chosen for use in the Topical Challenge phase of the main test were 100% (maximal non irritant concentration (MNIC)) and 50% (1/2 MNIC) in liquid paraffin.


Main Study

Skin Reactions Observed After Topical Challenge

100%

No skin reactions were noted at the topical challenge site of the test or control group animals at the 24 and 48 -hour observations.

50% in liquid paraffin

No skin reactions were noted at the topical challenge site of the test or control group animals at the 24 and 48 -hour observations.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of the test, the test material produced a 0% sensitisation rate and was classified as a non-sensitiser to guinea pig skin.
Executive summary:

Introduction.

The study was performed to assess the contact sensitisation potential of the test material ‘Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear’ in the albino guinea pig. The method was designed to meet the requirements of the following:

• OECD Guidelines for the Testing of Chemicals No. 406 "Skin Sensitisation" (adopted 7 July 1992)

• Method B6 Acute Toxicity (Skin Sensitisation) of Commission Directive 96/54/EC

Method.

Ten test and five control animals were used for the main study. Two phases were involved in the main test; an induction of a response by intradelmal injection and topical application and a topical challenge of that response.

Based on the results of sighting tests, the concentrations of test material for the topical induction and topical challenge phases were selected as:

- Intradermal Induction: 100%

- Topical Induction: 100%

- Topical Challenge: 100 % and 50% in liquid paraffin.

Conclusion.

Under the conditions of the test, the test material produced a 0% sensitisation rate and was classified as a non-sensitiser to guinea pig skin. The test material did not meet the criteria for classification as a sensitiser according to Regulation 1272/2008/EC.