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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:
The study was performed between 08 October 2007 and 08 November 2007.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007

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:
Date of inspection: January 2007; Date of certificate: February 2007
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
justification for the use of another test: unfeasible

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
482-130-1
EC Name:
-
Cas Number:
848301-87-1
Molecular formula:
Main general molecular formula: CnH(2n+2)
IUPAC Name:
Paraffin waxes (Fischer-Tropsch), full range, C15-50 - branched and linear
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): Paraffin waxes (Fischer-Tropsch), full range, C15-50 - branched and linear
- Physical state: Turbid liquid
- Lot/batch No.: Not reported
- Storage condition of test material: Room temperature in the dark

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Female albino Dunkin Hartley guinea pigs were supplied by Charles River (F-69592 L’Arbresle).
- Age at study initiation: 5 weeks old.
- Weight at study initiation: At the start of the main study the animals were in the weight range of 299 to 360 g
- Housing: The animals were housed in groups of two or three in makrolon cages furnished with woodflakes. The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
Free access to mains tap water and food (SDS, Essex UK, CM8 3AB) was allowed throughout the study. The diet, drinking water and bedding were routinely analysed and were considered not to contain any contaminant that could reasonably be expected to affect the purpose or integrity of the study.
- Acclimation period: At least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Set to achieve limits of 20 to 25 °C
- Humidity (%): Set to achieve limits of 30 to 53 %.
- Air changes (per hr): At least ten changes per hour.
- Photoperiod (hrs dark / hrs light): The lighting was controlled by a time switch to give twelve hours continuous light (07:00 to 19:00) and twelve hours darkness.

IN-LIFE DATES: From: 0 To: 26

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
other: olive oil and liquid paraffin
Concentration / amount:
Sighting Tests:
Intrademeral Induction: 100 %, 50 %, 25 %, 12.5 %, 6.25 % and 3.125 % by successive dilution (v/v) in olive oil
Topical Induction: 100 %, 50 %, 25 % and 12.5 % by successive dilution (v/v) in liquid paraffin
Topical Challenge: 100 %, 50 %, 25% and 12.5 % by successive dilution (v/v) in liquid paraffin

Main study:
Test animals
Intradermal induction: 1:1 preparation of Freund's Complete Adjuvant plus isotonic sodium chloride; 100 % substance (undiluted); 50 % substance (in 1:1 preparation of Freund's Complete Adjuvant plus isotonic sodium chloride)
Topical induction: 100 % substance (undiluted)
Topical challenge: 100 % substance (undiluted) and 50 % (v/v) in liquid paraffin

Control animals
Negative control:
Intradermal induction: 100 % Vehicle (olive oil); 1:1 preparation of Freund's Complete Adjuvant plus isotonic sodium chloride; 100 % (undiluted); 50 % (in 1:1 preparation of Freund's Complete Adjuvant plus isotonic sodium chloride)
Topical induction: 100 % (undiluted)
Topical challenge: 100 % (undiluted) and 50 % (v/v) in liquid paraffin


Positive control:
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: olive oil and liquid paraffin
Concentration / amount:
Sighting Tests:
Intrademeral Induction: 100 %, 50 %, 25 %, 12.5 %, 6.25 % and 3.125 % by successive dilution (v/v) in olive oil
Topical Induction: 100 %, 50 %, 25 % and 12.5 % by successive dilution (v/v) in liquid paraffin
Topical Challenge: 100 %, 50 %, 25 % and 12.5 % by successive dilution (v/v) in liquid paraffin

Main study:
Test animals
Intradermal induction: 1:1 preparation of Freund's Complete Adjuvant plus isotonic sodium chloride; 100 % substance (undiluted); 50 % substance (in 1:1 preparation of Freund's Complete Adjuvant plus isotonic sodium chloride)
Topical induction: 100 % substance (undiluted)
Topical challenge: 100 % substance (undiluted) and 50 % (v/v) in liquid paraffin

Control animals
Negative control:
Intradermal induction: 100 % Vehicle (olive oil); 1:1 preparation of Freund's Complete Adjuvant plus isotonic sodium chloride; 100 % (undiluted); 50 % (in 1:1 preparation of Freund's Complete Adjuvant plus isotonic sodium chloride)
Topical induction: 100 % (undiluted)
Topical challenge: 100 % (undiluted) and 50 % (v/v) in liquid paraffin

Positive control:
No. of animals per dose:
Sighting tests:
2 animals for induction concentrations.
3 animals for topical challenge concentrations.

Main study:
11 test animals and 5 control animals.
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 sites 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. 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 7, the scapular region of all test and control animals was shaved.
On Day 8 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 formulation. 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.

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 8, 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.

B. CHALLENGE EXPOSURE
The flanks of each animal were previously clipped free of hair 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 using the scale shown below.
Any other reactions were also recorded.

Scales for Evaluation of Skin Reactions
EVALUATION OF ERYTHEMA
No visible modification: 0
Slight or patches of erythema: 1
Moderate confluent erythema: 2
Intense erythema and swelling: 3
EVALUATION OF OEDEMA
No visible modification: 0
Slight oedema: 1
Moderate oedema: 2
Severe oedema: 3

Challenge controls:
5 female control animals were used for the topical challenge (100 % and 50 % in liquid paraffin).
Positive control substance(s):
yes
Remarks:
α-Hexylcinnamaldehyde (CAS [101-86-0])

Results and discussion

Positive control results:
Under the conditions of the test, the Positive control material, α-Hexylcinnamaldehyde, was classified sensitisers according to EU labelling regulation

Please refer to attached background material for two skin sensitisation studies on the positive control substance, α-Hexylcinnamaldehyde, confirming the validity of the postive control substance as a sensitiser and the validity of the study method.

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:
11
Clinical observations:
No skin reaction
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: 11.0. Clinical observations: No skin reaction.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
100 %
No. with + reactions:
0
Total no. in group:
11
Clinical observations:
No skin reaction
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: 11.0. Clinical observations: No skin reaction.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
50 %
No. with + reactions:
0
Total no. in group:
11
Clinical observations:
No skin reaction
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: 11.0. Clinical observations: No skin reaction.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
50 %
No. with + reactions:
0
Total no. in group:
11
Clinical observations:
No skin reaction
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: 11.0. Clinical observations: No skin reaction.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
100 %
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
No skin reaction
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: other: control. Dose level: 100%. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: No skin reaction.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
100 %
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
No skin reaction
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: control. Dose level: 100%. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: No skin reaction.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
50 %
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
No skin reaction
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: other: control. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: No skin reaction.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
50 %
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
No skin reaction
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: control. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: No skin reaction.
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
50% α-hexylcinnamaldehyde
No. with + reactions:
10
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Remarks:
The most recent positive control test was carried out on 16/10/2007
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
50% α-hexylcinnamaldehyde
No. with + reactions:
9
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Remarks:
The most recent positive control test was carried out on 16/10/2007
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
25% α-hexylcinnamaldehyde
No. with + reactions:
10
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Remarks:
The most recent positive control test was carried out on 16/10/2007
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
25% α-hexylcinnamaldehyde
No. with + reactions:
10
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Remarks:
The most recent positive control test was carried out on 16/10/2007

Any other information on results incl. tables

Preliminary Sighting Tests

Skin Reactions Observed After Intradermal Induction

A slight erythema was noted at the intradermal induction sites of the preliminary sighting test animals following treatment with the test material at the concentration of 100 % in two animals and at the concentration of 50 % in one animal. No skin reactions were noted at the intradermal inducion sites of the preliminary sighting test animals following treatment with the test material at the concentrations of 25 %, 12.5 %, 6.25 % and 13.125 %..

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.

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.

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.

Applicant's summary and conclusion

Interpretation of results:
other: EU GHS criteria not met
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.
The test material did not meet the criteria for classification as a sensitiser according to EU labelling regulations Commission Directive 2001/59/EC. No symbol and risk phrase are required.
Executive summary:

Introduction.

The study was performed to assess the contact sensitisation potential of the test material 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 17 July 1992)
  • Method B6 Acute Toxicity (Skin Sensitisation) of Commission Directive 96/54/EC

Method

Eleven 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 intradermal 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 EU GHS criteria.