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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:
01 July - 15 August 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
Cross-reference
Reason / purpose for cross-reference:
read-across: supporting information
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
01 July - 15 August 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
Reason / purpose for cross-reference:
read-across source
Positive control results:
The latest reliability check (performed less than 6 months before the study) shows a sensitisation rate of 90%.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
50% test substance in corn oil
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
50% test substance in corn oil
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
corn oil
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
corn oil
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
50% test substance in corn oil
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
50% test substance in corn oil
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
corn oil
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
corn oil
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
20% alpha-hexylcinnamicaldehyde
No. with + reactions:
8
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Remarks:
Positive control reliability check carried out July/August 2014
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
20% alpha-hexylcinnamicaldehyde
No. with + reactions:
8
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Remarks:
20% alpha-hexylcinnamicaldehyde Positive control reliability check carried out July/August 2014

Signs of irritation during induction:
For skin effects caused by the intradermal injections and epidermal exposure during the induction phase see appendix.

Evidence of sensitisation of the challenge concentration:

No skin reactions were evident after the challenge exposure in the experimental and control animals.

Toxicity / Mortality: No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

Body weights: Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

Interpretation of results:
GHS criteria not met
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
In a guinea pig maximisation test, conducted in accordance with OECD 406 and GLP, Hydrocarbons, C8-C11, n-alkanes, isoalkanes, <2% aromatics was tested at a challenge concentration of 50% v/v in corn oil. Intradermal induction was performed at a concentration of 20% v/v, and topical induction 50% test substance in corn oil. No skin reactions were observed in any test or control group animals at challenge indicating that the substance is not sensitising to skin.

Executive summary:

This data is being read across from the source study that tested Hydrocarbons, C8-C11, n-alkanes, isoalkanes, <2% aromatics based on analogue read across.

Assessment of Contact Hypersensitivity to Hydrocarbons, C8-C11, n-alkanes, isoalkanes, <2% aromatics in the Albino Guinea Pig (Maximization Test).

The study was carried out based on the guidelines described in:

OECD No. 406 (1992) "Skin Sensitization"

EC No 440/2008; B6: "Skin Sensitization: Guinea-Pig Maximization Test (GPMT)"

EPA OPPTS 870.2600 (2003) “Skin Sensitization”

JMAFF Guidelines (2000), including the most recent revisions.

The study was based on the method described by Magnusson and Kligman, "Allergic Contact Dermatitis in the Guinea Pig - Identification of Contact Allergens" (1970).

Test substance concentrations selected for the main study were based on the results of a preliminary study. In the main study, ten experimental animals were intradermally injected with a 20% concentration and epidermally exposed to a 50% concentration. Five control animals were similarly treated, but with vehicle alone (corn oil). Two weeks after the epidermal application all animals were challenged with a 50% test substance concentration and the vehicle. No skin reactions were evident after the challenge exposure in the experimental and control animals. There was no evidence that Hydrocarbons, C8-C11, n-alkanes, isoalkanes, <2% aromatics had caused skin hypersensitivity in the guinea pig, since no responses were observed in the experimental animals in response to a 50% test substance concentration in the challenge phase. This result indicates a sensitization rate of 0 per cent.

Based on these results Hydrocarbons, C8-C11, n-alkanes, isoalkanes, <2% aromatics does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the:

- Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2007) (including all amendments),

- Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (including all amendments).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
(1992)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
(2008)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
(2003)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nohsan, Notification No. 8147, April 2011; including the most recent partial revisions.
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Acceptable guinea pig maximisation test that followed sound scientific principles.

Test material

Constituent 1
Chemical structure
Reference substance name:
Hydrocarbons, C8-C11, n-alkanes, isoalkanes, <2% aromatics
EC Number:
940-733-1
Molecular formula:
C8H18 to C11H24
IUPAC Name:
Hydrocarbons, C8-C11, n-alkanes, isoalkanes, <2% aromatics
Test material form:
other: Clear colourless liquid

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River France, L’arbresle Cedex, France.
- Age at study initiation: Young adult animals (approx. 4 weeks old)
- Weight at study initiation: 277 g
- Housing: Group housing of maximally 5 animals per labeled cage.
- Diet (e.g. ad libitum): Complete breeding diet for guinea pigs (SSNIFF® MS-Z, V2273; SSNIFF® Spezialdiäten GmbH, Soest, Germany). Hay (TecniLab-BMI BV, Someren, The Netherlands) was provided at least twice a week.
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

IN-LIFE DATES: 01 July - 15 August 2014

Study design: in vivo (non-LLNA)

Induction
Route:
intradermal and epicutaneous
Vehicle:
corn oil
Concentration / amount:
100, 50, 20 and 10% for the intradermal induction and for the epidermal induction.
50% for the challenge phase.
Challenge
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
100, 50, 20 and 10% for the intradermal induction and for the epidermal induction.
50% for the challenge phase.
No. of animals per dose:
Test animals: 10
Control animals: 5
Details on study design:
RANGE FINDING TESTS: (4 animals, age: between 4 and 9 weeks old)

Intradermal injections:
A series of four test substance concentrations was used, the highest concentration being the maximum concentration that could technically be injected. Each of two animals received two different concentrations in duplicate (0.1 mL/site) in the clipped scapular region. The injection sites were assessed for irritation 24 and 48 hours after treatment.
For results see appendix.

Epidermal application:
A series of four test substance concentrations was used, the highest concentration being the maximum concentration that could technically be applied. Two different concentrations were applied (0.5 mL each) per animal to the clipped flank, using Metalline patches# (2x3 cm) mounted on Medical tape# which were held in place with Micropore tape# and subsequently Coban elastic bandage#. The animals receiving intradermal injections were treated with the lowest concentrations and two further animals with the highest concentrations. After 24 hours, the dressing was removed and the skin cleaned of residual test substance using water. The treated skin areas were assessed for irritation 24 and 48 hours after exposure.
#. Suppliers: Lohmann & Rauscher B.V., Almere, The Netherlands (Metalline) and 3M, St. Paul, Minnesota, U.S.A. (Medical tape, Micropore and Coban).
For results see appendix.

MAIN STUDY
INDUCTION - Experimental animals
Day 1 The scapular region was clipped and three pairs of intradermal injections (0.1 mL/site) were made in this area as follows:
A) A 1:1 w/w mixture of Freunds' Complete Adjuvant (Difco, Detroit, U.S.A.) with water for injection (B.Braun Melsungen AG, Melsungen. Germany).
B) The test substance at a 20% concentration.
C) A 1:1 w/w mixture of the test substance, at twice the concentration used in (B) and Freunds' Complete Adjuvant.
Note: One of each pair was on each side of the midline and from cranial A) to caudal C).
Day 3 The dermal reactions caused by the intradermal injections were assessed for irritation.
Day 8 The scapular area between the injection sites was clipped and subsequently treated with 0.5 mL of a 50% test substance concentration using a Metalline patch (2x3 cm) mounted on Medical tape, which was held in place with Micropore tape and subsequently Coban elastic bandage. The dressing was removed after 48 hours exposure, the skin cleaned of residual test substance using water and the dermal reactions caused by the epidermal exposure were assessed for irritation.
INDUCTION - Control animals
The control animals were treated as described for the experimental animals except that, instead of the test substance, vehicle alone was administered.

CHALLENGE - All animals
Day 22 One flank of all animals was clipped and treated by epidermal application of a 50% test substance concentration and the vehicle (0.1 mL each), using Patch Test Plasters (Curatest®, Lohmann, Almere, The Netherlands). The patches were held in place with Micropore tape and subsequently Coban elastic bandage. The dressing was removed after 24 hours exposure and the skin cleaned of residual test substance and vehicle using water. The treated sites were assessed for challenge reactions 24 and 48 hours after removal of the dressing.
After termination, animals were sacrificed using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and an intra-peritoneal injection of Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands).
Challenge controls:
Not applicable
Positive control substance(s):
yes
Remarks:
(the results of the latest reliability check, performed in July/August 2014 with Alpha- Hexylcinnamaldehyde, are reported)

Results and discussion

Positive control results:
The latest reliability check (performed less than 6 months before the study) shows a sensitisation rate of 90%.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
50% test substance in corn oil
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
50% test substance in corn oil
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
corn oil
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
corn oil
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
50% test substance in corn oil
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
50% test substance in corn oil
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
corn oil
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
corn oil
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
20% alpha-hexylcinnamicaldehyde
No. with + reactions:
8
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Remarks:
Positive control reliability check carried out July/August 2014
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
20% alpha-hexylcinnamicaldehyde
No. with + reactions:
8
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Remarks:
20% alpha-hexylcinnamicaldehyde Positive control reliability check carried out July/August 2014

Any other information on results incl. tables

Signs of irritation during induction:
For skin effects caused by the intradermal injections and epidermal exposure during the induction phase see appendix.

Evidence of sensitisation of the challenge concentration:

No skin reactions were evident after the challenge exposure in the experimental and control animals.

Toxicity / Mortality: No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

Body weights: Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
In a guinea pig maximisation test, conducted in accordance with OECD 406 and GLP, Hydrocarbons, C8-C11, n-alkanes, isoalkanes, <2% aromatics was tested at a challenge concentration of 50% v/v in corn oil. Intradermal induction was performed at a concentration of 20% v/v, and topical induction 50% test substance in corn oil. No skin reactions were observed in any test or control group animals at challenge indicating that the substance is not sensitising to skin.

Executive summary:

Assessment of Contact Hypersensitivity to Hydrocarbons, C8-C11, n-alkanes, isoalkanes, <2% aromatics in the Albino Guinea Pig (Maximization Test).

The study was carried out based on the guidelines described in:

OECD No. 406 (1992) "Skin Sensitization"

EC No 440/2008; B6: "Skin Sensitization: Guinea-Pig Maximization Test (GPMT)"

EPA OPPTS 870.2600 (2003) “Skin Sensitization”

JMAFF Guidelines (2000), including the most recent revisions.

The study was based on the method described by Magnusson and Kligman, "Allergic Contact Dermatitis in the Guinea Pig - Identification of Contact Allergens" (1970).

Test substance concentrations selected for the main study were based on the results of a preliminary study. In the main study, ten experimental animals were intradermally injected with a 20% concentration and epidermally exposed to a 50% concentration. Five control animals were similarly treated, but with vehicle alone (corn oil). Two weeks after the epidermal application all animals were challenged with a 50% test substance concentration and the vehicle. No skin reactions were evident after the challenge exposure in the experimental and control animals. There was no evidence that Hydrocarbons, C8-C11, n-alkanes, isoalkanes, <2% aromatics had caused skin hypersensitivity in the guinea pig, since no responses were observed in the experimental animals in response to a 50% test substance concentration in the challenge phase. This result indicates a sensitization rate of 0 per cent.

Based on these results Hydrocarbons, C8-C11, n-alkanes, isoalkanes, <2% aromatics does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the:

- Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2007) (including all amendments),

- Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (including all amendments).

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