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Description of key information

Skin sensitisation

Under the conditions of the study the test material is not considered to be a skin sensitiser.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 November 1987 to 19 December 1987
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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:
not specified
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Study conducted prior to adoption of LLNA guideline by the OECD.
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment: The test material was warmed to 40 °C following formulation prior to dosing
Species:
guinea pig
Strain:
Dunkin-Hartley
Remarks:
(albino)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: Test animals 409 g (group mean) and control animals 413 g (group mean)
- Housing: Guinea-pigs were housed in suspended cages with wire mesh floors.
- Diet: ad libitum, vitamin C enriched guinea-pig diet
- Water: ad libitum access to tap water
- Acclimation period: Yes; the report states that all animals were acclimated to the laboratory environment
- Indication of any skin lesions: None reported

ENVIRONMENTAL CONDITIONS
- Temperature: Approximately 21 °C
- Humidity: 30 to 70 % (relative)
- Air changes: Approximately 15 per hour
- Photoperiod: 12 hours of artificial light in each 24 hour period
Route:
intradermal and epicutaneous
Vehicle:
paraffin oil
Concentration / amount:
Intradermal: 10 % (w/w)
Topical: 40 % (w/w)
Day(s)/duration:
Interdermal injections took place on Day 1. One week later the topical application was applied for 48 hours.
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
paraffin oil
Concentration / amount:
20 % (w/w) applied to anterior site
10 % (w/w) applied to posterior site
Day(s)/duration:
2 weeks after the induction period, animals were exposed to the challenge dose for 24 hours
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
20 test animals and 10 control animals
Details on study design:
RANGE FINDING TEST
The intradermal and topical irritancy of a range of dilutions of the test material were investigated to identify (a) irritant test material concentrations suitable for the induction phase of the main study and (b) non-irritant concentrations by the topical route of administration for the challenge phase. Concentrations of 10 and 40 % (w/w) in liquid paraffin were the maximum practical concentrations that could be prepared and dosed intradermally and topically respectively. The following were selected:
-Induction: Intradermal injection: 10 % (w/w) in liquid paraffin; Topical application: 40 % (w/w) in liquid paraffin.
-Challenge: 20 and 10 % (w/w) in liquid paraffin.

MAIN STUDY
A. INDUCTION EXPOSURE
- Intradermal injections
A 4 x 6 cm area of dorsal skin on the scapular region of the guinea-pig was clipped free of hair with electric clippers. Three pairs of intradermal injections (0.1 mL each) were made simultaneously into the area. Injectables were prepared as follows:
1. Freund’s complete adjuvant was diluted with an equal volume of water for irrigation
2. The test material at 10 % (w/w) in liquid paraffin
3. The test material at 10 % (w/w) in a 50:50 mixture of Freund’s complete adjuvant and liquid paraffin

- Topical application
One week after the injections, the same 4 x 6 cm interscapular area was clipped and shaved free of hair. A 2 x 4 cm patch of Whatman No. 3 paper was saturated with the test material at 40 % (w/w) in liquid paraffin. The patch was placed on the skin and covered by a length of impermeable plastic adhesive tape (5 cm width). This in turn was firmly secured by elastic adhesive bandage (5 cm width) wound round the torso of the animal and fixed with impervious plastic adhesive tape. The dressing was left in place for 48 hours.
Control animals were treated similarly to the test animals with the exception that the test material was omitted from the application preparations.

B. CHALLENGE EXPOSURE
The test and control animals were challenged topically two weeks after the induction period using the test material at 20 and 10 % (w/w) in liquid paraffin.
Hair was removed by clipping and then shaving an area from the left flank of each guinea-pig. A 2 x 2 cm patch of Whatman No. 3 paper was saturated with approximately 0.2 mL of 20 % (w/w) test material in liquid paraffin and applied to an anterior site on the flank. The test material at 10 % (w/w) in liquid paraffin was then applied in a similar manner to the posterior site. The patches were sealed to the flank for 24 hours under strips of impermeable plastic adhesive tape secured by elastic adhesive bandage wound round the trunk and secured.
The challenge sites were evaluated 24, 48 and 72 hours after removal of the patches and scored using the following scale:
- Erythema and eschar formation:
0 = No erythema
1 = Slight erythema (barely perceptible)
2 = Well-defined erythema
3 = Moderate erythema
4 = Severe erythema (beet redness) to slight eschar formation (injuries in depth)

- Oedema formation:
0 = No oedema
1 = Slight oedema (barely perceptible)
2 = Well-defined oedema (edges of area well-defined by definite raising)
3 = Moderate oedema (raised approximately 1 mm)
4 = Severe oedema (raised more than 1 mm and extending beyond the area of exposure)
Challenge controls:
Yes, 20 and 10 % (w/w) in liquid paraffin
Positive control substance(s):
no
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
20 and 10 % (w/w) in liquid paraffin (anterior and posterior sites, respectively)
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
None specified
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
20 and 10 % (w/w) in liquid paraffin (anterior and posterior sites, respectively)
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
None
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
20 and 10 % (w/w) in liquid paraffin (anterior and posterior sites, respectively)
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
None
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
20 and 10 % (w/w) in liquid paraffin (anterior and posterior sites, respectively)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
None
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
20 and 10 % (w/w) in liquid paraffin (anterior and posterior sites, respectively)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
None
Remarks on result:
no indication of skin sensitisation
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
negative control
Dose level:
20 and 10 % (w/w) in liquid paraffin (anterior and posterior sites, respectively)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
None
Remarks on result:
no indication of skin sensitisation
Group:
positive control
Remarks on result:
other: not reported

The numerical scores awarded to the dermal reactions elicited by the challenge were all 0. There were no dermal reactions seen in any of the test or control animals at any time point.

Interpretation of results:
other: Not classified in accordance with EU criteria
Conclusions:
Under the conditions of this study the test material is not considered to be a skin sensitiser.
Executive summary:

The skin sensitisation potential of the test material was investigated in accordance with the standardised guidelines OECD 406 and EU Method B6 using the guinea-pig maximisation test.

Preliminary investigations were performed to assess the intradermal and topical irritancy of a range of dilutions of the test material. The doses selected for the induction part of the main study were 10 % (w/w) in liquid paraffin for intradermal injection and 40 % (w/w) in liquid paraffin for topical application. In the challenge portion of the study, 20 and 10 % (w/w) in liquid paraffin were applied to an anterior site and posterior site, respectively.

Twenty female guinea-pigs were assigned to the test group and ten to the control group for the main study. During induction, three pairs of intradermal injections (0.1 mL each) were made simultaneously: Freund’s complete adjuvant diluted with an equal volume of water for irrigation; the test material at 10 % (w/w) in liquid paraffin; and the test material at 10 % (w/w) in a 50:50 mixture of Freund’s complete adjuvant and liquid paraffin. One week after the injections, the same area was exposed to the test material at 40 % (w/w) in liquid paraffin for 48 hours. Control animals were treated similarly to the test animals with the exception that the test material was omitted from the application preparations.

The test and control animals were challenged topically two weeks after the induction period using the test material at 20 and 10 % (w/w) in liquid paraffin for 24 hours. The challenge sites were evaluated 24, 48 and 72 hours after removal of the patches.

The numerical scores awarded to the dermal reactions elicited by the challenge were all 0. There were no dermal reactions seen in any of the test or control animals at any time point.

Under the conditions of the study the test material is not considered to be a skin sensitiser.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The skin sensitisation potential of the test material was investigated in accordance with the standardised guidelines OECD 406 and EU Method B6 using the guinea-pig maximisation test. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Preliminary investigations were performed to assess the intradermal and topical irritancy of a range of dilutions of the test material. The doses selected for the induction part of the main study were 10 % (w/w) in liquid paraffin for intradermal injection and 40 % (w/w) in liquid paraffin for topical application. In the challenge portion of the study, 20 and 10 % (w/w) in liquid paraffin were applied to an anterior site and posterior site, respectively.

Twenty female guinea-pigs were assigned to the test group and ten to the control group for the main study. During induction, three pairs of intradermal injections (0.1 mL each) were made simultaneously: Freund’s complete adjuvant diluted with an equal volume of water for irrigation; the test material at 10 % (w/w) in liquid paraffin; and the test material at 10 % (w/w) in a 50:50 mixture of Freund’s complete adjuvant and liquid paraffin. One week after the injections, the same area was exposed to the test material at 40 % (w/w) in liquid paraffin for 48 hours. Control animals were treated similarly to the test animals with the exception that the test material was omitted from the application preparations.

The test and control animals were challenged topically two weeks after the induction period using the test material at 20 and 10 % (w/w) in liquid paraffin for 24 hours. The challenge sites were evaluated 24, 48 and 72 hours after removal of the patches.

The numerical scores awarded to the dermal reactions elicited by the challenge were all 0. There were no dermal reactions seen in any of the test or control animals at any time point.

Under the conditions of the study the test material is not considered to be a skin sensitiser.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to skin sensitisation.

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