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Description of key information

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:
From June 25, 1997 to Aug. 04, 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
, adopted by the council on July 17, 1992
Deviations:
yes
Remarks:
; the test group was treated without Freund's complete adjuvant in the third test site of the intradermal induction, due to high viscosity of the preparation
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
, Directive 96/54/EEC, July 30, 1996
Deviations:
yes
Remarks:
; the test group was treated without Freund's complete adjuvant in the third test site of the intradermal induction, due to high viscosity of the preparation
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The study was run before the requirements for LLNA testing entered into force.
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Nederland B.V., Woundenbergseweg 55, The Netherlands
- Age at study initiation: 4-6 wk
- Weight at study initiation: 379-455 g
- Housing: Individually in Makrolon type-3 cages with standard softwood bedding
- Diet: Pelleted standard Nafag Ecosan 845 25W4 guinea pig breeding/maintenance diet, ad libitum
- Water: Community tap water from Itingen, ad libitum and additional once weekly supply of ascorbic acid (approx. 1 g/L) via drinking water
- Acclimation period: Seven days for animals of control and test groups. No acclimatisation for pretest animals.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 40-70 %
- Air changes: 10-15 airchanges per h
- Photoperiod: 12 h dark/12 h light


Route:
intradermal and epicutaneous
Vehicle:
water
Concentration / amount:
Intradermal injection - 5% v/v in bi-distilled water,
Epicutaneous/topical application - 25% v/v in bi-distilled water
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
5% v/v in bi-distilled water under occlusive dressing
No. of animals per dose:
Test group: 10 animals; Control group: 5 animals
Details on study design:
RANGE FINDING TESTS: The intradermal and topical irritancy of a range of aqueous dilutions of test material was investigated to identify the following:
- irritant concentrations (maximum tolerated dose) of the test material suitable for the induction phase of the main study,
- non-irritant concentration by the topical route of administration for the challenge phase


MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: Intradermal injections - once (three pairs), topical application - once
- Exposure period: 48 h (topical application)
- Test groups:
Intradermal injections - 6 X 8 cm area of dorsal skin on the scapular region of the animals was clipped free of hair. Three pairs of intradermal injections (0.1 mL/site) were made at the border of a 4 X 6 cm area in the clipped region as follows:
1. 1:1 (v/v) mixture of Freund's complete adjuvant and physiological saline
2. Test material, 5 % v/v in bi-distilled water
3. Test material, 5 % v/v in bi-distilled water
Topical application - One wk after the injections, the scapular area (6 X 8 cm) area was clipped and shaved free of hair. A 2 X 4 cm patch of filter paper was saturated with test material and placed over the injection sites. The patch was covered with aluminium foil and firmly secured by an elastic plaster wrapped around the trunk and secured with impervious adhesive tape.
- Control group: During the induction period the control animals were treated similarly to the test animals with following exceptions:
- 1:1 (v/v) mixture of bi-distilled water in a 1:1 (v/v) mixture of Freund's complete adjuvant and physiological saline was used for third pair of intradermal injection
- the test material was replaced with bi-distilled water in all other cases.
- Site: Scapular region
- Frequency: Topical application was made one wk after intradermal injections
- Concentrations: Intradermal injection - 5 % v/v in bi-distilled water; topical application - 25 % v/v in bi-distilled water
- Evaluation: After 24 and 48 h of patch removal

B. CHALLENGE EXPOSURE
- No. of exposures: Once
- Day of challenge: 22
- Exposure period: 24 h
- Test group: The animals were challenged topically two wk after the induction period. Hair was clipped and shaved from a 5 X 5 cm area on the left and right flank of each animal. Two patches (2 X 2 cm) of filter paper were saturated with test material/bi-distilled water and applied to left and right flank, respectively using the same method as for topical application.
- Control group: The animals of the control group were challenged similarly to test group.
- Concentrations: 5 % v/v in bi-distilled water
- Evaluation: After 24 and 48 h of patch removal
Challenge controls:
Five animals challenged similarly to test group
Positive control substance(s):
yes
Remarks:
Alpha-hexylcinnamaldehyde (Historical control) tested in RCC project 901596 (Jan 06, 1997 to Feb 13, 1997)
Positive control results:
70 % of the animals of the test group were observed with positive skin reactions after treatment with a non-irritant concentration of positive control (25 % v/v). No reactions were observed in control group. This strong sensitisation elicted by positive control demonstrates the capability of the laboratory to identify positive dermal sensitisers.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
5% in bi-distilled water
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
No erythematous or oedematous reactions were observed
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
5% in bi-distilled water
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
No erythematous or oedematous reactions were observed
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
5 % in bi-distilled water
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No erythematous or oedematous reactions were observed
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 5 % in bi-distilled water. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No erythematous or oedematous reactions were observed.
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
5 % in bi-distilled water
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No erythematous or oedematous reactions were observed
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 5 % in bi-distilled water. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No erythematous or oedematous reactions were observed.
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Remarks on result:
not measured/tested

Results of the induction phase of the main study:

- Skin reactions after intradermal injection on test day 1:

Control and test group: A normal local symptoms were observed in the animals of both the groups.

- Skin reactions after epidermal application on test day 8:

Control group: No erythematous or oedematous reaction was observed in animals treated with bi-distilled water only.

Test group: No erythematous or oedematous reaction was observed in animals treated with 25 % test substance in bi-distilled water.

Mortality: No deaths were observed during the course of the treatment period.

Clinical signs (systemic): No symptoms of systemic toxicity were observed in the animals.

Body weights: All animals were within the range of physiological variability known for guinea pigs of this strain and age, except for some pretest animals which lost weight during the test substance treatment phase.

Interpretation of results:
GHS criteria not met
Conclusions:
Under the study conditions, the test material, applied at a concentration of 5% in bi-distilled water, was not sensitising to skin.
Executive summary:

The allergenic potential of the test substance was evaluated in a Guinea-pig Maximisation Test according to OECD Guideline 406 and the EU Method B.6. The study was performed in 15 (10 test and 5 control) male albino guinea pigs. The procedure consisted of two parts: induction and challenge exposures. Doses were determined on the basis of preliminary investigations.

Induction exposure:

Intradermal injections- three pairs of intradermal injections (1:1 (v/v) mixture of Freund's complete adjuvant and physiological saline, 5 % v/v of test material in bi-distilled water, and 5 % v/v of test material in bi-distilled water) were made simultaneously at the border of a 4 x 6 cm area of dorsal skin on the scapular region of the animals. Topical application - one week after the injections, a 2 x 4 cm occlusive patch (25% v/v in distilled water) was placed on the skin and left for 48 h. Test sites were evaluated after 24 and 48 h of patch removal.

Challenge exposure:

The animals were challenged topically two weeks after the induction period using 5% v/v test substance in bi-distilled water. A 2 x 2 cm occlusive patch with approximately 0.1 mL of the test material was applied.

The challenge sites were evaluated 24 and 48 h after patch removal. The skin reactions were evaluated using Draize scoring system. No positive skin reactions were observed in both control and treatment groups after challenging with 5% test material. Under the study conditions, the test material, applied at a concentration of 5% in bi-distilled water, was not sensitising to skin (Arcelin, 1997).

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

The allergenic potential of the test substance was evaluated in a Guinea-pig Maximisation Test according to OECD Guideline 406 and the EU Method B.6. The study was performed in 15 (10 test and 5 control) male albino guinea pigs. The procedure consisted of two parts: induction and challenge exposures. Doses were determined on the basis of preliminary investigations.

Induction exposure:

Intradermal injections- three pairs of intradermal injections (1:1 (v/v) mixture of Freund's complete adjuvant and physiological saline, 5 % v/v of test material in bi-distilled water, and 5 % v/v of test material in bi-distilled water) were made simultaneously at the border of a 4 x 6 cm area of dorsal skin on the scapular region of the animals. Topical application - one week after the injections, a 2 x 4 cm occlusive patch (25% v/v in distilled water) was placed on the skin and left for 48 h. Test sites were evaluated after 24 and 48 h of patch removal.

Challenge exposure:

The animals were challenged topically two weeks after the induction period using 5% v/v test substance in bi-distilled water. A 2 x 2 cm occlusive patch with approximately 0.1 mL of the test material was applied.

The challenge sites were evaluated 24 and 48 h after patch removal. The skin reactions were evaluated using Draize scoring system. No positive skin reactions were observed in both control and treatment groups after challenging with 5% test material. Under the study conditions, the test material, applied at a concentration of 5% in bi-distilled water, was not sensitising to skin (Arcelin, 1997).

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The substance was negative in a guinea pig maximisation test, therefore no classification for sensitization is required according to CLP (EC 1272/2008) criteria.