Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study; performed under GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
inspected February 1996; signature: July 1996
Type of study:
guinea pig maximisation test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
gas under pressure: refrigerated liquefied gas
Details on test material:
- Physical state: liquid
- Storage condition of test material: In the original container, in a refrigerator at approx. 4 °C.
- Other: safety precaution: Gloves, goggles and face mask were obligatory to ensure the health and safety of the personnel.

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: Ibm: GOHI; SPF-quality (Himalayan spotted)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: recognised animal source
- Age at study initiation: 4-6 weeks
- Weight at study initiation: pretest: 291 - 307 g; main test: 298 - 422g
- Housing: Individually in Makrolon type-4 cages with standard softwood bedding
- Diet: certified pellet diet ad libitum
- Water: tap water ad libitum
- Acclimation period: One week for the control and test group under test conditions after health examination. No acclimatization for the animals of the pretest.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22
- Humidity (%): 37-70
- Air changes (per hr): 10-15
- Photoperiod:12 hours light / 12 hours dark

IN-LIFE DATES: From: 28/10/98 To: 07/12/98

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
corn oil
Concentration / amount:
Intradermal induction: 5%
Epicutaneous induction: 100%
Epicutaneous challenge: 100%
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
Intradermal induction: 5%
Epicutaneous induction: 100%
Epicutaneous challenge: 100%
No. of animals per dose:
Control Group: 5 animals
Test Group: 10 animals
Intradermal Pretest: 1 animal
Epidermal Pretest: 2 animals
Details on study design:
RANGE FINDING TESTS:
INTRADERMAL INJECTIONS:
Four intradermal injections (0.1 ml/site) of a 1:1 (v/v) mixture of Freund's Complete Adjuvant/physiological saline were made into the shaved neck of one guinea pig. One week later intradermal injections (0.1 ml/site) were made into the clipped flank of the same guinea pig at concentrations of 5, 3 and 1 % of the test article in com oil. The resulting dermal reactions were assessed 24 hours later. For intradermal induction application in the main study a 5 % test article concentration was selected.
EPIDERMAL APPLICATIONS:
Four intradermal injections (0.1 mil site) of a 1:1 (v/v) mixture of Freund's Complete Adjuvant/physiological saline were made into the shaved neck of two guinea pigs. One week later both flanks of each of the guinea pigs were clipped and shaved just prior to the application. Thereafter 4 patches of filter paper (2 x 2 cm) were saturated with the test article at A = 100 %, B = 75 %, C = 50 % and D = 25 % and applied to the clipped and shaved flanks. The volume of test article applied was approximately 0.2 ml. Corn oil was used for the dilutions. The patches were covered by a strip of aluminium foil and firmly secured by elastic plaster wrapped around the trunk and covered with impervious adhesive tape. This procedure ensured the intensive contact of the test article. The dressings were removed after an exposure period of 24 hours. Approximately 21 hours after removal of the dressing the application site was depilated with an approved depilatory cream to clean the application site, so that possible erythema reactions were clearly visible at that time. The depilatory cream was placed on the patch sites and surrounding areas, and left on for 3-5 minutes. It was then thoroughly washed off with a stream of warm, running water. The animals were then dried with a disposable towel, and returned to their cages.

MAIN STUDY
A. INDUCTION EXPOSURE
Intradermal: An area of dorsal skin from the scapular region (approximately 6 x 8 cm) 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:
Test Group:
1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
2) The test article, at 5 % in corn oil.
3) The test article at 5% in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
Control group:
1) 1: 1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
2) Corn oil
3) 1:1 (w/w) mixture of com oil in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.

Epicutantaneous: On test day 7 and approximately 26.5 hours prior to the epidermal application the scapular area (approximately 6 x 8 ern) of the animals of the test group was clipped, shaved free of hair and the test area was pretreated with a 10% dilution of Sodium-Lauryl-Sulfate (SLS) in paraffinum perliquidum as no primary irritation had been observed in the pretest. The SLS was massaged into the skin with a glass rod without bandaging. This 10 % concentration of SLS enhances sensitization by provoking a mild inflammatory reaction (Magnusson and Kligman 1970). On test day 8, a 2 x 4 cm patch of filter paper was saturated with the undiluted test article and placed over the injection sites of the test animals. The volume of test article applied was approximately 0.3 mi. The patch was covered with aluminium foil and firmly secured by an elastic plaster wrapped around the trunk of the animal and secured with impervious adhesive tape. The dressings were left in place for 48 hours. The epidermal application procedure described ensured intensive contact of the test article. The guinea pigs of the control group remained untreated during the epidermal induction. Reaction sites were assessed for erythema and oedema 24 and 48 hours after removal of the dressing, using the numerical grading system according to Draize.

B. CHALLENGE EXPOSURE
The test and control guinea pigs were challenged two weeks after the epidermal induction application. The test and control guinea pigs were treated in the same way. Hair was clipped and shaved from a 5 x 5 cm area on the left flank of each guinea pig just prior to the application. One patch (3 x 3 em) of filter paper was saturated with the highest non-irritating concentration of 100% (left flank) using the same method as for the epidermal application. The volume of test article applied was approximately 0.2 mi. The dressings were left in place for 24 hours. Approximately 21 hours after removal of the dressing the test sites treated with the test article. Approximately 24 and 48 hours after the removal of the dressing the application sites were assessed for erythema and oedema using the numerical scoring system according to Draize.
Challenge controls:
corn oil
Positive control substance(s):
yes
Remarks:
alpha-hexylcinnamaldehyde

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
100%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No symptoms of systemic toxicity observed
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. Clinical observations: No symptoms of systemic toxicity observed.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
100%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No symptoms of systemic toxicity observed
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. Clinical observations: No symptoms of systemic toxicity observed.
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:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: corn oil . No with. + reactions: 0.0. Total no. in groups: 5.0.
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:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: corn oil. No with. + reactions: 0.0. Total no. in groups: 5.0.

Any other information on results incl. tables

CLINICAL SIGNS, SYSTEMIC

No symptoms of systemic toxicity were observed in the test organisms.

 

BODY WEIGHTS

The one organism in the control group lost body weight; 0.8 %; during the acclimatization period. One organism in the test group showed a weak loss of body weight 1.9 %; during the treatment period. The body weight of the other animals was within the range commonly recorded for animals of this strain and age.

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 this study, the test material is considered to be non-sensitising.
Executive summary:

The study was performed to the requirements of OECD Guideline 406 and EU Method B.6 in accordance with GLP to assess the skin sensitisation potential of the test material in Ibm: GOHI; SPF-quality female guinea pigs following the guinea pig maximisation method. The test was validated in a historic positive control group using alpha-hexylcinnamaldehyde positive control substance documented in the study report. In a pretest, the intradermal application concentration of 5% was selected for the main test as at this concentration only mild irritation was observed. For the epicutaneous induction and challenge application, a concentration of 100% was selected for the main test as no dermal reaction in any animal at any concentration was observed in the pretest. In the main study, a group of 15 test animals (10 test animals treated with test material, 5 animals serving as an irritation control) received induction doses by intradermal injection and topical application. The intradermal injections were made within the boundaries of a 4 -cm x 6 -cm area, one row of three injections on each side of the midline. Each animal received the following: 0.1 ml of a 1:1 dilution of Freund's Complete Adjuvant in physiological saline; 0.1ml of 5% test material; medial site: 0.1ml 5% test material in Freund's Complete Adjuvant in physiological saline. The control group received the vehicle instead of the test material. On Day 7 test animals backs were shaved and pretreated with a 10% w;w sodium lauryl sulfate (SLS) suspension in paraffinum perliquidum before the topical induction application. Undiluted test material was applied over the injection sites of the test animals. The patch was covered with aluminium foil and firmly secured by a elastic plaster wrapped around the trunk of the animal and secured with impervious adhesive tape. The dressings were left in place for 48 hours. The test and control guinea pigs were challenged two weeks after the epidermal induction application. None of the test animals exhibited a dermal reaction to the challenge application of the test or control materials. Based on these results, the test material is not considered to be a skin sensitiser in guinea pigs.