Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 July - 26 August, 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent

In vivo test system

Test animals

Species:
guinea pig
Strain:
Pirbright-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann, Borchen, Germany
- Age at study initiation: ca. 6 weeks
- Weight at study initiation: 350.0 - 399.6 g
- Housing: with 2-3 animals in Makrolon cages No.4.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): set at 30-70%, but the humidity was high in general and rose up to 80 % sometimes.
- Air changes (per hr): 8
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 19 July 2004 To: 20 August 2004

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
other: sesame oil
Concentration / amount:
PILOT:
Intradermal: 0.1 ml of 5 %, 2.5 %, 1% and 0.5 % (w/w) dilution in sesame oil
Dermal: soaked patch with 75 %, 50 %, 25 % and 10 % (w/w) in sesame oil
Challenge: Duhring chamber with 60 % and 50 % (w/w) in sesame oil

MAIN TEST:
Intradermal: 0.1 ml of 5 % (w/w) solution in sesame oil
Dermal: soaked patch with a 75 % (w/w) solution in sesame oil
Challenge: Duhring chamber with a 60 % (w/w) solution in sesame oil
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: sesame oil
Concentration / amount:
PILOT:
Intradermal: 0.1 ml of 5 %, 2.5 %, 1% and 0.5 % (w/w) dilution in sesame oil
Dermal: soaked patch with 75 %, 50 %, 25 % and 10 % (w/w) in sesame oil
Challenge: Duhring chamber with 60 % and 50 % (w/w) in sesame oil

MAIN TEST:
Intradermal: 0.1 ml of 5 % (w/w) solution in sesame oil
Dermal: soaked patch with a 75 % (w/w) solution in sesame oil
Challenge: Duhring chamber with a 60 % (w/w) solution in sesame oil
No. of animals per dose:
Pilot: total of 5 animals were used.
Main test: total of 15 animals: 10 in the test group, 5 in the control group
Details on study design:
RANGE FINDING TESTS:
Intradermal: one guinea pig was shorn on the right and left flank over an area of ca. 4x6 cm. 24 hours later this animal received 0.1 ml of various concentrations of test substance intradermally by disposable syringe. Skin reactions were assessed 24 hours later.
Dermal: soaked patches of 2x4 cm with various concentrations of the test substance in sesame oil were applied.
Challenge: Duhring chambers were filled with a soaked patch of 2x2 cm with various concentrations of test substance in sesame oil and applied to the skin. 24 hours before the flanks of the animals had been shorn. The gauze patch and the Durhing chamber were applied each to one flank of a guinea pig, covered with Blenderm and bandaged on with Acrylastic. After 24 hours the dressing was removed and skin reactions assessed.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 6 intradermal injections (3 per flank) and 2 dermal applications (1 per flank)
- Exposure period: day 1 intradermal injections, day 8 dermal applications
- Test groups: 10 animals
- Control group: 5 animals (sesame oil)
- Site: left and right shoulder region (4x6 cm) below the shoulder blade
- Duration: 48 hours of dermal application
- Concentrations: injection 1: 0.1 ml FCA/sesame oil (1:1 v/v); Injection 2: 0.1 ml 5 % dilution of test substance in sesame oil (or sesame oil in control animals); Injection 3: 0.1 ml 5 % dilution of test substance (or sesame oil in controls) in FCA (1:1 v/v); Dermal application: 75% dilution of test substance in sesame oil (or sesame oil in the controls)

B. CHALLENGE EXPOSURE
- No. of exposures: each animal was challenged with the test substance and with the vehicle (sesame oil)
- Day(s) of challenge: day 22
- Test groups: 10 animals
- Control group: 5 animals
- Site: left (test substance) and right flank (vehicle)
- Concentrations: 60 % dilution of the test substance in sesame oil
- Evaluation (hr after challenge): 48 hrs and 72 hrs

OTHER:
Classification of erythema: 0 (no reddening), 1 (slight reddening), 2 (easily detectable erythema), 3 (medium erythema), 4 (strong erythema to slight formation of scurf)
Classification of edema: 0 (no swelling), 1 (slight swelling), 2 (easily recognizable swelling), 3 (medium edema, ca. 1 mm), 4 (strong edema, >1 mm)
Grading system: 0-8 % (slight), 9-28 % (slight to moderate), 29-64 % (moderate), 65-80 % (strong), 81-100 % (very strong)
Challenge controls:
No rechallenge done.
Positive control substance(s):
yes
Remarks:
2-mercaptobenzothiazole

Results and discussion

Positive control results:
Sensitisation rate of 70 % after intradermal injection of a 1.5 % dilution, dermal application with a 50 % solution and challenge with a 40 % solution.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
60%
No. with + reactions:
2
Total no. in group:
10
Clinical observations:
None
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 60%. No with. + reactions: 2.0. Total no. in groups: 10.0. Clinical observations: None.
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
sesame oil only
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
None
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: sesame oil only. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: None.
Reading:
2nd reading
Hours after challenge:
72
Group:
test group
Dose level:
60%
No. with + reactions:
2
Total no. in group:
10
Clinical observations:
None
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 60%. No with. + reactions: 2.0. Total no. in groups: 10.0. Clinical observations: None.
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
sesame oil only
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
None
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: negative control. Dose level: sesame oil only. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: None.

Any other information on results incl. tables

Intradermal induction (5 % solution): caused slight erythema (10/10) and no (8/10) to slight (2/10) edema formation after 24 h. Later on crust formation was observed at the injection sites with FCA. Control animals showed no erythema and edema.

Dermal application (75 % solution): caused no (6/10) to slight (4/10) erythema and no edema after 48 h. Control animals showed no erythema and edema.

Challenge (60 % solution): caused slight erythema at one animal after 24 h and slight edema in another animal after 24 h. After 48 h the same reactions were observed at these two animals. No reactions were observed in the remaining animals of the test group and the control group.

Body weight development was positive and within normal range.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
Besides crust formation at the injection sites after intradermal application with FCA no other systemic or toxic reactions were observed. The intradermal exposure and the use of Duhring chambers for the challenge represent a worst case situation. The sensitisation grade of 20 % leads to a slight to moderate sensitisation grade for the test substance in this system.
According to EU classification, the performed adjuvant test did not generate a response of 30 %, so no classification is necessary.
Executive summary:

The skin sensitising potential of the substance was assessed in ten female guinea pigs of the strain Pirbright White. The test was performed according to the protocol of Magnusson and Kligman, the challenge application was executed with Duhring chambers to aim for better exposure conditions. The test protocol is in compliance with OECD Guideline 406.

The maximum compatible doses for the intradermal and dermal application as well as the subirritative dose for the challenge were determined in a pilot experiment.

In the main experiment 10 female guinea pigs were treated intradermally with 0.1 ml of a 5 % (w/w) dilution of the test substance in sesam oil. For the dermal induction a 75 % (w/w) dilution of the test substance in sesam oil was used. Intradermal application of the 5 % (w/w) dilution of the tests ubstance in sesame oil and the topical application of the 75 % (w/w) dilution of the test substance caused very slight erythema and edema formation.

In the challenge the test substance elicited a very slight erythema at one animal after 24 hours. A very slight edema was obsrved after 24 hours in another animal. After 48 hours the same reactions were observed at these two animals. No erythema or edema formation were observed in the remaining animals of the test group and in the control group 24 hours and 48 hours post application. Thus, the sensitisation rate for the tests substance was 20 %. The substance has therefore to be regarded as mildly sensitising under the applied test conditions when exposed to the skin of experimental animals.