Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21. Sep. 1987 to 29. Oct. 1987
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1987
Report Date:
1987

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
1981
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
An in vivo skin sensitisation study (different from LLNA test) is already available.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Remarks:
(DUHA KFM)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Kleintierfarm Madörin, 4414 Füllinsdorf, CH
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals: not specified
- Age at study initiation: 7 to 8 weeks
- Weight at study initiation: males: 383 to 447 g; females: 425 to 486 g
- Housing: individually in Makrolon type-3 cages with standard softwood bedding ("Lignocell", Schill AG, 4132 Muttenz, CH)
- Diet: ad libitum; Pelleted standard Kliba 342, batch 38/87 guinea pig breeding/maintenance diet ("Kliba", Klingentalmühle AG, 4303 Kaiseraugst, CH); subjected to analysis for contaminants (results included in report)
- Water: ad libitum; community tap water from Itingen, subjected to analyses for contaminants (results included in report)
- Acclimation period: one week under test conditions after veterinary health examination
- Indication of any skin lesions: not specified
- Identification: by unique cage number and corresponding ear tags
- Randomisation: randomly selected at time of delivery

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 40 to 70 %
- Air changes: 10 to 15 per hour
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
other: 50:50 Freunds' complete adjuvant and physiological saline
Concentration / amount:
0 %
Day(s)/duration:
single injection
Route:
intradermal
Vehicle:
physiological saline
Concentration / amount:
1 %
Day(s)/duration:
single injection
Route:
intradermal
Vehicle:
other: 50:50 Freunds' complete adjuvant and physiological saline
Concentration / amount:
1 %
Day(s)/duration:
single injection
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
25 %
Day(s)/duration:
48 hours
Challengeopen allclose all
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
Left flank: 25 %
Right flank: 0 %
Day(s)/duration:
24 hours
No.:
#2
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
Left flank: 0 %
Right flank: 25 %
Day(s)/duration:
24 hours
No. of animals per dose:
Preliminary study: 6 animals (intradermal testing: 1 male and 1 female; epidermal testing: 2 males and 2 females)
Control: 10 animals (5 males, 5 females)
Test: 20 animals (10 males, 10 females)
Total: 36 animals (18 males, 18 females)
Details on study design:
RANGE FINDING TESTS: PRELIMINARY STUDY
- The objective of this investigation was to identify irritant test item concentrations for the induction phase of the main study. In addition, a suitable non-irritant concentration of test item, by topical administration, was identified for the challenge application.
- INTRADERMAL INJECTIONS: 0.1 ml was administrated into the clipped flank of two guinea pigs (1 male and 1 female) at concentrations of 0.1, 0.3, 0.5, 1.0, 3.0 and 5.0 % test item in physiological saline and assessed for dermal reactions (erythema, oedema and diameter in mm) after 24 hours.
- EPIDERMAL APPLICATIONS: 2 x 2 cm patches of filter paper were saturated with concentrations of 3, 5, 10 and 25 % test item in physiological saline and applied to the clipped, shaved flanks of four guinea pigs. 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. The dressings were removed after 24 hours and the reaction sites were assessed for erythema and oedema. Further examinations of the sites were performed 24 and 48 hours after the removal of the dressing.
- RESULTS AND CONCLUSIONS: following intradermal injections, no erythema was recorded at any concentration at either 0 or 24 hours, and oedema was observed in 1 animal at 1.0 % test item, and both animals at concentrations of 3.0 and 5.0 % 24 hours after injections. 1.0 % was the lowest concentration to produce positive reaction. Following epidermal applications, no erythema and no oedema was observed among any animals or any test item concentrations 24 hours after application of the test item. 25 % was the highest concentration of test item tested, and it produced no positive reactions.

MAIN STUDY

A. INDUCTION EXPOSURE

I) INTRADERMAL INJECTIONS: 6 x 8 cm area was clipped free of hair and administered 3 pairs of 0.1 ml intradermal injections per site at the border of a 4 x 4 cm area within the clipped area.
- No. of exposures: 3 pairs of injections
- Test groups: 10 males and 10 females
- Control group: 5 males and 5 females
- Site: clipped dorsal skin from the scapular region
- Frequency of injections: once
- Concentrations: 1st pair: Freunds' complete adjunct 50:50 with physiological saline; 2nd pair: 1 % test item in physiological saline; 3rd pair: 1 % test item in Freunds' complete adjunct 50:50 with physiological saline (control group: 0 % test item in 2nd and 3rd pair of injections).
- Duration: day 1

II) EPIDERMAL APPLICATIONS: one week after the intradermal injections, the same region was re-clipped and a a patch of 4 x 4 cm filter paper was saturated with 25 % test item in physiological saline (control group: 0% test item) and placed over the injection sites on the test animals, covered with aluminium foil and firmly secured by elastic plaster wrapped around the trunk of the animal and secured with impervious adhesive tape for 48 hours.
- No. of exposures: 1
- Test groups: 10 males and 10 females
- Control group: 5 males and 5 females
- Site: clipped dorsal skin from the scapular region, as for intradermal injections
- Frequency of applications: once
- Exposure period: 48 hours
- Concentrations: 25 %
- Duration: days 8 to 10

B. CHALLENGE EXPOSURES: EPIDERMAL APPLICATIONS
- No. of exposures: 2
- Timing of challenge: two and four weeks after the epidermal applications
- Exposure periods: 24 hours
- Test groups: 10 males and 10 females
- Control group: 5 males and 5 females
- Site: left and right flanks
- Concentration: 25 % test item
- Evaluation: 0, 24 and 48 hours after removal of dressing
- Duration: days 22 to 38

I) FIRST CHALLENGE: hair from a 5 x 5 cm area on the left and right flanks of all animals was clipped and a 2 x 2 cm patch of filter paper was saturated with 25 % test item in physiological saline (non-irritant concentration) and applied to the left flank of all animals (test and control) and secured with a dressing for 24 hours. A second 2 x 2 cm patch of filter paper, saturated in vehicle only (physiological saline), was simultaneously applied to the right flanks of all animals. Test sites were assessed for erythema and oedema locally at 0, 24 and 48 hours after the removal of the dressings.

II) SECOND CHALLENGE: hair was re-clipped and a 2 x 2 cm patch of filter paper, saturated with 25 % test item in physiological saline, was applied to the right flank of each test animal with a secured dressing for 24 hours. A second patch of filter paper, saturated with vehicle (physiological saline), was simultaneously applied to the left flank of each test animal. A patch of filter paper saturated in vehicle only (physiological saline) was applied to the left flank of each control animal for 24 hours, and the right flank received no treatment. Test sites were assessed for erythema and oedema locally at 0, 24 and 48 hours after the removal of the dressings.
Challenge controls:
1st challenge: controls received same treatment as test animals (application of 25 % test item to the left flank; vehicle to the right flank)
2nd challenge: controls received vehicle to left flank
Positive control substance(s):
yes
Remarks:
DINITRO-CHLORO-BENZOL (DNCB) in a concurrent study

Results and discussion

Positive control results:
According to the results observed the positive control is considered to be a skin sensitiser.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
intradermal induction 1 % in physiological saline
No. with + reactions:
1
Total no. in group:
20
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
intradermal induction 1 % in physiological saline
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
no indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
24
Group:
test group
Dose level:
intradermal induction 1 % in physiological saline
No. with + reactions:
1
Total no. in group:
20
Reading:
rechallenge
Hours after challenge:
48
Group:
test group
Dose level:
intradermal induction 1 % in physiological saline
No. with + reactions:
1
Total no. in group:
20
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0 % in physiological saline
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:
negative control
Dose level:
0 % in physiological saline
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
0 % in physiological saline
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
0 % in physiological saline
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:
positive control
Dose level:
intradermal induction 0.5 % in ethanol
No. with + reactions:
15
Total no. in group:
15
Remarks on result:
positive indication of skin sensitisation

Any other information on results incl. tables

Local symptoms: among both control and the area surrounding the injection sites of the first and second pairs of intradermal injections was found to show erythema and oedema on days 2 to 7; necrosis on days 8 to 18; then exfoliation on days 19 to 30. Discolouration was observed from day 23 to 25. Additional necroses of the injection site areas of the first and second pairs of injections were observed on days 31 to 39 (termination).

Table 1: preliminary test reactions following intradermal injection of the test item

Animal no. Sex Concentration (%) Reaction readings after 24 hours
Erythema (0 - 4) Oedema (0 - 4) Diameter (mm)
891 male  5 0 1 0
892 female 0 1 0
891 male  3 0 1 0
892 female 0 1 0
891 male  1 0 1 0
892 female 0 0 0
891 male  0.5 0 0 0
892 female 0 0 0
891 male  0.3 0 0 0
892 female 0 0 0
891 male  0.1 0 0 0
892 female 0 0 0

Table 2: preliminary test reactions following dermal application of the test item

Animal no. Sex Concentration (%) Reaction readings after 24 hours
Erythema (0 - 4) Oedema (0 - 4)
893 male  25 0 0
10 0 0
5 0 0
3 0 0
894 female 25 0 0
10 0 0
5 0 0
3 0 0
895 male 25 0 0
10 0 0
5 0 0
3 0 0
896 female 25 0 0
10 0 0
5 0 0
3 0 0

Animal no.  Sex Erythema/oedema readings after removal of bandages
0 hours 24 hours 48 hours
Erythema  Oedema  Erythema  Oedema  Erythema  Oedema 
731 male 0 0 0 0 0 0
732 male 0 0 0 0 0 0
733 male 0 0 0 0 0 0
734 male 0 0 0 0 0 0
735 male 0 0 0 0 0 0
746 female 0 0 0 0 0 0
747 female 0 0 0 0 0 0
748 female 0 0 0 0 0 0
749 female 0 0 0 0 0 0
750 female 0 0 0 0 0 0

Applicant's summary and conclusion

Interpretation of results:
other: not classified according to the CLP Regulation (EC) no. 1272/2008
Conclusions:
The test item was found to be not skin sensitiser.
Executive summary:

Skin sensitisation potential was evaluated in an in vivo experimental study performed according to the Guinea Pig Maximisation Test (GPMT) outlined in the OECD Guideline 406 (1981), and the EU Method B.6 (1984).

In a preliminary study, the lowest concentration to produce a positive reaction following intradermal injection was 1.0 % test item, therefore this concentration was selected for intradermal injection in the main study. No skin reactions were observed following epidermal exposure, therefore, the highest concentration tested (25 %) was selected for epidermal application.

The main study comprised an induction followed by two challenges with the test item. During the induction, 10 male and 10 female guinea pigs were administered 3 pairs of 0.1 ml intradermal injections along the lateral borders of a 4 x 4 cm area of a clipped, dorsal skin of the scapular region. The three pairs of injections contained the following: 1st: Freunds' complete adjuvant 50:50 with physiological saline; 2nd: 1 % test item in physiological saline; 3rd: 1 % test item in Freunds' complete adjuvant 50:50 with physiological saline. An additional 5 male and 5 female control guinea pigs were administered the same with the omission of the test item. One week later, the scapular region of all animals was re-clipped and a 4 x 4 cm patch of filter paper, saturated in 25 % test item, was secured to the same site (left flank) with aluminium foil, elastic plaster and impervious adhesive tape for 48 hours. The right flank (control) was treated similarly with physiological saline only. Skin was monitored for erythema and oedema 0, 24 and 48 hours after application. Two weeks later, the first challenge was performed: the flanks of both the test and control animals were clipped (5 x 5 cm area). A 2 x 2 cm patch of filter paper saturated with 25 % test item was applied to the left flank, and another saturated with physiological solution to the right flank, with aluminium foil, elastic plaster and impervious adhesive tape for 24 hours, at which time the dressing was removed and skin was monitored for erythema and oedema 0, 24 and 48 hours after application. Two weeks later, the second challenge was performed, however, the left and right flanks was inverted and the controls received treatment of vehicle only.

No mortality, systemic symptoms or adverse changes in body weights were observed during the study period.

The maximum % of animals with positive reactions was 5 % (at 24 h after the first challenge; at 24 and 48 h after the second challenge).