Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Feb - Apr 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline conform GLP study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

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)
Type of study:
guinea pig maximisation test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: lbm: GOHI; SPF (synonym: Himalayan spotted)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd., Biotechnology & Animal Breeding Division, Füllinsdorf, Switzerland
- Age at study initiation: 5-7 weeks
- Weight at study initiation: 360-370 g (pretest group); 410-436 g (control and test group)
- Housing: individually in Makrolon type-4 cages
- Diet (e.g. ad libitum): pelleted standard Provimi Kliba 3418 guinea pig breeding / maintenance diet, containing Vitamin C, ad libitum
- Water (e.g. ad libitum): community tap water, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20+/-3°C
- Humidity (%): 330-70%
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 4. Feb. To: 15. Mar. 2002

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
polyethylene glycol
Remarks:
PEG 300
Concentration / amount:
1% in PEG 300 intradermal induction
50% in PEG 300 epidermal induction
15% in PEG 300 epidermal challenge
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
polyethylene glycol
Remarks:
PEG 300
Concentration / amount:
1% in PEG 300 intradermal induction
50% in PEG 300 epidermal induction
15% in PEG 300 epidermal challenge
No. of animals per dose:
control: 5
test group: 10
Details on study design:
1. Induction
- intradermal injections / performed on test day 1
An area of dorsal skin from the scapular region (approx. 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 inthe clipped region as follows:
Test Group:
a) 1:1 (v/v) mixture of Freund´s Complete Adjuvant and physiological saline
b) The test item at 1% in PEG 300
c) The test item at 1% in a 1:1 (v/v) mixture of Freund´s Complete Adjuvant and physiological saline
Control Group:
a) 1:1 (v/v) mixture of Freund´s Complete Adjuvant and physiological saline
b) PEG 300
c) 1:1 (w/w) mixture of PEG 300 in 1:1 (v/v) mixture of Freund´s Complete Adjuvant and physiological saline

- epidermal applications / performed on test day 8
One week after the injections, the scapular area (approx. 6 x 8 cm) was again clipped and shaved free of hair prior to the application. A 2 x 4 cm patch of flter paper was saturated with the test item (50% in PEG 300) and placed over the injection sites of the test animals. The amount of test item preparation applied was approx. 0.3 g. 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 occlusive dressings were left in place for 48 hours. The epidermal application procedure described ensured intensive contact of the test item.
The guinea pigs of the control group were treated as described above with PEG 300 only, applied at a volume of approx. 0.3 mL.
The reactionsites were assessed 24 and 48 hours after removal of the bandage for erythema and edema to method of Magnusson and Kligman.

2. Challenge / performed on test day 22
The test and control guinea pigs were challenged two weeks after the epidermal induction aplication and were treated in the same way.
Hair was clipped and shaved from a 5 x 5 cm area on the left and right flank of each guinea pig just prior to the application. Two patches (3 x 3 cm) of filter paper were saturated with the test item at the highest tested non-irritating concentration of 15% (applied to the left flank) and the vehicle only (PEG 300 applied to the right flank) using the same method as for the epidermal application. The volume of the test item preparation and vehicle applied was approx. 0.2 mL. The dressings were left in place for 24 hours.
24 hours after removal of the dressing the test sites treated with the test item were depilated as described in the epidermal pretest.
The reaction sites were assessed 24 and 48 hours after removal of the bandage for erythema and edema according to the method of Magnusson and Kligman.
Positive control substance(s):
yes
Remarks:
2-Mercaptobenzothiazole

Results and discussion

Positive control results:
The intradermal induction of sensitization in the test group was perfomed in the nuchal region with a 5% dilution of the test item in mineral oil and in an emulsion of Freund´s Complete Adjuvant (FCA) / physiological saline. The epidermal induction of sensitization was conducted for 48 hours under occlusion with the test item at 50% in mineral oil one week after the intradermal induction. The animals of the control groups were intradermally induced with mieral oil and FCA / physiological saline and epidermally induced with mineral oil under occlusion. Two weeks after epidermal induction the control and test animals were challenged by epidermal application of the test item at 0.5% in mineral oil and mineral oil alone under occlusive dressing.
Cutaneous reactions were evaluated at 24 and 48 hours after removal of the dressing.
No toxic semptoms were evident in the guinea pigs of the control or test group. No deaths occurred. All 10 test animals showed discrete/patchy to intense erythema and swelling at the 24- and 48-hour reading after the challenge treatment with 2-Mercaptobenzothiazole at 0.5% (w/w) in mineral ol. No skin effects was observed in the control group.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
15%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
none
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 15%. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: none.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
15%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
none
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 15%. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: none.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
15%
No. with + reactions:
9
Total no. in group:
10
Clinical observations:
none
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 15%. No with. + reactions: 9.0. Total no. in groups: 10.0. Clinical observations: none.
Reading:
2nd reading
Hours after challenge:
448
Group:
test group
Dose level:
15%
No. with + reactions:
9
Total no. in group:
10
Clinical observations:
none
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 448.0. Group: test group. Dose level: 15%. No with. + reactions: 9.0. Total no. in groups: 10.0. Clinical observations: none.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
none
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 0. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: none.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
none
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 0. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: none.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
0
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 0. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
0
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 0. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.

Any other information on results incl. tables

1. Skin effects after intradermal induction - performed on test day 1

The expected and common findings were observed in the control and test group after the different applications using FCA intradermally. These findings consisted of erythema, edema, necrotizing dermatitis, encrustation and exfoliation of encrustion.

2. Skin effects after epidermal induction - performed on test day 8

Control group: No erythematous or edematous reaction was observed in the animals treated with PEG 300 only.

Test group: Discrete/patchy erythema was observed in all animals at the 24 and 48 -hour reading after treatment with the test item at 50% in PEG 300.

3. Skin effects after the challenge - performed on test day 22

Control group: No skin reactions were observed in the animals when treated with either PEG 300 only or when treated with the testitem at 15% in PEG 300.

Test group: Discrete/patchy erythema was observed in all animals at the 24- and 48 -hour reading after treatment with the test item at 15% in PEG 300. No skin reactions were observed in the animals treated with PEG 300 only.

4. Viability / Mortality / Macroscopic findings

One animal of the test group was found dead on test day 10 (i.e. day of removal of the dressing in the epidermal induction phase). At necropsy, no macroscopic findings were noted. The cause of death could not be established. The death was considered spontaneous and not treatment related.

5. Clinical signs, systemic

No signs of systemic toxicity were observed in the animals.

6. Body weights

Animals no. 494 of the control group and no. 505 of the test group showed a loss of body weight (2.5 to 3.9%) during the acclimatization period. They recovered between the treatment start and the end of the study.

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:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Based on the described findings the test item has to be classified as skin sensistizer.
Executive summary:

In order to assess the sensitizing potential a Maximization-Test was performed in 15 (10 test and 5 control) female albino guinea pigs.

The intradermal induction of sensitizing in the test group was performed in the nuchal region with 1% dilution of the test item in PEG 300 and in an emulsion of Freund´s Complete Adjuvant (FCA)/physiological saline. The epidermal induction of sensitizing was conducted for 48 h under occlusion with the test item at 50% in PEG 300 one week after the intradermal induction. The animals of the control group were intradermally induced with PEG 300 and FCA/physiological saline and epidermally induced with PEG 300 under occlusion.

Two weeks after epidermal induction the control and test animals were challenged by epidermal application of the test item at 15% in PEG 300 and PEG 300 alone under occlusive dressing.

Cutaneous reactions were evaluated at 24 and 48 h after removal of the dressing.

No toxic symptoms were evident in the guinea pigs of the control or test group.

Skin reactions after the challenge procedure:

- after 24 hours:

control animals - 0/5 (15% test item in PEG 300), - 0/5 (PEG 300 only)

test animals - 9/9 (15% test item in PEG 300. One animal of the test group was found dead on test day 10; at necropsy, no macroscopic findings were noted. The cause of death could not be established. The death was considered to be spontaneous and treatment unrelated), - 0/9 (PEG 300 only).