Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 07, 2004 - December 20, 2004
Reliability:
1 (reliable without restriction)

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
, 1992
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
, 1996
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2000-08-02
Type of study:
guinea pig maximisation test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): PI-23513
- Substance type: technical product
- Physical state: colourless, liquid
- Storage condition of test material: ambient temperature, dark and dry
No further details are given.

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland GmbH)
- Age at study initiation: 38 days
- Weight at study initiation: 258-293g; positive reference group: 247-314g
- Housing: The animals were kept in pairs in Makrolon cages (type IV). Granulated texture wood was used as breeding material in the cages. The cages were changed and cleaned twice a week.
- Diet: ssniff Ms-H V2233; ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 3°C
- Humidity: 55 +/- 15%
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
other: sesame oil
Concentration / amount:
a 10% solution of PI-23513 (first induction stage)
2mL undiluted PI-23513 (second stage of induction)
2mL of a 25% solution of the test item in vehicle
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: sesame oil
Concentration / amount:
a 10% solution of PI-23513 (first induction stage)
2mL undiluted PI-23513 (second stage of induction)
2mL of a 25% solution of the test item in vehicle
No. of animals per dose:
preliminary study: 8 (six animals for the topical administration and 2 animals for the intracutaneous administration)
main study: 15 animals were randomly allocated to 2 groups (5 animals in the vehicle reference group, and 10 animals in the treatment group)
Details on study design:
RANGE FINDING TESTS:
The aim of the preliminary study was to determine the appropriate dose level of the test item following intracutaneous and topical administration. The concentration used in the main study following the induction procedure should produce mild to moderate skin reactions following topical application and be adjusted to the highest level that can be well tolerated locally and generally following intracutaneous injection. For the challenge exposure a subirritating concentration was used which produced no evidence of skin irritation in non-sensitised animals.
The allocation of different test sites of the animals was alternated in order to minimise site-to-site variations in response.
The shoulder and the flank region of the animals were shaved or shaved and depilated. Animals, even if only slightly injured, were exchanged.
(a) intracutaneous: 0.1mL of the prepared test item was administered intracutaneously (shoulder region). 3 concentrations of the test item were injected intradermally into one, 3 further concentrations into a second animal.
(b) topical: The test area of 3 animals each was shaved or depilated. 2mL of the test preparation was spread over a filter paper and applied to the test area and held in contact by an occlusive dressing for 24 hours. 2 concentrations each were applied to the shaved flanks of 3 animals. The occlusive dressing and the filter paper containing the test item were removed after 24 or 48 hours and the application sites were assessed immediately, 24 and 48 or immediately and 24 hours (non-depilated) after removal of the filter paper for erythema and oedema.

MAIN STUDY
A. INDUCTION EXPOSURE
Stage 1; Day 0: The shoulder region of the guinea pigs was shaved. Within this area each animal received 2 intracutaneous injections each of (1) 0.1mL Freund's complete adjuvant (FCA) (diluted 1:1 with physiological saline), (2) 0.1mL of the test item and (3) 0.1mL of the test item in a 1:1 mixture (v/v) FCA/physiological saline
Stage 1; Day 6: As the test item was not irritant to the skin, the fur was shaved from the application area and the exposed skin was coated with 0.5mL sodium lauryl sulfate 10% in vasiline in order to induce a local irritation.
Stage 2; Day 7: 7 days after the intracutaneous injection, the shoulder region of the same animals was shaved again and treated topically using the patch-test technique (exposure time: 48 hours)
Stage 3; Day 21: 2 weeks after topical application the flanks of the same animals were shaved and depilated for a further topically application using the patch test technique. The filter paper containing the test item was applied to the left flank, the filter paper with the vehicle to the right flank. 21 hours after the filter paper had been removed, the treated skin was cleaned.

The skin reaction results of the first induction exposure were evaluated at 25 and 48 hours, of the second induction at 49 and 72 hours after begin of exposure.

B. CHALLENGE EXPOSURE
Days 23 and 24:
- 21 hours after removing the filter paper the challenge area was cleaned and cleared of hair
- 3 hours later the skin reaction was observed and recorded
- 24 hours after this observation a second observation (72 hours) was made and recorded


OTHER: Skin reactions were graded as follows:
0 = no visible change
1 = discrete or patchy erythema
2 = moderate and confluent erythema
3 = intense erythema and swelling

Other observations: mortality, clinical signs, body weight
Challenge controls:
Negative control group: sesame oil; 5 males The vehicle reference animals were treated in the same way as the animals of the test group, but received sesame oil instead of the test item. However, in stage 3 the left flank was treated with the test item, the right flank with the vehicle i.e. in the same way as the test group.
Positive control substance(s):
yes
Remarks:
Benzocaine (The positive control data are obtained from the historical background of the laboratory.Test performed April/May 2004)

Study design: in vivo (LLNA)

Statistics:
The body weight was analysed statistically using Student's t-test.

Results and discussion

Positive control results:
Animals treated with benzocaine in 40% ethanolic 0.9% NaCl solution exhibited a sensitising reaction in all animals in form of a discrete or patchy erythema or a moderate and confluent erythema.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
2mL of a 25% solution of the test item in vehicle
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 2mL of a 25% solution of the test item in vehicle. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
72
Group:
test group
Dose level:
2mL of a 25% solution of the test item in vehicle
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 2mL of a 25% solution of the test item in vehicle. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: negative control. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
1st reading
Hours after challenge:
48
Group:
positive control
Dose level:
benzocaine in 40% ethanolic 0.9% NaCl solution
No. with + reactions:
20
Total no. in group:
20
Clinical observations:
in 7 animals discrete or patchy erythema was observed, in 13 animals moderate and confluent erythema occured.
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 48.0. Group: positive control. Dose level: benzocaine in 40% ethanolic 0.9% NaCl solution. No with. + reactions: 20.0. Total no. in groups: 20.0. Clinical observations: in 7 animals discrete or patchy erythema was observed, in 13 animals moderate and confluent erythema occured..
Reading:
2nd reading
Hours after challenge:
72
Group:
positive control
Dose level:
benzocaine in 40% ethanolic 0.9% NaCl solution
No. with + reactions:
20
Total no. in group:
20
Clinical observations:
in 6 animals discrete or patchy erythema was observed, in 14 animals moderate and confluent erythema occured.
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 72.0. Group: positive control. Dose level: benzocaine in 40% ethanolic 0.9% NaCl solution. No with. + reactions: 20.0. Total no. in groups: 20.0. Clinical observations: in 6 animals discrete or patchy erythema was observed, in 14 animals moderate and confluent erythema occured..

Any other information on results incl. tables

The body weight gain of the treated animals was within the range of the vehicle reference during the experiment. Behaviour remained unchanged.

Table 1: Examination of PI-23513 in the skin sensitisation test in guinea pigs according to Magnusson and Klingman; skin reaction

Animal No.

1. stage

2. stage

3. stage

 

Hours after start of treatment

 

shoulder

shoulder

48

72

 

25

48

49

72

left

right

left

right

 

Group1: vehicle control

 

1

0

0

1

1

0

0

0

0

 

2

0

0

1

1

0

0

0

0

 

3

0

0

1

1

0

0

0

0

 

4

0

0

1

1

0

0

0

0

 

5

0

0

1

1

0

0

0

0

 

Group 2: PI-23513

 

6

2

2

1

1

0

0

0

0

 

7

2

2

1

1

0

0

0

0

 

8

2

2

1

1

0

0

0

0

 

9

2

2

1

1

0

0

0

0

 

10

2

3

1

1

0

0

0

0

 

11

2

2

1

1

0

0

0

0

 

12

2

2

1

1

0

0

0

0

 

13

2

3

1

1

0

0

0

0

 

14

2

3

1

1

0

0

0

0

 

15

2

2

1

1

0

0

0

0

 

0 = no visible change, 1 = discrete or patchy erythema, 2 = moderate and confluent erythema, 3 = intense erythema and swelling

Table 2: Examination of Benzocaine in the skin sensitisation test in guinea pigs according to Magnusson and Klingman, skin reaction

Animal No.

1. stage

2. stage

3. stage

 

Hours after start of treatment

 

shoulder

shoulder

48

72

 

25

48

49

72

left

right

left

right

 

Group3: positive control (Benzocaine)

 

P1

0

0

1

1

1

0

1

0

 

P2

0

0

1

1

1

0

2

0

 

P3

0

0

1

1

2

0

2

0

 

P4

0

0

1

1

1

0

2

0

 

P5

0

0

1

1

2

0

2

0

 

P6

0

0

1

1

2

0

2

0

 

P7

0

0

1

1

2

0

2

0

 

P8

0

0

1

1

2

0

2

0

 

P9

0

0

1

1

1

0

1

0

 

P10

0

0

1

1

2

0

2

0

 

P11

0

0

1

1

1

0

1

0

 

P12

0

0

1

1

2

0

2

0

 

P13

0

0

1

1

2

0

2

0

 

P14

0

0

1

1

2

0

2

0

 

P15

0

0

1

1

2

0

2

0

 

P16

0

0

1

1

2

0

2

0

 

P17

0

0

1

1

2

0

2

0

 

P18

0

0

1

1

1

0

1

0

 

P19

0

0

1

1

2

0

1

0

 

P20

0

0

1

1

1

0

1

0

 

0 = no visible change, 1 = discrete or patchy erythema, 2 = moderate and confluent erythema, 3 = intense erythema and swelling

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
Under the present test conditions, PI-23513 revealed no sensitising properties in guinea pigs in a test model according to MAGNUSSON and KLINGMAN.