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:
weight of evidence
Study period:
12 Nov - 05 Dec 1996
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
No analytical purity of test substance given.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report Date:
1996

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
adopted in 1992
Deviations:
yes
Remarks:
No purity of the test substance given.
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
Commission directive 92/69/EEC, 1992
Deviations:
not specified
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The test was done before LLNA as first-choice method for in-vivo testing was set into force.

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:Mollegaard Breeding and Research Centre A/S, Ejby, DK-4623 Lille Skensved
- Females nulliparous and non-pregnant: [not specified]
- Microbiological status of animals, when known: SPF
- Weight at study initiation: 315 - 343 g
- Housing: two to three animals per cage, in polycarbonate (macrolone type IV, floor area 1800cm²) cages, softwood sawdust bedding
- Diet: Altromin 3113 (Chr. Petersen A/S, Ringsted, Denmark), ad libitum
- Water: vitamin C enriched domestic quality water acidified to pH 2.5 with hydrochloric acid
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3
- Humidity (%): 55 ± 15
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
other: sesame oil and a 1:1 (w/w) mixture of Freund´s complete adjuvant and sesame oil
Concentration / amount:
10% (w/w)
Day(s)/duration:
single injection
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
other: ethanol/diethylphthalate 1:1
Concentration / amount:
25%
Day(s)/duration:
48 h
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
Challenge
Route:
epicutaneous, occlusive
Vehicle:
other: ethanol/diethylphthalate 1:1
Concentration / amount:
25% (w/w)
Day(s)/duration:
24 h
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
10 (control), 20 (test group)
Details on study design:
RANGE FINDING TESTS:
The aim of the range finding test was to identify the highest concentrations which caused mild-to-moderate skin irritation after intracutaneous and epicutaneous administration and the highest non-irritant concentration after epicutaneous administration. For the intracutaneous irritancy test two animals were treated with 4 concentrations of the test substance (1.25, 2.5, 5 and 10%) in ethanol/diethylphthalate 1:1 (w/w). All concentrations tested induced erythema (grade 1) 24 and 48 h after administration and 10% was selected as adequate concentration for the intradermal induction phase of the main study. For the epicutaneous irritancy test the clipped/shaved flanks of two animals were treated with test substance concentrations of 6.25, 12.5 and 25% (w/w) under occlusive dressings for 24 and 48 h. The test substance did not cause skin reactions at any concentration tested, therefore 25% (w/w) was chosen as adequate concentration for epicutaneous induction and challenge application of the main study.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 (intradermal and epicutaneous, respectively)
- Exposure period: single injection (intradermal) and 48 h (epicutaneous)

- Test groups:
Intradermal (3 pairs of injections):
Injection 1: a 1:1 mixture (v/v) FCA/water
Injection 2: 10% (w/w) test substance in sesame oil
Injection 3: 10% (w/w) test substance in a 1:1 mixture (w/w) FCA/sesame oil
Epicutaneous: 25% (w/w) test substance in a 1:1 mixture (w/w) ethanol/diethylphthalate

- Control groups:
Intradermal (3 pair of injections):
Injection 1: a 1:1 mixture (w/w) FCA/water
Injection 2: sesame oil
Injection 3: a 1:1 mixture (w/w) FCA/water
Epicutaneous: sesame oil

- Site: intradermal: shoulder region (intradermal + epicutaneous: cranial: injection 1 and 2; caudal: injection 3)
- Frequency of applications: every 7 days
- Duration: Day 0-9
- Concentrations: intradermal: 10%; epicutaneous: 25%

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: Day 20 - 21
- Exposure period: 24 h
- Test groups: test substance in vehicle (ethanol/diethylphthalate 1:1) and vehicle only
- Control group: test substance in vehicle (ethanol/diethylphthalate 1:1) and vehicle only
- Site: left anterior flank (test substance) and left posterior flank (vehicle)
- Concentration: 25%
- Evaluation (hr after challenge): 24 and 48 h

OTHER:
To provoke a mild irritation, pretreatment of the clipped skin with 10% sodium lauryl sulphate in petrolatum was carried out the day before induction by epicutaneous administration.

Challenge controls:
The control group is actually a challenge control.
Positive control substance(s):
yes
Remarks:
Formaldehyde: 0.1% (intradermal) and 5% (epicutaneous induction and challenge) in distilled water (w/w)

Results and discussion

Positive control results:
The positive control is a historical background data group from a study performed during 10 Oct - 07 Nov 1996 under same experimental conditions. In this study 90% of the animals responded positively.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
Induction: 0% Challenge: 0%
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
Induction: 0% Challenge: 25%
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
Induction: 10% Challenge: 0%
No. with + reactions:
2
Total no. in group:
20
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
Induction: 10% Challenge: 25%
No. with + reactions:
12
Total no. in group:
20
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
Induction: 0% Challenege: 0%
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
Induction: 0% Challenge: 25%
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
Induction: 10% Challenge: 0%
No. with + reactions:
1
Total no. in group:
20
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
Induction: 10% Challenge: 25%
No. with + reactions:
13
Total no. in group:
20

Any other information on results incl. tables

Table1. Individual irritation scores 24 and 48 h after challenge

Animal No.

24 h after challenge

48 h after challenge

Left anterior (25%)

Left posterior (vehicle)

Left anterior (25%)

Left posterior (vehicle)

Control group

1

0

0

0

0

2

0

0

0

0

3

0

0

0

0

4

0

0

0

0

5

0

0

0

0

6

0

0

0

0

7

0

0

0

0

8

0

0

0

0

9

0

0

0

0

10

0

0

0

0

Test group

1

0

0

0

0

2

0

0

0

0

3

0

0

0

0

4

0

0

1

0

5

0

0

0

0

6

0

0

0

0

7

2

1

2

1

8

0

0

0

0

9

2

0

1

0

10

1

0

0

0

11

2

0

2

0

12

2

0

2

0

13

2

0

3

0

14

1

0

2

0

15

0

0

2

0

16

2

0

2

0

17

2

0

1

0

18

2

0

1

0

19

2

0

3

0

20

2

1

2

0

0: no visible change

1: slight or discrete erythema

2: moderate and confluent erythema

3: intense erythema and swelling

Intradermal injections of Freund' s complete adjuvant mixed with vehicle or test substance elicited irritation. No skin reactions were observed following induction with either vehicle or test substance.

No animal died or showed clinical signs during the course of investigation and the mean value for body weight and the body weight gain were not affected by the treatment.

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Under the conditions of the guinea pig maximisation test the test substance produced mild to intense (grade 1-3) erythema in 12/40 animals 24 h after challenge and in 13/40 animals 48 h after challenge. Therefore, the test substance is considered to be skin sensitising.
Executive summary:

The dermal sensitizing potential was investigated according to OECD Guidelines No. 406, 1992. Thirty animals divided into a test group of 20 animals and a negative control group of 10 animals were included in the study. The study comprised an induction and a challenge phase. The animals in the test group were induced with the test article whereas the animals in the control group were induced with sesame oil or Ethanol/diethylphalate 1:1. The induction procedure included intradermal injections and a closed patch topical application one week apart. A 10% (w/w) test article concentration in sesame oil was used for the intradermal induction. A 25% (w/w) test article concentration in Ethanol/Diethylphthalate 1:1 was used for the topical

induction and for the challenge application. Under these experimental conditions evidence of delayed contact hypersensitivity was seen in 12 out of 20 animals. The substance is considered to be a sensitizer.