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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report Date:
1991

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
not specified
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Study is older than 12 years

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Reactive Orange 64

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Remarks:
Pirbright-White
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Versuchstierzucht Winkelmann, Borchen/Kreis Paderborn
- Age at study initiation: mean 379 g (336 to 416 g)
- Weight at study initiation: 5 to 8 weeks
- Housing: up to 5/cage
- Diet (ad libitum): Altromin® 3020 - Haltungsdiat fUr Meerschweinchen, Hersteller: Altromin GmbH, Lage
- Water (ad libitum): tap water
- Acclimation period: at least 7 days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): ca. 50
- Air changes (per hr): ca. 10
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From: 25. June To: 26. July 1991

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
physiological saline
Concentration / amount:
0.1 mL of a 5 % suspension/application
Day(s)/duration:
single application on Day 1
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
0.5 mL of a 50 % suspension
Day(s)/duration:
single application, one week after intradermal induction for 48 h
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Challengeopen allclose all
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
0.5 mL of a 25 % suspension
Day(s)/duration:
Day 22 for 24 h
Adequacy of challenge:
highest non-irritant concentration
No.:
#2
Route:
epicutaneous, semiocclusive
Vehicle:
physiological saline
Concentration / amount:
25% and 5% / 0.5 mL
Day(s)/duration:
1 week after challenge / 24 h
No. of animals per dose:
20 test animals, plus two control groups (10 animals each)
Details on study design:
RANGE FINDING TESTS:
Intradermal: 0 %; 1 %; 2.5 %; 5 %
Epidermal: 6 %, 12 %, 25 %, 50 %

MAIN STUDY
A. INDUCTION EXPOSURE
Intradermal application
- No. of exposures: 1
- Test groups:
Three pairs of intradermal injections of 0.1 ml volume were given in the shoulder region so that one of each pair lies on each side of the midline.
Injection 1 (cranial): a 1:1 mixture (v/v) FCA/ physiological saline
Injection 2 (medial): 5% suspension of the test material in physiological saline
Injection 3 (caudal): 5% suspension of the test material formulated in a 1:1 mixture (v/v) FCA/ physiological saline.
- Control group:
Three pairs of intradermal injections of 0.1 ml volume were given in the shoulder region so that one of each pair lies on each side of the midline.
Injection 1 (cranial): a 1:1 mixture (v/v) FCA/ physiological saline
Injection 2 (medial): physiological saline
Injection 3 (caudal): physiological saline in a 1:1 mixture (v/v) FCA/ physiological saline.
- Frequency of applications: 1

Epicutaneous application
- No. of exposures: 1
- Exposure period: 48 h
A hypoallergenic band-aid (2x4 cm) impregnated with 0.5 ml the test suspension (50 % test material in physiological saline (test group) or physiological saline (control)) was put on the injection sides of the intradermal application, covered with aluminium foil and fixed for 48 h using a Fermoflex tape (Transatlantic GmbH, Schwarzenbach). At the end of the exposure period the substance was removed using water.

B. CHALLENGE EXPOSURE
- No. of exposures: 2
- Day(s) of challenge: 21 days after intradermal induction 1 week later re-challenge
- Exposure period: 24 h
One day before exposure, the application sides have been clipped. Two hypoallergenic band-aids (2x4 cm) impregnated with 0.5 ml the test suspension (25 % test material in physioloigical saline) was applied to the left flank of all animals (test and control group) and fixed for 24 h using a Fermoflex tape (Transatlantic GmbH, Schwarzenbach). A control band-aids impregnated with 0.5 mL physiological saline was applied to right flank of each animal in the same manor. At the end of the application sides have been chemically depilated using Pilca-Crème (Olivin GmbH, Hamburg).
- Concentration: 25 %; re-challenge: 25% and 5%
- Evaluation (hr after challenge): 48 and 72 h
- Scoring: The skin reaction have been scored according to Draize.

OTHER:
The body weight of all animals was recorded one day before study inition and on the las day of the study.
Challenge controls:
10 animals previously treated with the vehicle were challenged together with the test group using the same test article concentration as used for the test group.
Positive control substance(s):
no

Results and discussion

Positive control results:
n.a.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
25 %
No. with + reactions:
7
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
0 %
No. with + reactions:
0
Total no. in group:
20
Reading:
2nd reading
Hours after challenge:
72
Group:
test group
Dose level:
25 %
No. with + reactions:
2
Total no. in group:
20
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
72
Group:
test group
Dose level:
0 %
No. with + reactions:
0
Total no. in group:
20
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
25 %
No. with + reactions:
0
Total no. in group:
10
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
25 %
No. with + reactions:
0
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
0 %
No. with + reactions:
0
Total no. in group:
10
Group:
positive control
Remarks on result:
other: not stated in the study report
Reading:
rechallenge
Hours after challenge:
48
Group:
test group
Dose level:
25 %
No. with + reactions:
8
Total no. in group:
20
Remarks on result:
not determinable
Remarks:
positive reactions occurred also in vehicle controls sites of test item induced animals and in the control group
Reading:
rechallenge
Hours after challenge:
48
Group:
test group
Dose level:
0 %
No. with + reactions:
4
Total no. in group:
20
Remarks on result:
not determinable
Reading:
rechallenge
Hours after challenge:
72
Group:
test group
Dose level:
25 %
No. with + reactions:
11
Total no. in group:
20
Remarks on result:
not determinable
Remarks:
positive reactions occurred also in vehicle controls sites of test item induced animals and in the control group
Reading:
rechallenge
Hours after challenge:
72
Group:
test group
Dose level:
0 %
No. with + reactions:
9
Total no. in group:
20
Remarks on result:
not determinable
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
25 %
No. with + reactions:
3
Total no. in group:
10
Remarks on result:
not determinable
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
0 %
No. with + reactions:
0
Total no. in group:
10
Reading:
rechallenge
Hours after challenge:
72
Group:
negative control
Dose level:
25 %
No. with + reactions:
0
Total no. in group:
10
Reading:
rechallenge
Hours after challenge:
72
Group:
negative control
Dose level:
0 %
No. with + reactions:
0
Total no. in group:
10
Reading:
rechallenge
Hours after challenge:
48
Group:
test group
Dose level:
5 %
No. with + reactions:
4
Total no. in group:
20
Remarks on result:
not determinable
Remarks:
positive reactions occurred also in vehicle controls sites of test item induced animals
Reading:
rechallenge
Hours after challenge:
48
Group:
test group
Dose level:
0 %
No. with + reactions:
2
Total no. in group:
20
Remarks on result:
not determinable
Remarks:
control sire of 5% rechallenge group
Reading:
rechallenge
Hours after challenge:
72
Group:
test group
Dose level:
5 %
No. with + reactions:
8
Total no. in group:
20
Remarks on result:
not determinable
Remarks:
positive reactions occurred also in vehicle controls sites of test item induced animals
Reading:
rechallenge
Hours after challenge:
72
Group:
test group
Dose level:
0 %
No. with + reactions:
7
Total no. in group:
20
Remarks on result:
not determinable
Remarks:
vehicle control site of 5% re-challenge group

Any other information on results incl. tables

Results of the main test

Clinical observations:
No clinical signs were observed in any animal during the study, but 2 and 3 days following the 2. induction all animals of the test group had red-coloured ears. One animal of the test groups died on day 2. Necropsy revealed a blood filled thorax and a pale liver. The body weight development of the remaining animals corresponded to the control group.

The individual erythema and edema scores after the challenge are shown in Table 2.

Table 2: Individual results of the 1. Challenge

Animal Nr.  Test material concentration
Erythema/Edema Score
0% 50%
48 h 72 h 48 h 72 h
Control group
1 0/0 0/0 0/0 0/0
2 0/0 0/0 0/0 0/0
3 0/0 0/0 0/0 0/0
4 0/0 0/0 0/0 0/0
5 0/0 0/0 0/0 0/0
6 0/0 0/0 0/0 0/0
7 0/0 0/0 0/0 0/0
8 0/0 0/0 0/0 0/0
9 0/0 0/0 0/0 0/0
10 0/0 0/0 0/0 0/0
Test group
21 0/0 0/0 0/0 0/0
22 0/0 0/0 0/0 0/0
23 0/0 0/0 0/0 0/0
24 0/0 0/0 0/0 0/0
25 0/0 0/0 0/0 0/0
26 0/0 0/0 0/0 0/0
27 0/0 0/0 0/0 0/0
28 0/0 0/0 0/0 0/0
29 0/0 0/0 0/0 0/0
30 0/0 0/0 0/0 0/0
31 0/0 0/0 0/0 0/0
32 0/0 0/0 0/0 0/0
33 0/0 0/0 0/0 0/0
34 0/0 0/0 0/0 0/0
35 0/0 0/0 0/0 0/0
36 0/0 0/0 0/0 0/0
37 0/0 0/0 0/0 0/0
38 0/0 0/0 0/0 0/0
39 0/0 0/0 0/0 0/0
40 + + + +

+ animal died during study period

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
In this study, conducted according to OECD 406, the test material can be considered as a weak dermal sensitiser.
Executive summary:

In a dermal sensitization study conducted according to OECD 406 with a suspension of the test material in physiological saline, male guinea pigs were tested using the Guinea Pig Maximisation Test according to Magnusson and Kligman. Intradermal application was performed using a 5 % suspension of the test material. Epicutaneous induction was performed occlusive with a 50 % suspension for 48 h. The challenge was conducted with a 25 % suspensions of the test material for 24 h 21 days after the intradermal induction. Skin reaction was scored according to Draize. The application sides have been examined 48 and 72 h following application. After 48 hour 35% of the animals showed a weak positive reaktion, after 72 h, only 10% of the animals showed a positive effect.

Thus a re-challenge with 25% and 5% test substance was conducted one week after the first challenge. However, the data of the re-challenge are not valid, as the vehicle control side of the test animals treated with 25% and 5% test substance showed also positive effects, about in the same range as the test substance treated sides. Furthermore, at 25%, also 3/10 animals of the negative control showed postive effects.

Based on the results obtained in this study, it can be assumed that the test substance might have a weak sensitising effect; however, the results can be challenged.