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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:
1989-03-28 to 1989-08-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report Date:
1989

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
adopted 12 May 1981
Deviations:
no
GLP compliance:
no
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Existing data from 1989

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Reactive Yellow 27

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Versuchstierzucht Winkelmann, Borchen, Germany
- Microbiological status of animals, when known: SPF
- Age at study initiation: 5 - 7 weeks
- Weight at study initiation: Mean: 348 g (from 314 to 393 g)
- Housing: Animals (5 animals per cage) were housed in type IV Makrolon® cages with low-dust granula (Ssniff Spezialdiäten GmbH, Soest, Germany). The animal room was cleaned once per week and disinfected once per month. Contamination of the feed and the animals was excluded.
- Diet (e.g. ad libitum): Diet "Altromin 3022 - Haltungsdiät für Meerschweinchen", ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: at least 7 days
- Indication of any skin lesions: Only healthy, symptom-free animals were used

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 50
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 hours dark/ 12 hours light

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
physiological saline
Concentration / amount:
5% in physiological saline
Day(s)/duration:
single application
Adequacy of induction:
other: based on the results of a pre-test
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
25% in physiological saline
Day(s)/duration:
single application, one week after intradermal induction
Adequacy of induction:
highest technically applicable concentration used
Challengeopen allclose all
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
Concentration: 25%
Amount: 0.5 mL
Day(s)/duration:
24 hours
Adequacy of challenge:
highest non-irritant concentration
No.:
#2
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
Concentration: 12 and 2.5%
Amount: 0.5 mL
Day(s)/duration:
24 hours
Adequacy of challenge:
not specified
No. of animals per dose:
20 guinea pigs in the test group, 10 in the control group
Details on study design:
RANGE FINDING TESTS:
- Intradermal application:
One guinea pig received intradermally 0.1 mL of the following test item concentrations: 0, 1, 2.5 and 5%. The injection sites were assessed 24 and 48 h after application. For results please refer to box "Any other information on results incl. tables".
- Epicutaneous application:
4 guinea pigs received under occlusive conditions each of the following test item concentrations: 3%, 6%, 12% and 25% for 24 h. The application sites were assessed after 48 and 72 h.
For results please refer to box "Any other information on results incl. tables".

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 formulated 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
One day before exposure, the application sites have been clipped and coated with 0.2 mL of a 10% preparation of sodium lauryl sulfate solution in paraffin oil.
A hypoallergenic band-aid (2 x 4 cm) impregnated with 0.5 mL of the test suspension (25 % test material in physiological saline (test group) or physiological saline (control)) was put on the injection sites of the intradermal application, covered with aluminum 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 physiological saline.


B. CHALLENGE EXPOSURE
- No. of exposures: 2
- Day(s) of challenge: 21 and 28 days after intradermal induction
- Exposure period: 24 h
One day before exposure, the application sites have been clipped. Two hypoallergenic band-aids 2 x 4 cm), impregnated with 0.5 mL of the test item suspension (25% test material in physiological saline), were applied to the left flank of all animals (test and first control group) and fixed for 24 h using Saniplast-bandage.
For the second challenge, animals (test and second control group) have been applied two hypoallergenic band-aids impregnated with 0.5 mL of the test item suspension (12 and 2.5% test material in physiological saline) were applied to the left flank and fixed using a Fermoflex tape (Transatlantic GmbH, Schwarzenbach). In the two concentrations and alternating from animal to animal, the patches were applied cranially or caudally. On the right flank two control plaster were applied, soaked with physiological saline (0.5 mL application volume). At the end of the exposure time, application sites have been chemically depilated using Pilca-Creme (Olivin GmbH, Hamburg, Germany).
- Concentrations: 25% (1. Challenge, 21 days after intradermal induction), 12 and 2.5% (2. Challenge, 28 days after intradermal injection)
- Evaluation (hr after challenge): 48 and 72 h
- Scoring: The skin reaction has been scored as proposed by Magnusson and Kligman (see Table 1 in box "Any other information on material and methods incl. tables")
Challenge controls:
10 animals previously treated with the vehicle were challenged together with the test group using the same test article concentration as for the test group.
Positive control substance(s):
not specified

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:
18
Total no. in group:
20
Clinical observations:
13 animals showed discrete or patchy erythema, 5 showed moderate and confluent erythema
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
72
Group:
test group
Dose level:
25%
No. with + reactions:
16
Total no. in group:
20
Clinical observations:
12 animals showed discrete or patchy erythema, two animals showed additionally scaly skin. 4 animals showed moderate and confluent erythema, two animals showed additionally scaly skin.
Remarks on result:
positive indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
25%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
25%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
48
Group:
test group
Dose level:
12%
No. with + reactions:
15
Total no. in group:
20
Clinical observations:
12 animals showed discrete or patchy erythema, some animals showed additionally parts of scaly (n=6) and encrustion skin (1 animal). 3 animals showed moderate and confluent erythema, with partly scaly skin. 1 animal only showed partly scaly skin
Remarks on result:
positive indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
72
Group:
test group
Dose level:
12%
No. with + reactions:
9
Total no. in group:
20
Clinical observations:
9 animals showed discrete or patchy erythema, with additionaly scaly skin. One animal showed only partly scaly skin.
Remarks on result:
positive indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
12%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
72
Group:
negative control
Dose level:
12%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
48
Group:
test group
Dose level:
2.5%
No. with + reactions:
12
Total no. in group:
20
Clinical observations:
10 animals showed discrete or patchy erythema, three of them showed additionally partly scaly skin. 2 animals showed moderate and confluent erythema, and adiditionallly partly encrustation of the skin (n=1) or scaly skin (n=1).
Remarks on result:
positive indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
72
Group:
test group
Dose level:
2.5%
No. with + reactions:
5
Total no. in group:
20
Clinical observations:
5 animals showed discrete or patchy erythema, 3 of them showed additionally partly scaly skin.
Remarks on result:
positive indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
2.5%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
72
Group:
negative control
Dose level:
2.5%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation

Any other information on results incl. tables

Results of the pre-test

- Intradermal induction

After 24 hours the skin around the injection side were dyed golden-yellow

 

- Topical induction

Table 2: Results for skin reaction after topical application in the pre-test

Animal No.  Test material concentration
3% 6% 12% 25%
48 h 72 h 48 h 72 h 48 h 72 h 48 h 72 h
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

Further observations:

No animals died during the experimantal phase and the body weight gain was comparable to the control animals.

Results of the main test

Table 3: Individual results of the 1. Challenge

Animal No.

Test material concentration

25%

48 h

72 h

1. Control group

1

0

0

2

0

0

3

0

0

4

0

0

5

0

0

6

0

0

7

0

0

8

0

0

9

0

0

10

0

0

Test group

21

1

1

22

2

1

23

1

1

24

1

1

25

1

1

26

2

2

27

2

2*

28

0

0

29

1

1

30

0

0

31

1

1*

32

2

2

33

1

1*

34

1

0

35

1

0

36

1

1

37

1

1

38

1

1

39

2

2*

40

1

1

* = Application site partly scaly

Table 4: Individual results of the 2. Challenge

Animal No.

Test material concentration

12%

2.5%

48 h

72 h

48 h

72 h

1. 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

 

 

 

 

 

21

1+*

1*

1*

1*

22

1*

1*

1

0

23

1

0

1

0

24

1

1

1

0

25

1

1

1

0

26

1*

0

0

0

27

2*

1*

2*

1*

28

1*

1

0

0

29

1

0

1

0

30

0

0

0

0

31

1*

0*

1*

0

32

2*

1*

1*

1

33

1

0

0

0

34

1

0

1

0

35

0

0

0

0

36

0*

0*

0

0

37

1*

1*

1

1

38

0

0

0

0

39

2*

1*

2+

1*

40

0

0

0

0

* = Application site partly scaly

+ = Application site partly encrusted

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
In conclusion, based on the results obtained from a dermal skin sensitisation test, the test item can be considered as a dermal sensitizer.
Executive summary:

In a dermal sensitization study conducted according to OECD 406, young female BOR : DHPW guinea pigs (20 animals/test group & 10 animals/control) were tested using the Guinea Pig Maximisation Test according to Magnusson and Kligman. The animals received intradermally 5% of the test item in physiological saline. One week after the intradermal induction, the animals received the second induction by applying the test item as a 25% suspension in physiological saline topically. Three weeks after the intradermal induction, the animals were challenged with a 25% suspension in physiological saline of the test item. The animals of the control group were not treated during the induction, but were treated once at each challenge. After the first challenge the treatment group showed erythema at the application site. Moreover, in the treatment groups scales of the skin were observed. In the control group no indication of skin sensitisation occurred.

During a second challenge the animals were treated with either 12% or 2.5% suspensions in physiological saline. During the re-challenge, erythema and encrustations of the skin were only observed in the treatment groups. No effects were observed in the control group animals. Therefore, based on the results obtained, the test item can be considered as a dermal sensitizer.