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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:
1990-06-19 to 1991-09-10
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)
Version / remarks:
adopted 12th May 1981
Deviations:
no
GLP compliance:
no
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Existing data from 1991

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Reactive Orange 12

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
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: 4 -7 weeks
- Weight at study initiation: Mean: 329 g (from 263 to 376 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 weekly and disinfected once per month. Contamination of the feed and the animals was excluded.
- Diet (e.g. ad libitum): Altromin 3020, 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 / 12

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:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
25% in physiological saline
Day(s)/duration:
one week after intradermal induction/48 hours
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:
three weeks after intradermal induction/24 hours
Adequacy of challenge:
other: highest technically possible concentration used
No.:
#2
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
Concentration: 6 and 12%
Amount: 0.5 mL
Day(s)/duration:
four weeks after intradermal induction/24 hours
Adequacy of challenge:
not specified
No. of animals per dose:
20 test animals, 10 per control group
Details on study design:
RANGE FINDING TESTS:
- Intradermal application:
One guinea pig was intradermally injected with suspensions of the 0, 1, 2.5 and 5 % of test material. The injection sides have been examined 24 and 48 h after application.
For results please refer to "Any other information on results incl. tables"
- Epicutaneous application:
0.5 mL of the test material suspension (3, 6, 12 and 25%) was applied to 4 guinea pigs under occlusive conditions for 24 h. The application sides have been examined after 48 and 72 h.
For results please refer to "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 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 untreated plaster (control)) was put on the injection sites 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 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 physioloigical saline), were applied to the left flank of all animals (test and first control group) and fixed for 24 h using a Fermoflex tape (Transatlantic GmbH, Schwarzenbach).
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 suspension (12 and 6% test material in physiological saline), to the left flank and fixed using a Fermoflex tape. In the two concentrations, 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-Crème (Olivin GmbH, Hamburg, Germany).
- Concentrations: 25% (1. Challenge, 21 days after intradermal induction), 12 and 6% (2. Challenge, 28 days after intradermal injection)
- Evaluation (hr after challenge): 48 and 72 h
- Scoring: The skin reaction have 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):
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:
20
Total no. in group:
20
Clinical observations:
all animals showed erythema, some animals showed encrustation of the skin
Remarks on result:
not determinable
Remarks:
positive negative controls
Reading:
2nd reading
Hours after challenge:
72
Group:
test group
Dose level:
25%
No. with + reactions:
17
Total no. in group:
20
Clinical observations:
all animals showed erythema, some animals showed encrustation and scaly skin.
Remarks on result:
not determinable
Remarks:
positive negative controls
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
25%
No. with + reactions:
7
Total no. in group:
10
Clinical observations:
7 animals showed erythema, two animals showed additionally encrustation of the skin
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
25%
No. with + reactions:
5
Total no. in group:
10
Clinical observations:
5 animals showed erythema
Reading:
rechallenge
Hours after challenge:
48
Group:
test group
Dose level:
12%
No. with + reactions:
16
Total no. in group:
20
Clinical observations:
16 animals showed erythema, some animals showed additionally encrustation of the skin
Remarks on result:
positive indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
72
Group:
test group
Dose level:
12%
No. with + reactions:
8
Total no. in group:
20
Clinical observations:
8 animals showed erythema, some animals showed encrustation of the 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
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
Remarks on result:
no indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
48
Group:
test group
Dose level:
6%
No. with + reactions:
13
Total no. in group:
20
Clinical observations:
13 animals showed erythema, some animals showed encrustation of the skin
Remarks on result:
positive indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
72
Group:
test group
Dose level:
6%
No. with + reactions:
5
Total no. in group:
20
Clinical observations:
5 animals showed erythema, some animals showed encrustation of the skin
Remarks on result:
no indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
6%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
72
Group:
negative control
Dose level:
6%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
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:
0
No. with + reactions:
0
Total no. in group:
10
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:
0
No. with + reactions:
0
Total no. in group:
20

Any other information on results incl. tables

Results of the pre-test

- Intradermal induction

After 24 and 48 hours no reactions were observed at 0%. At 1, 2.5 and 5% a yellow coloured area was observed (diameter at 1% = 0.3 cm; diameter at 2.5% = 0.4 cm, diameter at 5% = 0.8 cm).

 

- Epicutaneous induction

Table 2: Results for skin reaction after topical application

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

Table 2: Results for skin reaction after challenge (skin redness and edema formation)

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
41 0 0 0 0 0 0 0 0
42 0 0 0 0 0 0 0 0
43 0 0 0 0 0 0 0 0
44 0 0 0 0 0 0 0 0
 45 0 0 0 0 0 0 0 0

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 1
2 2 0
3 2 0
4 0 0
5 2 0
6 0 0
7 2* 1
8 2 1
9 2* 1
10 2 1
Test group
21

3

2

22

3*

2

23

3*

1#

24

4

2#

25

2

3#

26

3*

1#

27

2

1#

28

3*

1

29

2*

1

30

1*

1

31

1*

1#

32

3*

2#

33

4*

3 +#

34

1

1#

35

3*+

0 +#

36

2*+

0 +#

37

1*+

0*+#

38

3*+

1 +#

39

1*

1#

40

1

1#

* = Application site was stained red-blue

+ = Application site partly encrusted

# = Application site partly scaly

  

Table 4: Individual results of the 2. Challenge

Animal No.

Test material concentration

6%

12%

48 h

72 h

48 h

72 h

1. Control group

11

0

0

0

0

12

0

0

0

0

13

0

0

0

0

14

0

0

0

0

15

0

0

0

0

16

0

0

0

0

17

0

0

0

0

18

0

0

0

0

19

0

0

0

0

20

0

0

0

0

Test group

21

0

0

0

0

22

1

0

2

1*

23

1

0

1

0

24

0

0*+

1*+

0*+

25

0

0

0

0

26

1

1*

3*

2*

27

0

0

0

0

28

0

0

1

0

29

1

0

1

0

30

0

0

0

0

31

1

0

2

0

32

1

1

2

1

33

3

2

2*+

1*

34

1*

0*

1*

0*

35

1*+

0*+

2

0*+

36

1 +

1*+

3 +

1*+

37

1

1*

1*

1*

38

0

0*

2

2*+

39

1

0

1

0

40

1

0

2

1*

* = Application site partly scorched

+ = 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 both groups, control and treatment group, showed erythema at the application site. Moreover, in the treatment groups encrustations of the skin were observed. 

During a second challenge the animals were treated with either 12% or 6% 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.