Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1995-07-15 to 1995-09-14
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The reliability of the original study used in Read Across was 1. Justification for Read Across is given in Section 13 of IUCLID.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Principles of method if other than guideline:
The relative humidity in the animal room transiently exceeded the target range of 40-70 %.
The concentrations used for the irritation screen for challenge were based only on the results of the irritation screen for induction since the test article stained the skin blue in the induction procedure.
GLP compliance:
yes (incl. certificate)
Type of study:
Buehler test
Justification for non-LLNA method:
Existing data from 1995

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vivo test system

Test animals

Species:
guinea pig
Strain:
Himalayan
Sex:
male
Details on test animals and environmental conditions:
Test Animals:
- Source: BRL, Biological Research Laboratories Ltd. Wölferstrasse 4, CH-4414 Füllinsdorf/Switzerland.
- Age at arrival: 5-7 weeks old.
- Weight at arrival: 368-498 grams.
- Housing: individually in Makrolon type-3 cages (size: 22x37x15 cm) with autoclaved standard softwood bedding S 8/15, batch 231 (“Lignocel, Schill AG, CH-4132 Muttenz).
- Diet: ad libitum pelleted standard Kliba 342, Batch no. 69/95 guinea pig breeding/maintenance diet (“Kliba”, Klingentalmühle AG, CH-4303 Kaiseraugst).
- Water: ad libitum Comunitx tap-water from FDüllinsdorf. Once weekly additionally supply of ascorbic acid (1g/l).
- Acclimation period: at least 7 days.

ENVIRONMENTAL CONDITIONS
- Humidity: 52 – 86 %.
- Air changes: approximately 10 - 15 per hr.
- Photoperiod (hrs dark / hrs light): 12/12, music during light period.

Study design: in vivo (non-LLNA)

Induction
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
Each 25 mm Hill Top Chamber was saturated by the freshly prepared test substance solution (50 % in bidistilled water), applied with a spatula. Dose applied was nit determined.
Day(s)/duration:
three weekly 6-hour applications
Adequacy of induction:
other: highest concentration of induction screen (50 %) showed no adverse effect
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
50 % in bidistilled water
Day(s)/duration:
6 hours
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
20 guniea pigs in the test group, 10 in the control group
Details on study design:
Treatment Methods:
Patching method: The same patching method was used for induction, irritation screens and challenge. The animals’ fur was shaved on the day before exposure with a fine clipper blade. Closed patches were applied to the animals as follow:
Each 25 mm Hill top Chamber was saturated by freshly prepared substance solution, applied with a spatula. The dose applied was not determined.
The animals were put in the restrainer and the designated patch was applied to the clipped surface as quickly as possible after the test item has been applied. The patch appliance was occluded with a rubber dental dam. The rubber dental gum used for occlusion of the patches was of medium gauge 12-15 cm wide, depending on the animal size and the number of patches to be covered. The rubber dental dam was pulled snug om each side of the animal and secured with one more large size clip on each side of the restrainer. The rubber dental dam was placed under the front and back metal restraining bands and had snug contact with the animal over the entire dorsal surface.
The restrainers were adjusted to minimize movement of the animals during the exposure period. Six hours later, the rubber dental dams and patches were removed and the animals were taken away from the restrainers. After the animals were returned to their individuals cages.

Induction:
The inductions phase of sensitization testing consisted of three weekly 6 –hour applications of the test substance at the same site on the left shaved shoulder of the test animal.
The induction was performed with the test article at 50 % in bi-distilled water. After the last induction exposure, the animals were left untreated for 14 days before primary challenge. The responses were graded 24 hours (± 2 hours) after patch removal, according to the grading method described below. Any gross skin reactions were recorded without depilation.

Challenge:
The animals previously exposed during the induction period (i.e. test group) as well as the previously untreated control animals were challenged two weeks after the last induction exposure using the test article concertation of 50 % in bi-distilled water. The fur was clipped from the left posterior quadrant of the side and back of the animals. Patch sites for challenge are indicated below. The exposure period was 6 hours on a naïve skin site. The responses were graded at 24 and 48 hours after the patches have been removed according to the grading method described below.

Grading method:
The animals used for irritation screens and challenge were depilated eighteen to twenty-two (18-22) hours after patches had been removed, using an approved depilatory cream (VEET cream, Reckitt & Colman AG, CH-4123 Allschwil). The depilatory was placed on the patch sites and surrounding areas and left on for 3-5 minutes. It was then thoroughly washed off with a stream of warm running water. The animals were dried with a disposable towel, and returned to their cages.
Erythema and oedema were assessed as follows:
Erythema:
0 = no erythema
± = slight, patchy erythema (i.e. barely perceptible or questionable reaction)
1 = slight confluent erythema (i.e. a slight but definite reaction at the patch site) or moderate, but patchy erythema (i.e. moderate erythema involving at least 50% or more of the area of the patch site.)
2= moderate confluent erythema
3= severe erythema

Oedema (according to Draize)
0 = no oedema
1 = very slight oedema (barely perceptible)
2 = well-defined oedema (edges of area well-defined by definite raising)
3= moderate oedema (raised approx. 1mm)
4= severe oedema (raised more than 1 mm and extending beyond the area of exposure)
The grading method used for irritation screens, induction and challenge was identical. This one was performed 24 hours (± 2 hours) after removal of the patches for irritation screens, induction and challenge and repeated 24 hours (± 2 hours) later (48-hour grades) for irritation screen and challenge.
Challenge controls:
10 animals previously untreated were challenged together with the test group using the test aritcle concentration of 50 % in bi-distilled water.
Positive control substance(s):
no

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
50 % in bi-distilled water
No. with + reactions:
2
Total no. in group:
20
Clinical observations:
2 animals with slight confluent erythema
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
50 % in bi-distilled water
No. with + reactions:
2
Total no. in group:
20
Clinical observations:
2 animals with slight patchy erythema
Remarks on result:
no indication of skin sensitisation

Any other information on results incl. tables

Resultion of the irritation screening:

Table 1: Results of the induction screening

Irritancy results

 

24 hours reading

Concentrations (%) of the test substance

24 hours reading

Concentrations (%) of the test substance

Response grade (erythema)

50

25

15

10

50

25

15

10

0

4*

4

4

4

4

4

4

4

±

0

0

0

0

0

0

0

0

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

 * = number of animals showing response

Grade 0 and ± are considered to be representative of insignificant response, whereas those of one or greater are considered to be significant. The results of the 24- and 48 hours reading were taken into consideration and in this case the test article at 50, 25, 15, and 10 % resulted in 4 scores of 0. Therefore the most representative concentration to stimulate a state of immune hypersensitivity is 50% in bi-distilled water.

Table 2: Results for Irritation screen for challenge

 

24 hours reading

Concentrations (%) of the test substance

24 hours reading

Concentrations (%) of the test substance

Response grade (erythema)

50

25

15

10

50

25

15

10

0

4*

4

4

4

4

4

4

4

±

0

0

0

0

0

0

0

0

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

 * = number of animals showing response

Grade 0 and ± are considered to be representative of insignificant response, whereas those of one or greater are considered to be significant. The results of the 24- and 48 hours reading were taken into consideration and in this case the test article at 50, 25, 15, and 10 % resulted in 4 scores of 0. Therefore the highest non-irritating concentration for the challenge was determined as the concentration of 50% in bi-distilled water.

 

Table 3: Primary Sensitization Results (Incidence Tables)

 

Erythema score

Test group

20 animals

Control group

10 animals

 

24 hrs

48 hrs

24 hrs

48 hrs

0

18

18

10

10

±

0

2

0

0

1

2

0

0

0

2

0

0

0

0

3

0

0

0

0

No. with grades ≥ 1

2

0

0

0

No. Tested

20

20

20

20

Total**

2

0

** The Total is the number of animals showing a grade ≥ 1 at either 24 or 48 hours.

 

10 % of the animals of the test group were observed with positive reactions (grade 0 and ± are considered to be non-significant responses, whereas those of 1 and greater are considered to be significant) after challenge performed with the highest non-irritating concentration of the test item at 50 % in bi-distilled water.

No reactions were observed in the control group treated in the same conditions during the challenge phase.

Significant responses in at least 15 % of the test group is considered positive and would be warrant for classification. Therefore the test item tested as described, is considered to be a non-sensitizer.

Applicant's summary and conclusion

Interpretation of results:
other: not classified for skin sensitisation according to the CLP Regulation (EC) No.1272/2008.
Conclusions:
The test item tested as described in this report, is considered to be a non-sensitizer.
Executive summary:

In a dermal sensitization study with the test substance in bi-distilled water (50 % w/w), young Himalayan guinea pigs (20 test group & 10 control/males) were tested using the modified Buehler test (OECD 406). Twenty male guinea pigs were treated topically with the test article 50 % in bi-distilled water once a week for a three week induction phase. Two weeks after the final induction the animals were challenged with the same test substance used for induction at 50 % in bi-distilled water. Th e animals of the control group were not treated during induction but were treated once at challenge.  

Only two animals of the test group showed a slight confluent erythema at the first (24 h) and a slight, patchy erythema at the second reading (48 h). These findings were considered to be not significant. 

Therefore in this study, the test material is not a dermal sensitizer.