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Toxicological information

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:
March to June 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report Date:
1994

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
OECD Guidelines for Testing of Chemicals, Number 406 "Skin Sensitization", adopted by the Council on July 17, 1992 (reported Paris, April 29, 1993).
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
Directive 92/69, EEC B.6. "Acute Toxicity - Skin Sensitization", July 31, 1992
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Study available is over 12 years old

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Basic Blue 123 Cl

In vivo test system

Test animals

Species:
guinea pig
Strain:
Himalayan
Remarks:
Ibm: GOHI (Himalayan spotted)
Sex:
male
Details on test animals and environmental conditions:
ANIMALS
- Strain: Ibm: GOHI; SPF-quality guinea pigs (Himalayan spotted)
- Source: BRL, Biological Research Laboratories Ltd., Wölferstrasse 4, 4414 Füllinsdorf/Switzerland
- Females (if applicable) nulliparous and non-pregnant: NA
- Age at study initiation:approx. 6 - 8 weeks
- Weight at study initiation: 313 - 432 g
- Housing: individually
- Diet (e.g. ad libitum): pelleted standard Kliba 342 guinea pig breeding/maintenance diet (Klingentalmuhle AG,CH-4303 Kaiseraugst), ad libitum.
- Water (e.g. ad libitum): Community tap water ad libitum. Once weekly additional supply of ascorbic acid (1 g/L) via the drinking water
- Acclimation period: One week for the control and test group under test conditions alter health examination.
No acclimatization for the animals of the intradermal pretest and epidermal pretest.
Only animals without any visual signs of illness were used for the study.
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23
- Humidity (%): 36.5 - 78
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From: 30-MAR-1994 to 02-MAY-1994

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
water
Remarks:
and Freund's Complete Adjuvant/ physiological saline (1:1)
Concentration / amount:
Test item in bi-distilled water: 5% / 0.1 mL / site
Test item / Freund's Complete Adjuvant/ physiological saline (1:1): 5% / 0.1 mL / site
Day(s)/duration:
one administration on Day 1
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
other: vaselinum album
Concentration / amount:
25%
Day(s)/duration:
Day 8 / 48 hours
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: vaselinum album
Concentration / amount:
5%
Day(s)/duration:
Day 22 / 24 hours
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Control group - 10 animals
Test group - 20 animals
Intradermal pretest - 2 animal
Epidermal pretest - 4 animals
Details on study design:
RANGE FINDING TESTS:
INTRADERMAL:
Intradermal injections (0.1 ml/site) were made into the clipped flank of two guinea-pigs at concentrations of 5, 3 and 1% of the test article in bi-distilled water.
The resulting dermal reactions were assessed 24 hours later. For intradermal induction application a 5% test article dilution was selected.

EPIDERMAL:
Both flanks of each of 4 guinea pigs were clipped and shaved just prior to the application. Thereafter 4 patches of filter paper (2 x 2 cm) were saturated with the test article at A = 25% (this concentration used was found to be the most qualifled to assure an optimum technical application procedure), B = 15%, C = 10% and D = 5% of the test article in vaselinum album and applied to the clipped and shaved flanks. The patches were covered by a strip of aluminum foil and firmly secured by elastic plaster wrapped around the trunk and covered with impervious adhesive tape. This procedure ensured the intensive contact of the test article. The dressings were removed alter an exposure period of 24 hours.
21.5 hours after removing of the dressing the application site was depilated with an approved depilatory cream (VEET Cream, Reckitt & Colman AG, CH-4123 Allschwil) to clean the application site from staining produced by the test article, so that possible erythema reactions were clearly visible at that time.
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, runn-ing water. The animals were then dried with a disposable towel, and returned to their cages. The reaction sites were assessed 24 and 48 hours after removal of the bandage for erythema and oedema on a numerical basis according to Draize.
For the epidermal induction the test article at 25% in vaselinum album was selected.
For the challenge procedure the test article at 5% in vaselinum album was selected.


MAIN STUDY
INDUCTION
INTRADERMALINDUCTION - PERFORMED ON TEST DAY 1
An area of dorsal skin from the scapular region (approximately 6 x 8 cm) was clipped free of hair. Three pairs of intradermal injections (0.1 ml/site) were made at the border of a 4 x 6 cm area in the clipped region as follows:
Test Group:
1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
2) The test article, diluted to 5% with bi-distilled water.
3) The test article diluted to 5% by emulsion in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.

Control Group:
1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
2) Bi-distilled water.
3) 1:1 (w/w) mixture of bi-distilled water in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline


EPIDERMAL INDUCTION - PERFORMED ON TEST DAY 8
On test day 7 and approximately 24 hours prior to the epidermal application the scapular area (approximately 6 x 8 cm) was clipped, shaved free of hair and the test area was pretreated with 10 % Sodium-Lauryl-Sulfate (SLS) in paraffinum perliquidum. The SLS was massaged into the skin with a glass rod without bandaging. This 10% concentration of SLS enhances sensitization by provoking a mild inflammatory reaction.
On test day 8, a 2 x 4 cm patch of filter paper was saturated with the test article (25% in vaselinum album) and placed over the injection sites of the test animals. The patch was covered with aluminum foil and firmly secured by an elastic plaster wrapped around the trunk of the animal and secured with impervious adhesive tape. The dressings were left in place for approximately 48 hours. The epidermal application procedure described ensured intensive contact of the test article.
The guinea-pigs of the control group were treated as described above with vaselinum album only.
Reaction sites were assessed for erythema and oedema 24 and 48 hours after removal of the dressing, using the numerical grading system according to Draize.


CHALLENGE - PERFORMED ON TEST DAY 22
The test and control guinea-pigs were challenged two weeks after the epidermal induction application. The test and control guinea-pigs were treated in the same way.
Hair was clipped and shaved from a 5 x 5 cm area on the left and right flank of each guinea-pig just prior to the application. Two patches (2 x 2 cm) of filter paper were saturated with the highest non-irritating concentration of 25% (left flank) and the vehicle only (vaselinum album, applied to the right flank) using the same method as for the epidermal application. The dressing were left in place for 24 hours.
22 hours after removing of the dressing the test sites were depilated with an approved depilatory cream (VEET Cream, Reckitt & Colman AG, CH-4123 Allschwil). The cream was placed on the patch sites for 3-5 minutes and then washed off with a stream of warm running water. When the application sites were clean and any stains from the test article removed the animals were dried with a disposable paper towel and returned to their cages.


READING OF CHALLENGE REACTIONS
The challenge site was evaluated 24 and 48 hours after the removal of the patch using the numerical scoring system according to Draize. The readings were made under artificial fluorescent light (daylight spectrum).

OBSERVATIONS
In addition to the sensitizing reactions the following observations and data were recorded during the test and observation period:
Viability/Mortality daily during the observation period
Clinical Signs (local/systemic) daily during the observation period
Skin reactions at the times specified during the induction and challenge periods.
Body Weights at the beginning of the acclimatization period, at day one and at the termination of the test

Positive control substance(s):
yes

Results and discussion

Positive control results:
50% of the animals were positive after treatment with a nonirritant test substance concentration of 25%

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
5%
No. with + reactions:
13
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
5%
No. with + reactions:
14
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
5%
No. with + reactions:
0
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
5%
No. with + reactions:
0
Total no. in group:
10
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
vehicle
No. with + reactions:
0
Total no. in group:
20
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
vehicle
No. with + reactions:
0
Total no. in group:
20
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
vehicle
No. with + reactions:
0
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
vehicle
No. with + reactions:
0
Total no. in group:
10
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
25% 4-aminobenzoic acid ethyl ester
No. with + reactions:
10
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
25% 4-aminobenzoic acid ethyl ester
No. with + reactions:
9
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation

Any other information on results incl. tables

No deaths occurred during the study, no signs of systemic toxicity were evident in the guinea pigs of the control or test group. Body weight development during the study remained unaffected.

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The test article applied at a concentration of 5% in vaselinum album is considered to be a sensitizer when used under the described test conditions.
Executive summary:

In order to assess the cutaneous allergenic potential of the test substance, the Maximization-Test in accordance with OECD Guideline No. 406 and the Directive 96154/EEC, B.6 was carried out in 30 (20 test and 10 control) male guinea pigs.

In this study 13 and 14 of the animals of the test group were observed with positive skin reactions after treatment with a non-irritant test substance concentration of 5% in vaselinum album after 24 and 48 hours, respectively. No skin reactions were observed in the control group. Therefore the test substance at concentration of 5% in vaselinum album is considered to be a sensitizer when described under the test conditions.