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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:
From January 30 to March 02, 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
July 17, 1992
Deviations:
yes
Remarks:
Ethanol was used as vehicle and was additionally applied in the third pair of intradermal injections performed in the test group.
GLP compliance:
yes (incl. QA statement)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
A LLNA study has not been conducted because adequate data from guinea pig Maximisation test study are already available

Test material

Constituent 1
Reference substance name:
Vat Red 013
IUPAC Name:
Vat Red 013
Test material form:
solid

In vivo test system

Test animals

Species:
guinea pig
Strain:
Himalayan
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Ibm: GOHI; SPF-quality guinea pigs delivered by BRL
- Age at the beginning of acclimatation period: 5 - 7 weeks
- Weight at study initiation: Control and Test Group, 324 - 457 g; Pretest, 343 - 440 g
- Housing: individually in Makrol on type-3 cages with autoclaved standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz).
- Diet (e.g. ad libitum): pelleted standard Kliba 342, Batch no. 67/94 guinea pig breeding/maintenance diet ("Kliba", Klingentalmühle AG, CH-4303 Kaiseraugst), ad libitum.
- Water (e.g. ad libitum): community tap water from Füllinsdorf, ad libitum
- Acclimation period: one week for the control and test group under test conditions after health examination. Three days for the animals of the pretest
- Indication of any skin lesions: only animals without any visual signs of illness were
used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): between 20 - 22.5 °C
- Humidity (%): 56 - 78 %.
- Air changes (per hr): 10-15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour light, 12-hour dark cycle (approx. 100 Lux) between the hours of 6.00 a.m and 6.00 p.m.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
other: Freund's Complete Adjuvant, physiological saline and ethanol
Concentration / amount:
0.1 ml of 5 % test substance in ethanol and 0.1 ml of 5% by emulsion in a 1:1 (w/w) mixture of ethanol in Freund's Complete Adjuvant:physiological saline (1:1, v/v)
Day(s)/duration:
Day 1
Route:
epicutaneous, occlusive
Vehicle:
other: vaselinum album
Concentration / amount:
25 % of test substance
Day(s)/duration:
Day 8
Challenge
Route:
epicutaneous, occlusive
Vehicle:
other: vaselinum album
Concentration / amount:
15 % of test substance
Day(s)/duration:
Day 22
No. of animals per dose:
Main test
Test group: 20 animals
Control group: 10 animals
Pretest
Intracutaneous: 2 animals
Epicutaneous: 4 animlas
Details on study design:
RANGE FINDING TESTS:
The objective of this investigation was to identify a maximally tolerated concentration of the test article suitable for the induction phase of the main study. In addition, a suitable non-irritant concentration of the test article, by the topical route of administration, was identified for the challenge application.
The procedure employed for these investigations was as follows:
INTRADERMAL INJECTIONS
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 ethanol.
The resulting dermal reactions were assessed 24 hours later. For intradermal induction application a 5 % test article dilution was selected.
EPIDERMAL APPLICATIONS:
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 = 30 % (this concentration used was found to be the most qualified to assure an optimum technical application procedure), B = 25 %, C = 15 % and D = 10 % 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 after an exposure period of 24 hours. 21 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, running 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.

MAIN STUDY
A. INDUCTION EXPOSURE
Intradermal injections / 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 ethanol.
3) The test article diluted to 5% by emulsion in a 1:1 (w/w) mixture of ethanol in Freund's Complete Adjuvant:physiological saline (1:1, v/v)
Control Group:
1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
2) ethanol
3) 1:1 (w/w) mixture of ethanol in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.

Epidermal applications / performed on test day 8
One week after the injections, the scapular area (approximately 6 x 8 cm) wasagain clipped and shaved free of hair. 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 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.

B. CHALLENGE EXPOSURE/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-1rritating concentration of 15 % (left flank) and the vehicle only (vaselinum album, applied to the right flank) using the same method as for the epidermal application. The dressings were left in place for 24 hours.
21 hours after removal of the dressing the test sites treated with the test article were depilated with an approved depilatory cream (VEET Cream, Reckitt & Col man 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.
24 and 48 hours after the removal of the dressing the application sites were assessed for erythema and oedema using the numerical scoring system according to Draize.
Positive control substance(s):
yes
Remarks:
2-MERCAPTOBENZOTHIAZOL

Results and discussion

Positive control results:
In this study 60 % of the animals were positive after treatment with a nonirritant test substance concentration of 25 % in mineral oil. According to the rating of allergenicity by Magnusson and Kligman (1969) the test article 2-MERCAPTOBENZOTHIAZOL is considered to be a moderate sensitizer (grade III) when described under the test conditions

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
15 % test susbtance in vaselinum album
No. with + reactions:
0
Total no. in group:
20
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
15 % in vaselinum album
No. with + reactions:
0
Total no. in group:
20

Applicant's summary and conclusion

Interpretation of results:
other: CLP criteria not met
Conclusions:
Not skin sensitizer
Executive summary:

Method

To assess the allergenic potential of the test substance in albino guinea pigs the Maximization-Test of B. Magnusson and A.M.

Kligman (1969) was used, according to the OECD guideline 406. Ten males were used as control group and 20 males were used as test group.

The highest non-irritating test article concentration used for challenge application was 15 % in vaselinum album (w/w) dilution of the original test article.

Observations

In this study none of the animals of the test group were observed with positive skin reactions after treatment with a non-irritant test substance concentration of 15 % in vaselinum album. No skin reactions were observed in the control group.

Conclusion

The test article at concentration of 15 % in vaselinum album is considered to be a non-sensitizer when described under the test conditions.