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Description of key information

Skin Sens 1B (H317), according to the CLP Regulation (EC) No 1272/2008

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
other: data on Acid Blue 225_constituent 1
Adequacy of study:
key study
Study period:
From May 24th to July 01st, 1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Justification for type of information:
The experiment was conducted on one of the substance components. It should be noted that the lot tested was characterized by the Acid Blue 225_constituent 1 as main component; however, the impurity profile resulted to be similar to that characterizing the substance under assessment (details are given in the document attached to IUCLID section 13).
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
adopted on July 17, 1992
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
GLP compliance:
yes (incl. QA statement)
Type of study:
Buehler test
Justification for non-LLNA method:
Already available test.
Species:
guinea pig
Strain:
Himalayan
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd. Wölferstrasse 4, CH-4414 Füllinsdorf/Switzerland.
- Microbiological status of animals: SPF-quality guinea pigs.
- Age at study initiation: 5-7 weeks
- Weight at study initiation: test and control animals 348 - 440 g. Animals used for Irritation Screen 378 - 445 g.
- Housing: individually in Makrol on type-3 cages with autoclaved standard softwood bedding S 8/15, batch 231 ("Lignocel", Schill AG, CH-4132 Muttenz).
- Diet: pelleted standard Kliba 342, Batch nos. 62/94 and 63/94 guinea pig breeding/ maintenance diet ("Kliba", Klingentalmühle AG, CH-4303 Kaiseraugst) ad libitum.
- Water: tap-water from Füllinsdorf, ad libitum. Once weekly additional supply of ascorbic acid (1 g/1) via the drinking water.
- Acclimation period: 7 days for the control and test group under test conditions after health examination.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 - 25 °C
- Relative humidity: 54.5 - 82 %
- Air changes: 10-15 air changes per hour.
- Photoperiod: 12 hours artificial fluorescent light (approx. 100 Lux) /12 hours dark
- Other: music during the light period.
Route:
epicutaneous, occlusive
Vehicle:
petrolatum
Remarks:
vaselinum album
Concentration / amount:
25 %
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
petrolatum
Remarks:
vaselinum album
Concentration / amount:
25 %
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Control Group: 10 animals
Test group: 20 animals
Irritation Screen for Induction and for challenge: 4 + 4 animals
Details on study design:
IRRITATION SCREEN FOR INDUCTION
- No of animals: 4 guinea pigs.
- Concentrations: 5, 10, 15 and 25 % in vaselinum album.
- Exposure period: 6 hours.
- Grading: at 24 and 48 hours after removal of the patches.

IRRITATION SCREENING FOR CHALLENGE
- No of animals: 4 guinea pigs.
- Concentrations: 5, 10, 15 and 25 % in vaselinum album.
- Exposure period: 6 hours.
- Grading: at 24 and 48 hours after removal of the patches.

INDUCTION
- Skin preparation: the fur was clipped from the left shoulder of each animal in the test group.
- Exposure period: patches were applied over a period of 3 weeks. Each test animal received one patch per week which remained in place for 6 hours each. The repeated application was performed at the same site. The interval between exposure was one week.
- Untreatment period: after the last induction exposure the animals were left untreated for 2 weeks (14 days) before the challenge.
- Grading: at 24 hours after the patches have been removed. However, as the test article stained the skin blue, it was not possible to determine whether erythema was present. Any gross skin reactions were recorded without depilating the skin.

CHALLENGE
- Treatment: animals previously exposed during the induction period were challenged two weeks after the last induction exposure with test item.
- Skin preparation: the fur was removed from the left posterior quadrant of the side and back of the animals.
- Exposure period: 6 hours.
- Grading: at 24 and 48 hours after removal of the patches.

TREATMENT AND OBSERVATION METHODS
- Patching Method: the same patching method was used for induction, irritation screens, challenge and rechallenge.
- Skin preparation: the animals fur was shaved on the day before exposure with a fine clipper blade.
- Patch: a 25 mm Hill Top Chamber was saturated with the test article dilution which was applied with a spatula. The animals were put in the restrainer and the designated patch was applied to the clipped surface as quickly as possible after the test substance had been applied.
- Type of dress: the patch appliance was occluded with a rubber dental dam. The rubber dental dam 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 on each side of the animal and secured with one or 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.
- Exposure period: 6 hours (+ 15 minutes) later, the rubber dental dams and patches were removed and the animals were taken away from the restrainers. After that the animals were returned to their individual cages.

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 then 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 approximately 1 millimeter)
4 = severe oedema (raised more than 1 millimeter 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 the irritation screens, induction and challenge and repeated 24 hours (+ 2 hours) later (48-hour grades) for irritation screen and challenge. The grading method used for rechallenge and challenge was identical.

OBSERVATIONS
- Mortality/Viability: daily during treatment/observation period.
- Clinical signs: daily, during the treatment and observation period.
- Grading of skin response score: skin responses were graded during the induction and challenge period.
- Body weights: at the beginning of the acclimatization, at day one (induction start) and at the termination of the test.

NECROPSY
Necropsy was performed by experienced prosectors in one animal of the control group which was found dead on test day 28 and in one animal of the test group which was found dead on test day 1.
No necropsy was performed in the animals euthanized at termination of observation.
Positive control substance(s):
yes
Remarks:
1-chloro-2,4-dinitrobenzene in ethanol
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
25 %
No. with + reactions:
8
Total no. in group:
19
Hours after challenge:
48
Group:
negative control
No. with + reactions:
0
Total no. in group:
9
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
0.05 % in ethanol, respondig to an induction at 0.5 %
No. with + reactions:
10
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation

In the study 42 % of the test animals were observed with significant response at either 24 or 48-hour challenge reading.

MORTALITY / VIABILITY / MACROSCOPIC FINDINGS

One animal (no. 652) of the control group was found dead on test day 28. At necropsy, a dark red discoloration of the uncollapsed lungs was observed as well as several dark red foci (D = 5 mm).

One animal (no. 678) of the test group was found dead on test day 1. At necropsy the lungs were not collapsed with several dark red foci (D = 4 mm).

CLINICAL SIGNS (LOCAL/SYSTEMIC)

Neither local nor systemic symptoms were observed during the study.

BODY WEIGHTS

Two animals of the control group and one animal of the test group lost weight during the acclimatization period. The lost of weight should be considered to be of incidental nature.

Interpretation of results:
other: Skin Sens 1B (H317), according to the CLP Regulation (EC) No 1272/2008
Conclusions:
Skin sensitising
Executive summary:

The purpose of the skin sensitizing study was to assess the possible allergenic potential of test substance when administered to the skin of albino guinea pigs. For this purpose the "Buehler-Test" modified by Ritz, H. L. and Buehler, E. V. (1980) was used, in accordance with OECD guideline 406 and EU method B.6. Ten females remained untreated and were used as control animals to be treated at challenge. Twenty test females were induced three times during induction and challenged with the test substance.

In the study 42 % of the test animals were observed with significant response at either 24 or 48-hour challenge reading.

The response of at least 15 % positive animals is considered positive "R43" following the commission 93/21/EEC, Commission Directive of April 27, 1993 adapting to technical progress for the 18th time Council Directive 67/548/EEC on the approximation of the laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances.

Therefore, the test substance in concentration of 25 % in vaselinum album is considered to be a sensitizer.

Conclusion

At the challenge, more than 15 % of animals showed positive reactions responding to a topical induction dose of 25 %, therefore, the substance meets the criteria to be classified as skin sensitising, sub-catgory 1B, according to the CLP Regulation (EC) No 1272/2008.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

There are no specific data about the skin sensitisation potential of Acid Blue 225, therefore the available information on Acid Blue 225_constituent 1 have been taken into consideration. It should be noted that the lot tested was characterized by the Acid Blue 225_constituent 1 as main component. The data can be considered as adequate and the approach can be considered as suitable (details are given in the document attached to IUCLID section 13).

The purpose of the skin sensitizing study was to assess the possible allergenic potential of Acid blue 225_constituent 1 when administered to the skin of albino guinea pigs. For this purpose the "Buehler-Test" modified by Ritz, H. L. and Buehler, E. V. (1980) was used, in accordance with OECD guideline 406 and EU method B.6. Ten females remained untreated and were used as control animals to be treated at challenge. Twenty test females were induced three times during induction and challenged with the test substance.

In the study 42 % of the test animals were observed with significant response at either 24 or 48-hour challenge reading. The response of at least 15 % positive animals is considered positive, therefore, the test substance in concentration of 25 % in vaselinum album is considered to be a sensitizer.

Justification for classification or non-classification

According to the CLP Regulation (EC) No 1272/2008, 3.4.2.2 skin sensitisation section, skin sensitizer means a substance that will lead to an allergic response following skin contact. Effects seen in either humans or animals will normally justify classification in a weight of evidence approach for skin sensitizers.

On the basis of results obtained in the Buehler assay, the substance shall be classified in sub-category 1B when ≥ 15 % animals give positive results responding at > 20 % topical induction dose.

In the experiments performed on the constituent 1, at the challenge, more than 15 % of animals showed positive reactions responding to a topical induction dose of 25 %.

Therefore, the substance meets the criteria to be classified as skin sensitising, sub-catgory 1B, according to the CLP Regulation (EC) No 1272/2008.

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