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Description of key information

Not skin sensitising.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From April 25th to May 26th, 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
29th April 1993
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
yes
Remarks:
a mixture of FC: physiological saline was chosen instead of FCA only in order to decrease the site effects of FCA when applied alone
GLP compliance:
yes (incl. certificate)
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.
Species:
guinea pig
Strain:
Himalayan
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Biological Research Laboratories Ltd. Switzerland.
- Females: nulliparous and non-pregnant.
- Age at beginning of acclimatization period: 5 - 7 weeks.
- Weight at study initiation: 342 - 491 g.
- Housing: individually in Makrolon type-3 cages with autoclaved standard softwood bedding ("Lignocel", Schill AG).
- Diet: pelleted standard Kliba 342, Batch no. 68/95 guinea pig breeding/maintenance diet ("Kliba", Klingentalmühle AG), ad libitum.
- Water: community tap water from Füllinsdorf, 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 after health examination. No acclimatization for the animals of the pretest. Only animals without any visual signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature: between 21 - 22 °C
- Relative humidity: between 46-60 %
- Air changes: 10-15 air changes per hour and continuously monitored environment.
- Photoperiod: 12-hour light, 12-hour dark cycle (approx. 100 Lux) between the hours of 6.00 a.m and 6.00 p.m.
- Other: music was played during the daytime light period.
Route:
intradermal and epicutaneous
Vehicle:
water
Concentration / amount:
5 % (intradermal injection)
50 % (epidermal application)
Vehicle:
water
Concentration / amount:
15 %
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
10 females as control group and 20 females as test group.
Details on study design:
PREPARATION OF TEST MATERIAL
The test article and vehicle were placed into a glass beaker on a tared Mettler PM 480 balance and weight/weight dilutions were prepared. Homogeneity of the test article in a suitable vehicle was maintained during treatment using a magnetic stirrer. The preparations were made immediately prior to each dosing.

RANGE FINDING TESTS
- N. of animals: 6 females, nulliparous and non-pregnant.
- Weight at study initiation: 342 - 491 g; pretest 355 - 431 g.
- Intradermal injection: injections of 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 application: 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 50 % (this concentration used was found to be the most qualified to assure an optimum technical application procedure), 25 %, 15 % and 10 % of the test article in bi-distilled water 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.
- Post-exposure: 21 hours after removal of the dressing the application site was depilated with an approved depilatory cream 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.
- Scoring system: 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 - APPLICATION ON DAY 1
- No. of exposures: three pairs of intradermal injections (0.1 ml/site) were made.
- Site: an area of dorsal skin from the scapular region (approximately 6 x 8 cm) was clipped free of hair. The injections were made at the border of a 4 x 6 cm area in the clipped region.
- Test groups: application day 1
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: application day 1
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 a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline

EPIDERMAL APPLICATIONS - APPLICATION ON DAY 8
- No. of exposures:
- Day(s) after first injection: 7 days.
- Site: the scapular area (approximately 6 x 8 cm) was again clipped and shaved free of hair.
- Test groups: a 2 x 4 cm patch of filter paper was saturated with the test article (50 % in bi-distilled water) 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 epidermal application procedure described ensured intensive contact of the test article.
- Exposure period: the dressings were left in place for 48 hours.
- Control group: the guinea-pigs of the control group were treated as these of test group with bidistilled water only.
- Evaluation: 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
- Day(s) of challenge: 22th, two weeks after the epidermal induction application.
- Site: 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.
- Test groups: 2 patches ( 2 x 2 cm) of filter paper were saturated with the highest non-irritating concentration of 15 % (left flank) and the vehicle only (bi-distilled water applied to the right flank), using the same method as for the epidermal application.
- Control group: the test and control guinea-pigs were treated in the same way.
- Exposure period: the dressings were left in place for 24 hours.
- Evaluation: 21 hours after removal of the dressing the test sites treated with the test article were depilated with an approved depilatory cream. 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. Approximately 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.

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 daily during the observation period (local/systemic)
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

PATHOLOGY
Necropsy was performed by experienced prosectors in one animal of the epidermal pretest group and one animal of the test group, which were found dead on test day 21 and 22, respectively.
No necropsies were performed on the animals sacrificed at termination of the observation period.
The surviving animals were sacrificed with an intraperitoneal injection of Narcoren (Rhone Merieux GmbH) at a dose of at least 5.1 ml /kg body weight (equivalent to 810 mg sodium pentobarbitone/kg body weight) and discarded.

POSITIVE CONTROL
The sensibility and reliability of the experimental technique used was assessed by use of substances which are known to have skin sensitization properties in the guinea pig strain. The positive controls were performed with 4-Aminobenzoic acid ethyl ester (testing laboratory project 392051) and 2-mercaptobenzothiazol (testing laboratory project 392231) and ran from 20-Feb-1995 to 23-Mar-1995 and from 06-Mar-1995 to 13-Apr-1995 respectively.

READINGS AND SCORING
The following parameters were recorded:
Erythema (E) - 0 to 4 numerical scores
Oedema (Oe) - 0 to 4 numerical scores
Diameter (D) - mm (for the intradermal pretest)
Erythema and oedema were assessed using the following numerical grading system according to Draize (Draize J.H., Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics, Association of Food and Drug Officials of the United States, Austin, Texas, 1959):

Erythema and eschar formation
No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema 3
Severe erythema (beet redness) to slight eschar formation (injuries in depth) 4

Oedema formation
No oedema 0
Very slight oedema (barely perceptible) 1
Slight oedema (edges of area well-defined by definite raising) 2
Moderate oedema (raised approximately 1 millimeter) 3
Severe oedema (raised more than 1 millimeter and extending beyond the area of exposure) 4
Positive control substance(s):
yes
Remarks:
4-aminobenzoic acid ethyl ester and 2-mercaptobenzothiazol
Positive control results:
4-AMINOBENZOIC ACID ETHYL ESTER
In this study 30 % and 35 % of the animals of the test group were observed with positive skin reactions after treatment with a non-irritant test substance concentration of 30 % in mineral oil. No skin reactions were observed in the control group. Therefore the test article at concentration of 30 % in mineral oil is considered to be a sensitizer when described under the test conditions. According to the rating of allergenicity by Magnusson and Kligman the test article is a moderate sensitizer.

2-MERCAPTOBENZOTHIAZOL
In this study 95 % and 90 % of the animals of the test group were observed with positive skin reactions at the 24- and 48-hour reading respectively after treatment with a non-irritant test substance concentration of 5 % in peanut oil. The test article at concentration of 5 % in peanut oil is considered to be a sensitizer when described under the test conditions. According to the rating of allergenicity by Magnusson and Kligman the test article is an extreme sensitizer.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
15 %
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
1 animal was found dead on test day 22
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
15 %
No. with + reactions:
0
Total no. in group:
19

SKIN EFFECTS AFTER INTRADERMAL INDUCTION PERFORMED ON TEST DAY 1

A normal evolution of the local symptoms was observed in the animals of the control and test group after the intradermal injections.

 

SKIN EFFECTS AFTER EPIDERMAL INDUCTION PERFORMED ON TEST DAY 8

- Test group: as the test article stained the skin yellow, it was not possible to determine whether erythema was present. However, no oedema was observed.

- Control group: no erythematous or oedematous reaction was observed in the animals treated with bi-distilled water only.

SKIN EFFECTS AFTER THE CHALLENGE PERFORMED ON TEST DAY 22

No positive reactions were observed in the animals either when treated with bidistilled water alone or when treated with the test article at 15 % in bidistilled water. Yellow discoloration was noted directly after removal of the patch. To remove discoloration all animals were depilated approximately 3 hours prior to challenge reading.

VIABILITY / MORTALITY / MACROSCOPIC FINDINGS

One animal of the epidermal pretest group and one animal of the test group were found dead on test day 21 and 22, respectively. At necropsy the lungs of the both animals were dark red discoloured.

CLINICAL SIGNS, SYSTEMIC

No symptoms of systemic toxicity were observed in the animals.

BODY WEIGHTS

One animal of the control group lost incidentally weight during the acclimatization period. No loss of weight was noted during the treatment period.

Interpretation of results:
other: not classified, according to the CLP Regulation
Conclusions:
Non 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. Ten females were used as control group and 20 females were used as test group as described in the OECD guideline 406.

Results

The highest non-irritating test article concentration used for challenge application was 15 % in bidistilled water.

0 % of the animals were positive after treatment with the highest non-irritant test substance concentration of 15 % in bidistilled water. Therefore the test article considered to be a non sensitizer.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), 3.4 Respiratory or skin sensitisation section, skin sensitizer means a substance that will lead to an allergic response following skin contact.

Based on the available information, the substance can be considered as non skin sensitising.

In conclusion, Acid Yellow 017 is not classified as skin sensitizer, according to the CLP Regulation (EC 1272/2008).