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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:
1998-03-09 to 1998-04-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Principles of method if other than guideline:
Reference: Ritz & Bühler (1980). Procedure for conducting the guinea pig assay.
GLP compliance:
yes
Type of study:
Buehler test
Justification for non-LLNA method:
The Murine Local Lymph Node Assay (LLNA) is the first-choice method for in vivo testing according to the REACH Regulation. However, this reliable Buehler test was performed before entry into force of the REACH Regulation.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report):T002113
- Substance type:beige powder
- Physical state:solid
- Analytical purity:99.9% (w/w)
- Impurities (identity and concentrations): no data
- Composition of test material, percentage of components:no data
- Isomers composition:no data
- Purity test date:no data
- Lot/batch No.: RT002113PFP121
- Expiration date of the lot/batch:1998-09-23
- Stability of test article: stable under storage conditions
- Stability of test article dilution: unknown in bidistilled water, is excluded from the statement of compliance
- Storage condition of test material:in the original container at room temperature (approx 20°C), away from direct sunlight

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: Ibm:GOHI (synonym Himalayan spotted)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BRL, Biological research Laboratories Ltd., Wölferstrasse 4, CH-4414 Füllinsdorf, Switzerland; 34 females (nulliparous and non-pregnant)
- Age at delivery/acclimatization start: 4-6 weeks
- Weight at delivery/acclimatization start: 304-384g (test and control animals); 318-368g (animals used for irritation screen
- Housing:standard laboratory conditions, individually in Makrolon type-4 cages with standard softwood bedding
- Diet (e.g. ad libitum): pelleted standard Nfag Ecosan 845 25W4, batch no. 112/97 guinea pig/breeding/maintenance diet, ad libitum. Results of analyses for contaminants are archived.
- Water (e.g. ad libitum): Community tap water from Füllinsdorf, ad libitum. once weekly additinal supply of ascorbic acid (appox 1g/l) via the drinking water was provided. Results of bacteriological, chemical and contaminen analyses are archived.
- Acclimation period:one week for the control and test group under test conditions after health examination. However, contrary to the test group the control group remained untreated during the 3 induction weeks. No acclimatization for the animals used in the irritation screen for induction and challenge. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22+/-3°C
- Humidity (%):40-70% (values above 70% during cleaning process)
- Air changes (per hr):10-15 air changes per hour
- Photoperiod (hrs dark / hrs light):12-hour light, 12-hour dark cycle. Music was played during the light period.

IN-LIFE DATES: From: 1998-03-09 To: 1998-04-08

Study design: in vivo (non-LLNA)

Induction
Route:
epicutaneous, occlusive
Vehicle:
other: bi-distilled water
Concentration / amount:
Concentration: 75%
Amount: 1 x 0.5 mL/25 mm Hill Top Chamber


Concentration of test material and vehicle used for each challenge:
Two weeks after the final induction application the animals
were challenged with the same test article used for
induction but at a concentration of 25% in bi-distilled
water.
Day(s)/duration:
three times a week for a 3 week induction phase (a total of 9 applications) on days 1, 3, 5, 8, 10, 12, 15, 17 and 19
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Challenge
Route:
epicutaneous, occlusive
Vehicle:
other: bi-distilled water
Concentration / amount:
Concentration: 25%
Amount: 1 x 0.5 mL/25 mm Hill Top Chamber


Concentration of test material and vehicle used for each challenge:
Two weeks after the final induction application the animals
were challenged with the same test article used for
induction but at a concentration of 25% in bi-distilled
water.
Day(s)/duration:
two weeks after final inducation application
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
First challenge:
-Number of animals in test group: 20
-Number of animals in negative control group: 10
Irritation screen: 4 animals
Details on study design:
RANGE FINDING TESTS:
The animal's fur was shaved on the day before exposure with a fine clipper blade. Closed patches were applied to the animals as follows:
During the induction phase the test article at 75% was settled directly in a 25 mm Hill Top Chamber in order to saturate it. During the challenge procedure 0.5 ml of the freshly prepared test article solution was settled in a 25 mm Hill Top Chamber. The animals were put in the restrainer and the designated patch was applied to the clipped surface as quickly as possible after the test article had been applied. The patch applianc was occluded with a rubber dental dam. The rubber dental dam used for occlusion of the patches was off medium gauge 12-5cm 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.
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 animalswere taken away from the restrainers. After that the animals were returned to their individual cages.

-irritation screen for induction and challenge (performed during the acclimatization period): four different concentrations were used on each animal for a 6-hour exposure period. The test article concentration of 75% in bidistilled water was considered to be the most qualified to assure an optimum technical application procedure.the application sites were assessed for erythema and oedema 24 and 48 hours after removal of the patches.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 9
- Exposure period: total of 3 weeks
- Test groups: 75%
- Control group:y
- Site: fur was clipped from the left shoulder of each test animal , repeated applicatioon was performed at the same site
- Frequency of applications:3 patches per week, interval of 2 days
- Duration: 6 hours
- Concentrations: 75%
-after the last induction exposure the animls were left untreated for 10 days before the challenge. The skin responses were graded approx 24 hours after the patches have been removed. Any gross skin reactions were recorded without depilation.

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge:day 29, one occassion
- Exposure period:6 hours
- Test groups:25%
- Control group:y
- Site:left posterior quadrant of the side and back of the animals
- Concentrations: 25%
- Evaluation (hr after challenge):25 and 48 hrs after the patches have been removed

OTHER:
GRADING METHOD: the animals used for irritation screen and challenge were depilated approx 21 hours after the patches had been removed, using an approved depilatory cream (VEET Cream). The depilatory cream was placed on the patch sites and surrounding areas, and left on for up to 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. Draize scoring system was followed. The grading method used for irritation screen, induction and challenge was identical. It was perforemd 24 hours (+/- 2 hours) after removal of the patches for the irritation screen, induction and challenge and repeated 24 hours (+/-2 hours) later (48-hour grades) for the irritation screen and the challenge.
PATCHING METHOD: the same patching method was used for induction, irritation screen and challenge.
Positive control substance(s):
yes
Remarks:
alpha-hexylcinnamaldehyde (RCC project 902046) from 1997-11-04 to 1997-12-12

Results and discussion

Positive control results:
65% of the animals of the test group were observed with positive skin reactions (grade of 0 and +/- are considered to be non-significant repsonses, whereas those of 1 and greater are considered to be significant) after challenge performed with the highest non-irritating concnetration of alpha-hexylcinnamaldehyde at 10% in PECD400. No skin reactions were observed in the control group treated in the same conditions during the challenge phase. A response of at least 15% positive animals is considered positive R43: may cause sensitization by skin contact according to the Commission Directive 96/54/EEC, July 30, 1996 adapting to technical progress for the 22nd 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 article alpha-hexylcinnamaldehyde applied at a concentration of 10% in PEG 400 is considered to be a sensitizer when used under the described test conditions.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
25 %
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
no symptoms of systemic toxicity were observed in the animals; as there were no deaths durign the course of the treatment period no necropsies were performed
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
25 %
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
no symptoms of systemic toxicity were observed in the animals; as there were no deaths durign the course of the treatment period no necropsies were performed
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
10% in PEG 400
No. with + reactions:
13
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:
10% in PEG 400
No. with + reactions:
10
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:
bi-distilled water
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
bi-distilled water
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation

Any other information on results incl. tables

Irritation screen:

the results of the 24 -hour reading were taken into consideration and in this case the test article at 75% and 50% resulted in 3 scores of 1. No skin reactions were observed in the other concentrations. Therefore, the most representative concentration to stimulate a state of immune hypersensitivity was 75% in bidistilled water used in the induction phase. The highest non-irritating concentration is determined as the concentration which induces responses in the four guinea pigs no more severe than two grades of +/- and two of 0. Therefore, the highest non-irritating concentration for the challenge was determined as the concentration of 25% in bidistilled water.


Induction:

irritant reaction was observed on and after the sixth induction application in relation with the repetitive topical application of the test article at 75% on the same skin site. From the sicth induction application to the eigth application, one or two animals were observed with slight confluent erythema and three or four animals with patchy erythema. In the last induction twelve animals were also observed with slight confluent erythema.

challenge:

no skin effect was noted after the challenge procedure


Other observations:

-there were no deaths during the course of the treatment period

-no symptoms of systemic toxicity were observed in the animals

-the body weight of the animals was within the range commonly recorded for animals of this strain and age.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
T002113 applied at a concentration of 25% in bi-diistilled water is considered not to be a sensitizer when used under the described test conditions.