Registration Dossier

Administrative data

Description of key information

The test substance is not considered to be a skin sensitizer.

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:
weight of evidence
Study period:
April to June 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
Directive 92/69 EEC B6
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Data were already avilable
Species:
guinea pig
Strain:
Himalayan
Sex:
male
Details on test animals and environmental conditions:

TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd. Wölferstrasse 4, 4414 Füllinsdorf/Switzerland
- Age at study initiation:5-8 weeks
- Weight at study initiation: Control and Test Group: 341-489 g. Pretest 317-435g
- Housing: Individually
- Diet (e.g. ad libitum): pelleted standard Kliba 342, Batch no 68/95 guinea pig breeding/maintenance diet, ad libitum
- Water (e.g. ad libitum): Community tap water from F$üllinsdorf, ad libitum. Once weekly additional supply of ascorbic acid (1g/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 visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23°C
- Humidity (%): 48-60%
- Air changes (per hr):10-15
- Photoperiod (hrs dark / hrs light): 12-hour lighs, 12 hour dark cyle

Route:
intradermal
Vehicle:
water
Concentration / amount:
5% / 0.1 mL
Day(s)/duration:
Day 1
Adequacy of induction:
highest technically applicable concentration used
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
25% / saturated filter paper (2*4 cm²)
Day(s)/duration:
Day 8 for 48 h
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
15%
Day(s)/duration:
Day 22 for 24 h
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Control group: 10 males in control group
Test group: 20 males in test group
Intradermal Pretest: 2 males.
Epidermal pretest: 4 males




I
Details on study design:
RANGE FINDING TESTS:
Intradermal: 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 in bi-distilled water was selected.

Epidermal: 5, 10, 15, and 25% in bi-distilled water.
25% in bi-distilled water was most qualified concentration to assure an optimum technical application procedure
For the epidermal induction and challenge procedure the test article at 25% in bi-distilled water was selected for the induction application and that of a concentration of 15% was selected for the challenge application.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 1 intrdermal + 1 epidermal
- Exposure period: Day 1; Day 8 to 10
- Test groups: 1
- Control group: 1
- Site: neck
- Frequency of applications:
Intradermal on both sides of the neck
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:25% in bidistilled water; or only bidistilled water
- Duration: 48 h
- Concentrations: 5 and 25%

B. CHALLENGE EXPOSURE
- No. of exposures: 1 test item + 1 vehicle
- Day(s) of challenge: Day 22
- Exposure period: 24 h
- Test groups: 1
- Control group: 1
- Site: flanks
- Concentrations: 0 and 15%
- Evaluation (hr after challenge): 24 and 48 h after patch removal

OTHER:
Approximately 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 & 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.

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


Positive control substance(s):
yes
Remarks:
4-aminobenzoic acid ethyl ester and 2- mercaptobenyothiazol
Positive control results:
For validation of sensitivity of the Maximization-Test of B. Magnusson and A.M. Kligman (1969) as well as the sensitivity of the test system used, a known sensitizer (2-MERCAPTOBENZOTHIAZOL) was selected as a positive control.
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. No skin reactions were observed in the control group. The results obtained with the test article at 10% in peanut oil were not taken into consideration since the both control group were observed with erythematous reactions.
For validation of sensitivity of the Maximization-Test of B. Magnusson and A.M. Kligman (1969) as well as the sensitivity of the test system used, a known mild sensitizer was selected as a positive control (4-AMIN0BENZ0IC 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.
The response of at least 30% positive animals is considered positive "R43" following the "Commission Directive 93/21/EEC, 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".
The test article 2-MERCAPTO-BENZOTHIAZOL at concentration of 5% in peanut oil is considered according to the rating of allergenicity by Magnusson and Kligman as an extreme sensitizer.
The test article 4-AMINOBENZOIC ACID ETHYL ESTER at concentration of 30% in mineral oil is considered according to the rating of allergenicity by Magnusson and Kligman as a moderate sensitizer.

Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
15%
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
15%
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
no indication of skin sensitisation
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:
15% in bi-distilled water.
No. with + reactions:
0
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
15% in bi-distilled water.
No. with + reactions:
0
Total no. in group:
10
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
vehicle, bi-distilled water
No. with + reactions:
0
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
vehicle, bi-distilled water
No. with + reactions:
0
Total no. in group:
10
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
5% 2-mercapto-benzothiazol
No. with + reactions:
19
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:
5% 2-mercapto-benzothiazol
No. with + reactions:
18
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation

VIABILITY / MORTALITY / MACROSCOPIC FINDINGS: As there were no deaths during the course of the treatment period no necropsies were performed.

CLINICAL SIGNS, SYSTEMIC: No symptoms of systemic toxicity were observed in the animals.

BODY WEIGHTS: Two out of 10 animals of the control group (nos. 325, 330) and one out of 20 animals of the test group (no. 346) incidentally lost weight during the acclimatization period. No weight loss was observed during treatment period.

Interpretation of results:
GHS criteria not met
Conclusions:
The test article at concentration of 15% in bi-distilled water is considered to be a non-sensitizer in guinea pigs
Executive summary:

To assess the allergenic potential of the substance in albino guinea pigs the Maximization-Test of Magnusson and Kligman was used according to OECD test guideline 406. Ten males were used as control group and 20 males were used as test group.

Before the test procedure a pretest was performed in order 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 pretest was performed with intradermal (0.1 ml/site, at concentrations of 1, 3 and 5% of the test article in bi-distilled water) and epidermal induction (5, 10, 15 and 25% of test material in bi-distilled water).

The results of the pretest were:

- For intradermal induction application a 5% test article dilution in bi-distilled water was selected.

- For the epidermal induction procedure, the test article at 25% in bi-distilled water was selected and for the challenge procedure, a concentration of 15% in bi-distilled was selected for the challenge application.

In this study 0% 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 bi-distilled water. No skin reactions were observed in the control group. According to the rating of allergenicity by Magnusson and Kligman the test article is considered a non-sensitizer.

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

There are two studies available for this endpoint. Both of them were performed according to Guideline OECD 406 and were GLP compliant.

In the study of Hagemann (1993) the test material used for testing was "roh trocken" meaning "crude". The sample tested had a content of 91% of active substance and inside the report it is defined that purity is 27%. In a more recent study done by Arcelin (1995) the test material is described as "gereignit" meaning "purified". The composition of the tested sample is not known. When purified, it is assumed that the purity of the sample will be higher than the one used in Hagemann (1993) test.

Under the test conditions of Arcelin (1995), EC 215-397-8 at concentrations of 15% in bi-distilled water is considered to be a non-sensitizer. Similar results are obtained with testing conditions of Hagemann (1993) at concentration 20% in vaseline.

There is also one study done with humans in 1972, Shelansky (1972). This is a human repeated insult patch test and the tested material gave a negative result on sensitisation when tested at 4% in 200 individuals covering a wide range of ages. The human patch test does not give any information concerning classification.

The substance to register has a higher purity than the test item used in the Hagemann’s study (27%) and therefore closer to the test substance used in Arcelin’s study. It can be concluded that the test substance is not skin sensitizer.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the above stated assessments of the skin sensitisation potential, the substance is classified as non-sensitising according to CLP (Regulation (EC) No 1272/2008.