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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:
1995
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
Directive 92/69 EEC B6
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
GLP compliance:
yes
Type of study:
guinea pig maximisation test
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 and epicutaneous
Vehicle:
physiological saline
Concentration / amount:
First induction:
Test:
1- FCA: Phys. saline 1:1
2- 5% Bi-distilled water
3- 5% FCA: Phys. saline 1:1
Control:
1- FCA: Phys.saline 1:1
2- Bi-distilled water
3- FCA:Ph. sal. 1:1/bi-ditilled water
Second induction
Bi-distilled water 25%
Challenge:
Bi-distilled water 15%
Route:
epicutaneous, semiocclusive
Vehicle:
physiological saline
Concentration / amount:
First induction:
Test:
1- FCA: Phys. saline 1:1
2- 5% Bi-distilled water
3- 5% FCA: Phys. saline 1:1
Control:
1- FCA: Phys.saline 1:1
2- Bi-distilled water
3- FCA:Ph. sal. 1:1/bi-ditilled water
Second induction
Bi-distilled water 25%
Challenge:
Bi-distilled water 15%
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:

MAIN STUDY
A. INDUCTION EXPOSURE

- No. of exposures:three pairs of intradermal injections
- Exposure period: 7 days
- Test groups:

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.

- Site: dorsal skin from the scapular region (approximaterly 6 X 8 cm) was clipped free of hair.
- Frequency of applications: 1 week
- Duration: 24h and 48h.


B. CHALLENGE EXPOSURE

- No. of exposures: 1
- Day(s) of challenge:
- Exposure period: 24h -48h
- 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. Two 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 he right flank) using the same method as for the epidermal application.
- Concentrations:
- Evaluation (hr after challenge): The test animals were considered to show no evidence of contact hypersensitivity
if the dermal reactions to the challenge application were identical to or less
marked and/or persistent than the reactions observed in the control animals.

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.

Reading:
other: Intradermal induction
Hours after challenge:
0
Group:
test chemical
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
Evolution of local symptoms
Remarks on result:
other: Reading: other: Intradermal induction. . Hours after challenge: 0.0. Group: test group. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: Evolution of local symptoms.
Reading:
other: intrademal induction
Hours after challenge:
0
Group:
negative control
Clinical observations:
Evolution of local symptoms
Remarks on result:
other: Reading: other: intrademal induction. . Hours after challenge: 0.0. Group: negative control. Clinical observations: Evolution of local symptoms.
Reading:
other: epidermal induction
Hours after challenge:
24
Group:
test chemical
Dose level:
25% in bi-distilled water
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
Skin effects (erythema and edema). Due to orange-red discoloration produced by the test substance a possible erythema reaction could not be determined.
Remarks on result:
other: see Remark
Remarks:
Reading: other: epidermal induction. . Hours after challenge: 24.0. Group: test group. Dose level: 25% in bi-distilled water. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: Skin effects (erythema and edema). Due to orange-red discoloration produced by the test substance a possible erythema reaction could not be determined..
Reading:
other: epidermal induction
Hours after challenge:
48
Group:
test chemical
Dose level:
25% in bi-distilled water
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
Skin effects (erythema and edema). Due to orange-red discoloration produced by the test substance a possible erythema reaction could not be determined.
Remarks on result:
other: see Remark
Remarks:
Reading: other: epidermal induction. . Hours after challenge: 48.0. Group: test group. Dose level: 25% in bi-distilled water. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: Skin effects (erythema and edema). Due to orange-red discoloration produced by the test substance a possible erythema reaction could not be determined..
Reading:
other: epidermal induction
Hours after challenge:
24
Group:
negative control
Dose level:
vehicle, bi-distilled water
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Skin effects (erythema and edema)
Remarks on result:
other: Reading: other: epidermal induction. . Hours after challenge: 24.0. Group: negative control. Dose level: vehicle, bi-distilled water. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: Skin effects (erythema and edema).
Reading:
other: epidermal induction
Hours after challenge:
48
Group:
negative control
Dose level:
vehicle, bi-distilled water
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Skin effects (erythema and edema)
Remarks on result:
other: Reading: other: epidermal induction. . Hours after challenge: 48.0. Group: negative control. Dose level: vehicle, bi-distilled water. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: Skin effects (erythema and edema).
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
15% in bi-distilled water.
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
Skin effects (erythema and edema)
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 15% in bi-distilled water.. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: Skin effects (erythema and edema).
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
vehicle, bi-distilled water
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
Skin effects (erythemand edema)
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: vehicle, bi-distilled water. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: Skin effects (erythemand edema).
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
15% in bi-distilled water.
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
Skin effects (erythema and edema)
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 15% in bi-distilled water.. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: Skin effects (erythema and edema).
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
vehicle, bi-distilled water
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
Skin effects (erythema and edema)
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: vehicle, bi-distilled water. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: Skin effects (erythema and edema).
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
Clinical observations:
Skin effects (erythema and edema)
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: vehicle, bi-distilled water. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: Skin effects (erythema and edema).
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
vehicle, bi-distilled water
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
Skin effects (erythema and edema)
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: vehicle, bi-distilled water. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: Skin effects (erythema and edema).
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
Clinical observations:
Skin effects (erythem and edema)
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 15% in bi-distilled water. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: Skin effects (erythem and edema).
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
Clinical observations:
Skin effects (erythema and edema)
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 15% in bi-distilled water. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: Skin effects (erythema and edema).

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:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
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.
The response of at least 30% positive animals is considered positive "R43" may cause sensitizer by skin contact 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".
Therefore the test article at concentration of 15% in bi-distilled water is considered to be a non-sensitizer when described under the test conditions.
According to the rating of allergenicity by Magnusson and Kligman the test article is a non-sensitizer.
Executive summary:

To assess the allergenic potential of the substance in albino guinea pigs the Maximization-Test of B. Magnusson and A.M.Kligman (1969) was used. 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 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.

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 1556 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 substance is not skin sensitizer.


Justification for selection of skin sensitisation endpoint:
The study is a GLP compliant and has Klimisch score 1

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

This endpoint is not an information requirement of Annex IX of REACH Regulation.

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 Directive 67/548/EEC and according to CLP (Regulation (EC) No 1272/2008 of the European Parliament and of the Council) as implementation of UN-GHS in the EU.