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Administrative data

Description of key information

The test substance is not a skin sensitiser.

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 August 16, 2006 to September 09, 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Study predates LLNA method availability.
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: LAB-ALL Bt. Budapest, 1174 Hunyadi u. 7.
- Animal health: Only animals in acceptable health condition were used for the test.
- Body weight range at treatment: 320-370 g
- Housing: Animals were housed in macrolon cages, size III., with 3 or 2 animals/cage (42 x 42 x 19 cm).
- Bedding: Laboratory bedding, SSNIFF Lignocel 3-4 Fasern
- Diet: PURINA Base – Lap gr. diet, ad libitum
- Water: Tap water from self-service watering system, ad libitum containing 50 mg/100 ml Ascorbic acid
- Acclimation period: Female: 21 d
- Animal identification: The animals were marked individually with ear punching. The cages were marked with individual identity cards with information about study code, sex, cage number, dose group and individual animal numbers.
- Randomization: All animals were sorted according to weight on the day before the start of the treatment period. After this the animals were allocated to the test groups. The result of the randomisation was checked according to the actual body weights assuring an acceptable homogeneity and variability among the groups.

ENVIRONMENTAL CONDITIONS
- Temperature: 20±3°C
- Humidity: 30-70%
- Photoperiod: 12 h light/dark cycle

IN-LIFE DATES: From: To: August 16, 2006 to September 09, 2006
Route:
intradermal
Vehicle:
physiological saline
Concentration / amount:
1% / 4 * 0.1 mL
Day(s)/duration:
Day 1
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
25% / 0.5 mL

25% was the highest administrable concentration. The test item at concentrations of 50 and 75 % were not suitable for the dermal treatment, because of the high density and viscosity of the formulation.
Day(s)/duration:
Day 8 / 48 h
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
25% /0.5 mL
Day(s)/duration:
Day 22 / 24h
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Number of animals in test group: 10
Number of animals in control group: 5
Details on study design:
Preliminary Study

Justification of the dosages:

A series of test substance concentrations were tested to identify any primary irritation by intra-dermal injection and dermal application. Four dose levels were tested in the preliminary dose range finding study to identify any primary irritation by intra-dermal injection and two dose levels by dermal application.

Approximately 24 h prior to the test the hair was removed from the right and left surface of the animals (approximately 5 x 5 cm). The hair removal was performed carefully by electric clippers to ensure it was closely shaven.

The test substance was applied at concentrations of 0.01, 0.1, 1, 5, 10 and 25% (w/v). For the intra-dermal application 0.1 mL formulated test substance was injected at concentrations of 0.01, 0.1, 1 and 5% (w/v). Two different concentrations were applied per animal to the hairless skin on the right and left flanks, and two animals were employed per concentration tested.

The intra-dermal treatment of test substance caused local irritation at concentrations of 0.1, 1 and 5 %. At concentrations of 0.1 and 1% very slight erythema were found in the animals 1h after the patch removal. At concentration of 5 % moderate erythema, slight oedema and necrosis were observed in both animals 1h after the patch removal. At concentrations of 0.01 % no changes in primary irritation were observed.

For the dermal application 0.5 mL formulated test substance was applied onto the skin of the animals at concentrations of 10 and 25% (w/v). A closed patch exposure was employed by means of an occlusive bandage. Two animals per concentration were tested.

The test substance at concentrations of 50 and 75% was not suitable for the dermal treatment, because of the high density and viscosity of the formulation. It was found that 0.5 mL of test formulation at concentrations of 10 and 25% (w/v) produced no reaction on the skin of guinea pigs.

As a result of these preliminary experiments, test substance was administered at concentrations of 1% for intra-dermal treatment. The intra-dermal treatmentwas performed as described below in main study-1. For dermal exposure test substance was applied at concentrations of 25%. Control animals were treated with vehicle (i.e., physiological saline solution (NaCl 0.9 %)) only. Before the dermal exposure the test area was painted with 0.5 mL of 10 % sodium dodecyl sulphate in Vaseline 24 h prior topical induction application, in order to create a local irritation. For the challenge exposure, all animals of the treatment and control group were treated with concentrations of 25%.

Time of exposure: 24 h. Local effects were examined and scored 1, 24, 48 and 72 h after patch removal.

Main Study
STUDY DESIGN
The treatments, concentrations of the test substance and the times of observations were performed in the manner as described in the below table in section of 'Any other information on materials and methods incl. tables'.

Control animals were treated similarly to test animals, except that during the induction phase, the test substance was omitted.

Induction involved two main procedures: intra-dermal treatments (i.e., main Study I) and dermal exposure (i.e., main Study II) with closed patch technique. The intra-dermal and the dermal induction treatments were observed and recorded.

Main Study I: Intra-dermal Induction Exposure

Before starting the exposure an area of approximately 5 x 5 cm on the scapular region of animals was clipped free of hair and shaven close with care.

Intra-dermal treatment

Test groups:
A row of three injections, six in all, was made on each side of test animals, as follows:
2 injections with 0.1 mL of Freund's complete adjuvant (i.e., FCA) mixed with physiological saline solution (1:1),
2 injections with 0.1 mL of the test substance (1 %) homogenized in physiological saline solution,
2 injections with 0.1 mL of test substance (1 %) mixed with physiological saline solution and homogenized in Freund's complete adjuvant (1:1).
Control group:
The control animals were treated similarly as the test group however, the vehicle, without the test substance was used for injections, as follows:
2 injections with 0.1 mL mix of Freund's complete adjuvant and physiological saline solution (1:1)(v/v),
2 injections with 0.1 mL of physiological saline solution,
2 injections with 0.1 mL of 50 w/v % physiological saline solution, in a 1:1 mixture (v/v) of Freund's complete adjuvant and physiological saline solution.

Main study II: Dermal Induction Exposure

6 d after the intra-dermal injections, in all animals the test area was painted with 0.5 mL of 10% sodium dodecyl sulphate in Vaseline 24 h prior to topical induction application, in order to create a local irritation.
Approximately 24 h after the painting, the test animals were exposed to test substance on the other hand the control animals were treated with physiological saline solution, as vehicle.

Closed patch was applied in the following manner: in case of the test animals 0.5 mL of test substance (at concentration of 25%) was spread on the surface prepared previously and covered with a standard (5x5 cm) size of porous gauze patch.
Control animals were treated dermally with 0.50 mL of physiological saline solution, as vehicle and the dressing was prepared and applied as for the test animals. The exposed areas were covered for 48 h with porous gauze fastened with "Leucoplast" (Closed Patch Test).

Main study III: Challenge Exposure

Two weeks after the dermal treatment the animals were exposed to the challenge dose, dermally. 24 h before the challenge treatment the left and the right flank areas (5x5 cm) of each animal were prepared for application. The challenge was performed as a dermal exposure (Closed Patch Test).
Left shaved flank areas of the animals (both the test and the control) were treated with 0.5 mL of the test substance at concentration of 25%. The right shaved flank areas were treated with 0.5 mL of physiological saline solution, in all cases. Implementation was done as described above for dermal Induction Exposure. Time of exposure was 24 h.

OBSERVATION AND SCORING

The dermal irritation scores (in case of the preliminary study (primary irritation) and in cases of induction dermal exposures) were evaluated according to the scoring system by Draize (1977) presented in the following table.

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 eschar formation (injuries in depth)................. .4

Oedema
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 mm) ..............................................................3
Severe oedema (raised more than 1 mm and extending beyond area of exposure)...4
Positive control substance(s):
yes
Remarks:
2-mercaptobenzothiazole (tested in another study)
Positive control results:
After the challenge treatment positive response was observed in 60% of the treated animals.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
25% dermal application
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
25% dermal application
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
25% dermal application
No. with + reactions:
0
Total no. in group:
5
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
25% dermal application
No. with + reactions:
0
Total no. in group:
5
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
Intra-dermal induction: 0.1 %, Epi-dermal induction: 75 %, Epidermal challenge: 25 %
No. with + reactions:
6
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
Intra-dermal induction: 0.1 %, Epi-dermal induction: 75 %, Epidermal challenge: 25 %
No. with + reactions:
5
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation

MAIN STUDY

Test group

After the challenge with test substance at concentration of 25%, positive response was not observed on the animals of the test group. The mean of the scores was 0.00 according to the 24 and 48 h results. On the opposite (right) side treated with vehicle no reaction was found.

Control group

Five control animals were exposed to vehicle during induction treatments and they were treated with the test substance on the challenge day only. No visible changes were found at the 24 and 48 h examinations. During the challenge exposure, the test substance at concentration of 25% did not evoke primary irritation.

BODY WEIGHT

There were no notable differences in body weight between the test animal group and the control group.

FORMULATION

The test substance was mixed with physiological saline solution and used for dermal induction and challenge treatments at concentration of 25% (w/v). The formulation was stirred with a magnetic stirrer before and during the treatment.

 

For intra-dermal treatment the test substance was used at concentration of 1 % (w/v).

Interpretation of results:
GHS criteria not met
Conclusions:
Under the study conditions, the test substance showed no evidence of sensitizing properties.
Executive summary:

A guinea pig maximization test was conducted to evaluate the skin sensitization potential of the test substance according to OECD Guideline 406, EPA OPPTS 870.2600 and EU Method B.6, in compliance with GLP.

Based on the results of a preliminary study, 1 and 25% of test substance in physiological saline solution were selected as intradermal and dermal induction doses. The non-irritating concentration used for challenge application was 25% test substance in physiological saline solution. None of the animals of the test group showed skin reactions 24 and 48 h after removing the dressings.

Under the study conditions, the test substance showed no evidence of sensitizing properties.

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

A guinea pig maximization test was conducted to evaluate the skin sensitization potential of the test substance according to OECD Guideline 406, EPA OPPTS 870.2600 and EU Method B.6, in compliance with GLP. Based on the results of a preliminary study, 1 and 25% of test substance in physiological saline solution were selected as intradermal and dermal induction doses. The non-irritating concentration used for challenge application was 25% test substance in physiological saline solution. None of the animals of the test group showed skin reactions 24 and 48 h after removing the dressings. Under the study conditions, the test substance showed no evidence of sensitising properties (Stahl, 2006c).


Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the results of a guinea pig maximization test, the test substance does not need to be classified for skin sensitisation potential according to the EU CLP criteria (EC 1272/2008).