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Description of key information

Skin sensitisation: equivalent to OECD 406. guinea pig. Not sensitising. Reliability = 1

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
This study was conducted in 1995, prior to the inception of REACh regulations.
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: Not reported
- Weight at study initiation: Males: 337-385 g; Females: 308-399 g
- Housing: Individually in polycarbonate cages equipped with a polypropylene bottle. Calibrated and dust-free sawdust was provided as litter
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±2°C
- Humidity (%): 30-70%
- Air changes (per hr): 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 hrs dark/12 hrs light
Route:
intradermal and epicutaneous
Vehicle:
other: sterile iostonic saline solution (0.9% NaCl)
Concentration / amount:
0.5 mL
Route:
epicutaneous, occlusive
Vehicle:
other: sterile iostonic saline solution (0.9% NaCl)
Concentration / amount:
0.5 mL
No. of animals per dose:
Control group: 5/sex/dose
Treated group: 10/sex/dose
Details on study design:
RANGE FINDING TESTS:
By intradermal route (Determination of the Minimum Irritant Concentration (M.I.C.)): 24 hours before treatment the dorsal region of the animals was clipped, the test substance was prepared in an appropriate vehicle, intradermal administration of the test substance (volume 0.1 mL) at increasing concentrations was performed in order to determine the minimum concentration which caused irritation. Evaluation of the potential cutaneous reactions was performed at 24 and 48 hours after injection.
By cutaneous route (Determination of the Minimum Irritant Concentration (M.I.C.) and Maximum Non-Irritant Concentration (M.N.I.C.)): 24 hours before treatment the dorsal region of the animals was clipped, 0.5 mL of the test substance in its original form was applied to a dry gauze pad of approximately 4 cm2 and then held in place by an occlusive dressing for 24 hours. Potential cutaneous reactions were evaluated at 24 and 48 hours after removal of the gauze pads.

MAIN STUDY
INDUCTION EXPOSURE

Intradermal route: On day 1, 6 intradermal injections were made into a clipped area (4 cm x 2 cm) in the scapular region, using a needle mounted on a 1 mL glass syringe. Three injections of 0.1 mL were injected into each side of the animal, as follows:
Control group: Freund's complete adjuvant diluted to 50% (v/v) with a sterile isotonic saline solution (0.9% NaCl); vehicle; a mixture of 50/50 (w/v) Freund's complete adjuvant diluted to 50% (v/v) with a sterile isotonic aqueous NaCl solution and the vehicle.
Treated group: Freund's complete adjuvant diluted to 50% (v/v) with a sterile isotonic saline solution (0.9% NaCl); test substance at a concentration of 1 % (w/w) in the vehicle; a mixture 50/50 (w/v) of Freund's complete adjuvant diluted to 50% (v/v) with a sterile isotonic saline solution (0.9% NaCl), and, the test substance at a concentration of 1 % (w/w) in the vehicle.

Cutaneous route: On day 7, the scapular area was clipped. As the test substance was shown to be non-irritant after occlusive cutaneous treatment during preliminary test, the animals were treated with 0.5 mL of sodium lauryl sulphate (10%) in Vaseline to provoke local irritation. On day 8, a cutaneous application on the 6 injection areas (4 cm x 2 cm) of the scapular region was performed. The control group received application of 0.5 mL of the vehicle. The treated group received application of 0.5 mL of a non-irritant concentration of the test substance in its original form. The test substance and the vehicle were prepared on a dry gauze pad which was then applied to the scapular region and held in place for 48 hours by means of an adhesive hypoallergenic dressing and an adhesive anallergenic waterproof plaster. One hour after removal of the occlusive dressing, cutaneous reactions were recorded.

CHALLENGE EXPOSURE: At the end of the rest period on day 22, the test substance was applied at the Maximum Non-Irritant Concentration (M.N.I.C.), i.e. in its original form.
On day 22, the animals from both groups received an application of 0.5 mL of the M.N.I.C. of the test substance on the posterior right flank, and 0.5 mL of the vehicle on the posterior left flank. This application was performed using a 1 mL plastic syringe. The test substance and vehicle were prepared on a dry gauze pad, and then applied to a 4 cm2 (2 cm x 2 cm) clipped area of the skin. The gauze pad was held in contact with the skin for 24 hours by means of an occlusive, hypo allergenic dressing and an adhesive anallergenic waterproof plaster. Twenty-four and 48 hours after removal of the dressing from the challenge application site, both flanks of the treated and control animals were observed in order to evaluate cutaneous reactions.
Positive control substance(s):
yes
Remarks:
Dinitro 2,4 chlorobenzene
Positive control results:
At a challenge concentration of 1% (w/w), the positive control induced positive skin sensitisation reactions in 95% of the guinea pigs.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
None observed
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: None observed.
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
None observed
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: None observed.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
100%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
None observed
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 100%. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: None observed.
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
100%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
None observed
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 100%. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: None observed.

At the end of the induction period, on day 10, after removal of the dressing, signs of irritation in control and treated groups were observed at the intradermal injection sites.

Interpretation of results:
GHS criteria not met
Conclusions:
This study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).
The test substance was not sensitising.
Executive summary:

Thirty guinea-pigs (15 males and 15 females) were allocated to 2 groups: a control group 1 (5 males and 5 females) and a treated group 2 (10 males and 10 females). The sensitization potential of the test substance was evaluated after a 10-day induction period during which time the animals were treated with sterile isotonic saline solution (0.9% NaCl) (control group) or the test substance (treated group). On day 1, in presence of Freund's complete adjuvant, 0.1 ml of the test substance at a concentration of 1% (w/w) in the vehicle was administered by intradermal route. On day 8, 0.5 mL of the test substance in its original form was applied by cutaneous route during 48 hours by means of an occlusive dressing. After a period of 12 days without treatment, a challenge cutaneous application of 0.5 mL of the vehicle (left flank) and 0.5 mL of the test substance in its original form (right flank) were administered to all animals. The test substance and the vehicle were prepared on a dry gauze pad then were applied to the skin and held in place for 24 hours by means of an occlusive dressing. Cutaneous reactions on the challenge application sites were then evaluated 24 and 48 hours after removal of the dressing. After the final scoring period, the animals were killed. Due to the absence of cutaneous reactions, no skin samples were taken from the challenge application sites from all the animals. The sensitivity of the guinea-pigs in experimental conditions was checked in a recent study with a positive sensitizer, dinitro-2,4-chlorobenzene. During induction period the test substance was applied at 0.1 % (day 1) and 5% (day 8) concentrations. At cutaneous challenge application, 1 % (w/w) was tested on the right flank. No clinical signs and no deaths were noted during the study. After 24 and 48 hours following removal of the dressing of the cutaneous challenge application of the test substance, no cutaneous reactions were recorded. The guinea pigs which were used in a recent study showed a satisfactory sensitisation response in 95% animals using a positive sensitizer. Under the experimental conditions and according to the maximization method established by Magnusson and Kligman, no cutaneous reactions attributable to the sensitisation potential of the test substance in its original form were observed in guinea pigs.

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

A Magnusson-Kligman maximization test (equivalent to OECD 406) was conducted with guinea pigs to determine the potential for the test substance to invoke dermal skin sensitisation reactions. After 24 and 48 hours following removal of the dressing of the cutaneous challenge application of the test substance, no cutaneous reactions were observed. Based on the results of this study, the test substance is not considered to be a contact skin sensitizer.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

There was no evidence of dermal sensitisation in guinea pigs. The substance does not need to be classified for skin sensitisation according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.