Registration Dossier

Administrative data

Description of key information

Under the conditions of the study, the test material is not considered to be 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:
24 April 2017 to 25 May 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The rare earth substances are known to give false positives in the LLNA studies. The Maximisation study was therefore deemed to be more appropriate for investigating the skin sensitisation potential of this substance.
Species:
guinea pig
Strain:
Dunkin-Hartley
Remarks:
Albino
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 3 or 4 weeks old
- Weight at study initiation: 264 to 297 g
- Housing: The animals were housed in groups of 3 at the maximum in polycarbonate containers, the flooring of which was covered with dust-free cuttings and the top fitted with a stainless steel lid with a feeding device and drinking device of 500 mL.
- Diet: ad libitum
- Water: tap water from public distribution system ad libitum
- Acclimation period: minimum period of 5 days, under stabling and nutritional conditions identical to those of the test.

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 25°C
- Humidity: 30 to 70%
- Air changes: at least ten changes per hour
- Photoperiod: lighting was controlled by a time switch to give twelve hours continuous light (07.00 to 19.00) and twelve hours darkness.
Route:
other: intradermal and topical
Vehicle:
other: olive oil (intradermal) and liquid paraffin (topical)
Concentration / amount:
Intradermal: 70 %, 0.1 mL
Topical: 80 %, 0.5 mL
Day(s)/duration:
Intradermal induction on Day 0, Topical induction on Day 8 for 48 hours.
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
No.:
#1
Route:
other: Topical
Vehicle:
other: liquid paraffin
Concentration / amount:
80 and 40 %, 1 sample cup
Day(s)/duration:
On day 21, 10 days after removal of topical induction. The challenge was applied for 24 hours.
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Negative control group: 5
Treatment group: 10
Details on study design:
PRELIMINARY STUDIES:
- Determination by intradermal injection of the Maximal Non Necrotising Concentration (MNNC):
Two animals received a volume of 0.1 mL of the test material, on both sides of the spine, at 4 concentrations: diluted at 80, 50, 30 and 20% in olive oil view to determine the MNNC. A macroscopic evaluation of the cutaneous reactions was conducted 24 hours after the injections. Due to the absence of necrosis between the tested concentrations of 80 and 50%, the same animals received a volume of 0.1 mL of the test material, on both sides of the spine at 2 new concentrations: diluted at 70 and 60% in physiological saline in view to determine the MNNC. A macroscopic evaluation of the cutaneous reactions was conducted 24 hours after the injections.
- Determination by topical application of the Pre-Maximal Non Irritant Concentration (Pre-MNIC):
The test material was applied on the dorso-lumbar zone of two guinea pigs shorn beforehand, with occlusive dressing for 24 hours, at 4 different concentrations: diluted at 80, 60, 50 and 30% in liquid paraffin. After removal of the occlusive dressing, the treated areas were rinsed with liquid paraffin. A macroscopic evaluation of the cutaneous reactions was conducted 24 hours after removal of the dressing.
- Determination by topical application of the Maximal Non Irritant Concentration (MNIC):
Three guinea pigs were treated according to the same treatment as animals from Group 1 (control) for the induction phase (i.e. olive oil and liquid paraffin). During the challenge phase, the animals were treated with the test material placed onto the selected treatment sites and covered with an occlusive dressing for a period of 24 hours at 4 different concentrations: diluted at 80, 60, 50 and 30% in liquid paraffin. After removal of the occlusive dressing, the treated areas were rinsed with liquid paraffin. A macroscopic evaluation of the cutaneous reactions was conducted 24 and 48 hours after removal of the occlusive dressing.

MAIN STUDY

A. INDUCTION EXPOSURE

1st Intradermal Induction:
- Day 0 : After shearing the scapular zone, three pairs of intradermal injections (ID) of 0.1 mL were performed on the scapular zone in such a way as an injection on each pair is placed to either side of the spine as follows:
Group 1 (control):
2 ID: Freund’s Complete Adjuvant diluted at 50% in olive oil
2 ID: olive oil
2 ID: a mixture with equal volumes v/v (Freund’s Complete Adjuvant at 50% and olive oil)
Group 2 (Treated):
2 ID: Freund’s Complete Adjuvant diluted at 50% in olive oil
2 ID: test material at 70% in olive oil
2 ID: a test mixture in equal volumes v/v (Freund’s Complete Adjuvant at 50% and the test material undiluted (100%))

2nd Topical Induction:
- Day 7: The scapular zone of all the animals in each group, shorn beforehand, was brushed with a solution of sodium lauryl sulphate at 10% in thick vaseline, in order to create a local irritation.
- Day 8: A topical application under occlusive dressing (25 x 25 mm non-woven swab of 4-layer patch from held in contact with the skin by means of 50 mm wide hypoallergenic micropore™ adhesive tape from 3M and Blenderm™ from 3M) for 48 hours was performed on the injection sites of each animal.
Group 1 (control): 0.5 mL of liquid paraffin.
Group 2 (treated): 0.5 mL of the test material at 80% in liquid paraffin.
- Day 9: After removal of the occlusive dressing, the treated areas were rinsed with liquid paraffin. Residue of black colouration not preventing the erythema quotation was noted.

Rest phase: The animals of both groups were left for 10 days.

B. CHALLENGE EXPOSURE
- Day 21: The experimental procedure of this phase was identical for both groups Group 1 (Control) and Group 2 (Treated) submitted to this experimentation: on the previously shorn dorso-lumbar zone, an application, under occlusive dressing, was performed for 24 hours:
1 sample cup containing the test material diluted at 80% (MNIC) and 1 sample cup containing the test material diluted at 40% in liquid paraffin (1/2 MNIC).

- Day 22: After removal of the occlusive dressing, the treated areas were rinsed with liquid paraffin.
- Day 23: 1st reading time – 24 hours after the patch removal.
- Day 24: 2nd reading time – 48 hours after the patch removal.

INTERPRETATION OF RESULTS
The test material will be regarded as a sensitiser if 30% or more of the test animals show a sensitisation response. In accordance with the Regulation (EC) No 1272/2008, the test material will be classified in category 1. The signal word “Warning” and hazard statement: H317 “May cause an allergic skin reaction” are required. In accordance with the Regulation (EC) No. 286/2011, the positive test material will be classified in sub-category 1A or 1B in accordance with the following:
- Sub-category 1A: ≥ 30% responding at ≤ 0.1% intradermal induction dose or ≥ 60% responding at > 0.1 to ≤ 1% intradermal induction dose
- Sub-category 1B: ≥ 30 to < 60% responding at > 0.1 to ≤ 1% intradermal induction dose or ≥ 30% responding at > 1% intradermal induction dose
Challenge controls:
The test material at 80 and 40% was applied in the challenge to the control group.
Positive control substance(s):
yes
Remarks:
α-hexylcinnamaldehyde (HCA)
Positive control results:
The results of the testing on the reference substance show that it must be categorised as sub-category 1B.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
80/40 %
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
80/40 %
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
80/40 %
No. with + reactions:
0
Total no. in group:
5
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
80/40 %
No. with + reactions:
0
Total no. in group:
5

PRELIMINARY STUDIES

- MNNC determination:

Necrosis in all animals was observed at the tested concentration of 80%. 24 hours after the injections, no cutaneous reaction was noted at the tested concentrations of 70, 60, 50, 30 and 20%.

The first induction of Group 2 was carried out by intradermal injection at the maximal non necrosing concentration of 70%.

 

- Pre MNIC determination:

24 hours after the removal of the occlusive dressings, no cutaneous reaction was noted whatever the tested concentration. In view of these results, the concentration selected was 80% for the 2nd induction of Group 2 and the MNIC determination began at the concentration of 80%.

 

- MNIC determination:

24 and 48 hours after the removal of the occlusive dressings, no cutaneous reaction was noted whatever the tested concentration. In view of this result, the concentrations selected were 80% (MNIC) and 40% (1/2 MNIC).

 

MAIN STUDY

- Induction phase Group 2: No cutaneous reaction was noted in the animals 24 hours after the first induction. Dryness of the skin with slight black colouration was noted in all animals (10/10) 24 hours after the second induction.

- Induction phase Group 1: No cutaneous reaction was noted 24 hours after the first induction. Dryness of the skin was noted in all animals (5/5) 24 hours after the second induction.

- Challenge phase Groups 1 & 2: In the treated group (treatment dose of 80%), no macroscopic cutaneous reactions attributable to allergy were noted after the challenge phase. In the control group (associated with the treatment dose of 80%), no macroscopic cutaneous intolerance reactions were recorded after the challenge phase. In the treated group (treatment dose of 40%), no macroscopic cutaneous reactions attributable to allergy were noted after the challenge phase. In the control group (associated with the treatment dose of 40%), no macroscopic cutaneous intolerance reactions were recorded after the challenge phase.

 

WEIGHT EVOLUTION: No abnormality was recorded in the body weight gain of both groups.

MORTALITY: No mortality was registered during the main test.

Interpretation of results:
other: Not sensitising in accordance with EU criteria
Conclusions:
Under the conditions of the study, the test material is not considered to be a skin sensitiser.
Executive summary:

The sensitising potential of the test material to the skin was investigated in accordance with the standardised guidelines OECD 406 and EU Method B.6 under GLP conditions, using a guinea pig maximisation test with intradermal and topical administrations.

According the results of the pre-tests, the induction phase (intradermic injection at 70% and topical application at 80%) was conducted with the test material to 10 female Dunkin-Hartley Guinea pigs with a 10-day rest phase. The challenge phase conducted under occlusive dressing for 24 hours, consisted of a single topical application of the test material diluted at 80 and 40% in liquid paraffin. The experimental protocol was established according to the Magnusson and Kligman method (J. Invest. Dermatol. 1969. 52, 268-276).

In the treated group (treatment dose of 80%), no macroscopic cutaneous reactions attributable to allergy were noted after the challenge phase. In the control group (associated with the treatment dose of 80%), no macroscopic cutaneous intolerance reactions were recorded after the challenge phase. In the treated group (treatment dose of 40%), no macroscopic cutaneous reactions attributable to allergy were noted after the challenge phase. In the control group (associated with the treatment dose of 40%), no macroscopic cutaneous intolerance reactions were recorded after the challenge phase.

Under the conditions of the study, the test material is not considered to be a skin sensitiser.

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

The sensitising potential of the test material to the skin was investigated in accordance with the standardised guidelines OECD 406 and EU Method B.6 under GLP conditions, using a guinea pig maximisation test with intradermal and topical administrations. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

According the results of the pre-tests, the induction phase (intradermic injection at 70% and topical application at 80%) was conducted with the test material to 10 female Dunkin-Hartley Guinea pigs with a 10-day rest phase. The challenge phase conducted under occlusive dressing for 24 hours, consisted of a single topical application of the test material diluted at 80 and 40% in liquid paraffin. The experimental protocol was established according to the Magnusson and Kligman method (J. Invest. Dermatol. 1969. 52, 268-276).

In the treated group (treatment dose of 80%), no macroscopic cutaneous reactions attributable to allergy were noted after the challenge phase. In the control group (associated with the treatment dose of 80%), no macroscopic cutaneous intolerance reactions were recorded after the challenge phase. In the treated group (treatment dose of 40%), no macroscopic cutaneous reactions attributable to allergy were noted after the challenge phase. In the control group (associated with the treatment dose of 40%), no macroscopic cutaneous intolerance reactions were recorded after the challenge phase.

Under the conditions of the study, the test material is not considered to be a skin sensitiser.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to skin sensitisation.