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Administrative data

Description of key information

Under the conditions of this study, the test material was found not 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:
05 December 2016 to 05 January 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
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.
Test material information:
Composition 1
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: YBF1002/16
- Expiration date of the lot/batch: 17 October 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, darkness

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: the test material was used freshly prepared in physiological saline.
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 3 to 4 weeks old
- Weight at study initiation: 303 to 350 g
- Housing: In groups of 3
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: Minimum of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 25°C
- Humidity: 30 to 70%
- Air changes: At least 10 per hour
- Photoperiod: twelve hours continuous light (07.00 to 19.00) and twelve hours darkness
Route:
other: Intradermal and Topical
Vehicle:
other: Physiological saline (Intradermal) and Distilled water (Topical)
Concentration / amount:
Intradermal: 20 %/ 0.1 mL
Topical: 80 %/ 0.5 mL
Day(s)/duration:
Intradermal induction took place on Day 0. On Day 8, animals received a topical induction application which was covered for 48 hours.
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
No.:
#1
Route:
other: Topical
Vehicle:
other: Distilled water
Concentration / amount:
80 and 40 % / 1 sample cup
Day(s)/duration:
On day 21 the challenge dose was applied for 24 hours
Adequacy of challenge:
not specified
No. of animals per dose:
5 in the negative control, 10 in the treated group.
Details on study design:
RANGE FINDING TESTS:
- Determination by intradermal injection of the Maximal Non Necrotizing 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%, 20% and 10% in physical 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):
This test, which allowed evaluating the irritancy potential of the test material, defined whether an application of sodium lauryl sulphate would be needed during topical induction phase. 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%, 50%, 20% and 10% in distilled water. After the removal of the occlusive dressing, the treated areas were rinsed with distilled water. 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. physiological saline and distilled water). 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%, 50%, 20% and 10% in distilled water. After the removal of the occlusive dressing, the treated areas were rinsed with distilled water. A macroscopic evaluation of the cutaneous reactions was conducted 24 and 48 hours after removal of the occlusive dressing.

MAIN STUDY

A. INDUCTION EXPOSURE
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 physiological saline
-2 ID: physiological saline
-2 ID: a mixture with equal volumes v/v: Freund’s Complete Adjuvant at 50% and physiological saline
GROUP 2 (Treated):
-2 ID: Freund’s Complete Adjuvant diluted at 50 % in physiological saline
-2 ID: test material at 20% in physiological saline
-2 ID: a test mixture in equal volumes v/v: Freund’s Complete Adjuvant at 50% and the test item at 40% in physiological saline

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 (25mm x 25mm non-woven swab of 4-layer patch held in contact with the skin by means of 50 mm wide hypoallergenic adhesive tape) for 48 hours was performed on the injection sites of each animal.
GROUP 1 (control): 0.5 mL of distilled water.
GROUP 2 (treated): 0.5 mL of the test material at 80 % in distilled water.
Day 10: The occlusive dressing was removed and the treated areas were rinsed with distilled water.

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 1 (Control) and 2 (Treated) submitted to this experimentation: on the previously shorn dorso-lumbar zone, an application, under occlusive dressing, was performed during 24 hours:
1 sample cup containing the test material diluted at 80 % (MNIC) and 1 sample cup containing the test item diluted at 40 % in distilled water (1/2 MNIC).

Day 22: The occlusive dressing was removed and the treated areas were rinsed with distilled water.
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.
Challenge controls:
No, the experimental procedure of this phase was identical for both groups 1 (Control) and 2 (Treated).
Positive control substance(s):
yes
Remarks:
α-hexylcinnamaldehyde (HCA)
Positive control results:
The number of animals sensitised was ≥ 70%.
Therefore, under the experimental conditions, the reference substance a-Hexylcinnamaldehyde must be classified in category 1 “Skin sensitisation” sub-category 1B in accordance with the Regulation EC No. 1272/2008 on classification, labelling and packaging of substances and mixtures. The signal word “Warning” and hazard statement H317 “May cause an allergic skin reaction” are required.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
80 %
No. with + reactions:
1
Total no. in group:
10
Clinical observations:
Intense erythema was noted in three animals, moderate erythema was noted in five animals and discrete erythema was noted in two animals 24 hours after the first induction.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
40 %
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
40 / 80 %
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
40 / 80 %
No. with + reactions:
0
Total no. in group:
5
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
40 / 80 %
No. with + reactions:
0
Total no. in group:
5

Preliminary studies

- MNNC determination:

Necrosis to slight necrosis was observed in the animals at the tested concentrations of 80 and 50%. 24 hours after the injections, moderate to discrete erythema was observed in the animals at the tested concentrations of 20 and 10 %. The first induction of the Group 2 has been carried out by intradermal injection at the maximal non necrosing concentration of 20 %.

- 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 the 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:

Intense erythema was noted in three animals (3/10), moderate erythema was noted in five animals (5/10) and discrete erythema was noted in two animals (2/10) 24 hours after the first induction. Discrete erythema was noted in four animals (4/10) and dryness of the skin was noted in all the animals (10/10) 24 hours after the second induction.

- Induction phase Group 1:

No cutaneous reaction was noted during the induction phase.

- Challenge phase Groups 1 & 2:

Overall results of the challenge phase with the test material (readings at 24 and 48 hours) are given in Table 1. In the treated group (treatment dose of 80 %), a very slight erythema was noted in 10 % (1/10) of the treated animal 24 hours after the challenge phase. No macroscopic cutaneous reactions attributable to allergy were noted 48 hours 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.

Table 1: Macroscopic evaluation (readings at 24 and 48 hours) of cutaneous reactions

Groups

Reading time

Concentrations

Incidence

% of positive responses1

% of animal sensitised

0

1

2

3

Group 1 Control

24 h

80 %

5

0

0

0

0

-

48 h

80 %

5

0

0

0

0

-

24 h

40 %

5

0

0

0

0

-

48 h

40 %

5

0

0

0

0

-

Group 2 Treated

24 h

80 %

9

1

0

0

10

10

48 h

80 %

10

0

0

0

0

0

24 h

40 %

10

0

0

0

0

0

48 h

40 %

10

0

0

0

0

0

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 aim of the study was to evaluate the possible sensitisation of the test material using the guinea pig maximisation test. The study was performed in accordance with the standardised guidelines OECD 406 and EU method B.6 under GLP conditions.

According to the results of the pre-tests, the test material was applied to 10 Guinea pigs during the the induction phase (intradermic injection at 20% and topical application at 80%). Induction was followed by a 10-day rest phase.

On day 0 after shearing the scapular zone, three pairs of intradermal injections of 0.1 mL were performed on the scapular zone, including the test material at 20% in physiological saline. On day 7 the scapular zone was brushed with a solution of sodium lauryl sulphate at 10 % in thick Vaseline to create a local irritation and then on day 8 a topical application under occlusive dressing was applied for 48 hours on the injection sites of each animal. The occlusive dressing was removed on day 10 and the treated areas were rinsed with distilled water. The challenge phase was conducted under occlusive dressing for 24 hours and consisted of a single topical application of the test material diluted at 80 % and at 40 % in distilled water. The challenge was applied on day 21, removed on day 22 and then observed on days 23 and 24 (24 and 48 hours after removal respectively).

In the treated group (treatment dose of 80 %), a very slight erythema was noted in 10 % (1/10) of the treated animals 24 hours after the challenge phase.No macroscopic cutaneous reactions attributable to allergy were noted 48 hours 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 this study, the test material does not have to be classified in category 1 as a skin sensitiser, in accordance with the Regulation EC No. 1272/2008 on classification, labelling and packaging of substances and mixtures.

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

The aim of the study was to evaluate the possible sensitisation of the test material using the guinea pig maximisation test. The study was performed in accordance with the standardised guidelines OECD 406 and EU method B.6 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

According to the results of the pre-tests, the test material was applied to 10 Guinea pigs during the the induction phase (intradermic injection at 20% and topical application at 80%). Induction was followed by a 10-day rest phase.

On day 0 after shearing the scapular zone, three pairs of intradermal injections of 0.1 mL were performed on the scapular zone, including the test material at 20% in physiological saline. On day 7 the scapular zone was brushed with a solution of sodium lauryl sulphate at 10 % in thick Vaseline to create a local irritation and then on day 8 a topical application under occlusive dressing was applied for 48 hours on the injection sites of each animal. The occlusive dressing was removed on day 10 and the treated areas were rinsed with distilled water. The challenge phase was conducted under occlusive dressing for 24 hours and consisted of a single topical application of the test material diluted at 80 % and at 40 % in distilled water. The challenge was applied on day 21, removed on day 22 and then observed on days 23 and 24 (24 and 48 hours after removal respectively).

In the treated group (treatment dose of 80 %), a very slight erythema was noted in 10 % (1/10) of the treated animals 24 hoursafter the challenge phase.No macroscopic cutaneous reactions attributable to allergy were noted 48 hours after the challenge phase.In the control group (associated with the treatment dose of 80 %), nomacroscopiccutaneous 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 %), nomacroscopiccutaneous intolerance reactions were recorded after the challenge phase.

Under the conditions of this study, the test material was found not 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.