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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:
05 December 2015 and 05 January 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
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.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: CEACK1/16
- 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: freshly prepared in olive oil for the intradermal injections and in liquid paraffin for the topical applications.
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: 297 to 362 g
- Housing: in groups up to 3 in polycarbonate containers, the floor of which was covered with dust-free cuttings and the top fitted with a stainless steel lid.
- 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 ten per hour
- Photoperiod: twelve hours continuous light (07.00 to 19.00) and twelve hours darkness.
Route:
other: intradermal and topical
Vehicle:
other: Olive oil (intradermal), liquid paraffin (topical)
Concentration / amount:
5 % / 0.1 mL (intradermal)
40 % / 0.5 mL (topical)
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:
other: Non-irritant substance but skin pre-treated with SLS prior to topical induction
No.:
#1
Route:
other: topical
Vehicle:
other: liquid paraffin
Concentration / amount:
40 and 20 % / 1 sample cup
Day(s)/duration:
On day 21 for 24 hour exposure
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Control group: 5 animals
Treatment group: 10 animals
Details on study design:
RANGE FINDING TESTS:

- DETERMINATION BY INTRADERMAL INJECTION OF THE MAXIMAL NON NECROTISING CONCENTRATION (MNNC)
This test was conducted for the purpose of defining a MNNC of the test material which, on intradermal injection during the induction phase, does not risk causing too great a lesion (non-necrotising concentration), should be well-tolerated systemically and should be the highest to cause mild-to-moderate skin irritation. Two animals received a volume of 0.1 mL of the test material, on both sides of the spine, at 4 concentrations: diluted at 5, 2, 1 and 0.5 % in olive oil 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 sulfate 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 40, 30, 20 and 10 % in liquid paraffin. After the 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)
This test was carried out for the purpose of determining the MNIC of the test material without risk of an irritant effect during the challenge phase. 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 40, 30, 20 and 10 % in 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
- 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 5% in olive oil,
2 ID: a test mixture in equal volumes v/v (Freund’s Complete Adjuvant at 50% and the test material at 10% in olive oil)

- Topical Induction: On day 7 the scapular zone of all the animals in each group, shorn beforehand, was brushed with a solution of sodium lauryl sulfate at 10% in thick vaseline, in order to create a local irritation. On day 8 a topical application under occlusive dressing (25 mm x 25 mm 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) recieved 0.5 mL of liquid paraffin
Group 2 (treated) recieved 0.5 mL of the test material at 40% in liquid paraffin.
On day 10 the occlusive dressing was removed and the treated areas were rinsed with liquid paraffin.

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

B. CHALLENGE EXPOSURE
On day 21 the experimental procedure of this phase was identical for both 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 during 24 hours:
1 sample cup containing the test material diluted at 40% in liquid paraffin (MNIC) and 1 sample cup containing the test material diluted at 20% in liquid paraffin (1/2 MNIC).
The occlusive dressing was removed on day 22 and the treated areas were rinsed with liquid paraffin.
The first reading was taken on day 23, 24 hours after the patch removal and the second reading was on day 24, 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:
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
Positive control substance(s):
yes
Remarks:
α-hexylcinnamaldehyde
Positive control results:
Under these experimental conditions, the reference substance α-Hexylcinnamaldehyde must be classified in category 1 “Skin sensitisation” sub-category 1B. 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 chemical
Dose level:
20 / 40 %
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
20 / 40 %
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
20 / 40 %
No. with + reactions:
0
Total no. in group:
5
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
20 / 40 %
No. with + reactions:
0
Total no. in group:
5

PRELIMINARY STUDIES

- MNNC Determination

24 hours after the injections, moderate erythema was observed in the animals at the tested concentration of 5%. No cutaneous reaction was noted in the animals at the tested concentrations of 2, 1 and 0.5%. The first induction of the Group 2 has been carried out by intradermal injection at the maximal non necrosing concentration of 5%.

- 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 40% for the 2nd induction of the Group 2 and the MNIC determination began at the concentration of 40%.

- 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 40% (MNIC) and 20% (1/2 MNIC).

 

MAIN STUDY

- Induction phase Group 2: Moderate erythema was noted in three animals (3/10), discrete erythema was noted in four animals (4/10) and no cutaneous reaction was noted in the other three animals (3/10) 24 hours after the first induction. Dryness of the skin was noted in all animals (10/10) after the second induction.

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

- Challenge phase Groups 1 & 2: 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. In the treated group (treatment dose of 20%), no macroscopic cutaneous reactions attributable to allergy were noted after the challenge phase. In the control group (associated with the treatment dose of 20%), no macroscopic cutaneous intolerance reactions were recorded after the challenge phase. Results of the challenge are summarised in Table 1.

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

Groups

Reading time (hrs)

Concentrations (%)

Incidence

% of positive responses 1

% of animal sensitised

0

1

2

3

Group 1: control

24

40

5

0

0

0

0

-

48

40

5

0

0

0

0

-

24

20

5

0

0

0

0

-

48

20

5

0

0

0

0

-

Group 2: treated

24

40

10

0

0

0

0

0

48

40

10

0

0

0

0

0

24

20

10

0

0

0

0

0

48

20

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 skin sensitisation potential of the test material was determined in accordance with the standardised guidelines OECD 406 and EU Method B.6, using the guinea pig maximisation test under GLP conditions.

According the results of the pre-tests, the test material was applied to 10 Guinea pigs during the induction phase (intradermic injection at 5% and topical application at 40%). Induction was followed with a 10-day rest phase. During the challenge phase, animals received a topical application of test material (at 40% and at 20% in liquid paraffin) under an occlusive dressing for a period of 24 hours.

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.

In the treated group (treatment dose of 20%), no macroscopic cutaneous reactions attributable to allergy were noted after the challenge phase. In the control group (associated with the treatment dose of 20%), no macroscopic cutaneous intolerance reactions were recorded after the challenge phase.

The positive control met the acceptability criteria so the study was considered to be valid.

Under the conditions of this 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 skin sensitisation potential of the test material was determined in accordance with the standardised guidelines OECD 406 and EU Method B.6, using the guinea pig maximisation test 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 the results of the pre-tests, the test material was applied to 10 Guinea pigs during the induction phase (intradermic injection at 5% and topical application at 40%). Induction was followedwith a 10-day rest phase. During the challenge phase, animals received a topical application of test material (at 40% and at 20% in liquid paraffin) under an occlusive dressing for a period of 24 hours.

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.

In the treated group (treatment dose of 20%), no macroscopic cutaneous reactions attributable to allergy were noted after the challenge phase.In the control group (associated with the treatment dose of 20%), nomacroscopiccutaneous intolerance reactions were recorded after the challenge phase.

The positive control met the acceptability criteria so the study was considered to be valid.

Under the conditions of this 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.

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