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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been conducted according to GLP and valid methods, therefore it is considered to be adequate, reliable and relevant for classifcation.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The sulfosuccinate substances might give false positive reactions in the LLNA test.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Stolzenseeweg 32-36, 88353 Kißlegg, Germany
- Age at start of administration: 25 days
- Weight at start of administration: 251 – 295 g (excluding positive control group); 316 – 370 g (positive control group)
- Housing: In pairs in MAKROLON cages (MZK 80/25); Granulated textured wood (Granulat A2, J. BRANDENBURG, 49424 Goldenstedt, Germany) was used as bedding material in the cages. The cages were changed and cleaned twice a week.
- Diet (e.g. ad libitum): Commercial diet, ssniff® Ms-H (ssniff Spezialdiäten GmbH, 59494 Soest, Germany , ad libitum
- Water (e.g. ad libitum): Tap water in drinking bottles was offered ad libitum.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3°C (maximum range)
- Humidity (%): 55% ± 15% (maximum range)
- Air changes (per hr): Not provided
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: November 16, 2012 To: February 23,2013

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
other: lntracutaneous(stage 1) and Topical (stage 2)
Vehicle:
other: aqua ad iniectabilia
Concentration / amount:
Preliminary test:
Stage1: intracutaneous: 10, 5, 1, 0.5, 0.1 and 0.01% suspension of the test item in aqua ad iniectabilia
Stage 2: topical: 100, 75, 50, 25, 10, 5 and 1% suspension of the test item in aqua ad iniectabilia
Stage 3: topical: 100, 75, 50, 25, 10, 5 and 1% suspension of the test item in aqua ad iniectabilia

Main test:
Stage1: intracutaneous: 0.5% suspension of the test item in aqua ad iniectabilia
Stage 2: topical: 25% suspension of the test item in aqua ad iniectabilia
Stage 3: topical: 10% suspension of the test item in aqua ad iniectabilia
Challengeopen allclose all
Route:
other: Topical (stage 3)
Vehicle:
other: aqua ad iniectabilia
Concentration / amount:
Preliminary test:
Stage1: intracutaneous: 10, 5, 1, 0.5, 0.1 and 0.01% suspension of the test item in aqua ad iniectabilia
Stage 2: topical: 100, 75, 50, 25, 10, 5 and 1% suspension of the test item in aqua ad iniectabilia
Stage 3: topical: 100, 75, 50, 25, 10, 5 and 1% suspension of the test item in aqua ad iniectabilia

Main test:
Stage1: intracutaneous: 0.5% suspension of the test item in aqua ad iniectabilia
Stage 2: topical: 25% suspension of the test item in aqua ad iniectabilia
Stage 3: topical: 10% suspension of the test item in aqua ad iniectabilia
No. of animals per dose:
Preliminary test: 10 animals in total
8 animals for the topical administration
2 animals for the intracutaneous administration

Main test: 15 animals in total (20 animals historical data)
10 test group
5 vehicle control group
20 positive control group (historical)
Details on study design:
MAIN STUDY
A. INDUCTION EXPOSURE

Stage 1: Intradermal injections Day 0
- No. of exposures: 3 (pairs of injections)
- Exposure period: Single injections
- Test groups: 10 animals, 0.5% concentration test item in aqua ad iniectabilia
Three pairs of intradermal injections of 0.1 mL were given in the shoulder region which was cleared of hair so that one of each pair lay on each side of midline.
(1) Freund's complete adjuvant
(diluted 1 : 1 with 0.9% NaCl )
(2) the test item (concentration see above)
(3) the test item in a 1:1 mixture (v/v) FCA/physiological saline
In injection 3, the final concentration of the test item was equal to that used in injection 2.
Injections (1) and (2) were given close to each other and nearest the head, while (3) was given towards the caudal part of the test area.
- Control group:
Vehicle control: 5 animals:
(1)Freund's complete adjuvant (diluted 1 : 1 with 0.9% NaCl )
(2) aqua ad iniectabilia
(3) aqua ad iniectabilia in a 1:1 mixture (v/v) FCA/physiological saline
Positive control: 20 animals (historical data): 10% (v/v) α-hexyl cinnamic aldehyde solution
- Site: Shoulder region
- Frequency of applications: Single injections
- Duration: Single injections (Day 0) and six days interval before stage 2 (Day 7)
- Concentrations: 0.5% concentration test item in tap water

Stage 2: Topical Day 7
- No. of exposures: 1 (patch)
- Exposure period: 48 hours
- Test groups: 10 animals: 25% concentration test item in aqua ad iniectabilia
- Control group:
Vehicle control: 5 animals: aqua ad iniectabilia
Positive control: 20 animals (historical data): undiluted α-hexyl cinnamic aldehyde
- Site: Shoulder region
- Frequency of applications: 1
- Duration: 48 hours
- Concentrations: 25% concentration test item in aqua ad iniectabilia


B. CHALLENGE EXPOSURE
Stage 3: Topical: Day 21
- No. of exposures: 1 (patch)
- Day(s) of challenge: Day 21
- Exposure period: 24 hours
- Test groups: The filter paper containing the test item (10% suspension of the test item in aqua ad iniectabilia) was applied to the left flank, the filter paper with the vehicle to the right flank of the animal.
- Control group:
Vehicle control: The left flank was treated with the test item (10% concentration in aqua ad iniectabilia), the right flank with the vehicle i.e. in the same way as the test group.
Positive control: The filter paper containing 0.01% α-hexyl cinnamic aldehyde solution was applied to the left flank, the filter paper with the vehicle to the right flank of the animal.
- Site: Flanks
- Concentrations: 10% suspension of the test item in tap water
- Evaluation (hr after challenge):
21 hours after removing the filter paper the challenge area was cleaned and cleared of hair if necessary; three hours later (at 48 hours from the start of challenge application) the skin reaction was observed and recorded; 24 hours after this observation a second observation (72 hours) was made and recorded.

OTHER:
MAGNUSSON/KLIGMAN grading scale tor the evaluation of challenge patch test reactions
0 = no visible change
1 = discrete or patchy erythema
2 = moderate and confluent erythema
3 = intense erythema and swelling

Challenge controls:
- Vehicle control: The left flank was treated with the test item (10% concentration in aqua ad iniectabilia), the right flank with the vehicle i.e. in the same way as the test group.
- Positive control: The filter paper containing 0.01% α-hexyl cinnamic aldehyde solution was applied to the left flank, the filter paper with the vehicle to the right flank of the animal.
Positive control substance(s):
yes
Remarks:
α-hexyl cinnamic aldehyde

Results and discussion

Positive control results:
Animals of the same strain treated with α-hexyl cinnamic aldehyde in sesame oil exhibited a sensitising reaction in all animals in form of a moderate and confluent erythema (grade 2).

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
10% suspension of the test item in aqua ad iniectabilia (2mL)
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
test group
Dose level:
10% suspension of the test item in aqua ad iniectabilia (2mL)
No. with + reactions:
0
Total no. in group:
10
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
aqua ad iniectabilia (2mL)
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: aqua ad iniectabilia (2mL). No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
aqua ad iniectabilia (2mL)
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: negative control. Dose level: aqua ad iniectabilia (2mL). No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
1st reading
Hours after challenge:
48
Group:
positive control
Dose level:
0.01% α-hexyl cinnamic aldehyde
No. with + reactions:
20
Total no. in group:
20
Clinical observations:
moderate and confluent erythema left flank
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: positive control. Dose level: 0.01% α-hexyl cinnamic aldehyde . No with. + reactions: 20.0. Total no. in groups: 20.0. Clinical observations: moderate and confluent erythema left flank.
Reading:
2nd reading
Hours after challenge:
72
Group:
positive control
Dose level:
0.01% α-hexyl cinnamic aldehyde
No. with + reactions:
20
Total no. in group:
20
Clinical observations:
moderate and confluent erythema left flank
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: positive control. Dose level: 0.01% α-hexyl cinnamic aldehyde . No with. + reactions: 20.0. Total no. in groups: 20.0. Clinical observations: moderate and confluent erythema left flank.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the present test conditions, test item containing 39.80% active ingredient, was found to be not sensitising to guinea pigs in a test model according to MAGNUSSON and KLIGMAN.
Executive summary:

The purpose of this study was to determine the potential of the test item, containing 39.80% active ingredient, in guinea pigs in a test model according to MAGNUSSON and KLIGMAN.

From a preliminary experiment, it was decided to use a 0.5% concentration in aqua ad iniectabilia for the 1st (intracutaneous) stage, a 25% suspension in aqua ad iniectabilia for the 2nd (topical) induction stage and a 10% suspension in aqua ad iniectabilia for the challenge.

A 0.5% suspension in aqua ad iniectabilia chosen for the 1st (intracutaneous) induction stage revealed a discrete or patchy erythema in all 10 animals 24 and 48 hours after administration. 2 mL of a 25% suspension of the test item in aqua ad iniectabilia/animal chosen for the 2nd (topical) induction stage revealed a moderate and confluent erythema 48 hours and a discrete or patchy erythema 72 hours after start of exposure in all 10 animals. The challenge with 2 mL of a 10% suspension of the test item in aqua ad iniectabilia /animal - the maximum non-irritating concentration - revealed no skin irritation in any animal and, thus, the test item had no sensitising properties.

The vehicle control revealed no skin reactions.

Animals of the same strain treated with α-hexyl cinnamic aldehyde in sesame oil exhibited a sensitising reaction in all animals in form of a moderate and confluent erythema (grade 2).

Behaviour of the animals remained unchanged. Body weights were not affected

Under the present test conditions, test item containing 39.8% active ingredient, was found to be not sensitising to guinea pigs in a test model according to MAGNUSSON and KLIGMAN.