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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Remarks:
Repeated Applications Buehler Test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 December 2015 to 10 February 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

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
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
Buehler test
Justification for non-LLNA method:
The LLNA is the study of choice for skin sensitisation. As detailed in the OECD 429 guideline, despite the advantages of the LLNA, it should be recognised that there are certain limitations that may necessitate the use of TG 406. Chemical groups such as metal salts, organometal, unsaturated compounds and surfactants have been known to be linked to false positive. The Substance is considered to be amphiphilic in nature and therefore would likely have surfactant characteristics, which are known to be falsely positive in the LLNA. Therefore a Guinea Pig Study, OECD 406, was selected to decrease the uncertainty of possible falsely positive effects.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Apperance: Yellow liquid
- Storage conditions: Room temperature
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
For the purpose of the study, the test item was prepared immediately prior to dosing in liquid paraffin for the topical applications (pre-tests). This vehicle was chosen as it produced the most suitable formulation at the required concentration. Indeed, the preparation of the test item at 50 % in liquid paraffin (w/w) was a thick yellow solution.

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 3, 4 or 5 weeks old
- Weight at study initiation: between 269 g and 371 g
- Housing: The animals were housed individually or in groups of 2 in polycarbonate cages. The flooring of the cages was covered with dust-free wood shavings and the top fitted a stainless steel lid containing a feeding device and drinking device of 500 mL.
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: Minimum acclimatization period of 5 days under housing and diet conditions identical to those of the test.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C ± 3°C
- Humidity (%): from 30 % to 70 %
- Air changes (per hr): At least 10 cycles per hour
- Photoperiod (hrs dark / hrs light): Circadian cycle (12 hrs day/ 12 hrs darkness)

Study design: in vivo (non-LLNA)

Induction
Route:
epicutaneous, occlusive
Vehicle:
paraffin oil
Remarks:
Liquid paraffin
Concentration / amount:
10 % in liquid paraffin
Day(s)/duration:
3 local applications were performed on Day 0, Day 7 and Day 14 during 6 hours under occlusive dressing.
Adequacy of induction:
not specified
Challengeopen allclose all
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
paraffin oil
Remarks:
Liquid paraffin
Concentration / amount:
5 % in liquid paraffin
Day(s)/duration:
6 hours
Adequacy of challenge:
highest non-irritant concentration
No.:
#2
Route:
epicutaneous, occlusive
Vehicle:
paraffin oil
Remarks:
liquid paraffin
Concentration / amount:
5 % in liquid paraffin
Day(s)/duration:
6 hours
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Control group: 10 animals
Test material group: 20 animals
Details on study design:
RANGE FINDING TESTS/ PRELIMINARY STUDY
This test was carried out with a reduced number of animals, for the purpose of determining the maximal non-irritating material concentration to be used for the challenge phase. Furthermore, this test evaluates the irritant potential of the test material, and defines, if possible, a mild to moderate irritant concentration to be used for the topical induction phase.
Three guinea pigs were treated with the test material placed onto the selected treatment sites and covered with an occlusive dressing (25 mm x 50 mm non-woven swab of 4-layer patch held in contact with the skin by means of 50 mm wide hypoallergenic micropore™ adhesive tape and Blenderm) for a period of 6 hours at 4 different concentrations: 100 %, and diluted at 75 %, 50 % and 25 % in liquid paraffin.
Washing of the skin after removal of the dressing was done with liquid paraffin.
The animals treated at the concentrations of 100 %, and diluted at 75 %, 50 % and 25 % received 0.5 mL of the corresponding preparation.
A macroscopic evaluation of the cutaneous reactions was conducted 24 and 48 hours after removal of the occlusive dressings. The skin reaction was observed and recorded according to the grades described hereafter.
Due to the cutaneous reactions, three other guinea pigs were treated with the test material placed onto the selected treatment sites and covered with an occlusive dressing (25 mm x 50 mm non-woven swab of 4-layer patch held in contact with the skin by means of 50 mm wide hypoallergenic micropore™ adhesive tape and Blenderm™ from 3M) for a period of 6 hours at 4 different concentrations: diluted at 10 %, 5 %, 2 % and 1 % in liquid paraffin. Washing of the skin after removal of the dressing was done with liquid paraffin.
The animals treated at the diluted concentrations of 10 %, 5 %, 2 % and 1 % received 0.5 mL of the corresponding preparation.
A macroscopic evaluation of the cutaneous reactions was conducted 24 and 48 hours after removal of the occlusive dressings. The skin reaction was observed and recorded according to the grades described hereafter.

MAIN STUDY
A. INDUCTION EXPOSURE
After shearing of the scapular zone, the 3 local applications were performed on days 0, 7 and 14 during 6 hours under occlusive dressing (25 mm x 50 mm non-woven swab of 4-layer patch held in contact with the skin by means of 50 mm wide hypoallergenic micropore™ adhesive tape from and Blenderm™).
The animals of the control group received 0.5 mL of liquid paraffin and the animals of the treated group received 0.5 mL of the test item diluted at 10 % in liquid paraffin.
Washing of the skin after removal of the dressing was done with liquid paraffin.

The animals of both groups were then left untreated for 13 days (rest phase).

B. CHALLENGE EXPOSURE
The experimental procedure of this phase was identical for both groups Group 1 (Control) and Group 2 (Treated) according to this approach: On the previously shorn dorso-lumbar zone, an application on either side of the spine, under occlusive dressing (25 mm x 50 mm non-woven swab of 4-layer patch held in contact with the skin by means of 50 mm wide hypoallergenic micropore™ adhesive tape and Blenderm™), was performed during 6 hours:
- 1 area containing 0.5 mL of the test material diluted at 5 % (MNIC = maximal non-irritant concentration) on the left flank and one area containing 0.5 mL of liquid paraffin on the right flank.
Washing of the skin after removal of the dressing was done with liquid paraffin.
In view to confirm or infirm the results, a second challenge phase was performed under the same experimental conditions with a new negative control group.

The animals of both groups were then left untreated for 7 days (rest phase).

C. RE-CHALLENGE EXPOSURE
The experimental procedure of this phase was identical for both groups Group 1 (Control) and Group 2 (Treated) according to the following approach. On the previously shorn dorso-lumbar zone, an application on either side of the spine, under occlusive dressing (25 mm x 50 mm non-woven swab of 4-layer patch held in contact with the skin by means of 50 mm wide hypoallergenic micropore™ adhesive tape and Blenderm™), was performed during 6 hours:
- 1 area containing 0.5 mL of the test item diluted at 5% (MNIC = maximal non-irritant concentration) on the left flank and one area containing 0.5 mL of liquid paraffin on the right flank.
The treated areas used for the re-challenge were below the treated areas used for the 1st challenge.
No washing of the skin was necessary after removal of the dressing.

OTHER:
PREPARATION OF ANIMALS
Before the experimentation process, animals were identified individually by marking with picric acid and by means of a numbered ring on the edge of one ear.

The animals were carefully shorn before each test item application:
- On the inter-scapular zone for the induction phase,
- On the dorso-lumbar zone for the challenge phase and the re-challenge phase.
At least 3 hours before the first reading (challenge phase and re-challenge phase) they were shorn a second time in this dorso-lumbar zone.

The animals were weighed at the beginning, before the second induction, after the reading at 48 hours and at the end of the study.
Challenge controls:
The animals of the control groups were subject to the same experimental procedure as the treatment groups.
Positive control substance(s):
yes
Remarks:
a-Hexylcinnamaldehyde

Results and discussion

Positive control results:
The results of the positive control show that the reference substance a-Hexylcinnamaldehyde caused skin sensitisation and therefore confirms the validity of the test.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
liquid paraffin
No. with + reactions:
1
Total no. in group:
10
Clinical observations:
Discrete erythema
Remarks on result:
other: As irritation was observed in the control group animals, only reactions in the test group animals that exceeded the most severe reactions seen in the control group animals were attributed to skin sensitization.
Remarks:
No cutaneous reaction was recorded at 48 and 72 hours.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
5 % in liquid paraffin
No. with + reactions:
5
Total no. in group:
20
Clinical observations:
Discrete erythema
Remarks on result:
other: This value was not attributed to skin sensitization as irritation was observed in the control group animals.
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
5 % in liquid paraffin
No. with + reactions:
1
Total no. in group:
20
Clinical observations:
Discrete erythema
Reading:
rechallenge
Hours after challenge:
24
Group:
test group
Dose level:
5 % in liquid paraffin
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No cutaneous reactions observed
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
50%
No. with + reactions:
5
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
50%
No. with + reactions:
5
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
25%
No. with + reactions:
3
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
25%
No. with + reactions:
2
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation

Any other information on results incl. tables

Preliminary Study

- MNIC determination:

24 hours after removal of the patches, an intense erythema was noted in all animals (3/3) at the tested concentrations of 100 %, 75 % and 50 %. Intense erythema in one animal (1/3) and moderate erythema in two animals (2/3) were also noted at the tested concentration of 25 %.

 

48 hours after removal of the patches, an intense erythema was noted in all animals (3/3) associated with dryness in two animals (2/3), at the tested concentration of 100 %. An intense erythema was noted in all animals (3/3) associated with dryness in all animals (3/3) at the tested concentrations of 75 % and 50 %. Moderate erythema was noted in two animals (2/3) associated with dryness in one animal (1/2) and a discrete erythema associated with dryness was noted in the last animal (1/3) at the tested concentration of 25 %.

 

24 hours after removal of the patches, a moderate erythema was noted in one animal (1/3) and a discrete erythema was noted in two animals (2/3) at the tested concentration of 10 %.

48 hours after removal of the patches, discrete erythema was noted in two animals (2/3) at the tested concentration of 10 %.

 

24 and 48 hours after removal of the patches, no cutaneous reaction was noted at the tested concentrations of 5 %, 2 % and 1 %.

 

In view of these results, the concentration selected for the 3 inductions of the main study was 10 % and the concentration selected for the challenge phase was 5 % (MNIC).

 

Main Study:

- Induction phase

No cutaneous reaction was recorded after the first induction. Moderate erythema was noted in eighteen animals (18/20) and discrete erythema was noted in two animals (2/20) after the second induction. Moderate erythema was noted in all animals (20/20) after the third induction.

 

No cutaneous reaction was recorded during the induction phase in the control group.

 

- First challenge phase:

The concentration selected for the induction phase and the challenge was based on the result of a pre-test.

Readings were performed 24, 48 and 72 hours after removal of the patches.

 

In the treated group (treatment concentration of 5 %), a discrete erythema in 25 % (5/20) and 5 % (1/20) of the animals at 24 and 48 hours after the challenge phase, respectively, was recorded. No cutaneous reaction was recorded at 72 hours.

In the control group (associated with the treatment concentration of 5 %), a discrete erythema in 10 % (1/10) of the animals at 24 hours after the challenge was recorded. No cutaneous reaction was recorded at 48 and 72 hours.

As irritation was observed in the control group animals, only reactions in the test group animals that exceeded the most severe reactions seen in the control group animals were attributed to skin sensitization. So, a sensitization reaction was noted in 5 % (1/20) of the animals from the treated group, 48 hours after the challenge phase, on the area challenged with the test item at 5 %.

 

In the treated group on the treated area with liquid paraffin,no macroscopic cutaneous reaction attributable to allergy was recorded during the examination following the removal of the occlusive dressing (challenge phase).

No cutaneous reaction was recorded in animals from the control groups after the challenge phase, on the treated area with liquid paraffin (control item).

 

In view to confirm or infirm these results, a second challenge phase was performed under the same experimental conditions with a new negative control group after a rest phase of 7 days.

 

- Second challenge phase:

In the treated group (treatment concentration of 5 %), no macroscopic cutaneous reactions attributable to allergy was recorded during the examination following the removal of the occlusive dressing (rechallenge phase).

In the control group (associated with the treatment concentration of 5 %),no cutaneous reaction was observed during the examination following the removal ohe occlusive dressing.

 

In the treated group on the treated area with liquid paraffin,no macroscopic cutaneous reactions attributable to allergy was recorded during the examination following the removal of the occlusive dressing (rechallenge phase).

No cutaneous reaction was recorded in animals from the control groups after the rechallenge phase, on the treated area with liquid paraffin (control item).

 

Weight Evolution

No abnormalities and no differences in the body weight between the control and the treated group were observed.

 

Mortality

No mortality occured during the main test.

 

Clinical Signs

No abnormal clinical signs related to the administration of the test item were observed.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test material was considered to be a non-sensitiser under the conditions of this study in accordance with EU criteria.
Executive summary:

The potential of the test material to act as a sensitiser was investigated in a GLP study in accordance with the standardised guidelines OECD 406, EU Method B.6 and US EPA OPPTS 870.2600.

After induction of 20 guinea-pigs by 3 topical applications with the test material applied diluted at 10 % in liquid paraffin under occlusive dressing and a 13-day rest phase, the challenge phase, under occlusive dressing for 6 hours, consisted of a single topical application of the test material at 5 % in liquid paraffin and of a negative control (liquid paraffin). The concentration selected for the induction phase and the challenge based on the result of a pre-test. Readings were performed 24, 48 and 72 hours after removal of the patches.

In the treated group (treatment concentration of 5 %), a discrete erythema in 25 % (5/20) and 5 % (1/20) of the animals at 24 and 48 hours after the challenge phase, respectively, was recorded. No cutaneous reaction was recorded at 72 hours. In the control group (associated with the treatment concentration of 5 %), a discrete erythema in 10 % (1/10) of the animals at 24 hours after the challenge was recorded. No cutaneous reaction was recorded at 48 and 72 hours. As irritation was observed in the control group animals, only reactions in the test group animals that exceeded the most severe reactions seen in the control group animals were attributed to skin sensitization. So, a sensitization reaction was noted in 5 % (1/20) of the animals from the treated group, 48 hours after the challenge phase, on the area challenged with the test material at 5 %. In the treated group on the treated area with liquid paraffin, no macroscopic cutaneous reaction attributable to allergy was recorded during the examination following the removal of the occlusive dressing (challenge phase). No cutaneous reaction was recorded in animals from the control groups after the challenge phase, on the treated area with liquid paraffin (control material). In view to confirm or infirm these results, a second challenge phase was performed under the same experimental conditions with a new negative control group after a rest phase of 7 days. In the treated group (treatment concentration of 5 %), no macroscopic cutaneous reactions attributable to allergy was recorded during the examination following the removal of the occlusive dressing (rechallenge phase). In the control group (associated with the treatment concentration of 5 %), no cutaneous intolerance reaction was observed during the examination following the removal of the occlusive dressing. In the treated group on the treated area with liquid paraffin, no macroscopic cutaneous reactions attributable to allergy was recorded during the examination following the removal of the occlusive dressing (rechallenge phase). No cutaneous reaction was recorded in animals from the control groups after the rechallenge phase, on the treated area with liquid paraffin (control material).

In conclusion, the test material was considered to be a non-sensitiser under the conditions of this study in accordance with EU criteria.