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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and OECD guideline conformant
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and OECD guideline conformant
Justification for type of information:
See read across justification document attached in section 13.2
Reason / purpose:
read-across source
Related information:
Composition 1
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
GLP compliance:
yes (incl. certificate)
Remarks:
testing lab.
Type of study:
Buehler test
Justification for non-LLNA method:
Structural similar compouds (such as prenyl acetat) showed fales positive results in the LLNA or inconclusive results in in vitro studies.
Test material information:
Composition 1
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: HARLAN, Kreuzelweg 53, 5961 NM HORST, The Netherlands
- Age at study initiation: 3-4 weeks
- Weight at D0 of study: 241-295 g
- Housing: in groups of 2
- Diet: SAFE 106 ad libitum
- Water: ad libitum
- Acclimation period: >5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 (+/- 3)
- Humidity (%): 30-70
- Air changes (per hr): >10
- Photoperiod (hrs dark / hrs light): 12 / 12
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
Animals in treated group were sensitized and challenged with 0.5ml of 100% (undiluted) test substance (MNIC determined in pre-study), control animals were treated with 0.5ml of liquid paraffin for sensitisation and challenged with 100% (undiluted) test substance.
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
Animals in treated group were sensitized and challenged with 0.5ml of 100% (undiluted) test substance (MNIC determined in pre-study), control animals were treated with 0.5ml of liquid paraffin for sensitisation and challenged with 100% (undiluted) test substance.
No. of animals per dose:
Total animals: 30
Group 1 (negative control): 10 female guinea pigs
Group 2 (treated with test substance): 20 female guinea pigs
Details on study design:
RANGE FINDING TESTS:
A preliminary study was carried out to dertermine the MNIC. Three guinea pigs were treated with concentrations of 100%, 75%, 50%, and 25% of test substance in liquid paraffin. Application was performed epicutaneously under occlusive dressing for 6 hours. Cutaneous reaction was determined 24h and 48h after removal of occlusive dressings. No irritating skin reaction could be found at all test concentrations. In view of these results, the concentration selected was 100% for the 3 inductions of the main study and the concentration selected was 100% (MNIC) for the challenge phase.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3
- Exposure at D0, D7, D14
- Site: Scapular zone
- Duration: 6h under occlusive dressing (50 x 25mm gauze patches hydrophilic Codex of 8-layer Gazin from Lohmann & Rauscher held in contact with the skin by means of 50mm wide hypoallergenic micropore adhesive tape from 3M and Blenderm from 3M)
- Concentrations: 100% test substance (treated group), liquid paraffin (control group)

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day of challenge: D28
- Exposure period: 6h under occlusive dressing (50 x 25mm gauze patches hydrophilic Codex of 8-layer Gazin from Lohmann & Rauscher held in contact with the skin by means of 50mm wide hypoallergenic micropore adhesive tape from 3M and Blenderm from 3M)
- Concentrations: 100% test substance (MNIC) on the left flank or 0% test substance in liquid paraffin on the right flank
- Evaluation (hr after challenge): 24h, 48h after removal of occlusive dressing

Grading was perfomed according to OECD 406:
0 = No visible change
1 = Discrete or patchy erythema
2 = Moderate and confluent erythema
3 = Intense erythema and swelling

The number of animals showing visible cutaneous reactions were determined and recorded at 24 and 48 hours after challenge.
The classification "sensitising" results if at least 15% of test animals showed cutaneous reactions at the time of evaluation. The evaluation is based on the criteria of the OECD Harmonized Integrated Classification System in place on the date of report signature.

Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 0%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 0%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
100%
No. with + reactions:
1
Total no. in group:
20
Clinical observations:
Discrete erythema
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 100%. No with. + reactions: 1.0. Total no. in groups: 20.0. Clinical observations: Discrete erythema.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
100%
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 100%. No with. + reactions: 0.0. Total no. in groups: 20.0.
Reading:
1st reading
Group:
positive control
Remarks on result:
other: no positive control reported

Induction phase:

No cutaneous reaction was recorded 24 hours after each induction in the treated group. No cutaneous reaction was recorded during the induction phase in the control group.

         Animals showing cutaneous reactions
   1st induction  2nd induction  3rd induction
 Control group (10 animals)  0  0  0
 Treated group  0  0  0

 

Challenge phase:

In the treated group (treatment concentration 100% of test item), a discrete erythema was noted in only one animal (1/20) at the reading time 24 hours. No macroscopic cutaneous reactions attributable to allergy were observed 48 hours after the removal of the occlusive dressing.

In the negative control group (treatment concentration of 100% liquid paraffin during induction phase and treatment concentration 100% of test item during challenge phase), no cutaneous reactions were observed at 24 or 48 hours after removal of occlusive dressing.

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

The animals showed normal weight gain during the time of the study.

                Grading scale    
 Groups  Reading time  Concentrations  0  1  2  3  % positive responses  % of animals sensitized
 Control           24 hours 100%   10  0  0  0  0  
 48 hours 100%  10  0  0  0  0  
 24 hours 0%  10  0  0  0  0  
 48 hours 0%  10  0  0  0  0  
 Treated           24 hours 100%  19  1  0  0  5  5
 48 hours 100%  20  0  0  0  0  0
 24 hours 0%  20  0  0  0  0  0
 48 hours 0%  20  0  0  0  0  0

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
GLP compliance:
yes (incl. certificate)
Remarks:
testing lab.
Type of study:
Buehler test
Justification for non-LLNA method:
Structural similar compouds (such as prenyl acetat) showed fales positive results in the LLNA or inconclusive results in in vitro studies.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Citronellyl acetate
- Physical state: yellowish liquid
- Analytical purity: 97.1%
- Lot/batch No.: 00011277LO
- Expiration date of the lot/batch: 05 August 2016
- Storage condition of test material: room temperature, under light exclusion

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: HARLAN, Kreuzelweg 53, 5961 NM HORST, The Netherlands
- Age at study initiation: 3-4 weeks
- Weight at D0 of study: 241-295 g
- Housing: in groups of 2
- Diet: SAFE 106 ad libitum
- Water: ad libitum
- Acclimation period: >5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 (+/- 3)
- Humidity (%): 30-70
- Air changes (per hr): >10
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
Animals in treated group were sensitized and challenged with 0.5ml of 100% (undiluted) test substance (MNIC determined in pre-study), control animals were treated with 0.5ml of liquid paraffin for sensitisation and challenged with 100% (undiluted) test substance.
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
Animals in treated group were sensitized and challenged with 0.5ml of 100% (undiluted) test substance (MNIC determined in pre-study), control animals were treated with 0.5ml of liquid paraffin for sensitisation and challenged with 100% (undiluted) test substance.
No. of animals per dose:
Total animals: 30
Group 1 (negative control): 10 female guinea pigs
Group 2 (treated with test substance): 20 female guinea pigs
Details on study design:
RANGE FINDING TESTS:
A preliminary study was carried out to dertermine the MNIC. Three guinea pigs were treated with concentrations of 100%, 75%, 50%, and 25% of test substance in liquid paraffin. Application was performed epicutaneously under occlusive dressing for 6 hours. Cutaneous reaction was determined 24h and 48h after removal of occlusive dressings. No irritating skin reaction could be found at all test concentrations. In view of these results, the concentration selected was 100% for the 3 inductions of the main study and the concentration selected was 100% (MNIC) for the challenge phase.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3
- Exposure at D0, D7, D14
- Site: Scapular zone
- Duration: 6h under occlusive dressing (50 x 25mm gauze patches hydrophilic Codex of 8-layer Gazin from Lohmann & Rauscher held in contact with the skin by means of 50mm wide hypoallergenic micropore adhesive tape from 3M and Blenderm from 3M)
- Concentrations: 100% test substance (treated group), liquid paraffin (control group)

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day of challenge: D28
- Exposure period: 6h under occlusive dressing (50 x 25mm gauze patches hydrophilic Codex of 8-layer Gazin from Lohmann & Rauscher held in contact with the skin by means of 50mm wide hypoallergenic micropore adhesive tape from 3M and Blenderm from 3M)
- Concentrations: 100% test substance (MNIC) on the left flank or 0% test substance in liquid paraffin on the right flank
- Evaluation (hr after challenge): 24h, 48h after removal of occlusive dressing

Grading was perfomed according to OECD 406:
0 = No visible change
1 = Discrete or patchy erythema
2 = Moderate and confluent erythema
3 = Intense erythema and swelling

The number of animals showing visible cutaneous reactions were determined and recorded at 24 and 48 hours after challenge.
The classification "sensitising" results if at least 15% of test animals showed cutaneous reactions at the time of evaluation. The evaluation is based on the criteria of the OECD Harmonized Integrated Classification System in place on the date of report signature.

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 0%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 0%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
100%
No. with + reactions:
1
Total no. in group:
20
Clinical observations:
Discrete erythema
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 100%. No with. + reactions: 1.0. Total no. in groups: 20.0. Clinical observations: Discrete erythema.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
100%
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 100%. No with. + reactions: 0.0. Total no. in groups: 20.0.
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Remarks on result:
other: no positive control reported

Any other information on results incl. tables

Induction phase:

No cutaneous reaction was recorded 24 hours after each induction in the treated group. No cutaneous reaction was recorded during the induction phase in the control group.

         Animals showing cutaneous reactions
   1st induction  2nd induction  3rd induction
 Control group (10 animals)  0  0  0
 Treated group  0  0  0

 

Challenge phase:

In the treated group (treatment concentration 100% of test item), a discrete erythema was noted in only one animal (1/20) at the reading time 24 hours. No macroscopic cutaneous reactions attributable to allergy were observed 48 hours after the removal of the occlusive dressing.

In the negative control group (treatment concentration of 100% liquid paraffin during induction phase and treatment concentration 100% of test item during challenge phase), no cutaneous reactions were observed at 24 or 48 hours after removal of occlusive dressing.

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

The animals showed normal weight gain during the time of the study.

                Grading scale    
 Groups  Reading time  Concentrations  0  1  2  3  % positive responses  % of animals sensitized
 Control           24 hours 100%   10  0  0  0  0  
 48 hours 100%  10  0  0  0  0  
 24 hours 0%  10  0  0  0  0  
 48 hours 0%  10  0  0  0  0  
 Treated           24 hours 100%  19  1  0  0  5  5
 48 hours 100%  20  0  0  0  0  0
 24 hours 0%  20  0  0  0  0  0
 48 hours 0%  20  0  0  0  0  0

Applicant's summary and conclusion