Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. Scientific justification is attached by the applicant.

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:
equivalent or similar to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nousan, Notification No 8147 (2000)
Deviations:
no
Principles of method if other than guideline:
Method employed in this study for the detection of delayed contact hypersensitivity was the guinea-pig maximization test described by B. Magnusson and A.M. Kligman - "The identification of contact allergens by animal assay, the guinea pig maximisation method"; Invest. Dermatol. 1969. 52, 268-276
GLP compliance:
yes (incl. certificate)
Remarks:
inspected April 2015; signature: October 2015
Type of study:
guinea pig maximisation test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: Liquid.
- Storage condition of test material: Approximately 4°C in the dark
- Other: Colourless 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: Recognised supplier
- Age at study initiation: Young adult (approximately 3- 4 weeks old).
- Weight at study initiation: 250 – 282 g (mean weight: 266 g).
- Housing: Individually or group housing of 2 animals per labelled polycarbonate containers, the flooring of which was covered with dust-free cuttings and the top fitted with a stainless steel lid.
- Diet (e.g. ad libitum): Complete maintenance diet for guinea pigs (details in the full study report).
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: The acclimatization period was at least 5 days before the start of treatment under laboratory conditions; identical to the test

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25°C
- Humidity (%): 30 – 70%
- Air changes (per hr): at least 10 per hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark

IN-LIFE DATES: From: To: 21-09-2015 to 22-10-2015

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
olive oil
Remarks:
olive oil for the intradermal injections and in liquid paraffin for the topical applications
Concentration / amount:
Preliminary irritation testing: A series of test substance concentrations were tested. Practical feasibility of administration determined the highest starting-concentration for each route. The starting- and subsequent concentrations were taken from the series: 100% (undiluted), 50%, 20%, 10%, 5%, 2%, 1%. This determined the Maximal Non Necrotizing Concentration (MNNC); Pre-Maximal Non Irritant Concentration (Pre-MNIC) and the the Maximal Non Irritant Concentration (MNIC) respectively.
Final concentrations for definitive testing based on preliminary irritation study:
- Intradermal: 20% test material in Olive Oil
- Topical: 100% test material (undiluted)
- Challenge: 20% in liquid paraffin
% are percent v/v of test substance in vehicle. Vehicle: Olive oil (jntradermal induction) and liquid paraffin (topical), was chosen on the basis of maximising the solubility of the test substance.
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
olive oil
Remarks:
olive oil for the intradermal injections and in liquid paraffin for the topical applications
Concentration / amount:
Preliminary irritation testing: A series of test substance concentrations were tested. Practical feasibility of administration determined the highest starting-concentration for each route. The starting- and subsequent concentrations were taken from the series: 100% (undiluted), 50%, 20%, 10%, 5%, 2%, 1%. This determined the Maximal Non Necrotizing Concentration (MNNC); Pre-Maximal Non Irritant Concentration (Pre-MNIC) and the the Maximal Non Irritant Concentration (MNIC) respectively.
Final concentrations for definitive testing based on preliminary irritation study:
- Intradermal: 20% test material in Olive Oil
- Topical: 100% test material (undiluted)
- Challenge: 20% in liquid paraffin
% are percent v/v of test substance in vehicle. Vehicle: Olive oil (jntradermal induction) and liquid paraffin (topical), was chosen on the basis of maximising the solubility of the test substance.
No. of animals per dose:
Test group: 10; Control group: 5
Details on study design:
RANGE FINDING TESTS:
A preliminary irritation study was conducted in order to select test substance concentrations to be used in the main study. Pre-MNNC: Two received a volume of 0.1 mL of the test item, on both sides of the spine, at 4 concentrations: undiluted (100%) and diluted at 50%, 20% and 10% in olive oil in view to determine theMNNC with macroscopic evaluation at 24 hours. PreMNIC: Two were treated with test item was applied on the dorso-lumbar zone of two guinea pigs shorn beforehand, with occlusive dressing for 24 hours, at 4 different concentrations: undiluted (100%) and diluted at 50%, 20% and 1 0% in liquid paraffin. A macroscopic evaluation of the cutaneous reactions was conducted 24 hours after dressing removal. MNIC: This test was carried out for the purpose of determining the MNIC of the test item without risk of an irritant effect during the challenge phase. Three 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 item placed onto the selected treatment sites and covered with an occlusive dressing for a period of 24 hours at 4 different concentrations: diluted at 20%, 10%, 5% and 2% in liquid paraffin. A macroscopic evaluation of the cutaneous reactions was conducted 24 and 48 hours after dressing removal.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 1 intradermal induction; 1 epidermal induction (topical booster)
- Exposure period: Day 0 intradermal induction and Day 8 topical induction (topical booster)
- Test groups: duplicate injections as follows: 2 ID: Freund's Complete Adjuvant diluted at 50% in olive oil; 2 ID: test item at 20% in olive oil ; 2 ID: a test mixture in equal volumes v/v of FCA at 50% and the test item at 40% in olive oil
- Control group: Vehicle and FCA only.
- Site: intradermal induction – three pairs of injections in clipped scapular region;
- Frequency of applications:
- Duration: 0-7 days. On day 8 - 48 hours for epidermal induction. The dressing was removed after 48 hours exposure
- Concentrations: Intradermal induction: A) A 1:1 w/w mixture of Freunds' Complete Adjuvant with water for injection; B) The test substance at a 20% concentration ; C) A 1:1 w/w mixture of the test substance, at twice the concentration used in (B) and Freunds' Complete Adjuvant. Topical induction: The scapular area between the injection sites was clipped and subsequently treated with 0.5 mL of a 100% test substance concentration using occlusive dressing.
The control group were treated as described for the experimental group except that, instead of the test substance, the vehicle was administered.

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 24 hours (topical challenge)
- Exposure period: Day 22 the dressing was removed after 24 hours exposure and the skin cleaned of residual test substance and vehicle using water. The treated sites were assessed for challenge reactions 24 and 48 hours after removal of the dressing.
- Test groups: 1; test substance 20% in liquid paraffin vehicle.
- Control group: 1; vehicle only
- Site: One flank (clipped)
- Concentrations: 20% using occlusive dressing.
- Evaluation (hr after challenge): 24 and 48 hours after dressing removal (at Day 23 and 24).
The control group were treated as described for the experimental group except that, instead of the test substance, the vehicle was administered.

OTHER: Mortality, toxicity and body weights along with irritation were examined as part of the study.
Challenge controls:
(Naive) negative control groups consisting of 5 females were exposed to the vehicle in the induction and challenge, consistent the main study with the difference that instead of test substance only the vehicle was administered.
Positive control substance(s):
yes
Remarks:
Alpha-Hexylcinnamicaldehyde (20%)

Results and discussion

Positive control results:
A reliability check was performed (three tests are listed at 6, 12 and 18 months of the study and presented in the full study report) to check the sensitivity of the test system and the reliability of the experimental techniques used. The study used the same conditions as the main study using Alpha-Hexylcinnamicaldehyde (at 6.25 and 12.5 concentrations %v/v) as positive control..
The skin reactions observed in seven experimental animals in response to the > 20 % test substance concentration in the challenge phase were considered indicative of sensitisation, based on the absence of any response in the control animals. These results lead to a sensitisation rate of 80% to the 12.5 %w/w concentration.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
20%
No. with + reactions:
1
Total no. in group:
10
Clinical observations:
One reported; maximum score = 1 (Discrete or patchy erythema)
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 20%. No with. + reactions: 1.0. Total no. in groups: 10.0. Clinical observations: One reported; maximum score = 1 (Discrete or patchy erythema).
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
20%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
None reported; maximum score = 0 (no visible change)
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 20%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: None reported; maximum score = 0 (no visible change).
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
None reported; maximum score = 0 (no visible change)
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: 5.0. Clinical observations: None reported; maximum score = 0 (no visible change).
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
None reported; maximum score = 0 (no visible change)
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: 5.0. Clinical observations: None reported; maximum score = 0 (no visible change).

Any other information on results incl. tables

Table 1: Preliminary irritation study

Skin reactions after intradermal injection (24 hours)

Number

100%v/v

50%v/v

20%v/v

10%v/v

1

Ne

Ne

2

2

2

Ne

Ne

2

2

 

 

 

 

 

Ne = Necrosis

0 = No visible change

1 = Discrete or patchy erythema

2 = Moderate and confluent erythema

3 = Intense erythema and swelling

MNNC: 20% in olive oil

 

Table 2: Preliminary irritation study

Skin reactions after topical application (24 hours)

Number

100%v/v

50%v/v

20%v/v

10%v/v

1

2

1

0

0

2

2

0

0

0

 

 

 

 

 

0 = No visible change

1 = Discrete or patchy erythema

2 = Moderate and confluent erythema

3 = Intense erythema and swelling

Pre-MNIC: 100%

 

Table 3. Preliminary irritation study

Number

Hours

20%v/v

10%v/v

5%v/v

2%v/v

1

24

0

0

0

0

 

48

0

0

0

0

2

24

0

0

0

0

 

48

0

0

0

0

3

24

0

0

0

0

 

48

0

0

0

0

 

 

 

 

 

 

MNIC: 20% in liquid paraffin

 

Table 4.

 

 

20% MNIC

 

Liquid Paraffin

 

 

Number

24 hours

48 hours

24 hours

48 hours

Control

1

0

0

0

0

 

2

0

0

0

0

 

3

0

0

0

0

 

4

0

0

0

0

 

5

0

0

0

0

Test group

1

0

0

0

0

 

2

0

0

0

0

 

3

0

0

0

0

 

4

0

0

0

0

 

5

0

0

0

0

 

6

0

0*

0

0

 

7

1

0

0

0

 

8

0

0

0

0

 

9

0

0

0

0

 

10

0

0

0

0

Where:

* = Dryness reported

0 = No visible change

1 = Discrete or patchy erythema

2 = Moderate and confluent erythema

3 = Intense erythema and swelling

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this study, the test material is not considered to be a contact sensitizer.
Executive summary:

The study was performed according to a method equivalent to guideline OECD TG 406 and EU Method B.6 and consistent with Magnusson-Kligman Guinea Pig Maximisation test to assess the skin sensitisation potential of the test substance. Test substance concentrations selected for the main study were based on the results of a preliminary study. In the main study, after induction (intradermic injection at 20% and topical application at 100%) and a ten day rest phase of ten experimental animals were challenged with 20% test item in liquid paraffin along with parallel control challenged at 20% in liquid paraffin in five animals. The treated group (treatment dose of 20%), indicated a discrete erythema was noted in 10% (1/10) of the treated animals at the reading time 24 hours. No cutaneous reaction attributable to allergy was recorded at the reading time 48 hours. In the control group (associated with the treatment dose of 20%), no cutaneous intolerance reaction was recorded at 24 and 48 hours. No cutaneous reaction was recorded in animals from the treated and control groups after the challenge phase, on the treated area with liquid paraffin (control item). Under the conditions of this study, the test substance is not considered to be a contact skin sensitizer.