Reaction product with n-butanol of high-boiling stream resulting from the hydroxylation, oxidative cleavage and hydrolysis of vegetable oil saturated and unsaturated C16-C22 triglycerides.

Skin sensitisation

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: In according to OECD guideline, GLP without protocol deviation. With complete substance informations

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The choice of the test to be conducted was done in consideration of the the test material nature. It contains functional groups known in the literature to act as potential confounders.

Test material

Test material information

Constituent 1

Reference substance name:

Reaction product with n-butanol of high-boiling stream resulting from the hydroxylation, oxidative cleavage and hydrolysis of vegetable oil saturated and unsaturated C16-C22 triglycerides.

EC Number:

940-822-5

IUPAC Name:

Reaction product with n-butanol of high-boiling stream resulting from the hydroxylation, oxidative cleavage and hydrolysis of vegetable oil saturated and unsaturated C16-C22 triglycerides.

Test material form:

liquid: viscous

Details on test material:

Identity: FAV ES
batch No: P23_11_12+ P25_07_13
Expiry date: 23 May 2014
Storage conditions: Room temperature; protected from light

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

Animal supply and acclimatisation:
- Sex: female, non pregnant and nulliparous
- Age: 4 to 5 weeks
- Weight: 250 to 300 grams
- Supplier: Charles River Italie S.p.A Calco (Lecco); Italy
- Breeder: Charles River France Laboratories, Domaine des Oncins B.P. 0109, F 69592 L'Abresle Cedex, France
- Date of arrival: 23 January 2014
- Acclimatisation period: at least 5 days
- Veterinary health check: during acclimatisation period
- Identification: Permanent by tatoo on the ear, following randomisation at arrival

Animal Husbandry:
- Animals per cage: up to 5
- Housing: Noryl cage measuring 74,3x54,3x25cm
-Cage control: Daily inspected and changed as necessary (at least 3 times/week)
- Water: drinking water supplied to each cage via a water bottle, ad libitum
- Diet: 8GP17 (Mucedola S.r.L, Via G. Galilei, 4, 20019, Settimo Milanese (MI) Italy), ad libitum throughout the study
- Room lighting: Artificial (fluorescent tubes), daily light/dark cycle of 12/12 hours
- Air changes: Approximately 15 to 25 air changes per hour
- Temperature range: 22°C +- 2°C
- Relative humidity range: 55% +- 15%

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

corn oil

Induction 2

Concentration / amount:

The test item was dissolved/suspended as follows: Vehicle and adjuvant: Corn oil and 50% v/v emulsion of Freund’s complete adjuvant (FCA) in sterile water. FCA is a mixture of paraffin oil, an emulsifier and killed mycobacteria used to enhance the potential of the substance to cause a delayed contact hypersensitivity reaction. Concentration (Preliminary tolerance phase): - Induction by intradermal injection: 0.1 mL of a solution containing 100, 50, 20, 10, 5 and 1% of test material in corn oil - Topical application tolerance test: each animal was dosed with 2 concentrations of the test item, 1 on each flank. The undiluted test item and concentrations of 50, 20, 10 and 5% in corn oil were selected. Concentration (Main phase): - Induction by intradermal injection (tolerated by the test system: 10% in FCA or corn oil); - Induction by topical application: undiluted test item - Challenge phase: 50% in corn oil.

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

corn oil

Challenge 2

Concentration / amount:

The test item was dissolved/suspended as follows: Vehicle and adjuvant: Corn oil and 50% v/v emulsion of Freund’s complete adjuvant (FCA) in sterile water. FCA is a mixture of paraffin oil, an emulsifier and killed mycobacteria used to enhance the potential of the substance to cause a delayed contact hypersensitivity reaction. Concentration (Preliminary tolerance phase): - Induction by intradermal injection: 0.1 mL of a solution containing 100, 50, 20, 10, 5 and 1% of test material in corn oil - Topical application tolerance test: each animal was dosed with 2 concentrations of the test item, 1 on each flank. The undiluted test item and concentrations of 50, 20, 10 and 5% in corn oil were selected. Concentration (Main phase): - Induction by intradermal injection (tolerated by the test system: 10% in FCA or corn oil); - Induction by topical application: undiluted test item - Challenge phase: 50% in corn oil.

No. of animals per dose:

test group: 10 animals
control group: 5 animals

Details on study design:

Study design was divided in 2 distinct phases. The first of these was a dose-ranging screen which was used to determine suitable dose levels for use in the second phase. This second phase corresponded to the main study: determination of the sensitisation potential of the test item.

RANGE FINDING TESTS:
Intradermal injection tolerance test:
- Allocation: 1 group of 2 animals was allocated to the intradermal injection tolerance test.
- Preparation of the site: The hair over the scapulae was removed in the allocated animals using an electric clipper with suitable blade.
- Dosage: 6 sites were selected on each animals over the shaved scapulae. Each site was injected with a single concentration of the test item. Conc entrations of 100, 50, 20, 10, 5 and 1% in corn oil were selected. The undiluated test item was not injectable due to its high viscosity. The 50% co ncentration was injected with difficulty.
- dosing method: on day 1, a single intradermal injection of 0.1 mL of each concentration was administrated with a suitable graduated syringe.
- Observations: on day 2 and day 7 for any signs of reactions in according to the Draize scoring and any response not covered by the scoring scal e was separately described.
Topical application tolerance test:
- Allocation: 1 group of 5 animals
- Preparation of the site and F.C.A injection: the hair over scapulae was removed. Each animals was injected intradermally at the prepared site with 2 injections, each of 0.1 mL, of emulsified Freund's complete adjuvant.
- Dosage: 7 days after adjuvant injection, the flanks of each animal were clipped free of hair. Each animal wad dosed with 2 concentrations of the test item, 1 on each flank. The undiluated test item and concentrations of 50, 20, 10 and 5% in corn oil were selected.
Dosage method (day 7):
- A gauze patch measuring at least 20x20 mm was soaked with 0.2 mL of each selected concentration of the test item and placed onto the selected treatment site. When both site of the animal had been treated, they were covered with a strip of synthetic film and the trunk wrapped with an elastic adhesive bandage to maintain the test item in contact with the skin. It was removed after 24h and treated site gently cleaned by washing with lukewarm water.
- Observations: 24 and 48 hours after removal of the dressing.

MAIN STUDY
Induction-Intradermal injection:
- Allocation: test group of 10 animals and control group of 5 animals
- Preparation of site: on the day of dosing the hair over scapulae was removed over an area of approximately 20x40 mm using an electric clipper with a suitable blade.
- Dosage: 3 pairs of intradermal injections were made at the prepared slin site of each animal. All injections were made at the edge of the prepared site and the anterior and median injection were positioned close together and distant from posterior injections.
- Volume of injection: 0.1 mL
Induction-Topical application:
- Preparation of the site (day 8): 7 days after injection, the hair was removed on the area surrounding the injection sites by means of an electric clipper.
- Dosing method (day 8):
- animals of test group were treated with 0.4 mL of the test item at 100% concentration
- animals of control group were treated with the vehicule alone (corn oil)
- A gauze patch measuring 20x40 mm was soaked with the selected concentration of the test item or vehicule and then placed over the injection sites.
- The treatment site was covered with a strip of synthetic film and the trunk wrapped with an elastic adhesive bandage to maintain the test item in contact with the skin.
- After a contact period of 48 hours the dressing were removed and the sites gently cleaned by washing with lukewarm water.
- Observation:
- reaction to treatment was assessed approximately 24 hours after removal of the dressings.

Challenge controls:

On day 22 - Preparation of the site (day 22): all animals were prepared for challenge by clipping the flanks free of hair to expose areas of approximately 50x50 mm on each flank. - Dosages : All animals of both test and control groups were treated with 0.2 mL aliquots of the test item at 50% concentration in corn oil on the right flank, in the centre of the prepared skin site. The left flank of each animal was treated with 0,2 ml of the vehicule alone (corn oil). - Dosage method (day 22): A gauze patch measuring 20×20 mm was soaked with the test item (or vehicle) and then placed over the centre of the right (or left) flank. When both sites of the animal had been treated, they were covered with a strip of synthetic film and the trunk wrapped with an elastic adhesive bandage to maintain the test item and vehicle in contact with the skin (occlusive barrier). After a contact period of 24 hours, the dressings and patches were removed and the treated sites were washed with lukewarm water. - Observations: The treated sites were closely clipped to remove any hair that may have grown approximately 21 hours after the removal of the dressings. Observation of the treated sites was carried out approximately 24 hours and 48 hours after removal of the dressings.

Positive control substance(s):

no

Results and discussion

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

50% test item

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

no visible change

Remarks on result:

no indication of skin sensitisation

Results 2

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

Only vehicle (corn oil)

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

no visible change

Remarks on result:

no indication of skin sensitisation

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

50% of -Hexylcinnamaldehyde in corn oil

No. with + reactions:

3

Total no. in group:

5

Clinical observations:

Evidence of sensitization in 60% of the animals

Remarks on result:

positive indication of skin sensitisation

Any other information on results incl. tables

Main study-injection induction- individual results

Group function	Animal number	Dermal response
		Anterior site: (FCA emulsion)		Median site:  (Vehicle)		Posterior site: (Vehicle/FCA)
		Left	Right	Left	Right	Left	Right
7 CONTROL	59	3	3	1	1	3	3
	61	3	3	2	2	3	3
	63	3	3	2	2	3	3
	65	3	3	1	1	3	3
	67	3	3	1	1	3	3

Main study-injection induction-individual results

Group function	Animal number	Dermal response
		Anterior site: (FCA emulsion)		Median site:  (Vehicle)		Posterior site: (Vehicle/FCA)
		Left	Right	Left	Right	Left	Right
8 TEST	69	3	3	2c	2c	3c	3c
	71	3	3	1c	2c	2c	2c
	73	3	3	1c	1c	2c	2c
	75	3	3	1c	1c	2c	3c
	77	3	3	1c	1c	2c	2c
	79	3	3	1c	1c	2c	2c
	81	3	3	1c	1c	3c	3c
	83	3	3	1c	1c	2c	2c
	85	3	3	1c	1c	3c	3c
	87	3	3	1c	1c	3c	3c

Key: 0= No erythema

1= Very slight erythema

2= Well defined erythema

3= Moderate to severe erythema

4= Severe erythema (beef redness) to eschar formation preventing grading of erythema

c= Dark coloration of the injection site not preventing evaluation of erythema

Main study-topical induction-individual results

Group function	Animal number	Dermal response
7 CONTROL	59	0
	61	0
	63	0
	65	0
	67	0
8 TEST	69	2c
	71	1c
	73	1c
	75	1c
	77	1c
	79	2c
	81	1c
	83	1c
	85	1c
	87	1c
Key:
0= No visible change
1= Discrete or patchy erythema
2= Moderate and confluent erythema
3= Intense erythema and swelling
c= Slight dark coloration of the treated site not preventing evaluation of erythema

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

FAV ES does not elicit a sensitisation response in the guinea pig.

Executive summary:

The potential of the test item to induce and elicit delayed dermal sensitisation was assessed by a guinea pig model using the maximisation test of Magnusson and Kligman according to OECD and GLP guidelines.

The concentrations of the test item used in the main study were determined by the results of preliminary screening tests. The main sensitisation test was undertaken using a test group of 10 animals and a control group of 5 animals. In an attempt to induce sensitisation, test animals were intradermally injected with an emulsion of Freund’s complete adjuvant (anterior sites), the test item at 10% concentration in the selected vehicle (corn oil) (middle sites) and the test item at 10% concentration in an emulsion of Freund’s complete adjuvant (posterior sites). One week later, animals were boosted by topical application of the undiluted test item over the injection sites. Control group animals were treated in the same manner but the selected vehicle (corn oil) was used in place of the test item. Two weeks after the second induction stage, all animals were challenged by topical application of both the vehicle (corn oil) and the test item at 50% concentration.

At the challenge with the test item, no response was apparent in any animal of the test or control group. No reaction to the vehicle alone was observed in any animal. These results indicate that the test item, FAV-ES, does not elicit a sensitisation response in the guinea pig under the conditions of the test.