Reaction mass of methyl (9E,12E,15Z)-9,12,15-octadecatrienoate and methyl (9E,12Z,15E)-9,12,15-octadecatrienoate and methyl (9Z)-9-octadecenoate and methyl (9Z,12E,15E)-9,12,15-octadecatrienoate and methyl (9Z,12Z)-9,12-octadecadienoate

Skin sensitisation

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:

2015

Report date:

2015

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

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 (1970) in "Allergic Contact Dermatitis in the Guinea-Pig: Identification of contact allergens"; C.C. Thomas, USA.

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected March 2013; signature: May 2013

Type of study:

guinea pig maximisation test

Test material

Test material information

Constituent 1

Reference substance name:

methyl (9E,12E,15Z)-octadeca-9,12,15-trienoate; methyl (9E,12Z,15E)-octadeca-9,12,15-trienoate; methyl (9Z)-octadec-9-enoate; methyl (9Z,12E,15E)-octadeca-9,12,15-trienoate; methyl (9Z,12Z)-octadeca-9,12-dienoate

Molecular formula:

not applicable (reaction mass of constitutional isomers)

IUPAC Name:

methyl (9E,12E,15Z)-octadeca-9,12,15-trienoate; methyl (9E,12Z,15E)-octadeca-9,12,15-trienoate; methyl (9Z)-octadec-9-enoate; methyl (9Z,12E,15E)-octadeca-9,12,15-trienoate; methyl (9Z,12Z)-octadeca-9,12-dienoate

Test material form:

gas under pressure: refrigerated liquefied gas

Details on test material:

- Physical state: Liquid.
- Storage condition of test material: In refrigerator (2-8°C protected from light, container flushed with nitrogen
- Other: Colourless to pale yellow

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 4 weeks old).
- Weight at study initiation: 250 – 282 g (mean weight: 266 g).
- Housing: Group housing of maximally 5 animals per labelled Noryl cages containing sterilized sawdust as bedding material, shelters and play tunnels as cage enrichment.
- Diet (e.g. ad libitum): Complete maintenance diet for guinea pigs (details in the full study report). In addition, hay was provided at least twice a week.
- 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.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 – 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: 26-05-2015 to 03-07-2015

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

corn oil

Induction 2

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%
Final concentrations for definitive testing based on preliminary irritation study:
- Intradermal: 50% test material
- Topical: 100% test material (undiluted)
- Challenge: 50%
% are percent v/v of test substance in vehicle. Vehicle: corn oil, was chosen on the basis of maximising the solubility of the test substance.

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

corn oil

Challenge 2

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%
Final concentrations for definitive testing based on preliminary irritation study:
- Intradermal: 50% test material
- Topical: 100% test material (undiluted)
- Challenge: 50%
% are percent v/v of test substance in vehicle. Vehicle: corn oil, 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. Intradermal Injection: A series of four test substance concentrations was tested, the highest concentration being the maximum concentration that could technically be injected. Two animals received two different concentrations in duplicate (0.1 mL/site) in the clipped scapular region. The injection sites were assessed for irritation 24 and 48 hours after treatment. Epidermal application: A series of four test substance concentrations was tested, the highest concentration being the maximum concentration that could technically be applied. Two different concentrations were applied (0.5 mL each) per animal to the clipped flank, using Metalline patches (2x3 cm) mounted on medical tape and held in place with micropore tape and elastic bandage.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 1 intradermal induction; 1 epidermal induction (topical booster)
- Exposure period: Day 1 intradermal induction and Day 8 epidermal induction (topical booster)
- Test groups: Test substance in 1:1 mixture FCA and additional 100% (undiluted) epidermal induction.
- Control group: Vehicle and FCA only.
- Site: intradermal induction – three pairs of injections in clipped scapular region;
- Frequency of applications:
- Duration: 0-8 days. On day 8 - 48 hours for epidermal induction. The dressing was removed after 48 hours exposure, the skin cleaned of residual test substance using water and the dermal reactions caused by the epidermal exposure were assessed for irritation.
- Concentrations: Intradermal induction: A) A 1:1 w/w mixture of Freunds' Complete Adjuvant with water for injection; B) The test substance at a 50% concentration ; C) A 1:1 w/w mixture of the test substance, at twice the concentration used in (B) and Freunds' Complete Adjuvant. Epidermal 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 (epidermal 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 50% in vehicle.
- Control group: 1; vehicle only
- Site: One flank (clipped)
- Concentrations: 50% 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 (within 6 months of the study) 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 (20%) as positive control. Test substance concentrations selected for this study were: Intradermal induction: A 1% solution in water (w/w); Epidermal induction: A 50% solution in water (w/w) and Challenge: a 20% solution in water (w/w).
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 70% to the 20%w/w concentration.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

50%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

None reported; maximum score = 0 (no visible change)

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: None reported; maximum score = 0 (no visible change).

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

50%

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: 50%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: None reported; maximum score = 0 (no visible change).

Results 3

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).

Results 4

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

Test Organism Number	Test substance Concentration %	24 Hours after injection		48 Hours after injection
		Erythema	Necrosis	Erythema	Necrosis
8	100		4		5
	50	2		2
9	20	1		1
	10	1		1

Skin reactions after epidermal exposure

Test Organism Number	Test substance Concentration %	24 Hours after injection		48 Hours after injection
		Erythema	Oedema	Erythema	Oedema
1	100	2	0	1s	0
	50	0	0	0	0
2	100	1	0	0s	0
	50	0	0	0	0
3	20	0	0	0	0
	10	0	0	0	0
4	20	0	0	0	0
	10	0	0	0	0

s. Scaliness, k. Scabs, a. Small scabs

Grading erythema:

0 = No erythema

1 = Slight erythema (barely perceptible)

2 = Well-defined erythema

Grading Oedema:

0 = No oedema

1 = Slight oedema (barely perceptible)

 

Table 2: Challenge Readings

Test Organism number	Day 23		Day 24		Comments
	Test substance concentration 50%	Vehicle	Test substance concentration 50%	Vehicle
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
Experimental
1	0	0	0	0	not sensitized
2	0	0	0	0	not sensitized
3	0	0	0	0	not sensitized
4	0	0	0	0	not sensitized
5	0	0	0	0	not sensitized
6	0	0	0	0	not sensitized
7	0	0	0	0	not sensitized
8	0	0	0	0	not sensitized
9	0	0	0	0	not sensitized
10	0	0	0	0	not sensitized

Grading challenge reactions:

0 = No visible change

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 used, 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 consistent with Magnusson-Kligman maximisation test to assess the skin sensitisation potential of the test substance. The study was conducted using a preliminary irritation screen, a two-stage induction phase and a challenge phase. Preliminary irritation testing was for use in the induction phases of the study and the challenge phase of the study. The first stage of the induction phase involved intradermal injections of a 1% v/v mixture of the test substance in 10% aqueous DIPG, a 1% v/v mixture of the test substance 10% aqueous DIPG 50:50 with Complete Freund's Adjuvant, and 50% aqueous Complete Freund's Adjuvant alone into the dorsal area of each of 10 guinea pigs. After 7 days the second stage of the induction phase entailed the topical application of the undiluted test substance onto the shaved dorsal area of the test group animals using occlusive dressings for 48 hours. 14 days after the induction period, a challenge dose of the test substance 25% v/v in DIPG was selected for the challenge application to the left flank on each test animal using the same procedure as in the topical induction. The site was inspected at 24, 48 and 72 hours for the presence of erythema and oedema. Under the conditions of this study, the test material is not considered to be a contact skin sensitizer.