Oleic acid, compound with (Z)-N-octadec-9-enylpropane-1,3-diamine (2:1)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9 Oct 2003 - 7 Dec 2003

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

Principles of method if other than guideline:

The study was performed before the REACH guidance

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The study has been performed before the entry in force of Reach regulation.

Test material

Test material information

Constituent 1

Reference substance name:

Oleic acid, compound with (Z)-N-octadec-9-enylpropane-1,3-diamine (2:1)

EC Number:

251-846-4

EC Name:

Oleic acid, compound with (Z)-N-octadec-9-enylpropane-1,3-diamine (2:1)

Cas Number:

34140-91-5

Molecular formula:

C21H44N2.2C18H34O2

IUPAC Name:

oleic acid, compound with (Z)-N-octadec-9-enylpropane-1,3-diamine (2:1)

Test material form:

liquid: viscous

Details on test material:

Chemical registery number : CAS 34140-91-5 / EC 251-846-4
Chemical name : N-[(9Z)-octadec-9-en-1-yl]propane-1,3-diaminium di[(9Z)-octadec-9-enoate

Based on the qualitative and quantitative information on the composition, the sample used are representative of the boundary composition shared and agree by each registrant.

In vivo test system

Test animals

Species:

guinea pig

Strain:

Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
Hartley Crl: (HA) BR, Caesarian obtained, Barrier sustained - Virus Antibody Free (COBS - VAF).
- Source: Charles River Laboratories France, L’Arbresle, France.
- Age at study initiation: 1-2 months old
- Weight at study initiation: mean body weight ± standard deviation of 373 ± 21 g for the males and 378 ± 11 g for the females.
- Housing: individually in polycarbonate cages with stainless steel lid (48 cm x 27 cm x 20 cm) equipped with a polypropylene bottle. Each cage contained autoclaved sawdust (SICSA, Alfortville, France).
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days before the beginning of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 30 to 70%
- Air changes (per hr): 12 cycles/hour of filtered, non-recycled air.
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 9 October 2003 (start preliminary test) To: 7 Dec 2003 (sacrifice animals upon completion)

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

other: intradermal: corn oil,; topical (induction and challenge) Acetone

Induction 2

Concentration / amount:

Intradermal: 0.1% (w/w)
topical induction: 25% (w/w)
challenge: first challenge 10 (w/w), second challenge 5% and 1%

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

other: intradermal: corn oil,; topical (induction and challenge) Acetone

Challenge 2

Concentration / amount:

Intradermal: 0.1% (w/w)
topical induction: 25% (w/w)
challenge: first challenge 10 (w/w), second challenge 5% and 1%

No. of animals per dose:

Positive control group: five control and ten treated females
preliminary test: 4 males and 4 females
Control group: 5 males and 5 females and again 5 males and 5 females for second challenge treatment group
Treated group: 10 males and 10 females

Details on study design:

RANGE FINDING TESTS:
By intradermal route (tested concentrations: 25%, 10%, 5%, 1% and 0.1% (w/w)):
- intradermal injections of the dosage form preparations (0.1 mL) were performed in the interscapular region,
- local reactions were evaluated approximately 24, 48 hours and 6 days after the injections.

By cutaneous route: Under the conditions of the induction phase (tested concentrations: 100%, 25%, 10% and 5% (w/w)):
- a filter paper (approximately 8 cm2) was fully-loaded with a dosage form preparation and was then applied to the clipped area of the skin. The filter paper was held in place by means of an occlusive dressing for 48 hours,
- cutaneous reactions were evaluated 24 and 48 hours after removal of the dressing.

Under the conditions of the challenge phase (tested concentrations: 100%, 25%, 10% and 5% (w/w)):
- the filter paper of a chamber (Finn Chamber¿) was fully-loaded with a dosage form preparation. The chamber was then applied to the clipped area of the skin (one concentration per flank). The chamber was held in place by means of an occlusive dressing for 24 hours,
- cutaneous reactions were evaluated 24 and 48 hours after removal of the dressings.

MAIN STUDY
A. INDUCTION EXPOSURE
Three injections of 0.1 mL were made into each side of this interscapular region (i.e. three pairs of sites), as follows:
1 Anterior site: FCA at 50% (v/v) in 0.9% NaCl
2 Middle location: test item at 0.1% (w/w) in 0.9% NaCl /corn oil
3 Posterior site: test item at 0.1% (w/w) in the mixture FCA/0.9% NaCl (50/50)

Cuteneous route:
(No SLS needed as substance was irritating)
On day 8, fully loaded filter paper (approximately 8 cm2) with 25% (w/w), applied on interscapular region for 48 hours. On removal of the dressing (day 10), no residual test item was observed.
A local irritation was recorded in all the animals of both groups.

B. CHALLENGE EXPOSURE
- Day(s) of challenge: day 22
- Exposure period: 24 hours
- all animals:
- Posterior right flank: filter paper of a chamber (Finn Chamber) was fully-loaded with the test item
- Posterior left flank: vehicle
- Concentrations: 10% (w/w)
- Evaluation (hr after challenge): 24 and 48 after removal of the dressing
Following equivocal cutaneous reactions, a second challenge was performed after a rest period of 17 days.
- Day(s) of challenge: day 22
- Concentrations: 5% (w/w) to median left flank and 1% (w/w) to median right flank
(otherwise as above)

Challenge controls:

Yes, similar as treated group

Positive control substance(s):

yes

Remarks:

Mercaptobenzothiazole

Results and discussion

Positive control results:

Concentration :
induction phase: 1% (w/w) on day 1 (intradermal route); 20% (w/w) on day 8 (cutaneous route)
challenge phase: 20% (w/w) on day 22 (cutaneous route)
Vehicle : corn oil
Mercaptobenzothiazole at the concentration of 20% (w/w) induced positive skin sensitization reactions in 100% (10/10) guinea pigs.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

20%

No. with + reactions:

10

Total no. in group:

10

Remarks on result:

other: First reading. Hours after challenge: 24.0.positive control group 1. Dose level: 20% Mercaptobenzothiazole. No with. + reactions: 10.0. Total no. in groups: 10.0.

Results 2

Reading:

rechallenge

Hours after challenge:

24

Group:

negative control

Dose level:

5%

No. with + reactions:

3

Total no. in group:

10

Remarks on result:

other: Reading: rechallenge. . Hours after challenge: 24.0. Group:negative control group 1. Dose level: 5%. No with. + reactions: 3.0. Total no. in groups: 10.0.

Results 3

Reading:

rechallenge

Hours after challenge:

24

Group:

negative control

Dose level:

1%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: rechallenge. . Hours after challenge: 24.0. Group:negative control group 1. Dose level: 1%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Results 4

Reading:

rechallenge

Hours after challenge:

24

Group:

other: 2nd negative control group

Dose level:

5%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: rechallenge. . Hours after challenge: 24.0. Group:negative control group 2. Dose level: 5%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Results 5

Reading:

rechallenge

Hours after challenge:

24

Group:

other: 2nd negative control group

Dose level:

1%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: rechallenge. . Hours after challenge: 24.0. Group:2nd negative control group . Dose level: 1%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Results 6

Reading:

rechallenge

Hours after challenge:

24

Group:

test chemical

Dose level:

5%

No. with + reactions:

2

Total no. in group:

18

Remarks on result:

other: Reading: rechallenge. . Hours after challenge: 24.0. Group: test group. Dose level: 5%. No with. + reactions: 2.0. Total no. in groups: 18.0.

Results 7

Reading:

rechallenge

Hours after challenge:

24

Group:

test chemical

Dose level:

1%

No. with + reactions:

0

Total no. in group:

18

Remarks on result:

other: Reading: rechallenge. . Hours after challenge: 24.0. Group:test group . Dose level:1%. No with. + reactions: 0.0. Total no. in groups: 18.0.

Any other information on results incl. tables

CHOICE OF THE VEHICLE

The test item was not soluble in 0.9% NaCl: two phases were observed.

The vehicle chosen for intradermal injections was corn oil: a homogeneous dosage form preparation was obtained at the maximum concentration of 25%(w/w); the dosage form preparation at the concentration of 25% (w/w) passed freely through a needle and into the dermis.

For topical applications (induction phase and challenge application), the vehicle used was acetone: a homogeneous dosage form preparation was obtained at the maximum concentration of 25% (w/w).

PRELIMINARY STUDY

Administration by intradermal route: 25, 10 and 5% resulted to necrosis within 24 hours. At 1% necrosis became evident after 48 hours.

At 0.1% irritation was observed.

 

Application by cutaneous route for induction: 100% resulted to necrosis. At 25% irritation (discrete or patchy erythema) was observed, with crusts at 48 hours. At 10% and 5% no irritation was observed.

 

Application by cutaneous route under conditions of challenge phase (2 animals / concentration):

100% resulted to necrosis. 25% resulted to irritation in one of the two animals.

10% and 5% did not result to irritation in either animal.

MAIN STUDY:

Clinical examinations

Marked local reactions at the intradermal injection sites were noted in a few animals of the treated group between days 7 and 36. Therefore, two animals (female Nos. 83 and 90) were sacrificed on day 22 for ethical reasons.

No systemic clinical signs and no deaths related to treatment were observed during the study.

 

Body weight

The body weight gain of the treated animals was similar to that of controls (appendix 3).

 

Challenge phase- Scoring of cutaneous reactions

On removal of the dressing, no residual test item was observed.

 

First challenge application:

Control group 1
Sex	Animal	24 hours		48 hours
	number	LF	RF	LF	RF
Male	61	0	2	0	2/S
	62	0	1	0	1/S
	63	0	0	0	0
	64	0	2	0	2
	65	0	0	0	0
Female	76	0	0	0	0
	77	0	0	0	0
	78	0	0	0	1
	79	0	0	0	1
	80	0	0	0	0
Treated group 2
Sex	Animal	24 hours		48 hours
	number	LF	RF	LF	RF
Male	66	0	1	0	2
	67	0	0	0	1
	68	0	1	0	2
	69	0	1	0	2/S
	70	0	1	0	1/S
	71	0	1	0	0
	72	0	0	0	1
	73	0	1	0	2/S
	74	0	0	0	0
	75	0	0	0	0
Female	81	0	0	0	0
	82	0	0	0	1
	83	-	-	-	-
	84	0	0	0	0
	85	0	0	0	0
	86	0	2/S	0	3/S/A
	87	0	0	0	1/S
	88	0	0	0	0
	89	0	0	0	0
	90	-	-	-	-

LF : left flank (vehicle)

RF : right flank (test item at the concentration of 10% (w/w))

S : dryness of the skin

- : dead animal

A : crusts

In order to determine whether the observed cutaneous reactions are attributable to delayed contact hypersensitivity or to an irritant effect of the test item, a second challenge application was performed. For this second challenge application, lower concentrations (1% and 5% (w/w)) were chosen.

 

Results second challenge application:

Control group 1
Sex	Animal	Before treatment		24 hours		48 hours
	number	LF	RF	LF	RF	LF	RF
Male	61	0	0	1	0	0	0
	62	0	0	0	0	0	0
	63	0	0	1	0	0	0
	64	0	0	0	0	0	0
	65	0	0	0	0	0	0
Female	76	0	0	0	0	0	0
	77	0	0	0	0	0	0
	78	0	0	1	0	0	0
	79	0	0	0	0	0	0
	80	0	0	0	0	0	0
Treated group 1
Sex	Animal	Before treatment		24 hours		48 hours
	number	LF	RF	LF	RF	LF	RF
Male	66	0	0	0	0	0	0
	67	0	0	0	0	0	0
	68	0	0	0	0	0	0
	69	0	0	0	0	0	0
	70	0	0	0	0	0	0
	71	0	0	0	0	0	0
	72	0	0	1	0	1	0
	73	0	0	0	0	0	0
	74	0	0	0	0	0	0
	75	0	0	0	0	0	0
Female	81	0	0	0	0	0	0
	82	0	0	0	0	0	0
	83	-	-	-	-	-	-
	84	0	0	0	0	0	0
	85	0	0	0	0	0	0
	86	0	0	2	0	1/S	0
	87	0	0	0	0	0	0
	88	0	0	0	0	0	0
	89	0	0	0	0	0	0
	90	-	-	-	-	-	-
Control group 3
Sex	Animal	Before treatment		24 hours		48 hours
	number	LF	RF	LF	RF	LF	RF
Male	181	0	0	0	0	0	0
	182	0	0	0	0	0	0
	183	0	0	0	0	0	0
	184	0	0	0	0	0	0
	185	0	0	0	0	0	0
Female	186	0	0	0	0	0	0
	187	0	0	0	0	0	0
	188	0	0	0	0	0	0
	189	0	0	0	0	0	0
	190	0	0	0	0	0	0

LF : left flank (test item at the concentration of 5% (w/w))

RF : right flank (test item at the concentration of 1% (w/w))

S : dryness of the skin

- : dead animal

The cutaneous reactions were as follows:

Control group 1

On the left flank (test item at the concentration of 5%), a discrete erythema (grade 1) was noted in 3/10 animals at the 24-hour reading only.

On the right flank (test item at the concentration of 1%), no cutaneous reaction was recorded.

 

Treated group 2

On the left flank (test item at the concentration of 5%), a discrete or moderate erythema (grade 1 or 2) was observed in 2/18 animals at the 24-hour reading. A discrete erythema (grade 1), together with dryness of the skin in one animal, persisted at the 48-hour reading in these two animals.

On the right flank (test item at the concentration of 1%), no cutaneous reaction was noted.

 

Control group 3

No cutaneous reaction was recorded.

The persistent cutaneous reactions observed in 2/18 animals of the treated group after the second challenge application may be attributable to delayed contact hypersensitivity.

 

Conclusion:

Under our experimental conditions and according to the maximization method of Magnusson and Kligman, the test item INIPOL 002 (batch No. 11638106) induces cutaneous reactions which could be attributable to delayed contact hypersensitivity in 2/18 (11%) guinea pigs and should therefore be considered as a mild sensitizer.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Migrated information

Conclusions:

Testing of Oleyl-diamine, dioleate in a GPMT study resulted to positive reactions in 2/19 (11%) Guinea pigs. According to classification criteria, no classification is required.

Executive summary:

The potential of the test item to induce delayed contact hypersensitivity was evaluated in guinea pigs according to the maximization method of Magnusson and Kligman and to OECD (No. 406, 17th July 1992).

The study was conducted in compliance with the principles of Good Laboratory Practice Regulations.

 

Methods

Thirty guinea pigs were allocated to two groups: a control group 1 of five males and five females and a treated group 2 of ten males and ten females. An additional control group of five males and five females (group 3) was also used for the second challenge application.

On day 1, three pairs of intradermal injections were performed in the interscapular region of all animals:

• Freund's complete adjuvant (FCA) diluted to 50% (v/v) with 0.9% NaCl (groups 1 and 2),

• test item at the concentration of 0.1% in corn oil (treated group) or vehicle alone (control group 1),

• test item at the concentration of 0.1% in a mixture FCA/0.9% NaCl (50/50, w/w) (treated group) or vehicle at the concentration of 50% (w/v) in a mixture FCA/0.9% NaCl (50/50, v/v) (control group 1).

 

On day 8, the animals of the treated group received a topical application of the test item at the concentration of 25% (w/w) in acetone to the same test site, which was then covered by an occlusive dressing for 48 hours. The animals of the control group 1 received an application of vehicle under the same experimental conditions.

 

On day 22, all animals of groups 1 and 2 were challenged by a cutaneous application of the test item at the concentration of 10% (w/w) in acetone to the right flank. The test item was maintained under an occlusive dressing for 24 hours. The vehicle was applied to the left flank under the same experimental conditions. Skin reactions were evaluated approximately 24 and 48 hours after removal of the dressing.

 

As equivocal cutaneous reactions were noted after the first challenge, a second challenge application was performed on day 40 with the original control and treated groups and with a new control group of ten animals (group 3), which was free from any previous treatment. The test item was applied at the concentrations of 5% (w/w) to the median left flank and 1% (w/w) in acetone to the median right flank of the animals of all groups, under the same experimental conditions as for the first challenge application.

At the end of the study, the animals were killed without examination of internal organs.

No skin samples were taken from the challenge application sites.

 

Results

No systemic clinical signs and no deaths related to treatment were noted during the study.

 

After the first challenge application, in the control group, a discrete or moderate erythema was observed at the 24-hour reading in 1/10 and 2/10 animals, respectively. At the 48-hour reading, a discrete or moderate erythema, together with dryness of the skin in two animals, was recorded in 3/10 and 2/10 animals, respectively.

In the treated group, at the 24-hour reading, a discrete or moderate erythema, together with dryness of the skin in one animal, was noted in 6/18 and 1/18 animals, respectively. At the 48-hour reading, a discrete, moderate or intense erythema, together with dryness of the skin in five animals and crusts in one animal, was observed in 5/18, 4/18 and 1/18 animals, respectively.

 

After the second challenge application, the cutaneous reactions were as follows:

Control group 1

On the left flank (test item at the concentration of 5%), a discrete erythema was noted in 3/10 animals, at the 24-hour reading only.

On the right flank (test item at the concentration of 1%), no cutaneous reaction was recorded.

Treated group 2

On the left flank (test item at the concentration of 5%), a discrete or moderate erythema was observed in 2/18 animals at the 24-hour reading. A discrete erythema, together with dryness of the skin in one animal, persisted at the 48-hour reading in these two animals.

On the right flank (test item at the concentration of 1%), no cutaneous reaction was noted.

Control group 3

No cutaneous reaction was recorded.

The persistent cutaneous reactions observed in 2/18 animals of the treated group after the second challenge application may be attributable to delayed contact hypersensitivity.

 

Conclusion

Under our experimental conditions and according to the maximization method of Magnusson and Kligman, the test item induces cutaneous reactions which could be attributable to delayed contact hypersensitivity in 2/18 (11%) guinea pigs and should therefore be considered as a mild sensitizer.

However, according to the classification criteria laid down in EC regulation 1272/2008 (and subsequent adaptations), the test item should not be considered as a skin sensitizer.