9-Octadecenoic acid (Z)-, sulfonated, potassium salts

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From May 13, 2014 to July 25, 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

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: Food and Agricultural Materials Inspection Centre (FAMIC), 12 Nohsan, Notification No. 8147, April 2011; including the most recent partial revisions.

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

An LLNA study was not conducted as the substance is a surfactant. Surfactants have been shown to often give false-positive results in LLNA studies. Therefore GPMT studies are more adapted to this type of chemistry.

Test material

Test material information

Constituent 1

Reference substance name:

9-Octadecenoic acid (Z)-, sulfonated, potassium salts

EC Number:

271-843-1

EC Name:

9-Octadecenoic acid (Z)-, sulfonated, potassium salts

Cas Number:

68609-93-8

Molecular formula:

A generic formula cannot be provided for this UVCB substance. The alkyl chain length of the sulfonated fatty acids range from C12-C22, however the major alkyl chain is C18.

IUPAC Name:

9-Octadecenoic acid (Z)-, sulfonated, potassium salts

Test material form:

other: powder/flakes with lumps

Details on test material:

- Name of test material (as cited in study report): 9-Octadecenoic acid (Z)-, sulfonated, potassium salts
- Physical state: Beige-yellow powder/flakes with lumps (i.e., determined at WIL Research Europe B.V.)
- Analytical purity: 100%
- Water content: 0.5%
- pH: 6 at concentration of 1g/L
- Lot/batch No.: 7495382
- Expiration date of the lot/batch: December 31, 2015
- Storage condition of test material: At room temperature in the dark
- Stability in vehicle: Water: Yes
- Solubility in vehicle: Water: Completely miscible

In vivo test system

Test animals

Species:

guinea pig

Strain:

other: Dunkin Hartley strain (SPF-quality)

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River France, L’arbresle Cedex, France
- Age: Young adult animals (approximately 4 weeks old) were selected. Females were nulliparous and non-pregnant.
- Housing: Group housing of maximally 5 animals per labeled Noryl cage (i.e., Tecniplast; 74 cm x 54 cm x 25 cm height) containing sterilized sawdust as bedding material (i.e., Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and shelters (i.e., CS3B02A Play tunnels (90 mm x 5 mm x 125 mm), Datesand, Manchester, UK) as cage enrichment
- Diet: Complete maintenance diet for guinea pigs (SSNIFF® Spezialdiäten GmbH, Soest, Germany). In addition, hay (TecniLab-BMI BV, Someren, The Netherlands) was provided at least twice a week
- Water: Free access to tap water
- Acclimation period: At least 5 d
- Animal identification: Ear tattoo
- Health inspection: At least prior to dosing. It was ensured that the animals were healthy and that the skin to be treated was intact and free from any abnormality

ENVIRONMENTAL CONDITIONS
- Temperature: 18 to 24°C
- Humidity: 40 to 70%
- Air changes: 10 air changes/h
- Photoperiod: 12 h light/12 h dark cycle

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

other: water and Freunds' Complete Adjuvant were used for the intradermal applications and water for the dermal applications

Induction 2

Concentration / amount:

Induction:
Concentration for intradermal injection: 0.5%
Concentration for dermal application: 5%

Challenge:
Concentration for dermal application: 5%

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

other: water and Freunds' Complete Adjuvant were used for the intradermal applications and water for the dermal applications

Challenge 2

Concentration / amount:

Induction:
Concentration for intradermal injection: 0.5%
Concentration for dermal application: 5%

Challenge:
Concentration for dermal application: 5%

No. of animals per dose:

Number of animals in experimental group: 10 females
Number of animals in control group: 5 females

Details on study design:

Main study
The concentrations and induction method were selected based on the results of the preliminary irritation study.

INDUCTION - Experimental animals
Day 1: The scapular region was clipped and three pairs of intradermal injections (i.e., 0.1 mL/site) were made in this area as follows:
A) A 1:1 w/w mixture of Freunds' Complete Adjuvant (Difco, Detroit, U.S.A.) with water for injection (B.Braun Melsungen AG, Melsungen. Germany).
B) The test substance at a 0.5% concentration.
C) A 1:1 w/w mixture of the test substance, at twice the concentration used in (B) and Freunds' Complete Adjuvant.
Note: One of each pair was on each side of the midline and from cranial A) to caudal C).
Day 3: The dermal reactions caused by the intradermal injections were assessed for irritation.
Day 8: The scapular area between the injection sites was clipped and subsequently treated with 0.5 mL of a 5% test substance concentration using a Metalline patch (i.e., 2x3 cm) mounted on Medical tape, which was held in place with Micropore tape and subsequently Coban elastic bandage.
The dressing was removed after 48 h exposure, the skin cleaned of residual test substance using water and the dermal reactions caused by the epidermal exposure were assessed for irritation.

INDUCTION - Control animals
The control animals were treated as described for the experimental animals except that, instead of the test substance, vehicle alone was administered.

CHALLENGE - All animals
Day 22: One flank of all animals was clipped and treated by epidermal application of a 5% test substance concentration and the vehicle (i.e., 0.1 mL each), using Patch Test Plasters (Curatest, Lohmann, Almere, The Netherlands). The patches were held in place with Micropore tape and subsequently Coban elastic bandage.
The dressing was removed after 24 h exposure and the skin cleaned of residual test substance and vehicle using water. The treated sites were assessed for challenge reactions 24 and 48 h after removal of the dressing.
After termination, animals were sacrificed using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and an intra-peritoneal injection of Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands).

Observations
-Mortality/Viability: Twice daily
-Toxicity: At least once daily.
-Body weights: Prior to start and at termination of the study.
-Irritation: Skin reactions were graded according to the numerical scoring systems. Furthermore, a description of all other (local) effects was recorded for the epidermal treated skin sites.
To facilitate scoring, the epidermally treated skin-areas were clipped at least 3 h before the 48 h reading of these areas in the challenge phase.

Positive control substance(s):

yes

Remarks:

(alpha-hexylcinnamaldehyde, technical grade tested in another study)

Results and discussion

Positive control results:

After the challenge treatment with 20% concentration positive response was observed in 50% of the treated animals.

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

5% dermal application

No. with + reactions:

0

Total no. in group:

10

Results 2

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

5% dermal application

No. with + reactions:

0

Total no. in group:

10

Results 3

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

5% dermal application

No. with + reactions:

0

Total no. in group:

5

Results 4

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

5% dermal application

No. with + reactions:

0

Total no. in group:

5

Results 5

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

20% dermal application

No. with + reactions:

5

Total no. in group:

5

Any other information on results incl. tables

PRELIMINARY IRRITATION STUDY

The results of the intradermal injections and epidermal exposures for the selection of suitable test substance concentrations for the main study are described in following tables.

SKIN REACTIONS AFTER INTRADERMAL INJECTION

Animal	Conc %	24 h after injection		48 h after injection
number		Erythema	Necrosis	Erythema	Necrosis
		(grade)	(mm)	(grade)	(mm)
28	50		8		8
	20		8		8
29	10		8		8
	5		7		7
25	1		2		2
	0.5	2		1
30	0.2	1		1
	0.1	1		1

 

SKIN REACTIONS AFTER EPIDERMAL EXPOSURE

Animal	Conc. %	24 h after exposure		48 h after exposure
number		Erythema	Oedema	Erythema	Oedema
		(grade)	(grade)	(grade)	(grade)
26	50	np	1	n	1
	20	np	0	np	0
27	50	np	1	n	1
	20	np	0	np	0
28	10	np	0	np	0
	5	0s	0	0s	0
29	10	np	0	np	0
	5	0s	0	0s	0

s. Scaliness

np.  Signs of superficial necrosis

n.    Signs of necrosis

Based on the results, the test substance concentrations selected for the main study were a 0.5% concentration for the intradermal induction and a 5% concentration for the epidermal induction exposure. A 5% test substance concentration was selected for the challenge phase.

 

MAIN STUDY

Induction phase

The skin effects caused by the intradermal injections and epidermal exposure during the induction phase are given in following table:

Animal		Intradermal injection (i.e., Day 3)											Epidermal exposure (i.e., Day 10)
Number		A			B			C					D
Control		E	N		E	N		E	N				Erythema		Oedema
31		3			0			3						0		0
32		3			0			3						0		0
33		3			0			2						0		0
34		3			0			3						0		0
35		3			0			3						0		0
Experimental		E	N		E	N		E	N
36		3			1			3						0		0
37		3			1				4					0		0
38		3			1				4					0		0
39		3			0				4					0		0
40		2			1			3						0		0
41		3			1				3					0		0
42		3			1				3					0		0
43		3			1				5					0		0
44		3			1				4					0		0
45		3			0				4					0		0

A. 1:1 Mixture of Freunds' Complete Adjuvant and water for injection.

B. A 0.5% test substance concentration (Experimental); vehicle (Control).

C. 1:1 Mixture of Freunds' Complete Adjuvant and a 10% concentration (Experimental) or vehicle (Control).

D. A 5% test substance concentration (Experimental); vehicle (Control).

Skin effects intradermal injections:

E. Erythema (grade)

N. Signs of necrosis (mm in diameter)

 

Challenge phase

No positive skin reactions were evident after the challenge exposure in the experimental and control animals. Scaliness was noted for one experimental animal which was not considered a positive sign indicating sensitization.

Toxicity / Mortality

No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

Body weights

Body weights and body weight gain of experimental animals remained in the same range as controls over the study period .

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Conclusions:

Under the study conditions, the test substance showed no evidence of sensitising properties.

Executive summary:

A study was conducted to determine the skin sensitization potential of the test substance according to OECD Guideline 406 (guinea pig maximization test), in compliance with GLP. Based on the results of the preliminary study, 0.5 and 5% of test substance in water were selected as intradermal and dermal induction doses. The concentration used for challenge application was 5% test substance in water. None of the animals in the treatment group showed skin reactions 24 and 48 hours after removal of the dressings. Under the study conditions, the test substance showed no evidence of sensitising properties (Van Huygevoort, 2014).