Octadecanoic acid, 12-hydroxy-, reaction products with ethylenediamine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 02 August 2012 to 17 September 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Justification for conducting a Guinea Pig Maximisation Test rather than a Local Lymph Node Assay:

The test item is a complex substance which contains 12 -hydroxystearic acid (12 -HSA) (EC n° 203 -366 -1, CAS n°106 -14 -9) as an impurity. 12-HSA is an unbranched fatty acid of C18 chain length with an hydroxyl group in C12.

In the context of its REACH registration, 12-HSA was tested for sensitization in a Local Lymph Node Assay (LLNA) and the study produced a positive response (Sanders A., 2012).

For the following reasons, the response seen in the LLNA study with 12-HSA was considered as a false positive:
- From a structural point of view, 12-HSA does not contain electrophilic groups that could potentially trigger skin sensitization reactions. (Q)SAR analysis suggests that 12-HSA is not a sensitizer,
- 12-HSA has a history of safe use in consumer products with substantial skin contact (e.g. cosmetics, laundry and cleaning products), with no evidence of skin sensitization issues. Furthermore, experience from worker exposure to 12-HSA at both production and handling sites suggests that the substance does not cause sensitizing effects,
- During the validation of the LLNA study, the observation was made that this assay often overestimates the sensitization potential for some substances, e.g. surfactants, fatty acids, fatty alcohols and siloxanes (Ball et al., 2011). Testing conducted with a number of fatty acids showed that some cause positive effects in the LLNA test but are then negative in the GMPT study. The LLNA results are therefore considered to be false positives. This is the case for 12-HSA which was recently tested in a GPMT study and gave negative results.

For the above mentioned reasons, it was advisable to conduct a Guinea Pig Maximisation Test rather than a Local Lymph Nodes Assay for assessing the skin sensitization potential of the test substance.

Test material

Test material information

Constituent 1

Reference substance name:

Octadecanoic acid, 12-hydroxy-, reaction products with ethylenediamine

EC Number:

309-629-8

EC Name:

Octadecanoic acid, 12-hydroxy-, reaction products with ethylenediamine

Cas Number:

100545-48-0

Molecular formula:

No discrete molecular formula available for this UVCB substance

IUPAC Name:

Reaction products of 12-hydroxyoctadecanoic acid with ethane-1,2-diamine

Test material form:

solid: particulate/powder

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: breeder: Charles River Laboratories France, L’Arbresle, France
- Age at study initiation: Approximately 1 to 2 months old on the day of treatment
- Mean body weight at study initiation: The males had a mean body weight of 360 g (range: 284 g to 393 g) and the females had a mean body weight of 352 g (range: 276 g to 382 g)
- Fasting period before study: No
- Housing: Polycarbonate cages with stainless steel lid
- Diet: 106 pelleted diet (free access)
- Water: Tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: At least 5 days before the beginning of the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2°C
- Humidity: 50 ± 20%
- Air changes: Approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h

IN-LIFE DATES: 07 August 2012 to 17 September 2012.

Study design: in vivo (non-LLNA)

Induction

Route:

intradermal and epicutaneous

Vehicle:

other: Corn oil for intradermal induction injections; ethanol/water for topical induction dose and acetone for topical challenge dose

Concentration / amount:

Concentration for intradermal injection: 5%
Concentration for topical application: 25%

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Challenge

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

other: Corn oil for intradermal induction injections; ethanol/water for topical induction dose and acetone for topical challenge dose

Concentration / amount:

Concentration for topical application: 10%

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

Main test: 10 control animals and 20 treated animals (15 males and 15 females).

Details on study design:

RANGE FINDING TESTS: conducted to define the concentrations to be tested in the main study

-Intradermal exposure:
Hair over the scapulae was removed using electric clippers before treatment.
Intradermal administration of 0.1 ml of the test material (TM) was given at increasing concentrations. The dosage form preparation did not pass into the dermis at the concentrations of 10% and 25%. Therefore concentrations of 5% in corn oil was tested in 2 animals.
Evaluation of the potential cutaneous reactions: 24, 48 hours and 6 days after injection

- Epicutaneous exposure:
Hair over the scapulae was removed using electric clippers before treatment.
Each selected concentration was applied to a filter paper patch measuring 8cm2 for 48 hours under occlusive dressing . 2 concentrations (10% and 25% w/v in ethanol/drinking water treated by reverse osmosis (80/20) were tested in 2 animals.
Evaluation of the potential cutaneous reactions: 24 and 48 hours after patch removal.


MAIN STUDY

A. INDUCTION EXPOSURE
- No. of exposures: 1 intradermal, 1 epicutaneous
- Exposure period: epicutaneous: 48 hours

-> TEST GROUPS:
Intradermal exposure
Three injections of 0.1 mL were injected into each side of the animal as follows:
. Freund's complete adjuvant (FCA) diluted to 50% with sterile water
. TM at 5 % w/v in vehicle
. TM at 5% w/v in a 50/50 (v/v) mixture of FCA and sterile water

Epicutaneous exposure
Application of 8 cm2 patch saturated with the TM at 25% to the scapular region and held in place for 48 hours using an occlusive dressing.

-> CONTROL GROUP:
Intradermal exposure
Three injections of 0.1 mL were injected into each side of the animal as follows:
. Freund's complete adjuvant (FCA) diluted to 50% with sterile water
. vehicle
. A mixture 50/50 (v/v) FCA diluted to 50% with sterile water, and vehicle

Epicutaneous exposure
Application of 8 cm2 patch saturated with the vehicle to the scapular region and held in place for 48 hours using an occlusive dressing.

- Site:
Intradermal exposure
6 injections in the clipped area (3x 4cm) in the scapular region

Epicutaneous exposure
3x 4cm area over the scapulae

- Frequency of applications:
One intradermal injection and one epicutaneous application on Day 8 on the same site.
- Duration:
Epicutaneous exposure: 48 hours

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 22
- Exposure period: 24 hours

-> TEST GROUPS:
Challenge: TM at 10% in the vehicle on the right flank and vehicule on the left flank (occlusive epicutaneous application)

-> CONTROL GROUPS:
Same treatment as test group
- Site: posterior flanks
- Evaluation (hr after challenge): 24 , 48 hours after removal of the dressing according to the method of Draize.

Challenge controls:

Control animals received vehicle only.

Positive control substance(s):

yes

Remarks:

Mercaptobenzothiazole

Results and discussion

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

5% intradermal injection

No. with + reactions:

14

Total no. in group:

20

Clinical observations:

discrete or moderate erythema, associated with dryness of the skin

Remarks on result:

positive indication of skin sensitisation

Results 2

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

5% intradermal injection

No. with + reactions:

15

Total no. in group:

20

Clinical observations:

discrete or moderate erythema, associated with dryness of the skin and edema

Remarks on result:

positive indication of skin sensitisation

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

vehicle

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

vehicle

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Results 5

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

1% intradermal injection

No. with + reactions:

8

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Any other information on results incl. tables

The preliminary study indicated that the test material injected at a 5% concentration was reasonably well tolerated. Concentrations of 25% and 10% were selected for the topical induction and the challenge phase respectively.

Table 7.4.1/2 Individual skin reactions after challenge

Group	Sex	Animal number	Skin reactions (Hour after removal of dressings)
			24 hours		48 hours
			LF	RF	LF	RF
Control group	Male	Y40443	0	0	0	0
		Y40444	0	0	0	0
		Y40445	0	0	0	0
		Y40446	0	0	0	0
		Y40447	0	0	0	0
	Female	Y40473	0	0	0	0
		Y40474	0	0	0	0
		Y40475	0	0	0	0
		Y40476	0	0	0	0
		Y40477	0	0	0	0
Treated group	Male	Y40448	0	1	0	1
		Y40449	1	2/S	0	2/S/Oe
		Y40450	0	2	0	1
		Y40451	0	3/S/Oe	0	3/S/Oe
		Y40452	0	0	0	0
		Y40453	0	2	0	2
		Y40454	0	1	0	2/S
		Y40455	0	2	0	2/S
		Y40456	0	1	0	2
		Y40457	0	2	0	2/S
	Female	Y40478	0	2/S	0	2/Oe
		Y40479	0	2	0	2/S
		Y40480	0	0	0	1
		Y40481	0	0	0	0
		Y40482	0	1	0	2/Oe/S
		Y40483	0	1	0	2
		Y40484	0	0	0	0
		Y40485	0	0	0	0
		Y40486	0	0	0	0
		Y40487	0	1	0	2/Oe

LF = Left flank (vehicle)

RF = Right flank (test item at the concentration of 10%)

S= Dryness of the skin

Oe=Oedema

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

The test substance induced delayed contact hypersensitivity in 15 out of 20 (75%) guinea pigs with an intradermal induction dose of 5% .
Since more than 30% of the animals responded at an intradermal induction dose of more than 1%, the substance is considered as a moderate sensitizer and is therefore classified Skin Sensitizer category 1 and sub- category 1B according to Regulation (EC) No. 1272/2008 and its subsequent amendments on classification, labeling and packaging (CLP) of substances and mixtures.

Executive summary:

The potential of the test substance to induce delayed contact hypersensitivity was assessed in guinea pigs according to OECD guideline 406 and in compliance with Good Laboratory Practice.

The induction phase was realized both by the intradermal route on day 1 (Test material 5% in corn oil) and by the cutaneous route on day 8 (Test material 25% in ethanol/drinking water treated by reverse osmosis 80/20) in 2 groups of guinea pigs: 10 in the control group and 20 in the treated group. The challenge phase was realized on day 22 by cutaneous application of the test material at 10% in acetone. The cutaneous reactions were scored 24 and 48 hours after the challenge phase.

In the control group, at the 24 and 48 hour readings, no cutaneous reactions were observed. In the treated group, a discrete or moderate erythema (grade1 or 2) associated with dryness of the skin, was noted in 13 of the 20 animals at the 24 hour reading

At the 48 hour reading, a discrete or moderate erythema (grade 1 or 2) was observed in 14 of the 20 animals, associated with dryness of the skin for 6 of them and oedema for 4 of them. In addition, at the 24 and 48 hour readings, an intense erythema (grade 3), associated with oedema and dryness of the skin was observed in 1 of the 20 animals.

The persistent cutaneous reactions observed in 15 of the 20 animals of the test item-treated group were attributed to delayed contact hypersensitivity.

It was therefore concluded that the test substance was a skin sensitizer.