Reaction mass of N,N’-ethane-1,2-diylbis(12-hydroxyoctadecan-1-amide), Octadecanamide, 12-hydroxy-N-[2-[(1-oxooctadecyl)amino]ethyl]- and Octadecanoic acid, 12-hydroxy-, 1-hexyl-12-[[2-[(12-hydroxy-1-oxooctadecyl)amino]ethyl]amino]-12-oxododecyl ester

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

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 (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

In the context of its REACH registration, 12-HSA was tested for sensitization in a Local Lymph Node Assay (LLNA). 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 it 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.

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

IUPAC Name:

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

Constituent 2

Reference substance name:

100545-48-0

Cas Number:

100545-48-0

IUPAC Name:

100545-48-0

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material: Octadecanoic acid, 12-hydroxy-, reaction products with ethylenediamine
- Physical state: White powder
- Composition of test material, percentage of components: N,N'-ethane-1,2-diylbis(12-hydroxyoctadecan-1-amide): 64.4%; octadecanamide, 12-hydroxy-N-[2-[(1-oxooctadecyl)amino]ethyl]-: 14.3%; monoesterification reaction product of 12-hydroxystearic acid with N-N'-ethane-1,2-diylbis(12-hydroxyoctadecan-1-amide): 11.7%; residual amine (ethylenediamine and mono-reacted species 12HSA-EDA etc.): 1.8%; Residual acid (12-hydroxystearic acid + impurities including stearic acid): 2.4%
- Impurity: 12-Hydroxstearic acid (12-HSA) (EC No. 203-366-1; CAS No. 106-14-9)
- Lot/batch No.: ADDEP10002-182
- Expiry date: 31 May 2014
- Storage condition: At room temperature.

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

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

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

Induction 2

Concentration / amount:

Induction phase:
Concentration for intradermal injection: 5% *
Concentration for topical application: 25% *
*: highest to cause mild-to-moderate skin irritation based on preliminary assays

Challenge phase:
Concentration for topical application: 10%**
** highest non-irritant concentration based on preliminary assays

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

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

Challenge 2

Concentration / amount:

Induction phase:
Concentration for intradermal injection: 5% *
Concentration for topical application: 25% *
*: highest to cause mild-to-moderate skin irritation based on preliminary assays

Challenge phase:
Concentration for topical application: 10%**
** highest non-irritant concentration based on preliminary assays

No. of animals per dose:

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

Details on study design:

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2
- Exposure period: Topical induction: 48 h
- Site: Interscapular region
- Dose: Intradermal injection: On Day 1, 3 pairs (top, middle and down) of 5% test substance in corn oil were administered by intradermal injection in the clipped interscapular region using a sterile plastic syringe fitted with a sterile single use needle. Topical induction: On Day 8, a filter paper (approximately 8 cm2; held in place with occlusive dressing) was fully-loaded with 25% test substance in ethanol/drinking water treated by reverse osmosis (80/20), and then applied to the clipped interscapular region, over the intradermal injection sites for 48 h. The induction phase was followed by a 14-d rest period.
- Frequency of applications: Once intradermal, once topical
- Type of patch: Occlusive patch (i.e., a filter paper (approximately 8 cm2) fully-loaded with the dose formulations was held in place by means of an occlusive dressing).
- Observations: The cutaneous reactions were evaluated before treatment, 24 and 48 h after removal of the dressing.

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: Day 22
- Exposure period: 24 h
- Dose: On Day 22, a Finn Chamber filter paper (held in place with occlusive dressing) was fully-loaded with the 10% test substance in acetone, and then applied to the clipped interscapular region, over the intradermal injection sites for 24 h. The filter paper was held in place by means of an occlusive dressing for 48 hours..
- Site: Right flank (test substance) and left flank (vehicle)
- Type of patch: Occlusive patch (i.e., a Finn Chamber® filter paper fully-loaded with the dose formulations was held in place by means of an occlusive dressing).
- Observations: The cutaneous reactions were evaluated before treatment, 24, 48 h after removal of dressing.


OBSERVATIONS:
- Morbidity and mortality: Each animal was checked for mortality and morbidity at least once a day during the treatment and observation periods, including weekends and public holidays.
- Clinical signs: Each animal was observed at least once a day, at approximately the same time, for the recording of clinical signs.
- Cutaneous reactions: Cutaneous reactions were scored in each animal according to the following scale:
. no visible change ...............................................................................................................................0
. discrete or patchy erythema ..............................................................................................................1
. moderate and confluent erythema.....................................................................................................2
. intense erythema ...............................................................................................................................3

Any observations of edema or other lesions were recorded.
- Body weight: The body weight of each animal was recorded the day of group allocation, then on the first day of treatment and at sacrifice.

Challenge controls:

Control animals received vehicle only.

Positive control substance(s):

not required

Remarks:

mercaptobenzothiazole tested in another study

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:

13

Total no. in group:

20

Clinical observations:

discrete or moderate erythema, associated with dryness of the skin

Remarks on result:

other: see Remark

Remarks:

Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 5% intradermal injection. No with. + reactions: 13.0. Total no. in groups: 20.0. Clinical observations: discrete or moderate erythema, associated with dryness of the skin.

Results 2

Key result

Reading:

2nd reading

Hours after challenge:

48

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 and edema

Remarks on result:

other: see Remark

Remarks:

Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 5% intradermal injection. No with. + reactions: 14.0. Total no. in groups: 20.0. Clinical observations: discrete or moderate erythema, associated with dryness of the skin and edema.

Any other information on results incl. tables

- Mortality: No unscheduled deaths occurred.

- Clinical signs: No clinical signs were observed in any animals. Scabs and cracks were observed in all groups during the observation period, associated with wounds in some of them.

- Cutaneous reactions:

- In the control group, at the 24- and 48-h readings, no cutaneous reactions were observed on the left flank (treated with vehicle) and on the right flank (treated with test substance) of animals.

- In the test substance-treated group: At the 24-h reading: A discrete erythema (grade 1) was noted on the left flank (treated with the vehicle) of 1/20 animals. A discrete or moderate erythema (grade 1 or 2), associated with dryness of the skin, was observed on the right flank (treated with test substance) of 13/20 animals. At the 48-h reading, a discrete or moderate erythema (grade 1 or 2) was observed on the right flank (treated with test substance) of 14/20 animals, associated with dryness of the skin in six of them and edema in four of them.

In addition, at the 24- and 48-h readings, an intense erythema (grade 3), associated with edema and dryness of the skin, was observed on the right flank of one male.

The cutaneous reactions observed on the right flank (test substance-treated) of the test substance-treated group were of higher incidence and severity than those recorded on the right flank (test substance-treated during challenge phase) of the control group. Therefore, these findings were considered to be attributed to delayed contact hypersensitivity.

- Body weight: The body weight change of the test substance-treated animals was similar to that of controls.

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

Under the conditions of the study, the test substance was considered to induce delayed contact hypersensitivity in 15/20 (75%) guinea pigs.

Executive summary:

A guinea pig maximization study was conducted to evaluate the skin sensitization potential of the test substance according to OECD Guideline 406 and EU Method B.6 in compliance with GLP.

During the induction phase, 20 guinea pigs (15 males and 15 females) were exposed to 5% test substance in corn oil via interdermal injection and 25% test substance in ethanol/drinking water (80/20) via 48 h topical occlusive patches. During the induction phase, exposure occurred via 10% (in acetone) 24 h topical occlusive patches. No unscheduled deaths, changes in body weight or any clinical signs were observed during the study. Scabs and cracks were seen in all animals, associated with wounds in some of them. No cutaneous reactions were observed in the control group at the 24 and 48 h readings. Further, at the 24 h reading in the test substance-treated group, a discrete or moderate erythema, associated with dryness of the skin, was noted on the right flank (treated with test substance) of 13/20 animals. At the 48 h reading, a discrete or moderate erythema was observed on the right flank (treated with test substance) of 14/20 animals, associated with dryness of the skin in six of them and edema in four of them. In addition, at the 24 and 48 h readings, an intense erythema, associated with edema and dryness of the skin, was observed on the right flank of 1/20 animals. Overall, the cutaneous reactions on the right flank of the test substance-treated group were of higher incidence and severity than those recorded on the right flank of the control group during the challenge phase. Therefore, these findings were considered to be attributed to delayed contact hypersensitivity.

Under the conditions of the study, the test substance was considered to induce delayed contact hypersensitivity in 15/20 (75%) guinea pigs.