N-(2-hydroxypropyl)oleamide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 January 2013 - 18 March 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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:

A M&K test was performed due to the chemical structure of the substance to test (surfactant), this kind of chemical structure is known to potentially cause false positive in LLNA tests (Kreiling R 2008; Roberts DW 2016)

Test material

Test material information

Constituent 1

Reference substance name:

N-(2-hydroxypropyl)oleamide

EC Number:

203-828-2

EC Name:

N-(2-hydroxypropyl)oleamide

Cas Number:

111-05-7

Molecular formula:

C21H41NO2

IUPAC Name:

N-(2-hydroxypropyl)oleamide

Test material form:

other: waxy solid

Details on test material:

- Name of test material: N-(2-hydroxypropyl) Oleamide
- Physical state: beige waxy solid
- Lot/batch No.: T22221 without solvent
- Analytical purity: 100% dry matter
- Expiry date: 29 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: 322 g for the males (range: 278 g to 351 g) and 295 g for the females (range: 263 g to 326 g).
- Housing: the animals of the preliminary test were individually housed in polycarbonate cages with stainless steel lids and the animals of the main test were group housed by five from the same sex and group in stainless steel lids.
- Diet: 106 pelleted diet (free access).
- Water: tap water filtered with a 0.22 µm filter (free access).
- Acclimation period: for a period of 4 days (preliminary test) or 8 days (main test) before treatment.

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

IN-LIFE DATES: 05 February 2013 to 18 March 2013

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

other: intradermal and cutaneous

Vehicle:

other: Intradermal : Corn oil ; topical induction : Ethanol/water ; Challenge : Acetone

Induction 2

Concentration / amount:

Induction phase:
Concentration for intradermal injection: 10% *
Concentration for topical application: 75% *
*: highest to cause mild-to-moderate skin irritation based on preliminary assays

Challenge phase:
Concentration for topical application: 50%**
** highest non-irritant concentration based on preliminary assays

Challenge

Challenge 1

Route:

other: cutaneous, occlusive

Vehicle:

other: Intradermal : Corn oil ; topical induction : Ethanol/water ; Challenge : Acetone

Challenge 2

Concentration / amount:

Induction phase:
Concentration for intradermal injection: 10% *
Concentration for topical application: 75% *
*: highest to cause mild-to-moderate skin irritation based on preliminary assays

Challenge phase:
Concentration for topical application: 50%**
** highest non-irritant concentration based on preliminary assays

No. of animals per dose:

- preliminary test: 4 treated animals (2 males and 2 females)
- main test: 30 animals (15 males and 15 females).

Details on study design:

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2
- Exposure period: topical induction: 48h
- Site: interscapular region
- Frequency of applications: once intradermal, once topical

B. CHALLENGE EXPOSURE
- No. of exposure: 1
- Day(s) of challenge: 4 weeks between injection (induction) and challenge
- Exposure period: 24h
- Site: right flank (test item) and left flank (vehicle)
- Evaluation (hr after challenge): 24, 48 h after removal of dressing

Positive control substance(s):

not required

Remarks:

mercaptobenzothiazole tested in another study

Results and discussion

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

50%

No. with + reactions:

3

Total no. in group:

10

Clinical observations:

na

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 50%. No with. + reactions: 3.0. Total no. in groups: 10.0.

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

50%

No. with + reactions:

7

Total no. in group:

10

Clinical observations:

Dryness of the skin

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 50%. No with. + reactions: 7.0. Total no. in groups: 10.0. Clinical observations: Dryness of the skin.

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

50%

No. with + reactions:

20

Total no. in group:

20

Clinical observations:

Dryness of the skin, Edema

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 50%. No with. + reactions: 20.0. Total no. in groups: 20.0. Clinical observations: Dryness of the skin, Edema .

Results 4

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

50%

No. with + reactions:

19

Total no. in group:

20

Clinical observations:

Dryness of the skin, Edema, Masked reading by dryness in one animal

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50%. No with. + reactions: 19.0. Total no. in groups: 20.0. Clinical observations: Dryness of the skin, Edema, Masked reading by dryness in one animal .

Results 5

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

induction phase: 2.5% (w/w) intradermal route and 50% (w/w) cutaneous route Challenge phase : 50% (w/w) cutaneous route

No. with + reactions:

15

Total no. in group:

20

Remarks on result:

positive indication of skin sensitisation

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: other: CLP Regulation

Conclusions:

The test item induced delayed contact hypersensitivity in more than 30% of the animals.

According to the criteria of CLP Regulation, the test item should be classified as skin sensitizer (category 1 and sub-category 1B) and assigned the signal word "warning" and the hazard statement "H317: May cause an allergic skin reaction".

Executive summary:

The objective of this study was to evaluate the potential of the test item to induce delayed contact hypersensitivity in guinea pigs.

Methods

A preliminary test was first performed in order to determine the test item concentrations to be used in the main test.

In the main test, one treated group of ten males and ten females received the test item:

.   on day 1 by intradermal injections in the interscapular region at the concentration of 10%,

.   on day 8 by topical application to the clipped interscapular region at 75%,

.   on day 22 by topical application to the posterior right flank at 50%. The posterior left flank of the animals received the vehicle.

 

A control group of five males and five females received the vehicles:

.     corn oil on daythe interscapular region,

.     ethanol/drinking water treated by reverse osmosis (80/20) on daythe interscapular region,

.     acetone on day 22 to the posterior left flank. The posterior right flank received the test item at 50%.

 

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

.       Freund's Complete Adjuvant (FCA) diluted to 50% (v/v) with 0.9% NaCl,

.       test item in vehicle or vehicle alone,

.       test item in a mixture FCA/0.9% NaCl (50/50, w/w) or vehicle at 50% (w/v) in FCA/0.9% NaCl (50/50, v/v).

 

As in the preliminary test, the highest well-tolerated concentration was shown to be non‑irritant after topical application, 0.5 mL of sodium lauryl sulfate at 10% (w/w) in vaseline was applied to the induction site on day 7 in order to induce a local irritation.

 

On day 8, a filter paper (approximately 8 cm²) was fully-loaded with the dose formulations, and then applied to the clipped interscapular region, over the intradermal injection sites. The filter paper was held in place by an occlusive dressing for 48 hours. The control group animals received an application of the vehicle under the same experimental conditions. The presence of local irritation was checked (but not scored).

The induction phase was followed by a 14-day rest period.

 

On day 22, a Finn Chamber® filter paper was fully-loaded with the dose formulations. The chamber was held in contact with the skin by an occlusive dressing for 24 hours. The vehicle was applied to the left flank under the same experimental conditions. Cutaneous reactions were evaluated before treatment and 24 and 48 hours after removal of the dressing.

 

Each animal was observed once a day for mortality and clinical signs during the treatment and observation periods. Body weight was recorded on day 1 and at the end of each observation period.

On completion of the observation period, the animals were sacrificed then discarded without macroscopic post-mortem examination. For all animals, skin samples of the challenged application sites were preserved. If no histological examination is requested, the skin samples will be destroyed at the finalization of the study report. No microscopic examination was performed in first instance.

Results

No unscheduled deaths occurred during the main test.

No clinical signs indicative of systemic toxicity were observed in any animals.

The mean body weight of test item-treated animals was considered unaffected by the treatment with the test item.

 

In the control group, at the 24- and 48-hour readings, no cutaneous reactions were observed at the left flank (treated with vehicle). However, a discrete erythema was noted at the right flank (treated with test item) of 3/10 animals at the 24-hour reading and of 7/10 animals at the 48-hour reading.

Moreover, at the 48-hour reading, dryness of the skin was observed at the right flank (treated with test item) of 3/10 animals.

In the test item-treated group, no local reaction was noted at left flank (treated with vehicle) at any reading time-point.

At the 24-hour reading, a discrete erythema was observed in 3/20 animals at the right flank (treated with test item) associated with dryness of the skin in one of them. A moderate or intense erythema was noted in 17/20 animals, associated with edema for 13 of them and/or with dryness of the skin for 8 of them.

At the 48-hour reading,a discrete erythema was observed at the right flank (treated with test item) of 2/20 animals associated with dryness of the skin for one of them. A moderate or intense erythema associated with dryness was noted in 17/20 animals. In addition, oedema was observed for 12 of them. In one animal, the cutaneous reaction was masked by dryness of the skin.

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

Conclusion

The test item induced delayed contact hypersensitivity in more than 30% of the animals.

 

According to the criteria of CLP Regulation,the test item should be classified as skin sensitizer (category 1 and sub-category 1B) and assigned the signal word “warning” and the hazard statement “H317: May cause an allergic skin reaction”.