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Description of key information

Skin sensitisation (OECD 406): not sensitising

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study with acceptable restrictions. Split Adjuvant Test due to limited solubility, but no positive control included.
Justification for type of information:
Study was undertaken prior to the acceptance and development of the LLNA test.
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
yes
Remarks:
no positive control substance included
GLP compliance:
yes
Type of study:
split adjuvant test
Justification for non-LLNA method:
Study was undertaken prior to the acceptance and development of the LLNA test.
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Olac, Great Britain
- Age at study initiation: 2 months
- Weight at study initiation: 320-412 g
- Housing: 4 per cage in metal cages with wire-mesh floors (RUCO, Valkenswaard). Distal parts of both rear feet were wrapped with adhesive tape (Sleek, Smith and Nephew, England) to prevent skin injuries.
- Diet: Standard guinea pig diet including ascorbic acid (1600 mg/kg), obtained from Hope Farms, Woerden (LC 23-B, pellet diameter 4 mm), availability not specified. In addition, once a week hay was provided.
- Water: Not specified.
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.5-21.0
- Humidity (%): 50-80
- Photoperiod (hrs dark / hrs light): 12/12
Route:
intradermal and epicutaneous
Vehicle:
corn oil
Remarks:
SAT: test substance in corn oil only applied epicutaneously, not injected
Concentration / amount:
Induction: 0.2 mL of 25% (w/v) on day 0 and 2; 5% (w/v) on day 4 and 7
Challenge: 50 µL of 25, 5, 1 and 0% (w/v) on day 21
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Remarks:
SAT: test substance in corn oil only applied epicutaneously, not injected
Concentration / amount:
Induction: 0.2 mL of 25% (w/v) on day 0 and 2; 5% (w/v) on day 4 and 7
Challenge: 50 µL of 25, 5, 1 and 0% (w/v) on day 21
No. of animals per dose:
20 females in test group, 10 in control group
Details on study design:
RANGE FINDING TESTS:
Before challenge phase a primary irritation test was carried out with 4 guinea pigs in order to find a concentration for challenge not inducing substantial irritation. Four guinea pigs were shaved and their left flank was exposed for 24 hours to 25, 10, 2.5 and 1% (w/v) of the test substance in corn oil under occlusive conditions; application was done in plastic Square chambers (v.d. Bend, Brielle, The Netherlands) provided with aluminium foil to avoid contact with the compound. Two animals died from bandage-induced stress, but the remaining animals showed no skin response to any of the tested concentrations.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 4
- Test groups: test substance in corn oil epicutaneously under occlusive conditions on day 0, 2, 4 and 7 (on day 0 treatment of skin with dry ice for 5 seconds prior to application); additionally 2 i.d. injections of 0.1 mL FCA on day 4 on either side of sensitisation area.
- Control group: 2 i.d. injections of 0.1 mL FCA on day 4 on either side of sensitisation area.
- Site: Shaved area of 2x2 cm on the back just behind the right shoulder girdle, injections were done on either side of the sensitisation area. The animals were wrapped in a "window dressing" consisting of Lenkelast elastic bandage (Lohmann, Neuwied, FRG) into which a 2x2 cm opening had previously been cut. This opening was placed over the sensitisation site. After applications the site was covered with a Metalline-patch (Lohmann), which was held in place by covering with water-impermeable tape (Sleek, Smith and Nephew, England).
- Frequency of applications: 2 days
- Duration: 8 days
- Concentrations: Test group: 0.2 mL of 25% (w/v) on day 0 and 2, and 5 % (w/v) on day 4 and 7

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 21
- Exposure period: 24 hours
- Test groups: test substance in corn oil under occlusive conditions
- Control group: test substance in corn oil under occlusive conditions
- Site: Application of test substance to the shaved left flank by patches consisting of 1 cm² filter paper (Schleicher & Schull), attached to a piece of aluminium foil with petrolatum, the aluminium foil being mounted on non-irritating Micropore tape (3M Co. St. Paul, USA). The animals were then wrapped in Coban elastic bandage (Smith and Nephew, England). The dressing was fixed in place for 24 hours.
- Concentrations: 50 µL of 25, 5, 1 and 0% (w/v)
- Evaluation (hr after challenge): 48 and 72 hours (24 and 48 hours after removal of the bandage and a close shave of the skin)
Challenge controls:
Control group also serves as challenge control, as injection of these animals with FCA alone on day 4 of the induction should not elicit any sensitivities.
Positive control substance(s):
no
Positive control results:
No positive control was included.
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
25%
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 20.0.
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
5%
No. with + reactions:
1
Total no. in group:
20
Clinical observations:
Moderate and diffuse reaction; two other animals demonstrated red spots (scattered reaction) which were not considered positive.
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 5%. No with. + reactions: 1.0. Total no. in groups: 20.0. Clinical observations: Moderate and diffuse reaction; two other animals demonstrated red spots (scattered reaction) which were not considered positive..
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
1%
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 1%. No with. + reactions: 0.0. Total no. in groups: 20.0.
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
0%
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 0%. No with. + reactions: 0.0. Total no. in groups: 20.0.
Reading:
2nd reading
Hours after challenge:
72
Group:
test group
Dose level:
25%
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 20.0.
Reading:
2nd reading
Hours after challenge:
72
Group:
test group
Dose level:
5%
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 5%. No with. + reactions: 0.0. Total no. in groups: 20.0.
Reading:
2nd reading
Hours after challenge:
72
Group:
test group
Dose level:
1%
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 1%. No with. + reactions: 0.0. Total no. in groups: 20.0.
Reading:
2nd reading
Hours after challenge:
72
Group:
test group
Dose level:
0%
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 0%. No with. + reactions: 0.0. Total no. in groups: 20.0.
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
25%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
5%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Two animals demonstrated red spots (scattered reaction), which were not considered positive.
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 5%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: Two animals demonstrated red spots (scattered reaction), which were not considered positive..
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
1%
No. with + reactions:
0
Total no. in group:
9
Clinical observations:
One animal demonstrated red spots (scattered reaction) which were not considered positive. A second one could not be evaluated due to reddening from shaving
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 1%. No with. + reactions: 0.0. Total no. in groups: 9.0. Clinical observations: One animal demonstrated red spots (scattered reaction) which were not considered positive. A second one could not be evaluated due to reddening from shaving.
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
9
Clinical observations:
One animal could not be evaluated due to reddening from shaving.
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 0%. No with. + reactions: 0.0. Total no. in groups: 9.0. Clinical observations: One animal could not be evaluated due to reddening from shaving..
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
25%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: negative control. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
5%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: negative control. Dose level: 5%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
1%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: negative control. Dose level: 1%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: negative control. Dose level: 0%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Interpretation of results:
not sensitising
Remarks:
Migrated information
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The skin sensitising potential of (Z)-N-octadec-9-enylhexadecan-1-amide (CAS 16260-09-6) was evaluated in a Split Adjuvant Test performed according to OECD 406 due to the low solubility of the test substance (Weterings, 1986). In the induction phase of the study, test substance in corn oil was applied epicutaneously to the skin of the 20 female Dunkin-Hartley guinea pigs of the test group under occlusive conditions on Days 0, 2, 4 and 7. The concentration of the test substance in vehicle was 25% (w/v) on Days 0 and 2 as well as 5% (w/v) on Days 4 and 7 of induction treatment. In addition, 2 intradermal injections of 0.1 mL Freund's Complete Adjuvant (FCA) were performed on Day 4 on either side of sensitisation area in both the treatment group and the control group (consisting of 10 female animals). For challenge exposure on Day 21, the test substance at 25, 5, 1 and 0% (w/v) concentration in corn oil was applied for 24 h to the skin of the left flank of treated and control animals. Skin reactions were evaluated 24 and 48 h after removal of the dressing. Except for one animal showing moderate and diffuse reaction after challenge treatment with 5% of the test substance in corn oil, no positive reactions were observed after challenge application in the other animals. Therefore, a sensitisation rate of 5% was obtained, which, however, did not fulfil the criteria for a positive result in this study. The reliability of the study was limited since no positive control was included in the assay. However, the animals of the negative control group, which actually were challenge controls, did not show skin reactions attributed to skin sensitisation. Under the conditions of this assay, it was concluded that the test substance was not sensitising to skin.


Justification for selection of skin sensitisation endpoint:
There is only one study available.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The available data on skin sensitisation of (Z)-N-octadec-9-enylhexadecan-1-amide (CAS 16260-09-6) do not meet the criteria for classification according to Regulation (EC) No 1272/2008 or Directive 67/548/EEC and are therefore conclusive but not sufficient for classification.