Registration Dossier

Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP, fully OECD 406 compliant.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
This is the scientific justification for performing the OECD Test No. 406 for Skin Sensitization, i.e. the Guinea Pig Maximization Test (GPMT), instead of the required OECD Test No. 429, i.c. Skin Sensitisation according to the Local Lymph Node Assay.
In recently published articles in peer reviewed journals, see reference list, it is clearly demonstrated that surfactants are more likely to give rise to false positives in the LLNA.
Consequently, in the evaluation of such substances for sensitizing properties the LLNA test is not an appropriate assay and would not represent an optimum use of test animals. It is therefore recommended that the Guinea Pig Maximisation Test (GPMT) is used instead. This is also supported by the TG OECD 406 " In addition, test substance classes or substances containing functional groups shown to act as potential con founders (Basketter et al., 2009) may necessitate the use of guinea pig tests".

References: can be supplied upon request, since field is too short

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Chemical Name in report: N-Alkyl "oleyl" N,N-bishydroxyethylamine
- Substance type: Organic
- Physical state:light yellow liquid
- Analytical purity:98.5% (CoA)
- Impurities (identity and concentrations): free amine 2.78%, moisture 0.14%
- Purity test date: 8th March 2005
- Lot/batch No.: 9194, Arkema filing number GRL 0004/05
- Expiration date of the lot/batch: Not stated
- Stability under test conditions:stable
- Storage condition of test material:in a well ventilated place at room temperature

In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, L'Arbresle, France.
- Age at study initiation: 1-2 months
- Weight at study initiation: males 361 +/- 18 g, females 339 +/- 25g
- Housing: Polycarbonate cages with stainless steel tops. Autoclaved sawdust bedding (SICA, Alfortville, France)
- Diet (e.g. ad libitum): ad libitum, 106 pelleted diet (SAFE, Villemoisson, Epiny-sur-Orge, France
- Water (e.g. ad libitum): ad libitum via polypropylene bottle. Water filtered by a FG Millipor membrane (0.22 micron)
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 30 - 70
- Air changes (per hr): approximately 12 cycles of filtered, no-recycled air
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 19th April 2005 To: 27th May 2005

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
other: corn oil for intradermal induction. topical induction ethanol/water (80/20). Acetone for the challenge phase.
Concentration / amount:
Intradermal induction injections:
Freunds complete adjuvant (FCA) diluted to 50% (v/v) with 0.9% NaCl (both groups)
Test substance at a concentration of 0.1% in corn oil (treated group) of corn oil vehicle alone (control group)
Test substance at the concentration of 0.1% in a mixture of FCA/0.9% NaCl (50/50 w/w) (treated group) or corn oil vehicle at a concentration of
50% (w/v) in a mixture of FCA/0.9% NaCl (50/50, v/v)
Topical induction: 10% of the test substance in ethanol/water (80/20).
Challenge dosing:
1% (w/w) test substance in acetone
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: corn oil for intradermal induction. topical induction ethanol/water (80/20). Acetone for the challenge phase.
Concentration / amount:
Intradermal induction injections:
Freunds complete adjuvant (FCA) diluted to 50% (v/v) with 0.9% NaCl (both groups)
Test substance at a concentration of 0.1% in corn oil (treated group) of corn oil vehicle alone (control group)
Test substance at the concentration of 0.1% in a mixture of FCA/0.9% NaCl (50/50 w/w) (treated group) or corn oil vehicle at a concentration of
50% (w/v) in a mixture of FCA/0.9% NaCl (50/50, v/v)
Topical induction: 10% of the test substance in ethanol/water (80/20).
Challenge dosing:
1% (w/w) test substance in acetone
No. of animals per dose:
10 males and 10 females for the vehicle control group
20 males and 20 females for the test substance group
Details on study design:
MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: Intradermal injection single exposure at six injection points. Cutaneous single application on day 8.
- Exposure period: For cutaneous application exposure was for 48 hours.
- Test groups: Backs of guinea pigs were clipped for an area of 4cm x 2cm in their interscapular area. 6 intradermal injections on day 1, 2 of FCA at 50 %( v/v) in 0.9% NaCl in the anterior part of the clipped area. 2 injections of the test substance at 0.1% (w/w) in corn oil in the middle of the area. 2 injections of the test substance at 0.1% (w/w) in a mixture of FCA/0.9% NaCl (50/50).
No topical application of sodium lauryl sulfate was necessary on day 7 due to test substance being irritant. On day 8 all test animals were exposed to 10% (w/w) test substance fully loaded on a 8cm2 filter paper applied under an occlusive dressing on the intrescapular area for 48 hours.
- Control group:
- Site: The clipped intrescapular are of the back
- Frequency of applications: Intradermal injections on day 1 followed by cutaneous application on day 8.
- Duration: Cutaneous application 48 hours
- Concentrations: For the intradermal injection 6 intradermal injections on day 1, 2 of FCA at 50 %( v/v) in 0.9% NaCl in the anterior part of the clipped area. 2 injections of the corn oil vehicle in the middle of the area. 2 injections of a mixture of FCA/0.9% NaCl (50/50).
On day 8 all animals were exposed to an 8cm2 filter paper fully loaded with the ethanol/water (80/20) vehicle applied under an occlusive dressing on the intrescapular area for 48 hours.

B. CHALLENGE EXPOSURE
- No. of exposures: On day 22 the treated and the test animals received a single application of the test substance and the vehicle control.
- Day(s) of challenge: Day 22
- Exposure period: 24 hours - Chamber held in contact with the skin by an adhesive anallergenic waterproof plaster.
- Test groups: The filter paper of a chamber (Finn Chamber) was full loaded with the test substance at a concentration of 1%(w/w) in acetone, applied to the shaved area of the posterior left flank. The acetone vehicle was applied under the same condition to the skin of the posterior left flank.
- Control group: They received the same challenge exposure with test substance and vehicle control as the test group.
- Site: The shaved left and right posterior flanks
- Concentrations: 1% test substance (w/w) in acetone or acetone vehicle control
- Evaluation (hr after challenge): 24 and 48 hours after removal of the dressing with the challenge application.

OTHER:
Positive control substance(s):
yes
Remarks:
Mercaptobenzthiazole

Results and discussion

Positive control results:
1% (w/w) Mercaptobenzothiazole was used for the intradermal induction and 20% (W/W) for the cutaneous induction on day 8. followed by 22% (w/w) on the day 22 challenge. The vehicle was corn oil. There were 5 control animals and 10 treated animals.
Under the experimental conditions based on the Magnusson and Kligman method, the mercaptobenzthiazole positive control induced a positive skin sensitisation in 80% (8/10) guinea pigs.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
corn oil intradermal and ethanol/water cutaneous
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No clinical signs
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: corn oil intradermal and ethanol/water cutaneous. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No clinical signs.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
Corn oil intradermal, ethanol/water cutaneous
No. with + reactions:
2
Total no. in group:
10
Clinical observations:
Discrete erythema (grade 1)
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: Corn oil intradermal, ethanol/water cutaneous. No with. + reactions: 2.0. Total no. in groups: 10.0. Clinical observations: Discrete erythema (grade 1).
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
0.1% intradermal and 10% for cutaneous induction
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No clinical signs
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 0.1% intradermal and 10% for cutaneous induction. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: No clinical signs.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
0.1% for intradermal and 10% for cutaneous induction
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No clinical signs
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 0.1% for intradermal and 10% for cutaneous induction. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: No clinical signs.

Any other information on results incl. tables

Preliminary Study
Administration by intradermal route
 Animal   Concentration of the   Scoring after treatment     
 number   test item % (w/w)   24 hours   48 hours   6 days 
 male 301   25 + FCA   N   N   - 
     25   N   N   - 
     10 + FCA   N   N   - 
     10   I   N   - 
     5 + FCA   I   N   - 
     5   I   N   - 
 female 302   25 + FCA   N   N   - 
     25   N   N   - 
     10 + FCA   N   N   - 
     10   N   N   - 
     5 + FCA   I   N   - 
     5   I   N   - 
 male 305   2.5 + FCA   I   N   - 
     2.5   I   N   - 
     1 + FCA   I   N   - 
     1   I   I   - 
     0.5 + FCA   I   I   - 
     0.5   LI   LI   - 
 female 306   2.5 + FCA   I   N   - 
     2.5   I   N   - 
     1 + FCA   I   N   - 
     1   I   I   - 
     0.5 + FCA   I   I   - 
     0.5   I   I   - 
 male 309   0.5 + FCA   I   I   A 
     0.5   LI   LI   I 
     0.25 + FCA   I   I   I 
     0.25   LI   LI   LI 
     0.1 + FCA   I   I   I 
     0.1   LI   LI   LI 
 female 310   0.5 + FCA   I   I   A 
     0.5   LI   LI   I 
     0.25 + FCA   I   I   A 
     0.25   LI   LI   LI 
     0.1 + FCA   I   I   I 
     0.1   LI   LI   LI 
FCA : mixture Freund's Complete Adjuvant/0.9% NaCl (50/50, v/v)
N : necrosis
I : irritation
LI : slight irritation
A : crusts
- : sacrificed animals
In order to respect the criteria for the selection of concentrations (the concentration
 should be well-tolerated systemically and locally, intradermal injections should 
cause moderate irritant effect but no necrosis or ulceration of the skin),
 the concentration chosen for the main study was 0.1% (w/w).

Application by cutaneous route
Results were as follows:
Under the conditions of the induction phase
 Animal   Concentration of the   Scoring after removal of the dressing       
 number   test item % (w/w)   24 hours   48 hours     
 male 307   25   2   2     
 female 308   10   1   1     
Under the conditions of the challenge phase
 Animal   Concentration of the       Scoring after removal of the dressing       
 number   test item %       24 hours   48 hours     
 male 301   100   RF   LA   -     
     50 (w/w)   LF   3/A   -     
 female 302   100   RF   LA   -     
     50 (w/w)   LF   LA   -     
 male 305   25 (w/w)   RF   LN   -     
     10 (w/w)   LF   2/Oe   -     
 female 306   25 (w/w)   RF   LN   -     
     10 (w/w)   LF   2/Oe   -     
 male 309   5 (w/w)   RF   2/Oe   2/A     
     2.5 (w/w)   LF   0   0     
 female 310   5 (w/w)   RF   2/A   3/A     
     2.5 (w/w)   LF   1   1     
 male 311   1 (w/w)   RF   0   0     
     0.5 (w/w)   LF   0   0     
 female 312   1 (w/w)   RF   0   0     
     0.5 (w/w)   LF   0   0     
             
RF : right flank
LF : left flank
LA : scoring masked by crusts
A : crusts
LN : scoring masked by necrosis
Oe : oedema
- : sacrificed animals
On removal of the dressing, no residual test item was observed.
In order to respect the criteria for the selection of concentrations (the concentrations should be 
well-tolerated systemically and locally, cutaneous application for the induction should cause at most 
weak or moderate skin reactions or be the maximal practicable concentration, cutaneous application 
for the challenge phase should be the highest concentration which does not cause irritant effect), the
 concentration chosen for the topical application of the induction phase (day 8) was 10% (w/w). 
For the challenge application (day 22), it was 1% (w/w).

Application by cutaneous route
Results were as follows:
Under the conditions of the induction phase
 Animal   Concentration of the   Scoring after removal of the dressing       
 number   test item % (w/w)   24 hours   48 hours     
 male 307   25   2   2     
 female 308   10   1   1     
Under the conditions of the challenge phase
 Animal   Concentration of the       Scoring after removal of the dressing       
 number   test item %       24 hours   48 hours     
 male 301   100   RF   LA   -     
     50 (w/w)   LF   3/A   -     
 female 302   100   RF   LA   -     
     50 (w/w)   LF   LA   -     
 male 305   25 (w/w)   RF   LN   -     
     10 (w/w)   LF   2/Oe   -     
 female 306   25 (w/w)   RF   LN   -     
     10 (w/w)   LF   2/Oe   -     
 male 309   5 (w/w)   RF   2/Oe   2/A     
     2.5 (w/w)   LF   0   0     
 female 310   5 (w/w)   RF   2/A   3/A     
     2.5 (w/w)   LF   1   1     
 male 311   1 (w/w)   RF   0   0     
     0.5 (w/w)   LF   0   0     
 female 312   1 (w/w)   RF   0   0     
     0.5 (w/w)   LF   0   0     
             
RF : right flank
LF : left flank
LA : scoring masked by crusts
A : crusts
LN : scoring masked by necrosis
Oe : oedema
- : sacrificed animals
On removal of the dressing, no residual test item was observed.
In order to respect the criteria for the selection of concentrations (the concentrations should be 
well-tolerated systemically and locally, cutaneous application for the induction should cause at most 
weak or moderate skin reactions or be the maximal practicable concentration, cutaneous application 
for the challenge phase should be the highest concentration which does not cause irritant effect), the
 concentration chosen for the topical application of the induction phase (day 8) was 10% (w/w). 
For the challenge application (day 22), it was 1% (w/w).

MAIN STUDY

 

The test substance was not soluble in 0.9% NaCl: two phases were observed.

The vehicle chosen for intradermal injections was corn oil: a solution was obtained at the concentration of 25%; the dosage form preparation at the concentration of 25% (w/w) passed freely through a needle and into the dermis.

For topical applications, the vehicles used were a mixture ethanol/water (80/20) for the induction phase and acetone for the challenge application: solutions obtained whatever the proportion.

 

Clinical examinations

Marked local reactions at the intradermal injection sites were noted in a few animals of the treated group, between days 21 to 25.

No systemic clinical signs and no mortality were observed during the study.

 

Body weight

The body weight change of treated animals was similar to that of controls.

Challenge phase - Scoring of cutaneous reactions
Scoring of skin reactions was as follows
         Control group       
 Sex   Animal   24 hours   48 hours     
     number   LF   RF   LF   RF 
 Male   81   0   0   0   0 
     82   0   0   0   0 
     83   0   0   0   1 
     84   0   0   0   0 
     85   0   0   0   0 
 Female   96   0   0   0   1 
     97   0   0   0   0 
     98   0   0   0   0 
     99   0   0   0   0 
     100   0   0   0   0 
           
LF : left flank (vehicle)
RF : right flank (test item at the concentration of 1% (w/w))
    Treated Group      
 Sex   Animal   24 hours   48 hours     
     number   LF   RF   LF   RF 
 Male   86   0   0   0   0 
     87   0   0   0   0 
     88   0   0   0   0 
     89   0   0   0   0 
     90   0   0   0   0 
     91   0   0   0   0 
     92   0   0   0   0 
     93   0   0   0   0 
     94   0   0   0   0 
     95   0    0    0    0  
 Female   101   0   0   0   0 
     102   0   0   0   0 
     103   0   0   0   0 
     104   0   0   0   0 
     105   0   0   0   0 
     106   0   0   0   0 
     107   0   0   0   0 
     108   0   0   0   0 
     109   0   0   0   0 
     110   0   0   0   0 
           
LF : left flank (vehicle)
RF : right flank (test item at the concentration of 1% (w/w))
On removal of the dressing, no residual test item was observed.
After the challenge application, a discrete erythema (grade 1) was observed in 2/10 animals 
of the control group at the 48-hour reading.
No cutaneous reactions were observed in the treated group.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
Under our experimental conditions and according to the maximization method of Magnusson and Kligman, the test substnace CECAJEL 210 (batch No. 9194) does not induce delayed contact hypersensitivity in guinea pigs.
Executive summary:

The potential of the test item (Z) -2,2’-(Octadec-9-enylimino) bisethanol CAS 13127-82-7 to induce delayed contact hypersensitivity was evaluated in guinea pigs according to the maximization method of Magnusson and Kligman and to OECD (No. 406, 17th July 1992) and EC (96/54/EEC, B.6, 30th July 1996) guidelines. The study was conducted in compliance with the principles of Good Laboratory Practice Regulations.

 

Methods

 

Thirty guinea pigs were allocated to two groups: a control group of five males and five females and a treated group of ten males and ten females. On day 1, three pairs of intradermal injections were performed in the interscapular region of all animals:

• Freund's complete adjuvant (FCA) diluted to 50% (v/v) with 0.9% NaCl (both groups),

• test item at the concentration of 0.1% in corn oil (treated group) or vehicle alone (control group),

• test item at the concentration of 0.1% in a mixture FCA/0.9% NaCl (50/50, w/w) (treated group) or vehicle at the concentration of 50% (w/v) in a mixture FCA/0.9% NaCl (50/50, v/v) (control group).

On day 8, the animals of the treated group received a topical application of the test item at the concentration of 10% (w/w) in ethanol/water (80/20) to the same test site, which was then covered by an occlusive dressing for 48 hours. The animals of the control group received an application of the vehicle under the same experimental conditions.

On day 22, all animals of both groups were challenged by a cutaneous application of the test item at the concentration of 1% (w/w) in acetone to the right flank. The test item was maintained under an occlusive dressing for 24 hours. The vehicle was applied to the left flank under the same experimental conditions. Skin reactions were evaluated approximately 24 and 48 hours after removal of the dressing.

At the end of the study, the animals were killed without examination of internal organs. No skin samples were taken from the challenge application sites. No histological examination was performed.

 

Results

No systemic clinical signs and no deaths were noted during the study. No relevant cutaneous reactions were observed after the challenge application.

 

Conclusion

Under the experimental conditions and according to the maximization method of Magnusson and Kligman, the test substance (Z) -2,2’-(Octadec-9-enylimino) bisethanol CAS 13127-82-7d oes not induce delayed contact hypersensitivity in guinea pigs.