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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 January 2006 - 18 May 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report date:
2006

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
yes
Remarks:
please refer to "Principles of method if other than guideline" for further information
Principles of method if other than guideline:
1) Due to cleaning in the animal room, the humidity deviated from the upper limit (80%) of the acceptable range stated in the Protocol on 18 days in total (90% at maximum). Since these deviations were temporal and there were no abnormalities in clinical condition of animals, it was judged that they did not adversely affect the study results.

2) From around 12:30 on February 3, 2006 to around 9:00 on February 4, 2006, no data of temperature and humidity in the animal room were recorded for approximately 20.5 hours due to contact failure between the cartridge pen holder and chart of thermohygrograph (recorder). However, during this period, there were no abnormalities in the other animal rooms in the same facility. In addition, since no abnormal clinical signs were observed in animals at the time of finding or on that day, it was at least considered to have no effect on study results.

Otherwise there were no environmental factors that might have affected the reliability of the study results.
GLP compliance:
yes (incl. QA statement)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The study was conducted before the LLNA method became the preferred study-type for skin sensitisation testing.

Test material

Constituent 1
Chemical structure
Reference substance name:
(2S)-N,N'-dibutyl-2-(2-ethylhexanamido)pentanediamide
EC Number:
700-664-8
Cas Number:
486455-65-6
Molecular formula:
C21H41N3O3
IUPAC Name:
(2S)-N,N'-dibutyl-2-(2-ethylhexanamido)pentanediamide

In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Japan Laboratory Animals Inc
- Age at study initiation: 6 or 7 weeks of age
- Weight at study initiation: individual body weights were in the range of 276 to 435 g.
- Housing: The animals were housed individually or in groups of 2 animals in stainless-steel wire-mesh cages (W 266 × D 266 × H 200 mm: Riko Denki Co., Ltd.)
- Diet (e.g. ad libitum): RC4 pelleted diet (Oriental Yeast Co., Ltd.) using stainless-steel feeders available ad libitum
- Water (e.g. ad libitum): Tap water (Fujimi Water Union) via an automatic water supplying system available ad libitum
- Acclimation period: 20 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 23
- Humidity (%): 35 to 90
- Air changes (per hr): 11 to 14
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 26th January 2006 To: 11th May 2006

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
other: Intradermal induction: Paraffin. Topical induction and challenge: Polyethylene glycol 300
Concentration / amount:
Preliminary Study 1: 0.5, 1, 5, 10 % in vehicle
Preliminary Study 2: 10, 25, 50 % in vehicle
Preliminary Study 3: 1, 2.5, 5, 10, 25 % in vehicle

Main Test

Intradermal Induction: 1 and 2 % in vehicle
Topical Induction: 25 % in vehicle
Challenge: 5 and 10 % in vehicle

Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: Intradermal induction: Paraffin. Topical induction and challenge: Polyethylene glycol 300
Concentration / amount:
Preliminary Study 1: 0.5, 1, 5, 10 % in vehicle
Preliminary Study 2: 10, 25, 50 % in vehicle
Preliminary Study 3: 1, 2.5, 5, 10, 25 % in vehicle

Main Test

Intradermal Induction: 1 and 2 % in vehicle
Topical Induction: 25 % in vehicle
Challenge: 5 and 10 % in vehicle

No. of animals per dose:
Test article sensitization group: 10
Control group: 5
Preliminary studies: 6
Details on study design:
RANGE FINDING TESTS:

1) Preliminary Study 1 (for selection of dose concentrations for intradermal induction)

Two animals without abnormalities (Animal numbers: 1 and 2) were selected, fur of the dorsal skin on the scapula was clipped and shaved, and 0.1 mL of each test article formulation at various concentrations (10, 5, 1 and 0.5 w/w%) was injected intradermally to the 4 application sites on the head of the animal

Since intradermal injection was not possible at dose concentration of 10 w/w%, administration was done only at three concentrations (5, 1 and 0.5 w/w%). For the concentration of 5 w/w%, administration was associated with erythema of score 2 at 24 hours after administration in 2/2 animals and of score 3 at 48 hours after administration in 2/2 animals and one animal developed necrosis at the observation at 48 hours after administration. For the concentrations of 1 and 0.5 w/w%, erythema of score 2 was observed in 2/2 animals both at 24 and 48 hours after administration; however, there was no necrosis. On the basis of the results, the dose concentration for intradermal induction was set at 1 w/w%, the maximum concentration not causing necrosis.

2) Preliminary Study 2 (for selection of dose concentrations for topical induction)

Another two animals without abnormalities (Animal numbers: 3 and 4) were selected, fur of the left flank (3 × 7 cm area) was clipped and shaved, and the test formulations at various concentrations, 2 concentrations (25 and 10 w/w%) per animal, were applied for topical induction. For the application, 0.1 mL of each test article formulation was put on a patch of 1.5 cm in diameter (application side: lint sheet, Nishio Eisei Zairyo Co., Ltd.; cover side: Nichiyu Linseed Oil Paper; Nichiyu Co., Ltd.; 3M Reston® Self-Adhering Foam Pad, 3M Health Care), and the patch was applied to the application sites (located on the left side of the torso) and covered occlusively with a polyethylene film tape (3M Transpore TM Surgical Tape, 3M Health Care). The patch was removed 24 hours after application, and the application sites were cleaned with absorbent cotton soaked with polyethylene glycol 300 (Lot number: SDG7670).

Application at each concentration of 25 or 10 w/w% was associated with no skin reactions at 24 or 48 hours after removal of application. Therefore, 25 w/w%, the maximum concentration at which the application was possible, was selected for topical induction.

3) Preliminary Study 3 (for selection of dose concentration for challenge)

Two other animals without abnormalities (Animal numbers: 5 and 6) were selected, fur of the dorsal skin on the scapula (2 × 4 cm area) was clipped and shaved, and 0.1 mL of 1:1 emulsion of physiological saline and FCA was injected intradermaly to each of the 4 corners of the area. After 7 days, fur of the left and right flanks (3 × 7 cm areas) was clipped and shaved, and the test formulations at 5 concentrations (25, 10, 5, 2.5 and 1 w/w%) were applied. The 25% and 10% applications were on the left flank of the torso, whilst the 5%, 2.5% amd 1% applications were on the right flank. For application, 0.1 mL of each test article formulation was put on a patch of 1.5 cm in diameter (application side: lint sheet, Nishio Eisei Zairyo Co., Ltd.; cover side: Nichiyu Linseed Oil Paper; Nichiyu Co., Ltd.; 3M Reston® Self-Adhering Foam Pad, 3M Health Care), and the patch was applied to the application sites and covered occlusively with a polyethylene film tape (3M Transpore TM Surgical Tape, 3M Health Care). The patch was removed 24 hours after application, and the application sites were cleaned with absorbent cotton soaked with polyethylene glycol 300 (Lot number: SDG7670).

Application at 25 w/w% was associated with erythema of score 1 in 2/2 animals at 24 hours after removal of patches, while application at 10, 5, 2.5 or 1 w/w% was associated with no skin reactions in the observation at 24 or 48 hours after removal of patches. Therefore, 10 w/w%, the maximum no-irritation concentration, and 5 w/w% (half of 10 w/w%), were selected for challenge exposure. At 25 w/w%, skin reactions tended to be severer than in the preliminary study 2 (for selection of concentration for topical induction). Although the reason was unclear, administration of adjuvant might have increased the sensitivity to irritations.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 1 (to both intradermal and topical sites)
- Exposure period: 48 hours
- Test groups: Induction site A (intradermal): A 1:1 emulsion of physiological saline and FCA
Induction site B (intradermal): 1 w/w% Test article formulation
Induction site C (intradermal): A 1:1 emulsion of 2 w/w% test article formulation in FCA and physiological saline
Induction site D (topical): 25 w/w% Test article formulation
- Control groups: Induction site A (intradermal): A 1:1 emulsion of physiological saline and FCA
Induction site B (intradermal): Liquid paraffin
Induction site C (intradermal): A 1:1 emulsion of physiological saline and FCA
Induction site D (topical): Polyethylene glycol 300
- Sites: Sites A, B and C were located on both sides of the animal, close to the head, above the scapula. Site D is the between the other 6 sites.


B. CHALLENGE EXPOSURE
- No. of exposures: 1. No rechallenges required.
- Day(s) of challenge: 21
- Exposure period: 24 hours
- Test group: Animals number 1-10 (1101 - 1110) were the test group
- Control group: Animals 1 - 5 (2101 - 2105) were the control group
- Site: Left flank of the animal. Each animal had 3 sites, E, F and G. Animals 1, 4, 7 and 10 had the 10% test substance formulation at site E (closest to head), 5% substance formulation at site F and Polyethylene glycol 300 at site G (closest to tail). Animals 2, 5 and 8 had 5% substance formulation at site E, Polyethylene glycol 300 at site F and 10% test substance formulation at site G. Animals 3, 6 and 9 had Polyethylene glycol 300 at site E, 10% test substance formulation at site F and 5% substance formulation at site G.
- Evaluation (hr after challenge): 0, 24 and 48 hours after removal of patch.
Positive control substance(s):
yes
Remarks:
mercaptobenzothiazole (CAS No 149-30-4)

Results and discussion

Positive control results:
Historial data showed a positive result from the positive control substance.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
10 w/w% test article
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No abnormal findings
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 10 w/w% test article. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No abnormal findings.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
5 w/w% test article
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No abnormal findings
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 5 w/w% test article. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No abnormal findings.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
0 w/w% (PEG300 vehicle only)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No abnormal findings
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 0 w/w% (PEG300 vehicle only). No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No abnormal findings.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
10 w/w% test article
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
No abnormal findings
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 10 w/w% test article. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: No abnormal findings.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
5 w/w% test article
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
No abnormal findings
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 5 w/w% test article. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: No abnormal findings.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0 w/w% (PEG300 vehicle only)
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
No abnormal findings
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 0 w/w% (PEG300 vehicle only). No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: No abnormal findings.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
10 w/w% test article
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No abnormal findings
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 10 w/w% test article. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No abnormal findings.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
5 w/w% test article
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No abnormal findings
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 5 w/w% test article. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No abnormal findings.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
0 w/w% (PEG300 vehicle only)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No abnormal findings
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 0 w/w% (PEG300 vehicle only). No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No abnormal findings.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
10 w/w% test article
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
No abnormal findings
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 10 w/w% test article. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: No abnormal findings.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
5 w/w% test article
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
No abnormal findings
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 5 w/w% test article. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: No abnormal findings.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0 w/w% (PEG300 vehicle only)
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
No abnormal findings
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 0 w/w% (PEG300 vehicle only). No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: No abnormal findings.
Group:
positive control
Remarks on result:
positive indication of skin sensitisation

Any other information on results incl. tables

Skin Reactions at Intradermal Induction

Observation was done 24 hours after intradermal injection.

1) Test Article Sensitization Group

At the injection site (site A) of 1:1 emulsion of physiological saline and FCA, erythema and edema of score 3 were observed at right and left sites in all animals (10/10 animals). The mean score was 3.0 each for right and left sites.

For the injection site of 1 w/w% test formulation (site B), erythema of score 1 or 2 was observed in all animals in right and left sites. The mean score was 1.5 on left side and 1.6 on right side.

At the injection site (site C) of 1:1 emulsion of 2 w/w% test article suspension which was suspended in FCA and physiological saline, erythema and edema of score 3 was observed at right and left sites in all animals. The mean score was 3.0 at every injection site.

Given the above, since there was no necrosis at injection sites of the test article (sites B and C), the set concentrations were considered to be appropriate.

2) Control Group

At the injection areas of 1:1 emulsion of physiological saline and FCA, all animals (5/5 animals) developed erythema and edema of score 3 at both sites A and C on right and left sides. The mean score was 3.0 each at site A and C on right and left sides.

For the injection site of liquid paraffin (site B), erythema of score 1 was observed at right and left injection sites in all animals. The mean score was 1.0 at every injection area of both sides.

Skin Reactions at Topical Induction

In the test article sensitization group, at topical induction sites (substance for induction: 25 w/w% test formulation), erythema of score 1 was observed in all (10/10) animals immediately after the removal of application. The mean score was 1.0.

In the control group, erythema of score 1 was also observed in all (5/5) animals at topical induction sites (substance for induction: polyethylene glycol 300) immediately after the removal of application. The mean score was 1.0.

These skin reactions were considered to involve some effects of the petrolatum containing 10% SLS which was applied on the day before topical induction. The procedure for induction was judged to be appropriate since mild irritation reactions were observed in all animals at the area of topical induction.

Skin Sensitization

1) Test Article Sensitization Group

At the site of challenge exposure to test article at 10 and 5 w/w%, no skin reactions were observed at 24 or 48 hours after removal of challenge application in any of the 10 animals.

Mean score was 0 at all observation times and the positive reaction rate was 0% at concentrations of 10 and 5 w/w%.

At the site of challenge exposure to polyethylene glycol 300 (vehicle), no skin reactions were observed at 24 or 48 hours after removal of challenge application. Mean score was 0 at all observation times and the positive reaction rate was 0%.

2) Control Group

Similarly to the test article sensitization group, at the site of challenge exposure to the test article, no skin reactions were observed at 24 or 48 hours after removal of challenge application at concentrations of 10 or 5 w/w% in any of the 5 animals. Mean score was 0 at all observation times and the positive reaction rate was 0% at concentrations of 10 and 5 w/w%.

At the site of challenge exposure to polyethylene glycol 300 (vehicle), no skin reactions were observed at 24 or 48 hours after removal of challenge application. Mean score was 0 at all observation times and the positive reaction rate was 0%.

Clinical Signs

There were no abnormal clinical signs in any animal in any group during the observation period.

Body Weight

On Day 24, the final day of observation, 5/10 animals in the test article sensitization group and 2/5 animals in the control group showed a decrease in body weight in comparison with the previous value (Day 21). The degree of the decrease was 2 to 12 g, but there were no abnormalities in the clinical observation in any animal. The decreases in body weight were judged to be caused mainly by the procedure for occlusive application for challenge, and not at least to be test article-related.

Applicant's summary and conclusion

Interpretation of results:
other: Not classified according to EU criteria
Conclusions:
In the test article sensitization group, no skin reactions were observed at 24 or 48 hours after removal of challenge application in any animals at concentrations of 10 or 5 w/w% of the test substance. The positive reaction rate was 0 % for both 10 and 5 w/w %. In the control group, no skin reactions were observed at the areas of challenge application at concentrations of 10 or 5 w/w %, and the positive reaction rate was 0 % for both concentrations. On the basis of the results in the test article sensitization group and the control group, it was judged that the the test substance had no skin sensitization potential. At the site of challenge application with polyethylene glycol 300, no skin reactions were observed in any group.
There were no test article-related abnormalities in clinical observation or body weight during the observation period in any group.
On the basis of the results described above, it was concluded that the test substance had no skin sensitization potential under the conditions of this study.
Executive summary:

A skin sensitization study was conducted in Hartley albino guinea pigs by the Maximization Test, in order to evaluate the skin sensitization potential of the test material. Two test groups were provided: a test article sensitization group (10 animals) and a control group (5 animals). In the test article sensitization group, intradermal induction was done at 1 w/w %, topical induction was done at 25 w/w%, and then challenge exposure was done at 10 and 5 w/w% and by polyethylene glycol 300, the vehicle. In the control group, intradermal induction was done by liquid paraffin, topical induction was done by polyethylene glycol 300, and then challenge exposure was done in the same manner as for the test article sensitization group. Observation of skin reactions was performed at 24 and 48 hours after removal of challenge application to determine any skin sensitization potential. The results are described in the following.

Skin Sensitization

In the test article sensitization group, no skin reactions were observed at 24 or 48 hours after removal of challenge application at concentrations of 10 or 5 w/w % of the test substance in any animal. The positive reaction rate was 0 % for both 10 and 5 w/w %. In the control group, no skin reactions were observed at the site of challenge application at concentrations of 10 or 5 w/w %, and the positive reaction rate was 0 % for both concentrations. At the area of challenge by polyethylene glycol 300, no skin reactions were observed in any group.

Clinical Signs and Body Weight

There were no test article-related abnormalities in clinical observation or body weight during the observation period in any group. On the basis of the results described above, it was concluded that the test material had no skin sensitization potential under the conditions of this study.