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Diss Factsheets

Administrative data

Description of key information

Two in vivo skin sensitisation studies (Guinea Pig Maximisation method) have been conducted on the test material. None of the animals exhibited a positive response.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

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
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.
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
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

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

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.

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.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
18 May 2004 - 18 June 2004
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The report does not state whether the study was conducted to GLP and does not provide any data on purity.
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
GLP compliance:
no
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.
Species:
guinea pig
Strain:
Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan, Inc. (795 Shimofurusawa, Atsugi, Kanagawa Prefecture)
- Age at study initiation: 4-5 weeks of age
- Weight at study initiation:
Test group - Mean: 333.4 g (SD: 6.5)
Control group - Mean: 322.9 g (SD: 14.1)
- Housing: The animals were accommodated individually or 2 animals in stainless-steel cages (W 285 x D 390 x H 185 cm: Tokiwa Kagaku Kikai Co., Ltd.)
- Diet: Animals were allowed free access to RC4 pellet diet (Oriental Yeast Co., Ltd.) using stainless-steel feeders
- Water: Animals were allowed free access to tap water (Kawasaki waterwork Bureau) with the addition of chlorine using an automatic water supplying system.
- Acclimation period: period of 5 days in the study environment (4 days for the animals used in the preliminary study). During the quarantine/acclimation period, the animals were observed for clinical signs once every day and weighed at least once a week.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 26°C
- Humidity (%): 30 to 70%
- Air changes (per hr): air ventilation at 10 to 15 times per hour
- Photoperiod (hrs dark / hrs light): 12-hour lighting (at 200-600 lux).

IN-LIFE DATES: From: May 19th 2004 To: June 18th 2004
Route:
intradermal and epicutaneous
Vehicle:
other: See "preparation of test article" in materials and methods section
Concentration / amount:
Main Study: Intradermal Induction - 5% (w/w) test article
Epidermal Induction - 25% (w/w) test article
Challenge - 5% and 10% (w/w) test article

Preliminary Study: Prepared concentrations were as follows: a total of 4 concentrations, 5, 1, 0.5 and 0.1 (w/w)% for intradermal induction (10(w/w)% suspension was technically inapplicable for intradermal injection. ); a total of 4 concentrations, 25, 10, 5 and 1 (w/w)% for epidermal induction; and a total of 4 concentrations, 25, 10, 5 and 2.5 (w/w)%, for challenge.

Route:
intradermal and epicutaneous
Vehicle:
other: See "preparation of test article" in materials and methods section
Concentration / amount:
Main Study: Intradermal Induction - 5% (w/w) test article
Epidermal Induction - 25% (w/w) test article
Challenge - 5% and 10% (w/w) test article

Preliminary Study: Prepared concentrations were as follows: a total of 4 concentrations, 5, 1, 0.5 and 0.1 (w/w)% for intradermal induction (10(w/w)% suspension was technically inapplicable for intradermal injection. ); a total of 4 concentrations, 25, 10, 5 and 1 (w/w)% for epidermal induction; and a total of 4 concentrations, 25, 10, 5 and 2.5 (w/w)%, for challenge.

No. of animals per dose:
Main Study: 15 animals (10 test material; 5 control group)

Preliminary Study: 6 animals
Details on study design:
RANGE FINDING TESTS:

- Intradermal Induction
Two animals without abnormalities were selected, approximately 4 x 6 cm area of the dorsal skin from the scapular region was clipped and shaved, and 0.1 mL of each test suspension (4 concentrations at 5, 1, 0.5 and 0.1 (w/w) % suspension) was intradermal administrated.

- Epidermal Induction
Two animals without abnormalities were selected, and an area of both flanks skin of each were clipped and shaved. A patch of 2 x 2 cm (application side: lint sheet, Yabane Co., Ltd.; cover side: Dermicel, Johnson & Johnson Medical) was saturated with 0.2 mL of each test suspension (4 concentrations at 25, 10, 5 and 1 (w/w)% suspension) and applied to the shaved skin and covered occlusively with an elastic tape (Elastikon, Johnson & Johnson Medical). The patch was removed 48 hours after application.

- Challenge
Four animals without abnormalities were selected. On the day before intradermal induction, approximately 4 x 6 cm area of dorsal skin from the scapular region was clipped and shaved. On the next day, emulsion (1:1) of FCA and physiological saline (A), liquid paraffin (B), and emulsion (1:1) of liquid paraffin FCA and physiological saline (C) was intradermal administered (0.1 mL / site) within the 2 x 4 cm area.

After 6 days, the same area was clipped and shaved and vaseline containing 10% sodium lauryl sulfate was applied. On the next day, a patch of 2 x 4 cm (application side: lint sheet, Yabane Co., Ltd.; cover side: Dermicel, Johnson & Johnson Medical) was saturated with 0.5 mL of PEG300 and placed over the injection sites and covered occlusively with a plastic film tape (Plasupport, HOGY medical Co., Ltd.).
The patch was removed 48 hours after application.
After 6 days, fur of both flanks (approximately 5 x 5 cm) were clipped with an electric clipper and further shaved with an electric shaver as closely as possible. On the next day, four patches of 2 x 2 cm (application side: lint sheet, Yabane Co., Ltd.; cover side: Dermicel, Johnson & Johnson Medical) were saturated with 0.2 mL of each test suspension (4 concentrations at 25, 10, 5 and 2.5 (w/w)%) and applied to the clipped/shaved flank skin and covered occlusively with a bandage tape (Elastikon, Johnson & Johnson Medical). The patch was removed 24 hours after application.

- Observation and Scoring of Skin Reactions
Administration sites were observed for skin reactions just after administration and 4 hours after administration for intradermal injection. The patch application sites were observed immediately after patch removal and 24 hours after, respectively.

MAIN STUDY
A. INDUCTION EXPOSURE
- Intradermal Induction (Day 1)
On the day before intradermal induction, approximately 4 x 6 cm area of scapular region was clipped with an electric clipper (Natsume Seisakusho Co., Ltd.) and further shaved with an electric shaver (Braun) as closely as possible. On the next day, 0.1 mL of each substance was intradermal
administered to the Area 1 to 3 within the 2 x 4 cm area.

- Epidermal Induction (Day 8)
On 6 days after intradermal induction, the same area as the Day 1 was clipped and shaved and vaseline containing 10% sodium lauryl sulfate was applied thoroughly. On the next day, patch of 2 x 4 cm (application side: lint sheet, Yabane Co., Ltd.; cover side: Dermicel, Johnson & Johnson Medical) was saturated with 0.2 mL of each substance and applied to the induction area and covered occlusively with a plastic film tape (Plasupport, HOGY medical Co., Ltd.). The patch was removed 48 hours after application. (see table 5)

- Challenge (Day 22)
Twenty days after the intradermal induction, fur of the right flank (approximately 5 x 5 cm) of animals were clipped with an electric clipper and further shaved with an electric shaver as closely as possible.
On the next day, 3 sites for challenge were provided for each animal. The patch of 2 x 2 cm(application side: lint sheet, Yabane Co., Ltd.; cover side: Dermicel, Johnson & Johnson Medical) was saturated with 0.2 mL of each substance and was fixed in place with a bandage tape (Elastikon, Johnson & Johnson Medical) for 24 hours for occlusive application of challenge. (see table 6)
Challenge controls:
Yes, see tables 5 and 6 in materials and methods for more information
Positive control substance(s):
no
Positive control results:
no data
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
5 & 10% (w/w) test article
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No signs of systemic toxicity
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 5 & 10% (w/w) test article. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No signs of systemic toxicity.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
5 & 10% (w/w) test article
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No signs of systemic toxicity
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 5 & 10% (w/w) test article. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No signs of systemic toxicity.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
5 & 10% (w/w) test article
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
No signs of systemic toxicity
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 5 & 10% (w/w) test article. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: No signs of systemic toxicity.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
5 & 10% (w/w) test article
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
No signs of systemic toxicity
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 5 & 10% (w/w) test article. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: No signs of systemic toxicity.

Skin Reactions

The results of observation for skin reactions after challenge are shown in the below tables.

Skin reactions of individual animals (Challenge)

Test group Substance for intradermal induction (concentration) Substance for epidermal induction (concentration) Substance for challenge exposure (concentration) Animal No.
 Score
24 hours a) 48 hours a)
Test article sensitization group Test article (5%) Test article (25%) Test article (10%) 50201 0 0
50202 0 0
50203 0 0
50204 0 0
50205 0 0
50206 0 0
50207 0 0
50208 0 0
50209 0 0
50210 0 0
Mean score 0.0 0.0
Number of animals with positive reactions b) 0/10 0/10
Positive reaction rate (%) 0 0
Test article (5%) 50201 0 0
50202 0 0
50203 0 0
50204 0 0
50205 0 0
50206 0 0
50207 0 0
50208 0 0
50209 0 0
50210 0 0
Mean score 0.0 0.0
Number of animals with positive reactions b) 0/10 0/10
Positive reaction rate (%) 0 0
PEG300 50201 0 0
50202 0 0
50203 0 0
50204 0 0
50205 0 0
50206 0 0
50207 0 0
50208 0 0
50209 0 0
50210 0 0
Mean score 0.0 0.0
Number of animals with positive reactions b) 0/10 0/10
Positive reaction rate (%) 0 0

a): Hours after removal of the application for challenge exposure

b): The number of animals with individual score of 1 or above

Skin reactions of individual animals (Challenge)

Test group Substance for intradermal induction (concentration) Substance for epidermal induction (concentration) Substance for challenge exposure (concentration) Animal No.
 Score
24 hours a) 48 hours a)
Control group Liquid Paraffin PEG300 Test article (10%) 50211 0 0
50212 0 0
50213 0 0
50214 0 0
50215 0 0
Mean score 0.0 0.0
Number of animals with positive reactions b) 0/5 0/5
Positive reaction rate (%) 0 0
Test article (5%) 50211 0 0
50212 0 0
50213 0 0
50214 0 0
50215 0 0
Mean score 0.0 0.0
Number of animals with positive reactions b) 0/5 0/5
Positive reaction rate (%) 0 0
PEG300 50211 0 0
50212 0 0
50213 0 0
50214 0 0
50215 0 0
Mean score 0.0 0.0
Number of animals with positive reactions b) 0/5 0/5
Positive reaction rate (%) 0 0

a): Hours after removal of the application for challenge exposure

b): The number of animals with individual score of 1 or above

In the test article sensitization group, no skin reactions were observed at the site of application for challenge with 10% test suspension, 5% test suspension or PEG300, and thus mean score was 0 and the positive reaction rate was 0%. In the control group, no skin reactions were observed at the site of application for challenge with 10% test suspension, 5% test suspension or PEG300, and thus mean score was 0 and the positive reaction rate was 0%.

Clinical Signs: There were no signs of systemic toxicity in any group during the observation period.

Body Weight: 8 animals in the test article sensitization group and 2 animals in the control group at the challenge observation period (Day 24), 1 animal in the test article sensitization group and 1 animal in the control group at the challenge observation period (Day 25) showed a loss of body weight. These body weight loss might be an effect of occlusive application for challenge, thus it was judged not to be related to the test article, although the cause of decrease was unclear.

Necropsy: No necropsy was performed, since there were no deaths during the course of the study.

Interpretation of results:
other: Not classified according to EU criteria
Conclusions:
In the test article sensitization group, no skin reactions were observed at the site of application for challenge with 10% test suspension, 5% test suspension or PEG300, and thus mean score was 0 and the positive reaction rate was 0%.
Based on the above results, it was concluded that the test material at a concentration of 10% suspension and 5% suspension should not be classified as a sensitizer.
Executive summary:

A skin sensitization study was conducted in Hartley strain guinea pigs by the Guinea Pig Maximization Test method, in order to evaluate the skin sensitization potential of the test material. The concentration of the test article was 5 (w/w) % for intradermal induction, 25 (w/w)% for epidermal induction, 10 and 5 (w/w)% for challenge.

Based on the results, it is concluded that the test material has no skin sensitization potential under the conditions of this study.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Key Study

A skin sensitisation 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 Sensitisation

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.

Supporting Study

A skin sensitisation study was conducted in Hartley strain guinea pigs by the Guinea Pig Maximization Test method, in order to evaluate the skin sensitization potential of the test material. The concentration of the test article was 5 (w/w) % for intradermal induction, 25 (w/w)% for epidermal induction, 10 and 5 (w/w)% for challenge.

Based on the results, it is concluded that the test material has no skin sensitization potential under the conditions of this study.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

By reference to the data summarised within this dataset, the substance does not meet the criteria for classification as a skin sensitiser according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.