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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:
Start of study: October 25 2001; End of study: February 7 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report date:
2002

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Remarks:
No deviation that affected the study reliability
GLP compliance:
yes (incl. QA statement)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
A study was conducted in 2002 according to OECD 406. This study was conducted before suitable In Vitro alternatives were available.

Test material

Constituent 1
Chemical structure
Reference substance name:
Xanthylium, 3,6-bis(ethylamino)-9-[2-(methoxycarbonyl)phenyl]-2,7-dimethyl-, molybdatetungstatephosphate
EC Number:
289-056-7
EC Name:
Xanthylium, 3,6-bis(ethylamino)-9-[2-(methoxycarbonyl)phenyl]-2,7-dimethyl-, molybdatetungstatephosphate
Cas Number:
85959-61-1
Molecular formula:
C162H180N12O18Cl6+O3.P2O5.xWO3.yMoO3
IUPAC Name:
heptakis(N-[(3E)-6-(ethylamino)-9-[2-(methoxycarbonyl)phenyl]-2,7-dimethyl-3H-xanthen-3-ylidene]ethan-1-aminium) dioxomolybdenumbis(olate) dioxotungstenbis(olate) phosphate
Test material form:
other: Pasty pigment containing water
Details on test material:
Name: BZ-0145B
Lot Number: 9302W
Purity: 100% (solid content 32.6%, and the remainder water)
Storage conditions: In a cool and dark place
Expiration: March 31, 2002

In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
female
Details on test animals and environmental conditions:
Hartley-strain white guinea pigs (48 females) were purchased from Japan Laboratory Animals, Inc. at 5 weeks of age and subjected to quarantine and acclimation to the study environment for 12 days (8 days for the animals used in the preliminary study). During this period, they were observed for general condition every day and weighed at least once a week. Thirty animals that showed regular body weight development without clinical abnormalities were selected and used in the main study. They were at 6 or 7 weeks of age and the body weights ranged from 334 to 431 g on the starting day of induction for senisitzation.

Animals were housed in an animal room in which the temperature was kept at 22.5 ± 3.5°C, relative humidity at 50 ± 20%, air ventilation at 10 to 15 times per hour, and 12-hour lighting per day (from 07:00 to 19:00). The animals were put in stainless wire-mesh cages individually or in groups of 2 animals, and allowed free access to pelleted diet RC4 and water using an automatic watering system.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
water
Remarks:
(for injection)
Concentration / amount:
Main Test:
Intradermal induction: 5%
Percutaneous induction: 25%
Challenge exposure: 5%
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
water
Remarks:
(for injection)
Concentration / amount:
Main Test:
Intradermal induction: 5%
Percutaneous induction: 25%
Challenge exposure: 5%
No. of animals per dose:
Main Test:
Test article sensitization group: 10
Positive control sensitization group: 5
Control group: 5
Details on study design:
PRELIMINARY STUDY TO DETERMINE TO DOSE CONCENTRATIONS OF TEST SOLUTION:
During the quarantine acclimation period, a preliminary study was conducted using some purchased animals to determine the dose concentrations of the test solution for intradermal induction, percutaneous induction and challenge exposure. For intradermal induction, 4 animals with no abnormalities were selected and the test solutions at various concentrations (each 0.1 rnL) administered intradermally to the skin above the scapula where hr was clipped and shaved. For percutaneous application, another 8 animals without abnormalities were selected. Test solution at each dose concentration (0/1 mL) was applied to the patch of 2 x 2 cm and the patch applied to the 8 animals. It was covered with polyethylene film tape for occlusive application. After 24 hours of application, the patches were removed and the application site cleaned using absorbent cotton soaked with water for injection. For both intradermal application and percutaneous application, skin reactions were judged (according to standards of Magnusson & Kligman: 1969) and the dose concentration for each application selected.

The results obtained in the preliminary study are shown below:
Test article BZ-0145B:
For intradermal application, administration was done at 25, 10, 5, 2.5 and 1% to 2 animals. Erythema and edema of score 3 with necrosis were observed at the concentrations of 10% and above. At the concentration of 5% or below, erythema of score 2 was observed in both animals.
For percutaneous application, administration was done at 25, 10, 5, 2.5 and 1% to 4 animals. Erythema of score 1 was observed at the concentrations of 25 and 10%. At the concentration of 5% or below, no skin reactions were observed in any animal.
Based on the results of the preliminary study described above, the following concentrations were selected for the main study: 5%, the maximum irritating concentration without necrosis, for intradermal induction; 25%, the maximum preparable concentration for percutaneous induction; 5%, the maximum non-irritating concentration, for challenge exposure.

MAIN TEST:
GROUP COMPOSITION:
Test groups were composed as follows:
Test article sensitization group (10 animals)
Positive control sensitization group (5 animals)
Control group (5 animals).

DESIGNATION OF STUDY DAYS:
Starting day of induction for sensitization (day of intradermal induction): Day 0 of induction
The day following intradermal induction and thereafter: Day 1 after induction, Day 2 after induction etc.

METHOD OF TREATMENT:
1) SENSITIZATION:
Intradermal induction for sensitization:
On the day before intradermal induction for sensitization, fur of the 4 x 6 cm area on the scapula was clipped using an electric clipper and shaved as short as possible using an electric shaver. On the next day, for all animals in all test groups, 0.1 mL of the substance for intradermal induction (shown in table of 'Substance for Induction' - see any other information on material and methods) was applied to the relevant areas of induction.

Percutaneous induction for sensitization:
Six days after induction for sensitization, the same area was clipped and shaved again, and 0.2 mL of petrolatum containing 10% sodium lauryl sulfate was applied open to the area (2 x 4 cm) for intradermal induction for sensitization for the animals in the positive control sensitization group and the control group. On the next day, the area was cleaned with absorbent cotton soaked with ether, 0.2 mL of the substance for percutaneous induction (shown in table of 'Substance for Induction' - see any other information on material and methods) applied to the area of induction using a patch of 2 x 4 cm, which was covered with polyethylene film tape for 48-hour occlusive application for percutaneous induction of sensitization. For the test article group, sodium lauryl sulfate was not applied since percutaneous induction was done at the concentration causing irritation.
For the control group, water for injection (the vehicle) was used in the same manner as for the sensitization groups.

2) CHALLENGE EXPOSURE:
On Day 20 after induction, the fur of the right and left abdominal flanks (area: 4 x 7 cm) of each animal in each group was clipped and shaved. On the next day, 0.1 mL of the substance for challenge exposure (shown in the table of 'Substance for Challenge' - see any other information on material and methods) was applied to a patch of 2 x 2 cm, which was applied to the application site. It was then covered with polyethylene film tape for 24-hour occlusive application. After the 24-hour application, the patch was removed and the application site cleaned using absorbent cotton soaked with water for injection.

OBSERVATION OF GENERAL CONDITION:
The animals were observed for general condition once a day from the starting day of induction for sensitization (Day 0) to the ending day of skin observation after challenge exposure (Day 24).

MEASUREMENT OF BODY WEIGHT:
The animals were weighed on the starting day of induction for sensitization (Day 0), day of percutaneous induction (Day 7), day of challenge exposure (Day 21) and ending day of observation (Day 24).

OBSERVATION OF SKIN REACTIONS:
Skin reactions were observed at 24 and 48 hours after removal of application for challenge exposure and the reactions observed were scored according the the evaluation table of skin reactions below:

Severity of skin reactions: Score:
No gross change 0
Sporadic or macular erythema 1
Moderate or diffuse erythema 2
Strong erythema and edema 3

After the end of the observation, the animals were euthanized by deep ether anesthesia.

EVALUATION:
Individual scores obtained according to the evaluation table were totaled, the mean score for each test group at each observation time calculated, and the positive sensitization rate calculated from the changes of at least score 1. The sensitizing potential of the test article was evaluated by comparing the group mean scores and the positive rates in each sensitization group and the control group.






Challenge controls:
See details on study design section.
Positive control substance(s):
yes
Remarks:
Mercaptobenzothiazole (MBT)

Results and discussion

Positive control results:
In the positive control sensitization group, all animals (5/5) showed erythema and edema of scores 1 to 3 in the site of challenge exposure to 15% MBT solution. The mean score was 2.4 each for 24 and 48 hours after removal of challenge application and the positive rate 100%.
In the site of challenge exposure to liquid paraffin, there were no skin reactions.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
5% 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% 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% 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% 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:
Water (vehicle)
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: Water (vehicle). 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:
Water (vehicle)
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: Water (vehicle). 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:
5% 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% 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% 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% 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:
15% MBT
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: 15% MBT. 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:
15% MBT
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: 15% MBT. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: No abnormal findings.
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
15% MBT
No. with + reactions:
5
Total no. in group:
5
Clinical observations:
No abnormal findings
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: 15% MBT. No with. + reactions: 5.0. Total no. in groups: 5.0. Clinical observations: No abnormal findings.
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
15% MBT
No. with + reactions:
5
Total no. in group:
5
Clinical observations:
No abnormal findings
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: positive control. Dose level: 15% MBT. No with. + reactions: 5.0. Total no. in groups: 5.0. Clinical observations: No abnormal findings.
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
Liquid paraffin
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: positive control. Dose level: Liquid paraffin. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: No abnormal findings.
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
Liquid paraffin
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: positive control. Dose level: Liquid paraffin. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: No abnormal findings.

Any other information on results incl. tables

Skin Sensitization Potential:

Results are shown in Tables 1 and 2 (see attached background material).

In the test article sensitization group, there were no skin reactions in the observation of the site of challenge exposure to the 5% solution of BZ-0145B after challenge exposure, the mean scores at 24 and 48 hours after removal of challenge application both zero, and thus the positive rate 0%. In the area of challenge exposure to water for injection (the vehicle), there were no skin reactions.

In the negative control group, there were no skin reactions in the site of challenge exposure to the 5% solution of BZ-0145B or the 15% solution of MBT.

General Condition:

Results are shown in Table 3 (see attached background material).

There were no abnormalities in the general condition during the observation period in any test group.

Body Weight:

Results are shown in Table 4 (see attached background material).

In the body weight of animals on day 7 after the start of induction, 1/5 animals in the positive control group showed a body weight decrease. In the body weight of the animals on the ending day of observation, 3/10 animals in the test article sensitization group and 3/5 animals in the control group showed a decrease. The degree of decrease was between 2 and 11 g. Its cause was unclear, but it was thought to be caused by stress from the treatment for intracutaneous induction or closed application at the time of challenge exposure, and this it was judged that it was not caused by administration of the test article or the positive control article.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Based on the results, the test article (BZ-0145B) did not induce skin sensitization under the conditions of the study.