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Diss Factsheets

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report date:
1995

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
GLP compliance:
yes
Type of study:
guinea pig maximisation test

Test material

Constituent 1
Chemical structure
Reference substance name:
6,13-dichloro-3,10-bis-{2-[4-fluoro-6-(2-sulfo-phenylamino)-1,3,5-triazin-2-ylamino]-propylamino}-benzo[5,6][1,4]oxazino[2,3-.b.]phenoxazine-4,11-disulphonic acid, lithium, sodium salt.
EC Number:
418-000-8
EC Name:
6,13-dichloro-3,10-bis-{2-[4-fluoro-6-(2-sulfo-phenylamino)-1,3,5-triazin-2-ylamino]-propylamino}-benzo[5,6][1,4]oxazino[2,3-.b.]phenoxazine-4,11-disulphonic acid, lithium, sodium salt.
Cas Number:
163062-28-0
Molecular formula:
CAS formula: C42 H34 Cl2F2 N14 O14 S4 .xLi. xNa
IUPAC Name:
dilithium(1+) disodium 6,13-dichloro-3,10-bis({[2-({4-fluoro-6-[(2-sulfonatophenyl)amino]-1,3,5-triazin-2-yl}amino)propyl]amino})-5,12-dioxa-7,14-diazapentacene-4,11-disulfonate
Details on test material:
- Name of test material (as cited in study report): FAT 40529/A
- Lot/batch No.: P 1/95
- Storage: Ambient conditions

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Como), Italy
- Age at study initiation: 5 to 6 weeks
- Weight at study initiation: 300-350g
- Housing: In groups of five animals, in stainless steel cages measuring 63x48x41cm, with a grid floor (Techniplast Gazzada S.a.r.l., Buguggiate, Varese, Italy). Cages were suspended over metal trays which held an absorbent material. This was inspected daily and changed as necessary.
- Diet (e.g. ad libitum): Commercially available laboratory diet (Altromin MSK, A. Rieper S.p.A., Bolzano, Italy), ad libitum
- Water (e.g. ad libitum): Supplied to each cage via a water bottle, ad libitum
- Acclimation period: at least six days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
other: water for the intradermal injection and the rechallange. Petrolatum for the topical induction and the challenge
Concentration / amount:
PRELIMINARY STUDY:
- Intradermal injections (0.1 mL/site) at concentrations of 0.1, 0.5, 1, 2, 5 % and 10% of the test article in sterile water.
- Epidermal applications: Patches of filter paper were saturated with concentrations of 1, 5, 10, 20 and 50% of the test article in petrolatum.
MAIN STUDY:
- Induction: Intradermal injections: 2 %, Topical applications: 50% in petrolatum
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: water for the intradermal injection and the rechallange. Petrolatum for the topical induction and the challenge
Concentration / amount:
PRELIMINARY STUDY:
- Intradermal injections (0.1 mL/site) at concentrations of 0.1, 0.5, 1, 2, 5 % and 10% of the test article in sterile water.
- Epidermal applications: Patches of filter paper were saturated with concentrations of 1, 5, 10, 20 and 50% of the test article in petrolatum.
MAIN STUDY:
- Induction: Intradermal injections: 2 %, Topical applications: 50% in petrolatum
No. of animals per dose:
10 animals for the vehicle control group and 20 animals for the test article group.
Details on study design:
PRELIMINARY TEST
Intradermal injection tolerance test:
Two animals were selected from those available and the hair over the scapulae was removed using an electric clipper with suitable blade. Six sites were selected on each of the animals and these injected intradermally with 0.1mL of the test substance. Each site was injected with a single concentration of the test substance. The two animals were treated with the substance at concentrations of 10, 5, 2, 1, 0.5, and 0.1% in sterile water. The treated sites were then examined after five days for any signs of reaction to treatment. Observed irritation was recorded using the Draize scoring.
Topical application tolerance test:
Five animals were selected from those available and the hair over the scapulae was removed using an electric clipper with suitable blade. Each animal was then injected intradermally at the prepared site with two injections, each of 0.1 mL, of emulsified Freund's complete adjuvant. At least seven days later the flanks of all animals were clipped free of hair. Each of the five animals was dosed with two concentrations of the test substance, one on either flank. A 0.2 mL aliquot of the selected concentration of the test substance was spread evenly over a gauze patch measuring approximately 20 x 20 mm. This was then placed onto the selected treatment site and secured in position using a patch of adhesive strapping. When both sites of the animal had been treated, they were covered with a strip of aluminum foil to act as an occlusive barrier and the trunk of the animal was wrapped with an elastic adhesive bandage to keep the test substance in contact with the skin. AII five animals were treated in this manner such that a total of five concentrations (50%, 20%, 10%, 5% and 1% in petrolatum) of the test substance were each dosed in duplicate. The adhesive dressings and gauze patches were removed after twenty four hours contact with the skin. Twenty four and forty eight hours after removal of the dressings, the treated sites were examined for signs of reaction to treatment.

MAIN TEST
Induction - intradermal injection:
Animals were allocated to treatment to give a test group of twenty animals and a control group of ten animals. 0n the day of dosing the hair was clipped from the scapular region of each animal over an area approximately 20 x 40 mm. Three pairs of intradermal injections were made at the prepared skin site of each animal. AII injections were made at the edge of the prepared site and the anterior and median injections were positioned close together and distant from the posterior injections. A volume of 0.1 mL was injected at each point. Animals of the test group were treated as follows: anterior with emulsified Freund's complete adjuvant, median with 2% test substance, posterior with 2% test substance in emulsified Freund's complete adjuvant. Animals of the control group were treated in the same manner except that the test substance was replaced by the vehicle alone. Skin reactions at the injection site was assessed approximately twenty-four hours after injection.
Induction - Topical application:
Six days after injection, the area surrounding the injection sites on each animal was clipped free of hair and 0.5 mL of sodium lauryl sulphate (10% in petrolatum) was spread evenly over the sites. This was intended to provoke irritation of the skin and enhance potential absorption of the test substance. The next day (Day 8 of the study), animals of the test group were treated with the test substance at 50% concentration in petrolatum. A gauze patch was covered with 0.4 mL of the substance and placed over the injection sites. This was covered with a strip of aluminum foil to serve as an occlusive barrier and the animal was then wrapped with a length of adhesive dressing to keep the gauze patch in contact with the skin. All animals of the test group were treated in the same manner. Animals of the control group were similarly treated with the vehicle alone.
After 48 hours the dressings were removed and the treated sites gently cleaned by washing with warm water. Reaction to treatment was assessed at approximately 24 hours after removal of the dressings.
Challenge:
Three weeks after the first induction, all animals were prepared for challenge by clipping the flanks free of hair to expose areas of approximately 50x50 mmon each flank. Patches of gauze (20x20 mm) were coated with 0.2mL aliquots of the test substance at 50% concentration in sterile water. These were placed on the right flank of each animal of both groups, in the center of the prepared skin sites. The left flank of each animal was treated similarly with the vehicle (water). The treated sites were covered with a strip of aluminum and each animal was then wrapped with a length of elastic bandage. After 24 hours, the patches were removed and another 21 hours later, the sites were closely clipped. Three hours later, the sites were examined for any signs of reaction, which was repeated 24 hours later. The challenge procedure was repeated one week later (20% in petrolatum).
Positive control substance(s):
no

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
9
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 0. No with. + reactions: 0.0. Total no. in groups: 9.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
9
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 0. No with. + reactions: 0.0. Total no. in groups: 9.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
50%
No. with + reactions:
5
Total no. in group:
9
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 50%. No with. + reactions: 5.0. Total no. in groups: 9.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
50%
No. with + reactions:
5
Total no. in group:
9
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 50%. No with. + reactions: 5.0. Total no. in groups: 9.0.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
0
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 0. No with. + reactions: 0.0. Total no. in groups: 20.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
0
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 0. No with. + reactions: 0.0. Total no. in groups: 20.0.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
50%
No. with + reactions:
11
Total no. in group:
20
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 50%. No with. + reactions: 11.0. Total no. in groups: 20.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
50%
No. with + reactions:
12
Total no. in group:
20
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50%. No with. + reactions: 12.0. Total no. in groups: 20.0.
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
9
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 24.0. Group: negative control. Dose level: 0. No with. + reactions: 0.0. Total no. in groups: 9.0.
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
9
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 48.0. Group: negative control. Dose level: 0. No with. + reactions: 0.0. Total no. in groups: 9.0.
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
20%
No. with + reactions:
0
Total no. in group:
9
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 24.0. Group: negative control. Dose level: 20%. No with. + reactions: 0.0. Total no. in groups: 9.0.
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
20%
No. with + reactions:
0
Total no. in group:
9
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 48.0. Group: negative control. Dose level: 20%. No with. + reactions: 0.0. Total no. in groups: 9.0.
Reading:
rechallenge
Hours after challenge:
24
Group:
test chemical
Dose level:
0
No. with + reactions:
0
Total no. in group:
18
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 24.0. Group: test group. Dose level: 0. No with. + reactions: 0.0. Total no. in groups: 18.0.
Reading:
rechallenge
Hours after challenge:
48
Group:
test chemical
Dose level:
0
No. with + reactions:
0
Total no. in group:
18
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 48.0. Group: test group. Dose level: 0. No with. + reactions: 0.0. Total no. in groups: 18.0.
Reading:
rechallenge
Hours after challenge:
24
Group:
test chemical
Dose level:
20%
No. with + reactions:
3
Total no. in group:
18
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 24.0. Group: test group. Dose level: 20%. No with. + reactions: 3.0. Total no. in groups: 18.0.
Reading:
rechallenge
Hours after challenge:
48
Group:
test chemical
Dose level:
20%
No. with + reactions:
1
Total no. in group:
18
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 48.0. Group: test group. Dose level: 20%. No with. + reactions: 1.0. Total no. in groups: 18.0.

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test substance may act as a sensitiser in the guinea pig.
Executive summary:

In a GLP compliant sensitivity study using the Maximization-Test, performed according to OECD guideline 406, guinea pigs were treated with the test substance. Twenty animals were treated with the test substance and ten animals with only the vehicle. The concentrations of the test substance used in the main study were determined by the results of the preliminary study. The intradermal induction of sensitization in the test group was performed using 2% of the test substance in both the vehicle and an emulsion of Freund’s complete adjuvant. One week later this was boosted by the topical application of the test substance at 50% concentration over the injection sites. Animals of the control group were treated in the same manner but the selected vehicle was used. Two weeks after the second induction all animals were challenged by topical application of the vehicle and the test substance at 50% concentration. Since animals in the test group showed responses, which could be a result of irritation instead of sensitization, a rechallenge was performed using 20% concentration. 17 and 6% of the animals of the test group, 24 and 48 hours after the rechallenge, respectively, showed a response. This indicates that the test substance may elicit a sensitization response, however the response was below 30% and therefore no classification is needed.