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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:
From October 5, 2004 to October 29, 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report date:
2004

Materials and methods

Test guidelineopen allclose all
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:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Study predates LLNA method.

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
457-630-8
EC Name:
-
Cas Number:
2138836-26-5
Molecular formula:
Hill formula: C29H23FN8Na4O16S5
IUPAC Name:
tetrasodium 3-amino-4-[(1E)-2-[4-({4-fluoro-6-[phenyl({2-[2-(sulfonatooxy)ethanesulfonyl]ethyl})amino]-1,3,5-triazin-2-yl}amino)-2-sulfonatophenyl]diazen-1-yl]-5-hydroxynaphthalene-2,7-disulfonate
Test material form:
solid: particulate/powder

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: Crl:HA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland GmbH, Stolzenseeweg 32-36, 88353 Kißlegg, Germany
- Body weight range at start of study: 347-421 g
- Housing: In transparent macrolon cages (type IV) on soft wood granulate in an air-conditioned room, 3 or 2 animals per cage
- Diet: Ssnif Ms-H (V 2233), ad libitum
- Water: Tap water in plastic bottles, ad libitum
- Acclimation period: At least 5 d
- Animal identification: Fur marking with KMnO4 and cage numbering
- Randomization procedure: Computer generated algorithm (archived with raw data) Randomization schemes 2004.0061

ENVIRONMENTAL CONDITIONS
- Temperature: 22±3°C (short lasting deviations are permissible, e.g., during cleaning processes)
- Humidity: 50±20% (short lasting deviations are permissible, e.g., during cleaning processes)
- Photoperiod: 12 h light/dark cycle

IN-LIFE DATES: From: To: October 5, 2004 to October 29, 2004

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
water
Concentration / amount:
5% / 4 x 0.1 mL
Day(s)/duration:
Day 1
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
25% / 0.5 mL
Day(s)/duration:
Day 8 for 48 h
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
25% / 0.5 mL
Day(s)/duration:
Day 22 for 24 h
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Number of animals in test group: 10
Number of animals in control group: 5
Details on study design:
TEST PROCEDURE
The following preparations were used for the intradermal injections:
Control group
1.) 50% Freund´s Complete Adjuvant emulsion
Original Freund's Complete Adjuvant (Sigma Chemical Company) was mixed immediately before use with an equal volume of deionized water.
2.) Deionized water (i.e., vehicle)
3.) 50% Freund´s Complete Adjuvant emulsion mixed with an equal volume of the vehicle
Treatment group
1.) 50% Freund's Complete Adjuvant emulsion
Original Freund's Complete Adjuvant (Sigma Chemical Company) was mixed immediately before use with an equal volume of deionized water.
2.) 5% test substance in deionized water.
3.) 5% test substance in a 50% Freund's Complete Adjuvant emulsion
For the intradermal injections of the test substance in 50% Freund's adjuvant, test substance was dissolved in deionized water and then mixed with an equal volume of Freund's Original Adjuvant [percentages w/v].
For the dermal treatments, test substance was suspended in deionized water [percentages w/v]

MAIN TEST FOR THE SENSITIZING PROPERTIES
Chronological description of the test procedure indicating the day, at which procedure was carried out:
Study Day 1:
The body weight of the animals was determined. The guinea pigs were shaved mechanically over a dorsal area of 4 x 6 cm in the vicinity of the shoulders.

Intradermal induction treatment.
Two intradermal injections per animal of each preparation (i.e. 50% Freund's Adjuvants, 5% test substance in deionized water and 5% test substance in 50% Freund's Adjuvants) was given in the treatment group. The injection sites were all within a dorsal area of 2 x 4 cm. Likewise control group was given two intradermal injections per animal of each preparation (i.e. 50% Freund's Adjuvants, deionized water and equal volume of deionized water and 50% Freund's Adjuvants).
Study Day 2 - 7:
The administration area was examined for local tolerance. Systemic toxic effects were recorded, when apparent.
Study Day 8:
Dermal induction treatment.
An amount of 0.5 mL of the test substance preparation (treatment group) or the vehicle (control group) was administered to a 2 x 4 cm cellulose patch. This patch covered the area where the intradermal injection had been placed. The administration area was then kept under an occlusive bandage covered with an impermeable film and an elastic bandage for 48 h.
Treatment group: 25% test substance in deionized water
Control group: Deionized water
Study Day 10:
Occlusive bandage was removed, irritant effects were recorded, when apparent.
Study Day 11 - 22:
No treatment of control or treatment group.
Test animals were kept under observation.
Study Day 22:
Dermal challenge treatment
One area of approximately 5 x 5 cm on the left flank was shaved mechanically.
An amount of 0.5 mL of the test substance preparation was administered to a 2 x 2 cm cellulose patch. The administration area was then kept for 24 h under an occlusive bandage covered with an impermeable film and an elastic bandage.
Treatment and control group (left flank): 25% test substance in deionized water
Study Day 23:
Occlusive bandage was removed. Any remnants of the test substance were carefully washed off with warm water.
Study Day 24:
Examination of the skin approximately 24 h after removal of the patches.
Study Day 25:
Examination of the skin approximately 48 h after removal of the patches.
Body weight of the test animals was determined.
Positive control substance(s):
yes
Remarks:
alpha-hexylcinnamaldehyde tested bi-annually

Results and discussion

Positive control results:
After the challenge treatment positive response was observed

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
25% dermal application
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
25% dermal application
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
25% dermal application
No. with + reactions:
0
Total no. in group:
5
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
25% dermal application
No. with + reactions:
0
Total no. in group:
5
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
20%
No. with + reactions:
8
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
20%
No. with + reactions:
15
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation

Any other information on results incl. tables

DETERMINATION OF THE TOLERANCE OF THE INTRADERMAL INJECTIONS

 

Intradermal injections with the 5.0% test substance preparation caused well-defined edema and encrustions. Slight edema was observed after intradermal injections with the 1.0 % test substance preparation. Intradermal injections with the 0.2 % test substance preparation caused slight edema on Day 2 of the study.

 

Based on this preliminary test, a 5% test substance preparation was selected for the intradermal injections in the main test.

 

Due to the dark red color of the test substance the treated skin of the animals could not be assessed for erythema in the 5% and 1% test substance preparation.

 

DETERMINATION OF THE PRIMARY NON-IRRITATING CONCENTRATION

 

No signs of irritation occurred after administration of the different test substance concentrations.

 

Based on these results, a concentration of 25% test substance in deionized water was chosen for the challenge at Day 22.

 

MAIN TEST

 

Body weight gains and clinical signs:The body weight gains of the animals were not impaired. The treated animals showed no clinical signs of intoxication throughout the study.

 

 

Intradermal induction treatment:Intradermal injections with Freund's Adjuvant (i.e., without test substance) caused severe erythema and edema as well as indurations and encrustations. Intradermal injections with Freund's Adjuvant (i.e., with test substance) caused well-defined edema as well as indurations and encrustations The administration sites treated with the test substance (i.e., in the vehicle) showed well-defined edema and encrustions. Intradermal injections of the vehicle alone exhibited no signs of irritation.

 

Due to the dark red color of the test substance the treated skin of the animals could not be assessed for erythema.

 

Due to these strong irritation reactions of the skin, 10% sodium dodecylsulfate was not administered at Day 7.

 

Dermal induction treatment

 

Control Group: After the removal of the patches at Day 10, well-defined erythema and edema, indurated and encrusted skin were observed at the sites previously treated with Freund's Adjuvant. The administration sites treated with the vehicle alone showed no signs of irritation.

 

Treatment Group: After the removal of the patches at Day 10, well-defined edema, indurated and encrusted skin were observed at the sites previously treated with Freund's Adjuvant. The administration sites treated with the test substance showed well-defined edema and encrustions.

 

Due to the dark red color of the test substance the treated skin of the animals could not be assessed for erythema.

 

Dermal challenge treatment:No skin reactions were observed in the control and the treatment group 24 and 48 h after removal of the occlusive bandage.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the study conditions, the test substance showed no evidence of sensitizing properties.
Executive summary:

A guinea pig maximization test was conducted to evaluate the skin sensitization potential of the test substance according to OECD Guideline 406 and EU Method B.6, in compliance with GLP.

Based on the results of a preliminary study, 5% and 25% of test substance in deionized water were selected as intradermal and dermal induction doses. The highest non-irritating concentration used for challenge application was 25% test substance in deionized water.

None of the animals of the test group showed skin reactions 24 and 48 h after removing the dressings.

Under the study conditions, the test substance showed no evidence of sensitizing properties.