Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1996
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Report date:
1996

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

Test material

Constituent 1
Chemical structure
Reference substance name:
Tris(2-hydroxyethyl)ammonium 7-{4-[4-(2-cyanoamino-4-hydroxy-6-oxidopyrimidin-5-ylazo)benzamido]-2-ethoxy-phenylazo}naphthalene-1,3-disulfonate
EC Number:
421-440-3
EC Name:
Tris(2-hydroxyethyl)ammonium 7-{4-[4-(2-cyanoamino-4-hydroxy-6-oxidopyrimidin-5-ylazo)benzamido]-2-ethoxy-phenylazo}naphthalene-1,3-disulfonate
Cas Number:
778583-04-3
Molecular formula:
Hill formula: C47 H66 N12 O19 S2 CAS formula: C29 H21 N9 O10 S2 x 3 C6 H15 NO3
IUPAC Name:
tris(tris(2-hydroxyethyl)azanium) 2-(cyanoamino)-5-{2-[4-({4-[2-(6,8-disulfonaphthalen-2-yl)diazen-1-yl]-2-methoxyphenyl}carbamoyl)phenyl]diazen-1-yl}-6-hydroxypyrimidin-4-olate

In vivo test system

Test animals

Species:
guinea pig
Strain:
Himalayan
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd., Wolferstrasse 4, 4414 Füllinsdorf / Switzerland
- Age at study initiation: 6-8 weeks
- Weight at study initiation: 296 - 397 g
- Housing: Individually in Makrolon type-3 cages with standard softwood bedding
- Diet (e.g. ad libitum): Pelleted standard Nafag Ecosan 845 25W4 guinea pig breeding/maintenance diet, ad libitum
- Water (e.g. ad libitum): Community tap water from Itingen, ad libitum.
- Acclimation period: Control and test group: 1 week; pretest animals: no acclimatation

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 40-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To:

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
water
Concentration / amount:
Concentration of test material and vehicle used at induction: - intradermal: 0.1 ml /site
A) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline B) test substance diluted to 5 % (w/w) in bidistilled water C) test substance diluted to 5 % (w/w) by emulsion in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline - epidermal: 0.3 ml test substance 50 % (w/w) in bi-distilled water
Concentration of test material and vehicle used for each challenge: - epidermal: 25 % (w/w) in bi-distilled water
Challengeopen allclose all
Route:
epicutaneous, semiocclusive
Vehicle:
water
Concentration / amount:
Concentration of test material and vehicle used at induction: - intradermal: 0.1 ml /site
A) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline B) test substance diluted to 5 % (w/w) in bidistilled water C) test substance diluted to 5 % (w/w) by emulsion in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline - epidermal: 0.3 ml test substance 50 % (w/w) in bi-distilled water
Concentration of test material and vehicle used for each challenge: - epidermal: 25 % (w/w) in bi-distilled water
No. of animals per dose:
Number of animals in test group: 20 Number of animals in negative control group: 10
Details on study design:
RANGE FINDING TESTS:
A pretest with 6 animals (intradermal: 2 animals, epidermal: 4 animals) was conducted to identify a maximally tolerated concentration of the test article suitable for the induction phase of the main study. In addition, a suitable non-irritant concentration of the test article, by the topical route of administration, was identified for the challenge application.
All animals were sensitized with intradermal application of a 1:1 suspension of FCA/physiological saline one week before the treatments.
Intradermal injections: 1, 3, and 5% in bidistilled water
Epidermal application: 10, 15, 25, 50% in bidistilled water
Reactions were evaluated 24h (intradermal injection) and 24 and 48h (epidermal application) after beginning of the treatment.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3 pairs of intradermal application with FCA, test item, and test item + FCA; 1 epicutaneous application with test item
- Exposure period: epicutaneous application: 48h
- Site: scapular region
- Frequency of applications: day 1: intradermal application; day 8: epicutaneous application
- Test item concentration: intradermal application: 5%; epicutaneous application: 50%

B. CHALLENGE EXPOSURE
- No. of exposures:
- Day(s) of challenge: on day 22
- Exposure period: approximately 24h
- Site: left and right flank
- Concentration: 25% (highest non-irritating concentration)
- Evaluation (hr after challenge): 24 and 48h after chellenge

OTHER:
- The following observations and data were recorded during the test and observation period: Viability/Mortality, Clinical Signs (local/systemic), Skin reactions, Body Weights.
Records were maintained of all additional and standard observations.
Positive control substance(s):
yes
Remarks:
alpha-hexylcinnamaldehyde, 2- mercaptobenzothiazole

Results and discussion

Positive control results:
ALPHA-HEXYLCINNAMALDEHYDE
In the positive control study 65% (at 24-hour reading) and 60% (at 48-hour reading) of the animals of the test group were observed with positive skin reactions after treatment with a non-irritant test substance concentration of 15% alpha-hexylcinnamaldehyde in PEG 400.
No skin reactions were observed in the control group.

2- MERCAPTOBENZOTHIAZOLE
In this positive control study 90% (at the 24-hour reading) and 100% (at the 48-hour reading) of the animals of the test group were observed with positive skin reactions after treatment with a non-irritant test substance concentration of 50% in mineral oil.
No skin reactions were observed in the control group.

In vivo (non-LLNA)

Resultsopen allclose all
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:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
25 %
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: 25 %. 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:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
25 %
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: 25 %. No with. + reactions: 0.0. Total no. in groups: 20.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0 %
No. with + reactions:
0
Total no. in group:
10
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: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
25 %
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 25 %. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0 %
No. with + reactions:
0
Total no. in group:
10
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: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
25 %
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 25 %. No with. + reactions: 0.0. Total no. in groups: 10.0.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
The substance applied at a concentration of 25% in bi-distilled water is considered to be a non-sensitizer.
Executive summary:

In order to assess the cutaneous allergenic potential of the substance, the Maximization-test in accordance with OECD Guideline No. 406 and the Directive 92/69 EEC B.6 was carried out in 30 (20 test and 10 control) male albino guinea pigs.

The intradermal induction of sensitization was carried out with a 5% dilution of the test article in bi-distilled water and in an emulsion with Freund's Complete Adjuvant (FCA)/physiological saline. The epicutaneous induction of sensitization was conducted under occlusion with the test article at 50% in bi-distilled water. Two weeks after the epicutaneous induction application the challenge was completed by epicutaneous application of the test article at 25% in bi-distilled water under occlusive dressing. The animals of the control group were induced with bi-distilled water and FCA/physiological and challenged similarly as those of the test group. Cutaneous reactions, i.e erythema and eschar, as well as oedema formation were evaluated at 24 and 48 hours after removal of the dressing.

No reactions were observed 24 and 48h after the challenge period.

In this study 0% of the animals of the test and control group were observed with positive skin reactions after treatment with a non-irritant test substance concentration of 25% in bi-distilled water.

Therefore, the substance applied at a concentration of 25% in bi-distilled water is considered to be a non-sensitizer when used under the described test conditions.