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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:
1989-06-05 to 1989-07-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report date:
1989

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
1981
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
1984
Principles of method if other than guideline:
Summary of protocol amendments
1. Two extra animals were used to clarify the toxicity of the test article: one animal for epidermal application and one for intradermal injection. The first animal was found dead a few hours after dosing.
2. The maximum measured humidity during the study period was 85 %, which is 15 % above the range as stated in the protocol. Meteorological conditions outside could have affected this effect.
3. The intradermal injections of animal 477 were also assessed 48 hours after injection.
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
A guinea pig maximisation test was already available.

Test material

Constituent 1
Chemical structure
Reference substance name:
A mixture of isomers of: 1,1'-[(3,5(or 2,4 or 4,6 or 2,6)-dihydroxy-o(or m or p)-phenylene)bis(azo-meta-phenyleneazo{1-[3-(dimethylamino)propyl]-1,2-dihydro-6-hydroxy-4-methyl-2-oxopyridine-5,3-diyl})]dipyridinium-dichloride-dihydrochloride; 1-(1-[3-(dimethylamino)propyl]-5-{3-[x-(4-{1-[3-(dimethylamino)propyl]-1,6-dihydro-2-hydroxy-4-methyl-6-oxo-5-pyridinio-3-pyridylazo}phenylazo)-2,4(or 2,6 or 3,5 or 4,6)-dihydroxyphenylazo]phenylazo}-1,2-dihydro-6-hydroxy-4-methyl-2-oxo-3-pyridyl)pyridinium-dichloride-dihydrochloride (where x is variable)
EC Number:
404-540-1
EC Name:
A mixture of isomers of: 1,1'-[(3,5(or 2,4 or 4,6 or 2,6)-dihydroxy-o(or m or p)-phenylene)bis(azo-meta-phenyleneazo{1-[3-(dimethylamino)propyl]-1,2-dihydro-6-hydroxy-4-methyl-2-oxopyridine-5,3-diyl})]dipyridinium-dichloride-dihydrochloride; 1-(1-[3-(dimethylamino)propyl]-5-{3-[x-(4-{1-[3-(dimethylamino)propyl]-1,6-dihydro-2-hydroxy-4-methyl-6-oxo-5-pyridinio-3-pyridylazo}phenylazo)-2,4(or 2,6 or 3,5 or 4,6)-dihydroxyphenylazo]phenylazo}-1,2-dihydro-6-hydroxy-4-methyl-2-oxo-3-pyridyl)pyridinium-dichloride-dihydrochloride (where x is variable)
Cas Number:
159405-95-5
Molecular formula:
C50H56Cl4N14O6
IUPAC Name:
1'-[3-(dimethylamino)propyl]-5'-(2-{3-[2-(4-{2-[3-(2-{1'-[3-(dimethylamino)propyl]-6'-hydroxy-4'-methyl-2'-oxo-1',2'-dihydro-1λ⁵-[1,3'-bipyridin]-1-ylium-5'-yl}diazen-1-yl)phenyl]diazen-1-yl}-2,6-dihydroxyphenyl)diazen-1-yl]phenyl}diazen-1-yl)-6'-hydroxy-4'-methyl-2'-oxo-1',2'-dihydro-1λ⁵-[1,3'-bipyridin]-1-ylium 1'-[3-(dimethylamino)propyl]-5'-(2-{4-[2-(4-{2-[4-(2-{1'-[3-(dimethylamino)propyl]-6'-hydroxy-4'-methyl-2'-oxo-1',2'-dihydro-1λ⁵-[1,3'-bipyridin]-1-ylium-5'-yl}diazen-1-yl)phenyl]diazen-1-yl}-2,6-dihydroxyphenyl)diazen-1-yl]phenyl}diazen-1-yl)-6'-hydroxy-4'-methyl-2'-oxo-1',2'-dihydro-1λ⁵-[1,3'-bipyridin]-1-ylium tetrahydrochloride tetrachloride
Test material form:
solid

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 Wiga, Sulzfeld / West-Germany
- Age at study initiation: approx. 12 weeks
- Weight at study initiation: 370 - 512 gram
- Housing: group housing of 2 animals per cage with wire-mesh floors
- Diet (e.g. ad libitum): free access to standard guinea pig diet, including ascorbic acid (1600 mg/kg);
- Water (e.g. ad libitum): free access to tap-water, diluted with decalcified water.
- Acclimation period: at least five days under test conditions after physical examination.

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 3 °C
- Humidity: 30 - 85 %
- Air changes (per hr): 7.5 - 15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
physiological saline
Concentration / amount:
0.5 %
Day(s)/duration:
day 1
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
25%
Day(s)/duration:
day 8 / 48 h
Adequacy of induction:
other: 24 h prior to application, test area was pretreated with 10 % SDS in petrolatum; 25 % was chosen as maximum tolerated concentration.
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
25 %, 10 %, 5 %
Day(s)/duration:
day 22 / 24 h
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:

Intradermal injections
Four intradermal injections (0.1 ml/site) were made within 2 hours after test substance preparation into the clipped shoulder region of one guinea pig at a concentration of 5 % (w/w) of the test substance in milli-ROwater. This animal was found dead a few hours later. Therefore a second animal was intradermally injected approx. 26 hours after test substance preparation in a similar manner with a 2.5% (w/w) test article concentration in milli-RO water. Also this animal died within a few hours after dosing. A third animal was intradermally injected approx. 24.5 hours after test substance preparation in a similar manner with a 0.5 % (w/w) test article concentration in milli-RO water. The resulting dermal reactions were assessed 24 and 48 hours later. The parameters Erythema/Necrosis (scored/if present yes or no) Diameter (size in millimeters) were assessed.

Epidermal applications
The first intracutaneously injected animal was also treated epicutaneously at the shaved left flank with 0.5 ml of a 50 % (w/w) concentration of the test substance in milli-RO water within 2 hours after preparation using a Metalline patch (Lohman, Neuwied / West Germany) mounted on Micropore tape and held in place with Coban elastic bandage. for 24 hours. Due to the death of this animal, another animal was added an epicutaneously applied in a similar manner with a 25 % (w/w) test article concentration in milli-RO water approx. 26 hours after preparation. The treated skin was assessed for erythema and oedema 24 and 48 hours after removal of the dressings on a numerical basis according to the scale described below.
Four animals were shaved on the left flank and exposed for 24 hours to 50 %, 25 %, 10 % and 5 % (w/w) test substance concentrations in milli-RO water (0.05 ml/concentration) within 2 hours after preparation, occlusively administered by means of Square chambers mounted on Micropore tape and fixed in place by means of Coban elastic bandage. This procedure ensured the intensive contact of the test substance. The reaction sites were assessed for erythema and oedema on a numerical basis according to the scale described below, 24 and 48 hours after removal of the dressings.

Results
Two animals died within a few hours after treatment, probably caused by intradermal injections of a 5 % and 2.5 % test article concentration. Three of the five survived animals lost body weight. The 25 % test article concentration was chosen as a maximum tolerated concentration for the main study.


MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2
- Exposure period: interdermal induction: 21 days prior to challange; epidermal induction: 14 days prior to challenge

- Test groups:
Intradermal injections:
On day 1 an area of the dorsal skin from the scapular region (approximately 4 × 6 cm) was clipped free of hair. Three pairs of intradermal injections (0.1 ml/site) were made 2-2.5 hours after test substance preparation at the border of a 2 × 4 cm area in the clipped region as follows:
A) The test substance dissolved to 0.5 % (w/w) with physiological saline.
B) Freunds' Complete Adjuvant, 50:50 with distilled water for injection.
C) The test substance, at twice the concentration used in (A), emulsified in a 50:50 mixture of Freunds' Complete Adjuvant.

Epidermal applications
Seven days after the intradermal injections, the scapular area (approximately 6 × 8 cm) was again clipped and shaved free of hair. A 2 × 4 cm patch of Metalline mounted on Micropore tape was treated with 0.5 ml of the test substance (25 % (w/w) in water) and placed over the injection sites of the test animals, within 1 hour after test substance preparation. The Micropore tape was firmly secured, wrapped around the trunk of the animal and secured with Coban elastic bandage. The dressings were left in place for approximately 48 hours. The epidermal application procedure described ensured intensive contact of the test substance even if it is insoluble in the vehicle used.
Approximately 24 hours prior to the epidermal application the test area was pretreated with 10 % Sodium-Dodecyl-Sulfat (SDS) in petrolatum. The SDS was massaged into the skin with a spatula without bandaging. This concentration of SDS enhances sensitization by provoking a mild inflammatory reaction.

- Control group: guinea pigs of the control group were treated as described above by the intradermal and epidermal inductions with the omission of test substance.


B. CHALLENGE EXPOSURE
- Test groups:
Hair was clipped and shaved from a 5 × 5 cm area on the left flank of each guinea-pig. The following series of 3 test substance concentrations and the vehicle were applied 4-5 hours after preparation, using Square chambers attached to Micropore tape:
a = 25 % in milli-RO water.
b 10 % in milli-RO water.
c = 5 % in mill i-RO water.
d = milli-RO water.
A volume of 0.05 ml of each concentration or the vehicle was placed into a Square chamber. The patches were placed on the shaved area, the Micropore tape firmly secured around the trunk of the animals and held in place by Coban elastic bandage.
- Control group: same as test groups
- Site: left flank
- Concentrations: see above
- Evaluation (hr after challenge): the sites were assessed for redness and swelling 24 and 48 hours after removal of the dressings.
Challenge controls:
Freunds Complete Adjuvant (FCA) was added to attract circulating immunocompetent cells to the injected area.
Positive control substance(s):
yes
Remarks:
formaldehyde

Results and discussion

Positive control results:
Clearly positive results were observed in the experimental animals after the challenge with 0.25 % (w/w) FORMALDEHYDE in milli-RO water (study conducted in March 1989).

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
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
0 %
No. with + reactions:
0
Total no. in group:
20
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
5 %
No. with + reactions:
5
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
5 %
No. with + reactions:
10
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
10 %
No. with + reactions:
11
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
10 %
No. with + reactions:
17
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
25 %
No. with + reactions:
17
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
25 %
No. with + reactions:
20
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0 %
No. with + reactions:
0
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
5 %
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:
negative control
Dose level:
10 %
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:
negative control
Dose level:
25 %
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
other: not specified
Group:
positive control
Dose level:
intradermal induction 1 % w/w, epidermal induction 1 % w/w, challenge 0.25 % w/w
No. with + reactions:
13
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Reading:
other: not specified
Group:
positive control
Dose level:
intradermal induction 1 % w/w, epidermal induction 1 % w/w, challenge 0.1 % w/w
No. with + reactions:
3
Total no. in group:
20
Reading:
other: not specified
Group:
positive control
Dose level:
intradermal induction 1 % w/w, epidermal induction 1 % w/w, challenge 0 % w/w
No. with + reactions:
0
Total no. in group:
10

Any other information on results incl. tables

Number of test animals with positive skin reactions to challenge (summary result of all positive reaction grade 2 or higher at 24 and 48 hours):

 Test item concentration 25 %   10 % 5 %  0 % 
Experimental group  20  17   13  0
Sensitization rate   100  85  65  0
Control group   0  0  0  0

Applicant's summary and conclusion

Interpretation of results:
other: classified as skin sensiting cat. 1A within the CLP Regulation (EC 1272/2008)
Conclusions:
Test substance is skin sensitising.
Executive summary:

The purpose of the study was to assess the potential of the test substance to induce skin sensitization in the guinea pig, following OECD guideline 406. The intracutaneous route of administration (first induction at 0.5 % w/w in physiological saline and 50:50 FCA mixture) was selected in order to obtain optimal contact between the test substance and elements of the immunosystem. Freunds Complete Adjuvant (FCA) was added to attract circulating immunocompetent cells to the injected area. Approximately 24 h before dermal application, the site was treated with 10 % SDS in petrolatum. The dermal route of administration (second induction at 25 % w/w in water and challenge at 25 %, 10 %, 5 % w/w in water) was selected because the test substance may accidentally come into contact with the skin during manufacture, handling and use.

Under the conditions used in this study, the substance resulted in a sensitization rate of 100 per cent after intracutaneous and epicutaneous application to the guinea pig.