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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:
1989-1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report date:
1990

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
GLP compliance:
yes (incl. QA statement)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
This skin sensitisation study was carried out in 1989-1990, before the entry into force of REACH regulation and the amendments of Annex VII.

Test material

Constituent 1
Chemical structure
Reference substance name:
Disodium 7-amino-4-hydroxy-3-[[4-[(4-sulphonatophenyl)azo]phenyl]azo]naphthalene-2-sulphonate
EC Number:
228-589-1
EC Name:
Disodium 7-amino-4-hydroxy-3-[[4-[(4-sulphonatophenyl)azo]phenyl]azo]naphthalene-2-sulphonate
Cas Number:
6300-50-1
Molecular formula:
C22H17N5O7S2.2Na
IUPAC Name:
disodium 7-amino-4-hydroxy-3-({4-[(4-sulfonatophenyl)diazenyl]phenyl}diazenyl)naphthalene-2-sulfonate
Test material form:
solid

In vivo test system

Test animals

Species:
guinea pig
Strain:
Himalayan
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd., Wolferstrasse 4, CH-4414 Fullinsdorf
- Females (if applicable) nulliparous and non-pregnant: not specified
- Microbiological status of animals, when known: not specified in the report
- Age at study initiation: 8 weeks
- Weight at study initiation: 367-408 g
- Housing: Individually in Makrolon type-3 cages with standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz).
- Diet (e.g. ad libitum): Pelleted standard Kliba 342, Batches 54/89 and 55/89 guinea pig breeding/ maintenance diet ("Kliba", Klingentalmuhle AG, CH-4303 Kaiseraugst) ad libitum.
- Water (e.g. ad libitum): Community tap water from Itingen, ad libitum
- Acclimation period: One week under test conditions after veterinary examination.
- Indication of any skin lesions: none

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+-3 (hourly monitored environment)
- Humidity (%): relative humidity 40-70 %
- Air changes (per hr): Air-conditioned with 10-15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light/12 hours dark, music/light period

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
physiological saline
Concentration / amount:
5%, 3%, 1%
Day(s)/duration:
1
Adequacy of induction:
other: preliminary study
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
25%, 15%, 10%, 5%
Day(s)/duration:
1 (exposure) + 2 (observation)
Adequacy of induction:
other: preliminary study
Route:
intradermal
Vehicle:
physiological saline
Concentration / amount:
5% test article
Adequacy of induction:
other: main study
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
15% test material
Day(s)/duration:
Application one week after the injections. 1 day (exposure) + 2 days (observation after exposure)
Adequacy of induction:
other: main study
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
10%
Day(s)/duration:
The test and control guinea-pigs were challenged two weeks after the epidermal induction application. 1 day (exposure) + 2 days (observation)
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
10 females for the control group and 20 females for the test group
Details on study design:
RANGE FINDING TESTS:
The objective of this investigation was to identify irritant test article concentrations 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.
The procedure applied for these investigations was as follows:
Intradermal injections:
Intradermal injections (0.1 ml/site) were made into the clipped flank of two guinea-pigs at concentrations of 5%, 3% and 1% of the test article in physiological saline. The resulting dermal reactions were assessed 24 hours later.
Epidermal applications:
Patches of filter paper ( 2 x 2 cm) were saturated with concentrations of 25%, 15%, 10% and 5% of the test article in physiological saline and applied to the clipped and shaved flanks of each of four guinea-pigs. The patches were covered by a strip of aluminum foil and firmly secured by elastic plaster wound round the trunk and covered with impervious adhesive tape. This procedure ensured intensive contact of the test article with the guinea pig skin. The dressings were removed after an exposure period of 24 hours and the reaction sites were assessed for erythema and edema on a numerical basis according to the scale described above. Further examination of the sites was performed 24 and 48 hours after removal of the dressings.
Prior to the first reading the test sites were depilated to facilitate the evaluation of a possible reaction.
The allocation of the different test sites on the animals was alternated in order to minimize site to site variation in responsiveness.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3
- Exposure period: 1 week
- Test groups:
1) Freunds' complete adjuvant 50:50 with bi-distilled water.
2) The test article, diluted to 5 % with physiological saline.
3) The test article at the concentration used in (2), emulsified in a 50:50 mixture of Freunds' complete adjuvant and the vehicle used in (2).
- Control group: The animals of control group were treated similar to the animals of the test group with the omission of the test article, but on the application site 3 the control animals were treated similar to injection site 1.
- Site: right and left
- Frequency of applications: not specified
- Duration: --
- Concentrations: 5%

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 1
- Exposure period: 1
- Test groups: Hair was clipped and shaved from a 5 x 5 cm area on the left and right flank of each guinea-pig. Two patches ( 2 x 2 cm) of filter paper were saturated with a) non-irritant concentration (10 % in physiological saline) of the test article and b) with the vehicle only and applied to the left (a) flank and right (b) flank respectively using the same method as for the epidermal application.
- Control group: The control animals were treated in the same way as described above.
- Site: --
- Concentrations: 10%
- Evaluation (hr after challenge): 24 and 48

Challenge controls:
Erythema and edema reactions
Positive control substance(s):
yes
Remarks:
Formaldehyde-solution

Results and discussion

Positive control results:
PROJECT 237060: Test for contact hypersensitivity in the albino guinea pig with FORMALDEHYDE-SOLUTION (HCHO). The guinea pig maximization test. Positive Control.

SUMMARY AND CONCLUSION
"Allergic Contact Dermatitis in the Guinea Pig: Identification of Contact Allergens" Magnusson B. Kligman A. M., 1970 published by C. C. Thomas, Springfield, Illinois, U. S. A.
According to the procedures used in this experiment (run during April 1989), clear positive results were observed in the HCHO treated animals after the epidermal challenge application.
POSITIVE ERYTHEMA REACTIONS AFTER THE FIRST CHALLENGE PROCEDURE:
after 24 hours, positive 6 / total 10
% positive of total 60
For the induction period a 10 % dilution of HCHO in bi-distilled water, and for the challenge procedure a 5 % dilution of HCHO was used.
According to the results observed it is considered that HCHO does possess a strong skin sensitizing (contact allergenic) potential in the guinea pig strain used (Dunkin-Hartley albino guinea pigs; DOHA KFM. Kleintierfarm Madoerin AG, CH-4414 Fuellinsdorf).
The positive control article HCHO (Formaldehyde-solution) was delivered by Fluka AG, 9470 Buchs, Switzerland (Article No. 4003 ), and the purity was described to be at least 37%.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
10%
No. with + reactions:
0
Total no. in group:
9
Clinical observations:
no erythema, no oedema
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
10%
No. with + reactions:
0
Total no. in group:
9
Clinical observations:
no erythema, no oedema
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
vehicle only
No. with + reactions:
0
Total no. in group:
9
Clinical observations:
no erythema, no oedema
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
vehicle only
No. with + reactions:
0
Total no. in group:
9
Clinical observations:
no erythema, no oedema
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
10%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
no erythema, no oedema
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
10%
No. with + reactions:
0
Total no. in group:
18
Clinical observations:
Females no. 383 and 387 died on day 25 of test. No toxic symptoms were evident in the guinea pigs of neither the control nor test group.
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
vehicle only
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
no erythema, no oedema
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
vehicle only
No. with + reactions:
0
Total no. in group:
18
Clinical observations:
no erythema, no oedema Females no. 383 and 387 died on day 25 of test. No toxic symptoms were evident in the guinea pigs of neither the control nor test group.
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
10% with HCHO
No. with + reactions:
6
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation

Any other information on results incl. tables

SENSITIZING EFFECTS

CONTROL GROUP: No positive reactions were evident after the first challenge application neither when treated with physiological saline on the right nor when treated

with a 10% test article dilution on the left flank.

TEST GROUP: No positive reactions were evident after the first challenge application neither when treated with physiological saline on the right nor when treated with a 10% test article dilution on the left flank.

Due to the unequivocal findings observed after the first challenge, no second challenge was performed.

MORTALITY / VIABILITY: Females no. 362 and 387 died spontaneously on day 24 respectively 25 of test and female no. 383 died in extremis on day 25 of test.

MACROSCOPICAL FINDINGS: Females no. 362, 383 and 387- Lungs: not collapsed. Discoloration, dark-red.

SYMPTOMS, LOCAL

CONTROL GROUP: Application area around the injection sites 1 and 3 was found to show erythema and edema from day 2 to 4; necroses were observed from day 5 to 12; desiccation from day 13 to 15 and exfoliation from day 13 to 25 (termination of test). In addition discoloration was observed on days 23 to 25 on the first challenge application area.

TEST GROUP: Application area around the injection site 1 was found to show erythema and edema from day 2 to 4; necroses were observed from day 5 to 12; desiccation from day 12 to 15 and exfoliation from day 13 to 25 (termination of test).

Application area around the injection sites 2 and 3 was found to show edema and discoloration from day 2 to 5; further local symptoms were similar to the symptoms observed around the injection site 1 but around the injection site 2 exfoliation ended at day 24 of test. In addition discoloration was observed on epidermal induction application area from day 10 to 25 and on first challenge application area from day 23 to 25.

On day 9 of the test no observation could be performed because the animals were treated semi-occlusively.

SYMPTOMS, SYSTEMIC

After intracutaneous induction application on day 1 of test, the test animals showed red discolored urine up to day 4 of test.

Female no. 383 showed dyspnea and emaciation on day 24 of test.

No further systemic symptoms were observed during the study.

BODY WEIGHTS

The body weight gain of the remaining animals was not affected during the study.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
From the results described above no allergenic potency of the test article was concluded. The results were interpreted according to the rating of Magnusson and Kligman (1970).
According to the CLP Regulation, this test article is considered to be not a sensitizer.
Executive summary:

The purpose of this skin sensitization study was to assess the allergenic potential of the test substance when administered to the skin of albino guinea pigs.

For this purpose the Maximization-Test of B. Magnusson and A.M. Kligman (1970) was used. Ten animals (10 females) were treated as control group and twenty animals (20 females) were treated as test group.

Prior to the first challenge reading of the reactions, the skin was depilated to clean the application site from staining produced by the test article, so that the reactions (erythema) were clearly visible at that time.

Due to the unequivocal findings observed after the first challenge, no second challenge was performed.