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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:
Sept to Dec 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
Directive 96/54/CEE, B.6 "Acute Toxicity - Skin Sensitization", July 30, 1996.
GLP compliance:
yes (incl. QA statement)
Remarks:
Switzerland GLP
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Data from a reliable in vivo test conducted before the enforcement of Commission Regulation (EU) 640/2012 of 06 July 2012 amending, for the purpose of its adaptation to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) are available.

Test material

Constituent 1
Chemical structure
Reference substance name:
reaction mass of: 7-amino-3,8-bis-[4-(2-sulfoxyethylsulfonyl)phenylazo]-4- hydroxynaphthalene-2-sulfonic acid, Na/K salt; 7-amino-3-[4-(2-sulfoxyethylsulfonyl)phenylazo]-4-hydroxy-8-[4-(2-sulfoxyethylsulfonyl)-2- sulfophenylazo]naphthalene-2-sulfonic acid, Na/K salt; 7-amino-8-[4-(2-sulfoxyethylsulfonyl)-phenylazo]-4-hydroxy-3-[4-(2-sulfoxyethylsulfonyl)- 2-sulfophenylazo]naphthalene-2-sulfonic acid, Na/K salt; 7-amino-3,8-bis-[4-(2-sulfoxyethylsulfonyl)-2-sulfophenylazo]-4-hydroxynaphthalene-2- sulfonic acid, Na/K salt
EC Number:
429-070-4
EC Name:
reaction mass of: 7-amino-3,8-bis-[4-(2-sulfoxyethylsulfonyl)phenylazo]-4- hydroxynaphthalene-2-sulfonic acid, Na/K salt; 7-amino-3-[4-(2-sulfoxyethylsulfonyl)phenylazo]-4-hydroxy-8-[4-(2-sulfoxyethylsulfonyl)-2- sulfophenylazo]naphthalene-2-sulfonic acid, Na/K salt; 7-amino-8-[4-(2-sulfoxyethylsulfonyl)-phenylazo]-4-hydroxy-3-[4-(2-sulfoxyethylsulfonyl)- 2-sulfophenylazo]naphthalene-2-sulfonic acid, Na/K salt; 7-amino-3,8-bis-[4-(2-sulfoxyethylsulfonyl)-2-sulfophenylazo]-4-hydroxynaphthalene-2- sulfonic acid, Na/K salt
Cas Number:
214362-06-8
Molecular formula:
C26H(25-y-x)KyN5NaxO16S5 / C26H(25-y-x)KyN5NaxO19S6 / C26H(25-y-x)KyN5NaxO19S6 / C26H(25-y-x)KyN5NaxO22S7
IUPAC Name:
octapotassium octasodium 7-amino-4-hydroxy-3,8-bis(2-{2-sulfonato-4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl)naphthalene-2-sulfonate 7-amino-4-hydroxy-3,8-bis(2-{4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl)naphthalene-2-sulfonate 7-amino-4-hydroxy-3-(2-{2-sulfonato-4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl)-8-(2-{4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl)naphthalene-2-sulfonate 7-amino-4-hydroxy-8-(2-{2-sulfonato-4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl)-3-(2-{4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl)naphthalene-2-sulfonate
Test material form:
solid: particulate/powder
Details on test material:
Test item name: Reactive Brown 49
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: TVR50
- Expiration date of the lot/batch: 01-SEP-2004

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In the original container at room temperature (approx. 20 °C) away from direct sunlight.
- Stability under test conditions: Stable in bi-distilled water, FCA and saline for at least 24 h

In vivo test system

Test animals

Species:
guinea pig
Strain:
Himalayan
Remarks:
Ibm: GOHI; (Himalayan spotted)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd, Biotechnology & Animal Breeding Division, Wölferstrasse 4, CH-4414 Füllinsdorf/ Switzerland
- Age at study initiation: 4 - 6 weeks
- Weight at study initiation: Pretest groups: 341 - 364 g ; Control and test group : 344.408 g
- Housing: Individually in Makrolon type-4 cages with standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz).
- Diet: Pelleted standard Nafag Ecosan 845 25W4, batch nos. 52/98 and 76/98 guinea pig breeding / maintenance diet ("Nafag", Nähr- und Futtermittel AG, CH-9202 Gossau), ad libitum. Results of analyses for contaminants are archived at RCC.
- Water: Community tap water from Füllinsdorf, ad libitum. Once weekly additional supply of ascorbic acid (approx. 1 g/l) via the drinking water was provided. Results of bacteriological, chemical and contaminant analyses are archived at RCC.
- Acclimation period: One week for the control and test group under test conditions after health examination. No acclimatization for the animals of the pretest. Only animals without any visible signs of illness were used.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 23
- Humidity (%): 36 - 79
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12-h light, 12-h dark cycle

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
water
Remarks:
bidistilled water
Concentration / amount:
5% ca. 0.1 mL/site
Day(s)/duration:
Day 1
Adequacy of induction:
highest technically applicable concentration used
Route:
epicutaneous, occlusive
Vehicle:
water
Remarks:
distilled water
Concentration / amount:
50% ca. 0.3 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
Remarks:
bidistilled water
Concentration / amount:
10% ca. 0.2 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 negative control group: 5
Details on study design:
INDUCTION
INTRADERMAL INJECTIONS / PERFORMED ON TEST DAY 1
An area of dorsal skin from the scapular region (approximately 6 x 8 cm) was clipped free of hair. Three pairs of intradermal injections (0.1 ml/site) were made at the border of a 4 x 6 cm area in the clipped region.
Test Group:
1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
2) The test article, at 5 % in bi-distilled water.
3) The test article at 5 % in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.

Control Group:
1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
2) Bi-distilled water
3) 1:1 (w/w) mixture of bi-distilled water in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.

EPIDERMAL APPLICATIONS / PERFORMED ON TEST DAY 8
One week after the injections, the scapular area (approximately 6 x 8 cm) was again clipped and shaved free of hair prior to the application. A 2 x 4 cm patch of filter paper was saturated with the test article (50 % in bi-distilled water) and placed over the injection sites of the test animals. The volume of test article applied was approximately 0.3 ml. The patch was covered with aluminum foil and firmly secured by an elastic plaster wrapped around the trunk of the animal and secured with impervious adhesive tape. The dressings were left in place for 48 h. The epidermal application procedure described ensured intensive contact of the test article.
The guinea pigs of the control group were treated as described above with bi-distilled wateronly.
Reaction sites were assessed for erythema and oedema 24 and 48 hours after removal of the dressing, using the numerical grading system according to Draize.

CHALLENGE / PERFORMED ON TEST DAY 22
The test and control guinea pigs were challenged two weeks after the epidermal induction application. The test and control guinea pigs were treated in the same way.
Hair was clipped and shaved from a 5 x 5 cm area on the left and right flank of each guinea pig just prior to the application. Two patches (3x3 cm) of filter paper were saturated with the highest non-irritating concentration of 10 % (left flank) and the vehicle only (bi-distilled water applied to the right flank) using the same method as for the epidermal application. The volume of test article applied was approximately 0.2 ml. The dressings were left in place for 24 h.
Approximately 21 h after removal of the dressing the test sites treated with the test article were depilated as described in the epidermal pretest.
Approximately 24 and 48 h after the removal of the dressing the application sites were assessed for erythema and oedema using the numerical scoring system according to Draize.
Positive control substance(s):
yes
Remarks:
alpha-hexylcinnamaldehyde

Results and discussion

Positive control results:
The intradermal induction of sensitization was performed with a 5 % dilution of the alpha-hexylcinnamaldehyde in polyethylene glycol (PEG 400) and in an emulsion of Freund's Complete Adjuvant (FCA) / physiological saline. The epidermal induction of sensitization was conducted under occlusion with the test article at 50 % in PEG 400. Two weeks after the epidermal induction application the first challenge was completed by epidermal application of the test article at 1 % in PEG 400 under occlusive dressing. Due to equivocal findings and weak effects after the first challenge, a second challenge was performed two weeks later using the test article at 5 % and 3 % in PEG 400. The animals of the control group I were induced with PEG 400 and FCA/physiological saline and challenged similarly to those of the test group. An additional control group (control group H) pretreated with FCA/physiological saline was also treated in the same conditions as the test group during the second challenge. Cutaneous reactions, i.e. erythema and eschar, as well as oedema formation were evaluated at 24 and 48 h after removal of the dressing.

There were 90% positive at 5% concentration and 40% at 3% concentration after 24 h challenge while 80% positive at 5% concentration and 30% at 3% concentration after 48 h challenge respectively.

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:
8
Clinical observations:
Two animals of the test group were found dead on test day 15
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:
8
Clinical observations:
Two animals of the test group were found dead on test day 15
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
0%
No. with + reactions:
0
Total no. in group:
8
Clinical observations:
Two animals of the test group were found dead on test day 15
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
10%
No. with + reactions:
0
Total no. in group:
8
Clinical observations:
Two animals of the test group were found dead on test day 15
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
5
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
10%
No. with + reactions:
0
Total no. in group:
5
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
5
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
10%
No. with + reactions:
0
Total no. in group:
5
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
5%
No. with + reactions:
9
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
5%
No. with + reactions:
8
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
3%
No. with + reactions:
4
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
3%
No. with + reactions:
3
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation

Any other information on results incl. tables

Signs of irritation during induction:
As the test article stained the skin red, it was not possible to determine whether erythema was present or not after application of the test article at 50 % in bi-distilled water. However, no oedema was observed in any animals after epidermal induction performed.

Evidence of sensitisation of each challenge concentration: 10% in bi-distilled water : 0/8 animals show a sensitisation.

MORTALITY: two animals were found dead on day 15 ; the origin of the death could not be established.During the preliminary test, a light erythematous was observed at the concentrations 15%, 25% and 50%. This is the reason why the concentration of 10% was established for the main study.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Reactive Brown 49 is considered not to be a sensitizer.
Executive summary:

Skin sensitization study of FAT 40571/A was carried out using Guinea pig maximization test according to OECD 406 and EU B.6 guideline. The intradermal induction of sensitization was performed with a 5 % dilution of the test article in bi-distilled water and in an emulsion of Freund's Complete Adjuvant (FCA) / physiological saline. The epidermal induction of sensitization was conducted under occlusion with the test article at 50 % in bi-distilled water. Two weeks after the epidermal induction application the challenge was completed by epidermal application of the test article at 10 % in bi-distilled water under occlusive dressing. The animals of the control group were induced with bi-distilled water and FCA/physiological saline and challenged similarly to those of the test group. Cutaneous reactions, i.e. erythema and eschar, as well as oedema formation were evaluated at 24 and 48 h after removal of the dressing.

None of the surviving animals of the test group were observed with erythematous reactions after 24 h (0/8) and 48 h (0/8) post treatment with a non-irritant test article concentration of 10 % in bi-distilled water. No skin reactions were observed in the control group. No toxic symptoms were evident in the guinea pigs of the control or test group. Two animals of the test group were found dead on test day 15. At necropsy, congestion in the lungs, distended stomach and clay-colored liver were noted. A cause of death could not be established.

Therefore, the test substance is considered not to be a skin sensitizer.