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Diss Factsheets

Administrative data

Description of key information

The test substance 1-amino-4-({3-[(2-hydroxyethyl)sulfamoyl]-4,5-dimethylphenyl}amino)-9,10- dioxo-9,10-dihydroanthracene-2-sulfonic acid, sodium salt was shown to be a sensitizer in the Guinea Pig Maximization test.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
yes
Remarks:
(two test substance concentrations were applied at the first and second challenge)
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
yes
Remarks:
(two test substance concentrations were applied at the first and second challenge)
GLP compliance:
yes (incl. QA statement)
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.
Specific details on test material used for the study:
Identification: TECTILON BLAU 4R-01 (FAT 20'033/H)
Description: Blue solid.
Batch Number: 672001.29
Purity/formulation: Active ingredients: 72 %
Stability of test article: Stable; expiration date: January 1994.
Stability of test article dilution: Unknown in FCA/physiological saline (1:1), in water and in Vaselinum album; excluded from the Statement of Compliance.
Storage Conditions: Room temperature
Safety precautions: Gloves, goggles and face mask were obligatory to assure personnel health and safety.
Species:
guinea pig
Strain:
Himalayan
Remarks:
White spotted
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd. Wolferstrasse 4, CH-4414 Fullinsdorf/Switzerland.
- Age at acclimatization initiation: 6 - 8 weeks
- Weight at acclimatization initiation: 395 - 479 g (control and test group), 424 - 480 g (pretest)
- Housing: Individually in Makrolon type-3 cages (size: 22 x 37 x 15 cm) with standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz).
- Diet: Pelleted standard Kliba 342, Batch 74/92 guinea pig breeding/ maintenance diet ("Kliba", Klingentalmuhle AG, CH-4303 Kaiseraugst) (ad libitum)
- Water: Community tap water (ad libitum); Once weekly additional supply of ascorbic acid (1 g/L) via the drinking water
- Acclimation period: One week
- Identification: By unique cage number and corresponding ear tags.
- Randomization: Randomly selected at time of delivery
- Animal distribution: Ten males for the control group and 20 males for the test group. Two males were used for the intracutaneous pretest and 4 males for the epicutaneous pretest

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 40-70 %
- Air changes: 10-15 air changes/h
- Photoperiod: 12 h/12 h (music during the light period)

IN-LIFE DATES: January 06, 1993, to February 23, 1993
Route:
intradermal
Vehicle:
other: Freund's complete adjuvant 50:50 with physiological saline and Bidistilled water.
Concentration / amount:
5 %
Day(s)/duration:
1
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, semiocclusive
Vehicle:
other: Vaselinum album
Concentration / amount:
25 %
Day(s)/duration:
7
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
No.:
#1
Route:
epicutaneous, semiocclusive
Vehicle:
other: vaselinum album
Concentration / amount:
15 % and 25 %
Day(s)/duration:
22
Adequacy of challenge:
highest non-irritant concentration
No.:
#2
Route:
epicutaneous, semiocclusive
Vehicle:
other: Vaselinum album
Concentration / amount:
15 % and 25 %
Day(s)/duration:
36
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Preliminary study:
Intradermal injections group: 2 animals
Epicutaneous applications group: 4 animals

Main study:
Control group: 10 animals
Test group: 20 treated animals
Details on study design:
PRETEST:

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 substance in bi-distilled water. The resulting dermal reactions were assessed 24 h later. For intracutaneous induction application a 5% test substance dilution in bi-distilled water was selected.

EPIDERMAL APPLICATIONS:
The both flanks of each of 4 guinea pigs were clipped and shaved just prior to the application. Thereafter patches of filter paper (2 x 2 cm) were covered with a thin layer of the test substance in vaselinum album at concentrations of 25, 15, 10 and 5% 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 wrapped around the trunk and covered with impervious adhesive tape. This procedure ensured the intensive contact of the test substance. The dressings were removed after an exposure period of 24 h and the reaction sites were assessed for erythema and edema on a numerical basis according to Draize score. Examination of the sites were performed 24 and 48 h after removal of the dressings.
After removal of the dressing, the application site was depilated with an approved depilatory cream to clean the application site from staining produced by the test substance, so that possible erythema reactions were clearly visible at that time. The depilatory was placed on the path sites and surrounding areas and left on for fifteen (15) minutes or less. It was then thoroughly washed off with a stream of warm, running water. The animals were then dried with a disposable towel and returned to their cages.
The allocation of the different test sites on the animals was alternated in order to minimize site to site variation in responsiveness. Due to the results observed in the epidermal pretest at the 25% concentration, for the epidermal induction a 25% test substance dilution and for the challenge 25% and 15% concentration was selected.

MAIN STUDY
INDUCTION
Intradermal injections:
An area of dorsal skin from the scapular region (approx. 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 as follows:

Test group:
1) Freund's complete adjuvant 50:50 with physiological saline.
2) The test substance, diluted to 5% with bi-distilled water.
3) The test substance diluted to 5% by emulsion in a 50:50 mixture of Freund's complete adjuvant and physiological saline.

Control Group:
1) Freund's complete adjuvant 50:50 with physiological saline.
2) Bi-distilled water.
3) Freund's complete adjuvant 50:50 with physiological saline.

Epidermal applications:
On Day 7 of the test and approx. 24 h prior to the epidermal application the scapular area (approx. 6 x 8 cm) was clipped, shaved free of hair and the test areas were pretreated with 10% Sodium lauryl sulfate (SLS) in petrolatum oil because no primary irritation concentration could be determined in the corresponding pretest. The SLS was massaged into the skin with a glass rod without bandaging. This SLS concentration enhances sensitization by provoking a mild inflammatory reaction.
On test Day 8 a 2 x 4 cm patch of filter paper was covered with a thin layer of the selected test substance concentration (25% in vaselinum album) and placed over the injection sites of the test animals. 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 approx. 48 h. The epidermal application procedure described ensured intensive contact of the test substance.
The guinea-pigs of the control group were treated as described above with the omission of test substance (vaselinum album only). Reaction sites were assessed for erythema and oedema 24 and 48 h after removal of the dressing, using the numerical grading system according to Draize.

FIRST CHALLENGE
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. Three patches (2 x 2 cm) of filter paper were covered with a thin layer of a) test substance concentrations, 15% and 25% in vaselinum album and b) with vaselinum album only and applied to the a) anterior left flank and posterior right flank respectively left flank, and b) anterior right flank using the same method as for the epidermal application. The dressings were removed approx. 24 h later. The sites were assessed for erythema and edema 24 and 48 h after removal of the dressing, using the numerical scoring system according to Draize.
After removal of the dressing, each application site was depilated with an approved depilatory cream to clean the application site from staining produced by the test substance, so that possible erythema reactions were clearly visible at that time. The depilatory was placed on the patch sites and surrounding areas and left on for fifteen (15) minutes or less. It was then thoroughly washed off with a stream of warm, running water. The animals were then dried with a disposable towel and returned to their cages.

SECOND CHALLENGE
A second challenge was performed two weeks after the first challenge. The treatment procedure for the animals of the test group was similar as described for the first challenge. The test substance concentrations of 15% and 25% were applied on the left flank, posterior and anterior, respectively. The control animals were treated with vaselinum album only on the left flank.


OBSERVATIONS:
In addition to the sensitizing reactions the following observations and data were recorded during the test and observation period:
Viability/Mortality: Daily
Body Weights: At acclimatization start, at Day 1 and at termination of test
Clinical Signs (local/systemic): Daily
Skin reactions: At the time of reactions readings during induction and challenge period.
Necropsy: No necropsy was performed in all animals euthanized at termination of observation. All animals were euthanized at the end of the test period with an intraperitoneal injection of Pentobarbitone (>800 mg/kg bw) and discarded.
Challenge controls:
First challenge: Test substance (25% (anterior left flank); 15% (posterior right flank)) and Vaseline album (anterior right flank)
Second challenge: Vaseline album (right flank)
Positive control substance(s):
yes
Remarks:
2-mercaptobenzothiazole tested in another study
Positive control results:
50 % (5/10) showed positive reactions following challenge phase.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
25 % epidermal application (anterior left flank)
No. with + reactions:
1
Total no. in group:
20
Clinical observations:
very slight erythematous reaction, grade 1
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
15 % epidermal application (posterior right flank)
No. with + reactions:
3
Total no. in group:
20
Clinical observations:
very slight erythematous reaction, grade 1
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
25 % epidermal application (anterior left flank)
No. with + reactions:
1
Total no. in group:
20
Clinical observations:
very slight erythematous reaction, grade 1
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
15 % epidermal application (posterior right flank)
No. with + reactions:
6
Total no. in group:
20
Clinical observations:
very slight erythematous reaction, grade 1
Reading:
rechallenge
Hours after challenge:
24
Group:
test chemical
Dose level:
25 % epidermal application (anterior left flank)
No. with + reactions:
9
Total no. in group:
20
Clinical observations:
erythematous reactions, grade 1 to 2; slight edema, grade 1
Reading:
rechallenge
Hours after challenge:
24
Group:
test chemical
Dose level:
15 % epidermal application (posterior left flank)
No. with + reactions:
8
Total no. in group:
20
Clinical observations:
erythematous reactions, grade 1 to 2; slight edema, grade 1
Reading:
rechallenge
Hours after challenge:
48
Group:
test chemical
Dose level:
25 % epidermal application (anterior left flank)
No. with + reactions:
6
Total no. in group:
20
Clinical observations:
erythematous reactions, grade 1 to 2
Reading:
rechallenge
Hours after challenge:
48
Group:
test chemical
Dose level:
15 % epidermal application (posterior left flank)
No. with + reactions:
4
Total no. in group:
20
Clinical observations:
erythematous reactions, grade 1 to 2
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
25 %
No. with + reactions:
5
Total no. in group:
10
Clinical observations:
Erythematous reaction (Grade 2)
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
25 %
No. with + reactions:
5
Total no. in group:
10
Clinical observations:
Erythematous reaction (Grade 2)
Remarks on result:
positive indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
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
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation

SKIN EFFECTS AFTER THE EPIDERMAL INDUCTION:


5 % (only one animal) of the test animals showed a positive oedema reaction, grade 1, at the 24 h reading when treated with a 25 % test substance dilution. Due to the dark-blue discolored skin of the test substance treated animals no possible erythema reactions could be detected.


 


CONTROL GROUP:


No positive skin reactions were observed in the animals treated with vaselinum album only.


 


SKIN EFFECTS AFTER THE CHALLENGES:


CONTROL GROUP:


No positive reactions were evident after the first challenge application, neither when treated with the vehicle alone nor when treated with the nonirritating test substance concentration of 15 % and with the test substance at 25 % in vaselinum album. After the second challenge no skin reactions were observed with vaselinum album alone.


 


VIABILITY/MORTALITY:


No death occurred during the entire study period.


 


CLINICAL SIGNS, LOCAL


CONTROL GROUP:


After induction injection at the application sites 1 and 3 erythema and oedema were observed between test 2 and 5 d; necrosis between test 6 and 8 d; encrustation between test 10 and 13 d and exfoliation between test 14 and 42 d (termination of the test). At the injection site 2 only erythema and oedema reactions were observed between 2 and 5 d of the test period. In addition after first epidermal challenge with the test substance discoloration was observed between 23 and 27 d of the test.


 


TEST GROUP:


After induction injection discoloration and oedema were observed between 2 and 5 d at the application site 3, erythema and oedema in the same period at the application site 1. For the both application sites necroses were observed between 6 and 8 d; encrustation between days 10 and 13 and exfoliation between 14 and 42 d of the study period. At the injection site 2 discoloration and oedema were observed between 2 and 5 d; necroses between 6 and 8 d, encrustation between 10 and 13 d and exfoliation between 14 and 17 d of the study period. In addition after the epidermal induction, first and second challenge with the test substance discoloration was observed between Days 10 to 23, 23 to 27 or 37 and 42, respectively. On 9 d of test, no observation could be performed because the animals were bandaged semiocclusively.


 


CLINICAL SIGNS, SYSTEMIC:


No systemic symptoms were observed in the animals.


 


BODY WEIGHTS:


Eight of 10 control animals, 16 of 20 test animals and all animals used for the intradermal and epidermal pretest lost weight during the acclimation period.

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The test substance was considered to induce delayed contact hypersensitivity in 40 and 45 % of the animals after the second challenge when treated with the test substance concentrations of 15 and 25 %, respectively.
Executive summary:

A guinea pig maximization study was conducted to evaluate the skin sensitization potential of the test substance (of ca. 72% purity) 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 % were selected as intradermal and topical induction doses respectively. The highest non-irritating concentration used for challenge application was 15 %. However, an additional concentration of 25 % was applied in the challenge procedure because only 1/4 animals showed a positive skin reaction during the epidermal pre-test. A second challenge was performed two weeks after the first one. The treatment procedure used for the animals in the test group was the same as for the first challenge except for the application site for the 15 % concentration. In the first challenge, one animal showed an erythema score of grade 1 at 24 and 48 h when treated at 25 %. At 15 %, 3 animals showed positive reactions (score 1) after 4 h and 6 animals (score 1) after 48 h. After the second challenge, when treated with the 25 %, a total of 9 animals showed positive erythema reactions (score 1: seven animals; score 2: two animals) and 2 animals showed positive oedema reactions (score 1) after 24 h. After 48 h, only 6 animals showed positive erythema responses (score 1: five animals; score 2: one animal). After treatment with the 15 % test substance concentration, a total of 8 animals showed positive erythema responses at the 24 h reading of the second challenge (score 1: six animals; score 2: two animals). In addition, 2 animals showed oedema responses of score 1 at the same time. At the 48 h reading of the second challenge, 3 animals showed an erythema score of 1 and one animal with a score of 2. Under the study conditions, the test substance was considered to induce delayed contact hypersensitivity in 40 and 45 % of the animals after the second challenge when treated with the test substance concentrations of 15 and 25 %, respectively. The test substance is classified as Skin sensitizer Category -1B according Regulation (EC) No 1272/2008 of CLP.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Two sensitization studies were performed on the test substance 1-amino-4-({3-[(2-hydroxyethyl)sulfamoyl]-4,5-dimethylphenyl}amino)-9,10 - dioxo-9,10-dihydroanthracene-2-sulfonic acid, sodium salt including a Guinea Pig Maximization Test (OECD 406) and a Buehler Test. In the Maximization Test, the highest non-irritating test article concentrations used for challenge application was 15 %. Additionally, a 25 % test substance concentration was applied because one animal out of 4 showed positive erythema reactions (grade 1) after 24 hours in the epidermal pretest. In this study 40 % and 45 % of the animals were found to have positive reactions after the second challenge when treated with the test substance concentrations of 15 % and 25 %, respectively. In the Buehler test no signs of irritation were noted during the induction and after challenge in the treated group. The macroscopic examinations did not show evidence of any pathological lesion of delayed hypersensitivity in the 20 guinea pigs of the treated group. No cutaneous abnormalities were noted in the 10 guinea pigs of the control group. From the results obtained under the experimental conditions employed, the test article did not provoke any reaction of cutaneous sensitization. However, the authors note that test article has tinted the skin thus making observation of erythema imprecise.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

- skin sensitisation:


Based on the above stated assessment of the skin sensitisation potential in the Maximization Test, the substance should be classified as a skin sensitizer Catagory 1B according CLP (Regulation (EC) No 1272/2008 of The European Parliament and of The Council) as implementation of UN-GHS in the EU.


- respiratory sensitisation:


As no data on respiratory sensitization is available for the substance a classification is not possible according to CLP (Regulation (EC) No 1272/2008 Of The European Parliament and of The Council)as implementation of UN-GHS in the EU.