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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:
From 31 January, 1994 to 24 February, 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report date:
1994

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
Justification for non-LLNA method:
Data from a reliable in vivo test (non-LLNA method) conducted before the enforcement of Commission Commission Regulation (EU) 2017/706 of 19 April 2017 amending Annex VII to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) as regards skin sensitisation are available.

Test material

Constituent 1
Chemical structure
Reference substance name:
Pentasodium 4-amino-6-[[5-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-2-sulphonatophenyl]azo]-3-[(2,5-disulphonatophenyl)azo]-5-hydroxynaphthalene-2,7-disulphonate
EC Number:
269-505-3
EC Name:
Pentasodium 4-amino-6-[[5-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-2-sulphonatophenyl]azo]-3-[(2,5-disulphonatophenyl)azo]-5-hydroxynaphthalene-2,7-disulphonate
Cas Number:
68259-02-9
Molecular formula:
C25H19ClN10O16S5.5Na
IUPAC Name:
pentasodium 4-amino-6-({5-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-2-sulfonatophenyl}diazenyl)-3-[(2,5-disulfonatophenyl)diazenyl]-5-hydroxynaphthalene-2,7-disulfonate
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: Pirbright White Strain (Tif: DHP)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: CIBA-GEIGY Limited, Animal Production, 4332 Stein / Switzerland
- Weight at study initiation: 323 to 395 g
- Housing: Housed individually in Macrolon cages (Type 3)
- Diet: Standard guinea pig pellets (NAFAG No. 845, Gossau SG), ad libitum
- Water: Fresh water, ad libitum
- Acclimation period: 5 d

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3
- Humidity: 30 to 70 %
- Photoperiod: 12 h light/12 h dark

IN-LIFE DATES: From January 31, 1994 to February 24, 1994

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
physiological saline
Concentration / amount:
5 %
Day(s)/duration:
Day 0
Adequacy of induction:
highest technically applicable concentration used
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
50 %
Day(s)/duration:
Day 8
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Challenge
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
20 %
Day(s)/duration:
Day 21
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Preliminary study:
10 and 20 % (epidermal application on contralateral flanks): 1 animal/sex
30 and 50 % (epidermal application on contralateral flanks): 1 animal/sex

Main study:
Control group: 5 male animals
Test group: 10 animals (5/sex)
Details on study design:
RANGE FINDING TESTS:
Intradermal induction: The concentration for the intradermal injections was selected on account of the solubility of the test substance in standard vehicles and its local and systemic tolerability in a pretest. The 5 % concentration of the test substance in physiological saline (w/v) has been used for intradermal injection since this concentration could be injected and was well tolerated.
Epidermal Applications (induction and challenge): The concentrations (10, 20, 30 and 50 %) for the epidermal applications were selected on account of the primary irritation potential of the test substance. On each animal, two concentrations of the test substance were applied simultaneously on the left and right flanks. A naive skin site served as control (not reported). Seven days before application of the test substance, two pairs of intradermal injections of FCA/saline mixture 1:1 (v/v; 0.1 mL per injection) were made simultaneously into the shaved neck of the animals. Skin reactions were observed (at both 24 and 48 h observations) with 50 % test substance in physiological saline.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2
- Exposure period: Topical induction: 48 h
- Test groups: Intradermal injection: On Day 0, three pairs of intradermal injections (0.1 mL per injection) of test substance in physiological saline were made simultaneously into the left and right side of the clipped neck of the test animals.
- FCA/saline mixture 1:1 (v/v)
- 5 % test substance in physiological saline (w/v)
- 5 % test substance in the FCA/saline mixture (w/v)
Topical induction: On Day 8, 50 % test substance in physiological saline was applied on a filter paper patch to the neck of the animals (patch 2 x 4 cm²; approximately 0.4 g per patch; occluded administration for 48 h).
- Control group: Animals were treated as described above with the omission of test substance (physiological saline instead of test substance for both intradermal and topical inductions).
- Frequency of applications: Once intradermal, once epidermal

B. CHALLENGE EXPOSURE
- No. of exposures: Single
- Day(s) of challenge: Day 21
- Exposure period: 24 h
- Test and control groups: Animals were tested on one flank with test substance in physiological saline and on the other flank with the vehicle alone (patch 2 x 2 cm²; approximately 0.2 g per patch; occluded administration for 24 h).
- Concentrations: 20 %
- Observations: The skin reactions were evaluated before treatment, 24, 48 h after removal of dressing.

OBSERVATIONS:
Induction reactions: - After the intradermal and the epidermal induction application irritant reactions are normally induced by the adjuvant and the high test substance concentration. Because most of the reactions are treatment related and not test substance related, the reactions are only described in special cases.
Challenge reactions: 24 and 48 h after removing the dressings, the challenge reactions were graded according to the Draize scoring scale.
General: The body weight was recorded at start and end of the test.

Pathology: Macroscopical examination: At the end of the test period all controls and treated animals were bled under ether anesthesia and subjected to detailed necropsy. The following organs and tissues were preserved in neutral buffered 4% formalin:
skin application site
skin remote site
Microscopical examination: After the fixation, organ samples listed below were taken, embedded in paraplast, sectioned at 3-5 μ, stained with hematoxylin and eosin, and subjected to a microscopical examination.
skin application site
skin remote site
Organs and tissues other than the skin are not of toxicological relevance for this type of study, and, therefore, the macroscopically reported liver findings were not examined microscopically.
Presentation of pathology data: The histopathological lesions observed were graded as to degree of severity according to the following criteria:
Grade "1" : Minimal (slight). Includes histopathological change that is a noticeable but not prominent feature of the tissue.
Grade "2" : Moderate. Includes histopathological change that is a prominent but not dominant feature of the tissue.
The incidence "0" was listed when no finding was reported.
Challenge controls:
None
Positive control substance(s):
yes
Remarks:
(mercaptobenzothiazole tested in another study)

Results and discussion

Positive control results:
None

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
20 % epidermal application
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
Erythema of grade 1 in 9/10 animals and grade 2 in 1/10 animals
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 20 % epidermal application. No with. + reactions: 10.0. Total no. in groups: 10.0. Clinical observations: Erythema of grade 1 in 9/10 animals and grade 2 in 1/10 animals.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
20 % epidermal application
No. with + reactions:
9
Total no. in group:
10
Clinical observations:
Edema of grade 1
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 20 % epidermal application. No with. + reactions: 9.0. Total no. in groups: 10.0. Clinical observations: Edema of grade 1.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
20 % epidermal application
No. with + reactions:
4
Total no. in group:
10
Clinical observations:
Erythema of grade 1; four animals could not be evaluated due to blue staining by test substance
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 20 % epidermal application. No with. + reactions: 4.0. Total no. in groups: 10.0. Clinical observations: Erythema of grade 1; four animals could not be evaluated due to blue staining by test substance.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
20 % epidermal application
No. with + reactions:
8
Total no. in group:
10
Clinical observations:
Edema of grade 1
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 20 % epidermal application. No with. + reactions: 8.0. Total no. in groups: 10.0. Clinical observations: Edema of grade 1.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
Vehicle control
No. with + reactions:
0
Total no. in group:
5
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
Vehicle control
No. with + reactions:
0
Total no. in group:
5
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
Test article control
No. with + reactions:
1
Total no. in group:
5
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
Test article control
No. with + reactions:
0
Total no. in group:
5
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
Intradermal induction: 5 %, Epidermal induction: 50 %, Challenge: 30 %
No. with + reactions:
20
Total no. in group:
20
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
Intradermal induction: 5 %, Epidermal induction: 50 %, Challenge: 30 %
No. with + reactions:
20
Total no. in group:
20

Any other information on results incl. tables

- Under the experimental conditions, 100 % and 80 % of the animals of the test group showed skin reactions 24 and 48 h after removing the dressings, respectively. One animal of the control group showed reactions 24 h after removing the dressings. In three males and in one female (animals of the test group) a possible erythema reaction could not be evaluated because of blue staining ( test substance-related). Blue staining impeded also the evaluation in the other animals. Therefore, histopathological examination of the skin application and remote site was performed in all animals of the study.

- Microscopy: Microscopical examination of the skin showed a positive reaction in treated animals. Detailed reporting of pathology findings is tabulated below.

Table 1: Occurrence and severity of skin changes in individual animals

 Animal number  Sex  Group

       Application site
         Epidermal acanthosis  Inflammatory cell infiltration  Congestion
 #1  Male  Control  0  1  0
 #2  Male  Control  0  2  0
 #3  Male  Control  1  1  0
 #4  Male  Control  0  1  0
 #5  Male  Control  0  1  0
 #6  Male  Test  1  2  1
 #7  Male  Test  0  1  0
 #8  Male  Test  2  2  1
 #9  Male  Test  2  2  1
 #10  Male  Test  2  2  0
 #11  Female  Test  1  1  0
 #12  Female  Test  0  1  0
 #13  Female  Test  1  2  1
 #14  Female  Test  1  1  0
 #15  Female  Test  1  1  0

 

Acanthosis was observed at the skin application site of 4/5 treated animals, versus 2/5 in control group animals. This lesion was minimal in control animals and treated females and minimal to moderate in treated males. Acanthosis is a thickening of the epidermis as a result of hyperplasia of the malpighian layer, especially the prickle cell layer. An inflammatory cell infiltration was seen in the dermis and subcutis of all control and treated animals. However, at the application site of treated males the severity of this lesion was increased, when compared to control animals. The inflammatory cell infiltration consisted essentially of lymphohistiocytic cells and rare neutrophils. Eosinophils were present in a comparable number between control and treated animals at the skin application site. Minimal congestion of the dermis was detected at the skin application site of 3/5 treated males and of 1/5 treated females.

- Macroscopy: Mottled liver was observed in 2/5 males in control group and 1/5 males and 2/5 females in treatment group. No other symptoms of systemic toxicity were observed.

- Body weight: Body weights were not affected by test substance treatment.

- Mortality: No unscheduled deaths occurred during the study period.

PRELIMINARY STUDY:

- At the 24 h reading of epidermal application: Erythema was noted at 30 % (in 2/2 animals) and 50 % (in 2/2 animals) concentrations of the test substance. No skin reactions were observed at 10 and 20 % concentrations.

- At the 48 h reading of epidermal application: Erythema was noted at 30 % (in 1/2 animals) and 50 % (in 2/2 animals) concentrations of the test substance. No skin reactions were observed at 10 and 20 % concentrations.

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The test substance was found to be a sensitiser.
Executive summary:

A guinea pig maximization study was conducted to evaluate the skin sensitisation potential of the test substance (of ca. 100 % 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 50 % were selected as intradermal and epidermal induction doses, respectively. The highest non-irritating concentration used for occluded epidermal application during challenge exposure was 20 %. Skin reactions were graded at 24 and 48 h after removal of the dressing.

In the test substance treated group, 10/10 and 8/10 of the animals showed skin reactions 24 and 48 h after removing the dressing, respectively. One animal of the control group showed reactions 24 h after removing the dressing. In three males and one female, possible erythema reactions were not evaluable because of blue staining ( test substance related). Blue staining impeded also the evaluation in the other animals. Therefore, histopathological examination of the skin application and remote site was performed in all animals. Microscopical examination of the skin showed higher incidence and severity of positive reactions (acanthosis, inflammatory cell infiltration and congestion) in treated animals when compared to controls. No unscheduled deaths, changes in body weight or any clinical signs were observed. Macroscopic examination revealed mottled liver in both treatment (3/10) and control (2/5) animals.

Under the study conditions, the test substance was found to induce delayed contact hypersensitivity in 100 and 85 % of the test animals at 24 and 48 h observations, respectively.