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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 December 21, 1988 to January 14, 1989.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The test procedures are well documented and scientifically acceptable.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Principles of method if other than guideline:
Intradermal injection and topical skin application (Magnusson-Kligman), protocol P-252.
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The test was performed before the validation of LLNA OECD method and is considered to be valid and acceptable for the assessment.

Test material

Constituent 1
Chemical structure
Reference substance name:
Disodium 4,4'-bis[[4-anilino-6-[(2-carbamoylethyl)(2-hydroxyethyl)amino]-1,3,5,-triazin-2-yl]amino]stilbene-2,2'-disulphonate
EC Number:
248-420-5
EC Name:
Disodium 4,4'-bis[[4-anilino-6-[(2-carbamoylethyl)(2-hydroxyethyl)amino]-1,3,5,-triazin-2-yl]amino]stilbene-2,2'-disulphonate
Cas Number:
27344-06-5
Molecular formula:
C42H46N14O10S2.2Na
IUPAC Name:
disodium 4,4'-bis[[4-anilino-6-[(2-carbamoylethyl)(2-hydroxyethyl)amino]-1,3,5,-triazin-2-yl]amino]stilbene-2,2'-disulphonate

In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Davidson's Mill Farm, NJ.
- Number of animals: 15.
-Weight: 250-320 g.
- Acclimation period: 9 days.
- Housing: in cage, five per suspended stainless steel gang cages with mesh floors.
- Diet: ad libitum, Purina Guinea Pig.
- Water: ad libitum.
- Identification: ear tag bearing, with an unique identification number.

ENVIRONMENTAL CONDITIONS
- Temperature: 65-74 F.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
other: water and Freunds Complete Adjuvant
Concentration / amount:
A. INDUCTION EXPOSURE
0.05 ml in water (5 %).
0.05 % in 50 % aqueous Freunds Complete Adjuvant.
0.05 % in 50 % aqueous Freunds Complete Adjuvant at multiple site.

B. CHALLENGE
0.1 ml.
Challengeopen allclose all
Route:
epicutaneous, open
Vehicle:
other: water and Freunds Complete Adjuvant
Concentration / amount:
A. INDUCTION EXPOSURE
0.05 ml in water (5 %).
0.05 % in 50 % aqueous Freunds Complete Adjuvant.
0.05 % in 50 % aqueous Freunds Complete Adjuvant at multiple site.

B. CHALLENGE
0.1 ml.
No. of animals per dose:
10 animals.
Details on study design:
SITE
A. INDUCTION EXPOSURE
Intradermal injection
- Test site: anterior left and right shoulder.
- Frequency of application: single application.
- Type of application: intradermal injection.
- Date of application: December 21, 1988.

Topical Induction (Booster)
- Test site: anterior left and right shoulder.
- Frequency of application: 7 days after first application on shoulders, by Hilltop Chamber.
- Type of application: intradermal injection.
- Date of the first topical application: December 28, 1988.
- Exposure period: 48 hours for the first application.

B. CHALLENGE
- Test site: shaved flanks.
- Frequency of application: 14 days after the induction.
- Type of application: topical application by Hilltop Chamber.
- Date of the challenge dose: January 11, 1989.
- Exposure period: 24 hours for the second application.
Challenge controls:
Evaluation:
- at 72 and 72 hours after the initial topical application;
- at 48 and 72 hours after challenge.

SCORING:
No evidence of any effect: 0
(Barely Perceptible) - minimal faint (light pink) uniform or spotty erythema: 0.5
(Mild) - Pink uniform erythema covering most of contact site: 1
(Moderate) - Pink-red erythema uniform in entire contact area: 2
(Marked) - Bright red erythema with accompanying edema, petachiae or papules: 3
(Severe) - Deep red erythema with vesiculation or weeping, with or without edema: 4

The sensitization potential (Sensitization Rate) of the test product was determined by determining the ratio of positive responses with scores greater than 0.5, 48 hours after patch removal to the total number of animals tested.
The degree of sensitization (erythema index) was calculated by adding the scores of 1 or more and dividing by the number of animals exhibiting a positive response.
See table 2 for further details.
Positive control substance(s):
yes

Results and discussion

Positive control results:
CHALLENGE SCORE
0/0.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
other: Sensitization Rate
Hours after challenge:
48
Group:
test chemical
Dose level:
0.1 ml
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: other: Sensitization Rate. . Hours after challenge: 48.0. Group: test group. Dose level: 0.1 ml. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
other: Sensitization Rate
Hours after challenge:
72
Group:
test chemical
Dose level:
0.1 ml
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: other: Sensitization Rate. . Hours after challenge: 72.0. Group: test group. Dose level: 0.1 ml. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
other: Erythema Index
Hours after challenge:
48
Group:
test chemical
Dose level:
0.1 ml
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: other: Erythema Index. . Hours after challenge: 48.0. Group: test group. Dose level: 0.1 ml. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
other: Erythema Index
Hours after challenge:
72
Group:
test chemical
Dose level:
0.1 ml
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: other: Erythema Index. . Hours after challenge: 72.0. Group: test group. Dose level: 0.1 ml. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
other: all parameters
Group:
negative control
Remarks on result:
not measured/tested
Reading:
other: all parameters
Group:
positive control
Remarks on result:
not measured/tested

Any other information on results incl. tables

RESULTS

General: apart from the slight irritation observed after the topical booster application, all guinea pigs appeared active and healthy throughout the test period. There were no signs of gross toxicity, adverse pharmacologic effects or abnormal behaviour. All animals gained weight.

Induction: barely perceptible to mild erythema was observed at all sites 24 hours after the topical booster application. By 48 hours all sites were clear.

Challenge: there was no evidence of erythema at any sites on any of the test animals 24 or 48 hours after challenge. Similarly, no irritation occurred on any of the dosed sites on the naive Guinea Pigs.

Erythema after induction and challenge

ANIMAL N. INDUCTION SCORE CHALLENGE SCORE
Test animal 24 h 48 h 24 h 48 h
3359 0.5/0 0/0 0/0 0/0
3360 0/0.5 0/0 0/0 0/0
3361 0.5/0.5 0/0 0/0 0/0
3362 0.5/0.5 0/0 0/0 0/0
3363 0.5/0.5 0/0 0/0 0/0
3364 1/0 0/0 0/0 0/0
3365 1/0.5 0/0 0/0 0/0
3366 0.5/0.5 0/0 0/0 0/0
3367 0.5/1 0/0 0/0 0/0
3368 0.5/1 0/0 0/0 0/0
NAIVE CONTROL ANIMALS
3369 0/0 0/0
3370 0/0 0/0
3371 0/0 0/0
3372 0/0 0/0
3373 0/0 0/0

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information According to the CLP Regulation. Criteria used for interpretation of results: EU
Conclusions:
Non sensitising.
Executive summary:

Method

The potential of the test material to provoke delayed skin sensitization reactions was assessed after intradermal injection and topical skin insult applications of the test item.

Induction: after acclimating to the laboratory environment, 10 healthy, male, Hartley Strain, guinea pigs were injected ID with approximately 0.05 ml of test product in water, 0.05 ml 5 % test material in 50 % aqueous Freunds Complete Adjuvant and 0.05 ml 50 % aqueous Freunds Complete Adjuvant at multiple sites in the shaven anterior left and right shoulder areas. 

Topical induction (booster): 7 days later, a Hilltop Chamber containing 0.1 ml of test material was placed adjacent to each previously injected area for 48 hours at which time the chambers were removed. The sites were scored for erythema 24 and 48 hours after patch removal (72 and 96 hours after initial topical application). 

Challenge: 14 later, both flanks of each guinea pig were shaved and each received a topical application of 0.1 ml test material per site on a Hilltop Chamber. The chamber was secured in place for 24 hours, then removed. 24 and 48 hours later (48 and 72 hours after challenge), the sites were scored for a sensitization response.

Results

General: apart from the slight irritation observed after the topical booster application, all guinea pigs appeared active and healthy throughout the test period. There were no signs of gross toxicity, adverse pharmacologic effects or abnormal behaviour. All animals gained weight.

Induction: barely perceptible to mild erythema was observed at all sites 24 hours after the topical booster application. By 48 hours all sites were clear.

Challenge: there was no evidence of erythema at any sites on any of the test animals 24 or 48 hours after challenge. Similarly, no irritation occurred on any of the dosed sites on the naive Guinea Pigs.

Discussion and conclusion

According to the CLP Regulation, a substance is classified as skin sensitiser when in the Guinea pig maximisation test the response of at least 30 % of the animals is considered as positive.

There are not positive results reported in this appropriate animal test.

In conclusion, the test item can be classified as NON sensitising, according to the CLP (EC 1278/2008) Regulation.