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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:
Experiment start date - 16 March 1999; Experiment end date - 28 April 1999; Study completion date - 04 May 1999.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report date:
1999

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 (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.

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
429-240-8
EC Name:
-
Cas Number:
212652-59-0
Molecular formula:
Hill formula: C25 H22 F N8 Na3 O13 S4 CAS formula: C25 H25 F N8 O13 S4 · 3 Na
IUPAC Name:
trisodium 3-amino-4-[2-(4-{[4-({2-[2-(ethenesulfonyl)ethoxy]ethyl}amino)-6-fluoro-1,3,5-triazin-2-yl]amino}-2-sulfonatophenyl)diazen-1-yl]-5-hydroxynaphthalene-2,7-disulfonate
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): FAT 40574/B
- Physical state: Solid
- Analytical purity: Approx 75%
- Lot/batch No.: WP 23/99
-
Specific details on test material used for the study:
Identity: FAT 40574/B
Batch: WP 23/99
Purity: Approx. 75 %
Appearance: Solid, dark-red powder
Storage: At room temperature at about 20 °C
Expiration Date: 08 February 2006

In vivo test system

Test animals

Species:
guinea pig
Sex:
male
Details on test animals and environmental conditions:
Source: RCC Ltd, Biotechnology & Animal Breeding Division, Wolferstrasse 4, CH-4414 Füllinsdorf / Switzerland
Number of animals for main study / pretest: 15 male / 5 male animals
Age at delivery: 4 - 6 weeks
Age at pretest start/beginning of acclimatization period: 4 - 6 weeks
Body weight at pretest start: Pretest groups: 323 - 384 g
Body weight at beginning of acclimatization period: Control and test group - 317-420 g
Identification: By unique cage number and corresponding ear tags.
Acclimatization: One week for the control and test group under test conditions after health examination. No acclimatization for the animals of the pretests. Only animals without any visible signs of illness were used for the study.

Conditions: The animals were accommodated under standard laboratory conditions. The animal room was air-conditioned with 10-15 air changes per hour and the air was continuously monitored with a range for room temperature of 20-28 °C and for relative humidity between 32-84 %. The animals were provided with an automatically controlled light cycle of 12 hours light and 12 hours dark. Fluorescent "Gold" lamps (Silvania Gold F40T1260) were present in the room used for the study. Music was played during the light period.

Accommodation: 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. 112/98 and 23/99, guinea pig breeding / maintenance diet ("Nafag", Nähr- und Futtermittel AG, CH-9202 Gossau), ad libitum.

Water: Community tap water from Füllinsdorf, ad libitum.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
polyethylene glycol
Remarks:
PEG 400
Concentration / amount:
5 % of test material
Day(s)/duration:
01
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
polyethylene glycol
Remarks:
PEG 400
Concentration / amount:
25 % test material
Day(s)/duration:
08
Adequacy of induction:
highest technically applicable concentration used
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
polyethylene glycol
Remarks:
PEG 400
Concentration / amount:
0.5% of test material
Day(s)/duration:
22
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:
MAIN STUDY
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 as follows:
Test Group: 1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
2) The test article, at 5 % in PEG 400.
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) PEG 400
3) 1:1 (w/w) mixture of PEG 400 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 (25 % in PEG 400) and placed over the injection sites of the test animals. The amount of test article preparation applied was approximately 0.3 g. 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 occlusive dressings were left in place for 48 hours. The epidermal application procedure described ensures intensive contact of the test article.
The guinea pigs of the control group were treated as described above with PEG 400 only, applied at a volume of approximately 0.3 ml.
The reaction sites were assessed 24 and 48 hours after removal of the bandage for erythema and oedema according to the method of Magnusson and Kligman.

CHALLENGE / PERFORMED ON TEST DAY 22
The test and control guinea pigs were challenged two weeks after the epidermal induction application and 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 ( 3 x 3 cm) of filter paper were saturated with the test article at the highest non-irritating concentration of 0.5 % (left flank) and the vehicle only (PEG 400 applied to the right flank) using the same method as for the epidermal application. The volume of test article preparation and vehicle applied was approximately 0.2 ml. The dressings were left in place for 24 hours. Twenty-one hours after removal of the dressing the test sites treated with the test article were depilated as described in the epidermal pretest. The reaction sites were assessed approximately 24 and 48 hours after removal of the bandage for erythema and oedema according to the method of Magnusson and Kligman.
Positive control substance(s):
yes
Remarks:
2-MERCAPTOBENZOTHIAZOLE

Results and discussion

Positive control results:
In this study 90 % (at the 24-hour reading) and 100 % (at the 48-hour reading) of the animals of the test group were observed with very slight to moderate/severe erythematous reactions after treatment with a non-irritant test article concentration of 10 % in mineral oil. No skin reactions were observed in the control group.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
5 % intradermal induction, 25 % epicutaneous, 0.5 % challenge.
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:
test chemical
Dose level:
5 % intradermal induction, 25 % epicutaneous, 0.5 % challenge.
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
No. with + reactions:
0
Total no. in group:
5
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:
5
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
5 % intradermal induction; 50 % epidermal induction; 10 % epidermal challenge
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 % intradermal induction; 50 % epidermal induction; 10 % epidermal challenge
No. with + reactions:
10
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation

Any other information on results incl. tables

Maximum concentration not causing irritating effects in preliminary test: 25 %
Signs of irritation during induction:
Possible erythema reaction could not be determined due to substance-related red staining of the skin. However, no oedema was observed.

Evidence of sensitisation of each challenge concentration:
Concentration 0.5 %: 0/10 (0 %) animals showed sensitization.

MORTALITY/CLINICAL SIGNS:No mortality and no clinical signs were noted in this study.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the test conditions FAT 40574/B applied at a concentration of 0.5 % in PEG 400 is considered not to be a skin sensitizer.
Executive summary:

In order to assess the cutaneous allergenic potential of FAT 40574/B, the Maximization-Test was performed in 15 (10 test and 5 control) male albino guinea pigs, in accordance with OECD Guideline No. 406 and the Directive 96/54/EEC, B.6 in a GLP certified laboratory. The intradermal induction of sensitization in the test group was performed with a 5 % dilution of the test article 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 25 % in PEG 400 one week after the intradermal induction. The animals of the control group were intradermally induced with PEG 400 and FCA/physiological saline and epidermally induced with PEG 400 under occlusion. Two weeks after epidermal induction the control and test animals were challenged by epidermal application of the test article at 0.5 % in PEG 400 and PEG 400 alone under occlusive dressing. Cutaneous reactions were evaluated at 24 and 48 hours after removal of the dressing. No toxic symptoms were evident in the guinea pigs of the control or test group. No deaths occurred. 0/10 guinea pigs showed positive reaction in test groups and 0/5 guinea pigs showed positive reaction in control groups. In this study none of the animals of the control and test group were observed with skin reactions after challenge treatment with a non-irritant test article concentration of 0.5 % in PEG 400. Under the test conditions FAT 40574/B applied at a concentration of 0.5 % in PEG 400 is considered not to be a skin sensitizer.