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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:
1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
None

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report date:
1993

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
yes
Remarks:
The classification Guidelines were added to the protocol, Section I of the chemical Law from August 1st, 1990" were omitted. The study was reported in January 1993 instead of November 1992; Males instead of females were delivered for the study.
Principles of method if other than guideline:
None
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
None

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of Tetrasodium 2-[{4-[{4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-5-sulfonatonaphthalen-1-yl}diazenyl]-7-sulfonatonaphthalen-1-yl}diazenyl]benzene-1,4-disulfonate and Tetrasodium 2-[{4-[{4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-5-sulfonatonaphthalen-1-yl}diazenyl]-6-sulfonatonaphthalen-1-yl}diazenyl]benzene-1,4-disulfonate
EC Number:
916-837-8
Molecular formula:
C29H16Na4ClN9O12S4
IUPAC Name:
Reaction mass of Tetrasodium 2-[{4-[{4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-5-sulfonatonaphthalen-1-yl}diazenyl]-7-sulfonatonaphthalen-1-yl}diazenyl]benzene-1,4-disulfonate and Tetrasodium 2-[{4-[{4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-5-sulfonatonaphthalen-1-yl}diazenyl]-6-sulfonatonaphthalen-1-yl}diazenyl]benzene-1,4-disulfonate
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Identification: FAT 40032/E
Description: Brown powder
Batch Number: OP 11
Purity/Formulation: Contents of active substance ingredients: 75.7 %
Stability of test article: Stable; expiration date: August 1997.
Storage Conditions: At room temperature

In vivo test system

Test animals

Species:
guinea pig
Strain:
Himalayan
Sex:
male/female
Details on test animals and environmental conditions:
Test system: Ibm: GOHI; SPF-quality guinea pigs (synonym: Himalayan spotted)
Rationale: Recognized by the International guidelines as the recommended test system, (e.g. OECD, EEC)
Source: BRL, Biological Research Laboratories Ltd. Wölferstrasse 4 CH-4414 Füllinsdorf.
Number of animals for main study / pre-test: 30 males / 6 males
Age at acclimatization start: 5-6 weeks
Body weight at acclimatization start: Control and test group 343 - 417 g; Pretest: 324 - 408 g
Identification: By unique cage number and corresponding ear tags.
Randomization: Randomly selected at time of delivery.
Acclimatization: One week for the control and test group under test conditions after veterinary examination. Only animals without any visual signs of illness were used for the study.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
physiological saline
Concentration / amount:
Test group:
1) Freund's complete adjuvant 50:50 with physiological saline.
2) The test article, diluted to 5 % with physiological saline.
3) The test article diluted to 5 % by emulsion 1n 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) Physiological saline.
3) Freund's complete adjuvant 50:50 with physiological saline.
Day(s)/duration:
Test Day 01
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
25 % in physiological saline
Day(s)/duration:
Test Day 07
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
Challengeopen allclose all
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
25 %
Day(s)/duration:
Test Day 22
Adequacy of challenge:
highest non-irritant concentration
No.:
#2
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
25 %
Day(s)/duration:
Test day 36
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
None
Details on study design:
PRE-TEST
The objective of this investigation was to identify a maximally tolerated concentration of the test article suitable for the induction phase of the main study. In addition, a suitable non-irritant concentration of the test article, by the topical route of administration, was identified for the challenge application.
The procedure employed for these investigations was as follows:

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 article in physiological saline. The resulting dermal reactions were assessed 24 hours later. For
intracutaneous induction application a 5 % test article dilution was selected.

Epidermal applications:
Patches of filter paper ( 2 x 2 cm) were saturated with concentrations of 5 %, 10 %, 15 %, and 25 % of the test article in physiological saline 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 article. The dressings were removed after an exposure period of 24 hours and the reaction sites were assessed 24 and 48 hours after removal of the bandage for erythema and oedema on a numerical basis according to Draize described above. After removal of the dressing, the application site was depilated with an approved depilatory cream* (VEET Cream, Reckitt & Colmann AG, CH-4005 Basel) to clean the application site from red staining produced by the test article, so that possible erythema reactions were clearly visible at that time.

MAIN STUDY:
Induction:
Intradermal Injections:
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) Freund's complete adjuvant 50:50 with physiological saline.
2) The test article, diluted to 5 % with physiological saline.
3) The test article diluted to 5 % by emulsion 1n 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) Physiological saline.
3) Freund's complete adjuvant 50:50 with physiological saline.

Epidermal applications:
On test day 7 and approximately 24 hours prior to the epidermal application the scapular area (approximately 6 x 8 cm) was clipped, shaved free of hair and the test area was pre-treated with 10 % Sodium-Lauryl-Sulfate (SLS) 1N petrolatum oil because no primary irritation concentration could be determined in the corresponding pre-test. The SLS was massaged into the skin with a glass rod without bandaging. This SLS-concentrat1on enhances sensitization by provoking a mild inflammatory reaction. On test day 8 a 2 x 4 cm patch of filter paper was saturated with the test article (25 % in physiological saline) 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 approximately 48
hours. 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 the omission of test article (physiological saline only). 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.

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. Two patches ( 2 x 2 cm) of filter paper were saturated with a non-1rr1tant concentration of 25 % (left flank), and the vehicle only (physiological saline, applied to the right flank) using the same method as for the epidermal application. The dressings were removed approximately 24 hours later. The sites were assessed for erythema and oedema 24 and 48 hours after removal of the dressing, using the
numerical scoring system according to Draize. After removal of the dressing, the application site was depilated with an approved depilatory cream* (VEET Cream, Recklett & Colmann AG, CH-4005 Basel) to clean the application site from staining produced by the test article, 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.
Erythema and oedema reactions are described 1n the tables under Appendix A.

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 with the exception that the applications on
flanks of all the guinea-pigs were inverted. The control animals were treated with physiological saline on the left flank.
Challenge controls:
None
Positive control substance(s):
yes

Results and discussion

Positive control results:
None

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
25 %
No. with + reactions:
7
Total no. in group:
20
Clinical observations:
Erythema observed in 7 animals out of 20.
Remarks on result:
other: First Challenge
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
25%
No. with + reactions:
7
Total no. in group:
20
Clinical observations:
Erythema observed in 7 animals out of 20.
Remarks on result:
other: First Challenge
Key result
Reading:
rechallenge
Hours after challenge:
24
Group:
test chemical
Dose level:
25%
No. with + reactions:
16
Total no. in group:
20
Clinical observations:
Erythema observed in 16 animals out of 20
Remarks on result:
other: Second Challenge
Key result
Reading:
rechallenge
Hours after challenge:
48
Group:
test chemical
Dose level:
25%
No. with + reactions:
15
Total no. in group:
20
Clinical observations:
Erythema observed in 15 animals out of 20
Remarks on result:
other: Second Challenge
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
FAT 40'032/E (left flank)
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: first challenge
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
FAT 40'032/E (left flank)
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: first challenge
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
vehicle only (right flank)
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: first challenge
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
vehicle only (right flank)
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: first challenge
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
vehicle only (right flank)
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: second challenge
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
vehicle only (right flank)
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: second challenge
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
25% dilution of 2-MERCAPTOBENZOTHIAZOL 1n acetone
No. with + reactions:
5
Total no. in group:
10

Any other information on results incl. tables

CLINICAL SIGNS:

Control group:

Erythema and oedema were observed at the injection sites 1 to 3 during days 2 to 5 of the observation. In addition, necroses were observed at the injection sites 1 and 3 between days 6 and 11 of the study. Following encrustation and exfoliation were observed at different observation days until test day 33. Around the epidermal induction application area fissures were observed from test day 10 to 18. After the first challenge the skin treated with the test article was discolored between days 23 and 28 of the study.

Test Group

Erythema and oedema were observed at the injection sites 1 to 3 during days 2 to 5 of the observation. In addition, necroses were observed at the injection sites between days 6 to 11 of the study. Following encrustation and exfoliation were observed at different observation days until test day 33. After the epidermal induction application, discoloration and fissures of the treated skin was observed between days 10 to 26 and 10 to 18 respectively. In addition, discolourated skin was observed after the first challenge between days 23 and 27 and after the second challenge between day 37 and termination of the study (day 38). On day 9 of test no observation could be performed because the animals were bandaged semi-occlusively.

Systemic:

No systemic symptoms were observed in the animals.

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
FAT 40032/E is considered to be a sensitizer.
Executive summary:

A study was performed to assess the allergenic potential of FAT 40'032/E in albino guinea pigs. The Maximization-Test of B. Magnusson and A.M. Kligman (1969) was used. Ten males were used as control group and 20 males were used as test group. The study was conducted between August 31 and October 15, 1992 at the BRL Laboratories in CH-4414 Füllinsdorf. Due to the equivocal findings observed after the first challenge, a second challenge was performed. The highest non-irritating test article concentration used for the both challenge applications was 25 %. No toxic symptoms were evident in the guinea pigs of the control or test group. No death occurred.

From the results described above a moderate to strong allergenic potency of the test article FAT 40'032/E was concluded. The results were interpreted according to the rating of Magnusson and Kligman (1969). The response of at least 30* positive animals is considered positive "M3" following the commission 91/325/EEC, Commission Directive of 1 March 1991 adapting to technical progress for the twelfth time Council Directive 67/548/EEC on the approximation of the laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances. In this study 35 % to 80 % of the animals were positive after treatment with a non-irritant test substance concentration of 25 %. According to EEC (European Economic Community) classification criteria described in guidelines 91/325/EEC (EC Official Journal Nr. L 180. July 08, 1991) and 67/548, June 27, 1967 (official journal 196 of August 16, 1967), this test article is considered to be a sensitizer.