Trichloro-12,24-dihydro-5H-naphtho[2,3-h]naphth[2'',3'':6',7']indolo[2',3':6,7]anthra[2,1,9-mna]acridine-5,10,13,18,25-pentone

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 23, 1992 to February 3, 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

OECD guideline 406, adopted May 12, 1981, adapted July 17, 1992

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

Annex V, Part B of Council Directive 79/831/EEC (Commission Directive 84/449/EEC of April 25. 1984)

Deviations:

not specified

Principles of method if other than guideline:

The maximisation test has been selected, because it is one of the recommended test in the OECD guideline 406, adopted May 12, 1981, adapted July 17, 1992, as well as in Annex V, Part B of Council Directive 79/831/EEC (Commission Directive 84/449/EEC of April 25. 1984), and because the sensitivity of the method is well known. The test has been performed according to the original protocol of Magnusson and Kligman.

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Study available is over 12 years old

Test material

Test material information

Constituent 1

Reference substance name:

Reaction product of 3,9-dibromobenzanthrone condensed with 2 equivalents of 1-aminoanthraquinone, subsequently further condensed under oxidative conditions

EC Number:

944-232-9

Molecular formula:

Not available - UVCB substance

IUPAC Name:

Reaction product of 3,9-dibromobenzanthrone condensed with 2 equivalents of 1-aminoanthraquinone, subsequently further condensed under oxidative conditions

Test material form:

solid

In vivo test system

Test animals

Species:

guinea pig

Strain:

Pirbright-Hartley

Sex:

male/female

Details on test animals and environmental conditions:

Test System
The albino guinea pig is the recommended species for skin sensitisation studies.
Animal strain:
Pirbright White Strain (Tif: DHP)Breeder: CIBA-GEIGY Limited, Animal Production, 4332 Stein, Switzerland
Date of acclimatisation: November 26, 1992
Group Size and Husbandry
The test was performed on 10 male and 10 female guinea pigs in the test group and 5 males and 5 females in the control group, respectively, initially weighing between 313 to 400 g.
The animals were house individually in Macrolon cages (Type 3), assigned to the different groups by means of random numbers generated by the random number generator, identified by individual ear tags, kept at a constant room temperature of 22 ± 3°C, at a relative humidity of 30 to 70% and a 12 hours light cycle day.
The animals received ad libitum standard guinea pig pellets – NAFAG No. 845, Gossau SG and fresh water.

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

arachis oil

Concentration / amount:

5% FAT 45019/E in oleum arachidis (w/v), 5% FAT 45019/E in the adjuvant/saline mixture (w/v)

Day(s)/duration:

One administration

Adequacy of induction:

not specified

Induction 2

Route:

epicutaneous, occlusive

Vehicle:

petrolatum

Concentration / amount:

50%

Day(s)/duration:

48 hours

Adequacy of induction:

non-irritant substance, but skin pre-treated with 10% SDS

Challenge

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

petrolatum

Concentration / amount:

50% in Vaseline

Day(s)/duration:

24 hours

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

The test was performed on 10 male and 10 female guinea pigs in the test group and 5 males and 5 females in the control group, respectively.

Details on study design:

Induction procedure (weeks 1 and 2)
The induction was a two-stage operation. First intradermal injections (into the neck region); second, closed patch exposure over the injection sites one week later.
First induction week, intradermal injection
Three pairs of intradermal injections (0.1 ml per injection) were made simultaneously into the shaved neck of the guinea pigs as follows:
Adjuvant/saline mixture 1:1 (v/v)
Test article FAT 45019/E in oleum arachidis (w/v)
Test article FAT 45019/E in the adjuvant saline mixture (w/v)
Second induction week, epidermal applicationIn the second week of induction FAT 45019/E was incorporated in Vaseline (w/w) and applied on a filter paper patch to the neck of the animals (patch 2x4 cm; approx. 0.4 g paste per patch; occluded administration for 48 hours).
Rest period
During weeks 3 and 4 no treatments were performed.
Challenge (week 5)
The animals were tested on the flank with FAT 45019/E in Vaseline (w/w) and the vehicle alone (patch 2x2 cm; approx. 0.2 g paste per patch; occluded administration for 24 hours).
Control group
A control group of 10 animals (5 m/5f) was treated with adjuvant and the vehicle during the induction period. During the challenge period the group was treated with the vehicle as well as with the test article to check the maximum sub-irritant concentration of the test article in adjuvant treated animals.

Concentrations
Intradermal Induction
The concentration for the intradermal injections was selected on account of the solubility of the test article in standard vehicles and its local and systemic tolerability in a pre-test. The following concentrations of test article have been prepared for intradermal injection:1, 3 and 5% in oleum arachidis
Since 5% FAT 45019/E in oleum arachidis could be injected and well tolerated, this concentration was used for the intradermal induction.
5% FAT 45019/E in oleum arachidis (w/v)
5% FAT 45019/E in the adjuvant/saline mixture (w/v)
Epidermal Application (induction and challenge)
The concentrations for the epidermal applications were selected on account of the primary irritation potential of the test article. The following concentrations of FAT 45019/E have been examined on separate animals for the determination of the maximum sub-irritant concentration:10, 20, 30 and 50% in Vaseline
The test concentrations did not induce erythema reactions, therefore the following concentration was selected:
Epidermal induction
Concentration of test article: 50%
Vehicle: Vaseline
No skin irritation was observed in the pre-test.Therefore the application site was pre-treated with 10% sodium-laurylsulfate (open application) 24 hours prior to the epidermal induction application
Epidermal challenge
Concentration of test article: 50%
Vehicle: Vaseline
Observations and recordsInduction reactions
After the intradermal and the epidermal induction application irritant reactions are normally induced by the adjuvant, the high test article concentration, or the sodium lauryl sulfate pre-treatment. Because most of the reactions are treatment related and not compound related, the reactions are only described in special cases in the section of results.
Challenge reactions
Twenty four and forty eight hours after removing the dressings, the challenge reactions were graded according to the Draize scoring scale.
General
The sensitising potential of FAT 45019/E was classified according to the grading of Magnusson and Kligman. The body weight was recorded at start and end of the test.

Challenge controls:

A control group of 10 animals (5 m/5 f) was treated with adjuvant and the vehicle during the induction period. During the challenge period the group was treated with the vehicle as well as with the test article to check the maximum subirritant concentration of the test article in adjuvant treated animals.

Positive control substance(s):

yes

Remarks:

Potassium-dichromate

Results and discussion

Positive control results:

Reported in table form - see Overall remarks

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

50%

No. with + reactions:

17

Total no. in group:

20

Clinical observations:

None specified

Remarks on result:

positive indication of skin sensitisation

Results 2

Key result

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

50%

No. with + reactions:

10

Total no. in group:

20

Clinical observations:

None specified

Remarks on result:

positive indication of skin sensitisation

Results 3

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

other: vehicle control

Dose level:

vehicle

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

None specified

Results 4

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

other: vehicle control

Dose level:

vehicle

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none specified

Any other information on results incl. tables

Number of positive animals per group after occlusive

epidermal application

Control group:
	After 24 hours	After 48 hours
Vehicle control Test article	0/10 0/10	0/10 0/10
Test group:
	After 24 hours	After 48 hours
Vehicle control Test article	0/20 17/20	0/20 10/20

 

Challenge reactions after epidermal application (CONTROL GROUP)

DRAIZE Score 24 hours after removal of the dressing

Vehicle control
Male animals	151	152	153	154	155
Erythema score: Edema score:	0 0	0 0	0 0	0 0	0 0
Female animals	166	167	168	169	170
Erythema score: Edema score:	0 0	0 0	0 0	0 0	0 0
Test article control
Male animals	151	152	153	154	155
Erythema score: Edema score:	0 0	0 0	0 0	0 0	0 0
Female animals	166	167	168	169	170
Erythema score: Edema score:	0 0	0 0	0 0	0 0	0 0

DRAIZE Score 48 hours after removal of the dressing

Vehicle control
Male animals	151	152	153	154	155
Erythema score: Edema score:	0 0	0 0	0 0	0 0	0 0
Female animals	166	167	168	169	170
Erythema score: Edema score:	0 0	0 0	0 0	0 0	0 0
Test article control
Male animals	151	152	153	154	155
Erythema score: Edema score:	0 0	0 0	0 0	0 0	0 0
Female animals	166	167	168	169	170
Erythema score: Edema score:	0 0	0 0	0 0	0 0	0 0

 

Challenge reactions after epidermal application (TEST GROUP)

DRAIZE Score 24 hours after removal of the dressing

Vehicle control
Male animals	156	157	158	159	160	161	162	163	164	165
Erythema score: Edema score:	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0
Female animals	171	172	173	174	175	176	177	178	179	180
Erythema score: Edema score:	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0
Test article
Male animals	156	157	158	159	160	161	162	163	164	165
Erythema score: Edema score:	1 0	1 0	2 0	1 0	1 0	1 0	1 0	1 0	1 0	0 0
Female animals	171	172	173	174	175	176	177	178	179	180
Erythema score: Edema score:	1 0	1 0	1 0	1 0	1 0	0 0	1 0	2 0	1 0	0 0

DRAIZE Score 48 hours after removal of the dressing

Vehicle control
Male animals	156	157	158	159	160	161	162	163	164	165
Erythema score: Edema score:	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0
Female animals	171	172	173	174	175	176	177	178	179	180
Erythema score: Edema score:	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0
Test article
Male animals	156	157	158	159	160	161	162	163	164	165
Erythema score: Edema score:	0 0	0 0	1 0	0 0	1 0	0 0	1 0	1 0	0 0	0 0
Female animals	171	172	173	174	175	176	177	178	179	180
Erythema score: Edema score:	1 0	0 0	1 0	0 0	1 0	1 0	1 0	0 0	1 0	0 0

 

Evaluation of the primary skin irritation potential

Procedure: On each animal 2 concentrations of FAT 45019/E were applied simultaneously on the left and right flank. A naïve skin site served as control.

5 days before application of FAT 45019/E two pairs of intradermal injections of an adjuvant/saline mixture 1:1 (v/v; 0.1 ml per injection) were made simultaneously into the shaved neck of the guinea pigs.

	Score 24 hours		Score 48 hours
	after removing the dressing
Concentrations of FAT 45019/E in Vaseline (w/v;%)
Animal No./sex	10% er/ed	20% er/ed	10% er/ed	20% er/ed
1 male 2 female	0/0 0/0	0/0 0/0	0/0 0/0	0/0 0/0
Animal No./sex	30% er/ed	50% er/ed	30% er/ed	50% er/ed
3 male 4 female	0/0 0/0	0/0 0/0	0/0 0/0	0/0 0/0

ed = edema, er = erythema

 

Individual animal bodyweights in g - males

CONTROL GROUP			TEST GROUP
Animal No.	Weight		Animal No.	Weight
	At start	At end		At start	At end
151 152 153 154 155	355 364 339 359 372	564 540 462 410 504	156 157 158 159 160 161 162 163 164 165	372 349 390 383 347 385 354 362 400 382	521 517 559 499 452 454 523 465 555 532
Mean Std. Dev.	358 12.3	496 61.6		372 18.5	508 39.2

 

Individual animal bodyweights in g - females

CONTROL GROUP			TEST GROUP
Animal No.	Weight		Animal No.	Weight
	At start	At end		At start	At end
166 167 168 169 170	373 352 318 338 346	470 543 487 507 417	171 172 173 174 175 176 177 178 179 180	350 343 354 348 347 343 350 322 351 313	411 503 459 502 465 484 476 406 488 413
Mean Std. Dev.	345 20.1	485 46.6		342 13.6	461 37.7

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

85% of the animals were sensitised by the substance under the experimental conditions employed.
According to the maximisation grading, the substance showed an extreme grade of skin-sensitising (contact allergenic) potential in albino guinea pigs.
In accordance with the CLP Regulation, the substance is classified as a skin sensitiser category 1B

Executive summary:

At the request of the Textile Dyes Division of CIBA-GEIGY Limited, a sensitisation test in albino guinea pigs was performed to determine the contact allergenic potency of FAT 45019/E in albino guinea pigs.

This test was based on the OECD Guideline No. 406, adopted May 12, 1981, adapted July 17, 1992, by the OECD council, and on Annex V, Part B of Council Directive 79/831/EEC (Commission Directive 84/449/EEC of April 25, 1984).

 

85% of the animals were sensitised by FAT 45019/E under the experimental conditions employed.

According to the maximisation grading FAT 45019/E showed an extreme grade of skin-sensitising (contact allergenic) potential in albino guinea pigs.