Reaction mass of trisodium 4-amino-3-[[4-[[4-[(2-amino-4-hydroxyphenyl)azo]phenyl]amino]-3-sulphonatophenyl]azo]-5-hydroxy-6-(phenylazo)naphthalene-2,7-disulphonate and trisodium 4-amino-3-[[4-[[4-[(4-amino-2-hydroxyphenyl)azo]phenyl]amino]-3-sulphonatophenyl]azo]-5-hydroxy-6-(phenylazo)naphthalene-2,7-disulphonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

October 01st to November 15th, 1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Remarks:

The test was conducted by means of Read Across approach. The reliability of the source study report is 1. Further information was attached at section 13

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

adopted May 12th, 1981

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Adequate data from a previously performed Guinea Pig Maximisation Test were available.

Test material

Test material information

Constituent 1

Reference substance name:

Direct Black 168:1 - Similar Substance 01

IUPAC Name:

Direct Black 168:1 - Similar Substance 01

In vivo test system

Test animals

Species:

guinea pig

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd.
- Age at study initiation: 8 weeks.
- Weight at study initiation: 296 - 385 g.
- Housing: individually housed in Makrolon type-3 cages with standard softwood bedding.
- Diet: pelleted standard Kilba 342 guinea pig breeding, ad libitum.
- Water: community tap water, ad libitum. Once weekly additional suppy of ascorbic acid via the drinking water.
- Acclimation: one week under test conditions after veterinary examination.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 40 - 70 %
- Air changes: 10-15 air changes per hr
- Photoperiod: 12 hrs dark / 12 hrs artificial fluorescent light.
- Other: music during the light period, room was air-conditioned.

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

physiological saline

Concentration / amount:

5 %

Induction 2

Route:

intradermal

Vehicle:

other: emulsion in a 50:50 mixture of Freund's complete adjuvant and physiological saline.

Concentration / amount:

5 %

Induction 3

Route:

epicutaneous, occlusive

Vehicle:

petrolatum

Remarks:

vaselinum album

Concentration / amount:

25 %

Day(s)/duration:

2

Challenge

Challenge 1

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

petrolatum

Remarks:

vaselinum album

Concentration / amount:

25 %

Day(s)/duration:

1

Challenge 2

No.:

#2

Route:

epicutaneous, occlusive

Vehicle:

petrolatum

Remarks:

vaselinum album

Concentration / amount:

10 %

Day(s)/duration:

1

Challenge 3

No.:

#2

Route:

epicutaneous, occlusive

Vehicle:

petrolatum

Remarks:

vaselinum album

Concentration / amount:

25 %

Day(s)/duration:

1

No. of animals per dose:

20 in test group, 10 in control group.

Details on study design:

TEST ARTICLE PREPARATION
The test article and vehicle were placed into a glass beaker on a tared balance and w/w dilution was prepared. Homogeneity of the test article in physiological saline for the intracutaneous applications was maintained during treatment using a magnetic stirrer. Vaselinum album was used as vehicle for the epicutaneous applications and homogeneity of the test article in vaselinum album was maintained with a spatula. The preparations were made immediately prior to each dosing.

RANGE FINDING TESTS
INTRADERMAL INDUCTION
- No. of animals: two.
- Concentrations: 1, 3 and 5 % w/v in physiological saline.
- Amount: intradermal injections of 0.1 ml/site were made into the clipped flank.
- Assessement of dermal reactions: 24 hours after the injection.

EPICUTANEOUS INDUCTION EXPOSURE
- No. of animals: four.
- Concentrations: 5, 10, 15 and 25 % w/v in vaselinum album.
- Exposure period: 24 hours.
- Application of test material and dressing: patches of filter paper (2 x 2 cm) were saturated with test article in vaselinum album and applied to the clipped and shaved flanks of each animal. 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.
- Assessement of dermal reactions: immediately and then 24 and 48 hours after patch removal.
- Other: prior to first reading the test sites were depilated to clean them from black staining produced by the test article. The allocation of the different test sites on the animals was alternated in order to minimise site to site variation in responsiveness.

MAIN STUDY
INTRADERMAL INDUCTION EXPOSURE
- Site: an area of dorsal skin from the scapular region (approximately 6 x 8 cm) was clipped free of hair.
- Test groups: 3 pairs of intradermal injections (0.1 ml/site) were made at the border of a 4 x 6 cm area.
Freund's complete adjuvant 50:50 with physiological saline.
The test article, diluted to 5 % with physiological saline.
The test article diluted to 5 % by emulsion in a 50:50 mixture of Freund's complete adjuvant and physiological saline.
- Control group: 3 pairs of intradermal injections (0.1 ml/site) were made at the border of a 4 x 6 cm area.
Freund's complete adjuvant 50:50 with physiological saline.
Physiological saline.
Freund's complete adjuvant 50:50 with physiological saline.

EPICUTANEOUS INDUCTION EXPOSURE
- Site: on day 7 of the test and approximately 24 hours prior to the epidermal application the scapular area (approximately 6 x 8 cm) was clipped, shaved free of hair.
- Pretreatment: 24 hours prior to the epidermal application the scapular area (approximately 6 x 8 cm) was clipped, shaved free of hair and the test areas were pretreated with 10 % Sodium-Lauryl-Sulfate (SLS) in petrolatum-oil, because no primary irritation concentration could be determined in the corresponding pretest (range finding test). The SLS was massaged into the skin with a glass rod without bandaging. This SLS-concentration enhances sensitization by provoking a mild inflammatory reaction.
- Test groups: 25 % of test item in vaselinum album.
- Control: the guinea-pigs of the control group were treated as the test groups animals with the omission of test article.
- Application in test group: on day 8 of test a 2 x 4 cm patch of filter paper was saturated with the test article and placed over the injection sites of the test animals. - Dressing: the patch was covered by aluminum foil and firmly secured by an elastic plaster wrapped around the trunk of the animal and secured with impervious adhesive tape.
- Exposure period: 48 hours.
- Evaluation: reaction sites were assessed for erythema and edema immediately, 24 and 48 hours after removal of the dressing, using the numerical grading system according to Draize.

FIRST CHALLENGE EXPOSURE
- Days after induction: 2 weeks after the first challenge.
- Application in test and control group: 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 test article and vehicle and applied to the left flank and right flank respectively (the same method as for the epidermal application was used).
- Test groups: non-irritant concentration, i.e. 25 %, of the test article and with vaselinum album; application of paper with vaselinum album only and applied to the left flank and right flank.
- Dressing: same as in the induction phase.
- Exposure period: 24 hours.
- Evaluation: immediately and after 24 and 48 hours of patch removal, erythema and edema were assessed under artificial fluorescent light (daylight spectrum).

SECOND CHALLENGE EXPOSURE
- Days after first challenge: 2 weeks after the first challenge.
- Application in test and control group: similar as described for the first challenge with the exception that the flanks of all guinea-pigs were changed and one additional concentration was used.
- Test groups: 10 and 25 % of test item in vaselinum album
- Control: animals were treated with vaselinum album on the left flank.
- Evaluation: immediately and after 24 and 48 hours of patch removal, erythema and edema were assessed under artificial fluorescent light (daylight spectrum).

OBSERVATIONS
- Mortality/Viability: daily.
- Body Weights: at acclimatization start, start of application and termination of test.
- Clinical Signs (local/systemic): daily.
- Necropsy: no necropsy was performed in the animals euthanized at termination of observation. All animals were euthanized at the end of the test period with an intraperitoneal injection of Pentobarbital and discarded.

READINGS AND SCORING
Erythema and edema were assessed using the numerical grading system according to Draize.
Erythema and eschar formation:
No erythema ................................................................0
Very slight erythema (barely perceptible)....................1
Well-defined erythema..................................................2
Moderate to severe erythema.......................................3
Severe erythema (beet redness) to slight
eschar formation (injuries in depth) .............................4

Edema formation:
No edema .....................................................................0
Very slight edema (barely perceptible).........................1
Slight edema (edges of area well-defined
by definite raising).........................................................2
Moderate edema (raised approximately
1 millimeter)....................................................................3
Severe edema (raised more than 1 millimeter
and extending beyond the area of exposure)...............4

Positive control substance(s):

yes

Remarks:

formaldehyde solution is tested twice a year for sensitivity check of the guinea pig strain. The most recent test was run 4 months before the present study.

Results and discussion

Positive control results:

Formaldehyde produced a 70 % sensitisation rate.

In vivo (non-LLNA)

Results

Results 1

Reading:

rechallenge

Hours after challenge:

48

Group:

test chemical

Dose level:

25 %

No. with + reactions:

0

Total no. in group:

20

Results 2

Reading:

rechallenge

Hours after challenge:

48

Group:

negative control

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Results 3

Reading:

rechallenge

Hours after challenge:

48

Group:

positive control

Dose level:

15 %

No. with + reactions:

7

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Any other information on results incl. tables

MAIN TEST

3 out of 20 animals of the first and second challenge showed positive erythema reactions after the 24-hour reading when treated with a 25 % test article dilution. No positive erythema reactions were observed when the animals were treated with vaselinum album alone.

2 out of 20 animals after the second challenge showed positive erythema reaction after the 24- and 48-hour reading when treated with a 10 % test article dilution.

Mortality / viability

No death occurred.

Clinical signs, local

Application area around the injection site 1 was found to show erythema and edema from day 2 to 5; necroses were observed from day 6 to 14; encrustations from day 12 to 19 and exfoliation from day 18 to 39. Around the injection site 2 edema were observed from day 2 to 5; staining from day 2 to 8; necroses from day 6 to 14; encrustation from day 12 to 19 and exfoliation from day 18 to 32.

Around the injection site 3 edema were observed on days 2 and 3, staining from day 2 to 8, necroses from day 4 to 14, encrustations from day 12 to 19 and exfoliation from day 18 to 39. In addition epidermal induction application area, first challenge and second challenge application areas showed staining respectively from day 10 to 28, 23 to 28 and 37 to 39. On day 9 of test no observation could be performed because the animals were bandaged semi-occlusively.

Clinical signs, systemic

No systemic symptoms were observed.

Body weight

Females no. 533 and 536 of epidermal pretest and females no. 514, 517 and 524 of test group lost slightly weight during the acclimatization period.

They had recovered again during the remaining study period. The body weight gain of the further animals was not affected adversely during the study.

CONTROL GROUP

No positive reactions were evident after the first and second challenge application, neither when treated with vaselinum album nor when treated with a 25 % the test article dilution.

Application area around the injection sites 1 and 3 was found to show erythema and edema from day 2 to 6; necroses were observed from day 7 to 14; encrustations from day 12 to 20 and exfoliation from day 20 to 39 (termination of test).

In addition around the injection site 2 erythema and edema were observed on day 2 of test and first challenge application area showed staining from day 23 to 28.

RESULTS OF THE FIRST CHALLENGE

Control group - test article treated (25 % in vaselinum album) - Left flank

Animal no	Sex	Erythema/Edema readings after removal of bandage
		Immedietely		24 hours		48 hours
		E	O	E	O	E	O
501	F	0	0	0	0	0	0
502	F	0	0	0	0	0	0
503	F	1	0	0	0	0	0
504	F	1	0	0	0	0	0
505	F	0	0	0	0	0	0
506	F	1	0	0	0	0	0
507	F	0	0	0	0	0	0
508	F	0	0	0	0	0	0
509	F	1	0	0	0	0	0
510	F	0	0	0	0	0	0

Control group - vehicle (vaselinum album) - Right flank

Animal no	Sex	Erythema/Edema readings after removal of bandage
		Immedietely		24 hours		48 hours
		E	O	E	O	E	O
501	F	0	0	0	0	0	0
502	F	0	0	0	0	0	0
503	F	0	0	0	0	0	0
504	F	0	0	0	0	0	0
505	F	0	0	0	0	0	0
506	F	0	0	0	0	0	0
507	F	0	0	0	0	0	0
508	F	0	0	0	0	0	0
509	F	0	0	0	0	0	0
510	F	0	0	0	0	0	0

Test group - test article (25 % in vaselinum album) - Left flank

Animal no	Sex	Erythema/Edema readings after removal of bandage
		Immedietely		24 hours		48 hours
		E	O	E	O	E	O
511	F	0	0	0	0	0	0
512	F	1	0	1	0	0	0
513	F	1	0	0	0	0	0
514	F	0	0	0	0	0	0
515	F	0	0	0	0	0	0
516	F	1	0	0	0	0	0
517	F	1	0	1	0	0	0
518	F	1	0	0	0	0	0
519	F	0	0	0	0	0	0
520	F	0	0	0	0	0	0
521	F	0	0	0	0	0	0
522	F	1	0	0	0	0	0
523	F	1	0	0	0	0	0
524	F	1	0	1	0	0	0
525	F	1	0	0	0	0	0
526	F	1	0	0	0	0	0
527	F	1	0	0	0	0	0
528	F	0	0	0	0	0	0
529	F	1	0	0	0	0	0
530	F	1	0	0	0	0	0

Test group - vehicle (vaselinum album) - Right flank

Animal no	Sex	Erythema/Edema readings after removal of bandage
		Immedietely		24 hours		48 hours
		E	O	E	O	E	O
511	F	0	0	0	0	0	0
512	F	0	0	0	0	0	0
513	F	0	0	0	0	0	0
514	F	0	0	0	0	0	0
515	F	0	0	0	0	0	0
516	F	0	0	0	0	0	0
517	F	0	0	0	0	0	0
518	F	0	0	0	0	0	0
519	F	0	0	0	0	0	0
520	F	0	0	0	0	0	0
521	F	0	0	0	0	0	0
522	F	0	0	0	0	0	0
523	F	0	0	0	0	0	0
524	F	0	0	0	0	0	0
525	F	0	0	0	0	0	0
526	F	0	0	0	0	0	0
527	F	0	0	0	0	0	0
528	F	0	0	0	0	0	0
529	F	0	0	0	0	0	0
530	F	0	0	0	0	0	0

RESULTS OF THE SECOND CHALLENGE

Control group - vehicle (vaselinum album) - Left flank.

Animal no	Sex	Erythema/Edema readings after removal of bandage
		Immedietely		24 hours		48 hours
		E	O	E	O	E	O
501	F	0	0	0	0	0	0
502	F	0	0	0	0	0	0
503	F	0	0	0	0	0	0
504	F	0	0	0	0	0	0
505	F	0	0	0	0	0	0
506	F	0	0	0	0	0	0
507	F	0	0	0	0	0	0
508	F	0	0	0	0	0	0
509	F	0	0	0	0	0	0
510	F	0	0	0	0	0	0

Test group - test material (25 % in vaselinum album) - Right flank.

Animal no	Sex	Erythema/Edema readings after removal of bandage
		Immedietely		24 hours		48 hours
		E	O	E	O	E	O
511	F	0	0	0	0	0	0
512	F	1	0	0	0	0	0
513	F	0	0	0	0	0	0
514	F	0	0	0	0	0	0
515	F	0	0	0	0	0	0
516	F	0	0	0	0	0	0
517	F	2	1	1	0	0	0
518	F	0	0	0	0	0	0
519	F	0	0	0	0	0	0
520	F	0	0	0	0	0	0
521	F	1	0	1	0	0	0
522	F	0	0	0	0	0	0
523	F	1	0	0	0	0	0
524	F	2	1	2	1	0	0
525	F	0	0	0	0	0	0
526	F	1	0	0	0	0	0
527	F	0	0	0	0	0	0
528	F	0	0	0	0	0	0
529	F	0	0	0	0	0	0
530	F	0	0	0	0	0	0

Test group - test material (10 % in vaselinum album) - Anterior left flank.

Animal no	Sex	Erythema/Edema readings after removal of bandage
		Immedietely		24 hours		48 hours
		E	O	E	O	E	O
511	F	0	0	0	0	0	0
512	F	0	0	0	0	0	0
513	F	0	0	0	0	0	0
514	F	0	0	0	0	0	0
515	F	0	0	0	0	0	0
516	F	0	0	0	0	0	0
517	F	2	1	1	0	1	0
518	F	0	0	0	0	0	0
519	F	0	0	0	0	0	0
520	F	0	0	0	0	0	0
521	F	1	0	0	0	0	0
522	F	0	0	0	0	0	0
523	F	1	0	0	0	0	0
524	F	1	0	1	0	1	0
525	F	0	0	0	0	0	0
526	F	0	0	0	0	0	0
527	F	1	0	0	0	0	0
528	F	0	0	0	0	0	0
529	F	0	0	0	0	0	0
530	F	0	0	0	0	0	0

Test group - Vehicle (vaselinum album) - Posterior left flank.

Animal no	Sex	Erythema/Edema readings after removal of bandage
		Immedietely		24 hours		48 hours
		E	O	E	O	E	O
511	F	0	0	0	0	0	0
512	F	0	0	0	0	0	0
513	F	0	0	0	0	0	0
514	F	0	0	0	0	0	0
515	F	0	0	0	0	0	0
516	F	0	0	0	0	0	0
517	F	0	0	0	0	0	0
518	F	0	0	0	0	0	0
519	F	0	0	0	0	0	0
520	F	0	0	0	0	0	0
521	F	0	0	0	0	0	0
522	F	0	0	0	0	0	0
523	F	0	0	0	0	0	0
524	F	0	0	0	0	0	0
525	F	0	0	0	0	0	0
526	F	0	0	0	0	0	0
527	F	0	0	0	0	0	0
528	F	0	0	0	0	0	0
529	F	0	0	0	0	0	0
530	F	0	0	0	0	0	0

Applicant's summary and conclusion

Interpretation of results:

other: not classified, according to the CLP Regulation (EC 1272/2008)

Conclusions:

The substance is not a skin sensitiser to albino quinea pigs.

Executive summary:

For the assessement of the skin sensitisation potential of the test material to albino guinea pig the Guinea Pig Maximisation test was performed, according to the OECD Guideline 406. Twenty females were initially exposed to the test substance by intradermal injection (5 % in vaselinum album) and epidermal application (25 % in vaselinum album) (induction exposure). Following a period of 14 days (induction period), the animals were exposed to a challenge dose (25 %). A second challenge was performed two weeks after the first challenge; the animals were treated with an additional concentration of 10 % in vaselinum album. The extent and degree of skin reaction to the challenge exposures in the test animals was assessed immediately and after 24 and 48 hours following challenge exposure. The skin reactions and the number of animals presenting these skin reactions were compared with that demonstrated by control animals which underwent sham treatment during induction and received the challenge exposures.

The animals in the control group showed no reaction at the substance after both the challenge exposures. At the first challenge and second challenge exposure three out of twenty animals (15 %) of the test group presented positive erythema reactions after the 24 - hour reading, that were not present after 48 hours, when treated with a 25 % test article dilution. Two out of 20 animals (10 %) after the second challenge showed positive erythema reaction after the 24- and 48- hour reading when treated with a 10 % test article dilution. The test group presented a skin sensitisation rate of 10 -15 % in comparison with the control group that presented 0 % of rate.

Conclusion

The substance is considered to be a non skin sensitiser to albino quinea pigs.