Trisodium [5-acetamido-4-hydroxy-3-[[2-hydroxy-4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]naphthalene-2,7-disulphonato(5-)]cuprate(3-)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 14th, 1992 to October 16th, 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

EG-Guideline B.6. Acute Toxicity Sensitization of the Skin of the Directive 84/449/EWG: Commission Directive of 25 April 1984 adapting to technical progress for the sixth time Council Directive 67/548/EWG on the approximation of the laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

OECD-Guideline for testing of chemicals, 406 "Skin Sensitization", Adopted 12 May 1981

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Available study data is over 12 years old.

Test material

Test material information

Constituent 1

Reference substance name:

Trisodium [5-acetamido-4-hydroxy-3-[[2-hydroxy-4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]naphthalene-2,7-disulphonato(5-)]cuprate(3-)

EC Number:

263-856-6

EC Name:

Trisodium [5-acetamido-4-hydroxy-3-[[2-hydroxy-4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]naphthalene-2,7-disulphonato(5-)]cuprate(3-)

Cas Number:

63105-49-7

Molecular formula:

C20H14CuN3O15S4.3Na C20H14CuN3Na3O15S4

IUPAC Name:

trisodium [5-acetamido-4-hydroxy-3-[[2-hydroxy-4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]naphthalene-2,7-disulphonato(5-)]cuprate(3-)

Test material form:

solid: particulate/powder

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

Test species: Pirbright-White guinea pig
Sex: female
Strain: Hoe: DHPK (SPFLac)
Origin: HOECHST AG, Kastengrund, SPF breeding colony
Body weight at start of study: mean 304 g (= 100.0 %) range 280 g (- 7.8 %) to 329 g (+ 8.2 %)
Numer of animals: 15
Randomisation schemes: 393/92
Animal maintenance: in fully air-conditioned rooms in Makrolon cages (Type 4) on soft wood granulate, in groups of 5 animals
Ambient temperature: 22 ± 3 °C
Rel. atmospheric humidity: 55 ± 20 %
Lighting time: 12 hours daily
Acclimatisation: at least 5 days
Diet: Altromin 3112 for guinea pigs and rabbits, ad libitum
Water: tap water in plastic bottles, ad libitum
Animal identification: fur-marking with KMn04 and cage numbering

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

physiological saline

Concentration / amount:

5%

Day(s)/duration:

one administration on day 1

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Induction 2

Route:

intradermal

Vehicle:

other: 1:1 preparation of Freund's Complete Adjuvant and physiological saline

Concentration / amount:

5%

Day(s)/duration:

one administration on day 1

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Induction 3

Route:

epicutaneous, occlusive

Vehicle:

physiological saline

Concentration / amount:

25%

Day(s)/duration:

Day 8/ 48 hous

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Challenge

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

physiological saline

Concentration / amount:

25%

Day(s)/duration:

Test Day 22/24 hours

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

10 animals in the treatment group and 5 animals in the control group were used.

Details on study design:

Preparation of test concentrations
The following vehicles were used:
- Freund's Complete Adjuvant (Behringwerke AG, Marburg)
- isotonic saline (sterile, pyrogen-free; Fresenius AG, Bad Homburg)
Freund's Complete Adjuvant was mixed immediately before use with an equal volume of physiological saline. This 50 % adjuvant solution was administered to the animals by intradermal injection.
For the dermal and intradermal treatments, Remazol-Brilliantviolett 5R neu was applied in isotonic saline [percentages w/v].
For the intradermal injections of the test substance in 50 % Freund's adjuvant, Remazol-Brilliantviolett 5R neu was diluted with isotonic saline and then mixed with an equal volume of Freund's Original Adjuvant [percentages wjv].
The concentrations for the maximisation test cannot be standardised. Suitable concentrations are established in preliminary tests. The selected concentration of the test substance depends on the individual phases of the study.

Determination of the primary non-irritant concentration
In a dermal-occlusive test for primary skin irritation, each of the following test concentrations was applied to the left flank of two guinea pigs:
25% in isotonic saline
5% in isotonic saline
1% in isotonic saline
The hair on the left flank of the animals was removed mechanically. 0.5 ml of the test substance preparation was applied to a 2 x 2 cm cellulose patch, which was then fixed to the left flank and covered occlusively for 24 hours with a bandage and film. 24 hours after removal of the patches, the treated skin areas were examined for erythema and oedema.

Determining of the tolerance of intradermal injections
To determine the tolerance of intradermal injections, each of the following preparations was administered twice by intradermal injection to 3 guinea pigs. The injection sites (sites 1, 2 and 3) were all within a dorsal area measuring 2 x 4 cm in the vicinity of the shoulder.
site appl. vol. in ml conc. in % vehicle
1 2 X 0.1 5.0 isotonic saline
2 2 X 0.1 1.0 isotonic saline
3 2 X 0.1 0.2 isotonic saline

The injection sites (site 1, 2 and 3) were all within a dorsal area measuring 2 x 4 cm in the vicinity of the animals' shoulder.

Main test for sensitisinq properties
Chronological description of the test procedure indicating the day, at which procedure was carried out, on the left margin of the page:

Day 0
The body weights of animals were determined.
The guinea pigs were shaved mechanically over a dorsal area of 4 x 6 cm in the vicinity of the shoulders.

Day 1
Intradermal induction treatment
Two intradermal injections per animal of the following preparations.
The injection sites (site 1, 2 and 3) were all within a dorsal area of 2 x 4 cm. The injection sites were left uncovered.
Treated group:
Site 1: 2 X 0.1 ml 50% Freund's Adjuvant
Site 2: 2 X 0.1 ml 5 % solution of test substance in isotonic saline
Site 3: 2 X 0.1 ml 5 % solution of test substance in 50% Freund's adjuvant

Control and escort groups:
Site 1: 2 x 0.1 ml 50% Freund's Adjuvant
Site 2: 2 x 0.1 ml isotonic saline
Site 3: 2 x 0.1 ml 50% Freund's Adjuvant

Days 1-7
The application area was examined for local tolerance. Any systemic toxic effects were recorded.

Day 8
Dermal induction treatment
0.5 ml of the test substance preparation or the vehicle was applied to a 2 x 4 cm cellulose patch. This patch covered the area where the intradermal injection had been placed. The application area was then kept for 48 hours under an occlusive bandage with an impermeable film and an elastic bandage.
Treated group : 25% test substance in isotonic saline
Control and escort group : isotonic saline

Day 10
Occlusive bandage removed.
Irritant effects recorded.

Days 11-21
No treatment of control or treated group.
Test animals kept under observation.

Days 15-18
Challenge treatment of escort group, carried out in same way as that of control and treated groups (see days 22 - 25).

Day 22
Dermal challenge treatment
One area of approx. 5 x 5 cm on the left flank was shaved mechanically.
0.5 ml of the test substance preparation was applied to a 2 x 2 cm cellulose patch. The application area was then kept for 24 hours under an occlusive bandage with an impermeable film and an elastic bandage.
Treated and control groups (left flank):
25% Remazol-Brilliantviolett 5R neu in isotonic saline

Day 23
Occlusive bandage removed.

Day 24
Skin examined.

Day 25
Skin examined.

Challenge controls:

yes (escort group)

Positive control substance(s):

yes

Remarks:

Positive controls were done bi-annually

Results and discussion

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

25%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

The treated area was discoloured slightly blue.

Results 2

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

25%

No. with + reactions:

0

Total no. in group:

9

Clinical observations:

The treated area was discoloured slightly blue. One animal was found dead on day 20. A substance related effect was not obvious.

Remarks on result:

no indication of skin sensitisation

Results 3

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

25%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

The treated area was discoloured slightly blue.

Results 4

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

25%

No. with + reactions:

0

Total no. in group:

9

Clinical observations:

The treated area was discoloured slightly blue. One animal was found dead on day 20. A substance related effect was not obvious.

Remarks on result:

no indication of skin sensitisation

Any other information on results incl. tables

Body weight gains

Animal No.	Body weight at start of study (g)	Body weight at end of study (g)	Increase (%)
Control group:
1 2 3 4 5	317 270 303 306 325	342 317 351 325 349	+ 8 + 17 + 16 + 6 + 7
Treated group:
6 7 8 9 10	280 329 321 310 299	303 349 354 344 *	+ 8 + 6 + 10 + 11 +
11 12 13 14 15	302 307 285 293 312	336 338 310 299 353	+ 11 + 10 + 9 + 2 + 13

*found dead at day 20 of the study.

 

Scoring of dermal reactions – individual data

Challenge treatment, escort group

Remoazol-Brilliantviolett 5R neu 25% in isotonic saline (day 15)

Treat area: left flank

Scoring of dermal reactions
	Animal No.:	16	17	18	19	20
24 hours	Erythema Oedema Skin surface – slight blue discoloured	0 0   X	0 0   X	0 0   X	0 0   X	0 0   X
	Animal No.:	16	17	18	19	20
48 hours	Erythema Oedema Skin surface – slight blue discoloured	0 0   X	0 0   X	0 0   X	0 0   X	0 0   x

 

Challenge treatment: Remazol-Brilliantviolett 5R neu 25% in isotonic saline (day 22)

Treated area: left flank

Time of observation: 24 hours (day 24)

Scoring of dermal reactions
Control animals	1	2	3	4	5
Erythema Oedema Skin surface – slight blue discoloured	0 0   X	0 0   X	0 0   X	0 0   X	0 0   X
Treated animals	6	7	8	9	10	11	12	13	14	15
Erythema Oedema Skin surface – slight blue discoloured	0 0   X	0 0   X	0 0   X	0 0   X		0 0   X	0 0   X	0 0   X	0 0   X	0 0   X

 

Time of observation: 48 hours (day 25)

Scoring of dermal reactions
Control animals	1	2	3	4	5
Erythema Oedema Skin surface – slight blue discoloured	0 0   X	0 0   X	0 0   X	0 0   X	0 0   X
Treated animals	6	7	8	9	10	11	12	13	14	15
Erythema Oedema Skin surface – slight blue discoloured	0 0   X	0 0   X	0 0   X	0 0   X		0 0   X	0 0   X	0 0   X	0 0   X	0 0   X

 

The skin of none treated animals showed a positive reaction during the observation period after the challenge.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the present study, none of the animals of the treatment group showed a positive skin response after the challenge procedure.
Based on the results of this study Remazol-Brilliantviolett 5R neu showed no evidence for sensitizing properties. The test substance is not classifiable according to CLP criteria.

Executive summary:

Testing for sensitizing properties of Remazol-Brilliantviolett 5R neu was performed in female Guinea pigs according to the method of MAGNUSSON & KLIGMAN.

 

Intradermal induction was performed using 5% Remazol-Brilliantviolett 5R neu in isotonic saline. Dermal induction and challenge treatment were carried out with 25% Remazol-Brilliantviolett 5R neu in isotonic saline.

 

Based on the results of this study Remazol-Brilliantviolett 5R neu showed no evidence for sensitizing properties.

The test substance is not classified in accordance with CLP criteria.