Nickel, [29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]-, chlorosulfonyl derivs., reaction products with 2-[(4-aminophenyl)sulfonyl]ethyl hydrogen sulfate monosodium salt, sodium salts

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 28th, 1992 to August 28th, 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:

EC-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:

Study available is over 12 years old.

Test material

Test material information

Constituent 1

Reference substance name:

Nickel, [29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]-, chlorosulfonyl derivs., reaction products with 2-[(4-aminophenyl)sulfonyl]ethyl hydrogen sulfate monosodium salt, sodium salts

EC Number:

305-644-9

EC Name:

Nickel, [29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]-, chlorosulfonyl derivs., reaction products with 2-[(4-aminophenyl)sulfonyl]ethyl hydrogen sulfate monosodium salt, sodium salts

Cas Number:

94891-43-7

Molecular formula:

C32 H16 N8 Ni . C8 H11 N O6 S2 . 2 Na

IUPAC Name:

Nickel, [29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]-, chlorosulfonyl derivs., reaction products with 2-[(4-aminophenyl)sulfonyl]ethyl hydrogen sulfate monosodium salt, sodium salts

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

No further details specified in the study report.

In vivo test system

Test animals

Species:

guinea pig

Strain:

Pirbright-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:
x = 320 g (= 100.0 %)
x min 273 g (- 14.7 %)
x max = 348 g (+ 8.8 %)
n 15

Randomisation schemes: 386/92
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:

unchanged (no vehicle)

Concentration / amount:

50% Freund's Adjuvent

Day(s)/duration:

1 administration on Day 1

Adequacy of induction:

other: Caused slight oedema

Induction 2

Route:

intradermal

Vehicle:

other: saline

Concentration / amount:

5%

Day(s)/duration:

One injectrion on Day 1

Adequacy of induction:

other: Caused slight oedema

Induction 3

Route:

intradermal

Vehicle:

other: 50% Freund's adjuvent

Concentration / amount:

5%

Day(s)/duration:

One injection on Day 1

Adequacy of induction:

other: Caused slight oedema

Induction 4

Route:

epicutaneous, occlusive

Vehicle:

other: saline

Concentration / amount:

25%

Day(s)/duration:

48 hours

Adequacy of induction:

other: No signs of irritation occurred after application of the different test concentrations.

Challenge

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

other: saline

Concentration / amount:

25%

Day(s)/duration:

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-Brillantgrün 6B was applied in isotonic saline [percentages w/v].
For the intradermal injections of the test substance in 50 %Freund's adjuvant, Remazol-Brillantgrün 6B was diluted with isotonic saline and then mixed with an equal volume of Freund's Original Adjuvant [percentages w/v].
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. (ml) conc. (%) 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 sensitising properties
Chronological description of the test procedure indicating the day, at which procedure was carried out:
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-Brillantgrün 6B in isotonic saline

Day 23
Occlusive bandage removed.

Day 24
Skin examined.

Day 25
Skin examined.
Body weights of test animals determined.

Evaluation
Erythema and oedema are major clinical indicators of an allergic reaction. The decisive criterion for evaluation of the sensitising properties of a test substance is the number of sensitised test animals, not the intensity of the dermal reaction.
The substance is considered to be sensitising if 30 % of the animals in the treated group definitely show a positive reaction.

Challenge controls:

Treatment of the animals with Freund's Adjuvant can lower the threshold value for primary irritation determined in preliminary tests. For this reason, the five animals in the escort group which had been treated with Freund's Adjuvant were treated with 25 % Remazol-Brillantgrün 68 in isotonic saline. As no reactions were observed in these animals, a concentration of 25 % Remazol-Brillantgrün 6B in isotonic saline was chosen for the challenge at day 22.

Positive control substance(s):

no

Results and discussion

Positive control results:

Not specified

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

25%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No signs of irritation were observed. Intensive turquoise discolored skin was noted

Remarks on result:

no indication of skin sensitisation

Results 2

Key result

Reading:

2nd reading

Hours after challenge:

72

Group:

test chemical

Dose level:

25%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No signs of irritation were observed. Intensive turquoise discolored skin was noted

Remarks on result:

no indication of skin sensitisation

Results 3

Key result

Reading:

1st reading

Hours after challenge:

48

Group:

negative control

Dose level:

25%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

No signs of irritation were observed

Remarks on result:

no indication of skin sensitisation

Results 4

Key result

Reading:

2nd reading

Hours after challenge:

72

Group:

negative control

Dose level:

25%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

No signs of irritation were observed

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	273 328 339 340 293	352 399 407 431 388	+ 29 + 22 + 20 + 27 + 32
Treated group:
6 7 8 9 10 11 12 13 14 15	345 293 348 336 288 320 296 329 325 343	420 373 415 443 362 375 385 393 391 449	+ 22 + 27 + 19 + 32 + 26 + 17 + 30 + 20 + 20 + 31

 

Scoring of dermal reactions – individual data

Challenge treatment, escort group

Remazol-Brillantgrün 6B 25% in isotonic saline (day 15)

Treated are: left flank

 

Scoring of dermal reactions

	Animal No.:	16	17	18	19	20
48 hours p.a.	Erythema Oedema Light turquoise discoloured	0 0 X	0 0 X	0 0 X	0 0 X	0 0 X
	Animal No.:	16	17	18	19	20
72 hours p.a.	Erythema Oedema Light turquoise discoloured	0 0 X	0 0 X	0 0 X	0 0 X	0 0 X

 

Challenge treatment: Remazol-Brillantgrün 6B 25% in isotonic saline (day 22)

Treated area: left flank

 

Time of observation: 48 hours after treatment (day 24)

Scoring of dermal reactions

Control animals	1	2	3	4	5
Erythema Oedema Light turquoise 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 Light turquoise 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	0 0 X

 

Time of observation: 72 hours after treatment (day 25)

Scoring of dermal reactions

Control animals	1	2	3	4	5
Erythema Oedema Light turquoise 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 Light turquoise 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	0 0 X

 

The skin of none of the treated animals (25%) 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 ten animals of the treatment group showed a positive skin response after the challenge procedure.
Based on the results of this study Remazol-Brillantgrün 6B showed no evidence for sensitizing properties.

Executive summary:

Testing for sensitizing properties of Remazol-Brillantgrün 6B was performed in female Guinea pigs according to the method of MAGNUSSON & KLIGMAN.

Intradermal induction was performed using 5.0% Remazol-Brillantgrün 6B in isotonic saline. Dermal induction and challenge treatment were carried out with 25% Remazol-Brillantgrün 6B in isotonic saline.

Based on the results of this study there is no evidence for sensitizing properties of Remazol-Brillantgrün 6B.