Cuprate(4-), [C-(aminosulfonyl)-C-[[[2-[[4-chloro-6-[(2,5-disulfophenyl)amino]-1,3,5-triazin-2-yl]amino]ethyl]amino]sulfonyl]-29H,31H-phthalocyanine-C,C-disulfonato(6-)-N29,N30,N31,N32]-, sodium salts

Endpoint summary

Description of key information

Skin sensitisation

The test material produced a 0% (0/10) sensitisation rate and was classified as a non-sensitiser to guinea pig skin.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5 April 1993 to 27 June 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The GMPT study is historically available to the registrant as this study was conducted originally for use with the EU NONS scheme. As this study is available and is adequate for classification it is considerd unjustifiable to repeat the animal test to prepare the LLNA study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

OECD Guidelines for the Testing of Chemicals (1981), No . 406 "Skin Sensitisation''

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

Method B6 in Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/ 548/EEC).

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Available study data is +12 years old and is K1 quality.

Specific details on test material used for the study:

No further details specified in the study report.

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

Specification
Twenty-one female, albino Dunkin-Hartley guinea pigs were supplied by David Hall Limited, Burton-on-Trent, Staffordshire, U.K. At the start of the main study the animals weighed 354 - 445g , and were approximately eight to twelve weeks old. After a minimum acclimatization period of five days, each animal was selected at random and given a number unique within the study which was written on a small area of clipped rump using a black indelible marker-pen.

Husbandry
The animals were housed in groups of up to three in solid-floor polypropylene cages furnished with softwood shavings. Free access to mains tap water and food (Guinea Pig FD1 Diet, Special Diet Services Limited, Witham, Essex, U.K.) was allowed throughout the study.
The animal room was maintained at a temperature of 20 – 24 °C and relative humidity of 44 - 67%. The rate of air exchange was approximately 15 changes per hour and the lighting was controlled by a time switch to give 12 hours light and 12 hours darkness.

Route:

intradermal

Vehicle:

water

Concentration / amount:

1% w/v test material

Day(s)/duration:

Day 1/7 Days

Adequacy of induction:

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

Route:

intradermal

Vehicle:

water

Concentration / amount:

1% w/v test material in 1:1 Freund's Complete Adjuvant

Day(s)/duration:

Day 1/7 Days

Adequacy of induction:

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

Route:

intradermal

Vehicle:

water

Concentration / amount:

Freund;s Complete Adjuvant 1:1 ratio with vehicle

Day(s)/duration:

Day 1/7 Days

Adequacy of induction:

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

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

25% w/w

Day(s)/duration:

Day 7/48 hours

Adequacy of induction:

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

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

25% w/w

Day(s)/duration:

Day 21/24 hours

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

A group of fifteen guinea pigs was used for the main study, ten test and five control.

Details on study design:

PROCEDURE
Selection of Concentrations for Main Study (Sighting Tests)
The concentrations of test material to be used at each stage of the main study were determined by 'sighting tests' in which groups of guinea pigs were treated with various concentrations of test material. The procedures were as follows:
a) Selection of Concentration for Intradermal Induction
Two animals were intradermally injected with preparations of test material (1% or 5% w/v in distilled water). The highest concentration that did not cause local necrosis, ulceration or systemic toxicity, was selected for the intradermal induction stage of the main study.
b) Selection of Concentration for Topical Induction
Two guinea pigs (intradermally injected with Freund's Complete Adjuvant seventeen days earlier) were treated with four preparations of the test material (25%, 10%, 5% and 2% w/w in distilled water).
The highest concentration producing only mild to moderate dermal irritation after a 48-hour occlusive exposure, was selected for the topical induction stage of the main study.
c) Selection of Concentration for Topical Challenge
Two preparations of the test material (25% and 10% w/w in distilled water) were applied occlusively to the flanks of two guinea pigs for a period of 24 hours. These guinea pigs did not form part of the main study but had been treated identically to the control animals of the main study, up to Day 14.
The highest non-irritant concentration of the test material and one lower concentration were selected for the topical challenge stage of the main study.

Main Study
A group of fifteen guinea pigs was used for the main study, ten test and five control. The bodyweight of each animal was recorded at the start and end of the study.
Two main procedures were involved in the maximisation test; (a) an induction of a response and (b) a challenge of that response.

a) Induction
Induction of the Test Animals: Shortly before treatment on Day 0 the hair was removed from an area approximately 40 mm x 60 mm on the shoulder region of each animal with veterinary clippers.
A row of three injections (0.1 ml each) was made on each side of the mid-line. The injections were:
i) Freund's Complete Adjuvant plus distilled water in the ratio 1:1.
ii) a 1% (w/v) dilution of test material in distilled water.
iii) a 1% (w/v) dilution of test material in a 1:1 preparation of Freund's Complete Adjuvant plus distilled water.
Approximately 24 and 48 hours after intradermal injection the degree of erythema at the test material injection sites were evaluated according to the following scale from Draize J.H. (1959) Association of Food and Drug Officials of the United States, Austin, Texas, "The Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics".

On day 6, the nuchal region was clipped and shaved. A volume of 0.5 ml of sodium laurylsulphate (10% w/w in petrolatum) was applied in order to provoke an inflammatory response. The treatment sites remained non-occluded.

One week later (Day 7), the same area on the shoulder region used previously for intradermal injections was clipped again and treated with a topical application of the test material formulation (25% w/w in distilled water). The test material formulation (0.2 - 0.3 ml) was applied on filter paper (WHATMAN No.4: approximate size 40 mm x 20 mm) which was held in place by a strip of surgical adhesive tape (BLENDERM: approximate size 60 mm x 25 mm) and covered with an overlapping length of aluminium foil. The patch and foil were further secured by a strip of elastic adhesive bandage (ELASTOPLAST: approximate size 250 mm x 35 mm) wound in a double layer around the torso of each animal. This occlusive dressing was kept in place for 48 hours.

Erythematous reactions were quantified one and twenty-four hours following removal of the patches using the Draize scale from Draize J.H. {1959) Association of Food and Drug Officials of the United States, Austin, Texas, “The Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics”.

Induction of the Control Animals: Intradermal injections were administered using an identical procedure to that used for the test animals, except that the injections were:
i) Freund's Complete Adjuvant plus distilled water in the ratio 1:1.
i i) distilled water.
iii) Freund's Complete Adjuvant plus distilled water in the ratio 1:1.
The topical applications followed the same procedure as for the test animals except that the vehicle alone was applied to the filter paper. Skin reactions were quantified as for the test animals.

b) Challenge
Shortly before treatment on Day 21, an area, approximately 50 mm x 70 mm on both flanks of each animal, was clipped free of hair with veterinary clippers.

A quantity of 0.1 - 0.2 ml of the test material formulation (25% w/w in distilled water) was applied to the shorn right flank of each animal on a square of filter paper (WHATMAN No.4: approximate size 20 mm x 20 mm) which was held in place by a strip of surgical adhesive tape (BLENDERM: approximate size 40 mm x 50 mm). To ensure that the maximum non-irritant concentration was used at challenge, the test material at a concentration of 10% (w/w) in distilled water was also similarly applied to a separate skin site on the right shorn flank. The vehicle alone was similarly applied to the left shorn flank. The patches were occluded with an overlapping length of aluminium foil and secured by a strip of elastic adhesive bandage (ELASTOPLAST: approximate size 250 mm x 75 mm) wound in a double layer around the torso of each animal.
After 24 hours, the dressing was carefully cut using blunt tipped scissors, removed and discarded. The challenge sites were swabbed with cotton wool soaked in distilled water and diethyl ether to remove residual material. The vehicle sites were similarly swabbed. The position of the treatment sites was identified by using a black indelible marker-pen.
Prior to the 24-hour observation the flanks were clipped using veterinary clippers to remove regrown hair.

Evaluation of Skin Reactions
Approximately 24 and 48 hours after challenge dressing removal erythematous reactions were quantified using the Draize scale.

Challenge controls:

Two preparations of the test material (25% and 10% w/w in distilled water) were applied occlusively to the flanks of two guinea pigs for a period of 24 hours. These guinea pigs did not form part of the main study but had been treated identically to the control animals of the main study, up to Day 14.
The highest non-irritant concentration of the test material and one lower concentration were selected for the topical challenge stage of the main study.

Positive control substance(s):

no

Positive control results:

Not applicable

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

25%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

Pale blue-coloured staining was commonly noted at the challenge sites of test and control group animals.

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

25%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

Pale blue-coloured staining was commonly noted at the challenge sites of test and control group animals

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

10%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

Pale blue-coloured staining was commonly noted at the challenge sites of test and control group animals

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

10%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

Pale blue-coloured staining was commonly noted at the challenge sites of test and control group animals

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

25% and 10% vehicle control

No. with + reactions:

0

Total no. in group:

5

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

25% and 10% vehicle control

No. with + reactions:

0

Total no. in group:

5

JPR BLUE 100: MAGNUSSON & KLIGMAN MAXIMISATION STUDY IN THE GUINEA PIG

INDIVIDUAL SKIN REACTIONS IN TEST ANIMALS AT CHALLENGE

CHALLENGE CONCENTRATIONS: 25% AND 10% (w/w)             VEHICLE: DISTILLED WATER

Animal Number

Skin Reactions (Hours After Removal of Dressing)

24 hours

48 hours

25%

10%

Vehicle control

25%

10%

Vehicle control

Er

Oe

Er

Oe

Er

Oe

Er

Oe

Er

Oe

Er

Oe

1 2 3 4 5 6 7 8 9 10

0STA 0STA 0STA 0STA 0STA 0STA 0STA 0STA 0STA 0STA

0 0 0 0 0 0 0 0 0 0

0STA 0STA 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0

0STA 0STA 0STA 0STA 0STA 0STA 0STA 0STA 0STA 0STA

0 0 0 0 0 0 0 0 0 0

0STA 0STA 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0

Er = erythema                                     STA = pale blue coloured staining

Oe = oedema

 

JPR BLUE 100: MAGNUSSON & KLIGMAN MAXIMISATION STUDY IN THE GUINEA PIG

INDIVIDUAL SKIN REACTIONS IN CONTROL ANIMALS AT CHALLENGE

CHALLENGE CONCENTRATIONS: 25% AND 10% (w/w)             VEHICLE: DISTILLED WATER

Animal Number

Skin Reactions (Hours After Removal of Dressing)

24 hours

48 hours

25%

10%

Vehicle control

25%

10%

Vehicle control

Er

Oe

Er

Oe

Er

Oe

Er

Oe

Er

Oe

Er

Oe

11 12 13 14 15

0STA 0STA 0STA 0STA 0STA

0 0 0 0 0

0 0 0 0STA 0

0 0 0 0 0

0 0 0 0 0

0 0 0 0 0

0STA 0STA 0STA 0STA 0STA

0 0 0 0 0

0 0 0 0STA 0

0 0 0 0 0

0 0 0 0 0

0 0 0 0 0

Er = erythema                                     STA = pale blue coloured staining

Oe = oedema

 

JPR BLUE 100: MAGNUSSON & KLIGMAN MAXIMISATION STUDY IN THE GUINEA PIG

INTRADERMAL SIGHTING TEST – SUMMARY OF RESULTS

VEHICLE: DISTILLED WATER

Animal Identification	Time of Observation	Concentration of Test Material (% w/v)	Evidence of Local Necrosis	Evidence of Systemic Toxicity
A	24 hours 48 hours 72 hours 7 days	1	None None None None	None None None None
B	24 hours 48 hours 72 hours 7 days	5	Eschar Eschar Eschar Eschar	None None None None

The concentration of the test material selected for the intradermal induction stage of the main study was 1% (w/v) in distilled water.

 

JPR BLUE 100: MAGNUSSON & KLIGMAN MAXIMISATION STUDY IN THE GUINEA PIG

TOPICAL SIGHTING TEST FOR INDUCTION APPLICATION

(48-HOUR EXPOSURE) – INDIVIDUAL SKIN REACTIONS

VEHICLE: DISTILLED WATER

Animal Identification	Concentration of Test Material (% w/w)	Skin Reactions (Hours After Removal of Patches)
		1		24		48
		Er	Oe	Er	Oe	Er	Oe
C	25* 10 5 2	1STA 1STA 0STA 0STA	0 0 0 0	0STA 0 0 0	0 0 0 0	0STA 0 0 0	0 0 0 0
D	25* 10 5 2	1STA 1STA 0STA 0STA	0 0 0 0	0STA 0 0 0	0 0 0 0	0 0 0 0	0 0 0 0

* Maximum attainable concentration suitable for topical application.

The concentration of the test material selected for the main study topical induction was 25% (w/w) in distilled water.

Er = erythema                                                     STA = slight blue coloured staining

Oe = oedema

 

JPR BLUE 100: MAGNUSSON & KLIGMAN MAXIMISATION STUDY IN THE GUINEA PIG

TOPICAL SIGHTING TEST FOR CHALLENGE APPLICATION

(24-HOUR EXPOSURE) – INDIVIDUAL SKIN REACTIONS

VEHICLE: DISTILLED WATER

Animal Identification	Concentration of Test Material (% w/w)	Skin Reactions (hours After Removal of Patches)
		1		24		48
		Er	Oe	Er	Oe	Er	Oe
E	25* 10	1STA ?s	0 0	0STA 0STA	0 0	0STA 0STA	0 0
F	25* 10	?s ?s	0 0	0STA 0STA	0 0	0STA 0STA	0 0

* Maximum attainable concentration suitable for topical application

The concentrations of the test material selected for the main study topical challenge were 25% and 10% (w/w) in distilled water

Er = erythema                                      STA = slight blue coloured staining

Oe = oedema                                       ?s = blue coloured staining prevents accurate evaluation of erythema

 

 

JPR BLUE 100: MAGNUSSON & KLIGMAN MAXIMISATION STUDY IN THE GUINEA PIG

INTRADERMAL INDUCTION – INDIVIDUAL SKIN REACTIONS IN TEST ANIMALS

INTRADERMAL INDUCTION CONCENTRATION: 1% (w/v)                                   VEHICLE: DISTILLED WATER

Animal Number	Skin Reactions (Hours After Removal of Dressing)
	24 hours		48 hours
	Left side	Right side	Left side	Right side
1 2 3 4 5 6 7 8 9 10	?s ?s ?s ?s ?s ?s ?s ?s ?s ?s	?s ?s ?s ?s ?s ?s ?s ?s ?s ?s	?s ?s ?s ?s ?s ?s ?s ?s ?s ?s	?s ?s ?s ?s ?s ?s ?s ?s ?s ?s

?s = blue coloured staining prevents accurate evaluation of erythema

 

JPR BLUE 100: MAGNUSSON & KLIGMAN MAXIMISATION STUDY IN THE GUINEA PIG

INTRADERMAL INDUCTION – INDIVIDUAL SKIN REACTIONS IN CONTROL ANIMALS

VEHICLE: DISTILLED WATER

Animal Number	Skin Reactions (Hours After Removal of Dressing)
	24 hours		48 hours
	Left side	Right side	Left side	Right side
11 12 13 14 15	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0

 

 

JPR BLUE 100: MAGNUSSON & KLIGMAN MAXIMISATION STUDY IN THE GUINEA PIG

TOPICAL INDUCTION – INDIVIDUAL SKIN REACTIONS IN TEST ANIMALS

INDUCTION CONCENTRATION: 25% (w/v)                                 VEHICLE: DISTILLED WATER

Animal Number	Skin Reactions (Hours After Removal of Dressing)
	1 hour		24 hours
	Er	Oe	Er	Oe
1 2 3 4 5 6 7 8 9 10	?s ?s ?s ?s ?s ?s ?s ?s ?s ?s	0 0 0 0 0 0 0 0 0 0	?s ?s ?s ?s ?s ?s ?s ?s ?s ?s	0 0 0 0 0 0 0 0 0 0

Er – erythema                    ?s = blue coloured staining prevents accurate evaluation of erythema

Oe = oedema

 

JPR BLUE 100: MAGNUSSON & KLIGMAN MAXIMISATION STUDY IN THE GUINEA PIG

TOPICAL INDUCTION – INDIVIDUAL SKIN REACTIONS IN CONTROL ANIMALS

VEHICLE: NOT REQUIRED, BLANK PATCH APPLIED

Animal Number	Skin Reactions (Hours After Removal of Dressing)
	1 hour		24 hours
	Er	Oe	Er	Oe
11 12 13 14 15	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0

Er – erythema

Oe = oedema

 

JPR BLUE 100: MAGNUSSON & KLIGMAN MAXIMISATION STUDY IN THE GUINEA PIG

INDIVIDUAL BODYWEIGHTS AND BODYWEIGHT GAINS OF TEST ANIMALS

Animal Number	Bodyweight (g)		Bodyweight (g) Increase
	Day 0	Day 24
1 2 3 4 5 6 7 8 9 10	411 421 354 403 445 372 441 416 395 444	614 585 760 648 606 505 588 646 593 666	203 164 406 245 161 133 147 230 198 222

 

JPR BLUE 100: MAGNUSSON & KLIGMAN MAXIMISATION STUDY IN THE GUINEA PIG

INDIVIDUAL BODYWEIGHTS AND BODYWEIGHT GAINS OF CONTROL ANIMALS

Animal Number	Bodyweight (g)		Bodyweight (g) Increase
	Day 0	Day 24
11 12 13 14 15	410 415 386 384 375	555 600 593 563 593	145 185 207 179 218

 

Interpretation of results:

GHS criteria not met

Conclusions:

The test material, JPR BLUE 100, produced a 0% (0/10) sensitisation rate and was classified as a NON-SENSITISER to guinea pig skin.
The test material was also classified as a non-sensitiser according to EEC labelling regulations. No risk phrase is therefore required.

Executive summary:

A study was performed to assess the skin contact sensitisation potential of the test material in the albino guinea pig. The method used followed that described in the OECD Guidelines for Testing of Chemicals (1981) No. 406 ''Skin Sensitisation" - Magnusson and Kligman Maximisation Test referenced as Method B6 in Commission Directive 92/ 69/ EEC (which constitutes Annex V of Council Directive 67/548/ EEC).

 

The results may be used as a basis for classification and labelling under Annex VI of Council Directive 67/ 548/ EEC (as adapted to technical progress by Commission Directive 91/ 325/ EEC).

 

Ten test and five control animals were used for the main study.

 

Based on the results of sighting tests, the concentrations of test material for the induction and challenge phases were selected as follows:

Intradermal Induction: 1% (w/v) in distilled water

Topical Induction: 25% (w/ w) in distilled water

Topical Challenge: 25% and 10% (w/w) in distilled water

 

The test material produced a 0% (0/10) sensitisation rate and was classified as a non-sensitiser to guinea pig skin. The test material was also classified as a non -sensitiser according to EEC labelling regulations. No risk phrase is required.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Skin sensitisation

A study was performed to assess the skin contact sensitisation potential of the test material in the albino guinea pig. 

Ten test and five control animals were used for the main study.

Based on the results of sighting tests, the concentrations of test material for the induction and challenge phases were selected as follows:

Intradermal Induction: 1% (w/v) in distilled water

Topical Induction: 25% (w/ w) in distilled water

Topical Challenge: 25% and 10% (w/w) in distilled water

The test material produced a 0% (0/10) sensitisation rate and was classified as a non-sensitiser to guinea pig skin.

Justification for classification or non-classification

The test material was classified as a non -sensitiser according to EEC labelling regulations. No risk phrase is required.