Reaction products of 7-amino-4-hydroxynaphthalene-2-sulfonic acid reacted with 2-((1,3,5-triazin-2-yl)amino)ethan-1-ol, coupled with diazotised 3-amino-4-hydroxybenzenesulfonic acid, subsequently coupled with diazotised 2-amino-5-methoxybenzenesulfonic acid, chelated with copper, sodium salt

Skin sensitisation

Some information in this page has been claimed confidential.

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1991-04-16 to 1991-06-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Study was already available

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Details on test material:

Direct Red 83:1

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

30 Albino Dunkin-Hartley guinea pigs:
Test: 20
Control: 10

Weight: 355 – 445 g
Approx. 8 – 12 weeks old
Minimum acclimatisation period of 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-22 °C
- Humidity (%): 47-63 °C
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hrs dark /12 hrs light

IN-LIFE DATES: From: 1991-04-16 to 1991-06-03

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

water

Concentration / amount:

1% / 4 x 0.1 mL

Day(s)/duration:

Day 1

Adequacy of induction:

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

Induction 2

Route:

epicutaneous, occlusive

Vehicle:

arachis oil

Concentration / amount:

50% (w/w) in arachis oil B.P. / 0.2 - 0.3 mL

Day(s)/duration:

Day 8 for 48 h

Adequacy of induction:

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

Challenge

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

arachis oil

Concentration / amount:

50% (w/w) in arachis oil B.P. / 0.1 - 0.2 mL

Day(s)/duration:

Day 22 for 24 h

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

Test: 20
Control: 10

Details on study design:

MAIN STUDY: A group of 30 guinea pigs was used for the main study, twenty test and ten 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 OF A RESPONSE
Day 0: A row of 3 injections (0.1mL each)
a) Freund´s complete adjuvant plus distilled water in ratio 1:1
b) A 1% (w/v) dilution of test material in distilled water
c) A 1% dilution of test material in a 1: a preparation of Freund´s Complete Adjuvant plus distilled water
On day 7:
Same area on shoulder region used previously for intra dermal injections was clipped again and treated with topical application of the test material formulation
(50% W/W in arachis oil B.P.)
The test material formulation (0.2 – 0.3 mL) was applied on filter paper (WHATMAN No. 4: approx. 40mmx20mm) which was held in place by a strip of surgical adhesive tape (BLENDERM: approx. 60mmx25mm) and covered with overlapping length of aluminium foil. Patch and oil were further secured by a strip of elastic adhesive bandage 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 1 and 24 hours following removal of the patches using a 0 – 3 scale.

INDUCTION OF THE CONTROL ANIMALS:
Same procedure for that used for the test animals except that injections were:
i) Freund's complete Adjuvant plus distilled water in the ratio 1:1
2i) distilled water
3i) 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 animals.

2) CHALLENGE
Shortly before treatment on Day 21, an area (approx 50mmx70mm) on both flanks of each animal, was clipped free of hair.
A quantity of 0.1 – 0.2 mL of the test material formulation was applied to the shorn right flank of each animal on a square of filter paper which was held in place by a strip of surgical adhesive tape. 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 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.

EVALUATION OF SKIN REACTION:
Skin Reactions Observed after topical challenge: Purple coloured staining was occasionally noted at test material sites during challenge. This did not affect evaluation of erythema.
No adverse reactions were noted at the test material and vehicle control sites of the test or control animals at the 24 and 48 hour observations


Skin Reactions Observed after topical challenge: Purple coloured staining was occasionally noted at test material sites during challenge. This did not affect evaluation of erythema.
No adverse reactions were noted at the test material and vehicle control sites of the test or control animals at the 24 and 48 hour observations

Challenge controls:

Four preparations of the test material (50%, 25%, 10% and 5% w/w in arachis oil) were applied occlusively to the flanks of two guinea pigs for a period of 24 hours).

Positive control substance(s):

yes

Remarks:

2,4-Dinitrochlorobenze (DNCB)

Results and discussion

Positive control results:

2,4-Dinitrochlorobenze (DNCB), produced a 94% /17/18) sensitisation rate

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

50 %

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

Slight purple staining that did not hinder the erythema evaluation

Remarks on result:

no indication of skin sensitisation

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

50%

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

Slight purple staining that did not hinder the erythema evaluation

Remarks on result:

no indication of skin sensitisation

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

50%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

Slight purple staining that did not hinder the erythema evaluation

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

50%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

Slight purple staining that did not hinder the erythema evaluation

Results 5

Reading:

rechallenge

Hours after challenge:

24

Group:

negative control

Dose level:

50%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

Purple coloured staining was occasionally noted at test material site during challenge. No averse reaction on the test or control animals

Results 6

Reading:

rechallenge

Hours after challenge:

48

Group:

negative control

Dose level:

50%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

Purple coloured staining was occasionally noted at test material site during challenge. No averse reaction on the test or control animals

Results 7

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

1%

No. with + reactions:

17

Total no. in group:

18

Remarks on result:

positive indication of skin sensitisation

Results 8

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

1%

No. with + reactions:

11

Total no. in group:

18

Remarks on result:

positive indication of skin sensitisation

Any other information on results incl. tables

MAGNUSSON & KLINGMANN MAXIMISATION STUDY IN THE GUINEA PIG TABLE I: INDIVIDUAL SKIN REACTIONS IN TEST ANIMALS AT CHALLENGE CHALLENGE CONCENTRATION: 50%                                                                  VEHICLE ARACHIS OIL BP
Skin Reactions (hours after removal of Dressing)
24 hours                                                                    48 hours
Animal Number	Test	Vehicle	Test	Vehicle
1	0	0	0	0
2	0 STA	0	0 STA	0
3	0 STA	0	0 STA	0
4	0 STA	0	0 STA	0
5	0 STA	0	0 STA	0
6	0 STA	0	0 STA	0
7	0	0	0	0
8	0 STA	0	0 STA	0
9	0 STA	0	0 STA	0
10	0	0	0	0
11	0	0	0	0
12	0	0	0	0
13	0 STA	0	0 STA	0
14	0 STA	0	0 STA	0
15	0	0	0 STA	0
16	0	0	0	0
17	0 STA	0	0 STA	0
18	0	0	0	0
19	0 STA	0	0 STA	0
20	0	0	0	0
TABLE II: INDIVIDUAL SKIN REACTIONS IN CONTROL ANIMALS AT CHALLENGE CHALLENGE CONCENTRATION: 50%                                                                         VEHICLE ARACHIS OIL BP
21	0	0	0	0
22	0 STA	0	0 STA	0
23	0	0	0	0
24	0 STA	0	0 STA	0
25	0 STA	0	0 STA	0
26	0	0	0	0
27	0 STA	0	0 STA	0
28	0 STA	0	0 STA	0
29	0	0	0	0
30	0 STA	0	0 STA	0
STA= Purple-coloured staining

 

KAYARUS SUPRA RUBINE BLN: MAGNUSSON & KLINGMANN MAXIMISATION STUDY IN THE GUINEA PIG TABLE III: INTRADERMAL SIGHTING TEST –SUMMARY OF RESULTS
VEHICLE : DISTILLED WATER
Animal identification	Time of observation	Concentration of Test Material (%w/v)	Evidence of local necropsis	Evidence of systemic toxicity
A	24 hours	1	None	None
	48 hours		None	None
	72 hours		None	None
	7 days		None	None
B	24 hours	5	Eschar	None
	48 hours		Eschar	None
	72 hours		Eschar	None
	7 days		Eschar	None
The concentration of the test material selected for the intradermal induction stage of the main study was 1% w/v in distilled water
TABLE IV: TOPICAL SIGHTING TEST (48 HOURS EXPOSURE) INDIVIDUAL SKIN REACTIONS
VEHICLE: ARACHIS OIL BP
Animal identification	Concentration of Test Material (%w/w)	Skin reaction (hour (s) After Removal of Patches
		1	24	48
C	50<	1STA	1STA	1STA
	25	1STA	1STA	0 STA
	10	1STA	0 STA	0 STA
	5	1STA	0 STA	0 STA
D	50<	2STA	1STA	1STA
	25	2 STA	1STA	0 STA
	10	1STA	0 STA	0 STA
	5	1 STA	0 STA	0 STA
TABLE V: TOPICAL SIGHTING FOR THE CHALLENGE (24 HOURS EXPOSURE ) INDIVIDUAL SKIN REACTIONS VEHICLE: ARACHIS OIL B.P.
Animal identification	Concentration of Test Material (%w/w)	Skin reaction (hour (s) After Removal of Patches
		1	24	48
E	50<	1	0	0
	25	0	0	0
	10	0	0	0
	5	0	0	0
F	50<	1	0	0
	25	1	0	0
	10	0	0	0
	5	0	0	0
< maximum attainable concentration suitable for topical application STA= Slight purple-coloured staining
The concentration of the test material selected for the main study topical challenge stage of the main study was 50% w/v in arachis oil B.P.

 

KAYARUS SUPRA RUBINE BLN: MAGNUSSON & KLINGMANN MAXIMISATION STUDY IN THE GUINEA PIG TABLE VI: TOPICAL INDUCTION-INDIVIDUAL SKIN REACTIONS IN TEST ANIMALS
INDUCTION CONCENTRATION 50 % (w/w)                         VEHICLE: ARACHIS OIL B.P.
Animal Number	Skin reaction (hour (s) After Removal of Patches
	1	24
1	?S	?S
2	?S	?S
3	?S	?S
4	?S	0 STA
5	?S	?S
6	?S	?S
7	?S	?S
8	?S	?S
9	?S	?S
10	?S	0 STA
11	?S	?S
12	?S	?S
13	?S	?S
14	?S	?S
15	?S	?S
16	?S	0 STA
17	?S	?S
18	?S	0 STA
19	?S	?S
20	?S	?S
TABLE VII: TOPICAL INDUCTION-INDIVIDUAL SKIN REACTIONS IN CONTROL ANIMALS
21	0	0
22	0	0
23	0	0
24	0	0
25	0	0
26	0	0
27	0	0
28	0	0
29	0	0
30	0	0
STA= purple-coloured staining ?S: unable to score erythema due to dense purple-coloured staining

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Test material produced a 0% (0/20) sensitisation rate and was classified as a non-sensitiser to guinea pig skin. The test material was classified as a non-sensitiser according to CLP labelling regulations: No risk phrase is required.

Executive summary:

A skin sensitisation study was performed according to Magnusson-Kligman method using Freund's complete adjuvant technique to assess (evaluate) the sensitisation potential of Direct Red 83:1.

30 Albino Dunkin-Hartley guinea pigs were subjected to sensitisation procedures in two stage operation, i.e. an intra-dermal treatment and a topical application. 20 test and 10 control animals were used for the main study. Based on the results of sighting tests, the concentration of test material for the induction and challenge were selected as follows:

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

Topical induction: 50% (w/w) in arachis oil B.P.

Topical challenge: 50% (w/w) in arachis oil B.P.

Two main procedures were involved in the maximisation test; (a) an induction of a response and (b) a challenge of that response.

A row of 3 injections (0,1ml each) on each side of the mid-line.

a)      Freund´s complete adjuvant plus distilled water in ratio 1:1

b)      a 1% (w/v) dilution of test material in distilled water

c)      a 1% dilution of test material in a 1:a preparation of Freund´s Complete Adjuvant plus distilled water

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 animals.

 Shortly before treatment on Day 21, an area (approx. 50mmx70mm) on both flanks of each animal, was clipped free of hair.

A quantity of 0.1 – 0.2 mL (50% W/W in arachis oil B.P.) of a challenge dose  was applied to the shorn right flank of each animal on a square of filter paper which was held in place by a strip of surgical adhesive tape. Vehicle alone was similarly applied to the left shorn flank.

10 control guinea pigs were simultaneously exposed to vehicle during the sensitisation phase and they were treated with the test item only in the case of challenge.

The known contact sensitiser, 2,4-Dinitrochlorobenze (DNCB), produced a 94% /17/18) sensitisation rate. This was considered to be satisfactory sensitisation response for this material under the conditions of the test.

Skin reactions after topical induction:

Evaluation of erythema was not possible at all treatment sites of test animals one hour after patch removal and at fifteen treatment sites at the 24-h observation due to dense purple staining. This was also noted at the remaining treated skin sites at this time. This did not affect evaluation of erythema.

 

Skin Reactions Observed after topical challenge: Purple coloured staining was occasionally noted at test material sites during challenge. This did not affect evaluation of erythema.

No adverse reactions were noted at the test material and vehicle control sites of the test or control animals at the 24 and 48 hour observations

 

Bodyweight:

Bodyweight gains of guinea pigs in the test group, between Day 0 and Day 24, were comparable to those observed in the control group animals over the same period

Hence, Direct Red 83:1 produced a 0% (0/20) 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 CLP labelling regulations: No risk phrase is required.