(2,2'-(3,3'-dioxidobiphenyl-4,4'-diyldiazo)bis(6-(4-(3-(diethylamino)propylamino)-6-(3-(diethylammonio)propylamino)-1,3,5-triazin-2-ylamino)-3-sulfonato-1-naphtholato))dicopper(II) acetate lactate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From July 10, 1990 to August 17, 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1991

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

May 12, 1981

Deviations:

yes

Remarks:

Body weight of two animals below the required range, age at start approx. 10 weeks

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The Local Lymph Node Assay (LLNA; TG 429) was adopted in 2002; the test was performed in 1990.

Test material

Test material information

Constituent 1

Reference substance name:

(2,2'-(3,3'-dioxidobiphenyl-4,4'-diyldiazo)bis(6-(4-(3-(diethylamino)propylamino)-6-(3-(diethylammonio)propylamino)-1,3,5-triazin-2-ylamino)-3-sulfonato-1-naphtholato))dicopper(II) acetate lactate

EC Number:

407-240-9

EC Name:

(2,2'-(3,3'-dioxidobiphenyl-4,4'-diyldiazo)bis(6-(4-(3-(diethylamino)propylamino)-6-(3-(diethylammonio)propylamino)-1,3,5-triazin-2-ylamino)-3-sulfonato-1-naphtholato))dicopper(II) acetate lactate

Cas Number:

159604-94-1

Molecular formula:

C66H88Cu2N20O10S2.C3H5O3.C2H3O2

IUPAC Name:

7,7'-bis[4-(3-diethylaminopropylamino)-6-(3-diethylammoniopropylamino)-1,3,5-triazin-2-ylamino]-{μ-4,4'-dihydroxy-1:2k2O4:O4'-3,3'-[3,3'-dihydroxy-1:2k2O3:O3'-biphenyl-bisazo-1:2(N3,N4-η:N3',N4'-η)]dinaphthalene-2-sulphonato(6-)}dicuprate(2-), mixed (1:1) acetic/lactic acid salts

Test material form:

solid

In vivo test system

Test animals

Species:

guinea pig

Strain:

other: Himalayan

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: BRL Ltd, Basel, Switzerland
- Age at study initiation: approx. 11 weeks
- Weight at study initiation: 293 - 448 gram
- Housing: group housing of 2 animals per cage with wire-mesh floors
- Diet: ad libitum (standard guinea pig diet, including ascorbic acid (1600 mg/kg); in addition, once a week hay was provided)
- Water: ad libitum
- Acclimation period: at least 5 days before start of treatment under test conditions after physical examination.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C
- Humidity (%): 55 %
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light / 12 hours dark per day
- IN-LIFE DATES: From: To:

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

other: Freunds' Complete Adjuvant

Concentration / amount:

5 % (w/w) / 0.1 ml/site

Induction 2

Route:

intradermal

Vehicle:

other: Freunds' Complete Adjuvant, 50:50 with distilled water

Concentration / amount:

1 ml/site

Adequacy of induction:

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

Induction 3

Route:

intradermal

Vehicle:

physiological saline

Concentration / amount:

2.5 % (w/w) / 0.1 ml/site

Adequacy of induction:

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

Challenge

Challenge 1

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

water

Remarks:

milli-R0 water

Concentration / amount:

5%, 10%, 25% / 0.05 ml

Day(s)/duration:

24 hours

Adequacy of challenge:

highest non-irritant concentration

Challenge 2

No.:

#2

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

5%, 2%, 1% / 0.05 ml

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

Primary irritation experiments: 1 animal for the intradermal injection. Four animals and the intradermally injected animal for the epidermal applications.
Main study: experimental group: 20, control group: 10

Details on study design:

PRIMARY IRRITATION EXPERIMENTS: The objective of this investigation was to identify irritant test substance concentrations suitable for the induction phase of the main study. In addition, a suitable non-irritant concentration of the test substance, by the topical route of administration, was identified for the challenge application. Any systemic toxic effects may also be detected in the primary irritation experiments.

Intradermal injections:
Four intradermal injections (0.1 ml/site) were made into the clipped shoulder region of one guinea pig at a concentration of 5% (w/w) of the test substance in milli-RO water. The resulting dermal reactions were assessed 24 and 48 hours later.
The following parameters were recorded:
Erythema: scored according to the scale described below,
Necrosis: if present yes or no,
Diameter: (mm) of effect (erythema, necrosis or discolouration) .
Epidermal applications:
The intradermally injected animal was also treated epidernally at the shaved left flank with 0.5 ml of a 50% (w/w) concentration of the test substance in milli-RO water using a Metalline patch (Lohman, Neuwied, West Germany) mounted on Micropore tape (3M, St. Paul , U.S.A. ) and held in place with Coban elastic bandage (3M, St. Paul , U.S.A.). After 24 hours, the dressings and residual test article were removed using a moistened tissue. The treated skin was assessed for erythema and oedema 24 and 48 hours after bandage removal on a numerical basis according to the scale described below.
Four animals were shaved on the left flank and exposed to 0.05 ml of 50%, 25%, 10% and 5% (w/w) test substance concentrations in milli-R0 water, occlusively administered by means of Square chambers (v. d. Bend, Brielle, The Netherlands) mounted on Micropore tape and fixed in place by means of Coban elastic bandage. This procedure ensured the intensive contact of the test substance even if it is insoluble in the vehicle used. After 24 hours, the dressings and residual test article were removed using a moistened tissue.
The reaction sites were assessed for erythema and oedema on a numerical basis according to the scale described below, 24 and 48 hours after bandage removal. Immediately after the 24 hours skin reading the treated areas were shaved.

Erythema and eschar formation:
No erythema : 0
Slight erythema (barely perceptible): 1
Well-defined erythema: 2
Moderate erythema: 3
Severe erythema (beet redness) to slight eschar formation (injuries in depth): 4

Oedema:
No oedema: 0
Slight oedema (barely perceptible): 1
Well-defined oedema (edges of area well-defined by definite raising): 2
Moderate oedema (raised approximately 1 millimeter): 3
Severe oedema (raised more than 1 millimeter and extending beyond the area of exposure): 4

MAIN STUDY
Induction
Intradermal injections:
On day 1 an area of the dorsal skin from the scapular region (approximately 4 x 6 cm) was clipped free of hair. Three pairs of intradermal injections (0.1 ml/site) were made at the border of a 2 x 4 cm area in the clipped region as follows:
A) The test substance dissolved to 2.5% (w/w) with physiological saline.
B) Freunds' Complete Adjuvant (FCA, Difco, Detroit, U.S.A. ) , 50: 50 with distilled water for injection (pyrogen free, Ferensius AG, Bad Homburg, West-Germany) .
C) The test substance, at twice the concentration used in (A), emulsified in a 50:50 mixture of Freunds' Complete Adjuvant.

Epidermal applications:
Seven days after the intradermal injections, the scapular area (approximately 6 x 8 cm) was clipped and shaved free of hai r. A 2 x 4 cm patch of Metall ine mounted on Micropore tape was treated with 0.5 ml of the test substance (25% (w/ w) in milli-RO water) and placed over the injection sites of the test animals. The Micropore tape was firmly secured, wrapped around the trunk of the animal and secured with Coban elastic bandage. After 48 hours, the dressings and residual test article were removed using a moistened tissue. The epidermal application procedure described ensured intensive contact of the test substance even if it is insoluble in the vehicle used.
The guinea pigs of the control group were treated as described above by the intradermal and epidermal inductions with the omission of test substance.
Reaction sites were assessed for erythema and oedema immediately after removal of the dressings, using the numerical grading system described previously.

Challange: The test and control guinea pigs were challenged two weeks after the epidermal induction application.
Hair was clipped and shaved from a 5 x 5 cm area on the left flank of each guinea-pig. The following series of 3 test substance concentrations and the vehicle were applied using Square chambers attached to Micropore tape:
a_ 25% in milli-R0 water.
b_ 10% in milli-R0 water.
c_ 5% in milli-R0 water
d_ milli-R0 water.
A volume of 0.05 ml of each concentration or the vehicle was placed into a Square chamber. The patches were placed on the shaved area, the Micropore tape firmly secured around the trunk of the animals and held in place by Coban elastic bandage.
The dressings and residual test substance were removed after approximately 24 hours, using a moistened tissue.
The sites were assessed for redness and swelling 24 and 48 hours after removal of the dressings, using the numerical grading system described below (modified from Kligman A.M. , J. Invest. Dermatol. 47, 1966).
The test sites were shaved with an electric razor after the first reading.

no skin reaction: 0
red spots (scattered reactions): 1
Moderate bu confluent redness: 2
Redness and swelling: 3
Intense reddening and swelling: 4

Rechallenge: A second challenge was performed one week after the first challenge, due to inconclusive results.
The method was similar to the first challenge on all animals, but now the contralateral flank was applied. The following decreased test article concentrations 5%, 2% and 1% and the vehicle were applied. The concentrations were decreased because similar skin reactions were observed in experimental and control animals in the first challenge.
All animals were killed at the end of the test period by carbon dioxide asphyxiation.

In addition to the skin reactions the following observations and data were recorded:

Mortality/viability: once daily
Body weights: during acclimatisation and at termination of the study
Toxicity symptoms: daily

Challenge controls:

Challange: The test and control guinea pigs were challenged two weeks after the epidermal induction application.
Hair was clipped and shaved from a 5 x 5 cm area on the left flank of each guinea-pig. The following series of 3 test substance concentrations and the vehicle were applied using Square chambers attached to Micropore tape:
a_ 25% in milli-R0 water.
b_ 10% in milli-R0 water.
c_ 5% in milli-R0 water
d_ milli-R0 water.
A volume of 0.05 ml of each concentration or the vehicle was placed into a Square chamber. The patches were placed on the shaved area, the Micropore tape firmly secured around the trunk of the animals and held in place by Coban elastic bandage.
The dressings and residual test substance were removed after approximately 24 hours, using a moistened tissue.
The sites were assessed for redness and swelling 24 and 48 hours after removal of the dressings, using the numerical grading system described below (modified from Kligman A.M. , J. Invest. Dermatol. 47, 1966).
The test sites were shaved with an electric razor after the first reading.

no skin reaction: 0
red spots (scattered reactions): 1
Moderate bu confluent redness: 2
Redness and swelling: 3
Intense reddening and swelling: 4

Rechallenge: A second challenge was performed one week after the first challenge, due to inconclusive results.
The method was similar to the first challenge on all animals, but now the contralateral flank was applied. The following decreased test article concentrations 5%, 2% and 1% and the vehicle were applied. The concentrations were decreased because similar skin reactions were observed in experimental and control animals in the first challenge.

Results and discussion

In vivo (non-LLNA)

Results

Results 1

Group:

positive control

Dose level:

0.5 %

No. with + reactions:

19

Remarks on result:

other: No other information available

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

no test substance applied

No. with + reactions:

0

Total no. in group:

10

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

no test substance applied

No. with + reactions:

0

Total no. in group:

10

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

5 % in milli-R0 water

No. with + reactions:

7

Total no. in group:

20

Remarks on result:

other: reading after rechallenge

Results 5

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

5 % in milli-R0 water

No. with + reactions:

7

Total no. in group:

20

Remarks on result:

other: reading after rechallenge

Any other information on results incl. tables

PRIMARY IRRITATION EXPERIMENTS

No signs of systemic toxicity were observed during the primary irritation experiments, however body weight loss was noted in one of the five animals. The choice of milli-R0 water as vehicle in this test was based on the following:

• the test article dissolved or suspended well in milli-R0 water at the concentrations used.


• the test article is stable in milli-R0 water.

In accordance with Magnusson and Kligman (1969) and based on the findings in the primary irritation experiments, the following concentrations were selected for the induction and challenge phase:

Intradermal induction: 2.5% (w/w) in physiological saline Epidermal induction: 25% (w/w) in milli-R0 water. Challenge:

a = 25% (w/w) in milli-R0 water.

b = 10% (w/w) in milli-R0 water.

c = 5% (w/w) in milli-RO water.

d = milli-R0 water.

MAIN STUDY

INDUCTION

All experimental animals showed slight to well defined skin irritation after the 48 hours occluded epidermal induction exposure.

FIRST CHALLENGE

Severe skin reactions, characterised by small crusts, erythema and oedema were noted in nine control animals in response to the 25% concentration, in one control animal in response to the 10% concentration and in one animal in response to the 5% concentration.

Similar skin reactions were noted in sixteen experimental animals in response to the 25% concentration and in two experimental animals in response to the 10% concentration.

Based on these results it was concluded to carry out a second challenge.

SECOND CHALLENGE

CONTROL GROUP:

No skin reactions were evident after this challenge exposure.

EXPERIMENTAL GROUP:

Seven animals showed a positive reaction in response to the 5% concentration.

The reactions were characterised by redness and scaliness.

TOXICITY SYMPTOMS / MORTALITY

No symptoms of systemic toxicity were observed in the animals during the study.

No mortality occurred during the study.

BODY WEIGHTS

The average body weight gain of experimental and control animals was simi lar

However, one control (no. 334) and one experimental animal (no. 320) gained only 44 grams and 23 grams, respectively, during the study period.

 

Applicant's summary and conclusion

Interpretation of results:

other: Category 1B (indication of skin sensitising potential) based on CLP criteria

Conclusions:

Skin sens 1B, H317

Executive summary:

The purpose of the study was to obtain information on the potential of the test item to induce delayed contact hypersensitivity (skin sensitisation) in the guinea pig after intradermal and epidermal exposures.

This study was carried out in accordance with the OECD Guideline No. 406, "Skin Sensitisation", the EEC Directive 84/449/EEC, Part 8.6, "Skin Sensitisation" and in accordance with the method described by Magnusson and Kligman, "Allergic Contact Dermatitis in the Guinea Pig - Identification of Contact Allergens".

After identification of the slightly irritating and the non-irritating test article concentrations in the primary i rritation experiments, a main study was performed with the selected test article concentrations. The experimental animals were intradermally applied with a 2.5% concentration and epidermal ly with a 25% concentration, while the control animals were similar treated, but with the vehicle only. Immediately after the epidermal exposure, the skin irritation was scored. Thereafter, all animals were chal lenged with the following test article concentrations (25%, 10% and 5%) and the vehicle. This rst challenge reaction was inconclusive and a second challenge was carried out with the following test article concentrations (5%, 2% and 1%) and the vehicle. Both the challenge reactions were assessed 24 and 48 hours after bandage removal.

The epidermal exposure of the test item in the induction phase resulted in slight to well defined erythema. The epidermal exposure of he test item in the second challenge phase resulted in seven positive sensitisation reactions in response to the 5% test article concentration.

Under the conditions used in this study, the test item resulted in a sensitisation rate of 35 per cent.