Reaction products of diazotised 2-amin-6-nitrophenol-4-sulphonic Acid and 2-Amino-4-nitrophenol-6-sulfonic Acid, coupled with a mixture of 6-Amino-4-hydroxy-2-naphthalenesulfonic Acid and Acetylated 6-Amino-4-hydroxy-2-naphthalenesulfonic Acid, subsequently metallized with Chromium (III) Basic Sulfate and Acetic Acid, and at the end diazotised and coupled with p-Cresol, sodium salts

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

May 12, 1981

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

An in vitro or in chemico skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study (different from LLNA test) is available.

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

405-750-6

EC Name:

-

Molecular formula:

Not applicable for UVCB substance

IUPAC Name:

diiron(3+) dihydrogen tris(7-[2-(2-hydroxy-5-methylphenyl)diazen-1-yl]-2-[2-(3-nitro-2-oxido-5-sulfophenyl)diazen-1-yl]-3-sulfonaphthalen-1-olate) 7-acetamido-2-[2-(3-nitro-2-oxido-5-sulfophenyl)diazen-1-yl]-3-sulfonaphthalen-1-olate

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Remarks:

albino (SPF-quality)

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
-Source: Charles River Wiga, Sulzfeld / West Germany
-Age at start of treatment: approx. 11 weeks
-Body Weight at acclimatisation: 328 - 420 g
-Identification: tattoo
-Acclimatisation: at least five days under test conditions after physical examination, with the exception of the animals in the primary irritation experiments, these animals were four days in acclimatisation
-Housing: group housing of two animals per cage with wire-mesh floors (ITL, Bergen / The Netherlands).

ENVIRONMENTAL CONDITIONS
-Temperature: 21 ± 3°C
-Humidity: 30-70 %
-Air changes: 7.5-15 air changes per hr with air-conditioned (hourly monitored)
-Photoperiod: 12 hrs artificial fluorescent light / 12 hrs dark
-Diet: standard guinea pig diet, including ascorbic acid (1600 mg/kg); LC 23-B, pellet diameter 4mm. (Hope Farms, Woerden, The Netherlands). In addition, once a week hay was provided (Broekman Institute, Someren / The Netherlands).
-Water: tap-water diluted with decalcified water, ad libitum.

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

physiological saline

Concentration / amount:

5 % (w/w)

Day(s)/duration:

a single injection

Induction 2

Route:

intradermal

Vehicle:

other: 50:50 FCA distilled water

Concentration / amount:

0 %

Day(s)/duration:

a single injection

Induction 3

Route:

intradermal

Vehicle:

other: 50:50 mixture FCA

Concentration / amount:

10 %

Day(s)/duration:

a single injection

Induction 4

Route:

epicutaneous, occlusive

Vehicle:

water

Remarks:

milli-RO

Concentration / amount:

25 % (w/w)

Day(s)/duration:

48 h

Challenge

Challenge 1

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

water

Remarks:

milli-RO

Concentration / amount:

25 % (w/w)

Day(s)/duration:

24 h

Challenge 2

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

water

Remarks:

milli-RO

Concentration / amount:

10 % (w/w)

Day(s)/duration:

24 h

Challenge 3

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

water

Remarks:

milli-RO

Concentration / amount:

5 % (w/w)

Day(s)/duration:

24 h

Challenge 4

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

water

Remarks:

milli-RO

Concentration / amount:

0 % (w/w)

Day(s)/duration:

24 h

No. of animals per dose:

-20 for the experimental group.
-10 for the control group and 5 for the primary irritation test

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.
1) 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 h later. Necrosis (diameter, size in mm) was the recorded parameter. Erythema and oedema could not be scored, due to the presents of necrosis.
2) Epidermal applications: the animal was also treated epicutaneously at the shaved left flank with 0.5 ml of a 50% concentration of the test substance in milli-RO water using a Metalline patch mounted on Micropore tape and held in place with Coban elastic bandage for 24 h. The treated skin was assessed for erythema and oedema 24 and 48 h after removal of the dressings. Other four animals were shaved on the left flank and exposed for 24 h to 50 %, 25 %, 10 % and 5 % (w/w) test substance concentrations in milli-RO water (0.05 ml / concentration), occlusively administered by means of Square chambers 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. The reaction sites were assessed for erythema 24 and 48 h after removal of the dressings.
The erythema and oedema assessments were based on this scale:
-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 mm): 3
Severe oedema (raised more than 1 mm and extending beyond the area of exposure): 4

MAIN STUDY
Based on the findings in the primary irritation experiments, concentrations were selected for the induction and challenge phase. The 25% test substance concentration is chosen as the highest useable concentration, due to the fact that the 50% concentration was too clotty and discoloured the skin too black.
1) INDUCTION EXPOSURE
Intradermal injections:
On day 1 an area of the dorsal skin from the scapular region (approximately 4 * 6 cm) was clipped free of hair. Three pairs of intradermal injections (0.1 ml/site) were made at the border of a 2 * 4 cm area in the clipped region from the cranial region to the caudal.
Epidermal applications:
7 d after the intradermal injections, the scapular area (approximately 6 * 8 cm) was again clipped and shaved free of hair. Approx. 24 h prior to the epidermal application the test area was pre-treated with 10 % Sodium-Dodecyl-Sulfat (SDS) in petrolatum. The SDS was massaged into the skin with a spatula without bandaging. This concentration of SDS enhances sensitization by provoking a miId inflammatory reaction. A 2 * 4 cm patch of Metalline 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. The dressings were left in place for approximately 48 h. 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 under primary irritation experiments.
2) CHALLENGE EXPOSURE
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. Series of 3 test substance concentrations and the vehicle were applied using Square chambers attached to Micropore tape. 0.05 ml of each concentration and 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 sites after removal were assessed for redness and swelling 24 and 48 h after removal of the dressings, using this numerical grading system:
no skin reaction: 0
Red spots (scattered reactions): 1
Moderate but confluent redness: 2
Redness and swelling: 3
Intense reddening and swelling: 4
The test sites were shaved with an electric razor after the first reading. All animals were killed at the end of the test period by carbon dioxide asphyxiation.

OTHER OBSERVATIONS
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.

INTERPRETATION
The irritation and sensitisation scores and all other observations were recorded on data sheets and transcribed for compilation and analysis.
The results evident in test animals at the challenge application(s) were compared with the results evident in control animals. Positive skin reactions (grade 2 or more) were considered signs of sensitization, provided that such reactions were not observed in the control group.
The readings after the challenge applications were compared to assess the sensitisation rate i.e. the number of sensitised animals in proportion to the total number of animals of the experimental group at the same test substance concentration.

Challenge controls:

yes

Positive control substance(s):

yes

Remarks:

formaldehyde

Results and discussion

Positive control results:

Positive results were observed in the experimental animals after the challenge with 0.5 % (w/w) formaldehyde in milli-RO water.

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

0, 5, 10, 25 %

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

other: after the apllication for challenge

Results 2

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

0, 5, 10, 25 %

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

other: after the application for challenge

Any other information on results incl. tables

PRIMARY IRRITATION EXPERIMENTS

In the animal treated initially with intradermal injection (concentration 5 %) a very four very small necrotic areas of 1 mm diameter were recorded at 24 and 48 h after the application. For epidermal application (concentration of 50 %), this animal showed no irritant effects: erythema and oedema scores were 0 at 24 and 48 h after bandage removal.

In the other four animals (concentrations 5, 10, 25 and 50 %) erythema scores were 0, except in one animal at concertation of 50 % in which a slight erythema (grade 1) and scaliness were detected at 24 and 48 h respectively. No oedema was scored. Skin readings after bandage removal were difficult due to black discolouration by the test substance. No signs of systemic toxicity were observed during the primary irritation experiments, except of body weight loss of one animal.

INDUCTION

14 experimental animals showed skin irritation after the 48 hours occluded epicutaneous induction exposure.

CHALLENGE

-Control group: no positive skin reactions were evident after the challenge exposure.

-Experimental group: none of the animals showed a positive sensitisation reaction in response to any of the test substance concentrations.

Other minor reactions to treatment were characterised by red spots and scaliness.

SENSITISATION

These results lead to a sensitization rate of 0 %, which indicates that the test substance has weak sensitising properties in this test applying the rating of allergenicity described by Kligman A.M. (1966).

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 similar.

Applicant's summary and conclusion

Interpretation of results:

other: Not classified according to the CLP Regulation (EC 1272/2008)

Conclusions:

Under the conditions used in this study the substance resulted in a sensitisation rate of 0 % after intra-cutaneous and epi-cutaneous application to the guinea pig.

Executive summary:

The skin sensitisation potential was evaluated with an in vivo maximization test on guinea pigs, according to the OECD 406 (1981). A primary irritation experiment was performed in order to determine the test concentrations of the main study. Under the conditions used in this study the substance resulted in a sensitisation rate of 0 % after intra-cutaneous and epi-cutaneous application to the guinea pig.