Reaction products of diazotised 4,4-diaminodiphenylamine-2-sulfonic acid, subsequently coupled with 6-amino-4-hydroxynaphthalene-2-sulfonic acid, further diazotised and coupled with metaphenylendiamine, sodium salts

Dermal absorption

Administrative data

Endpoint:

dermal absorption in vivo

Type of information:

other: report on simlar substance

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Regarding the hypothesis for analogue approach, refer to the main endpoint summary.

Data source

Reference

Reference Type:

publication

Title:

EXCRETION OF RADIOACTIVITY FROM RATS AND RABBITS FOLLOWING CUTANEOUS APPLICATION OF TWO 14C-LABELED AZO DYES

Author:

Franklin D. Aldrich, William F. Busby and James G. Fox

Year:

1986

Bibliographic source:

Journal of Toxicology and Environmental Health, 18:347-355,1986

Materials and methods

Principles of method if other than guideline:

A p-phenylenediamine derived dye labelled with carbon-14, was applied to the shaved dorsal skin of male Fischer-344 rats and New Zealand rabbits. Application sites were protected with nylon gauze and elastic bandage assemblies. Following application of measured amounts of radiolabelled dye in 0.1 M pH 10.2 carbonate buffer, serial urine and faecal samples were obtained from individual animals in metabolism cages at 24, 48, 72, 96, 120, and 144 h. Aliquots of urine and faecal homogenates were assayed for radioactivity by scintillation counting.

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 4-amino-3,6-bis[[4-[(2,4-diaminophenyl)azo]phenyl]azo]-5-hydroxynaphthalene-2,7-disulphonate

EC Number:

229-208-1

EC Name:

Disodium 4-amino-3,6-bis[[4-[(2,4-diaminophenyl)azo]phenyl]azo]-5-hydroxynaphthalene-2,7-disulphonate

Cas Number:

6428-31-5

Molecular formula:

C34H29N13O7S2.2Na

IUPAC Name:

disodium 4-amino-3,6-bis({4-[(2,4-diaminophenyl)diazenyl]phenyl}diazenyl)-5-hydroxynaphthalene-2,7-disulfonate

Test material form:

solid: particulate/powder

Radiolabelling:

yes

Remarks:

[-phenyl-14C]

Test animals

Species:

other: rats and rabbits

Strain:

other: Rabbis: New Zealand White; rats: Fischer-344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
RABBITS
- Source: purchased from HARE, Hewitt, N.J.
- Weight at study initiation: 2.4-3.7 kg
- Diet: ad libitum; commercial rabbit food
- Water: ad libitum; tap water
- Acclimation period: 1 week in holding room

RAT
- Source: purchased from Charles River Laboratories, Wilmington, Mass
- Weight at study initiation: 250-275 g
- Diet: ad libitum; Ziegler NIH open-formula
- Water: ad libitum; tap water
- Acclimation period: 1 week in holding room

ENVIRONMENTAL CONDITIONS
All animals were housed and maintained according to accepted standards (Department of Health, Education and Welfare, 1978).
- Photoperiod: 12 hours

Administration / exposure

Type of coverage:

open

Doses:

DOSE PREPARATION
- Method for preparation of dose suspensions: dye was dissolved in 0.1M sodium carbonate buffer, pH 10.2. Radioactivity levels of the working solutions was 0.81 mCi/mL

Details on study design:

TEST SITE
- Barrier/bendage: multilayer barriers, with an inner layer of 260-mesh nylon (Nitex, Lambert Co., Boston, Mass.) 5 x 5cm; mesh pore size was 0.0023 in, with an open area of 36%. A supporting perimeter of 1/4-in-wide Molefoam (Scholl, Inc., Chicago, III.) was placed about the nylon mesh. This subassembly was covered by a 5 x 5 cm piece of 40 x 50 mesh copper gauze (Jelliff Corporation, Southport, Conn.). The barrier assembly was held together on all sides by 3/4-inwide strips of elastic adhesive bandages (Elastoplast, Beiersdorf, Inc., South Norwalk, Conn.). This assembly was designed to protect the treated skin area from contact with the sides of the animal's cage or by the animal during grooming. The barrier thus assures an intact dermal application site without occlusion and prevents loss or ingestion of any of the applied dye solution by the animal
- Animal preparation: preparation of rats and rabbits was essentially identical. Rats were placed under light ether anaesthesia; rabbits were anesthetized with ketamine hydrochloride, 44 mg/kg intramuscularly
- Preparation of test site: animals were shaved on the left dorsum with electric clippers (Oster size 40 surgical blade) with care to avoid cuts or bruises. A 2 x 2 cm area for dye application was demarcated in the shaved area with an India ink pen.
- Coverage: then the animals were wrapped with 3-in-wide Elastoplast bandage in which a 3.5 x 3.5 cm opening had been cut out; this aperture was centered over the demarcated area.

APPLICATON: the dye solutions were spread evenly within the demarcated areas with an automatic pipet equipped with disposable polyethylene tips.

Drying of the applied dye solutions was hastened by warm air from a small hair dryer. Dried dyes remained on the skin for the subsequent collection period.

POST APPLICATION
The barrier assemblies then were applied over the treated areas of skin, anchored in front and in back with %-in-wide strips of Elastoplast bandage long enough to encircle the animals completely and overlap the abdomen. Two more strips of 3A-\r\ Elastoplast were applied to the top and bottom of the barrier assemblies.
- Cage: Rabbits were then placed individually in stainless-steel metabolism cages for collection of urine and faeces; rats were placed individually in glass metabolism cages. The efficacy of the barrier system had been tested previously by applying fluorescein dye solution

COLLATION AND PREPARATION OF SAMPLES; RADIOASSAYS
- Collection of samples: urine and faeces were removed from the animals' cages at intervals of 24,48, 72, 96,120, and 144 h after application of dye solutions.
- Cage rinse: cages were rinsed with distilled water at each collection interval; this water was saved and assayed separately for radioactivity.
- Urine sample: individual urine volumes were recorded at each collection. Aliquots of 100 µL were added to 1.0 ml water and 12 ml ACS counting fluid (Amersham Corp., Arlington Heights, III.) in glass vials and counted in a Beckman LS-8100 liquid scintillation counter.
- Faecal sample: faecal samples were homogenized in water (~1 g faeces in 20 ml water) by successively processing with a Polytron homogenizer (Brinkmann
Instruments, Westbury, N.Y.). Aliquots of the resulting suspensions were counted directly in 1.0 ml water and 12 ml counting fluid.

Efficacy and accuracy of these direct counting procedures were verified in previous digestion experiments using Protosol (New England Nuclear, Boston, Mass.) as well as by constructing counting curves and using sample aliquot sizes that fell well within linear range.
Quenching was routinely corrected in each sample by internal standardization techniques with carbon-14-labeled toluene.

Results and discussion

Any other information on results incl. tables

From rats at 144h, none of the dermal dose of dye appeared in faeces; in urine, 0.04% of dose was present at that collection time. From dye -treated rabbits, cumulative excretion of radioactivity was 0.04% of total dermal dose in urine at 144h; negligible excretion (0.01% of dermal dose) appeared at 144 h in faeces.

Cumulative Excretion of Radioactivity from 4 Rats for Dye, 18,468,000 dpm Applied per Animal

	Urine average			Faeces average
Hours	dpm	§dpm^b	%Dose ± SE	dpm	§dpm^b	%Dose ± SE
24	1733	1733	0.01 ± 0.0	0	0	0
48	1237	2970	0.02 ± 0.0	89	89	0
72	962	3932	0.02 ± 0.0	393	482	0
96	979	4911	0.02 ± 0.0	240	482	0
120	661	5572	0.03 ± 0.0	304	1026	0
144	791	6363	0.04 ± 0.0	287	1313	0

Cumulative Excretion of Radioactivity from 4 Rabbits for Dye, 55,404,000 dpm Applied per Animal

	Urine average			Faeces average
Hours	dpm	§dpm^b	%Dose ± SE	dpm	§dpm^b	%Dose ± SE
24	7904	7904	0.01 ± 0.0	3269	3269	0.01 ± 0.0
48	8082	15,986	0.03 ± 0.01	0	3269	0.01 ± 0.0
72	3661	19,647	0.04 ± 0.01	0	3269	0.01 ± 0.0
96	1378	21,205	0.04 ± 0.01	0	3269	0.01 ± 0.0
120	2733	23,758	0.04 ± 0.01	0	3269	0.01 ± 0.0
144	824	24,582	0.04 ± 0.01	0	3269	0.01 ± 0.0

Applicant's summary and conclusion

Conclusions:

It was not found significant excretion of radioactivity from rats or rabbits that received dermal applications of test item labeled with carbon-14 in its phenyl nuclei, thus it can be concluded that dye is not absorbed by skin in the test conditions.

Executive summary:

Purpose

Purpose of the study was to establish if dye tested undergoes to formation of carcinogenic amine by skin absorption.

Starting from the consideration that skin penetration by the whole dye molecules is unlikely because of their polarity, ionized state in solution, avid binding to skin and molecular weight of ca 600, the test substance was labelled with carbon-14 in such way to consider the amount in feces and urine representative of the breackage of azo linkages, responsible of the amine formation.

Procedure

A p-phenylenediaminederived dye labelled with carbon-14, was applied to the shaved dorsal skin of male Fischer-344 rats and New Zealand rabbits. Application sites were protected with nylon gauze and elastic bandage assemblies. Following application of measured amounts of radiolabelled dye in 0.1 M pH 10.2 carbonate buffer, serial urine and faecal samples were obtained from individual animals in metabolism cages at 24, 48, 72, 96, 120, and 144 h. Aliquots of urine and faecal homogenates were assayed for radioactivity by scintillation counting.

Result

Cumulative excretion of radioactivity at 144 h was in rat urine 0.04% of total dermal dose appeared; in rat faeces, no radioactivity was recovered; while in rabbit urine, 0.04% of dermal dose was found; 0.01% appeared in rabbit faeces.

Conclusion

It was not found significant excretion of radioactivity from rats or rabbits that received dermal applications of test item labelled with carbon-14 in its phenyl nuclei, thus it can be concluded that dye is not absorbed by skin in the test conditions.