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

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

other: Publication

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1986

Materials and methods

Objective of study:

excretion

Test guideline

Qualifier:

no guideline available

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

Radiolabelling:

yes

Remarks:

14C

Test animals

Species:

other: rats and rabbits

Strain:

other: Rats: male Fisher-344; Rabbit: New Zealand

Sex:

male

Details on test animals or test system and environmental conditions:

Specific-pathogen-free male New Zealand white rabbits (2.4-3.7 kg) were purchased from HARE, Hewitt, N.J. Commercial rabbit food and tap water were furnished ad libitum.
Male Fischer-344 rats, weighing 250-275, were purchased from Charles River Laboratories, Wilmington,
Mass. Ziegler NIH open-formula diet and tap water were provided ad libitum.
Prior to treatment, all animals were acclimated 1 wk in holding rooms with a 12 h photoperiod. All animals were housed and
maintained according to accepted standards (Department of Health, Education and Welfare, 1978).

Administration / exposure

Route of administration:

dermal

Vehicle:

other: sodium carbonate buffer

Details on exposure:

Prior to preparation of the animals, sandwiched barrier mountings were fabricated.
The assembly of the barriers, with an inner layer of 260-mesh nylon (Nitex, Lambert Co., Boston, Mass.).
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 (W. F. Busby, Jr., M. E. Goldman, and G. N. Wogan, 1983, personal communication).
Rats were placed under light ether anesthesia; rabbits were anesthetized with ketamine hydrochloride, 44 mg/kg intramuscularly. 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. Then the animals were wrapped with 3-in-wide.
Elastoplast bandage in which a3.5 x 3.5 cm opening had been cut out; this aperture was centered over the demarcated area. 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.
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.
Rabbits were then placed individually in stainless-steel metabolism cages for collection of urine and feces; rats were placed individually in glass metabolism cages. The efficacy of the barrier system had been tested previously by applying fluorescein dye solution and barrier assemblies to rats, using technique similar to that described above. Rats and their cages were examined daily for 1 wk with long-wavelength ultraviolet light (Woods lamp). No stray fluorescence was found. In addition, cage washings and urineswere examined for fluorescence; none was found. Dyes and barriers remained fixed to animals throughout the specimen collection period.

Duration and frequency of treatment / exposure:

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. Cages were rinsed with distilled water at each collection interval; this water was saved and assayed separately for radioactivity.
Individual urine volumes were recorded at each collection. Aliquots of 100 |xl 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.
Fecal samples were homogenized in water (~1 g feces 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.

Doses / concentrations

Remarks:

Doses / Concentrations:
C.I. direct black 19-[phenyl-14C] was prepared by Sandoz, Incorporated, to a specific activity of 40 mCi/mM and a final radiochemical purity
of >80%. The dye were dissolved in 0.1 M sodium carbonate buffer, pH 10.2.
Radioactivity levels of the working solutions were 0.81 mCi/ml for DB19.

No. of animals per sex per dose / concentration:

6 male rats
4 male rabbits

Statistics:

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.

Applicant's summary and conclusion

Conclusions:

No bioaccumulation potential based on study results
From rats at 144 h, none of the dermal dose of DB19 appeared in feces; in urine, 0.04% of dose was present at that collection time.
From rabbits at 144 h cumulative excretion of radioactivity was 0.04% of total dermal dose in urine; negligible excretion (0.01 of dermal dose) appeared at 144 h in feces.

Executive summary:

Based on the results it could be concluded that Direct Black 19 is stable on skin and no breakage of azo linkages in dyes occurred.

Skin penetration by the whole dye molecules is unlikely because of their polarity, ionized state in solution, avoid binding to skin, and molecular weights(Loomis, 1980; Malkinson and Gehlmann, 1977).

The minimal excretions of radioactivity from both species receiving topical Direct Black 19 suggests that the dye yealding very little free p-phenylendiamine for absorption and excretions.