Disodium; 4-Amino-3-{4-[4-(2,4-diamino-phenylazo)-benzenesulfonylamino]-phenylazo}-5-hydroxy-6-phenylazo-naphthalene-2,7-disulfonate

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics, other

Remarks:

toxicokinetics assessment

Type of information:

other: assessment

Adequacy of study:

key study

Study period:

1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Materials and methods

Objective of study:

toxicokinetics

Test guideline

Qualifier:

no guideline required

Principles of method if other than guideline:

This toxicokinetic assessment is based upon the physico-chemical properties of the test substance, scientific literature and the results of toxicity studies.

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

421-880-6

EC Name:

-

Molecular formula:

C34H25N11Na2O11S3

IUPAC Name:

disodium 4-amino-6-[[4-(N-(4-((E)-(2,4-diaminophenyl)diazenyl)phenyl)sulfamoyl)phenyl)diazenyl)-5-hydroxy-3-((E)-(4-nitrophenyl)diazenyl)naphthalene-2,7-disulfonate

Test animals

Species:

rat

Sex:

male/female

Results and discussion

Any other information on results incl. tables

INFORMATION BASED ON PHYSICO-CHEMICAL PROPERTIES & LITERATURE

The Substance, C34H27N11 011 S3 , has a molecular weight of 861.9 for the free acid, is moderately soluble in water (>27% w/v) and has an estimated log P value of -3.1. The substance also includes a range of minor components, each of which is coloured and represents from 0.1% to 6.1% of the test material. Due to its high molecular weight and log P value, absorption of the intact molecule across the gastrointestinal mucosa would be expected to be both slow and limited. However, the presence of three azo bonds in substance confers the potential for cleavage of the molecule across any one or more of these bonds, particularly by intestinal microorganisms. The various resultant cleavage products would be expected to be more extensively absorbed, well-distributed and subject to further metabolism. Any intact molecule absorbed would similarly be subject to biotransformation; however, its high molecular weight would allow it to be eliminateci via bile, with subsequent excretion in faeces.

Cleavage products of substance may include substituted anilines, which would be subject to ring hydroxylation to produce substituted aminophenols, typically excreted in urine as glucuronide and sulphate conjugates. Alternatively, the amine moiety may be N-acetylated, which would promote urinary excretion, but it may also be metabolised by Noxidation to produce an aryl hydroxylamine, which could be responsible for the mild haemolytic anaemia, observed in the 28 day oral toxicity study.

Alternatively, a larger molecule may be metabolised to forma hydroxylamine metabolite, which could be derived by reduction of the nitrophenyl group.

Cleavage of the substituted naphthalene moiety from the substance would give a very polar metabolite which would be expected to be excreted rapidly in urine. In the remaining part of the cleaved substance molecule, the sulphonamide bridge linking the two aniline rings would be expected to remain intact, giving a metabolite which could be N-acetylated for excretion in urine. Hence, metabolites would be expected to be excreted predominantly in urine, although some high molecular weight conjugates may be eliminateci via bile, for subsequent excretion in faeces, together with

the unabsorbed test substance.

INFORMATION BASED ON TOXICITY STUDIES

The following studies have been considered:

Acute 0ral Toxicity Study in Rats

Acute Dermal Toxicity Study in Rats

Skin Irritation to the Rabbit

Eye Irritation to the Rabbit

Skin Sensitisation to the Guinea Pig

28 Day 0ral Toxicity Study in Rats

Assessment of Mutagenic Potential Using L5178Y Mouse Lymphoma Cells

In the 28 day oral toxicity study, the faeces of rats administered doses of 1000 and 150mg/kg/day were coloured black throughout the study, indicating that the dark brown coloured test substance was extensively eliminateci by this route, probably representing unabsorbed dose. No compound-related effects were seen at the 25 or 150mg/kg dose levels. However, some absorption from the l000mg/kg dose was apparent from histopathological changes in various organs, observations of tissue pigmentation, perturbations in blood clinical chemistry parameters and effects upon the

red blood cells, with reductions in haemoglobin concentration. Increased haemosiderin deposition was also apparent in the spleen, consistent with observations of mild haemolytic anaemia. Plasma alanine and aspartate aminotransferase activities and plasma total bilirubin levels were increased in both sexes, whilst plasma alkaline phosphatase activities were reduced in both sexes. These changes in marker enzyme profiles were consistent with the observations of centrilobular hypertrophy of the liver in almost all rats administered the highest dose level.

No additional toxicokinetic information could be derived from the acute oral study or from any of the dermal or eye studies above.

ABSORPTION, DISTRIBUTION & EXCRETION

The extent of absorption of the test substance cannot be assessed from the above reports, neither can any quantification be applied to excretion, other than to comment that the faeces were intensely coloured, indicating that a significant proportion of the dose was excreted by this route, whereas no discolouration of the urine was apparent.

From observations of widespread tissue pigmentation made during the 28 day oral toxicity study, the absorbed test substance and/or its metabolites were widely distributed. The majority of rats of both sexes had enlarged dark spleens and showed pigmentation of myocardial cells of the heart and in the cortical tubular epithelial cells of the kidney. Some males also showed dark colourations in the brain, epididymis, kidney and liver.

However, some of this colouration may be attributable to coloured impurities in substance orto any coloured metabolites absorbed or formed by biotransformation.

METABOLISM

n the gene mutation assay with L5178Y mouse lymphoma cells, no significant increases in mutant frequency were observed, either in the presence or absence of S9-mix, when tested up to a concentration of 625μg/ml, which represented the limit set by the toxicity of the test sample, therefore, no conclusions can be drawn on the metabolism of the substance from these data.

Applicant's summary and conclusion

Conclusions:

The intact substance molecule is likely to be poorly absorbed following oral administration or dermal application, which is consistent with observations of black coloured faeces in both of the oral toxicity studies. However, some cleavage of the substance molecule by intestinal microorganisms would be expected to result in more extensive absorption of the cleavage products, which would be both widely distributed and subject to further metabolism.
The extensive distribution was evident from pigment deposition in various tissues, although this was not associated with any degenerative changes.
In the 28 day oral administration study, symptoms of toxicity were evident only at the top dose level of 1000mg/kg/day, which represented a limit dose for this type of study. The incidence of mild haemolytic anaemia may have been caused by the formation of aryl hydroxylamines, by N-oxidation of substituted anilines, in turn produced by cleavage of the substance molecule.
The incidence of aniline-induced haemolytic anaemia is well-established; however, indications were seen in this 28 day study of a concurrent regenerative process to compensate for the incidence of haemolytic anaemia.
Microsomal enzymes have been shown, in the Ames's test, to possess the capacity to metabolise substance to a mutagenic metabolite.
The excretion of relatively low molecular weight, polar metabolites of substance in urine would be consistent with observations of the absence of any urinary discolouration.