disodium 4-amino-6-((4-((4-(2,4-diaminophenyl)azo)phenylsulfamoyl)phenyl)azo)-5-hydroxy-3-((4-nitrophenyl)azo)naphthalene-2,7-disulfonate

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

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

Objective of study:

toxicokinetics

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

Species:

rat

Sex:

male/female

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.

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.

Reference 2

Endpoint:

basic toxicokinetics, other

Remarks:

assessment of the toxicokinetics effects based on acute toxicity tests, long term exposure and reproductive/developmental toxicity test

Type of information:

other: Assessment on existing data

Adequacy of study:

key study

Study period:

May,2011-Novemebr 11, 2011

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The assessment is based on existing data on the potassium and sodium salt of Acid Black 210

Objective of study:

toxicokinetics

Principles of method if other than guideline:

assessment of the toxicokinetics effects based on acute toxicity tests, long term exposure and reproductive/developmental toxicity test

GLP compliance:

no

Species:

rat

Sex:

male/female

The test substance,Acid Black 210, potassium salt was applied to laboratory animals (mouse, rat, rabbit) during studies with different way of entry into organism (e.g. skin, stomach, eye).

Acute toxicity studies - Acid Black 210 - potassium salt

Non toxic for rats after single administration into the stomach. 

Acid Black 210 administered at the dose level of 2000 mg/kg caused no death of animals. No clinical signs of intoxication were observed in all six animals only changes of colour of urine (yellow) was recorded on the following day post treatment accompanied by black-coloured faeces.

No corrosive effects were observed on the skin of rabbit.

Very slight erythema was observed at the 1 hour after 4-hour exposure to the test substance in two animals. At the 24, 48 and 72 hours after exposure no irritation of the skin was observed in all animals. No clinical signs of systemic intoxication were observed.

No corrosive effects were observed on the eye of rabbit.

Slight changes of eye in all rabbitwere observedfrom 1 hour to 48 hours after application. In one rabbit changes persisted till the 5^thday after application. 

No clinical signs of systemic intoxication were observed.

Acid black 210 -sodium salt

The test substance is not a contact allergen in mouse.

The test substance, Acid Black 210, did not show a tendency to increase lymph node cell count, lymph node weight and ear weight in mice after application on skin of ears (it confirmed existing dermal irritation data).

 

Conclusion:

The test substance, Acid Black 210– CAS No.: 85223-29-6, was applied to laboratory animals (mouse, rat, rabbit) durin acute studies with different way of entry into organism (oral – by stomach, skin and eye).

The test substance applied to the stomach of the rat elicited no systemic effects.The substance applied on the skin of mouse elicited no effects at the site of application and no systemic toxic effects. The substance applied on the skin of rabbit elicited effects at the site of application that disappeared quickly without systemic toxic effects. The substance when applied to the mucous membrane (conjunctivae) of the rabbit elicited slight effects at the site of application (disappeared until the 6^thday) without systemic toxic effects.

Long-term toxicity study - Acid Black 210 - sodium salt

The Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test was performed with the test substance Acid Black 210, sodium salt.

The negative effect on growth of animals (slight decrease of body weight increments, food consumption and food conversion), significant negative effect on some haematological parameters (changes of red and white blood component), some biochemical parameters (changes of AST and ALT activity, creatinine, total cholesterol, phosphorus, and chloride)and biometry of organs (changes of spleen and brain weight)were observed.

The test substance had influence on macroscopical (changes of colour of brain, spleen, thyroid gland, epithelium of thoracic and abdomial cavity, mucosa, skeletal muscle and skin) and microscopical (brown dye in kidneys, spleen, thyroid gland, liver in both sexes and lymph nodes, ovaries in females) structure of organs and tissues.

The test substance treatment affected number of pups, sperm motility, pre-implantation and post-implantation losses.

 

The test substance administration had not adverse effect on mortality, parameters of functional observation and some reproduction parameters - course of mating, pregnancy and lactation, weights of reproductive organs and pituitary gland, spermiogenesis, macroscopical and microscopical structure of reproductive organs and pituitary gland of parental animals, number of post-natal losses of mothers, sex ratio, weight and development of pups.

The test substance, Acid Black 210, after repeated oral application at the highest dose level caused changes of growth of animals (changes of body weight increments, food consumption and conversion),haematological parameters(changes of red and white blood component),biochemical parameters(changes of AST and ALT activity, creatinine, total cholesterol, phosphorus, and chloride) andbiometry of organs(changes of spleen and brain weight).

The test substance had influence on macroscopical and microscopical structure of some organs and tissues (occurrence of pigment in kidneys, spleen, thyroid gland, liver and lymph nodes and ovaries, reversible increased occurrence of extramedular haemopoiesis and enlargement of marginal zone of periarteriolar lymphoid sheaths in spleen).

The test substance treatment affected the number of pups (decrease of the total number of live pups and average number of pups per litter), sperm motility (slower sperm motility), pre-implantation and post-implantation losses (decreased number of corpora lutea, uterus implantations and pups).

The highest incidence of statistically or biologically significant differences was recorded at the highest dose level while most of changes which were found at the middle and the lowest dose level had only mild  intensity without adverse alteration of animal organism.

 

Conclusion:

The results of the Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test with oral administration to the rats showed the negative effect ongrowth of animals, some haematological and some biochemical parameters of the blood, biometry of organs, macroscopical and microscopical structure of some organs and tissues,number of pups, sperm motility, pre-implantation and post-implantation losses.

Acid Black 210 – CAS No.: 85223-29-6 caused toxicological significance irreversible accumulation of pigment in kidneys, spleen, thyroid gland, liver and lymph nodes and ovaries of rats (at the dose level 150 and 450 mg/kg/day) – when it is applied orally.

  

ACCORDING TO THE RESULTS OF LITERATURE DATA

No relevant toxicokinetic references.

 

3.3 ACCORDING TO THE DATABASES DATA

No relevant toxicokinetic references.

Conclusions:

Interpretation of results (migrated information): no bioaccumulation potential based on study results
No systemic effects were described after acute administration of Acid Black 210 – CAS No.: 85223-29-6, to rats. The test substance is nontoxic after single administration into the stomach. The substance applied on the skin of mouse elicited no effects at the site of application and no systemic toxic effects. After skin or eye single application systemic toxic effects were not detected. The test substance is probably not absorbed through the stomach, skin or eye after single administration

The systemic effects were described after repeated oral administration of Acid Black 210 – CAS No.: 85223-29-6, to rats. The test substance had the negative effect on growth of animals, some haematological and some biochemical parameters of the blood, biometry of organs, macroscopical and microscopical structure of some organs and tissues, number pups, sperm motility, pre-implantation and post-implantation losses only at doses > 150 mg/kg bw7day (NOAEL).
Only at the highest tested dose of 450 mg/Kg bw Acid Black 210 – CAS No.: 85223-29-6 caused irreversible accumulation of dye in kidneys, spleen, thyroid gland, liver and lymph nodes and ovaries of rats by penetration into the stomach through the blood into different organs.

Executive summary:

Evaluation of toxicokinetics of the substance is required according to the 8.8.1 of Annex VII to the Directive (EC) No. 1907/2006 (REACH). 

Toxicokinetics of the test substance was evaluated from three sources:

-                 Experimental data of toxicological tests

-                 Literary data obtained from internet

-                 Data from toxicological databases – free and commercial     

Experimental toxicological data involved acute toxicity data, long term and reproductive/developmetnal toxicity.

Description of key information

low bioaccumulation potential

Key value for chemical safety assessment

Bioaccumulation potential:

low bioaccumulation potential

Additional information

Based on the evaluation of toxicity data (acute, long term and reproducive+developmental) Acid Black 210 shows a low potential for bioaccumulation.

Two assessment are available and both have comparable outcome.

In the first assessment, no systemic effects were described after acute administration of Acid Black 210 potassium salt to rats. The test substance is non toxic after single administration into the stomach. The substance applied on the skin of mouse elicited no effects at the site of application and no systemic toxic effects. After skin or eye single application systemic toxic effects were not detected. The test substance is probably absorbed through the stomach, skin or eye after single administration in a very low amount, not enough to provoque odservable effects.

The systemic effects were described after repeated oral administration of Acid Black 210 sodium salt to rats. The test substance had the negative effect on growth of animals, some haematological and some biochemical parameters of the blood, biometry of organs, macroscopical and microscopical structure of some organs and tissues, number of pups, sperm motility, pre-implantation and post-implantation losses only at doses > 150 mg/kg bw/day (NOAEL).

Only at the highest tested dose of 450 mg/Kg bw Acid Black 210 sodium salt caused irreversible accumulation of dye in kidneys, spleen, thyroid gland, liver and lymph nodes and ovaries of rats by penetration from the stomach through the blood into different organs.

The second assessment confirm that Acid Black 210 sodium salt 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 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.

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.

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.