Pentasodium 4-amino-6-[[5-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-2-sulphonatophenyl]azo]-3-[(2,5-disulphonatophenyl)azo]-5-hydroxynaphthalene-2,7-disulphonate

Endpoint summary

Link to relevant study record(s)

Reference

Endpoint:

basic toxicokinetics

Type of information:

other: Expert statement

Adequacy of study:

supporting study

Study period:

2014

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:

No ADME studies are available for FAT 40171 Y. Therefore, the toxicokinetic assessment of FAT 40171 Y is predicted based on its physico-chemical properties and available toxicological study data. The OECD QSAR application toolbox v3.2 was also utilized to make a qualitative prediction of metabolites formed in the liver and skin.

GLP compliance:

no

Details on absorption:

Oral route:
A combined repeated dose oral toxicity study with the reproduction/ developmental toxicity screening test was performed in Wistar rats. No mortality occurred in the control or any of the dose groups during the study period and there were no adverse effects of toxicological relevance on any of the parameters investigated (clinical signs, body weight, food consumption, organ weights, histopathology, reproduction, development of the pups, gross external abnormalities, macroscopic findings).

Although there were no obvious signs of toxicity exhibited during the study, dose-dependent macroscopic findings related to the test item were found to be dark and/or blue discolouration of several organs in males and females of the medium dose and high dose supplementary group as well as blue discolouration of all visible organs and tissues in males of the high dose supplementary group. Regarding the low dose group, kidneys of 5 females were also discoloured dark. Since the macroscopic finding of dark/ blue/ dark blue discolouration was presumably due to the dark blue colour of the test item used in the study, the blue discoloured organs were not subjected to microscopic examination. For dark discoloured organs, no histological correlate was seen at histopathological examination.

There were no clinical signs of toxicological relevance in the dose groups, when compared to the control groups. However, the skin of all male and female animals of the high dose supplementary group was observed to be discoloured blue from the 7th day of treatment until the end of the study. Blue coloured faeces were noted in both males and females of the low dose and the medium dose group from the 4th day of treatment and in both males and females of the high dose supplementary group from the 3rd day of treatment until the end of the study, respectively. Discolouration of the skin and the faeces was considered to be based on the properties of the blue coloured test item.

The discolouration of the organs investigated indicates that FAT 40171 Y and/or blue-coloured metabolites have been absorbed from the GI tract.

The OECD Toolbox predicts that FAT 40171 Y will undergo some metabolism which could produce smaller molecules which may retain the blue colour of the parent, especially if these molecules still contain the -azo part of the original substance in combination with an aromatic system. It is also known that metabolic activity in the intestine is capable of similar biotransformations as those occurring in the liver. Therefore it should not be assumed that blue discolouration of tissue is due to absorption of the FAT 40171 Y from the intestine. It could be due to smaller (but still blue coloured) metabolites of the parent that have been absorbed. Equally, it could also be due to parent that was absorbed and passed though the liver without transformation. This could be supported by the observation that there was a dose-response relationship for the amount of tissue/organs being coloured as dose was increased, suggesting that biotransformation mechanisms that would metabolise FAT 40171 Y to non-coloured metabolites, may become depleted thus allowing more of the coloured dye into the systemic circulation. However, this could apply to the either/both mechanisms for biotransformation in the intestine and the liver.

The lack of oral toxicity seen in the repeated dose study is supported by the acute oral toxicity study. No mortalities occurred in the control or dose groups during the study period and there were no observed substance related gross organ changes.

The OECD QSAR application toolbox was used to apply Lipinski's Rule of Five. FAT 40171 Y was predicted to be not orally bioavailable based on these rules.
These predictions are clearly incorrect, based on observation of blue coloured tissues!

The toolbox is also designed to predict possible metabolites that can be produced by phase 1(e.g. oxidation/reduction) and phase 2 (e.g. conjugation) biotransformations in the liver, based on the structure of the parent molecule. This biotransformation can occur during the first pass effect, but can also occur if the unmetabolised molecule passes into the systemic circulation and returns through the liver.

Inhalation route:
There is no information available regarding the absorption or toxicity of FAT 40171 Y via inhalation.

The vapour pressure of FAT 40171 Y is predicted to be very low indicating that inhalation exposure from volatilisation is unlikely to be a potential route of exposure.

The particle size distribution (L50D = 117.5 µm, L10D = 47.0 µm) of the test material indicates the presence of inhalable particles. In addition, the test indicated that approximately 1.5% of particles are within the range 5-15 µm, indicating the presence of a small number of respirable particles. REACH endpoint specific guidance (R.7c) states that respirable particles are < 15 µm. However, according to other REACH guidance (chapter R.14) respirable particles are regarded as being < 10 µm. In this case, there are only 0.5% of particles that are < 10 µm. No particles are < 5 µm. Particles that reach the alveolar region of the respiratory tract are likely to be absorbed in this case, considering that oral absorption looks to have occurred. Inhalable particles are likely to be cleared from the lungs by the mucociliary escalator, but then swallowed making them potentially available for absorption via the GI tract.

In conclusion, uptake via the inhalation route is expected to be very low to extremely low based on the particle size distribution.

Dermal route:
There is no information available regarding the absorption or toxicity of FAT 40171 Y following dermal exposure. Dermal absorption is influenced inter alia by water solubility, log Pow and molecular weight. Dermal absorption is predicted to be low based on the high molecular weight (a MW > 500 lowers the ability to be absorbed through skin).The high water solubility would potentially slow down movement across the stratum corneum but the log Pow of 3.96 favours absorption.

In conclusion, dermal absorption is expected to be low.

Details on distribution in tissues:

In the repeated dose oral toxicity study report, macroscopic examination indicated that most of the organs studied in many of the animals were discoloured dark, blue or dark blue. This indicates that FAT 40171 Y and/or its metabolites/ breakdown products are distributed extensively throughout the body. It is possible that only a very small amount of the test substance or metabolite (which is still blue coloured) can lead to the appearance of blue coloured tissue, but this cannot be ascertained quantitatively.

Details on excretion:

The repeated dose toxicity study reports that the faeces were stained a blue colour.

No blue colouration of the urine supports the absence of urinary excretion. It is not known what proportion of this is unabsorbed FAT 40171 Y or absorbed then excreted via bile, or whether the blue colour could be due only to absorbed/biliary excreted blue-coloured metabolites. There is no indication of any urinary excretion of FAT 40171 Y or any blue-coloured metabolites. Urinary excretion of non-coloured metabolites (e.g. M5) cannot be excluded, since the molecular weight would be low enough for urinary excretion to be favourable.

In conclusion, FAT 40171 Y and/or any blue-coloured metabolites are excreted via the GI tract. The exact composition/identity of the blue colour is not known. Urinary excretion is not observed for FAT 40171 Y or any blue-coloured metabolites, but would be possible for smaller fragments such as M5.

Details on metabolites:

Potential metabolites of FAT 40171 Y in the liver and skin have been predicted using OECD Toolbox. Seven metabolites were predicted for liver metabolism (3 are bioavailable and 4 not bioavailable) and four metabolites for skin metabolism (2 are bioavailable and 2 not bioavailable). Two metabolites were common in both liver and skin.

The OECD Toolbox has predicted that FAT 40171 Y will undergo phase I transformations (reductions, hydroxylation). Some of these transformations such as reductions may also occur in the intestine due to action by microflora. In skin the molecule remains intact, although hydroxylation (N-hydroxylamine group is present in M9) is indicated.

Only two of the metabolites were identified by the OECD QSAR application toolbox or by the websites PubChem and ChemSpider.

Toxicologically relevant metabolites:

The ‘smaller’ metabolites are formed as a result of breakage of the azo bond(s) via Phase I reduction transformation, either in the intestine and/or the liver. Metabolites M1 and M9 have almost identical structures to the parent molecule and therefore would be expected to have similar toxicity profiles to the parent.

Where there are no additional structural alerts (type and number, compared to parent) and the metabolites are similar in structure to the parent, it is expected that the toxicity will be similar to the parent. However, metabolites such as M5 and M7 have a relatively larger number of aromatic amino groups as part of their structure and therefore possible enhanced toxicity compared to the parent. It is known that certain aromatic amines are associated with human carcinogenicity. This does not mean that these metabolites are carcinogens, but the potential is considered to be there. Even though bioavailability is not indicated for M7 (OECD toolbox), it cannot be completely excluded.

In addition to the prediction from the toolbox, a visual assessment of the structure of the parent indicates there is the possibility of one of the metabolites being a triazine derivative, specifically 2-chloro-4, 6-diamino-S-triazine could form due to reduction processes either in the liver (Phase I) or via microflora in the intestine which are known to reduce nitro and azo groups. A quick search of TOXNET indicated three Ames tests (on TA100, 97 and 98 strains) all with negative results. No other data was indicated.

Conclusions:

The absorption, distribution, metabolism and excretion of FAT 40171 Y have been predicted in the absence of toxicokinetic studies.

FAT 40171 Y (and/or any blue-coloured metabolites) can undergo oral absorption, based on blue colouration of tissues and organs in the repeated dose oral study.

Uptake of FAT 40171 Y via the inhalation route is expected to be very low to extremely low, based on particle size distribution.

Dermal absorption of FAT 40171 Y is expected to be low.

There is wide distribution of FAT 40171 Y and/or any blue-coloured metabolites, based on blue colouration of tissues and organs in the repeated dose oral study. Distribution of non-coloured metabolites cannot be excluded.
There is insufficient information available to determine whether FAT 40171 Y is absorbed as is and then distributed in its unchanged form or metabolised (before and/or after absorption from GI tract) into metabolites that are themselves blue-coloured, which are then distributed.

The OECD toolbox predicts eleven metabolites; seven from liver metabolism and four from skin metabolism. The toolbox predicts that the parent molecule is likely to undergo at least phase I biotransformations in the liver (hydroxylations, breakage of the azo bond). Some of these transformations such as reductions may also occur in the intestine due to action by microflora. In skin the molecule remains intact although hydroxylation (N-hydroxylamine group formed) is indicated. Visual assessment of the parent also indicates the possibility of a chloro-diamino triazine metabolite, although toolbox does not predict this.

FAT 40171 Y and/or any blue-coloured metabolites are excreted via the GI tract. The exact composition/identity of the substances causing the blue colour is not known. Urinary excretion is not observed for FAT 40171 Y or any blue-coloured metabolites, but would be possible for smaller fragments such as M5.

Although there is clear evidence that FAT 40171 Y is being systemically absorbed following oral ingestion, there was no noticeable toxicity seen in the repeated dose oral toxicity study.

Description of key information

Expert statement

Additional information

The absorption, distribution, metabolism and excretion of FAT 40171 Y have been predicted in the absence of toxicokinetic studies.

FAT 40171 Y (and/or any blue-coloured metabolites) can undergo oral absorption, based on blue colouration of tissues and organs in the repeated dose oral study.

Uptake of FAT 40171 Y via the inhalation route is expected to be very low to extremely low, based on particle size distribution.

Dermal absorption of FAT 40171 Y is expected to be low.

There is wide distribution of FAT 40171 Y and/or any blue-coloured metabolites, based on blue colouration of tissues and organs in the repeated dose oral study. Distribution of non-coloured metabolites cannot be excluded.

There is insufficient information available to determine whether FAT 40171 Y is absorbed as is and then distributed in its unchanged form or metabolised (before and/or after absorption from GI tract) into metabolites that are themselves blue-coloured, which are then distributed.

The OECD toolbox predicts eleven metabolites; seven from liver metabolism and four from skin metabolism. The toolbox predicts that the parent molecule is likely to undergo at least phase I biotransformations in the liver (hydroxylations, breakage of the azo bond). Some of these transformations such as reductions may also occur in the intestine due to action by microflora. In skin the molecule remains intact although hydroxylation (N-hydroxylamine group formed) is indicated. Visual assessment of the parent also indicates the possibility of a chloro-diamino triazine metabolite, although toolbox does not predict this.

FAT 40171 Y and/or any blue-coloured metabolites are excreted via the GI tract. The exact composition/identity of the substances causing the blue colour is not known. Urinary excretion is not observed for FAT 40171 Y or any blue-coloured metabolites, but would be possible for smaller fragments such as M5.

Although there is clear evidence that FAT 40171 Y is being systemically absorbed following oral ingestion, there was no noticeable toxicity seen in the repeated dose oral toxicity study.