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Basic toxicokinetics

There are no experimental studies available in which the toxicokinetic behaviour of 7-amino-3-{(E)-[5-({4-(2-chloroethyl)butanoyl}amino)]diazenyl}-4-hydroxy-8-[(E)-(4-{2-(sulfonatooxy)ethyl}) diazenyl]naphthalene, polysulfonate, polysulfonyl, polyphenyl, sodium/potassium salt has been assessed.

The test substance represents an organic diazo acid dye based on a sulphobenzoic acid sodium/potassium salt with an average purity of 40 - 60% (main constituent: 42.04%). Impurities mainly comprise organic compounds (6 known coloured (20.66%) and 4 uncoloured (12.17%) by-products as well as 8.17% unknown coloured and 0.15% other unknown by-products). The remaining impurities of the test substance refer to inorganic salt compounds, including calcium bis(dihydrogenorthophosphate) (0.43%), disodium sulphate (4.3%), dipotassium sulphate (0.8%), disodium hydrogen phosphate (2.75%), dipotassium hydrogen phosphate (0.5%), calcium dichloride (0%), sodium chloride (3.65%) and potassium chloride (0.8%). The analysed amount of unsulphonated primary aromatic amines including diverse aniline derivates was below the detection limit of 1 ppm (typical concentration: < 0.0001%). All known organic by-products show a strong structural relationship to the main constituent and are therefore considered to exhibit similar toxicokinetic properties. Further details regarding the composition of the test substance are presented in CSR chapter 1.2 and IUCLID chapter 1.2 and 1.4, respectively.

In accordance with Annex VIII, Column 1, Item 8.8.1, of Regulation (EC) 1907/2006 and with Guidance on information requirements and chemical safety assessment Chapter R.7c: Endpoint specific guidance (ECHA, 2012), assessment of the toxicokinetic behaviour of the substance is conducted to the extent that can be derived from the relevant available information. This comprises a qualitative assessment of the available substance specific data on physicochemical and toxicological properties according to the relevant Guidance (ECHA, 2012).

The molecular weight of the main constituent of the test substance is 1007.45 g/mol for the free acid and 981.84 g/mol for the sodium/potassium salt (Müller, 2011). All other known constituents of the test substance comprise molecular weights in the range of 281.31 - 969.68 g/mol for the organic compounds as well as 74.55 - 310.18 g/mol for the inorganic compounds (Müller, 2011). The test substance is a red powder (Müller, 2011) with a water solubility of 393 g/L at 21 °C and pH 3.5 (Mollandin, 2012). The log Pow of the substance was determined to be < 0.3 at 25 °C (Mollandin, 2012) and the vapour pressure was estimated to be < 10E-05 Pa at 20 °C (Meinerling, 2012). The acid dissociation constants (pKa) of the substance were determined to be 4.413 (titration with 0.01 M NaOH) at 20 ± 0.4 °C, 6.948 (titration with 0.01 and 0.1 M NaOH) at 20 ± 0.5 °C and 9.972 (titration with 0.1 M NaOH) at 20 ± 0.5 °C (Fieseler, 2012).

Absorption

Absorption is a function of the potential for a substance to diffuse across biological membranes. The most useful parameters providing information on this potential are the molecular weight, octanol/water coefficient (log Pow) value and water solubility (ECHA, 2012).

Oral

In general, a moderate log Pow (-1 to 4) value in combination with the high water solubility suggests that any absorption will likely happen via passive diffusion, in case the substance does not contain ionisable groups and has a molecular weight < 500 g/mol (ECHA, 2012). The molecular weight of the main constituent of the test substance is higher than 1000 g/mol, indicating that the substance is not favourable for absorption. Furthermore, the sulphonic acid structure of the molecule makes it potentially ionisable and thus ready diffusion through biological membranes is not expected (ECHA, 2012).

The acid dissociation constant (pKa) of the test substance is in the range of 4.4 - 9.9, thus suggesting low ionisation and potential absorption in the stomach (pH1.35 to 3.5), but high ionisation and thus low absorption potential in the gut (pH 5-9). Since no significant absorption of the major constituent of the test substance via passive diffusion is anticipated due to its physicochemical properties, active transport mechanisms from the GI tract may potentially play a role in facilitating the uptake of the major constituent as well as structurally related constituent with high molecular weight. In contrast, for lower molecular weight constituents (below 500 g/mol), which mainly comprise uncoloured organic compounds, ready absorption via passive diffusion into the gastrointestinal tract is anticipated. Furthermore, absorption of the inorganic impurities of the test substance cannot be excluded, as the passage of small, water-soluble molecules like ions or carriage of ionic species with the passage of water is possible through aqueous pores (ECHA, 2012). Moreover, transport via ion channels and/or active transport mechanisms seems conceivable for inorganic impurities like sodium, calcium, sulphate, chloride and phosphate ionic species.

Besides physico-chemical properties, data on acute and repeated dose toxicity of a substance may also indicate a potential for absorption, e.g. if treatment-related systemic toxicity and coloration of urine and/or internal organs has been observed in treated animals (ECHA; 2012).

The acute oral toxicity study on FAT 40858/ A TE in rats resulted in a LD50 value > 5000 mg/kg bw and the occurrence of only slight clinical signs, which were fully reversible within the first day after application (Holalagoudar, 2012a). Moreover, data on the oral repeated dose toxicity in rats is available, indicating that administration of the test substance for 14 or 28 days did not result in any adverse effects up to and including the currently applied limit dose of 1000 mg/kg bw/day (Holalagoudar, 2012b; Holalagoudar, 2012c). Visual examination of urine from the 28-day study revealed a yellow to orange and rose or reddish colouration in animals of all dose groups (100, 300 and 1000 mg/kg bw/day), which was considered to be a result of excretion of the red-coloured test substance in the urine (Holalagoudar, 2012c). Furthermore, discolouration of organs was observed at macroscopic examination at and above 300 mg/kg bw/day, which was due to absorbed test substance and, under the conditions of this study, without toxicological significance (Holalagoudar, 2012c). In a combined repeated dose and reproductive/developmental toxicity screening test, gross pathology of parental animals revealed reddish discoloration of various organs (e.g. kidney, skin, and testis) in a dose-related manner among all treated groups, which was considered to be due to the specific red colour of the test substance.In accordance with the observed substance-related reddish discoloration of some organs at necropsy, brown and red-brown pigments were observed in reproduction organs and kidney as well as in a number of lymph nodes at microscopic examination. However, in view of the low degree of severity observed and in the absence of any changes indicating functional impairment of the organs, the pigmentation was not considered to be of toxicological relevance (Holalagoudar, 2012d).

Overall, the available data indicate that the substance has the potential for absorption after oral ingestion in a dose-dependent manner, although the physico-chemical properties in general impede absorption via passive diffusion Furthermore, no assumptions can be made regarding the actual amount absorbed based on these experimental data of the test substance. In general, these data demonstrate that absorption of the substance after repeated oral administration is likely to be associated with a low systemic toxicity and the absence of adverse toxic effects in the target organs of absorption.

Dermal

In general, the physical state may already be taken into consideration for a crude estimation of the absorption potential of a substance, which means that dermal uptake of liquids and substances in solution is higher than that of dry particulates, since dry particulates need to dissolve into the surface moisture of the skin before uptake can begin. Furthermore, the dermal uptake of substances with a high water solubility of > 10 g/L (and log Pow < 0) will be low, as the substance may be too hydrophilic to cross the stratum corneum. Log Pow values between 1 and 4 favour dermal absorption (values between 2 and 3 are optimal), in particular if water solubility is high. In contrast, log P values < 0 indicating poor lipophilicity will limit penetration into the stratum corneum and hence dermal absorption. Furthermore, log Pow values < –1 suggest that a substance is not likely to be sufficiently lipophilic to cross the stratum corneum, therefore dermal absorption is likely to be low (ECHA, 2012).

The test substance is a solid with very good solubility in water and a molecular weight exceeding 500 g/mol, thus indicating a low dermal absorption potential (ECHA, 2012). Since the log Pow of the substance is < 0.3, penetration into the stratum corneum and hence dermal absorption are expected to be limited (ECHA, 2012).

Apart from the physico-chemical properties, further criteria may apply to assume the dermal absorption potential of the substance.

In general, substances that show skin irritating or corrosive properties may enhance penetration by causing damage to the surface of the skin. Furthermore, if a substance has been identified as a skin sensitiser, then some uptake must have occurred although it may only have been a small fraction of the applied dose (ECHA, 2012).

The experimental animal and in vitro data on the test substance showed that no skin irritation and corrosion occurred, which excludes enhanced penetration of the substance due to local skin damage (Lütkenhaus, 2012; Lehmeier, 2012). However, the test substance was identified to be a skin sensitiser in the Local Lymph Node Assay (LLNA) in mice, indicating that a small fraction of the applied dose is able to be absorbed (Lütkenhaus, 2012).

Using the OECD toolbox (Version 3.0), the main constituent and 5 potential skin metabolites were identified as potential skin sensitizers due to protein binding most probably based on Michael Addition reactions. However, it has to be considered that dermal absorption of the main constituent, the respective skin metabolite and organic by-products is impeded by their chemical state as dry particulates first have to dissolve into the surface moisture of the skin before dermal uptake can occur (ECHA, 2012).

Furthermore, data on dermal toxicity may indicate whether a substance may be absorbed, if signs of systemic toxicity were clearly attributable to treatment (ECHA, 2012).

Consistent with the data on skin irritation and sensitisation, there is no indication for clinical signs of toxicity and any other treatment-related adverse effects from the acute dermal toxicity study with the substance, resulting in a dermal LD50 > 2000 mg/kg bw in rat. Thus, a low potential for acute dermal toxicity has been demonstrated, although no information on the actual amount of absorbed substance may be derived from these observations.

With regard to the low molecular weight (74.55 - 310.18 g/mol) and the good water solubility of the inorganic compounds, moderate dermal absorption is expected in general for the inorganic impurities. However, the ionic character of the inorganic constituents may hinder dermal absorption.

Overall, based on the available information, the dermal absorption potential of the test substance is predicted to be low.

Inhalation

As the vapour pressure of the test substance is very low (< 10E-05 Pa at 20 °C), the volatility is also negligible. Therefore, the potential for exposure and subsequent absorption via inhalation as vapour can be excluded. However, due to its physical appearance as powder, dust formation of the substance and inhalation of particles is conceivable.

In general, particles with an aerodynamic diameter < 100 μm have the potential to be inhaled, whereas only particles with an aerodynamic diameter < 50 μm can reach the thoracic region and those < 15 μm may enter the alveolar region of the respiratory tract (ECHA, 2012).

The particle size distribution of the test substance in its marketed form (fine granules) revealed that nearly 100% of the test substance has a particle size significantly exceeding 100 µm, as shown by a mean particle size of 157 and 529 µm for 10 and 90% of the particles, respectively, and a derived median aerodynamic diameter (MMAD) of 290.5 ± 1.6 µm (Smeykal, 2012). Only a very small proportion (< 0.2%) of the test substance showed a particle size of ca. 90 µm, which may potentially be inhaled, but may not reach the thoracic and alveolar region of the respiratory tract. Therefore, the potential of the test substance to induce systemic effects after inhalation is not considered to be likely.

Distribution and Accumulation

Distribution of a compound within the body depends on the physicochemical properties of the substance; especially the molecular weight, the lipophilic character and the water solubility. In general, the smaller the molecule, the wider is the distribution. If the molecule is lipophilic, it is likely to distribute into cells and the intracellular concentration may be higher than extracellular concentration, particularly in fatty tissues (ECHA, 2012).

Data from repeated dose studies in rats demonstrated that the coloured main constituent of the test substance as well as the coloured by-products are absorbed in the gastrointestinal tract and distributed widely in the body as indicated by the observation of substance-related red discolouration of various organs in males and females at macroscopic examination at 300 mg/kg bw/day as well as in all organs of both genders at the highest dose level of 1000 mg/kg bw/day (Holalagoudar, 2012c). Similar findings were reported in a combined repeated dose and reproductive/developmental toxicity screening test, showing reddish discoloration of various organs (e.g. kidney, skin, and testis) in a dose-related manner among all treatment groups (Holalagoudar, 2012d). Since discolouration was considered to be due to the specific red colour of the substance, it may be concluded that a substantial amount of the substance is absorbed and distributed in its unchanged form. Moreover, data from the preliminary range-finding study of an in vivo MNT test in mice receiving the test substance via the intraperitoneal route at 500 and 2000 mg/kg bw/day indicate systemic distribution of the test substance, as shown by significant clinical signs of toxicity, including reduction of spontaneous activity, constricted abdomen, piloerection, half eyelid closure, bradykinesia, kyphosis, opisthotonos, recumbency, abnormal breathing and muscle twitches. Additionally, the fur and urine of the animals were red coloured by the test substance (Donath, 2012). As the inorganic compounds of the test substance represent small water soluble ions which are most probably able to diffuse through pores and aqueous channels, rapid systemic distribution within the body is expected, particularly for those ions that are involved in several physiological processes (sodium, potassium, calcium, sulphate, chloride and phosphates).

However, as the substance exhibits a high water solubility and low log Pow, it is not expected to accumulate within the body, but is rather rapidly excreted via urine after absorption. This assumption is substantiated by data on repeated dose toxicity in rats showing red colouration of the urine after treatment with the substance.

Thus, distribution of the constituents of the test substance throughout the body seems feasible whereas accumulation is considered unlikely.

Metabolism

No data are available on the potential metabolism of the main compound and its organic by-products.

The potential metabolites following enzymatic metabolism of the main constituent of the test substance were predicted using the QSAR OECD toolbox (OECD, 2012). This QSAR tool predicts which metabolites may result from enzymatic activity in the liver and in the skin, and by intestinal bacteria in the gastrointestinal tract. Based on the rat liver S9 metabolism simulator, 5 potential metabolites were predicted for the main constituent of the test substance, which were attributable to partial or full cleavage of the azo bonds in the molecule. Simulation of potential skin metabolisms revealed that predicted metabolites were mainly attributable to chemical reactions including dehalogenation, hydroxylation of the major constituent as well as amid hydrolysis of the molecule, resulting in further break down products subject to dehalogenation and hydroxylation. Analysis with the microbial metabolism simulator provided 249 potential metabolites originating from the main constituent, mainly resulting from chemical reactions like azo bond cleavage, substitution, hydrolysis, cleavage of polycyclic aromatic ring structure and epoxidation of the azo bond. However, the presence of several exocyclic sulphonyl groups and the high water solubility most probably enable gastrointestinal dissolution and absorption and subsequent elimination of the non-metabolised main constituent via the urine and may therefore hinder further chemical reactions.

Furthermore, there is no indication that the test substance is activated to reactive intermediates under the relevant test conditions. The experimental studies performed on genotoxicity (Ames test, chromosome aberration assay in mammalian cells in vitro, in vivo mammalian erythrocyte micronucleus test) were negative, with and without metabolic activation (Wallner, 2012; Oppong-Nketiah, 2012; Donath, 2012).

Excretion

In general, characteristics favourable for urinary excretion are low molecular weight, good water solubility and ionisation of the molecule at pH of urine. In contrast, non-absorbed substances that have been ingested are excreted via faeces (ECHA, 2012).

Visual examination of urine from the 28-day study revealed a yellow to orange and rose or reddish colouration in animals of all dose groups (100, 300 and 1000 mg/kg bw/day), which was considered to be a result of excretion of the red-coloured test substance in the urine (Holalagoudar, 2012c). The observed excretion of the test substance in urine is in good agreement with its physico-chemical properties, including high water solubility and ionisation due to multiple sulphonate residues within the molecule of the main constituent and the structurally related by products. The inorganic constituents of the test substance are also eliminated via the urine due to their high water solubility, ionic character and small molecular size.

In conclusion, based on the physiochemical properties of the organic and inorganic constituents, excretion of the test substance is considered to occur mainly via the urine.