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EC number: -
CAS number: -
There are no experimental studies available in which the
toxicokinetic behaviour of
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 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,
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
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
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.
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
Overall, based on the available information, the dermal absorption
potential of the test substance is predicted to be low.
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
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
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
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).
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.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
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