Hexasodium 4,4'-[1,4-phenylenebis[imino(6-chloro-1,3,5-triazine-4,2-diyl)imino]]bis[5-hydroxy-6-[(2-sulphonatophenyl)azo]naphthalene-2,7-disulphonate]

Endpoint summary

Description of key information

Toxicokinetics of C.I. Reactive Red 120

 

A toxicokinetic assessment for C.I. Reactive Red 120 has been made based on the physical and chemical properties of the substance and the available toxicity studieson the substance.

A substance can enter the body via the lungs, the gastrointestinal tract, and the skin. To determine the absorption rate, the different routes need to be assessed individually.

The size of the molecule, log Kow and water solubility are important factors in uptake and distribution of chemicals.

 

C.I. Reactive Red 120 is a dye. Reactive dyes are anionic, water-soluble, narrow, flat molecules that attach themselves to their substrates by a chemical reaction that forms a covalent bond between the molecule of dye and that of the fibre.

Based on the data generated for C.I. Reactive Red 120, it can be concluded that the log Kow is low (< 0.3) and the water solubility is high (344.3 g/L). The molecular weight is 1338 Da (as free acid).

 

Oral absorption

In general, a compound needs to be dissolved before it can be taken up from the gastro-intestinal tract after oral administration.

(1) C.I. Reactive Red 120 has a high water solubility, therefore it is expected to dissolve into the gastrointestinal fluids, but uptake by passive diffusion is limited due to its low solubility in the GI lining.

(2) Based on the molecular weight, absorption is expected to be low.

(3) The substance has a low log Kow, which makes the compound very hydrophylic.

(4) The substance is polar and this will limit uptake (the presence of sulfonate groups on the molecule is known to reduce uptake (Levine 1991)).

(5) The substance may be reduced by bacteria in the gastro intestinal tract, but the metabolites formed will also be polar of nature, which will limit their uptake (see Annex)

 

In the available repeated dose-reproduction study (vivo Science 2018 see dossier) the substancewas associated with mortality and reduced fertility at the highest dose tested. The effects reported, however, only could be used to draw a preliminary conclusion on the NOAEL. The treatment related mortality and fertility effects were seen during the first 28 days of the study when the high dose was 1000 mg/kg bw. After reduction of this dose to 500 mg/kg bw, no additional adverse effects were reported. Red staining as observed in the urinary tract and the male reproductive system is indicative for some absorption of the substance (metabolites are expected not to be colored).

No mortality or systemic toxicity was observed at doses >4000 mg/kg bw in an acute oral toxicity studies available (Ciba 1974, see dossier).

Based on the study results and the physicochemical properties of the substance as discussed above, some absorption of the substance is anticipated. It cannot be excluded that the substance is metabolized in the gastro-intestinal tract (see Annex) and that metabolites are taken up. The red discoloration found, however, is indicative for absorption of the parent. Therefore the oral absorption is set at 50% in a worst case approach.

 

Dermal absorption

When the substance comes in contact with the skin, the first layer of the skin, the stratum corneum, forms a barrier for hydrophilic compounds. The substance has a log Kow < 0.3 and is very water soluble, suggesting that uptake in the stratum corneum will be limited. The high molecular weight and the polarity of the chromophore are expected to contribute further to a low absorption.

In vivoskin irritation studies show that the substance is not irritant and/or corrosive.The substance is a skin sensitizer and is therefore expected to have some potential for dermal absorption.

 

According to the criteria given in the REACH Guidance, 10% dermal absorption will be considered in cases where the MW >500 and log Pow <-1 or >4. The weight of evidence of the following factors indicates that the substance can be assumed to have a dermal absorption of 10%:

1) the molecular weight (1338) fulfils the criterion

2) the log Kow is within the stated range (< 0.3) and

3) skin irritation testing did not report any corrosive effects which would enhance absorption significantly.

(4) The substance is polar and this will limit uptake (the presence of sulfonate groups on the molecule is known to reduce uptake (Levine 1991)).

 

Inhalation

The vapour pressure of Reactive Red 120 could not be measured, but QSAR calculations show that it is expected to be negligible. The substance is therefore not expected to evaporate and become available via inhalation. Moreover, aerosol formation is not expected from the current uses. Therefore exposure of the respiratory tract is not likely. If, however, the test substance would reach the tracheobronchial region, it may likely dissolve within the mucus lining the respiratory tract due to its high water solubility, but uptake would be very low because the high hydrophilicity will prevent passage via bio-membranes.Based on these consideration, for risk assessment purposes the inhalation absorption of the substance is set at 10%.

 

The inhalation route are considered not relevant as exposure route and are therefore not further considered.

 

Bioavailability and metabolism

 

RR120 is a red powder, generally used in liquid formulations. The available studies cannot exclude that substance induces systemic toxicity. The substance, is not skin/eye/respiratory irritant. It is however a skin sensitizer.

As indicated above absorption of the substance is expected to be low. When absorbed, no bioaccumulation is expected, as the substance or its sulfonated metabolites are likely to be conjugated and cleared via the kidney.

Some azo dyes undergo cleavage of the azo-bond leading to the formation of aromatic amine metabolites and this represents a risk (see Annex to this document); the sulfonation ofC.I. Reactive Red 120, however is expected to lower susceptibility tocleavage of the azo bonds and formation of aromatic amines (Levine 1991), however this cannot be excluded.

In the studies conducted on repeated exposure, the presence of substance as such or a metabolite was not recorded in blood, but based on the effects observed, absorption can be expected.

RR120 is non-mutagenic based on a weight of evidence approach based on the results of the available in vitro studies. Positive results in a micronucleus test and a HPRT test, were not confirmed in repeats of these tests with a purer sample of CI Reactive Red 120 (Envigo 2017, Laus 2017). No influence on the outcome of the studies by metabolic activation was reported. Based on these findings, it is not expected that the formation of aromatic amines, that are potentially carcinogenic, is of concern.

 

Excretion

Excretion of C.I. Reactive Red 120 will be mainly via the feces (see the red discoloration of the GI-tract in the OECD 422 study), but as the red discoloration was also reported in the urinary system, renal clearance is expected to contribute to excretion as well. Any substance or metabolites that are absorbed will be cleared via the kidneys after conjugation.

 

 

 

 

Annex Cleavage of the azo linkage and reduction

(data from Environment Canada Health Canada July 2012)

 

As azo dyes are highly water soluble, they do not tend to accumulate in the body. Thus, it is likely that their toxicity might not be due to the dye itself, but rather to degradation metabolism of the dyes.

The azo linkage (N=N) is the most labile portion of an azo dye molecule and may easily undergo enzymatic breakdown in bacteria and mammals including humans.

Nevertheless some characteristics of the substance may influence the susceptible of cleavage, for example it has been noted that sulfonation of azo dyes may inhibit the release of aromatic amines. In vivo, azo reduction occurs by an enzyme-mediated reaction. In mammalian organisms azo-reductases are, with different activities, present in various organs like liver, kidney, lung, heart, brain, spleen and muscle tissues. The azo-reductase of the liver, followed by the azoreductase of the kidneys possesses the greatest enzymatic activity.

 

Azoreductases are also present and active in the microflora of the there is evidence that some molecules require gut flora reduction before they can be further metabolized by the liver. Nevertheless studies conducted on dyes have found that bacterial azoreductase activity is over 100 times more efficient than that of liver azoreductases and may constitute the primary pathway for azo reduction.

After cleavage of the azo-linkage, the component aromatic amines are absorbed in the intestine and excreted in the urine. Sulphonation of azo dyes appears to decrease toxicity by enhancing urinary excretion of the dye and its metabolites.

The polarity of azo dyes influences the metabolism and consequently the excretion. Sulfonation of azo dyes appears to decrease toxicity by enhancing urinary excretion of the dye and its metabolites. Sulphonated dyes, mainly mono-, di- and trisulphonated compounds are world-wide permitted for use in foods, cosmetics and as drugs for oral application. Highly sulphonated azo dyes are poorly absorbed from the intestine after oral intake. Practically a complete cleavage of the azo linkage takes place in the gastrointestinal tract. This results in sulphonic acids rather than aromatic amines. These acids are rapidly absorbed, modified by the liver and excreted in the bile and urine.

Several hundred species of bacteria are expected to be present in human skin, and a number of these have been shown to possess azoreductase.

 

Oxidation

In the mammalian liver, azo compounds are metabolized by cytosolic and microsomal enzymes. This is followed by microsomal oxidation and N-acetylation or O-esterification to form DNA adducts in the liver. At least three different types of azoreductase activity are found in the liver; they differ with respect to localization, substrate specificity, response to enzyme inducers and sensitivity to oxygen and carbon monoxide. Two of these types of activities are associated with the microsomal fraction and require cytochrome P450, while one activity is located in the cytosol of the liver.

 

Activation and conjugation

Three mechanisms for the metabolic activation of azo dyes have been identified. While they all require some type of metabolic activation to produce reactive electrophilic intermediates that can interact with cellular material, i.e., covalently bind to DNA or ribonucleic acid (RNA), they differ in terms of the sequence of metabolic reactions leading to the reactive intermediates. The three mechanisms are described below:

1 -Aromatic amine released by azo bond cleavage: metabolic activation involves N-hydroxylation followed by O-acylation, yielding acyloxy amines.

2 –Oxidation of a free aromatic amine group that is part of the azo dye structure: when an azo dye has a free aromatic amine group (or a similar N-methylated derivative), azo bond reduction is not always necessary for creation of a reactive intermediate. In such cases, depending on the azo dye structure, metabolic azo bond reduction can act as a detoxification mechanism/

3 -Activation of the azo dyes via direct oxidation of the azo linkage to highly reactive electrophilic diazonium salts: in the liver, certain azo dyes can undergo direct oxidation at the azo bond, without prior azo bond reduction, to generate reactive intermediates, including diazonium ion, which can further react with cellular DNA, RNA or protein. Different cytochrome P450 enzymes are involved in the oxidative processes, leading to the formation of various reactive metabolites. Although reduction and cleavage of the azo-linkage is the major metabolic pathway of azo dyes in mammals, other metabolic pathways may take place. Major routes of detoxifying metabolism of azo dyes and aromatic amines are ring hydroxylation and glucuronide conjugation.

 

 

 

 

 

References

 

Environment Canada, Health Canada Aromatic Azo- and Benzidine-Based Substances 

Draft Technical Background Document July 2012

Levine WG. 1991. Metabolism of azo dyes: Implication for detoxification and activation. Drug Metab Rev 23:253–309.

 

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

50

Absorption rate - dermal (%):

10

Absorption rate - inhalation (%):

10