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Toxicokineticsof C.I. Direct Violet 35


A toxicokinetic assessment for C.I. Direct Violet 35 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. Direct Violet 35 is a direct dye. Direct dyes are anionic, water-soluble, narrow, flat molecules capable of aligning with flat cellulose molecules via van der Waals forces. Direct dyes are bound to fibres through deposition in cavities. Principal uses of direct dyes are the continuous colouring of paper.


Based on the data generated forC.I. Direct Violet 35, it can be concluded that the log Kow is low (<-5.) and the water solubility is high (5-10%). The molecular weight is 778 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. Direct Violet 35has 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(Envigo 2017 see dossier) the substancewas associated with slight changes in the testes and epididymides of males treated at 40 mg/kg/day. This finding did not correlate with the decreased pregnancy rate in females at 40 mg/kg bw. Other effects found (mineralization of heart and kidneys) were considered not related to treatment.

In an acute oral toxicity study mortality was observed at 2000 mg/kg bw, but no mortality was seen and effects were limited to the purple staining of the faeces in 4 of 5 females at 300 mg/kg (Envigo 2017 see dossier).

The findings are indicative for at least some absorption of the substance when administered orally.


Based on the study results and the physicochemical properties as discussed above, limited or no absorption of the test substance is anticipated. The oral absorption is therefore set at 10%.


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 < -1 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 vitroskin irritation and corrosion studies show that the substance is not irritant and/or corrosive. In an acute dermal toxicity study no effects were found at 2000 mg/kg bw.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 (778) fulfils the criterion

2) the log P is considerably outside the stated range (<-5) 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)).



The very low vapour pressure (4.1E-12Pa) indicates that the substance is 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


DV 35 is a green organic solid, generally used in formulations. The substance shows systemic toxicity for all of the end-points evaluated: it is harmful for short-term exposure and effects on the male reproductive system are seen after prolonged exposure. The substance, is not skin or respiratory irritant. It is however a skin sensitizer and an eye irritant.

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. Direct Violet 35, however is expected to lower susceptibility tocleavage of the azo bonds and formation of aromatic amines (Levine 1991).

In the studies conducted of acute exposure and repeated exposure, adverse effects were recorded. The presence of substance as such, however, was not recorded in blood, but based on the effects observed, some absorption can be expected.

DV 35 is non-mutagenic based of the available in vitro studies. An Ames test using the Prival and Mitchell modification was negative (Envigo 2017 see dossier). Therefore it is not expected that the formation of aromatic amines that are potentially carcinogenic is of concern.



Excretion of C.I. Direct Violet will be mainly via the feces (see also results of the acute oral study). 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 urin. 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.



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.








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.


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