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EC number: 474-870-9 | CAS number: -
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
![](https://echa.europa.eu/o/diss-blank-theme/images/factsheets/A-REACH/factsheet/print_toxicological-information.png)
Basic toxicokinetics
Administrative data
- Endpoint:
- basic toxicokinetics
- Type of information:
- other: written assessment based on available toxicology studies
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: see 'Remark'
- Remarks:
- A written assessment of toxicokinetic behaviour is considered appropriate for the substance. The substance displays only minor toxicological effects in any of the studies proposed, and is deemed to be be not harmful for health effects. As such, it is deemed inappropriate in terms of animal welfare to conduct a toxicokinetic assessment when no harmful effects are predicted based on known toxicology. A written assessment has therefore been prepared to address this endpoint.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 021
- Report date:
- 2021
Materials and methods
- Objective of study:
- other: Assessment of toxicokinetic behaviour
- Principles of method if other than guideline:
- Written assessment based on toxicological profile.
- GLP compliance:
- no
Test material
- Reference substance name:
- -
- EC Number:
- 474-870-9
- EC Name:
- -
- Cas Number:
- 80156-97-4
- Molecular formula:
- Hill formula: C28H20ClN9Na4O16S5 CAS formula: C28H24ClN9O16S5.4Na
- IUPAC Name:
- tetrasodium 7-[(1E)-2-[2-(carbamoylamino)-4-{[4-chloro-6-({4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}amino)-1,3,5-triazin-2-yl]amino}phenyl]diazen-1-yl]naphthalene-1,3,6-trisulfonate
- Test material form:
- solid: particulate/powder
- Details on test material:
- Reactive Yellow 176 Ester
Reactive Yellow 176 Sulfato
Constituent 1
Test animals
- Species:
- other: Not applicable
Administration / exposure
- Details on exposure:
- Not applicable
- Duration and frequency of treatment / exposure:
- Not applicable
Doses / concentrations
- Remarks:
- Doses / Concentrations:
Not applicable
- No. of animals per sex per dose / concentration:
- Not applicable
- Positive control reference chemical:
- Not applicable
- Details on study design:
- Not applicable
- Details on dosing and sampling:
- Not applicable
- Statistics:
- Not applicable
Results and discussion
Metabolite characterisation studies
- Metabolites identified:
- not measured
- Details on metabolites:
- Not applicable
Any other information on results incl. tables
- Acute oral toxicity in rats
- Acute dermal toxicity in rats
- In vivo eye irritation
- Skin sensitization
- In vitro mutagenicity test in mammalian cells
- Subacute repeat dose toxicity study
- Subchronic repeat dose toxicity study
- Developmental toxicity study
1 Introduction
Toxicokinetic parameters such as uptake, distribution, metabolism and excretion form the essential toxicological profile of a substance. An approximate indication of the toxicokinetic pattern can be gained from the physico-chemical properties taking into account the molecular weight, the number of atoms (hydrogen bond donors and acceptors), the solubility in solvents, log KOW, etc. and the results of basic toxicity testing of the test article. The assessment of the toxicokinetic properties of Reactive Yellow 176 Ester given below is based on the results obtained for, the following toxicological endpoints:
All studies were carried out according to the principles of Good Laboratory Practice and/or met the requirements of the OECD and EU-Guideline for the Testing of Chemicals.
2 Substance Identity
The substance Tetrasodium 7-(4-(4-chloro-6-(4-(2-sulfooxyethanesulfonyl)phenylamino)-1,3,5-triazin-2-ylamino)-2-ureido-phenylazo)naphthalene-1,3,6-trisulfonate is a mono-constituent substance. It is the ester form of Reactive Yellow 176, which was registered first under EC number 303-153-4 with the trade name Remazol Gelb 3RS and is the reactive form during dyeing with respect to the 2-sulfatoethylsulfone moiety, and with that Reactive Yellow 176 Ester is the precursor of Reactive Yellow 176 Vinyl. Depending on temperature and humidity during synthesis, transport and storage, there will always a certain amount of the vinyl form of Reactive Yellow 176 be present in the batches for Reactive Yellow 176 Ester. In the current batch the content of Reactive Yellow 176 Vinyl was 4.6% (w/w), in former batches, it varied between 3 and 8% (w/w). In the dye bath (60°C, pH 10-11) all of the 2-sulfatoethylsulfone reacts to the vinyl sulfone.
Table 1. Substance identity of test substance
Common names: | Gelb Sulfato, Reactive Yellow 176 Ester, Reactive Yellow 176 Sulfato |
EC number: | 474-870-9 |
IUPAC name: | Tetrasodium 7-(4-(4-chloro-6-(4-(2-sulfooxyethanesulfonyl)phenylamino)-1,3,5-triazin-2-ylamino)-2-ureido-phenylazo)naphthalene-1,3,6-trisulfonate |
CAS number: | 80156-97-4 |
CAS name: | 1,3,6-Naphthalenetrisulfonic acid, 7-((2-((aminocarbonyl)amino)-4-((4-chloro-6-((4-((2-(sulfooxy)ethyl)sulfonyl)phenyl)amino)-1,3,5-triazin-2-yl)amino)phenyl)azo)-, tetrasodium salt |
SMILES: | C1(=CC=C(C=C1)[S](CCO[S]([O-])(=O)=O)(=O)=O)NC2=NC(=NC(=N2)Cl)NC5=CC=C(N=NC4=CC3=C(C=C(C=C3C=C4[S]([O-])(=O)=O)[S](=O)([O-])=O)[S]([O-])(=O)=O)C(=C5)NC(N)=O.[Na+].[Na+].[Na+].[Na+] |
Molecular formula: | Hill formula: C28H20ClN9Na4O16S5 CAS formula: C28H24ClN9O16S5.4Na |
Molecular weight: | 1026.3 g/mol |
3 Physico-chemical and ADME properties
3.1 Physico-chemical properties
The test substance is a solid with a molecular weight range of 1026.3 g/mol. It is characterised by a high water solubility of 396.7 g/L and a low partition coefficient (log Kow -6.4).
Table 2. Physicochemical properties
Endpoint | Reactive Yellow 176 Ester |
|
Physical state | Solid |
|
Melting point [°C] | > 300 |
|
Boiling point [°C] | > 500 (1380 calculated QSAR Toolbox) |
|
Vapour pressure [hPa] | 8.87 E-14 at 25°C |
|
Partition coefficient – log Kow | -6.4 at 20°C |
|
Water solubility [g/L] | 396.7 at 20°C |
|
Properties of ionic molecule* |
| |
miLogP | -1.55 | |
TPSA | 414.75 | |
Atom count (natoms) | 59 | |
Molecular weight [g/mol] | 934.30 | |
H-bond acceptor (nON) | 25 | |
H-bond donor (nOHNH) | 5 | |
No of violations | 2 | |
No of rotatable bonds (nrotb) | 15 | |
volume | 650.76 | |
Bioactivity score* |
| |
GPCR ligand | -2.10 | |
Ion channel modulator | -3.32 | |
Kinase inhibitor | -2.68 | |
Nuclear receptor ligand | -3.34 | |
Protease inhibitor | -1.52 | |
Enzyme inhibitor | -2.53 |
* Molinspiration Cheminformatics (https://www.molinspiration.com/cgi-bin/properties)
According to the modified “Lipinski Rule of Five”, the following criteria define the bioavailability of chemical substances:
Partition coefficient: log Kow > -0.4 or < 5.6 good bioavailability
Molecular weight: < 500 daltons good bioavailability
Atom count (natoms): < 70 good bioavailability
H-bond acceptor (nON): < 10 good bioavailability
H-bond donor (nOHNH): < 5 good bioavailability
3.2 ADME prediction
Reactive Yellow 176 Ester is a micro granulated solid at room temperature conditions. The melting point of the substance is > 300°C therefore a significant inhalation exposure to vapours is not expected. In view of the low n‑octanol/water partition coefficient, systemic bioavailability after oral exposure is very low, after dermal exposure it is not anticipated.
Evaluating Reactive Yellow 176 Ester and its active form Reactive Yellow 176 Vinyl using SwissADME (http://www.swissadme.ch) of the Swiss Institute of Bioinformatics produced the following results.
Table 3. Prediction of bioavailability using SwissADME
Prediction | Reactive Yellow 176 Ester | Reactive Yellow 176 Vinyl |
Physico-chemical Properties | ||
Formula | C28H20ClN9Na4O16S5 | C28H19ClN9Na3O12S4 |
Molecular weight | 1026.25 g/mol | 906.19 g/mol |
Num. heavy atoms | 63 | 57 |
Num. arom. heavy atoms | 28 | 28 |
Fraction Csp3 | 0.07 | 0 |
Num. rotatable bonds | 16 | 13 |
Num. H-bond acceptors | 21 | 17 |
Num. H-bond donors | 4 | 4 |
Molar Refractivity | 196.34 | 186.1 |
TPSA | 456.64 Ų | 381.83 Ų |
Lipophilicity | ||
Log Po/w (iLOGP) | -58.57 | -43.8 |
Log Po/w (XLOGP3) | 0.73 | 2.29 |
Log Po/w (WLOGP) | 7.25 | 7.83 |
Log Po/w (MLOGP) | 0.73 | 1.57 |
Log Po/w (SILICOS-IT) | -3.47 | -1.36 |
Consensus Log Po/w | -10.67 | -6.69 |
Water Solubility | ||
Log S (ESOL) | -5.94 | -6.41 |
Solubility | 1.19e-03 mg/ml | 3.55e-04 mg/ml |
Class | Moderately soluble | Poorly soluble |
Log S (Ali) | -9.9 | -9.95 |
Solubility | 1.30e-07 mg/ml | 1.03e-07 mg/ml |
Class | Poorly soluble | Poorly soluble |
Log S (SILICOS-IT) | -8.01 | -8.72 |
Solubility | 1.01e-05 mg/ml | 1.72e-06 mg/ml |
Class | Poorly soluble | Poorly soluble |
Pharmacokinetics | ||
GI absorption | Low | Low |
BBB permeant | No | No |
P-gp substrate | Yes | Yes |
CYP1A2 inhibitor | No | No |
CYP2C19 inhibitor | No | No |
CYP2C9 inhibitor | No | No |
CYP2D6 inhibitor | No | No |
CYP3A4 inhibitor | No | No |
Log Kp (skin permeation) | -12.04 cm/s | -10.20 cm/s |
Druglikeness | ||
Lipinski | No; 2 violations: MW>500, NorO>10 | No; 2 violations: MW>500, NorO>10 |
Ghose | No; 4 violations: MW>480, WLOGP>5.6, MR>130, #atoms>70 | No; 4 violations: MW>480, WLOGP>5.6, MR>130, #atoms>70 |
Veber | No; 2 violations: Rotors>10, TPSA>140 | No; 2 violations: Rotors>10, TPSA>140 |
Egan | No; 2 violations: WLOGP>5.88, TPSA>131.6 | No; 2 violations: WLOGP>5.88, TPSA>131.6 |
Muegge | No; 4 violations: MW>600, TPSA>150, Rotors>15, H-acc>10 | No; 3 violations: MW>600, TPSA>150, H-acc>10 |
Bioavailability Score | 0.17 | 0.17 |
Medicinal Chemistry | ||
PAINS | 1 alert: azo_A | 1 alert: azo_A |
Brenk | 3 alerts: diazo_group, sulfonic_acid_2, sulphate | 2 alerts: diazo_group, sulfonic_acid_2 |
Leadlikeness | No; 2 violations: MW>350, Rotors>7 | No; 2 violations: MW>350, Rotors>7 |
Synth. accessibility | 5.24 | 4.89 |
Profiling of Reactive Yellow 176 Ester and Vinyl shows that the substance and its active component has a low oral and a very low dermal bioavailability. Bioaccumulation of the substance or its metabolites in not anticipated.
3.3 Toxicokinetic behaviour of azo-dyes
The cleavage of azo-bonds has been described in many textbooks and scientific articles. In Industrial Dyes - Chemistry, Properties, Applications (Hunger, 2003), the following is stated: “With regard to metabolism, azo dyes are the most widely investigated class of dyes. According to metabolic pathway two groups of azo dyes can be distinguished: (1) water-soluble dyes, mostly bearing sulfo groups, and (2) solvent-soluble dyes with nonpolar substituents. By far the most predominant metabolic pathway for water-soluble azo dyes is cleavage of the azo linkage by azoreductase of the liver and extrahepatic tissue or by intestinal microflora in the body”. In Dye Application, Manufacture of Dye Intermediates and Dyes (Freeman & Mock (2007) an example of the metabolic breakdown of an azo dye by azo reductase enzymes is given.
This literature data has been supported by own data from toxicokinetic studies with Reactive Black 5, one of the most prominent reactive dyes, which has been very well investigated in metabolism studies and due to its typical basic structure of a reactive dye consisting of H-Acid coupled with two parabase esters can be used as general example for the behaviour of azo dyes. These studies are deliberately used as “generic” information, as the chemical mechanism behind the cleavage of azo-bonds is well known and the same for each chemical with azo-groups and does not have to be proven newly for each substance. According to A textbook of modern toxicology (Hodgson, 2004), the requirements for azo reduction are anaerobic conditions and NADPH. They are inhibited by CO, and presumably they involve CYP. The ability of mammalian cells to reduce azo bonds is rather poor, and intestinal microflora may play a role. Therefore, not the structure of the molecule plays a role in the rate of cleavage, but the origin of the cells exposed to the azo-dye.
Metabolism studies with Reactive Black 5 were conducted in four separate studies. Two with labelling of the parabase esters and two with labelling of the H-Acid.
3.3.1 ADME Studies of Reactive Black 5 with labelling of the benzene rings
There were two studies in rats conducted with Reactive Black 5 labelled at the parabase structures of the test item; one to investigate the kinetic behaviour of Reactive Black 5 and one to investigate its metabolism.
The ADME study results for the parabase section of the molecule are as follows:
Oral absorption: 28.6%
Excretion: 85% faeces; 15% urine
There was no unmodified Reactive Black 5 found in the excretion of metabolites. The metabolites excreted were 84% sulfate ester and 7% N-acetated sulfate ester.
The main metabolic route to excretion is considered to be via reductive cleavage of the two main metabolite azo groups: Amine direct excretion via acetylation (elimination of the mono azo group – as a possible intermediate of reduction – was not found in the faeces).
3.3.2 ADME Studies of Reactive Black 5 with labelling of the naphthalene structure
There were two studies in rats conducted with Reactive Black 5 labelled at the naphthalene structure of the test item; one study to investigate the kinetic behaviour of Reactive Black 5 and one to investigate its metabolism.
The ADME study results for the naphthalene section of the molecule are as follows:
Oral absorption: ca 1%
Excretion: 96% faeces; 1.5% urine
No unmodified Reactive Black 5 was present in the excretion of metabolites, of which there was at least 7 numbers in the faeces and at least 3 numbers in the urine. All metabolites were polar as all metabolites were deemed most likely to have at least one azo-link chain metabolised and removed.
On the basis of all four studies, the metabolic pathway was further extrapolated.
On the basis of this information, it is therefore concluded that the route of metabolism will include the formation of the naphthalene system and the parabase structure.
For Reactive Black 5, the naphthalene structure derived from “H-Acid” (4-amino-5-hydroxynaphthalene-2,7-disulfonic acid) was solely found in the faeces (seemed not to be absorbed) and it was not found in plasma or urine.
3.3.3 Assumed metabolic behaviour of Reactive Yellow 176 Ester
Reactive Yellow 176 can be considered to be metabolised by azo-reductase from intestinal bacteria by cleavage of the azo bond as described in literature and shown in the metabolism studies with Reactive Black 5. Following the same route of metabolisation is considered to be appropriate for the substance Reactive Yellow 176 leading to tetrasodium (2-amino-5-{[4-chloro-6-({4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}amino)-1,3,5-triazin-2-yl]amino}phenyl)urea and 7-aminonaphthalene-1,3,6-trisulfonate as metabolites.
3.4 Evaluation of Toxicokinetics – Absorption, distribution, metabolism and excretion
3.4.1 Absorption
According to ECHA Guidance R.7c (ECHA (2017c)), the smaller the molecule the more easily it may be taken up via oral route. Generally, oral absorption is favoured for molecular weights below 500 g/mol. As the Substance is a large molecule (> 800 g/mol), it will therefore be taken up at a very low rate following the oral route. This assumption is supported by the results of the acute oral toxicity study in rats, where no deaths and no clinical signs were noted at the limit dose of 2000 mg dye/kg bw. Yellow discolouration of the urine and faeces for 48 hours followed treatment. In the 90-day repeat dose study, yellow discolouration of some organs was seen in single animals of the high-dose group at necropsy leading to the assumption that a low amount of the substance was orally available. This discolouration was no longer present after the 4-week treatment free period.
For dermal absorption, molecular weights below 100 g/mol favours dermal uptake, molecules with a weight above 500 g/mol may be too large to be absorbed (ECHA 2017c). With molecular weights of the source and the target substances above 800 g/mol, the molecules are too large to allow absorption through skin. The high water solubility of the substances, would favour the dermal absorption rates under normal circumstances, as the substance must be sufficiently soluble in water to partition from the stratum corneum into the epidermis. However, the poor lipophilicity (log Kow < -1) will hinder penetration through the lipid rich environment of the stratum corneum and therefore dermal absorption is limited. Single dermal application of 2000 mg/kg body weight onto male and female rats produced no deaths or symptoms of systemic toxicity. Test item related discoloration of skin (5/5 male; 5/5 female), were noted on the treated skin surface for 2-9 days after treatment. Testing for sensitising properties was performed in guinea pigs according to the method of Magnusson & Kligman. Intradermal induction was done at 5% test substance concentration and dermal induction and challenge treatment at 50%. Despite the QSAR Toolbox giving some alerts for apparent skin sensitising properties, no evidence of skin sensitising properties was found in the guinea pig maximisation test, confirming a low dermal penetration rate of the substance.
As the source and target substances have a low volatility (melting point above 300°C), they are not available for inhalation as a vapour. Should particles of the solid form of the substances be inhaled, absorption through the respiratory tract epithelium is not expected due to the molecule sizes.
3.4.2 Distribution
In general, the smaller the molecule, the wider the distribution. Based on their molecular weight distribution and the hydrophilicity it is assumed, that if absorbed, the Substance is distributed within the aqueous compartment of the organism. Furthermore, an accumulation within adipose tissue can be excluded based on the low log Kow of all constituents of source and target substance. However, the substance distributes into the tissue water of most organs as can be seen from the yellow discolouration of stomach, salivary glands, prostate, epididymides, testes, cervical lymph nodes and/or uterus of one to tree rats of the high-dose group. This discolouration was no longer present after the 4-week treatment free period, indicating the reversibility of this effect and therefore excluding a bioaccumulation potential of the Substance. At microscopy, pigment storage was observed in the cortex of renal (mainly proximal) tubular cells of high-dose animals, resulting from re-resorption of excreted dye. This pigment storage was reduced in severity and number of animals after the 4-week recovery period, showing the reversibility of the effect.
3.4.3 Metabolism
The Substance is not expected to be bioavailable following oral, inhalative or dermal exposure at a relevant level as a result of their properties. As shown in oral acute to subchronic toxicity studies, the Substance is absorbed to some extent. Based on the results of in-vitro and in-vivo genotoxicity studies, it can be assumed that the Substance and its Structural Analogues are not enzymatically activated (toxified) during metabolism as the metabolic activated test substance showed no higher genotoxicity compared to the test substance without metabolic activation. Available data indicate that the Substance is susceptible to hydrolysis. It is likely that during passage of the GI-tract the azo bond is reduced via azo-reductase (see section 3.3). However, as shown in radio-labelled studies with Reactive Black 5, the naphthalenic structure is unlikely to be absorbed. Accordingly, the resulting other cleavage product (2-amino-5-{[4-chloro-6-({4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}amino)-1,3,5-triazin-2-yl]amino}phenyl)urea and 7-aminonaphthalene-1,3,6-trisulfonate is unlikely to be absorbed with a molecular weight of > 500 g/mol and a good water solubility.
3.4.4 Excretion
According to the physico-chemical properties of the substances, molecular weight and hydrophilic characteristics the main route of excretion is expected to be via faeces, as substances that are excreted in the urine tend to be water-soluble and of low molecular weight (below 300 g/mol in the rat). As seen from the pigment storage observed in the cortex of proximal renal tubular cells of high-dose animals, resulting from re-resorption of excreted dye at the end of the treatment phase in the 90-day repeat-dose toxicity study, a very low amount of the substance and/or its metabolites are also excreted via the kidneys.
4 Environmental fate
4.1 Behaviour of reactive dyes in water
“Reactive dyes are unique textile colorants because they contain reactive groups that bind to fibres through formation of a covalent bond. Approximately 40 types of reactive groups have been listed for the commercial dyes. Of these, the highest volume commercial products are those containing the 2 sulfatoethylsulfone moiety” (Weber, 1990). The reactive form of the dye, the vinyl sulfone, is generated in the dye bath by treatment with base. The dye-fibre adduct is formed in a subsequent reaction by Michael-type 1,4-nucleophilic addition. Competing with this reaction is hydrolysis to give the 2 hydroxyethylsulfone. This reaction is the same in each dye containing the 2-sulfatoethylsulfone moiety, independently of the remaining dye-structure, therefore it is a general mechanism as described in many textbooks and scientific articles. The rate of this transformation is dependent on temperature and pH. In all dyes containing the 2-sulfatoethylsulfone moiety tested so far, the half-life at pH 9 was extrapolated to about 2.5 hours at 25°C. However, as dyeing with these kind of reactive dyes takes place at a temperature of about 60°C at pH 10 to 11, the dye is completely hydrolysed at the end of the dyeing process.
In Dye Application, Manufacture of Dye Intermediates and Dyes (Freeman & Mock, 2007), the general structure and mechanism of reactive dyes is explained. “Reactive dyes are used mainly as colorants for cotton, although they are also suitable for nylon and woo1. They are water soluble, due to the presence of one or more
—SO3Na groups, and undergo fixation to polymer chains via covalent bond formation. […] Each dye is composed of five basic parts: SG—C—B—RG—LG. In this regard, SG = water solubilizing group (—SO3Na), C = chromogen (e.g., azo, anthraquinone), B = bridging or linking group (e.g., —NH—), RG = reactive group (e.g., chlorotriazine, vinylsulfone), and LG = leaving group (e.g., —Cl, —F, —SO4H). […] Although the most commonly used reactive systems involve the halotriazine and sulfatoethyl sulfone (vinyl sulfone) groups, halo-genated pyrimidines, phthalazines, and quinoxalines are also available. For all of these systems, alkali is used to facilitate dye—fiber fixation, and fixation occurs either by nucleophilic substitution or addition. The requirement for alkali in the application of reactive dyes to cotton leads to an undesirable side reaction, namely hydrolysis of the reactive groups before dye—fiber fixation can occur.”
In the case of dyes containing a 2-sulfatoethylsulfone moiety, the hydrolysis takes place as nucleophilic addition at this reactive group, as described in Color Chemistry (Zollinger, 2003). According to Zollinger, nucleophilic additions to reactive groups are often preceded by a general-base-catalysed elimination of a nucleofugic group (→ k1 path), followed by a specific-base-catalysed addition of the functional group HY of the textile fiber (→ k2 path). The most important function in this series is the [(sulfooxy)ethyl]sulfonyl reactive group, which, under dyeing conditions, readily dissociates to form reactive vinyl sulfones (—SO2CH=CH2)
This has been confirmed in own studies on abiotic degradation and a sediment/water simulation study with Reactive Yellow 176 Ester and labelled Reactive Black 5 as an example for the typical vinyl-sulfone dye.
4.2 Fate of reactive dyes in surface water
The environmental behaviour of Reactive Black 5, one of the most used vinyl sulfone dye, was investigated in two studies. In a study on abiotic degradation (hydrolysis as a function of pH; report OC-HL 62-1, 01-Mar-1989), the fate of Reactive Black 5 at different pH (1.2, 4, 7, 9, 11) and temperatures was investigated. The report states that at pH 7 and 50°C, after 2.4 hours the ester-dye is almost completely degraded. The main degradation product is the vinyl sulfone (75%). A second degradation product is found with 17% at 2.4 hours, which is most likely the 2-hydroxyethylsulfone. When Reactive Black 5 was applied to a sediment/water test system (report DB89/001, 06-Aug-1990), the test substance was rapidly degraded to the vinyl sulfone and further to the 2-hydroxyethylsulfone.
4.3 Assumed environmental fate of Reactive Yellow 176 Ester
A structural analogue, the vinylated metabase form of Reactive Yellow 176 Ester, showed no relevant biodegradation throughout the testing period of an “Manometric Respirometry Test” that in all essential parts is identical with the OECD 301F method. Hence, the test substance can be classified as ‘Not Readily Biodegradable’.
Studies on direct phototransformation in water are not available but it is assumed on the basis of chemical structure and nature of use that the substance is not degraded by direct photolysis.
It is concluded, therefore, that abiotic processes would contribute significantly to the depletion of the substance within the environment.
In the abiotic degradation study (hydrolysis), Reactive Yellow 176 Ester is hydrolytically stable at pH 4 with an extrapolated half-life of > 1 year at 25°C. At pH 7, the half-life period was extrapolated to 18.9 days and at pH 9 (25°C), the substance was hydrolytically instable with a half-life of < 1 day.
Possible abiotic degradation products for Reactive Yellow 176 Ester are driven by nucleophilic addition (neutral and basic environment) or substitution (acidic environment). The hydrolysis product derived from nucleophilic addition is identical to the structural analogue 04, while the product of the nucleophilic substitution corresponds to the structural analogue 02 (constituent 2).
The results of the OECD TG 309 (Fiebig & Goller, 2021) study for simulation testing on ultimate degradation in surface water shows a fast degradation of the test item in aerobic natural water. Transformation of Reactive Yellow 176 started directly after application. About 55 % transformation was reached within 3 days, after 14 days the detected concentration was < 10 % of the applied concentration. The test item was degraded to only one metabolite, the vinylsulphone form of Reactive Yellow 176 (SA04).
This metabolite was formed by an elimination of the terminal alkyl sulfate moiety. This metabolite was also formed in the abiotic controls at the end of the study in equal quantities. This result indicates that the degradation is driven by an abiotic process. The DTx values of Reactive Red 239 from this study are given in Table 4:
Table 4. DTx Values of Reactive Yellow 176
| DTx values in days [30µg/L] | DTx values in days [100µg/L] |
DT50 | 1.91 | 2.05 |
DT90 | 6.34 | 6.82 |
Thus, the OECD 309 study confirms the assumption that abiotic processes lead to rapid degradation of the substance in surface waters. Based on this finding and its high water solubility, low partition coefficient and fairly rapid hydrolysis rate at environmentally relevant pH, it can be concluded that it is unlikely that Reactive Yellow 176 Ester could potentially be persistent within the environment.
5 Conclusion
The results of ADME prediction and basic toxicity testing give no reason to anticipate unusual characteristics with regard to the toxicokinetics of Reactive Yellow 176 Ester. The data indicate that there is little or no dermal absorption. No signs of a systemic toxicity associated with absorption through skin have been observed. Based on exposure model from AG Textilien des Bundesinstituts fur Risikobewertung (BfR), the dermal penetration rate for dyes through the skin was found to be less than 2%.
Based on physico-chemical data and the results of oral toxicity studies, Reactive Yellow 176 Ester has a limited oral bioavailability. The substance is considered to have low volatility as evident from the vapour pressure measurement and the calculated melting point of > 300°C, so the potential for the generation of inhalable forms is low. The molecular weight is higher than 500 g/mol and the chromophore is negative charged. This together with the high water solubility and low partition coefficient value, indicate the substance is not able to cross the mucous layer of the respiratory tract. Due to the high water solubility, vapours if generated/inhaled, will be trapped in the mucus of the respiratory tract, thereby further limiting the absorption. Hence, the main route of exposure of the substance, if inhaled, will be due to swallowing of particles deposited in the nose/mouth. Therefore, the bioavailability for inhalation is considered the same as for oral intake.
Bioaccumulation of Reactive Yellow 176 Ester can most probably be excluded due to the available data. Based on the results of genotoxicity assays, a metabolisation towards genotoxic metabolites can also be excluded for mammalian species.
On the basis of the results, it is anticipated that the substance does not undergo significant metabolic activity; rather it is metabolised for excretion with little subsequent toxicity. The substance is therefore not considered to be of concern for ADME related effects.
It could be shown that Reactive Yellow 176 Ester is subject to rapid abiotic degradation in surface waters. Based on this finding and its high water solubility, low partition coefficient and fairly rapid hydrolysis rate at environmentally relevant pH, it can be concluded that it is unlikely that Reactive Yellow 176 Ester could potentially be persistent within the environment.
6 References
ECHA (2017c) Guidance on information requirements and chemical safety assessment, Chapter R.7c: Endpoint specific guidance.
Fiebig S, Goller S (2021): Aerobic Mineralization in Surface Water. Simulation Biodegradation Test, Noack Laboratorien GmbH, Study ID SO20481 / ASF19121, Sarstedt.
Freeman HS, Mock GN (2007): Dye Application, Manufacture of Dye Intermediates and Dyes. In: Kent J.A. (eds) Kent and Riegel’s Handbook of Industrial Chemistry and Biotechnology. Springer, Boston, MA.
Hodgson E (2004): A textbook of modern toxicology, third edition. John Wiley & Sons, Inc., Hoboken, New Jersey
Hunger K (2003): Industrial Dyes - Chemistry, Properties, Applications. WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Molinspiration Cheminformatics. Calculation of Molecular Properties and Bioactivity Score (https://www.molinspiration.com/cgi-bin/properties)
OECD QSAR Toolbox release 4.4.1, April 2020 (https://qsartoolbox.org/).
SwissADME: A free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules. Sci Rep 7: 42717 (http://www.swissadme.ch/)
Weber EJ, Sturrock PE, Camp SR. Reactive Dyes in the Aquatic Environment: A Case Study of Reactive Blue 19. Environmental research brief. U.S. Environmental Protection Agency, Environmental Research Laboratory, 1990:1-7
Zollinger H: Color Chemistry: Syntheses, Properties, and Applications of Organic Dyes and Pigments, 3rd revised edition, 2003:230-231
Applicant's summary and conclusion
- Conclusions:
- Interpretation of results (migrated information): no bioaccumulation potential based on study results
- Executive summary:
Toxicokinetic parameters such as uptake, distribution, metabolism and excretion form the essential toxicological profile of a substance. An approximate indication of the toxicokinetic pattern can be gained from the physico-chemical properties taking into account the molecular weight, the number of atoms (hydrogen bond donors and acceptors), the solubility in solvents, log KOW, etc. and the results of basic toxicity testing of the test article. The assessment of the toxicokinetic properties of Reactive Yellow 176 Ester given below is based on the results obtained for the following endpoints:
- Acute oral toxicity in rats
- Acute dermal toxicity in rats
- In vivo eye irritation
- Skin sensitization
- In vitro mutagenicity test in mammalian cells
- Subacute repeat dose toxicity study
- Subchronic repeat dose toxicity study
- Developmental toxicity study
- Abiotic degradation
- Ultimate degradation in surface water
The results of ADME prediction and basic toxicity testing give no reason to anticipate unusual characteristics with regard to the toxicokinetics of Reactive Yellow 176 Ester. The data indicate that there is little or no dermal absorption. No signs of a systemic toxicity associated with absorption through skin have been observed. Based on exposure model from AG Textilien des Bundesinstituts fur Risikobewertung (BfR), the dermal penetration rate for dyes through the skin was found to be less than 2%.
Based on physico-chemical data and the results of oral toxicity studies, Reactive Yellow 176 Ester has a limited oral bioavailability. The substance is considered to have low volatility as evident from the vapour pressure measurement and the calculated melting point of > 300°C, so the potential for the generation of inhalable forms is low. The molecular weight is higher than 500 g/mol and the chromophore is negative charged. This together with the high water solubility and low partition coefficient value, indicate the substance is not able to cross the mucous layer of the respiratory tract. Due to the high water solubility, vapours if generated/inhaled, will be trapped in the mucus of the respiratory tract, thereby further limiting the absorption. Hence, the main route of exposure of the substance, if inhaled, will be due to swallowing of particles deposited in the nose/mouth. Therefore, the bioavailability for inhalation is considered the same as for oral intake.
Bioaccumulation of Reactive Yellow 176 Ester can most probably be excluded due to the available data. Based on the results of genotoxicity assays, a metabolisation towards genotoxic metabolites can also be excluded for mammalian species.
On the basis of the results, it is anticipated that the substance does not undergo significant metabolic activity; rather it is metabolised for excretion with little subsequent toxicity. The substance is therefore not considered to be of concern for ADME related effects.
It could be shown that Reactive Yellow 176 Ester is subject to rapid abiotic degradation in surface waters. Based on this finding and its high water solubility, low partition coefficient and fairly rapid hydrolysis rate at environmentally relevant pH, it can be concluded that it is unlikely that Reactive Yellow 176 Ester could potentially be persistent within the environment.
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