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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: inherent biodegradability
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
1. Hypothesis for the analogue approach
The target substance Direct Black 155-Na salt, CAS No. 68877-33-8, Disodium 4-amino-3-[[[(2,4-diaminophenyl)diazenyl]phenyl]diazenyl]-5-hydroxy-6-(phenyldiazenyl)naphthalene-2,7-disulfonate (Direct Black RBB) is defined as a mono-constituent substance.

The available toxicological data on this substance are insufficient to fulfil the data requirements for a REACH Annex VIII dossier.

In order to prevent unnecessary animal testing, the occurring data gaps on toxicity studies might be filled by applying read-across from the similar substance (source) Direct Black 155-NaKLi salt, CAS No. 2196165-14-5, Sodium, potassium, lithium 4-amino-3-[{4-[(2,4-diaminophenyl)diazenyl]phenyl}diazenyl]-5-hydroxy-6-[phenyldiazenyl]naphthalene-2,7-disulfonate (Direct Black RBK) which is also defined as a mono-constituent substance.

Both substances, target and source, have the same molecular structure. The only difference between the source structure Direct Black 155-NaKLi salt (CAS No. 2196165-14-5) and target Direct Black 155-Na salt (CAS No. 68877-33-8) is the counter ion.

Both substances are synthetized using the same raw materials and following the same manufacturing process. They are identical in relation to the anionic chemical structure.

CAS No. 2196165-14-5 is the result of the precipitation at the final stage of the reaction with potassium chloride and neutralization with lithium hydroxide and sodium hydroxide, whilst in CAS No. 68877-33-8 the precipitating agent is sodium chloride and the neutralization agent is sodium hydroxide.

The read-across is based on the hypothesis that the source and target substances have similar toxicological and environmental fate properties because both molecules have the following similarities:


a) Identical raw materials and manufacturing process.
b) Similar impurities, in comparable amounts.
c) Structural similarity: sulphonated molecules, aromatic rings, azo bonds.
Both dyes have identical anionic structure, the same polyaromatic structures polysulphonated, linked with azo bonds.
d) Both have the same ionic functional groups (sulphonic, amino, phenol).
The substances in a solid state are salts and in water solution at neutral pH are the same polyanions solvated with water.
e) Both have affinity to the same type of substrates/molecules.
The substances are able to be adsorbed on the same type of substance, e.g. polysaccharides (cellulose), polyphenols (lignine) and proteins.
f) Both may release by reductive cleavage the same degradation products belonging to the same family (sulphonamines, diamines), of identical size and identical physicochemical properties
g) Both substances have similar physicochemical properties.


In summary, it is considered that both substances have the same mode of action with regard to the following endpoints:

• Biodegradation
• Genetic toxicity
• Skin Sensitisation

2. Source and target chemicals (Purity / Impurities)
Read-across is possible provided that there is no impact of impurities on the toxicological properties of the target and source chemicals. Both substances have similar composition, same degree of purity and impurities are comparable structurally and in content.
The composition and impurities of the target and source substances are shown in Table 1
(see attached document in Section 13).

3. Analogue approach justification
As per available data, both substances, source and target, have similar structure, physicochemical properties, metabolism, mechanistic considerations and biological activity (predicted and empirical).
Therefore, read-across is an appropriate approach for the toxicity data gap endpoints to be filled.
3.1 Structural Similarity

Both substances, target and source, are considered structurally similar. Both are polysulphonates and consequently are polyanions. They are also polyaromatic substances and contain azo bonds. As a result of common starting materials used during their synthesis, both substances contain aromatic ring structures that contain sulfonated salt functional groups. The alkali metal salts are expected to dissociate in aqueous media and as a result the solubility of these compounds is increased.

3.2 Physicochemical Property Similarity

The physico-chemical properties of both substances are shown in Table 2.

Due to similar chemical structure, the source and target substances are similar with respect to relevant physicochemical properties. As the members of the sulfonated azo compounds group, both substances are solids (at room temperature) with low values of log Kow at expected pH in the small intestine.
In general, sulfonated azo compounds are expected to be ionized at physiological pH and over the pH ranges within the GI tract. Due to similar properties of volatility, solubility and reactivity among others for both substances, source and target, a similar bioavailability is expected.
3.3 Metabolic Similarity

The potential for metabolic reduction of the azo bond to yield aromatic amines is typically the determining factor in the genotoxic mode of action for azo type substances (Brown and De Vito 1993).
The similarity hypothesis of the analogue approach is based on the consideration that after oral intake, both azo direct dyes are metabolically reduced through the action of azoreductase of microflora in the intestine to release the related aromatic amines. The ability of the azo bond to be reduced for a particular substance is influenced by its solubility (Golka et al. 2004).
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 (Ollgaard at al. 1998).

The source and target chemicals are structurally very close molecules and the expected metabolites via breakdown of the azo linkage are the same:
- Benzene-1,2-4-triyltriamine, EC 210-443-2, CAS 615-71-4
- P-phenylendiamine, EC 203-404-7, CAS 106-50-3
- 3,4,6-Triamino-5-hydroxynaphthalene-2,7-disulfonica cid, CAS 69762-07-8

In conclusion, the potential for both substances to undergo metabolic azo reductions to aromatic amine metabolites is regarded as similar.

3.4 Mechanistic Similarity
Certain azo dyes are mutagenic after reductive cleavage of the azo linkage to their aromatic amine metabolites. The azo linkage is the most labile portion of an azo molecule and the potential for azo compounds to become mutagens is often determined by their ability to undergo enzymatic breakdown in mammalian organisms or micro-organisms. (Brown and DeVito 1993).




Cleavage of aromatic azo bond can yield aromatic amine metabolites that can potentially bind to DNA
leading to gene mutations.

Biodegradation

As seen in Table 1, there is a clear chemical similarity between the source substance Direct Black 155-NaKLi salt and the target substance Direct Black 155-Na salt. Similarity on physicochemical properties is shown in Table 2.
The target and source substances are soluble salts that will be rapidly dissociated in water to the anionic component (identical for both substances) and the corresponding cations.
The degradation pathways, speed and degradation products are expected to be nearly identical, as the functional groups and reactivity are the same and the physico-chemical properties are very similar.
Consequently, the behaviour in regards of biodegradation is considered to be very similar for both substances and the read-across is regarded as feasible for this endpoint.

Sensitisation

The source substance and the target one have identical chemical anionic structure but vary in the cationic part.
The physicochemical properties (water solubility, molecular weight, particle size, log Know, pKa) are also similar. Therefore, a similar toxicokinetic behaviour is expected. The substances are expected to be dissociated shortly after absorption to the same anionic component and the cationic free ions (Na+, Li+ and K+ for the source substance and Na+ for the target). The absorption, distribution, degradation products and excretion will be comparable.
As a result, the sensitisation potential is assumed to be very similar, or equal concerning the anionic part of the molecule.
However, information on potential skin-sensitizing effect caused by the cationic part has to be assessed.
Potassium is the chief cation in the intracellular fluid of muscle and other cells. Potassium ion is a strong electrolyte that plays a significant role in the regulation of fluid volume and maintenance of the water-electrolyte balance. Potassium is the major cation (positive ion) inside animal cells, while sodium is the major cation outside animal cells. The concentration differences of these charged atoms cause a difference in electric potential between the inside and outside of cells, known as the membrane potential. The balance between potassium and sodium is maintained by ion pumps in the cell membrane. The cell membrane potential created by potassium and sodium ions allows the cell generates an action potential—a "spike" of electrical discharge. The ability of cells to produce electrical discharge is critical for body functions such as neurotransmission, muscle contraction, and heart function. Potassium is also an essential mineral needed to regulate water balance, blood pressure and levels of acidity.
The normal levels of potassium in the human blood are between 3.6-5.2 mmol/L. Potassium is non-sensitizer, but values of potassium higher than 5.5 mmol/L are considered critical for health, causing kidney disease and heart problems.
Sodium ion is a naturally occurring cation in the body with a blood plasma concentration of 140 mmol/L. It is excreted with the urine and does not cause any toxic effects when administered in low concentrations. Sodium is not a skin sensitizer.
Lithium is readily absorbed from the gastrointestinal tract and excreted via the kidneys. Dermal absorption of the cation is insignificant. Lithium is not a skin sensitizer and not genotoxic but is known to cause nephro- and neurotoxicity and is used as psychiatric medication (3).
As a conclusion of the evaluation, the cationic part of the substances (Na+ or Li+, K+ and Na+) is not contributing significantly to the sensitization potential. The organic part is the main driver of the sensitization potential On the other hand, with the sensitisation studies conducted on the source substance CAS No. 2196165-14-5 Direct Black 155-NaKLi salt, the contribution to sensitisation of the K+, Li+ and Na+ together with the organic part of the substance was tested (Table3).
In conclusion, the only difference between both substances is the counter ion, and an increase of sensitizing effects due to the presence of higher content of Na+ in the target chemical CAS No. 68877-33-8 is not expected.
Consequently, the read across from the studies conducted on the source chemical CAS No. 2196165-14-5 is regarded as feasible for this endpoint.

Genetic toxicity
Both the source substance Direct Black RBK and the target substance Direct Black RBB are assumed to be rapidly dissociated in the blood to anionic components and free cations (Na+, K+, Li+ cations and Na+ cations, respectively), which are then readily available in the body.
- Contribution of the cations
The contribution of K+, Li+ and Na+ to genetic toxicity has been evaluated based on available information.
The genotoxicity of sodium chloride has been assessed from different sources considered in the “Genetic Toxicity Assessment: Employing the Best Science for Human Safety Evaluation Part VI: When Salt and Sugar and Vegetables Are Positive, How Can Genotoxicity Data Serve to Inform Risk Assessment” review(3). The conclusion is that “genotoxicity of sodium chloride is a conditional property, in that under certain conditions (high doses leading perturbations in osmotic strength) it can elicit a genotoxic response, while under a different set of conditions (lower dose levels) such a response is not induced nor is it biologically plausible.” The effects were seen at concentrations of 5 mg/mL in different in vitro studies.
The source of K+ is in the form of KCl (potassium chloride). In the SIDS Initial Assessment Report of KCl(4), “no gene mutations were reported in bacterial tests, with and without metabolic activation. However, high concentrations of KCl showed positive results in a range of genotoxic screening assays using mammalian cells in culture. The action of KCl in culture seems to be an indirect effect associated with an increased osmotic pressure and concentration. Therefore KCl, do not have any direct relevance in the intact body were such concentrations cannot occur.” The lowest concentration where effects were seen is 5.5 mg/mL a Chromosome aberration test.
In the Version 02; December 2020 of the CLH report of lithium compounds(5) it is stated that “In summary, lithium compounds have been tested for mutagenicity, chromosome aberrations, sister chromatid exchanges, DNA damage in a number of in vitro and in vivo studies. Mainly negative results were obtained, but positive results were also reported, usually at high cytotoxic doses. According to Lagerkvist and Lindell (2002) a possible explanation for the observation of genotoxic effects at higher doses may be increased cell survival, since lithium inhibits apoptosis by inhibiting the enzyme glycogen synthase kinase-3 (GSK3). However, an aneugenic potential of lithium salts could not be excluded considering positive results obtained in in vitro micronucleus test associated with an increase of kinetochore positive micronuclei and an increase of damage mitosis. Moreover, no micronucleus test was performed in vivo to investigate this aneugenic potential.”
Lithium is a trace element in the environment and is present in soil, fresh water, salt water and plants. Lithium is readily absorbed from the gastrointestinal tract and excreted via the kidneys and it is known to cause nephron- and neurotoxicity and is used as psychiatric medication.
Li concentrations in the blood of rats range from 0.03 to 0.05 mmol/L (Neiri et al, 2012)(7). In the disseminated information in the ECHA website, different studies conducted with LiCl were reported(8). In a 2-year drinking water study with Wistar rats (1958, OECD 452) animals with a Li+ plasma level up to 2 mmol/L were not distinguishable from control animals.
The systemic NOAEL value of 80 mg/kg bw for repeated dose and reproduction/developmental toxicity is stablished for Direct Black RBK based on read-across from the OECD 422 study conducted with the analogue source substance Direct Black 19 ([REACH&colours Kft]; 2012; OECD 422 ; 70% of DYE; GLP; K=1; NOAEL 80 mg/kg bw/day)(9). This results in a NOAEL of approximately 24 mg/rat (rats weight ca. 330 g). The rats blood volume is 60 mL/Kg with a haematocrit of 43% resulting in 34 mL plasma/Kg (Wistar rat, Delerenco et al, 2002)(10) or 11 mL/rat, respectively 11 g/rat (assuming a specific density of 1). This results in 24 mg target chemical/L plasma assuming the test substance is readily absorbed via the gastrointestinal tract (worst case) and well distributed.
The Li+ concentration in Direct Black RBK is ≤0.2% (w/w). Referring to the NOAEL of 80 mg/Kg bw, this would result in 0.023 mmol/L (Mw of Li: 6.94) plus the naturally occurring Li+ plasma concentration of 0.05 mmol/L (Neiri et al, 2012) in a final Li+ plasma concentration of 0.073 mmol/L.
However, since in this estimation clearance parameter are not considered, it must be assumed that actually the Li+ concentration in plasma would be significantly lower than 0.073 mmol/L due to excretion process. The potential Li+ concentration in plasma is therefore regarded to be well within the safe range considering the NOAEL of 2 mmol/L plasma in the 2 year study (8).
In view of the quantities of K+, Li+ and Na+ present in the composition of Direct Black RBK, it is not expected that there is an effect produced by the cations on the results of the in vitro studies conducted on the substance. On the other side, the higher quantity of Na+ present in the composition of the target substance Direct Black RBB (2.2% vs. 1.85% w/w in Direct Black RBK) is neither expected to have an effect on the genotoxicity potential of the target substance. The main driver for genetic toxicity response will be the organic part.
- Contribution of the anions
The toxicity of source and target substances is expected to be driven by the organic anionic parts.
The organic anions of the target and source substances are identical and therefore they will have the same behavior in regards of absorption, distribution and interaction in the body, with the same amine metabolites. The expected toxicity effects of both substances are regarded as very similar
The potential metabolites are amines, as a result of the reductive cleavage of the azo bonds. Generally, the aromatic amines are moderately to highly soluble in water (6.4–238000 mg/L) due to the presence of one or multiple solubilizing functional groups, such as the amino functional group. Most of the aromatic amines are weak bases (pKa values of less than 5.5) that will be protonated at low pH but will be found in their neutral form under environmentally relevant pH (7–9). Given their hydrophilicity and ionic character tend to have low to very low experimental log K and distribution coefficient (log D) values. In our case some of the degradation products have simultaneously the occurrence in the whole structure of amine and sulfonate, which usually favours a diminution of the toxicity in almost all the target organs or aquatic organisms.
Generally stated, genotoxicity is associated with all aromatic amines with benzidine moieties, as well as with some aromatic amines with toluene, aniline and naphthalene moieties. The toxicity of aromatic amines depends strongly on the spatial structure of the molecule or –in other words– the location of the amino-group(s). For instance, whereas there is strong evidence that 2-naphthylamine is a carcinogen, 1-naphthylamine is much less toxic. The toxicity of aromatic amines depends furthermore on the nature and location of other substituents. As an example, the substitution with nitro, methyl or methoxy groups or halogen atoms may increase the toxicity, whereas substitution with carboxyl or sulphonate groups generally lowers the toxicity. As most soluble commercial azo dyestuffs contain one or more sulphonate groups, insight in the potential danger of sulphonated aromatic amines is particularly important. In an extensive review of literature data on genotoxicity and carcinogenicity of sulphonated aromatic amines, it was concluded that sulphonated aromatic amines, in contrast to some of their unsulphonated analogues, have generally no or very low genotoxic and tumorigenic potential(3).
The available tests and literature on Benzene-1,2,4-triyltriamine (CAS 615-71-4) and CAS 615-47-4 (as HCl salt) show that there is a light positivity on strain TA98 and strain TA 1538iIn the Ames test, but this positivity seems to be proved wrong by the Mouse sperm morphology test and by the IARC evaluation on the metabolic precursor 2-nitro-para-phenylenediamine (CAS 5307-14-2).
The available tests on p-phenylenediamine conclude that the substance is not mutagenic, although the Ames test showed mutagenic effect in strain TA98 with metabolic activation.
Metabolite CAS 69762-07-8 is a derivative of H Acid (EC 226-736-4, CAS 5460-093 Sodium hydrogen 4-amino-5-hydroxynaphthalne-2,7-disulphonate). The H acid monosodium salt is registered under REACH and is not classified. Several azo-colourants permitted as food additives like E110 (Sunset Yellow), E122 (Azorubine), E123 (Amaranth), E124 (Ponceaux R), E129 (Allura Red), E151 (Brilliant Black), E154 (Brown FK), are based on naphthalene mono-di-sulphonic acids with amino and(or hydroxy derivatives and none of them gave concern for genotoxicity. Other derivatives with existing negative data on bacteria gene mutation are: acid red 131 (CAS 70210-37-6), Acid Red 249 (CAS 6416-66-6), Acid Red 252 (CAS 70209-97-1), Acid Violet 54 (CAS 70210-05-8) and others. The capability of sulphonation to eliminate the activation to carcinogenic products is noted by Jung et al (Jung, 1992) and is illustrated by the fact that a property of most permitted synthetic azo dyes is sulphonation on all component rings. The article describes the toxicological main principle metabolic pathway of sulphonation as natural detoxification phase II pathway in the liver. The general aim of sulphonation is to make the substrate more soluble in water and usually less active pharmacologically. Sulphonated molecules are more readily eliminated in bile and urine.

In conclusion, due to the high structural similarity and physico-chemical properties of the source and target substances, their toxicokinetic behaviour is regarded as highly similar. In the genetic toxicity studies conducted on the source substance CAS No. 2196165-14-5, Direct Black RBK, the contribution to the genotoxicity of the K+, Li+ and Na+ was tested together with the contribution of the organic part, which is the main driver of mutagenicity. The potential metabolites that may lead to a mutagenic effect are the same for both substances, and therefore, read-across from available mutagenicity studies with Direct Black 155_NaKLi salt is considered adequate to predict the same behaviour and results for Direct Black 155_Na salt, as shown in Table 4.

In view of the quantities of K+, Li+ and Na+ present in the composition of Direct Black 155-NaKLi salt, it is not expected that there is an effect produced by the cations on the results of the in vitro studies conducted on the substance. The main driver for genetic toxicity response will be the organic part.
On the other side, the higher quantity of Na+ present in the composition of the target substance Direct Black 155-Na salt (2.2% vs. 1.85% w/w in Direct Black 155-NaKLi salt) is neither expected to have an effect on the genotoxicity potential of the target substance.

In conclusion, the only difference between the source structure (CAS No. 2196165-14-5) and the target chemical sodium salt (CAS No. 68877-33-8) is the counter ion, and they are not expected to contribute and an increase or change of genotoxic effects due to the presence of Na+ in the target chemical CAS No. 68877-33-8 is not expected. Consequently, read across to the source chemical CAS No. 2196165-14-5 is regarded as feasible.


4. Data matrix
See document attached to Section 13.

5.Conclusions on analogue approach hypothesis, C&L and PBT/vPvB assessment

As it has been discussed above, the similarity between the source and target substances is very high: molecular structure, functional groups, degradation products, physico-chemical properties and toxicological data are comparable.
The solubility, partition coefficient and pKa are similar, as expected for substances that are so similar from a structural and chemical point of view. Therefore, it could be assumed that the biodegradation in water can be assessed from the source substance, Direct Black 155-NaKLi salt.
On the other side, the likelihood of absorption, interaction in the body, degradation pathways and metabolites are expected to be similar for the source and target substances and lead to similar toxicity.
- Results of skin irritation studies with the source and the target substance show comparable results, they are not skin irritants. They also show similar behaviour for acute toxicity. This similar behaviour and the close structural similarity are the basis for considering adequate to assess the sensitisation potential of Direct Black 155-Na salt from the available data on Direct Black 155-NaKLi salt.
- The results on acute toxicity tests with the source and the target substances are comparable (LD50 > 2000 mg/Kg bw). The repeated dose toxicity and screening for reproduction toxicity is assessed from another structural analogue: LD50 = 80 mg/Kg bw for repeated dose toxicity and LD50 ) 80 mg/Kgbw for developmental toxicity. Due to the analogue structures and chemistry, the assessment of mutagenicity of Direct Black 155-Na salt from the available studies with Direct Black 155-NaKLi salt is considered feasible.

C&L: source and target substances are not classified for acute toxicity and repeated dose toxicity, they are not skin irritants and are classified for eye corrosion hazard (Eye Dam 1).The source substance shows a skin sensitizing potential that triggers classification as Skin Sens. 1 and the same classification is applied to the target substance. The source substance was found positive for mutagenicity in an Ames test, while negative results were obtained in a chromosome aberration and a micronucleus test. The same behaviour is assumed for the target substance (read-across).
In addition to the in vitro studies, to conclude on C&L for Direct Black 155_Na salt and Direct Black 155_NaKLi salt, an available in vivo mutagenicity study with the analogue substance Direct Black 19 (CAS 6428-31-5) is taken into account. Direct Black 19 showed negative results in an in vivo mammalian erythrocyte micronucleus test (OECD 474). Based on the structural and chemical similarity, as well as the composition of Direct Black 19 and Direct Black 155 (Na and NaKLi salts), read-across from the in vivo mutagenicity study with Direct Black 19 is used to predict the same behaviour for Direct Black 155 (Na and NaKLi salts).
Based on the available information, Direct Black 155_Na salt and Direct Black 155_NaKLi are not classified as mutagenic.

Both substances are not PBT and not vPvB; they both have a low potential for bioaccumulation.
Reason / purpose for cross-reference:
read-across source
Key result
Parameter:
% degradation (TOC removal)
Remarks:
50 ppm initial TOC
Value:
74.8
Sampling time:
28 d
Key result
Parameter:
% degradation (TOC removal)
Remarks:
100 ppm initial TOC
Value:
47.3
Sampling time:
28 d
Interpretation of results:
inherently biodegradable
Conclusions:
The test item presents a percentage of biodegradation of 74.8% (50 ppm initial TOC) and 47.3% (100 initial TOC ppm) after 28 days of testing.
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
1. Hypothesis for the analogue approach
The target substance Direct Black 155_Na salt, CAS No. 68877-33-8, Disodium 4-amino-3-[[[(2,4-diaminophenyl)diazenyl]phenyl]diazenyl]-5-hydroxy-6-(phenyldiazenyl)naphthalene-2,7-disulfonate (Direct Black RBB) is defined as a mono-constituent substance.

The available toxicological data on this substance are insufficient to fulfil the data requirements for a REACH Annex VIII dossier.

In order to prevent unnecessary animal testing, the occurring data gaps on toxicity studies might be filled by applying read-across from the similar substance (source) Direct Black 155-NaKLi salt, CAS No. 2196165-14-5, Sodium, potassium, lithium 4-amino-3-[{4-[(2,4-diaminophenyl)diazenyl]phenyl}diazenyl]-5-hydroxy-6-[phenyldiazenyl]naphthalene-2,7-disulfonate (Direct Black RBK) which is also defined as a mono-constituent substance.

Both substances, target and source, have the same molecular structure. The only difference between the source structure Direct Black 155-NaKLi salt (CAS No. 2196165-14-5) and target Direct Black 155-Na salt (CAS No. 68877-33-8) is the counter ion.

Both substances are synthetized using the same raw materials and following the same manufacturing process. They are identical in relation to the anionic chemical structure.

CAS No. 2196165-14-5 is the result of the precipitation at the final stage of the reaction with potassium chloride and neutralization with lithium hydroxide and sodium hydroxide, whilst in CAS No. 68877-33-8 the precipitating agent is sodium chloride and the neutralization agent is sodium hydroxide.

The read-across is based on the hypothesis that the source and target substances have similar toxicological and environmental fate properties because both molecules have the following similarities:


a) Identical raw materials and manufacturing process.
b) Similar impurities, in comparable amounts.
c) Structural similarity: sulphonated molecules, aromatic rings, azo bonds.
Both dyes have identical anionic structure, the same polyaromatic structures polysulphonated, linked with azo bonds.
d) Both have the same ionic functional groups (sulphonic, amino, phenol).
The substances in a solid state are salts and in water solution at neutral pH are the same polyanions solvated with water.
e) Both have affinity to the same type of substrates/molecules.
The substances are able to be adsorbed on the same type of substance, e.g. polysaccharides (cellulose), polyphenols (lignine) and proteins.
f) Both may release by reductive cleavage the same degradation products belonging to the same family (sulphonamines, diamines), of identical size and identical physicochemical properties
g) Both substances have similar physicochemical properties.


In summary, it is considered that both substances have the same mode of action with regard to the following endpoints:

• Biodegradation
• Genetic toxicity
• Skin Sensitisation

2. Source and target chemicals (Purity / Impurities)
Read-across is possible provided that there is no impact of impurities on the toxicological properties of the target and source chemicals. Both substances have similar composition, same degree of purity and impurities are comparable structurally and in content.
The composition and impurities of the target and source substances are shown in Table 1
(see attached document in Section 13).

3. Analogue approach justification
As per available data, both substances, source and target, have similar structure, physicochemical properties, metabolism, mechanistic considerations and biological activity (predicted and empirical).
Therefore, read-across is an appropriate approach for the toxicity data gap endpoints to be filled.

3.1 Structural Similarity

Both substances, target and source, are considered structurally similar. Both are polysulphonates and consequently are polyanions. They are also polyaromatic substances and contain azo bonds. As a result of common starting materials used during their synthesis, both substances contain aromatic ring structures that contain sulfonated salt functional groups. The alkali metal salts are expected to dissociate in aqueous media and as a result the solubility of these compounds is increased.

3.2 Physicochemical Property Similarity

The physico-chemical properties of both substances are shown in Table 2.

Due to similar chemical structure, the source and target substances are similar with respect to relevant physicochemical properties. As the members of the sulfonated azo compounds group, both substances are solids (at room temperature) with low values of log Kow at expected pH in the small intestine.
In general, sulfonated azo compounds are expected to be ionized at physiological pH and over the pH ranges within the GI tract. Due to similar properties of volatility, solubility and reactivity among others for both substances, source and target, a similar bioavailability is expected.

3.3 Metabolic Similarity

The potential for metabolic reduction of the azo bond to yield aromatic amines is typically the determining factor in the genotoxic mode of action for azo type substances (Brown and De Vito 1993).
The similarity hypothesis of the analogue approach is based on the consideration that after oral intake, both azo direct dyes are metabolically reduced through the action of azoreductase of microflora in the intestine to release the related aromatic amines. The ability of the azo bond to be reduced for a particular substance is influenced by its solubility (Golka et al. 2004).
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 (Ollgaard at al. 1998).

The source and target chemicals are structurally very close molecules and the expected metabolites via breakdown of the azo linkage are the same:
- Benzene-1,2-4-triyltriamine, EC 210-443-2, CAS 615-71-4
- P-phenylendiamine, EC 203-404-7, CAS 106-50-3
- 3,4,6-Triamino-5-hydroxynaphthalene-2,7-disulfonica cid, CAS 69762-07-8

In conclusion, the potential for both substances to undergo metabolic azo reductions to aromatic amine metabolites is regarded as similar.

3.4 Mechanistic Similarity
Certain azo dyes are mutagenic after reductive cleavage of the azo linkage to their aromatic amine metabolites. The azo linkage is the most labile portion of an azo molecule and the potential for azo compounds to become mutagens is often determined by their ability to undergo enzymatic breakdown in mammalian organisms or micro-organisms. (Brown and DeVito 1993).
Cleavage of aromatic azo bond can yield aromatic amine metabolites that can potentially bind to DNA leading to gene mutations.

Biodegradation

As seen in Table 1, there is a clear chemical similarity between the source substance Direct Black 155-NaKLi salt and the target substance Direct Black 155-Na salt. Similarity on physicochemical properties is shown in Table 2.
The target and source substances are soluble salts that will be rapidly dissociated in water to the anionic component (identical for both substances) and the corresponding cations.
The degradation pathways, speed and degradation products are expected to be nearly identical, as the functional groups and reactivity are the same and the physico-chemical properties are very similar.
Consequently, the behaviour in regards of biodegradation is considered to be very similar for both substances and the read-across is regarded as feasible for this endpoint.

Sensitisation

The source substance and the target one have identical chemical anionic structure but vary in the cationic part.
The physicochemical properties (water solubility, molecular weight, particle size, log Know, pKa) are also similar. Therefore, a similar toxicokinetic behaviour is expected. The substances are expected to be dissociated shortly after absorption to the same anionic component and the cationic free ions (Na+, Li+ and K+ for the source substance and Na+ for the target). The absorption, distribution, degradation products and excretion will be comparable.
As a result, the sensitisation potential is assumed to be very similar, or equal concerning the anionic part of the molecule.
However, information on potential skin-sensitizing effect caused by the cationic part has to be assessed.
Potassium is the chief cation in the intracellular fluid of muscle and other cells. Potassium ion is a strong electrolyte that plays a significant role in the regulation of fluid volume and maintenance of the water-electrolyte balance. Potassium is the major cation (positive ion) inside animal cells, while sodium is the major cation outside animal cells. The concentration differences of these charged atoms cause a difference in electric potential between the inside and outside of cells, known as the membrane potential. The balance between potassium and sodium is maintained by ion pumps in the cell membrane. The cell membrane potential created by potassium and sodium ions allows the cell generates an action potential—a "spike" of electrical discharge. The ability of cells to produce electrical discharge is critical for body functions such as neurotransmission, muscle contraction, and heart function. Potassium is also an essential mineral needed to regulate water balance, blood pressure and levels of acidity.
The normal levels of potassium in the human blood are between 3.6-5.2 mmol/L. Potassium is non-sensitizer, but values of potassium higher than 5.5 mmol/L are considered critical for health, causing kidney disease and heart problems.
Sodium ion is a naturally occurring cation in the body with a blood plasma concentration of 140 mmol/L. It is excreted with the urine and does not cause any toxic effects when administered in low concentrations. Sodium is not a skin sensitizer.
Lithium is readily absorbed from the gastrointestinal tract and excreted via the kidneys. Dermal absorption of the cation is insignificant. Lithium is not a skin sensitizer and not genotoxic but is known to cause nephro- and neurotoxicity and is used as psychiatric medication (3).
As a conclusion of the evaluation, the cationic part of the substances (Na+ or Li+, K+ and Na+) is not contributing significantly to the sensitization potential. The organic part is the main driver of the sensitization potential On the other hand, with the sensitisation studies conducted on the source substance CAS No. 2196165-14-5 Direct Black 155-NaKLi salt, the contribution to sensitisation of the K+, Li+ and Na+ together with the organic part of the substance was tested (Table3).

In conclusion, the only difference between both substances is the counter ion, and an increase of sensitizing effects due to the presence of higher content of Na+ in the target chemical CAS No. 68877-33-8 is not expected.
Consequently, the read across from the studies conducted on the source chemical CAS No. 2196165-14-5 is regarded as feasible for this endpoint.

Genetic toxicity
Both the source substance Direct Black RBK and the target substance Direct Black RBB are assumed to be rapidly dissociated in the blood to anionic components and free cations (Na+, K+, Li+ cations and Na+ cations, respectively), which are then readily available in the body.
- Contribution of the cations
The contribution of K+, Li+ and Na+ to genetic toxicity has been evaluated based on available information.
The genotoxicity of sodium chloride has been assessed from different sources considered in the “Genetic Toxicity Assessment: Employing the Best Science for Human Safety Evaluation Part VI: When Salt and Sugar and Vegetables Are Positive, How Can Genotoxicity Data Serve to Inform Risk Assessment” review(3). The conclusion is that “genotoxicity of sodium chloride is a conditional property, in that under certain conditions (high doses leading perturbations in osmotic strength) it can elicit a genotoxic response, while under a different set of conditions (lower dose levels) such a response is not induced nor is it biologically plausible.” The effects were seen at concentrations of 5 mg/mL in different in vitro studies.
The source of K+ is in the form of KCl (potassium chloride). In the SIDS Initial Assessment Report of KCl(4), “no gene mutations were reported in bacterial tests, with and without metabolic activation. However, high concentrations of KCl showed positive results in a range of genotoxic screening assays using mammalian cells in culture. The action of KCl in culture seems to be an indirect effect associated with an increased osmotic pressure and concentration. Therefore KCl, do not have any direct relevance in the intact body were such concentrations cannot occur.” The lowest concentration where effects were seen is 5.5 mg/mL a Chromosome aberration test.
In the Version 02; December 2020 of the CLH report of lithium compounds(5) it is stated that “In summary, lithium compounds have been tested for mutagenicity, chromosome aberrations, sister chromatid exchanges, DNA damage in a number of in vitro and in vivo studies. Mainly negative results were obtained, but positive results were also reported, usually at high cytotoxic doses. According to Lagerkvist and Lindell (2002) a possible explanation for the observation of genotoxic effects at higher doses may be increased cell survival, since lithium inhibits apoptosis by inhibiting the enzyme glycogen synthase kinase-3 (GSK3). However, an aneugenic potential of lithium salts could not be excluded considering positive results obtained in in vitro micronucleus test associated with an increase of kinetochore positive micronuclei and an increase of damage mitosis. Moreover, no micronucleus test was performed in vivo to investigate this aneugenic potential.”
Lithium is a trace element in the environment and is present in soil, fresh water, salt water and plants. Lithium is readily absorbed from the gastrointestinal tract and excreted via the kidneys and it is known to cause nephron- and neurotoxicity and is used as psychiatric medication.
Li concentrations in the blood of rats range from 0.03 to 0.05 mmol/L (Neiri et al, 2012)(7). In the disseminated information in the ECHA website, different studies conducted with LiCl were reported(8). In a 2-year drinking water study with Wistar rats (1958, OECD 452) animals with a Li+ plasma level up to 2 mmol/L were not distinguishable from control animals.
The systemic NOAEL value of 80 mg/kg bw for repeated dose and reproduction/developmental toxicity is stablished for Direct Black RBK based on read-across from the OECD 422 study conducted with the analogue source substance Direct Black 19 ([REACH&colours Kft]; 2012; OECD 422 ; 70% of DYE; GLP; K=1; NOAEL 80 mg/kg bw/day)(9). This results in a NOAEL of approximately 24 mg/rat (rats weight ca. 330 g). The rats blood volume is 60 mL/Kg with a haematocrit of 43% resulting in 34 mL plasma/Kg (Wistar rat, Delerenco et al, 2002)(10) or 11 mL/rat, respectively 11 g/rat (assuming a specific density of 1). This results in 24 mg target chemical/L plasma assuming the test substance is readily absorbed via the gastrointestinal tract (worst case) and well distributed.
The Li+ concentration in Direct Black RBK is ≤0.2% (w/w). Referring to the NOAEL of 80 mg/Kg bw, this would result in 0.023 mmol/L (Mw of Li: 6.94) plus the naturally occurring Li+ plasma concentration of 0.05 mmol/L (Neiri et al, 2012) in a final Li+ plasma concentration of 0.073 mmol/L.
However, since in this estimation clearance parameter are not considered, it must be assumed that actually the Li+ concentration in plasma would be significantly lower than 0.073 mmol/L due to excretion process. The potential Li+ concentration in plasma is therefore regarded to be well within the safe range considering the NOAEL of 2 mmol/L plasma in the 2 year study (8).
In view of the quantities of K+, Li+ and Na+ present in the composition of Direct Black RBK, it is not expected that there is an effect produced by the cations on the results of the in vitro studies conducted on the substance. On the other side, the higher quantity of Na+ present in the composition of the target substance Direct Black RBB (2.2% vs. 1.85% w/w in Direct Black RBK) is neither expected to have an effect on the genotoxicity potential of the target substance. The main driver for genetic toxicity response will be the organic part.
- Contribution of the anions
The toxicity of source and target substances is expected to be driven by the organic anionic parts.
The organic anions of the target and source substances are identical and therefore they will have the same behavior in regards of absorption, distribution and interaction in the body, with the same amine metabolites. The expected toxicity effects of both substances are regarded as very similar
The potential metabolites are amines, as a result of the reductive cleavage of the azo bonds. Generally, the aromatic amines are moderately to highly soluble in water (6.4–238000 mg/L) due to the presence of one or multiple solubilizing functional groups, such as the amino functional group. Most of the aromatic amines are weak bases (pKa values of less than 5.5) that will be protonated at low pH but will be found in their neutral form under environmentally relevant pH (7–9). Given their hydrophilicity and ionic character tend to have low to very low experimental log K and distribution coefficient (log D) values. In our case some of the degradation products have simultaneously the occurrence in the whole structure of amine and sulfonate, which usually favours a diminution of the toxicity in almost all the target organs or aquatic organisms.
Generally stated, genotoxicity is associated with all aromatic amines with benzidine moieties, as well as with some aromatic amines with toluene, aniline and naphthalene moieties. The toxicity of aromatic amines depends strongly on the spatial structure of the molecule or –in other words– the location of the amino-group(s). For instance, whereas there is strong evidence that 2-naphthylamine is a carcinogen, 1-naphthylamine is much less toxic. The toxicity of aromatic amines depends furthermore on the nature and location of other substituents. As an example, the substitution with nitro, methyl or methoxy groups or halogen atoms may increase the toxicity, whereas substitution with carboxyl or sulphonate groups generally lowers the toxicity. As most soluble commercial azo dyestuffs contain one or more sulphonate groups, insight in the potential danger of sulphonated aromatic amines is particularly important. In an extensive review of literature data on genotoxicity and carcinogenicity of sulphonated aromatic amines, it was concluded that sulphonated aromatic amines, in contrast to some of their unsulphonated analogues, have generally no or very low genotoxic and tumorigenic potential(3).
The available tests and literature on Benzene-1,2,4-triyltriamine (CAS 615-71-4) and CAS 615-47-4 (as HCl salt) show that there is a light positivity on strain TA98 and strain TA 1538iIn the Ames test, but this positivity seems to be proved wrong by the Mouse sperm morphology test and by the IARC evaluation on the metabolic precursor 2-nitro-para-phenylenediamine (CAS 5307-14-2).
The available tests on p-phenylenediamine conclude that the substance is not mutagenic, although the Ames test showed mutagenic effect in strain TA98 with metabolic activation.
Metabolite CAS 69762-07-8 is a derivative of H Acid (EC 226-736-4, CAS 5460-093 Sodium hydrogen 4-amino-5-hydroxynaphthalne-2,7-disulphonate). The H acid monosodium salt is registered under REACH and is not classified. Several azo-colourants permitted as food additives like E110 (Sunset Yellow), E122 (Azorubine), E123 (Amaranth), E124 (Ponceaux R), E129 (Allura Red), E151 (Brilliant Black), E154 (Brown FK), are based on naphthalene mono-di-sulphonic acids with amino and(or hydroxy derivatives and none of them gave concern for genotoxicity. Other derivatives with existing negative data on bacteria gene mutation are: acid red 131 (CAS 70210-37-6), Acid Red 249 (CAS 6416-66-6), Acid Red 252 (CAS 70209-97-1), Acid Violet 54 (CAS 70210-EO05-8) and others. The capability of sulphonation to eliminate the activation to carcinogenic products is noted by Jung et al (Jung, 1992) and is illustrated by the fact that a property of most permitted synthetic azo dyes is sulphonation on all component rings. The article describes the toxicological main principle metabolic pathway of sulphonation as natural detoxification phase II pathway in the liver. The general aim of sulphonation is to make the substrate more soluble in water and usually less active pharmacologically. Sulphonated molecules are more readily eliminated in bile and urine.

In conclusion, due to the high structural similarity and physico-chemical properties of the source and target substances, their toxicokinetic behaviour is regarded as highly similar. In the genetic toxicity studies conducted on the source substance CAS No. 2196165-14-5, Direct Black RBK, the contribution to the genotoxicity of the K+, Li+ and Na+ was tested together with the contribution of the organic part, which is the main driver of mutagenicity. The potential metabolites that may lead to a mutagenic effect are the same for both substances, and therefore, read-across from available mutagenicity studies with Direct Black 155_NaKLi salt is considered adequate to predict the same behaviour and results for Direct Black 155_Na salt, as shown in Table 4.

In view of the quantities of K+, Li+ and Na+ present in the composition of Direct Black 155-NaKLi salt, it is not expected that there is an effect produced by the cations on the results of the in vitro studies conducted on the substance. The main driver for genetic toxicity response will be the organic part.
On the other side, the higher quantity of Na+ present in the composition of the target substance Direct Black 155-Na salt (2.2% vs. 1.85% w/w in Direct Black 155-NaKLi salt) is neither expected to have an effect on the genotoxicity potential of the target substance.

In conclusion, the only difference between the source structure (CAS No. 2196165-14-5) and the target chemical sodium salt (CAS No. 68877-33-8) is the counter ion, and they are not expected to contribute and an increase or change of genotoxic effects due to the presence of Na+ in the target chemical CAS No. 68877-33-8 is not expected. Consequently, read across to the source chemical CAS No. 2196165-14-5 is regarded as feasible.


4. Data matrix
See document attached to Section 13.


5.Conclusions on analogue approach hypothesis, C&L and PBT/vPvB assessment

As it has been discussed above, the similarity between the source and target substances is very high: molecular structure, functional groups, degradation products, physico-chemical properties and toxicological data are comparable.
The solubility, partition coefficient and pKa are similar, as expected for substances that are so similar from a structural and chemical point of view. Therefore, it could be assumed that the biodegradation in water can be assessed from the source substance, Direct Black 155-NaKLi salt.
On the other side, the likelihood of absorption, interaction in the body, degradation pathways and metabolites are expected to be similar for the source and target substances and lead to similar toxicity.
- Results of skin irritation studies with the source and the target substance show comparable results, they are not skin irritants. They also show similar behaviour for acute toxicity. This similar behaviour and the close structural similarity are the basis for considering adequate to assess the sensitisation potential of Direct Black 155-Na salt from the available data on Direct Black 155-NaKLi salt.
- The results on acute toxicity tests with the source and the target substances are comparable (LD50 > 2000 mg/Kg bw). The repeated dose toxicity and screening for reproduction toxicity is assessed from another structural analogue: LD50 = 80 mg/Kg bw for repeated dose toxicity and LD50 ) 80 mg/Kgbw for developmental toxicity. Due to the analogue structures and chemistry, the assessment of mutagenicity of Direct Black 155-Na salt from the available studies with Direct Black 155-NaKLi salt is considered feasible.

C&L: source and target substances are not classified for acute toxicity and repeated dose toxicity, they are not skin irritants and are classified for eye corrosion hazard (Eye Dam 1).The source substance shows a skin sensitizing potential that triggers classification as Skin Sens. 1 and the same classification is applied to the target substance. The source substance was found positive for mutagenicity in an Ames test, while negative results were obtained in a chromosome aberration and a micronucleus test. The same behaviour is assumed for the target substance (read-across).
In addition to the in vitro studies, to conclude on C&L for Direct Black 155_Na salt and Direct Black 155_NaKLi salt, an available in vivo mutagenicity study with the analogue substance Direct Black 19 (CAS 6428-31-5) is taken into account. Direct Black 19 showed negative results in an in vivo mammalian erythrocyte micronucleus test (OECD 474). Based on the structural and chemical similarity, as well as the composition of Direct Black 19 and Direct Black 155 (Na and NaKLi salts), read-across from the in vivo mutagenicity study with Direct Black 19 is used to predict the same behaviour for Direct Black 155 (Na and NaKLi salts).
Based on the available information, Direct Black 155_Na salt and Direct Black 155_NaKLi are not classified as mutagenic.

Both substances are not PBT and not vPvB; they both have a low potential for bioaccumulation.
Reason / purpose for cross-reference:
read-across source
Key result
Parameter:
% degradation (TOC removal)
Remarks:
10 ppm initial TOC
Value:
12.5
Sampling time:
28 d
Key result
Parameter:
% degradation (TOC removal)
Remarks:
30 ppm initial TOC
Value:
8
Sampling time:
28 d
Interpretation of results:
not readily biodegradable
Conclusions:
The test item presents a percentage of biodegradation of 12.5% (10 ppm initial TOC) and 8.0% (30 ppm Initial TOC) after 28 days of testing.

Description of key information

There are two studies available for the substance with CAS 2196165-14-5 (Direct Black RBK - Na, Li, K salt) done according to the OECD 301A (readily biodegradability) and OECD 302B (inherent biodegradability). This substance has the identical chemical molecular structure in respect to the anionic organic components than the substance with CAS 68877-33-8 (Direct Black RBB Na salt).

One study has been conducted according to the OECD 301A. The results show a degradation of the substance after 28 days of 12.5% (10 ppm Initial TOC) and 8.0% (30 ppm Initial TOC). In view of the results, we can conclude that the colorant Direct Black RBK does not have a rapid biodegradation in the specific conditions of the 301A guideline. Another study has been conducted according to the OECD 302B (Zahn-Wellens). The results show a degradation of the substance after 28 days of 74.8% (50 ppm of initial TOC) and of 47.3% (100 ppm of initial TOC). It is to say that at concentrations of ca. 50 ppm of initial TOC this substance has an inherent biodegradability of 75%. Based on these results the substance with CAS 2196165-14-5 (Direct Black RBK - Na, Li, K salt) has been considered as "inherently biodegradable".

Both substances CAS 2196165-14-5 (Direct Black RBK - Na, Li, K salt) and CAS 68877-33-8 (Direct Black RBB Na salt) have the same anionic structure, therefore it is expected the same biodegradability concerning this organic part. Whereas the counterion of the substance CAS 68877-33-8 (Direct Black RBB Na salt) is the sodium, the ones of the substance with CAS 2196165-14-5 (Direct Black RBK - Na, Li, K salt) are sodium, potassium and lithium. The inorganic cations do not contribute to the biodegradation of the substance and therefore, it is justified to consider that the substance with CAS 68877-33-8 will have at most the same degree of degradation of the substance with CAS 2196165-14-5, which is "inherently biodegradable".

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable
Type of water:
freshwater

Additional information