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Short-term toxicity to fish

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Reference
Endpoint:
short-term toxicity to fish
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 RBB (Direct Black 155_Na salt CAS 68877-33-8) 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 Direct Black 19 (CAS No. 6428-31-5), Disodium, 4-amino-3,6-bis{[4-[(2,4-diaminophenyl)diazenyl]phenylene]diazenyl}-5-hydroxy-naphthale-ne-2, 7 –disulfonate, named as the “source” substance.

The read-across is based on the hypothesis that source and target substances have similar toxicological and ecotoxicological properties because they 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 they 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 materials and products, 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 regards to the following endpoints:

- Mutagenicity
- Repeated dose toxicity and screening for reproductive toxicity
- Short-term toxicity to fish

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Both substances are synthetized from the same raw materials and following similar manufacturing processes.

Read across is possible based on the structural similarity of both substances:

(a) the organic molecular structure of Direct Black RBB (Direct Black 155) constitutes the main part of the organic molecular structure of Direct Black 19
(b) the structure of Direct Black 19 is found as impurity in Direct Black RBB (Direct Black 155)
(c) the structure of Direct Black RBB (Direct Black 155) is found as impurity in Direct Black 19

The composition and impurities of the target and source substances are shown in table 1 (see report attached 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 appropiate 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

Identified physicochemical properties for both substances are presented in the Table 2.
Due to similar chemical structure, these 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 logKow 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 similar 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


Mutagenicity
For the analogue approach justification, it is assumed that Direct Black 155_Na salt is rapidly dissociated in the blood to anionic components and free Na+. In analogy, the source chemical Direct Black 19 is expected to be dissociated shortly after absorption to anionic components and the cations Na+ are also assumed to be readily available in the body.
Sodium ion, present in both substances, 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. Therefore, the toxicity of the substances is expected to be driven by the organic anionic parts.
The organic anions of the target and source substances, very similar in structure, are expected to have a similar behavior in regards of absorption, distribution and interaction in the body, resulting similar toxicity effects.
In regards of the metabolites, 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 1538 in 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 o54 (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.

The available data on mutagenicity for both the source and target is presented in the Table 3.

The source and target chemicals showed positive result in in vitro gene mutation studies in bacteria while negative results were obtained in respective in vitro gene mutation study in mammalian cells.
The target chemical Direct Black 155-Na salt is assumed to show the same behaviour as the source substance (positive for Ames test, negative for in vitro gene mutation in mammalian cells) based on read-across from the analogue substance Direct Black 155-NaLiK salt. On the other side, Direct Black 155-NaKLi salt also resulted not clastogenic/non aneugenic to human lymphocytes in an in vitro micronucleus study, and the same behaviour is assumed for the target substance Direct Black 155-Na salt.
Negative results in two in vitro mammalian cell tests covering both mutation and clastogenicity/aneugenicity endpoints should be considered as indicative of absence of in vivo genotoxic or carcinogenic potential (Kirkland et al., 2014). However, appropriate in vivo mutagenicity studies shall be considered in case of a positive result in one of the genotoxicity studies with the assessed substance.
An in vivo mutagenicity study with Direct Black 19 is available. Due to their similarity, and analogue results in in vitro tests, read across from the existing in vivo mutagenicity study carried on the source substance Direct Black 19 is regarded as feasible for the assessment of in vivo mutagenicity of the target substance Direct Black 155-Na.

Short-term toxicity and screening for reproductive toxicity

As described, both substances, source and target, have similar structure, physicochemical properties, metabolism, mechanistic considerations and biological activity.
It is assumed that Direct Black 155_Na salt (RBB) is rapidly dissociated in the blood to anionic components and free cations. In analogy, the source chemical Direct Black 19 is expected to be dissociated shortly after absorption and the cations Na+ are also assumed to be readily available in the body.
The organic anions of the target and source substances, very similar in structure, are expected to have a similar behavior in regards of absorption, distribution and interaction in the body, resulting in a similar toxicity.
The available short-term toxicity studies show that both substances are not acute toxic. (DL50 oral > 2000 mg/Kg).
In conclusion, the available study of short-term toxicity and screening for reproductive toxicity with the source substance Direct Black 19 is considered appropriate to assess the same endpoints for the target substance Direct Black 155_Na salt (RBB).

Short-term toxicity to fish

The source and target substances have recognized molecular structure similarity. They are soluble in water and have similar partition coefficient values. They are not hydrolysable and not readily biodegradable. A low potential to cross biological membranes is expected, based on the octanol-water partition coefficients. As a consequence, a similar behavior of the source and the target substances is expected in regards of fate and distribution in the environment.
There are available toxicity data to Daphnia and to Lemna minor for both the target and source substances showing comparable effect levels that do not trigger classification. It is expected that the effects on fish caused by both substances will also be similar
The short-term toxicity to fish was assessed in a ISO 7340 study with Direct Black 19 and a value of LC50 > 1000 mg/l was determined.
Read across to Direct Black RBB is considered feasible, based on the high similarity of the chemical structure of source and target substance, similar physicochemical parameters and comparable results in regards of other aquatic toxicity studies.

4. DATA MATRIX
See RA justification attachemnt in Section 13.

5. CONCLUSIONS ON ANALOGUE APPROACH HYPOTHESIS, C&L AND PBT/vPvB

Due to similar physicochemical properties, chemical degradation products, biodegradation products, and toxicity of both the target and the source substances it is justified to do the read-across approach between them. Also, from a structural point of view, both are aromatic, sulphonated and azo compounds, with close physicochemical and toxicological properties.
Based on the structural similarities joint with the available experimental data, it can be assessed that the target chemical will have a similar human and aquatic toxicity effect than the source chemical.
It can be assumed that the repeated dose toxicity of Direct Black RBB (Direct Black 155 sodium salt, target substance) can be assessed from Direct Black 19 sodium salt (source substance) and the NOAEL for repeated dose toxicity is about 80 mg/kg bw.
In relation to the reproductive toxicity, the structure of both the target and source substances don’t favor a positive response. The output of the study for screening the reproductive toxicity of the source substance indicates a NOAEL of 80 mg/kg bw. It can be assumed then, that the reproductive toxicity of Direct Black RBB (Direct Black 155 sodium salt) (target substance) can be assessed from Direct Black 19 sodium salt (source substance) and the NOAEL for reproductive toxicity is about 80 mg/kg bw.
Finally, it can be assumed that the toxicity to fish of Direct Black RBB (Direct Black 155_Nai salt, target substance) can be assessed from Direct Black 19 sodium salt (source substance) and the LC50 (fish) is >1000 mg/L.

C&L
Based on the available test, in vitro and in vivo, no classification for mutagenicity is warranted under Regulation 1272/2008 for both, the source and target substances.
Based on the available test, no classification for repeated dose toxicity and for toxicity to Reproduction is warranted under Regulation 1272/2008 for both, the source and target substances.
The source and the target chemicals have similar aquatic toxicity data and they are not classified according to Regulation 1272/2008.

PBT/vPvB assessment
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
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Conclusions:
The test performed on the analogue substance follows the ISO 7340 and shows no toxicity on the test organisms. The obtained value LC50 > 1000 mg/L is taken as valid as justified in the document that for read-across.

Description of key information

There is one test conducted on the structural analogue substance Direct Black 19 done, that follows the ISO 7340 and shows no toxicity on the tests organisms. LC50 (48h) value is >1000 mg/L. Direct Black 19 and Direct Black RBB have similar structures as shown in read-across justification. The results of all the studies support the effect of the substance to fish and the read-across approach between both structures.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Dose descriptor:
LC50
Effect concentration:
> 1 000 mg/L

Additional information