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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

The value of NOAEL (No Observed Adverse Effect Level) for the REPRODUCTION and for DEVELOPMENT OF PUPS was established as 80 mg/kg body weight/day. 

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
2021 (update)
Justification for type of information:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The target substance Direct Black RBK (Direct Black 155_NaKLi salt CAS 2196165-14-5) 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-hydroxynaphthale-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 RBK (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 RBK (Direct Black 155)
(c) the structure of Direct Black RBK (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 attachment 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 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.


Mutagenicity
For the analogue approach justification, it is assumed that Direct Black RBK is rapidly dissociated in the blood to anionic components and free Na+, K+, Li+. 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. Li+ and K+ are present in low amounts and their contribution to mutagenicity is not considered relevant, as explained in the REACH registration dossier of Direct Black 155_Na salt (see report_PF12337D2021RA_RBK-to-RBB). 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 54 (CAS 70210-05-8) and others. The capacity 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.


Several studies on mutagenicity are available for the source and the target substances. The results are presented in Table 3.
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 studies in mammalian cells. On the other side, Direct Black 155-NaKLi salt also resulted not clastogenic/non aneugenic to human lymphocytes in an in vitro micronucleus study.
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-NaKLi.

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 RBK 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 Diect Black RBK.

Short-term toxicity to fish

The source and target substances have 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 be also 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 155_NaKLi salt (RBK) 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 attachment in section 13.


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

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 RBK (Direct Black 155 sodium, potassium lithium 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 RBK (Direct Black 155 sodium, potassium lithium 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_NaKLi 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
Dose descriptor:
NOAEL
Effect level:
80 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see summary of effects in the source endpoint
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
80 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
other: not specified
Reproductive effects observed:
not specified
Conclusions:
The values of NOAEL for the reproduction was established to be 80 mg/kg bw/day
Executive summary:

The test substance,Direct Black 19,was tested for reproduction and subacute toxicity using the OECD Test Guideline No. 422:Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, Adopted by the Council on March 22nd1996. This study is used as source for read-across to Direct Black 155 RBK.


 


Results


Repeated dose toxicity part of study:


Repeated oral administration ofDirect Black 19to rats by gavage at the dose levels 20, 80 and 320 mg/kg/daydid not cause any mortality. No negative treatment-related effects were detected during functional observation of animals.


Body weight, food consumption, water consumption, clinical status of animals, urinalysis, weight of organs, macroscopical and microscopical structure of organs were not seriously affected by treatment of the test substance at the dose level 80 mg/kg/day.Slight decrease of food conversionin males, change of one haematological parameter –prolonged tromboplastin timein females (above historical control),statistically significant decrease of ALP activityin males were recorded at the dose level 80 mg/kg/day.


 


Food consumption, water consumption, clinical status of animals, relative weight of organs and macroscopical structure of organs were not seriously affected by treatment of the test substance at the dose level80mg/kg/day. Decreased body weight incrementsat the end of application period in males,decreased food conversionin males,changes of some haematological parameters(increase of total leucocyte count – above historical control in females),differences in biochemical blood parameters(statistically significant decrease of urea concentration, decrease ALP and concentration of inorganic phosphorus in males, statistically increase of urea concentration in females),changes of absolute weight of liver(statistically significant decrease of absolute weight of liver in males and statistically significant increase of absolute weight of liver in females), increased occurrence ofmicroscopical changes of brain, cerebellum, spleen and thyroid gland in males (deposits of pigment), ofkidneys, spleen and thyroid glandin females (deposits of pigment) and ofheart(sporadical regressive changes in myocardium) were detected at the dose level80 mg/kg/day.


 


Growth of animalsat the dose level320mg/kg/daywas influenced by the test substance treatment (decrease of body weight increments or fall of body weight, food consumption and food conversion).Clinical status of animalsafter application was also influenced by the test substance treatment (red-coloured bedding, test-substance coloured excrements, reversible change of colour of skin and irreversible change of colour of visible mucous membranes).Haematological examination(delayed significant increase of platelets count - above historical control and change of differential leucocyte count in males; reversible increase of total leucocyte count and delayed significant increase of PT value, delayed increase of APTT value in females),blood biochemical examination(reversible statistically significant and dose dependent decrease of ALP activity and urea concentration in males; delayed statistically significant increase of urea, albumin, inorganic phosphorus and potassium ions concentration in males; irreversible significant increase of urea concentration – above historical control and decrease of total protein concentration, reversible increase of AST activity – above historical control in females, delayed statistically significant increase of ALP activity and inorganic phosphorus in females),urinalysis(change of colour of urine in males),biometry of organs(irreversible increase of relative weight of testes, reversible statistically significant and dose dependent decrease of absolute and relative weight of liver, delayed statistically significant increase of absolute and relative weight of adrenal glands and relative weight of spleen, reversible decrease of absolute weight of heart in males; irreversible statistically significant and dose dependent increase of absolute and relative weight of liver, reversible and dose dependent decrease of absolute and relative weight of thymus),gross examination of organs and tissues(irreversible change of colour of mucous membranes, muscles, brain, spleen, thyroid gland, reversible change of colour of skin, subcutis and peritoneum in males; irreversible change of colour of mucous membranes, muscles, spleen, thyroid gland, reversible change of colour of skin, subcutis, brain, organs of thoracic and abdominal cavity and peritoneum, reducement of thymus in females) andhistological examination of organs and tissues(irreversible regressive changes in myocardium accompanied by reparative fibrosis and accumulation of pigmentophages in males and females, irreversible occurrence of pigment in brain, cerebellum, heart, spinal cord, kidneys, spleen and thyroid gland in males and females, focal atrophic or necrotic changes in skeletal muscle of females; reversible dystelectasis in lungs and activation of white pulp in spleen of males; reversible inflammation or necrosis of muscle fibres in larynx, atrophy of cortex in thymus in females) of animals at the dose level320mg/kg/dayrevealed significant changes attributable to the test substance administration.    


 


Reproduction part of study:


The course of mating, pregnancy and lactation of parental animals, number of femalesachieving pregnancy and bearing live pups, weights of reprodutive organs and pituitary gland, spermiogenesis and sperm parameters, macroscopical and microscopical structure of reproductive organs and pituitary gland of parental animals, number of pre-implantation, post-implantation and post-natal losses of mothers and number, weight, sex ratio and development of pups were not adversely affected by the test substance treatment at the dose levels20 and 80 mg/kg/day.


The course of mating, pregnancy and lactation of parental animals, number of females achieving pregnancy, absolute weight of reproductive organs and pituitary gland and relative weight of pituitary gland, spermiogenesis and sperm parameters, macroscopical and microscopical structure of reproductive organs and pituitary gland of parental animals, number of post-implantation and post-natal losses of mothers, sex ratio and development of pups were not adversely influenced by the test substance treatment at the dose level320 mg/kg/da


Evaluation of relative weights of reproductive organs(increase of relative weight of testes and epididymis),examination of number of pups(decrease of the total number of live pups and mean number of pups per litter),calculation of reproduction parameters(decreased number of females bearing live pups, increased number of stillborn pups),calculation of pre-implantation losses(decreased number of corpora lutea and uterus implantations) and evaluation of pup weights (decrease of mean litter weight, decrease of mean pup weight on day 4 after parturition) in animals of the dose level 320 mg/kg/day revealed adverse effects attributable to test substance.


 


Conclusion


Administration of the test substance,Direct Black 19, had not adverse effect on mortality, parameters of functional observation and on some reproduction parameters - course of mating, pregnancy and lactation, weights of reproductive organs and pituitary gland, spermiogenesis, macroscopical and microscopical structure of reproductive organs and pituitary gland of parental animals, number of post-implantation and post-natal losses of mothers, sex ratio and development of pups.  


Test-relatedreduction in body weight gains(reduced body weight increments in both sexes, fall of body weight in females, decreased food consumption and conversion in both sexes) were noted at the dose of 320 mg/kg/day. Somebiochemical parameters(changes of enzymes activity, urea, inorganic phosphorus, total protein and albumin concentration) and biometry of organs(changes of thymus, heart and spleen weight) were significantly changed especially in animals of the highest dose level


 


Theheartappeared to be atarget organfollowing repeated oral exposure of the test substance,Direct Black 19, at the highest dose level. Serious irreversible regressive lesions accompanied by accumulation of pigment were diagnosed in myocardium. In addition, similar changes were observed in muscle fibers of larynx andskeletal muscle. The above mentioned findings tended to be more serious in females than in males. The test had influence on macroscopical and microscopical structure of some other organs and tissues.


Irreversible accumulation of pigmentin brain, cerebellum, spinal cord, spleen, thyroid gland and kidneys was detected in animals of the middle and the highest dose level. Atrophic changes occurred in thymus of females at the highest dose level.


 


Daily oral administration of the test substance at the dose level 320 mg/kg/day also affected thenumber of live pups(decrease of the number of females bearing live pups, decrease of total number of live pups and mean number of pups per litter),early prenatal development of organism in uterus(increased pre-implantation losses),growth of pups(decreased mean litter weight, decreased mean pup weight on day 4 after parturition). However, male ability to produce sperm that can fertilise eggs and female ability to impregnate was not significantly changed.


     


The value of NOAEL (No Observed Adverse Effect Level) for REPEATED DOSE TOXICITY was established as 80 mg/kgbody weight/day both for MALES and FEMALES.


 


The value of NOAEL (No Observed Adverse Effect Level) for the REPRODUCTION and for DEVELOPMENT OF PUPS was established as 80 mg/kg body weight/day.  

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
80 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Justification for selection of Effect on fertility via oral route:

The submitted study completely assess the endpoint and is compliant with the official guideline.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
80 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Justification for selection of Effect on developmental toxicity: via oral route:

The submitted study completely assess the endpoint and is compliant with the official guideline.

Justification for classification or non-classification

No classification for toxicity to reproduction is warranted under Regulation 1272/2008.

Additional information