Disodium 7-amino-4-hydroxy-3-[[4-[(4-sulphonatophenyl)azo]phenyl]azo]naphthalene-2-sulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

There are’nt any available studies on humans for this endpoint.

The results of in vitro studies are negative:

- One Ames test (OECD 471, GLP compliant); no toxicity was observed.

- One In vitro Mammalian Chromosome Aberration test (OECD 473, GLP compliant)

- One In vitro Mammalian Cell Gene Mutation Test (OECD 476, GLP compliant) conducted on a analogue substance (read-across)

A QSAR prediction for mutagenicity gave negative results

All available in vitro tests in genetic toxicity (GLP compliant and scored Klimishch 1) showed negative results. Thus there is no need to carry out in vivo studies in genetic toxicity. There is no reason to believe that the negative results would not be relevant to humans.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

November 2006 to January 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Trinova Biochem GmbH (35394 Giessen, Germany)
- Suitability of cells: Regular checking of the properties of the strains regarding the membrane permeability and ampicillin resistance as well as spontaneous mutation rates is performed in RCC Cytotest Cell Research according to B. Ames and D. Maron and B. Ames. In this way it was ensured that the experimental conditions set down by Ames were fulfilled.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: The strain cultures were stored as stock cultures in ampoules with nutrient both + 5% DMSO in liquid nitrogen.

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Trinova Biochem GmbH (35394 Giessen, Germany)
- Suitability of cells: Regular checking of the properties of the strains regarding the membrane permeability and ampicillin resistance as well as spontaneous mutation rates is performed in RCC Cytotest Cell Research according to B. Ames and D. Maron and B. Ames. In this way it was ensured that the experimental conditions set down by Ames were fulfilled.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: The strain cultures were stored as stock cultures in ampoules with nutrient both + 5% DMSO in liquid nitrogen.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

No precipitation of the test item occurred up to the highest investigated dose

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: the solvent was chosen because of its solubility porperties

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine, 4-NOPD

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

The following materials were mixed in a test tube and poured onto the selective agar plates:
100 µL test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control)
500 µL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation)
100 µl Bacteria suspension (cf. test system, pre-culture of the strains)
2000 µL overlay agar

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
To evaluate the toxicity of the test item a pre-experiment was performed with strains TA 1535, TA 1537, TA 98, TA 100 and WP2 uvrA. Eight concentrations were tested for toxicity and mutation induction with three plates each. The experimental conditions in this pre-experiment were the same as described for the plate incorporation test.
Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.
- Any supplementary information relevant to cytotoxicity:
In the pre-experiment the concentration range of the test item was 3-5000 µg/plate. The pre-experiment is reported as experiment I since no relevant toxic effects were observed and 5000 µg/plate were chosen as maximal concentration

The concentration range included two logarithmic decades. The following concentrations of the active ingredient were tested in experiment II: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

DURATION
- Preincubation period: In the pre-incubation assay 100 µL test solution, 500 µL S9 mix/S9 mix substitution buffer and 100 µL bacterial suspension were mixed in a test tube and shaken at 37ºC for 60 minutes. After pre-incubation 2.0 mL overlay agar (45ºC) was added to each tube. The mixture was poured on selective agar plates.
- After solidification the plates were incubated upside down for at least 48 hours at 37ºC in the dark.

NUMBER OF REPLICATIONS: 3 plates for each strain and dose level

COUNTING
The colonies were counted using the Petri Viewer Mk2 wih the software program Ames Study Manager. Due to intense colour of the test item the revertant colonies were partly counted manually.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not cconsidered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: In experiment strain TA 1535 showed irregular background growth in the control plates, therefore strain TA 1535 had to be repeated with and without metabolic activation

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshits in hte genome of the strains used.
Therefore, the test item is considered to be non-mutagenic in this Salmonella Typhimirium and Eschirichia coli reverse mutation assay.

Executive summary:

The study was performed to investigate the potential of the test item to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella Typhimirium strains TA 1535, TA 1537, TA 98 and TA 100, and Eschirichia colin strain WP2 uvrA.

The assay was performed in five independent experiments. Pre-Experiment/Experiment I, and Experiment II and IIA were performed with and without liver microsomal activation. Pre-Experiment/Experiment I A was performed with metabolic activation, only. Each concentration, including the controls, was tested in triplicate. Since a wrong stock solution was prepared in Experiment II A (results are not reported) this part was repeated (Experiment II B) and was reported as part of experiment II. The test item was tested at the following concentrations of the active ingredient:

Pre-Experiment/EXperiment I and IA: 3; 10; 33; 100; 1000; 2500; and 5000 µg/plate

Experiment II and II B: 10; 33; 100; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.

No toxics effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metablilc activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix).

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

October 1989 to January 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted May 26, 1983

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian cell transformation assay

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: LMP; D-6100 Darmstadt (Germany)

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
Stocks of V79 cell line are stored in liquid nitrogen in hte cell bank of the laboratory, allowing the repeated use of the same cell culture batch in experiments.
Thawed stock cultures are propagated at 37°C in 80 cm2 plastic flasks (GREINER, D-7440 Niirtingen, F.R.G.). Seeding is done with about 5 X 10^ cells per flask in15 ml of MEM (minimal essential medium; SEROMED; D-1000 Berlin, F.R.G.) supplemented with 10 % fetal calf serum (FCS; Boehringer Mannheim, D-6800 Mannheim, F.R.G.). The cells are subcultured twice weekly. The cell cultures are incubated at 37°C and 4.5 % carbon dioxide atmospher
- Periodically checked for Mycoplasma contamination: before freezing, each batch of V79 is screened for mycoplasma contamination.
- Periodically checked for karyotype stability: before freezing, each batch og V79 is screened for karyotype stability
- Periodically 'cleansed' against high spontaneous background: not specified

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

PRE-EXPERIMENT FOR TOXICITY
The toxicity of the test article was determined in a pre-experiment in order to establish a concentration dependent plating efficiency relationship. The experimental conditions in this preexperiment were the same as described below for the experiment.
Toxicity of the test article was evidenced by a reduction in plating efficiency.

DOSE SELECTION
According to the results from this pre-experiment six concentrations (18 h interval) to be applied in the chromosomal aberration assay were chosen.
The highest dose level used was 10 mM unless limited by the solubility of the test article or that producing some indication of cytotoxicity (reduced plating efficiency and/or partial inhibition of mitosis).
If toxic effects were produced the highest dose level should reduce the plating efficiency to approximately 20-50 %. In addition, this concentration should suppress if possible mitotic activity (% cells in mitosis) by approximately 50 %, but not so great a reduction that insufficient scorable mitotic cells can be found.

Treatment was performed with the following concentrations:

without S9 mix:
7 h: 3.0; 4.0; 5.0; 6.0 mg/ml
18 h: 0.3; 1.5; 3.0; 4.0; 5.0; 6.0 mg/ml
28 h: 3.0; 4.0; 5.0; 6.0 mg/ml

with S9 mix:
7 h: 3.0; 4.0; 5.0; 6.0 mg/ml
18 h: 0.3; 1.5; 3.0; 4.0; 5.0; 6.0 mg/ml
28 h: 3.0; 4.0; 5.0; 6.0 mg/ml

According to the criteria mentioned above one (7 h and 28 h) and three concentrations (18 h) were selected to evaluate metaphases for cytogenetic damage.
In the pre-experiment for toxicity the colony forming ability of the V79 cells was reduced after treatment with the highest concentration . Higher concentrations than 6.0 mg/ml were not applied because of the 10 mM limitation.
In the main experiment, cultures after treatment with 6.0 mg/ml as highest concentration were evaluated for cytogenetic damage.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: no data
- Justification for choice of solvent/vehicle: ---

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Seeding of the Cultures
Two days old logarithmically growing stock cultures more than 50 % confluent were trypsinised and a single cell suspension was prepared. The trypsin concentration was 0.2 % in Ca-Mg-free salt solution (Trypsin: Difco Laboratories, Detroit, USA).
The Ca-Mg-free salt solution was composed as follows (per litre): NaCl 8000 mg, KCl 400 mg, Glucose 1000 mg, NaHCO3 350 mg.
The cells were seeded into Quadriperm dishes (Heraeus, D-6450 Hanau, F.R.G.) which contained microscopic slides (2 chambers per dish and test group). In each chamber 5 x 10E4 - 1 x 10E5 cells were seeded with regard to preparation time. The medium was MEM + 10 % FCS.

Treatment
After 4 8 h ( 7 h , 28h preparation interval) and 55 h (18 h preparation interval) the medium was replaced with serum-free medium containing the test article, either without S9 mix or with 20 |il/ml S9 mix. After 4h this medium was replaced with normal medium after rinsing twice with "saline G".
The "saline G" solution is composed as follows (per litre): NaCl 8000 mg, KCl 400 mg, Glucose 1100 mg, Na2HPO4*7H2O 290 mg, KH2PO4 150 mg and pH adjusted to 7.2
All incubations were done at 37° C in a humidified atmosphere with 4.5 % CO2.METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

Preparation of the Cultures
5, 15.5 and 25.5 h after the start of the treatment colcemid was added (0.2 µg/ml culture medium) to the cultures. 2.0 h (7 h interval) or 2.5 h later, (18 h and 28 h interval) the cells were treated on the slides in the chambers with hypotonic solution (0.4 % KCI) for 20 min at 37° C. After incubation in the hypotonic solution the cells are fixed with 3 + 1 absolute methanol + glacial acetic acid. All two slides per group were prepared. After fixation the cells were stained with giemsa (Merck, D-6100 Darmstadt, F.R.G.)

Analysis of Metaphase Cells
Evaluation of the slides were performed using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. At least 100 well spread metaphases per slide were scored for cytogenetic damage on coded slides. Only metaphases with characteristic chromosome number of 22 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined. In addition, the number of polyploid cells (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype) is scored.

Rationale for test conditions:

The chromosomal aberration assay is considered acceptable if it meets the following criteria:
a) the number of aberrations found in the negative and/or solvent controls fall within the laboratory historical control data range: 0.00 % - 4.00 %.
b) the positive control substances should produce significant increases in the number of cells with structural chromosome aberrations.

Evaluation criteria:

A test article is classified as mutagenic if it induces either a significant dose-related increase in the number of structural chromosomal aberrations or a significant positive response for at least one of the test points.
A test article producing neither a significant dose-related increase in the number of structural chromosomal aberrations nor a significant positive response at any one of the test points is considered non-mutagenic in this system.
This can be confirmed by means of the chi-square test. However, both biological and statistical significance should be considered together.

Statistics:

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the chi-square test. Evaluation was performed only for cells carrying aberrations exclusive gaps.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test article did not' induce structural chromosome aberrations as determined by the chromosomal aberration test in the V79 Chinese hamster cell line.
Therefore, the test item is considered to be non-mutagenic in this chromosomal aberration test.

Executive summary:

The test item was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro.

Preparation of chromosomes was done 7 h (high dose), 18 h (low, medium and high dose), and 28 h (high dose) after start of treatment with the test article. The treatment interval was 4 h.

In each experimental group two parallel cultures were used. Per culture 100 metaphases were scored for structural chromosomal aberrations.

The following dose levels were evaluated:

	without S9 mix	with S9 mix
7h	6.0 mg/ml	6.0 mg/ml
18h	0.3; 5.0; 6.0 mg/ml	0.3; 5.0; 6.0 mg/ml
28h	6.0 mg/ml	6.0 mg/ml

The concentration range of the test article applied had been determined in a pre-experiment using the plating efficiency assay as indicator for toxicity response.

Treatment with 6.0 mg/ml (ca. 10 mM) reduced the plating efficiency of the V79 cells. Also the mitotic index was reduced after treatment with the highest concentration at fixation intervals 7 h and 18 h in the presence of S9 mix.

There was no relevant increase in cells with structural aberrations after treatment with the test article at any fixation interval either without or with metabolic activation by S9 mix. Appropriate reference mutagens were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Guideline:

other: REACH Guidance on QSARs R.6

Principles of method if other than guideline:

Specific QSAR models developed for dyes. See technical documentation.

Key result

Genotoxicity:

negative

Remarks on result:

no mutagenic potential (based on QSAR/QSPR prediction)

Conclusions:

QSAR prediction: negative

Executive summary:

Applicability domain: in applicability domain

QSAR prediction: negative

Reliability score: 1.00

Number of similar structures: 1

Average similarity to nearest structures: 1.00

Occurrence frequency Negative: 1.00

Benigni Bossa alerts: none

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The target substance CAS 6300-50-1; EC 228-589-1; disodium 7-amino-4-hydroxy-3-[[4-[(4-sulphonatophenyl)azo]phenyl]azo]naphthalene-2-sulphonate 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 with CAS 1325-54-8, a substance of two constituents:
Tetrasodium 5-[[4-[(4-sulfonatophenyl)diazenyl]phenyl]diazenyl]-2-[2-[2-sulfonato-4-[[4-[(4-sulfonatophenyl)diazenyl]phenyl]diazenyl]phenyl]ethenyl]benzenesulfonate (53%) and
Hexasodium 2-((E)-2-sulfonato-4-((E)-(3-sulfonato-4-((E)-2-sulfonato-4-((E)-(4-((E)-(4- sulfonatophenyl)diazenyl)phenyl)diazenyl)styryl)phenyl)diazenyl)styryl)-5-((E)-(4-((E)-(4- sulfona-tophenyl)diazenyl)phenyl)diazenyl)benzenesulfonate (24%)
The read-across is based on the hypothesis that source and target substances have similar toxicological properties because both molecules have the following similarities:
 
a) Structural similarity: sulphonated molecules, aromatic rings, azo bonds.
Both dyes have polyaromatic structures polysulphonated, linked with azo bonds.

b) Both have ionic functional groups (sulphonic, amino, phenol).
The substances in a solid state are sodium salts and in water solution at neutral pH are polyanions solvated with water.
 
c) 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.
 
d) Their degradation products belong to the same family (sulphonamines, diamines), of similar size and similar physicochemical properties.
Based on the similarity analysis part of the project “Study of potential toxicity of dyes by read-across approach and QSAR modelling” developed by the Milano Chemometrics and QSAR Research Group, Dept. Earth and Environmental Sciences, University Milano-Bicocca in Italy, both substances are considered analogues on potential main metabolites.
The result of this similarity analysis is that the source and the target substances have two (2) common potential metabolites and each of them a distinct one.
 
 
In summary, it is considered that both substances have the same mode of action with regard to the following endpoints:
 
- repeated dose toxicity and reproductive/developmental toxicity (screening)
- mutagenicity


2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)

Read across is possible provided that there is no impact of impurities on the toxicological properties of the target and source chemicals. For both, impurities are comparable.

The composition and impurities of the target and source substances are shown in table 1 of the attached document to this record.

3. ANALOGUE APPROACH JUSTIFICATION
Both molecules, source and target are polysulphonates and consequently are polyanions. They are also polyaromatic substances and contain azo bonds. These are important traits of the molecule that determine its physicochemical properties, like solubility and partition coefficient n-octanol/water.
Such properties also play a critical role in determining the environmental fate of substances (including their potential for long-range transport), as well as their toxicity to humans and other life organisms.
Several physical and chemical properties--namely, melting point, water solubility, size, log Kow, Vapour pressure, Henry’s Law constant and acid dissociation constant (pKa)--are important in terms of ecological and human health assessment.
No experimental data on absorption, distribution and excretion is available for the target substances, but a few pieces of information are available for the target substance and the hydrolysis products. Sulphonates display low toxicity in general, but presence of other functional groups may increase its toxicity. Mathematical models were applied to the structures in order to study its toxicokinetics based on physicochemical properties. The bioavailability of both substances has been estimated with Advanced Chemistry Development, Inc. (ACD/Labs). The probability that target compound has % absorption (Oral) > 30% is 0.033; % absorption(Oral) > 70% is 0.008, and the probability that source compound has % absorption (Oral) > 30% is 0.033; % absorption(Oral) > 70% is 0.011(5). Therefore, it has to be expected a similar behaviour in life organisms and environment for both substances. On another hand, being both polyanions, their capability of crossing the life organisms’ membranes will be very limited.
Results from a combined repeated dose toxicity with the reproduction/developmental toxicity screening test (OECD 422) is available for the source substance. Effects at 300 mg/kg bw/day were considered not to represent an adverse effect. Therefore, a ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was considered to be 300 mg/kg bw/day (target organ: urogenital system, kidney). The predicted health effects using ACD/Labs(6) of the source substances confirms that the target organs will be the kidneys. One could expect that the target substance would behave similarly, with a NOAEL value in the same range. In the same test there were no treatment-related effects detected on the reproductive parameters investigated, therefore the ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 750 mg/kg bw/day. In another light, the prediction of the endocrine disrupting properties of both substances was calculated with ACD/Labs(7). The results were as “no binding to Estrogen Receptor alpha (LogRBA<-3)”, and the probability of Estrogen Receptor Binding is LogRBA > -3: 0.01 and LogRBA > 0: 0 for source substance and the probability of Estrogen Receptor Binding is LogRBA > -3: 0.02 and LogRBA > 0: 0 for target substance. Again, one could expect that the target substance would behave similarly, with a NOEL value in the same range.
Other experimental data obtained with the source and target substances indicate that both substances have low oral and dermal acute toxicity (LD50>2000 mg/kg bw). The substances are not irritating to skin and eye, are not sensitising, and are negative in many genotoxicity tests.
The degradation products of both molecules are the same ones or quite similar. p-Phenylendiamine and 4-aminobenzene sulphonic acid are common predicted reductive cleavage products. Each target and source have a distinct degradation product: a substituted naphthalene sulphonate for the target substance and a stilbene disulphonated for the source substance. All these structures are of similar size and similar physicochemical properties.
Generally, the aromatic amines are moderately to highly soluble (6.4–238000 mg/L) in water 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).
Neither the substances nor the amines that may release by reductive cleavage from both of them, are included in the lists of “Azodyes which can be split into carcinogenic amines pursuant to TRGS 614 (March 2001 edition)” Association TEGEWA e.V. (Germany) (9)(10) or in the “Report on Carcinogens, 14th edition” from the EPA - NTP (National Toxicology Program). 2016.(11) or in the Candidate List of substances of very high concern for Authorisation, published in accordance with Article 59(10) of the REACH Regulation. (12)


4. DATA MATRIX
Two data matrix are included in the attached document to this record: Matrix 1 (Toxicity data on the source and target substance) and Matrix 2 (Main potential metabolites data)

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Following the criteria as described Annex 1of Regulation (EC) No. 1272/2008, and based on the available data, the substance is not classified for genetic toxicity.