[x Sodium, y Potassium, z Lithium, x+y+z =2] 4-amino-3-[[[(2,4-diaminophenyl)diazenyl]phenyl]diazenyl]-5-hydroxy-6-(phenyldiazenyl)naphthalene-2,7-disulfonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro mutagenicity tests are available for Direct Black RBK.

Bacterial reverse mutation assay, Ames test, showed positive results both with and without metabolic activation.

The in vitro gene mutation test, mammalian cell gene mutation assay (OECD 476), did not show any mutagenic effect.

The in vitro Micronucleous Test in Human Lymphocytes (OECD 487), did not show any clastogenic and aneugenic to human lymphocytes effect.

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).  

Other tests, specially in vivo, should be considered to clarify this possible hazard in Direct Black RBK.

The analogue substance Direct Black 19 showed negative results in an in vivo micronucleous assay, chromosome aberration. This study is used as source to read-across to Direct Black RBK.

 

Based on these test results, the Direct Black RBK is considered not mutagenic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April May 2018

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:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Principles of method if other than guideline:

Ames plate pre-incubation method (Prival and Mitchell modification)

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, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Hamster liver homogenate metabolizing system (30% liver S9 in modified co-factors)

Test concentrations with justification for top dose:

The test item was tested using the following method. The maximum concentration was 5000 µg/plate (the OECD TG 471 maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the Prival and Mitchell method for testing azo dyes in the presence of flavin mononucleotide and hamster liver S9.

Vehicle / solvent:

Sterile distilled water ; Supplier: BAXTER; Batch number, (purity), expiry: 17H09BB1A, (N/A), expiry Jul 2020.
The test item was fully soluble in sterile distilled water at 50 mg/mL in solubility checks performed in-house. Sterile distilled water was therefore selected as the vehicle.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

congo red

mitomycin C

other: 2-Aminoanthracene (2AA) and 1,8-Dihydroxyanthraquinone (DAN)

Details on test system and experimental conditions:

Test for Mutagenicity: Experiment 1 – The Prival-Mitchell Modification to the Ames Test
The Prival-Mitchell modification to the standard Ames Test is necessary for the testing of azo dyes which can contain mutagenic aromatic amines which are not readily detected using the standard method (Prival and Mitchell (1982)). The modification differs in five key areas from the standard plate incorporation Ames Test:
• Uninduced hamster liver S9 rather than induced rat liver S9.
• 0.15 mL of S9 rather than the maximum of 0.05 mL of S9 in the standard Ames Test.
• The use of flavin mononucleotide (FMN), nicotinamide adenine dinucleotide (NADH), four times the standard amount of glucose-6-phosphate, and the inclusion of exogenous glucose 6 phosphate dehydrogenase in the co-factor mix.
• A 30 minute pre-incubation prior to the addition of the molten top agar.
• Vogel-Bonner plates containing 0.5% glucose instead of the standard 2% glucose.
Only the test item concentrations, vehicle and the positive control, Congo Red, were dosed using the Prival Mitchell modification.

Without Metabolic Activation: Measured aliquots (0.1 mL) of one of the bacterial cultures were dispensed into sets of test tubes followed by 0.5 mL of phosphate buffer and 0.1 mL of the test item formulation or vehicle. Each mixture was shaken gently at 37 ± 3 ºC for 30 ± 3 minutes. Then, 2 mL of molten, trace histidine supplemented, top agar was added to each tube. The mixture was vortexed and poured onto Vogel-Bonner minimal agar plates containing 0.5% glucose. Each concentration of the test item, appropriate positive control, and each bacterial strain, was assayed using triplicate plates.

With Metabolic Activation: The procedure was the same as described previously except that following the addition of the test item formulation and bacterial culture, 0.5 mL of hamster S9 mix was added to the molten trace amino-acid supplemented media instead of phosphate buffer. In addition, 0.1 mL of TA98 or TA100, 0.5 mL of uninduced hamster liver S9 and 0.1 mL of Congo Red at 50 µg/plate was dispensed into dosing tubes, incubated and overlaid onto 0.5% glucose Vogel Bonner plates as previously described.
The standard Ames positive controls were dosed using the pre-incubation method (previously described) where 0.1 mL of bacterial culture was mixed with 0.5 mL of rat liver S9-mix (phenobarbitone/beta-naphthoflavone) or phosphate buffer and 2 mL of amino-acid supplemented top agar before overlaying onto 2% glucose Vogel-Bonner agar plates.
The negative (untreated) controls were dosed using the plate incorporation method where 0.1 mL of bacterial culture was mixed with 2 mL of amino-acid supplemented top agar before overlaying onto 2% glucose Vogel-Bonner agar plates.

Incubation and Scoring: All of the plates were incubated at 37 ± 3 ºC for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Manual counts were performed at and above 500 µg/plate (absence of S9) and 1500 µg/plate (presence of S9) because of test item induced coloration. Sporadic manual counts were also performed due to spreading colonies which prevented an accurate automated count.

Test for Mutagenicity: Experiment 2 – The Prival-Mitchell Modification to the Ames Test
The second experiment was not performed because the OECD 471 test guideline permits non repetition of the experiment when a clear, positive response is obtained in the first experiment. Therefore, a second, confirmatory experiment (employing the Prival-Mitchell modification) was not required.

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and TA102 or a three-fold increase for TA1535 and TA1537 (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).
5.Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in the experiment was shown to be sterile. The test item formulation was also shown to be sterile. These data are not given in the report.
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The maximum dose level of the test item in the first experiment was selected as the OECD TG 471 recommended dose level of 5000 µg/plate.
There was no visible reduction in the growth of the bacterial background lawn at any test item dose level, either in the presence or absence of metabolic activation (30% liver S9 in modified co factors). Toxicity, in terms of reductions in revertant colony frequency, were noted to the majority of tester strains at the upper test item dose levels.
A black test item colouration was noted from 50 µg/plate (absence of S9) and 150 µg/plate (presence of S9) with an associated precipitate observed by eye from 500 µg/plate (absence of S9) and 1500 µg/plate (presence of S9). These observations did not prevent the scoring of revertant colonies.
The test item induced substantial increases in the frequency of TA100, TA102, TA98 and TA1537 revertant colonies in the presence of S9 only (30% liver S9 in modified co factors). The increases for TA98 were particularly large and showed a dose-related response following a bell-shaped curve with a maximum 15.4 fold increase over the concurrent vehicle control noted at 150 µg/plate. Furthermore, the individual colony counts from 5 µg/plate exceeded the maxima in-house untreated/vehicle historical data for the strain. The responses for TA100, TA102 and TA1537 were smaller but 2 fold increases were noted and in many instances individual colony counts were in excess of the in-house maxima historical control counts for each strain. A smaller response was also noted for TA98 in the absence of S9, however revertant colony counts were within the in-house untreated/vehicle control range.
Experiment 2 (The Prival-Mitchell Modification to the Ames Test)
The second experiment was not performed because the OECD 471 test guideline permits non repetition of the experiment when a clear, positive response is obtained in the first experiment. Therefore, a second, confirmatory experiment (employing the Prival-Mitchell modification) was not required.

Conclusions:

In this Reverse Mutation Assay ‘Ames Test’ (employing the Prival-Mitchell Modification) using strains of Salmonella typhimurium (OECD TG 471) the test item induced large, statistically significant and dose-related increases in the frequency of TA100, TA102, TA98 and TA1537 revertant colonies at the majority of the dose levels used predominantly with metabolic activation (30% liver S9 in modified co factors). Under the conditions of this test the test item was considered to be mutagenic.

Executive summary:

 

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008, the ICH S2(R1)guideline adopted June 2012 (ICH S2(R1) Federal Register. Adopted 2012; 77:33748-33749)and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

Salmonella typhimuriumstrains TA1535, TA1537, TA102, TA98 and TA100 were treated with the test item using the Ames plate pre-incubation method (Prival and Mitchell modification) at eight dose levels, in triplicate, both with and without the addition of a hamster liver homogenate metabolizing system (30% liver S9 in modified co‑factors). The dose range for Experiment 1 was pre-determined and was 1.5 to 5000 µg/plate. 

The OECD 471 test guideline permits non-repetition of the experiment when a clear positive response is obtained in the first experiment, therefore, with the Sponsor’s approval, testing was suspended at the end of Experiment 1.

The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The maximum dose level of the test item in the first experiment was selected as the OECD TG 471 recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any test item dose level, either in the presence or absence of metabolic activation (30% liver S9 in modified co‑factors), in the first mutation test. Toxicity, in terms of reductions in revertant colony frequency, were noted to the majority of tester strains at the upper test item dose levels.

A black test item colouration was noted from 50 µg/plate (absence of S9) and 150 µg/plate (presence of S9) with an associated precipitate observed by eye from 500 mg/plate (absence of S9) and 1500 µg/plate (presence of S9). These observations did not prevent the scoring of revertant colonies.

The test item induced substantial increases in the frequency of TA100, TA102, TA98 and TA1537 revertant colonies in the presence of S9 only. The increases for TA98 were particularly large and showed a dose-related response following a bell-shaped curve with a maximum 15.4 fold increase over the concurrent vehicle control noted at 150 µg/plate. Furthermore, the individual colony counts from 5 µg/plate exceeded the maxima in-house untreated/vehicle historical data for the strain. The responses for TA100, TA102 and TA1537 were smaller but 2 fold increases were noted and in many instances individual colony counts were in excess of the in-house maxima historical control counts for each strain. A smaller response was also noted for TA98 in the absence of S9, however revertant colony counts were within the in-house untreated/vehicle control range.

The test item was considered to be mutagenic under the conditions of this test.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May-June 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Cells used:
- Type and source of cells: The V79 cell stocks were obtained from Harlan CCR in 2010 and originated from Labor für Mutagenitätsprüfungen (LMP); Technical University; 64287 Darmstadt, Germany.
- Suitability of cells: The V79 cell line has been used successfully in in vitro experiments for many years. The high proliferation rate (doubling time 12 - 16 h in stock cultures) and a good cloning efficiency of untreated cells (as a rule more than 50 %) make it an appropriate cell line to use for this study type. The cells have a stable karyotype with a modal chromosome number of 22 (Howard-Flanders, 1981).
Cell culture: The stock of cells is stored in liquid nitrogen. For use, a sample of cells will be removed before the start of the study and grown in Eagles Minimal Essential (MEM) (supplemented with sodium bicarbonate, L-glutamine, penicillin/streptomycin, amphotericin B, HEPES buffer and 10% fetal bovine serum (FBS)) at approximately 37ºC with 5% CO2 in humidified air.
Cell cleansing: Cell stocks spontaneously mutate at a low but significant rate. Before a stock of cells is frozen for storage the number of pre-existing HPRT-deficient mutants must be reduced. The cells are cleansed of mutants by culturing in HAT medium for four days. This is MEM growth medium supplemented with Hypoxanthine (13.6 µg/mL, 100 µM). Aminopterin (0.0178 µg/mL, 0.4 µM) and Thymidine (3.85 µg/mL, 16 µM). After four days in medium containing HAT, the cells are passaged into HAT free medium and grown for four to seven days. Bulk frozen stocks of these “HAT” cleansed cells are frozen down prior to use in the mutation studies, with fresh cultures being removed from frozen before each experiment.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Lot Number 29/03/18 was used in the main test, and was pre-prepared in house (outside the confines of the study) following standard procedures.
- method of preparation of S9 mix: The S9 mix was prepared by mixing S9 with a phosphate buffer containing NADP (5 mM), G-6 P (5 mM), KCl (33 mM) and MgCl2 (8 mM) to give a 20% or 10% S9 concentration.
- concentration or volume of S9 mix and S9 in the final culture medium : The final concentration of S9 when dosed at a 10% volume of S9-mix was 2% for the Preliminary Toxicity Test and the Mutagenicity Test.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Prior to use, each batch of S9 is tested for its capability to activate known mutagens in the Ames test and the certificate of efficacy is presented in Appendix 2.

Test concentrations with justification for top dose:

The molecular weight of the test item was greater than 200, therefore, the maximum dose level was initially set as 2000 µg/mL, which was the maximum recommended dose level. The purity of the test item was 90.19% and was accounted for in the test item formulations.

Preliminary test: The concentrations of test item used were 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000, 2000 µg/mL for the 4-hours –S9 exposure group and 0.31, 0.63, 1.25, 2.5, 5, 10, 20, 30 and 40 µg/mL for the 4-hours +S9 exposure group.
At the end of the exposure period, precipitate of the test item was observed at and above 15.63 µg/mL in the absence of metabolic activation. Precipitate was observed at and above 10 µg/mL in the presence of metabolic activation.
The maximum concentration selected for the main mutagenicity experiment was therefore limited by the onset of precipitate.

Main experiment: 4-hour without S9: 0*, 0.13, 0.25, 0.5*, 1*, 2*, 4*, 8*, 16* µg/mL, EMS 500* and 750* µg/mL and 4-hour with S9 (2%) 0*,0.16, 0.31*, 0.63*, 1.25*, 2.5*, 5*, 10*, 20 µg/mL DMBA 1.0* and 2.0* µg/mL

Vehicle / solvent:

The vehicle control used in the Main Test and as solvent control was as follows:
Identity: MEM
Purity: Treated as 100%
Supplier: Gibco
Expiry: 31 March 2019
Batch number: 1945297

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9,10-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

Preliminary Cytotoxicity Test:
Several days before starting each experiment, a fresh stock of cells was removed from the liquid nitrogen freezer and grown up to provide sufficient cells for use in the test. The preliminary cytotoxicity test was performed on cell cultures plated out at 1 x 107 cells/225 cm2 flask approximately 24 hours before dosing. This was demonstrated to provide at least 20 x 106 available for dosing in each flask using a parallel flask, counted at the time of dosing. On dosing, the growth media was removed and replaced with serum-free MinimalEssential Medium (MEM). One flask per concentration was treated for 4-hours without metabolic activation and for 4-hours with metabolic activation (2% S9). The 4-hour +S9 exposure group of the preliminary toxicity was repeated due to a failed control culture.
In the repeat test the concentrations were revised to avoid excessive precipitation. The concentrations of test item used were 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000, 2000 for the 4-hours –S9 exposure group and 0.31, 0.63, 1.25, 2.5, 5, 10, 20, 30 and 40 µg/mL for the 4-hours +S9 exposure group.
Exposure was for 4 hours at approximately 37 °C with a humidified atmosphere of 5% CO2 in air, after which the cultures were washed twice with phosphate buffered saline (PBS) before being detached from the flasks using trypsin. Cells from each flask were suspended in MEM with 10% FBS, a sample was removed from each concentration group and counted using a Coulter counter. For each culture, 200 cells were plated out into three 25 cm2 flasks with 5 mL of MEM with 10% FBS and incubated for 7 days at approximately 37 °C in an
incubator with a humidified atmosphere of 5% CO2 in air. The cells were then fixed and stained and total numbers of colonies in each flask counted to give cloning efficiencies (CE).
Results from the preliminary cytotoxicity test were used to select the test item concentrations for the mutagenicity experiment.

Mutagenicity Test – Main Experiment:
Several days before starting each experiment, a fresh stock of cells was removed from the liquid nitrogen freezer and grown up to provide sufficient cells for use in the test. Cells were seeded at 1 x 107 cells/225 cm2 flask approximately 24 hours being exposed to the test or control items. This was demonstrated to provide at least 20 x 106 available for dosing in each flask using a parallel flask. Duplicate cultures were set up, both in the presence and absence of metabolic activation, with eight test item concentrations (0.13 to 16 µg/mL in the absence of metabolic activation and 0.16 to 20 µg/mL in the presence of metabolic activation), and vehicle and positive controls. Treatment was for 4 hours in serum free media (MEM) at 37 °C in an incubator with a humidified atmosphere of 5% CO2 in air.

At the end of the treatment period the flasks were washed twice with PBS, before cells were detached from the flasks with trypsin and then suspended in MEM with 10% FBS. A sample of each concentration group cell suspension was counted using a Coulter counter. Cultures were plated out at 2 x 106 cells/flask in a 225 cm2 flask to allow growth and expression of induced mutants, and in triplicate in 25 cm2 flasks at 200 cells/flask to obtain the cloning efficiency, for an estimate of cytotoxicity at the end of the exposure period. Cells were grown in MEM with 10% FBS and incubated at 37 °C in an incubator with a humidified atmosphere of 5% CO2 in air.

Cytotoxicity flasks were incubated for 7 days then fixed with methanol and stained with Giemsa. Colonies were manually counted and recorded to estimate cytotoxicity.

During the 7 Day expression period the cultures were sub-cultured and maintained on days 2 and 5 to maintain logarithmic growth. At the end of the expression period the cell monolayers were detached using trypsin, cell suspensions counted using a Coulter counter
and plated out as follows:
i) In triplicate at 200 cells/25 cm2 flask in 5 mL of MEM with 10% FBS to determine cloning efficiency. Flasks were incubated for 6 to 7 days, fixed with methanol and stained with Giemsa. Colonies were manually counted, counts were recorded for each culture and the percentage cloning efficiency for each dose group calculated.
ii) At 2 x 105 cells/petri dish (ten replicates per group) in MEM with 10% FBS supplemented with 11 µg/mL 6-Thioguanine (6-TG), to determine mutant frequency. The dishes were incubated for 7 days at 37 °C in an incubator with humidified atmosphere of 5% CO2 in air, then fixed with methanol and stained with Giemsa. Mutant colonies were manually counted and recorded for each dish.

The percentage cloning efficiency and mutation frequency per survivor were calculated for each dose group.
Fixation and staining of all flasks/petri dishes was achieved by aspirating off the media, washing with phosphate buffered saline, fixing for 5 minutes with methanol and finally staining with a 10% Giemsa solution for 5 minutes.

Evaluation criteria:

Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly positive if, in any of the experimental conditions examined:
i) At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
ii) The increase is considered to be concentration-related.
iii) The results are outside the range of the historical negative control data for the test item concentrations.
When all these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.

Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly negative if, in all of the experimental conditions examined:
i) None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
ii) There is no concentration related increase.
iii) The results for the test item concentrations are within the range of the historical negative control data.
The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.

There is no requirement for verification of a clearly positive or negative response.

In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgment and/or further investigations. Performing a repeat experiment possibly using modified experimental conditions (e.g. concentration spacing, S9 concentration, and exposure time) may be useful.

Statistics:

When there is no indication of any marked increases in mutant frequency at any concentration then statistical analysis may not be necessary. In all other circumstances comparisons will be made between the appropriate vehicle control value and each individual concentration, using Student’s t-test. Other statistical analysis may be used if they are considered to be appropriate.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no significant change in pH when the test item was dosed into media
- Effects of osmolality: did not increase by more than 50 mOsm
- Precipitation: precipitation observed in the preliminary cytotoxicity test.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
4-S9 MFS 10-6, Positive EMS 500 µg/mL: 171-346 (mean 261.30, st. dev 36.82);
4-S9 MFS 10-6, Positive EMS 750 µg/mL: 291-574 (mean 417.54, st. dev 63.80);
4+S9 MFS 10-6, Positive DMBA 1 µg/mL: 209-774 (mean 334.40, st. dev 114.17);
4+S9 MFS 10-6, Positive DMBA 2 µg/mL: 103-925 (mean 484.97, st. dev 174.87);

- Negative (solvent/vehicle) historical control data:
4-S9 MFS 10-6,Vehicle: 5-23 (mean 12.07, st. dev 4.44);
4+S9 MFS 10-6, Vehicle: 5-24 (mean 12.47, st. dev 3.95)

Conclusions:

The test item did not induce any toxicologically significant or concentration-related increases in mutant frequency per survivor in either the absence or presence of metabolic activation. The test item was therefore considered to be non-mutagenic to V79 cells at the HPRT locus under the conditions of this test.

Executive summary:

The purpose of this study is to assess the potential mutagenicity of a test item on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of the V79 cell line.

Chinese hamster (V79) cells were treated with the test item at eight concentrations, in duplicate, together with vehicle (MEM culture media) and positive controls in both the absence and presence of metabolic activation.  

The concentrations used in the main test were selected using data from the preliminary toxicity test.  The maximum dose levels were limited by precipitate.  The concentrations of test item plated for cloning efficiency and expression of mutant colonies were as follows:

Exposure Group Final concentration of test item (µg/mL)

4-hour without S9: 0.5, 1, 2, 4, 8, 16

4-hour with S9 (2%): 0.31, 0.63, 1.25, 2.5, 5, 10

The vehicle (MEM) controls gave mutant frequencies within the range expected of V79 cells at the HPRT locus.

The positive control substances induced marked increases in the mutant frequency within the historical control data, sufficient to indicate the satisfactory performance of the test system and of the activity of the metabolizing system.

The test item did not induce any toxicologically significant or concentration-related increases in the mutant frequency at any of the concentration levels in the main test, in either the absence or presence of metabolic activation.

With precipitating dose levels analyzed, both exposure groups met the requirements of the OECD 476 guideline.

The test item was shown to be non-mutagenic to V79 cells at the HPRT locus under the conditions of the test.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

29 July 2016

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

CELLS USED
- Normal cell cycle time (negative control): The average generation time (AGT) for human lymphocytes it is considered to be approximately 16 hours.
- Sex, age and number of blood donors: Preliminary Toxicity Test: female, aged 27 years. Main Experiment: male, aged 31 years.
- Whether whole blood or separated lymphocytes were used: whole blood from the peripheral circulation of a non-smoking volunteer (18-35)
- Mitogen used for lymphocytes: phytohaemagglutinin (PHA)

MEDIA USED
Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin,
amphotericin B and 10% fetal bovine serum (FBS), at approximately 37 ºC with 5% CO2 in
humidified air.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : The S9 Microsomal fractions were pre-prepared using standardized in-house procedures (outside the confines of this study). Lot No. PB/βNF S9 29/03/18 was used in this study.
- method of preparation of S9 mix : The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM).
- concentration or volume of S9 mix and S9 in the final culture medium : The final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2%.
- quality controls of S9: efficacy and sterility (certificate attached in the study)

Test concentrations with justification for top dose:

The molecular weight of the test item was given as greater than 500, therefore, the maximum dose level was 2000 µg/mL, the maximum recommended dose level. The purity of the test item was 90.19% and was accounted for in the test item formulations.
The solubility of the test item was investigated in the Envigo Research Limited HPRT Forward Mutation assay, Study number DG51XG. The test item was a fine suspension suitable for dosing in MEM at 20 mg/mL in solubility checks performed in-house. However, due to the presence of excessive precipitate and coloured media, the maximum dose level was further limited to 200 µg/mL. Prior to each experiment, the test item was accurately weighed, formulated in MEM and serial dilutions prepared.
There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al., 1991). The osmolality, pH and precipitate of the test item was investigated in the Envigo Research Limited HPRT Forward Mutation assay, Study number DG51XG.
The pH and osmolality readings are presented in the following table:

Concentration (µg/mL) 0 7.81 15.63 31.25 62.5 125 250 500 1000 2000
pH 7.44 7.41 7.43 7.45 7.46 7.44 7.41 7.44 7.54 7.57
Osmolality (mOsm) 313 313 318 316 313 315 312 313 316 318

The test item was formulated within two hours of it being applied to the test system; it is assumed that the test item formulation was stable for this duration. No analysis was conducted to determine the homogeneity, concentration or stability of the test item
formulation because it is not a requirement of the guidelines. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: MEM / water or saline or culture medium
- Justification for choice of solvent/vehicle: solubility of the substance in water
- Justification for percentage of solvent in the final culture medium:

Negative solvent / vehicle controls:

yes

Remarks:

MEM

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: demecolcine

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate for each dose level in the main experiment; single cultures for each dose level in the preliminary toxicity test.
- Number of independent experiments : 1 preliminary toxicity test; 1 main experiment

METHOD OF TREATMENT/ EXPOSURE:
- Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:
8.05-9.05 mL MEM, 10% (FBS)
0.1 mL Li-heparin
0.1 mL phytohaemagglutinin
0.75 mL heparinized whole blood

- Preliminary toxicity test: Three exposure groups were used:
i) 4-hour exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
iii) 24-hour continuous exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
The dose range of test item used was 0, 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, 100 and 200 µg/mL.

- Main experiment: Three exposure groups were used for Main Experiment:
i) 4-hour exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
iii) 24-hour continuous exposure to the test item without S9-mix, followed by a 24-hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
The dose range of test item used for all three exposure groups was 0, 1, 2, 4, 6, 8, 12 and 24 µg/mL

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 48 hours incubation at approximately 37 ºC, 5% CO2 in humidified air.
- Exposure duration/duration of treatment: 4h with and without metabolic activation (S9); 24h without metabolic activation (S9)
- Harvest time after the end of treatment (sampling/recovery times): the exposure was followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- If cytokinesis blocked method was used for micronucleus assay: Cytochalasin B was added at a final concentration of 4.5 µg/mL, after the exposure period, and then the cells were incubated for a further 24 hours.
- Methods of slide preparation and staining technique used including the stain used:
Cell Harvest: At the end of the Cytochalasin B treatment period the cells were centrifuged, the culture medium was drawn off and discarded, and the cells resuspended in MEM. The cells were then treated with a mild hypotonic solution (0.0375M KCl) before being fixed with fresh methanol/glacial acetic acid (19:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC prior to slide making.
Preparation of Microscope Slides:The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry with gentle warming. Each slide was permanently labeled with the appropriate identification data.
Staining:When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): A minimum of approximately 500 cells per culture were scored for the incidence of mononucleate, binucleate and multinucleate cells and the CBPI value expressed as a
percentage of the vehicle controls.
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): The micronucleus frequency in 2000 binucleated cells was analyzed per concentration (1000 binucleated cells per culture, two cultures per concentration). Cells with 1, 2 or more micronuclei were recorded as such but the primary analysis was on the combined data. Experiments with human lymphocytes have established a range of micronucleus frequencies acceptable for control cultures in normal volunteer donors. The criteria for identifying micronuclei were that they were round or oval in shape, non-refractile, not linked to the main nuclei and with a diameter that was approximately less than a third of the mean diameter of the main nuclei. Binucleate cells were selected for scoring if they had two nuclei of similar size with intact nuclear membranes situated in the same cytoplasmic boundary. The two nuclei could be attached by a fine nucleoplasmic bridge which was approximately no greater than one quarter of the nuclear diameter.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cytokinesis-block proliferation index

Evaluation criteria:

Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly negative if, in most/all of the experimental conditions examined:
1. None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is no dose-related increase.
3. The results in all evaluated dose groups should be within the range of the laboratory historical control data.
Providing that all of the acceptability criteria are fulfilled, a test item may be considered to be clearly positive, if in any of the experimental conditions examined, there is one or more of the following applicable:
1. At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is an increase which can be considered to be dose-related.
3. The results are substantially outside the range of the laboratory historical negative control data.
When all the criteria are met, the test item is considered able to induce chromosome breaks and/or gain or loss in this test system.
There is no requirement for verification of a clear positive or negative response.
In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations.

Statistics:

The frequency of binucleate cells with micronuclei was compared, where necessary, with the concurrent vehicle control value using the Chi-squared Test on observed numbers of cells with micronuclei. A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the frequency of binucleate cells with micronuclei was less than 0.05 and there was a dose-related increase in the frequency of binucleate cells with micronuclei.

Key result

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

1. Preliminary Toxicity Test:
The dose range for the Preliminary Toxicity Test was 0.78 to 200 µg/mL. The maximum dose was limited due to the presence of excessive precipitate and colouration to the media.
A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at and above 12.5 µg/mL in all three exposure groups. A black colouration to the media was observed at and above 1.56 µg/mL in the 4-hour exposure groups and at and above 3.125 µg/mL in the 24-hour continuous exposure group.
Microscopic assessment of the slides prepared from the exposed cultures showed that binucleate cells were present at up to 200 µg/mL in all three exposure groups. The test item induced no evidence of toxicity in any of the exposure groups.
The selection of the maximum dose level for the Main Experiment was based on the onset of the precipitating dose level for all three exposure groups which was determined to be 24 µg/mL.

2. Micronucleus Test – Main Experiment
The dose levels of the controls and the test item are given in the table below:
Group Final concentration of test item: DIRECT BLACK RBK (µg/mL)
4-hour without S9 0*, 1, 2, 4*, 6*, 8*, 12, 24, MMC 0.2*
4-hour with S9 (2%) 0*, 1, 2, 4*, 6*, 8*, 12, 24, CP 5*
24-hour without S9 0*, 1, 2, 4, 6*, 8*, 12*, 24, DC 0.075*

* = Dose levels selected for analysis of micronucleus frequency in binucleate cells
MMC = Mitomycin C
CP = Cyclophosphamide
DC = Demecolcine

The qualitative assessment of the slides determined that fairly precipitate was similar to that observed in the Preliminary Toxicity Test and that there were binucleate cells suitable for scoring at the maximum dose level of test item, 24 µg/mL, in all three exposure groups.
A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at and above 24 µg/mL in the 4-hour exposure groups only. No precipitate in the blood-free cultures was observed in the 24-hour exposure group, however, precipitate of the test item was observed in the blood cultures at the end of the exposure at and above 8 µg/mL in the 4-hour exposure groups and at and above 12 µg/mL in the 24-hour exposure group. A black colouration to the media was observed at and above 1 µg/mL in the exposure groups in the absence of metabolic activation (S9) and at and above 2 µg/mL in the exposure group in the presence of S9. Therefore, the blood culture precipitate observations were used to determine the maximum dose level for binucleate analysis.

In the 4-hour group in the absence of S9, no inhibition of cell proliferation was achieved at any dose level tested. Therefore, the maximum dose level selected for analysis of binucleate cells was the lowest precipitating dose level (8 µg/mL).
In the presence of S9 , again no dose-related inhibition of CBPI was observed at any dose level. The maximum dose level selected for analysis of binucleate cells was the lowest precipitating dose level (8 µg/mL).
In the 24-hour exposure group, no dose-related inhibition of CBPI was observed at any dose level. The maximum dose level selected for analysis of binucleate cells was the lowest precipitating dose level (12 µg/mL).

The assay was determined to be valid because:
• The vehicle control cultures had frequencies of cells with micronuclei within the expected range
• The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated
• Cell proliferation criteria in the solvent control was considered to be acceptable.
• The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.
• The required number of cells and concentrations was analyzed

The test item did not induce a statistically significant increases in the frequency of binucleate cells with micronuclei, either in the absence or presence of metabolic activation.

Remarks on result:

other: report in progress

Conclusions:

The test item, detailed above, did not induce a statistically significant increase in the frequency of binucleate cells with micronuclei in either the absence or presence of a metabolizing system.  The test item was therefore considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

Executive summary:

This report describes the results of an in vitro study for the detection of the clastogenic and aneugenic potential of the test item on the nuclei of normal human lymphocytes.  

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells at up to three dose levels, together with vehicle and positive controls.  Three exposure conditions in a single experiment were used for the study using a 4-hour exposure in the presence and absence of a standard metabolizing system (S9) at a 2% final concentration and a 24-hour exposure in the absence of metabolic activation.  At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B.

The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited on precipitate. The dose levels selected for the Main Test were as follows:

Group	Final concentration of test item DIRECT BLACK RBK (µg/mL)
4 -hour without S9	0, 1, 2, 4, 6, 8, 12, 24
4 -hour with S9 (2%)	0, 1, 2, 4, 6, 8, 12, 24
24 -hour without S9	0, 1, 2, 4, 6, 8, 12, 24

All vehicle (Eagle's minimal essential medium with HEPES buffer (MEM)) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes.

The positive control items induced statistically significant increases in the frequency of cells with micronuclei.  Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item was non-toxic and did not induce any statistically significant increases in the frequency of cells with micronuclei, using a dose range that included a dose level that was the lowest precipitating dose level.

The test item, DIRECT BLACK RBK was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Read-across from the structural analogue Direct Black 19 is performed to assess the in vivo mutagenicity hazard of Direct Black RBK.

An in vivo test followgin OECD 474 is available. The results are negative for mutagenicity. No cytotoxic effect of the test substance were observed. No potential of chromosomal damages were observed.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity chromosome aberration

Type of information:

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

Adequacy of study:

key study

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

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Interpretation of results: negative
No cytotoxic effect of the test substance were observed.
No potential of chromosomal damages were observed.

Executive summary:

This end point was assessed based on read-across from the structural analogue Direct Back 19.

An in vivo mutagenicity test following the OECD 474 and performed in GLP conditions is available fo the source substance. Micronuclei arise as a result of damage to the chromosomes or the spindle apparatus through a mutagenic active substance during the mitotic cell division in proliferating bone marrow cells. The results show no increased incidence of micronucleated polychromatic erythrocytes

The calculated frequency of the micronuleated polychromatic erythrocytes of the groups treated with the substance was between 0,17 and 0,42 %.The observed frequency corresponds to the data reported in the literature which is related to the spontaneous rate of occurrence of micronuclei in polychromatic erythrocytes.

Hence a potential of chromosomal damages or damage to the mitotic apparatus could be excluded for the tested substance. The same behaviour is expected for the target substance Direct Black 155- Na, K, Li salt.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Based on the mutagenicity test results, the substance is not classified for genotoxicity.