Disodium 4-amino-3-[[4-[(2,4-diaminophenyl)azo]phenyl]azo]-5-hydroxy-6-(phenylazo)naphthalene-2,7-disulphonate

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

[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

EC Number:

824-263-3

Cas Number:

2196165-14-5

Molecular formula:

C28H21N9O7S2.xNa.yK.zLi

IUPAC Name:

[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

Method

Species / strain

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:

Controls

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.

Results and discussion

Test results

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

Untreated negative 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: non mutagenic potential

Applicant's summary and conclusion

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.