Sodium bis[1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

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

Adequacy of study:

key study

Study period:

2015-11-17 to 2016-05-18

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

26 Sep 2014

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: In vitro Mammalian Cell Micronucleus Test, No B.49; No L 193

Version / remarks:

Commission Regulation (EC) No 640/2012 of 06 July 2012

Deviations:

no

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:

Reaction mass of sodium [1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and sodium bis[1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and sodium bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-)

IUPAC Name:

Reaction mass of sodium [1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and sodium bis[1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and sodium bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-)

Method

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELL LINE AND STORAGE
The V79 cell line is a permanent cell line derived from the Chinese hamster and has a
− high proliferation rate (doubling time of about 12 - 14 hours),
− high plating efficiency (≥ 90 %),
− stable karyotype (modal number of 22 chromosomes).
The V79 cell line has shown its suitability to detect aneugenic effects in the Micronucleus test in vitro either in the absence and presence of cytochalasin B.
Stocks of the V79 cell line (1-mL portions) were maintained at -196 °C in liquid nitrogen using 7 % (v/v) dimethyl sulfoxide (DMSO) in culture medium as a cryoprotectant. Each batch used for the cytogenetic experiments was checked for
− mycoplasma contamination,
− karyotype stability,
− plating efficiency (=colony forming ability) incl. vital staining.

CULTURE MEDIA
MEM (minimal essential medium with Earle's salts) containing a L-glutamine source supplemented with
− 10 % (v/v) fetal calf serum (FCS)
− 1 % (v/v) penicillin/streptomycin (10000 IU / 10000 μg/mL)
− 1 % (v/v) amphotericine B (250 μg/mL)

CELL CULTURE
Deep-frozen cell stocks were thawed at 37 °C in a water bath, and volumes of 0.5 mL were transferred into 25 cm² plastic flasks containing about 5 mL MEM supplemented with 10 % (v/v) FCS. Cells were grown with 5 % (v/v) CO2 at 37 °C and ≥ 90 % relative humidity and subcultured twice weekly. Cell monolayers were suspended in culture medium after detachment with 0.25 % (w/v) trypsin solution.

CELL CYCLE AND HARVEST TIME
The cell cycle of the untreated V79 cells lasts for about 12 - 14 hours under the selected culture conditions (last measurement based on the BrdU method of Speit et al.: 12 hours; May 2014). Thus, a harvest time of 24 hours is about 2 times the normal cell cycle length.
V79 cells are an asynchronous cell population, i.e. at the time of test substance treatment there are different cell stages (G1-, S-, G2-phase and mitosis). Since the effect on these cell stages may vary for different test substances, more than one harvest time after treatment may be appropriate.
Furthermore, substance-induced mitotic delay may considerably delay the first post-treatment mitosis. Therefore, delayed harvest times (e.g. 44 hours) and prolonged exposure periods (e.g. 24 hours treatment) may be required for the detection of several substances.

Cytokinesis block (if used):

actin polymerisation inhibitor cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction from phenobarbital and β-naphthoflavone induced Wistar rat livers

Test concentrations with justification for top dose:

PRETEST
- Justification: Following the requirements of the current OECD Guideline 487 a test substance with defined composition should be tested up to a maximum concentration of 2 mg/mL, 2 μL/mL or 10 mM, whichever is the lowest. When the test substance is not of defined composition, e.g. substance of unknown or variable composition, complex reaction products or biological materials (socalled UVCBs), or environmental extracts, the top concentration should be higher to increase the concentration of each of the components (e.g. 5 mg/mL). In case of toxicity, the top concentration should produce 55 ± 5 % cytotoxicity: reduction of the proliferation index (CBPI) to 45 ± 5 % of the concurrent vehicle control. For relatively insoluble test substances only one concentration should be tested showing turbidity or precipitation in culture medium at the end of exposure period. In the pretest for toxicity based on the content of the Cr-complex of the test substance in the 2600 μg/mL total substance was used as top concentration.
- Concentrations, with and without S9 mix: 20.3, 40.6, 81.3, 162.5, 325.0, 650.0, 1300.0, 2600.0 μg/mL

MAIN TEST
- Justification: The concentrations tested in this study were selected in accordance with the requirements set forth in the test guidelines and based on the results of a preliminary range finding test (experimental conduct with records and documentation in general accordance with the GLP principles).
- Concentrations 1ST Experiment, with and without S9 mix, 24 hour preparation interval: 5.00, 10.00, 20.00, 40.00, 80.00, 160.00 μg/mL
- Concentrations 2ST Experiment, without S9 mix, 24 hour preparation interval: 10.00, 20.00, 40.00, 80.00, 160.00 μg/mL
- Concentrations 2ST Experiment, with and without S9 mix, 44 hour preparation interval: 10.00, 20.00, 40.00, 80.00, 160.00 μg/mL

Vehicle / solvent:

PRETEST
- Vehicle used: culture medium
- Justification for choice of vehicle: Culture medium was the only feasible vehicle to reach a maximum concentration of 2600 μg/mL (homogeneous suspension) due to physico-chemical properties.

MAIN TEST
- Vehicle used: DMSO
- Justification for choice of vehicle: In the pretest, precipitates were found in all exposed test groups from the lowest applied concentration of 20 μg/mL onward. Following an additional solubility test with a lower top concentration it was decided to use dimethyl sulfoxide (DMSO) as vehicle in the main experiments of this study.

Controls

Untreated negative controls:

yes

Remarks:

without vehicle and S9 mix

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

EXPERIMENTAL PROCEDURE
- Method of application: in medium
- Preparation of test cultures: The stocks of cells (1.0-mL portions) were thawed at 37 °C in a water bath. 0.5 mL were pipetted into 25 cm² cell culture flasks containing 5 mL MEM (incl. 10 % [v/v] FCS). The flasks were subsequently incubated at 37 °C, 5 % (v/v) CO2 and relative humidity of ≥ 90 % until they have reached confluency of at least 50 % (duration about 2 – 4 days). The medium was replaced after about 24 - 30 hours to remove any dead cells. Prior to the preparation of the final test cultures, the cells may run through max. 15 routine passages. After the "last" routine passage, there was another passage to prepare test cultures.
- Seeding of the cells: A single cell suspension with the required cell count (3 - 5 E+5 cells per culture, depending on the schedule) was prepared in MEM incl. 10 % (v/v) FCS. 5 mL cell suspension was transferred into 25 cm² cell culture flasks using a dispenser. Subsequently, the test cultures were incubated at 37 °C, 5 % (v/v) CO2 and ≥ 90 % relative humidity. The cultures were visually checked for attachment and viability before treatment of the test cultures.
- Definition of test cultures: A test group consists of two separately treated flasks (Culture A and B). Each, two slides were prepared and, thus, four slides were available for scoring of each test group, in general.
- Treatment of test cultures: After the attachment period, about 20 - 24 hours after seeding, the medium was removed from the flasks and the treatment medium was added. The cultures were incubated for the respective exposure period at 37 °C, 5 % (v/v) CO2 and ≥ 90 % relative humidity.
- At the end of the 4-hour exposure period, the medium was removed and the cultures were rinsed twice with 5 mL HBSS (Hanks Balanced Salt Solution). Subsequently, 5 mL MEM (incl. 10 % [v/v] FCS) supplemented with CytB (final concentration: 3 μg/mL; stock: 0.6 mg/mL in DMSO; AppliChem, Cat.No. A7657) was added and the cultures were incubated at 37 °C, 5 % (v/v) CO2 and ≥ 90 % relative humidity for the respective recovery time. In the case of 24-hour continuous exposure, CytB was added to the treatment medium at start of treatment, and cell preparation was started directly at the end of exposure. At 44 hours preparation interval in the presence of S9 mix CytB was added 24 hours before preparation of the cultures.

DURATION
Exposure-, Recovery- and Harvest time: see Table 1.

SPINDLE INHIBITOR
actin polymerisation inhibitor cytochalasin B (CytB)

STAIN
4’,6-diamidino-2-phenylindole dihydrochloride and propidium iodide

NUMBER OF REPLICATIONS
2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED
- Cell harvest and preparation of slides: The cells were prepared based on the method described by Fenech, 1993. Just before preparation the culture medium was completely removed. Single cell suspensions were prepared from each test group by enzymatic dissociation. Then, the cell number per flask of each cell suspension was determined using a cell counter (CASY®, Roche Applied Science, Mannheim, Germany). Subsequently, 5xE+4 cells per slide were centrifuged at 600 rpm for 7 minutes onto labelled slides using a Cytospin centrifuge (Cellspin I, Tharmac, Waldsolms, Germany). At least two slides per flask were prepared. In the case of strongly reduced cell numbers below 10x1E4 cells per flask no slides were prepared. After drying, the slides were fixed in 90 % (v/v) methanol for 10 minutes.
- Staining: Before scoring, the slides were stained with a mixture of 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI; stock: 5 mg/mL; Sigma-Aldrich, Cat.No. D9542) and propidium iodide (stock: 5 mg/mL; Sigma-Aldrich, Cat.No. P4170) in Fluoroshield™ (Sigma-Aldrich, Cat.No. F6182) at a concentration of 0.25 μg/mL each. By the use of the combination of both fluorescence dyes it can be differentiated between DNA (DAPI; excitation: 350 nm, emission: 460 nm) and cytoplasm (PI; excitation: 488 nm, emission: 590 nm).

NUMBER OF CELLS EVALUATED
At least 1000 binucleated cells per culture, in total at least 2000 binucleated cells per test group

CRITERIA FOR MICRONUCLEUS IDENTIFICATION
see 'Evaluation criteria'.

DETERMINATION OF CYTOTOXICITY
- Method: relative population doubling (RPD), Proliferation Index (CBPI)
- Any supplementary information relevant to cytotoxicity (RPD): The RPD takes into account either cytotoxicity or cell proliferation over the whole incubation period until slide preparation when determination of cell numbers occurs. However, in the main experiments supplementation of culture medium with CytB blocks cell division. Thus, under the experimental conditions described, RPD is an indication of cell viability mainly for the time period before addition of CytB.

OTHER EXAMINATIONS:
- Cell morphology: At the end of the treatment period, all test groups were examined microscopically with regard to cell morphology, which is a further indication for cytotoxicity.
- pH value: Changes in the pH were apparent by a color change of the indicator in the culture medium (phenol red: normal range: about pH 6.7 - 8.3). The pH was measured at least for the top concentration and for the vehicle control with and without S9 mix.
- Osmolality: Osmolality was measured at least for the top concentration and for the vehicle control with and without S9 mix.
- Solubility: Test substance precipitation was checked immediately after start of treatment of the test cultures (macroscopically) and at the end of treatment (macroscopically / microscopically).

OTHER:
- Assessment of the slides: Dose selection for scoring for cytogenetic damage was based on the results of a previous check on slide and/or cell quality, number of analysable cells and nuclear fragmentation.

Evaluation criteria:

ACCEPTANCE CRITERIA
The in vitro micronucleus assay is considered valid if the following criteria are met:
- The quality of the slides allowed the evaluation of a sufficient number of analysable cells both in the control groups (vehicle/positive) and in at least three exposed test groups.
- Sufficient cell proliferation was demonstrated in the vehicle control.
- The number of cells containing micronuclei in the vehicle control was within the range of the laboratory’s historical negative control data (95 % control limit). Weak outliers can be judged acceptable if there is no evidence that the test system is not “under control”.
- The positive control substances both with and without S9 mix induced a distinct, statistically significant increase in the number of micronucleated cells in the expected range.

ASSESSMENT CRITERIA
A test substance is considered to be clearly positive if the following criteria are met:
• A statistically significant increase in the number of micronucleated cells was obtained.
• A dose-related increase in the number of cells containing micronuclei was observed.
• The number of micronucleated cells exceeded both the value of the concurrent vehicle control and the range of our laboratory’s historical negative control data (95% control limit)
A test substance is considered to be clearly negative if the following criterion is met:
• Neither a statistically significant nor dose-related increase in the number of cells containing micronuclei was observed under any experimental condition.
• The number of micronucleated cells in all treated test groups was close to the concurrent vehicle control value and within the range of our laboratory’s historical negative control data (95 % control limit).

Statistics:

The statistical evaluation of the data was carried out using an appropriate statistical analysis. The proportion of cells containing micronuclei was calculated for each test group. A comparison of the micronucleus rates of each test group with the concurrent vehicle control group was carried out for the hypothesis of equal proportions (i.e. one-sided Fisher's exact test).

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster Ovary (CHO)

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: Osmolality and pH values were not influenced by test substance treatment.
- Precipitation: Test substance precipitation in culture medium at the end of exposure period was observed in the absence of S9 mix at 80 μg/mL and above in the 1st Experiment and at 160 μg/mL in the 2nd Experiment. In addition, in the presence of metabolic activation precipitates in culture medium occurred at 10 μg/mL and above in both experiments (macroscopical assessment).

RANGE-FINDING/SCREENING STUDIES
The test substance was poorly soluble in all commonly used vehicles. Thus, test substance suspensions in culture medium were used in the pretest. Precipitation in culture medium was observed from the lowest applied concentration of 20.3 μg/mL onward 4 and 24 hours after start of exposure. No cytotoxicity was observed in the pretest when tested up to the highest required concentration of 2600 μg/mL. Based on the experience on solubility of the test substance in other in vitro test systems (internal data) the vehicle DMSO was used in both main experiments. However, precipitates in culture medium were still observed in both experiments. Therefore, in the main experiments of this micronucleus study concentrations at the border of solubility in culture medium were tested for cytogenetic damage.

GENOTOXICITY - MICRONUCLEUS ANALYSIS
In this study, no biologically relevant increase in the number of micronucleated cells was observed either without S9 mix or after the addition of a metabolizing system. In both experiments in the absence and presence of metabolic activation after 4 and 24 hours treatment with the test substance the values (0.3 – 0.9 % micronucleated cells) were close to the concurrent vehicle/negative control values (0.3 - 0.8 % micronucleated cells) and within the range of the 95 % control limit of our historical negative control data (0.0 - 1.0 % micronucleated cells).
Besides, in the 1st Experiment in the absence of S9 mix the micronucleus rates were concentration-related increased at 40, 80 and 160 μg/mL (0.5 %, 0.6 % and 0.9 % micronucleated cells, respectively). However, all values were clearly within the range of the 95 % control limit of our historical negative control data range and, therefore, this finding has to be regarded as biologically irrelevant.
The positive control substances ethyl methanesulfonate and cyclophosphamide induced statistically significant increased micronucleus frequencies in both independently performed experiments in at least one positive control group each. In this study, in the absence and presence of metabolic activation the frequency of micronucleated cells (1.6– 5.9 % micronucleated cells) was above the range of our historical negative control data (0.1 - 1.5 % micronucleated cells) and close to or within our historical positive control data range (2.3 – 13.8 % micronucleated cells). Unfortunately, in the 1st Experiment in the absence of S9 mix the positive control EMS did not show the expected increase at 500 μg/mL (1.3 % micronucleated cells). But the second positive control group of 600 μg/mL EMS led to the expected statistically significant increase of the micronucleus rate (2.3 % micronucleated cells).

CYTOTOXICITY - RELATIVE POPULATION DOUBLING
In both main experiments in the absence and presence of S9 mix no cytotoxicity indicated by reduced RPD of below 50 % of control was observed up to the highest applied test substance concentrations. These values were calculated based on cell numbers determined at the end of each experiment. In the pretest the parameter RPD is a valuable indicator of test substance toxicity. However, in the main experiments due to the use of the cytokinesis block method it is a measure of cell proliferation only until addition of cytochalasin B to the cultures. But, it also gives an useful information on cell loss due to test substance exposure.

CYTOTOXICITY - PROLIFERATION INDEX
No clearly reduced proliferative activity was observed either after 4 hours exposure interval in the absence and presence of S9 mix or after 24 hours continuous test substance treatment in the test groups scored for cytogenetic damage. However, slightly elevated cytostasis indicated by reduction of the CBPI was obtained in the absence of S9 mix at 160 μg/mL (25.4 % of control) in the 2nd Experiment.
Due to the use of DMSO either as vehicle for the test substance or as solvent for cytochalasin B a final concentration of 1.5 % (v/v) DMSO was reached in the treatment medium in the 2nd Experiment at 24 hours continuous treatment in the absence of S9 mix. Therefore, a negative control culture supplemented with cytochalasin B (0.5 % [v/v] DMSO) only was run in parallel. This culture showed only a slightly higher CBPI (absolute value: 2.19) than the vehicle control (absolute value: 2.12). Thus, based on these data it was confirmed that the applied DMSO concentration had no detrimental impact on the outcome of this experimental part.

CELL MORPHOLOGY
Cell attachment/morphology was adversely influenced (grade > 2) only in the 1st Experiment in the presence of metabolic activation at 160 μg/mL.

Any other information on results incl. tables

Table 2. Historical negative control data, Cytochalasin B Method, Period: December 2013 - December 2014

	Without S9 mix - all vehicles*	With S9 mix - all vehicles*
	Micronucleated Cells [%]	Micronucleated Cells [%]
Exposure / Sampling period	4 h / 24 h & 24 h / 24 h	4 h / 24 h & 4 h / 44 h
Mean	0.5	0.6
Minimum	0.2	0.1
Maximum	0.9	1.5
Standard Deviation	0.2	0.3
95% Lower Control Limit	0.1	0.0
95% Upper Control Limit	0.8	1.1
No. of Experiments	43	42

* = culture medium, DMSO 1% (v/v), acetone 1% (v/v), ethanol 1% (v/v)

 

Table 3. Historical positive control data, Cytochalasin B Method, Period: December 2013 - December 2014

	Without S9 mix Ethyl methanesulfonate (EMS) 300 - 500 μg/mL	With S9 mix Cyclophosphamide (CPP) 0.5 - 1.0 μg/mL
	Micronucleated Cells [%]	Micronucleated Cells [%]
Exposure / Sampling period	4 h / 24 h & 24 h / 24 h	4 h / 24 h & 4 h / 44 h
Mean	3.0	5.4
Minimum	2.3	2.6
Maximum	6.4	13.8
Standard Deviation	0.8	2.3
95% Lower Control Limit	1.4	0.7
95% Upper Control Limit	4.7	10.1
No. of Experiments	43	42

Applicant's summary and conclusion

Conclusions:

Negative