Disodium [4-hydroxy-3-[(2-hydroxy-4-nitrophenyl)azo]naphthalene-1-sulphonato(3-)][1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)]chromate(2-)

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:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Disodium [4-hydroxy-3-[(2-hydroxy-4-nitrophenyl)azo]naphthalene-1-sulphonato(3-)][1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)]chromate(2-)

EC Number:

271-351-7

EC Name:

Disodium [4-hydroxy-3-[(2-hydroxy-4-nitrophenyl)azo]naphthalene-1-sulphonato(3-)][1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)]chromate(2-)

Cas Number:

68541-71-9

Molecular formula:

C32H17CrN6O11S.2Na

IUPAC Name:

disodium [4-hydroxy-3-[(2-hydroxy-4-nitrophenyl)azo]naphthalene-1-sulphonato(3-)][1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)]chromate(2-)

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

None

Specific details on test material used for the study:

Identification: FAT 20011/E TE
Purity: 65 %
Molecular Weight: 791.55 g/mol
Lot No.: 1309023
Expiration Date: 30 September 2018
Description: Dark violet powder (BioReliance) Black powder (Sponsor)
Storage Conditions: Room Temperature, protected from light
Receipt Date: 02 March 2015

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Exponentially growing CHO-K1 cells were seeded in complete medium (McCoy's 5A medium containing 10 % fetal bovine serum, 1.5 mM L-glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin and 2.5 μg/mL Amphotericin B) for each treatment condition at a target of 5 x 105 cells/culture. The cultures were incubated under standard conditions (37 ± 1 °C in a humidified atmosphere of 5 ± 1 % CO2 in air) for 16-24 hours.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 was used as the metabolic activation system.

Test concentrations with justification for top dose:

Cytotoxicity (≥50% reduction in cell growth index relative to the vehicle control) was observed at dose levels 20, 200, 500, and 2000 μg/mL in the non-activated 4-hour exposure group, at dose levels ≥60 μg/mL in the S9-activated 4-hour exposure group, and at dose levels ≥600 μg/mL in the non-activated 20-hour exposure group.

Vehicle / solvent:

Water

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

For non-activated studies

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

For S9-activated studies

Details on test system and experimental conditions:

Chromosome Aberration Assays:
Seven to nine dose levels were tested using duplicate cultures at appropriate dose intervals based on the toxicity profile of the test substance. Precipitation of test substance dosing solution in the treatment medium was determined using unaided eye at the beginning and conclusion of treatment. The highest dose level evaluated for chromosome aberrations was based on visible precipitation of the test substance in treatment medium at the conclusion.

Treatment of Target Cells (Preliminary Toxicity Test and Chromosome Aberration Assays):
The pH of the highest dose level of dosing solution in the treatment medium was measured using pH meter. Treatment was carried out by re-feeding the cultures with 4.5 mL complete medium for the non-activated exposure or 4.5 mL S9 mix (3.5 mL culture medium + 1 mL of S9 cofactor pool) for the S9-activated exposure, to which was added 500 μL of test substance dosing solution or vehicle alone. In the definitive assay, positive control cultures were resuspended in either 5 mL of complete medium for the non-activated studies, or 5 mL of the
S9 reaction mixture (4 mL serum free medium + 1 mL of S9 cofactor pool), to which was added 50 μL of positive control in solvent.

After the 4 hour treatment period in the non-activated and the S9-activated studies, the treatment medium were aspirated, the cells were washed with calcium and magnesium free phosphate buffered saline (CMF-PBS), re-fed with complete medium, and returned to the incubator under standard conditions. For the non-activated 20 hour treatment group, cultures with visible precipitate were washed with CMF-PBS to avoid precipitate interference with cell counts.
For the definitive assay only, two hours prior to cell harvest, Colcemid® was added to all cultures at a final concentration of 0.1 μg/mL.

Collection of Metaphase Cells (Preliminary Toxicity Test and Chromosome Aberration Assayd)
For the preliminary toxicity test and chromosome aberration assays, cells were collected 20 hours (± 30 minutes), 1.5 normal cell cycles, after initiation of treatment to ensure that the cells are analyzed in the first division metaphase. Just prior to harvest, the cell cultures was visually inspected for the degree of monolayer confluency relative to the vehicle control. The cells were trypsinized and counted and the cell viability was assessed using trypan blue dye exclusion.
The cell count was determined from a minimum of two cultures to determine the number of cells being treated (baseline). The data was presented as cell growth inhibition in the treatment group compared to vehicle control. Cell growth was determined by Relative Increase in Cell Counts (RICC) as a measure of cytotoxicity (Fellows and O'Donovan 2007; Lorge et al., 2008). The cell counts and percent viability were used to determine cell growth inhibition relative to the vehicle control (% cytotoxicity).

RICC (%) = 100 - {[(mean viable cells treated - mean viable cells baseline)/(mean viable cells solvent - mean viable cells baseline)]X100}

For the definitive assay only, cells were collected by centrifugation, treated with 0.075M KCl, washed with fixative (methanol: glacial acetic acid, 3:1 v/v), capped and stored overnight or longer at 2 to 8 °C. To prepare slides, the cells were collected by centrifugation and the cells were resuspended in fresh fixative. The suspension of fixed cells was applied to glass microscope slides and air-dried. The slides were stained with Giemsa, permanently mounted, and identified by the BioReliance study number, dose level, treatment condition, harvest date, activation system, test phase, and replicate tube design.

Evaluation criteria:

The test substance was considered to have induced a positive response if:
• at least one of the test concentrations exhibited a statistically significant increase when compared with the concurrent negative control (p ≤0.05), and
• the increase was concentration-related (p ≤0.05), and
• results were outside the 95 % control limit of the historical negative control data.

The test substance was considered to have induced a clear negative response if none of the criteria for a positive response were met.

Statistics:

Statistical analysis was performed using the Fisher's exact test (p ≤0.05) for a pairwise comparison of the frequency of aberrant cells in each treatment group with that of the vehicle control. The Cochran-Armitage trend test was used to assess dose-responsiveness.

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Any other information on results incl. tables

Solubility Test:

Water was used as the vehicle based on the solubility of the test substance and compatibility with the target cells. In a solubility test conducted at BioReliance, the test substance formed a workable suspension in water at a maximum concentration of approximately 10 mg/mL.

Preliminary Toxicity Assay;

CHO cells were first exposed to nine dose levels of FAT 20011/E TE, ranging from 0.2 to 2000 μg/mL, as well as vehicle controls, in both the absence and presence of an Aroclor-induced S9 metabolic activation system for 4 hours, or continuously for 20 hours in the absence of S9 activation. The test substance formed workable suspensions in water at concentrations ≥ 0.06 mg/mL, while concentrations ≤ 0.02 mg/mL were soluble in water. Visible precipitate (the culture was opaque dark blue) was observed in treatment medium at the following dose levels:

Treatment Condition	Treatment Time	Visible precipitate
		At the beginning of Treatment period	At the conclusion of Treatment period
Non-activated	4 hr	≥200 µg/mL	≥200 µg/mL
	20 hr	≥200 µg/mL	≥200 µg/mL
S9-activated	4 hr	≥200 µg/mL	≥200 µg/mL

The osmolality in treatment medium was measured as follows:

Dose tested	Dose levels (µg/mL)	Osmolality (mmol/kg)
Vehicle	0	268
Highest soluble	60	270
Lowest precipitating	200	274
Highest	2000	292

The osmolality of the test substance dose levels in treatment medium is acceptable because it did not exceed the osmolality of the vehicle by more than 120%. The pH of the highest dose level of test substance in treatment medium was 7.46.

Cytotoxicity (50% reduction in cell growth index relative to the vehicle control) was observed at dose levels 20, 200, 500, and 2000 µg/mL in the non-activated 4-hour exposure group, at dose levels ≥60 µg/mL in the S9-activated 4-hour exposure group, and at dose levels ≥600 µg/mL in the non-activated 20-hour exposure group. Based on the results of the preliminary toxicity test, the dose levels selected for testing in the chromosome aberration assay were as follows:

Treatment Condition	Treatment Time	Recovery Time	Dose level (µg/mL)
Non-activated	4 hr	16 hr	2.5, 5, 10, 25, 60, 75, 100, 200, 300
	20 hr	0 hr	5, 10, 25, 60, 75, 100, 200, 300
S9-activated	4 hr	16 hr	5, 10, 25, 60, 75, 100, 200

Initial chromosome abberation assay:

Treatment Condition	Treatment Time	Visible precipitate
		At the beginning of Treatment period	At the conclusion of Treatment period
Non-activated	4 hr	≥ 75 µg/mL	≥ 75 µg/mL
	20 hr	≥ 75 µg/mL	≥ 75 µg/mL
S9-activated	4 hr	≥ 75 µg/mL	≥ 75 µg/mL

Repeat chromosome abberation assay:

Treatment Condition	Treatment Time	Visible precipitate
		At the beginning of Treatment period	At the conclusion of Treatment period
S9-activated	4 hr	≥ 55 µg/mL	≥ 55 µg/mL

Applicant's summary and conclusion

Conclusions:

FAT 20011/E TE was concluded to be negative for the induction of structural and numerical chromosome aberrations in the non-activated and S9-activated test systems in the in vitro mammalian chromosome aberration test using CHO cells.

Executive summary:

FAT 20011/E TE was tested in the chromosome aberration assay using Chinese hamster ovary (CHO) cells in both the absence and presence of an Aroclor-induced rat liver S9 metabolic activation system according to OECD Guideline 473.

A preliminary toxicity test was performed to establish the dose range for the chromosome aberration assay. The chromosome aberration assay was used to evaluate the clastogenic potential of the test substance. In both phases, CHO cells were treated for 4 and 20 hours in the non-activated test system and for 4 hours in the S9-activated test system. All cells were harvested 20 hours after treatment initiation. Dose formulations were adjusted for the purity of the test substance (65 %), using a correction factor of 1.54.

Water was used as the vehicle based on the solubility of the test substance and compatibility with the target cells. In a solubility test conducted at BioReliance, the test substance formed a workable suspension in water at a maximum concentration of approximately 10 mg/mL. Cyclophosphamide and mitomycin C were evaluated as the concurrent positive controls for treatments with and without S9, respectively.

In the preliminary toxicity assay, the doses tested ranged from 0.2 to 2000 μg/mL. Cytotoxicity (≥50 % reduction in cell growth index relative to the vehicle control) was observed at dose levels 20, 200, 500, and 2000 μg/mL in the non-activated 4-hour exposure group, at dose levels ≥ 60 μg/mL in the S9-activated 4-hour exposure group, and at dose levels ≥600 μg/mL in the non-activated 20-hour exposure group. Based on these findings, the doses chosen for the chromosome aberration assay ranged from 2.5 to 300 μg/mL for the non-activated 4-hour exposure group, from 5 to 200 μg/mL for the S9-activated 4-hour exposure group, and from 5 to 300 μg/mL for the non-activated 20-hour exposure group.

In the initial chromosome aberration assay, 55 ± 5 % cytotoxicity (reduction in cell growth index relative to the vehicle control) was not observed at any dose level in the non-activated 4-hour exposure group. Cytotoxicity was observed at 200 μg/mL in the S9-activated 4-hour exposure group and at dose levels ≥ 200 μg/mL in the non-activated 20-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed at dose levels ≥75 μg/mL in all three treatment groups. The highest dose analyzed under each treatment condition exceeded the limit of solubility in treatment medium at the conclusion of the treatment period, which met the dose limit as recommended by testing guidelines for this assay.

The percentage of cells with structural or numerical aberrations in the non-activated 4 and 20-hour exposure groups was not significantly increased relative to vehicle control at any dose level (p >0.05, Fisher's Exact test).

The percentage of cells with structural aberrations in the S9-activated 4-hour exposure group was statistically significantly increased (3.0 %) relative to vehicle control at 75 μg/mL (p ≤0.01, Fisher's Exact test). However, the Cochran-Armitage test was negative for a dose response (p > 0.05). The percentage of cells with numerical aberrations in the S9-activated 4-hour exposure group was not significantly increased relative to vehicle control at any dose level (p >0.05, Fisher's Exact test).

In order to confirm the positive response observed, the chromosome aberration assay was repeated in the S9-activated 4-hour exposure group at dose levels ranging from 25 to 100 μg/mL. In the repeat assay, 55 ± 5 % cytotoxicity (reduction in cell growth index relative to the vehicle control) was not observed at any dose level in the non-activated 4-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed at dose levels ≥55 μg/mL. The highest dose analyzed exceeded the limit of solubility in treatment medium at the conclusion of the treatment period, which met the dose limit as recommended by testing guidelines for this assay.

In the repeat assay, the percentage of cells with structural aberrations was not significantly increased relative to vehicle control at any dose level (p >0.05, Fisher's Exact test). The percentage of cells with numerical aberrations in the S9-activated 4-hour exposure group was statistically significantly increased (6.0 % and 5.7 %) relative to vehicle control at dose levels 25 and 50 μg/mL, respectively (p ≤0.05, Fisher's Exact test). However, the Cochran-Armitage test was negative for a dose response (p > 0.05). In addition, the percentage of cells with numerical aberrations was within the historical control range of 0.0 % to 9.5 % and also within the 95 % control limit of historical data. Therefore, the statistically significant induction was considered to have no biological relevance.

All vehicle control values were within historical ranges, and the positive controls induced significant increases in the percent of aberrant metaphases (p ≤0.01). Thus, all criteria for a valid study were met.

Conclusion

Based on the above findings, FAT 20011/E TE was considered to be negative for the induction of structural and numerical chromosome aberrations in the non-activated and S9-activated test systems in the in vitro mammalian chromosome aberration test using CHO cells.