Disodium 5,5'-[(1-methylethylidene)bis(4,1-phenyleneoxysulphonyl-2,1-phenyleneazo)]bis[6-amino-4-hydroxynaphthalene-2-sulphonate]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Acid Red 260 was considered to be not genotoxic.

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:

Study initiation date - 06 February 1995; Experiment start date - 08 March 1995; Experiment end date - 03 April 1995; Study completion date - 26 April 1995.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Code number: FAT 21021/D
Batch-Nr.: 9
Purity: ca. 70 %
Appearance: solid
Solubility: 30 g/l
Storage: room temperature
Expiration: 12/99

Target gene:

histidine auxotrophy for S. typhimurium.

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

The bacterial strains TA 1535, TA 98 and TA 100 were obtained from Ames (University of California, 94720 Berkeley, U.S.A.).The bacterial strain TA 1537 was obtained from BASF (D-67063 Ludwigshafen).
Regular checking of the properties of the strains with regard to membrane permeability and ampicillin resistance as well as spontaneous mutation rates is performed in CCR according to Ames et al. In this way it was ensured that the experimental conditions set down by Ames were fulfilled.
Storage: The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO (MERCK, D-64293 Darmstadt) in liquid nitrogen.

Metabolic activation:

with and without

Metabolic activation system:

S9 (Preparation by C C R)
The S9 liver microsomal fraction was obtained from the livers of 8 - 12 weeks old male Wistar rats, strain Hanlbm (BRL, CH-4414 Füllinsdorf weight approx. 220 - 320 g) which received a single i.p. injection of 500 mg/kg b.w. Aroclor 1254 (Antechnika, D-76275 Ettlingen, F.R.G.) in olive oil 5 days previously. After cervical dislocation the livers of the animals were re-moved, washed in 150 mM KCI and homogenised. The homogenate was diluted 1 + 3 in KCI and centrifuged at 9,000 g for 10 minutes at 4° C. A stock of the supernatant containing the microsomes was frozen in ampoules of 2, 3 or 5 ml and stored at -80° C. Small numbers of the ampoules are kept at -20° C for up to several weeks before use. The standardisation of the protein content was made using the analysis kit of Bio-Rad Laboratories, D-80939 München: Bio-Rad protein assay, Catalogue 500 000 6 (6). The protein concentration in the S9 preparation was 33.0 mg/ml (lot 310894) in the pre-experiment and in experiment I, and 37.2 mg/ml (lot 230195) in experiment I and II.

S9 Mix
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 15 % v/v. The composition of the co-factor solution was concentrated to yield the following concentrations in the S9 mix:
8 mM MgCl2
33 mM KCl
5 mM Glucose-6-phosphate
5 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment the S9 mix will be stored in an ice bath. The S9 mix preparation will be performed according to Ames et al.

Test concentrations with justification for top dose:

With and without microsomal activation: 33.3; 100; 333; 1000; 2500; and 5000 µg/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without metabolic activation - TA 1535 and TA 100

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

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

Remarks:

Without metabolic activation - TA 1537, TA 98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, 2-AA

Remarks:

With metabolic activation - TA 1535, TA 1537, TA98, TA 100

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments: Two

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium; in agar (plate incorporation).

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Background growth inhibition.

METHODS FOR MEASUREMENTS OF GENOTOXICIY : Concentration dependent increases in revertant colony numbers.

Evaluation criteria:

The generally accepted conditions for the evaluation of the results are:
- corresponding background growth on both negative control and test plates
- normal range of spontaneous reversion rates.

A test article is considered positive if either a dose related and reproducible increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced.
A test article producing neither a dose related and reproducible increase in the number of revertants nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.
A significant response is described as follows:
A test article is considered mutagenic if in strain TA 100 the number of reversions is at least twice as high and in strains TA 1535, TA 1537 and TA 98 at least three times higher as compared to the spontaneous reversion rate.
Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the above described enhancement factors or not.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in the second experiment without metabolic activation at a concentration of 2500.0 ug/plate and higher.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in the first experiment without metabolic activation at a concentration of 1000.0 ug/plate and higher

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

Toxic effects, evidenced by a reduction in the number of revertants, occurred in the strain TA 98 in the first experiment without metabolic activation at a concentration of 1000.0 µg/plate and higher and in strain TA 1537 in the second experiment without metabolic activation at a concentration of 2500.0 µg /plate and higher. The plates incubated with the test article showed normal background growth up to 5000.0 µg/plate with and without S9 mix in all strains used. Substantial and concentration dependent increases in revertant colony numbers were observed in strain TA 98 with metabolic activation following treatment with FAT 21021/D in both experiments. This increase started at 1000.0 µg/plate in both experiments. The factor of 3 required as described under "evaluation of results" was exceeded at 5000 µg/plate in experiment I and 2500 µg/plate in experiment 2. The other strains showed no relevant increases either with or without metabolic activation in both experiments. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Conclusions:

FAT 21021/D induced gene mutations by frameshifts in the genome of the strain TA 98. Therefore, is considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.

Executive summary:

A key study was performed to investigate the potential of FAT 21021/D according to the plate incorporation test (experiment I and II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100. The assay was performed in two independent experiments, both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test substance was tested at the following concentrations: 33.3, 100, 333.3, 1000, 2500, and 5000 µg/plate (active ingredient). Toxic effects, evidenced by a reduction in the number of revertants, occurred in the strains TA 98 and TA 1537 without metabolic activation. The plates incubated with the test article showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used substantial and concentration dependent increases in revertant colony numbers were observed in strain TA 98 with metabolic activation following treatment with FAT 21021/D in both experiments. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. Under the experimental conditions reported, the test article did induce gene mutations by frameshifts in the genome of the strain TA 98. Therefore, FAT 21021/D is considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiation date - 29 June 2015; Experimental starting date - 30 June 2015; Experimental end date - 09 July 2015; Study completion date - 23 October 2015.

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)

Deviations:

no

GLP compliance:

yes

Type of assay:

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

Specific details on test material used for the study:

Identification: FAT 21021/E TE
Batch: 72
Purity: ≥75 %
Physical state/ Appearance: Red solid
Expiry date:16 February 2020
Storage Conditions: room temperature in the dark.

Target gene:

HPRT gene

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

The CHO-K1-BH4 cell line is a proline auxotroph with a modal chromosome number of 20, a population doubling time of 12-14 hours, and a cloning efficiency generally greater than 80 % (Li et al., 1987). The CHO-K1-BH4 cells used in this study were obtained from A.W. Hsie, Oak Ridge National Laboratories (Oak Ridge, TN).

CHO cells were maintained in Ham's F12 medium supplemented with 3 mM L-glutamine and 5 % (v/v) heat-inactivated and dialyzed fetal bovine serum (F12FCM5) under standard conditions (37 ± 1 °C in a humidified atmosphere of 5 ± 1 % CO2 in air). All media contained antimycotics and antibiotics.

Metabolic activation:

with and without

Metabolic activation system:

Liver Homogenate
Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from male Sprague-Dawley rats that were injected intraperitoneally with Aroclor™ 1254 (200 mg/mL in corn oil) at a dose of 500 mg/kg, five days before sacrifice. The S9 (Lot No. 3408, Expiration Date: 03 February 2017) was purchased commercially from Moltox (Boone, NC). Upon arrival at BioReliance, the S9 was stored at -60 °C or colder until used. The lot of S9 was assayed for sterility and its ability to metabolize at least two pro-mutagens to forms mutagenic to Salmonella typhimurium TA100. The Record of Analysis is on file with the Testing Facility.

S9 Mix
The S9 mix was prepared on the day of use. The final concentrations of the components in the mix were as indicated below.
Component Final Concentration in Cultures
NADP (sodium salt) 0.8 mM
Glucose-6-phosphate 1 mM
Calcium chloride 2 mM
Potassium chloride 6 mM
Magnesium chloride 2 mM
Sodium Phosphate 10 mM
S9 homogenate 20 μL/mL

Test concentrations with justification for top dose:

FAT 21021/E was prepared in DMSO and evaluated in a preliminary toxicity assay at concentrations of 2.44, 4.88, 9.77, 19.5, 39.1, 78.1, 156, 313, 625 and 1250 μg/mL with and without S9. FAT 21021/E was evaluated in the definitive mutagenicity assay at concentrations of 1.25, 2.50, 5.00, 10.0, 15.0 and 19.5 μg/mL with and without S9.

Vehicle / solvent:

The vehicle used to prepare the test substance dose formulations, and also used as the vehicle control was DMSO.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without S9 activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With S9 activation

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration; Duplicate
- Number of independent experiments: One

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 10E6
- Test substance added in medium: in suspension.

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 5 ± 0.5 hours

FOR GENE MUTATION:
- Expression time: The cultures were subcultured for 7 days, at 2- to 3-day intervals, to maintain logarithmic growth and permit expression of the mutant phenotype.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Cloning efficiency.

METHODS FOR MEASUREMENTS OF GENOTOXICIY : Dose-dependent increase in mutant frequency.

Evaluation criteria:

Evaluation of Test Results
Statistical analyses were performed using the method of Snee and Irr (1981), with significance established at the 0.05 level. Once criteria for a valid assay were met, the responses observed in the assay were evaluated as follows. The test substance was considered to have produced a positive response if it induced a statistically significant and dose-dependent increase in mutant frequency (p ≤0.05) that exceeded the 95 % confidence limit of the historical vehicle control data from this laboratory. If only one criterion was met (a statistically significant or dose-dependent increase or an increase exceeding the historical control 95 % confidence interval), the results were considered equivocal. If none of these criteria were met, the results were considered to be negative. Other criteria also may be used in reaching a conclusion about the study results (e.g., comparison to historical control values, biological significance, etc.). In such cases, the Study Director used sound scientific judgment and clearly reported and described any such considerations.

Statistics:

The primary computer or electronic systems used for the collection of data or analysis included, but were not limited to, the following:


System Purpose
LIMS Labware System Test Substance tracking
Excel 2007 (Microsoft Corporation) Calculations
Kaye Lab Watch Monitoring system (Kaye GE) Environmental monitoring
BRIQS Deviation and audit reporting
ProtoCOL Colony Counter Data collection

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Solubility Test: DMSO was selected as the solvent of choice based on the solubility of the test substance and compatibility with the target cells. Following sonication at 34.0 °C for 35 minutes, the test substance formed a workable suspension in DMSO at a concentration of ~50 mg/mL in the solubility test conducted at BioReliance.

Preliminary Toxicity Assay
Results of the preliminary toxicity assay are presented in Table 1. FAT 21021/E TE was prepared in DMSO and evaluated in a preliminary toxicity assay at concentrations of 2.44, 4.88, 9.77, 19.5, 39.1, 78.1, 156, 313, 625 and 1250 μg/mL with and without S9. The maximum dose evaluated was based on the solubility of the test substance in the vehicle. The test substance formed clear solutions in DMSO from 0.244 to 0.977 mg/mL and workable suspensions in DMSO from 1.95 to 125 mg/mL. Visible precipitate was observed at concentrations ≥156 μg/mL at the beginning of treatment and at concentrations ≥19.5 μg/mL by the end of treatment. The pH of the cultures was adjusted at a concentration of 1250 μg/mL to maintain neutral pH, and the test substance had no adverse impact on the osmolality of the cultures [421, 429, 418 and 413 mmol/kg for the vehicle control, the highest concentration (1250 μg/mL), the lowest precipitating concentration (156 μg/mL) and the highest soluble concentration (78.1 μg/mL), respectively]. Adjusted relative survival was 37.90 and 8.42 % at a concentration of 1250 μg/mL with and without S9, respectively.

Definitive Mutagenicity Assay
Based on the results of the preliminary toxicity assay, FAT 21021/E was evaluated at concentrations of 1.25, 2.50, 5.00, 10.0, 15.0 and 19.5 μg/mL with and without S9. No visible precipitate was observed at the beginning of treatment; however, visible precipitate was observed at concentrations ≥10.0 μg/mL by the end of treatment, and the test substance had no adverse impact on the pH of the cultures. The average adjusted relative survival was 118.01 and 67.70 % at a concentration of 19.5 μg/mL with and without S9, respectively. Cultures treated at concentrations of 2.50, 5.00, 10.0, 15.0 and 19.5 μg/mL with and without S9 were chosen for mutant selection (cultures treated at a concentration of 1.25 μg/mL with and without S9 were excluded from evaluation of mutagenicity because a sufficient number of higher concentrations was available). No significant increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at any concentration evaluated with or without S9 (p >0.05). In contrast, the positive controls induced a significant increase in mutant frequency (p <0.01).
All positive and vehicle control values were within acceptable ranges, and all criteria for a valid study were met.

Conclusions:

The results indicate FAT 21021/E was negative in the In Vitro Mammalian Cell Forward Gene Mutation (CHO/HPRT) Assay with Duplicate Cultures, under the conditions and according to the criteria of the test protocol.

Executive summary:

The test substance, FAT 21021/E, was evaluated for its ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system (S9), as assayed by colony growth in the presence of 6-thioguanine (TG resistance, TGr). FAT 21021/E was prepared in DMSO and evaluated in a preliminary toxicity assay at concentrations of 2.44, 4.88, 9.77, 19.5, 39.1, 78.1, 156, 313, 625 and 1250 μg/mL with and without S9. The maximum dose evaluated was based on the solubility limitations of the test substance in the vehicle. Visible precipitate was observed at concentrations ≥156 μg/mL at the beginning of treatment and at concentrations ≥19.5 μg/mL by the end of treatment. The pH of the cultures was adjusted at a concentration of 1250 μg/mL to maintain neutral pH, and the test substance had no adverse impact on the osmolality of the cultures. Adjusted relative survival was 37.90 and 8.42 % at a concentration of 1250 μg/mL with and without S9, respectively. Based on these results, FAT 21021/E was evaluated in the definitive mutagenicity assay at concentrations of 1.25, 2.50, 5.00, 10.0, 15.0 and 19.5 μg/mL with and without S9. No visible precipitate was observed at the beginning of treatment; however, visible precipitate was observed at concentrations ≥10.0 μg/mL by the end of treatment, and the test substance had no adverse impact on the pH of the cultures. The average adjusted relative survival was 118.01 and 67.70% at a concentration of 19.5 μg/mL with and without S9, respectively. Cultures treated at concentrations of 2.50, 5.00, 10.0, 15.0 and 19.5 μg/mL with and without S9 were chosen for mutant selection (cultures treated at a concentration of 1.25 μg/mL with and without S9 were excluded from evaluation of mutagenicity because a sufficient number of higher concentrations was available). No significant increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at any concentration evaluated with or without S9 (p >0.05). In contrast, the positive controls induced a significant increase in mutant frequency (p <0.01). All positive and vehicle control values were within acceptable ranges, and all criteria for a valid study were met. These results indicate FAT 21021/E was negative in the In Vitro Mammalian Cell Forward Gene Mutation (CHO/HPRT) Assay with Duplicate Cultures, under the conditions and according to the criteria of the test protocol.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study Initiation Date - 26 October 2015; Experimental Starting Date - 16 November 2015; Experimental Completion Date - 31 December 2015; Study Completion Date - 10 February 2016.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Identification: FAT 21021/E TE
Batch: 72
Purity: ≥75 %
Physical state/ Appearance: Red solid
Expiry date: 16 February 2020
Storage Conditions: room temperature in the dark.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Chinese hamster ovary (CHO-K1) cells (repository number CCL 61) were obtained from American Type Culture Collection, Manassas, VA. In order to assure the karyotypic stability of the cell line, working cell stocks were not used beyond passage 15. The frozen lot of cells was tested using the Hoechst staining procedure and found to be free of mycoplasma contamination. This cell line has an average cell cycle time of 10-14 hours with a modal chromosome number of 20. The use of CHO cells has been demonstrated to be an effective method of detection of chemical clastogens.

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 10E5 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. The S9 was prepared from male Sprague-Dawley rats that were injected intraperitoneally with Aroclor™ 1254 (200 mg/mL in corn oil) at a dose of 500 mg/kg, five days before sacrifice. The S9 (Lot No. 3330, Exp. Date: 09 Sep 2016) was purchased commercially from MolTox (Boone, NC). Upon arrival at BioReliance, the S9 was stored at -60 °C or colder until used. Each bulk preparation of S9 was assayed for its ability to metabolize benzo(a)pyrene and 2-aminoanthracene to forms mutagenic to Salmonella typhimurium TA100. The S9 mix was prepared on the day of use and added to the test system at 20 % (v/v). The final concentrations of the components in the test system are indicated below:
Component Final Concentration in Cultures
NADP (sodium salt) 1 mM
Glucose-6-phosphate 1 mM
Potassium chloride 6 mM
Magnesium chloride 2 mM
S9 homogenate 20 μL/mL

Test concentrations with justification for top dose:

0.2. 0.6, 2, 6, 20, 60, 200 600, 2000 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMF
- Justification for choice of solvent/vehicle: DMF was used as the vehicle based on the solubility tests and compatibility with the target cells.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Duplicate
- Number of independent experiments
: One

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: FAT 21021/E: 150 for structural, 150 for numerical; Positive controls: 75 for structural, 150 for numerical.
- Test substance added in medium; in suspension.

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment:
Based on the results of the preliminary toxicity test, the dose levels selected for testing in the chromosome aberration assay were as follows:
Without metabolic activation (4 hr): 100, 250, 500, 1000, 1500, 2000 μg/mL
Without metabolic activation (20 hr): 25, 100, 200, 250, 300, 325, 350, 375, 400, 425, 450, 500 μg/mL
With metabolic activation (4 hr): 25, 50, 100, 250, 500 μg/mL
- Harvest time after the end of treatment: 20 hours.

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Methods of slide preparation and staining technique used including the stain used: To prepare slides, the cells were collected by centrifugation and the cells were resuspended in fresh fixative (methanol : glacial acetic acid 3:1 v/v). 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.
- Number of cells spread and analysed per concentration: The percentage of cells in mitosis per 500 cells scored (mitotic index) was determined and recorded for each coded treatment group selected for scoring chromosomal aberrations.
- Determination of polyploidy and endoreplication:
The percentage of cells with numerical aberrations (polyploid and endoreduplicated cells) was evaluated for 150 cells per culture (a total of 300 per dose level).


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative increase in cell count (RICC)

METHODS FOR MEASUREMENTS OF GENOTOXICIY
: frequency of cells with structural chromosomal aberrations.

Evaluation criteria:

Criteria for Determination of a Valid Test:

Vehicle Controls: The frequency of cells with structural chromosomal aberrations should ideally be within the 95 % control limits of the distribution of the historical negative control database. If the concurrent negative control data fall outside the 95 % control limits, they may be acceptable as long as these data are note extreme outliers (indicative of experimental or human error).

Positive Controls: The frequency of cells with structural chromosomal aberrations must be significantly greater than the concurrent vehicle control (p ≤ 0.05). In addition, the cytotoxicity response must not exceed the upper limit for the assay (60 %).

Cell Proliferation: The average viable cell count in the vehicle control at harvest must be ≥ 1.5-fold the average viable cell baseline value.
Test Conditions:
The test substance must be tested using a 4-hour treatment with and without S9, as well as a 20-hour treatment without S9. However, all three treatment conditions need not be evaluated in the case of a positive test substance response under any treatment condition.

Analyzable Concentrations:
At least 300 metaphases must be analyzed from at least three appropriate test substance concentrations. The number of metaphases scored may be reduced when high numbers of cells with chromosomal aberrations (≥10 % metaphases) are observed as with a positive test substance or the positive control substance.

Evaluation of Test Results:
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.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

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 specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS

- Effects of pH: The pH of the highest dose level of test substance in treatment medium was 7.4.
- Effects of osmolality: 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 %.

COMPARISON WITH HISTORICAL CONTROL DATA:
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.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxicity (≥50 % reduction in cell growth index relative to the vehicle control) was not observed at any dose in the non activated and S9-activated 4-hour exposure groups. Cytotoxicity was observed at doses ≥600 µg/mL in the non activated 20-hour exposure group.

Preliminary Toxicity Assay

In the preliminary toxicity assay, CHO cells were exposed to nine dose levels of FAT 21021/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 DMSO from 20 to 200 mg/mL and formed clear solutions in DMF from 0.02 to 6 mg/mL. Visible precipitate was observed in treatment medium at the following dose levels:

Treatment Condition	Treatment Time	Visible Precipitation
		At the beginning of treatment period	At the conclusion of treatment period
Non-activated	4 hr	≥ 2000 µg/mL	≥ 2000 µg/mL
	20 hr	≥ 2000 µg/mL	≥ 2000 µg/mL
S9-activated	4 hr	≥ 2000 µg/mL	≥ 200 µg/mL

The osmolality in treatment medium was measured as follows:

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 7.48.

Dose tested	Dose levels (µg/mL)	Osmolality (mmol/kg)
Vehicle	0	288
Highest soluble	600	293
Highest	2000	304

In the chromosome aberration assay, the test substance formed workable suspensions in DMF from 10 to 200 mg/mL and formed clear solutions in DMF from 2.5 to 5 mg/mL. Visible precipitate was observed in treatment medium at the following dose levels:

Treatment condition	Treatment Time	Visible Precipitation
		At the beginning of treatment period	At the conclusion of treatment period
Non-activated	4 hr	≥ 1000 µg/mL	≥ 1000 µg/mL
	20 hr	None	None
S9-activated	4 hr	None	≥ 100 µg/mL

Conclusions:

Under the conditions of the assay described in this report, FAT 21021/E was concluded to be negative for the induction of structural and numerical chromosome aberrations in both non-activated and S9-activated test systems in the in vitro mammalian chromosome aberration test using CHO cells.

Executive summary:

The test substance, FAT 21021/E, was tested in the chromosome aberration assay using Chinese hamster ovary (CHO) cells following OECD guideline 473 in compliance to GLP in both the absence and presence of an Aroclor induced rat liver S9 metabolic activation system. 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. Dimethyl formamide (DMF) was used as the vehicle. 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 not observed at any dose in the non activated and S9-activated 4-hour exposure groups. Cytotoxicity was observed at doses ≥600 µg/mL in the non activated 20-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed at 2000 µg/mL in the non-activated 4 and 20-hour exposure groups, and at dose levels ≥200 µg/mL in the S9-activated 4-hour exposure group. Based on these findings, the doses chosen for the chromosome aberration assay ranged from 100 to 2000 µg/mL for the non activated 4-hour exposure group, from 25 to 500 µg/mL for the S9 activated 4-hour and the non activated 20 hour exposure groups. In the chromosome aberration assay, 55 ± 5 % cytotoxicity (reduction in cell growth index relative to the vehicle control) was not observed at any dose in the non activated and S9-activated 4-hour exposure groups. Cytotoxicity was observed at doses ≥450 µg/mL in the non activated 20-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed at doses ≥1000 µg/mL in the non activated 4-hour exposure group; and at dose levels ≥100 µg/mL in the S9 activated 4-hour exposure group. The dose levels selected for microscopic analysis were 250, 500, and 1000 µg/mL for the non activated 4-hour exposure group; 25, 50, and 100 µg/mL for the S9 activated 4-hour exposure group, and 100, 300, and 450 µg/mL for the non activated 20-hour exposure group. No significant or dose dependent increases in structural or numerical (polyploid or endoreduplicated cells) aberrations were observed in any of the test substance treated groups (p > 0.05; Fisher’s Exact and Cochran-Armitage tests). However, in the S9-activated 4-hour exposure group, a statistically significant increase in numerical aberrations was observed in the untreated control group. The untreated control was included in order to compare with DMF and confirm that DMF was not too toxic. Moreover, the numerical aberrations was increased only in S9-activated 4-hour treatment while in the same experimental condition in non-activated 4 and 20-hour treatments, the values were within normal range. Since the induction of chromosome aberrations was compared to that of DMF and not with the untreated control, the Study Director, upon consultation with the Sponsor has concluded that these results had no impact on the study. 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. Under the conditions of the assay described in this report, FAT 21021/E was concluded to be negative for the induction of structural and numerical chromosome aberrations in both non-activated and S9-activated test systems in the in vitro mammalian chromosome aberration test using CHO cells.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

An in vitro study was performed to investigate the potential of Acid Red 260 to induce gene mutations according to OECD Guideline 471 and EU Method B.14 in compliance with GLP. The test article FAT 21021/D; was assessed for its potential to induce gene mutations according to the plate incorporation test using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100. A dose dependent increase in revertant colony numbers were observed in strain TA 98 with metabolic activation. There was no relevant increase with any of the other strains with and without metabolic activation. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did induce gene mutations by frameshifts in the genome of the strain TA 98. FAT 21021/E was tested in the chromosome aberration assay using Chinese hamster ovary (CHO) cells following OECD Guideline 473 in compliance to GLP in both the absence and presence of an Aroclor induced rat liver S9 metabolic activation system. 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, in two phases. All cells were harvested 20 hours after treatment initiation. No significant or dose dependent increases in structural or numerical (polyploid or endoreduplicated cells) aberrations were observed in any of the test substance treated groups (p >0.05; Fisher’s Exact and Cochran-Armitage tests). Hence, FAT 21021/E was considered to be not clastogenic in this chromosomal aberration assay. In a mammalian cell gene mutation assay (HPRT locus) conducted according to OECD Guideline 476, CHO cells cultured in vitro were exposed to the substance dissolved in culture medium at various concentrations. No significant increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at any concentration evaluated with or without S9 (p> 0.05). Hence, FAT 21021/E considered to be non-mutagenic in the HPRT locus using CHO cells of the Chinese Hamster.

Conclusion:
Acid Red 260 did lead to mutagenic effect with strain TA 98 with metabolic activation in the bacterial reverse mutation assay. However, the mutagenic response could not be reproduced in the mammalian cells when tested in the in vitro mammalian cell gene mutation assay. Acid Red 260 did not lead to clastogenic effects in the in vitro chromosmal aberration assay as well. Taking these results from mammalian cells into account the test substance is considered to be neither mutagenic nor clastogenic, hence not genotoxic.

Justification for classification or non-classification

Based on the available genotoxicity studies, the test substance does not need to be classified for genotoxicity according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.