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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An in vitro study was performed to investigate the potential of the test substance to induce gene mutations according to OECD Guideline 471 and EU Method B.14 in compliance with GLP. Under the study conditions, the test substance was considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay. A second study was conducted according to OECD guideline no. 476 and in accordance with GLP. In a mammalian cell gene mutation assay (HPRT locus), CHO cells cultured in vitro were exposed to FAT 41001/H TE dissolved in culture medium at various concentrations. In the described in vitro cell gene mutagenicity test under the experimental conditions reported, the test item is considered to be non-mutagenic in the HPRT locus using CHO cells of the Chinese Hamster. A third study was conducted with the test item according to OECD guideline no. 473 and in accordance with GLP. The test article FAT 41001/H was assessed for its potential toinduce structural chromosomal aberrations in CHO cells of the Chinese hamster in vitro in two independent experiments. The substanceis considered to be non-mutagenic in thischromosome aberration test. Results of the three studies indicate that the test item is not classified as genotoxic.


Justification for selection of genetic toxicity endpoint
All three in vitro studies (OECD 471, OECD 473 and OECD 476) were taken into consideration.

Short description of key information:
The test substance showed negative results in the Salmonella typhimurium reverse mutation assay (OECD 471), in a mammalian cell gene mutation assay (OECD 476) and in chromosome aberrations test (OECD 473).

Endpoint Conclusion: No adverse effect observed (negative)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1993
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
Qualifier:
according to guideline
Guideline:
EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Test No.: 930844
Test material: Cibacron Blau P-3R roh Lösung für Flüssighandelsform (FAT 41001/F)
Batch No.: Op.Nr.2 2 eingedampft
Purity: about 54 % (active ingredient)
Stability of the test material: see Expiration date
Expiration date: June 1997
Vehicle: Bidistilled water
Study initiation date: September 13, 1993
Experimental start date: September 21, 1993
Experimental termination date: November 15, 1993
Study termination date: December 15, 1993
Target gene:
histidine-requiring strains of Salmonella typhimurium
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
TA 98, TA 102, TA 1535 and TA 1537 - Prof. B. Ames, Berkeley, CA., U.S.A
TA 100 - M. Schüpbach, Hoffmann-La Roche Ltd., Basel, Switzerland.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Range in the cytotoxicity test: 20.58, 61.73, 185.19, 555.56, 1667.67, 5000 µg/plate

Range in the original mutagenicity test: 61.73, 185.19, 555.56, 1667.67, 5000 µg/plate

Range in the confirmatory mutagenicity test: 114.3, 342.93, 1028.78, 3086.33, 9259 µg/plate
Vehicle / solvent:
Bidistilled water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
bidistilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without microsomal activation for Salmonella TA 100, TA 1535
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
bidistilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without microsomal activation for TA 102
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without microsomal activation; Salmonella TA 98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without microsomal activation; Salmonella TA 1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with microsomal activation; Salmonella TA 100, 102, 98, 1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
bidistilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with microsomal activation; Salmonella TA 1535
Details on test system and experimental conditions:
Preparation of the bacterial cultures:
Inoculates from frozen master copies were set up monthly. They were grown in liquid NB-medium overnight and then plated on NB agar. After incubation, single colonies were taken from the plates, grown overnight in liquid NB-medium and then used for the experiment.

Preparation of the metabolic activation mixture:
Rat-liver microsomal fraction S9 was prepared in advance from male RAI rats (Tif: RAIf[SPF]), reared at the Animal Farm of CIBA- GEIGY, Sisseln, Switzerland. The animals (150-250 g) were treated with Aroclor 1254 (500 mg/kg, i.p.) 5 days prior to sacrifice. The livers were homogenized with 3 volumes of 150 mM KCl and the 9000x g supernatant (S9) was stored at approximately -80 °C for no longer than one year. The protein contents of the S9 fractions were 30.2 and 28.9 mg/ml.
The activation mixture (Ref. 6) contained:
Rat liver S9 fraction: 100 µl/ml
NADP: 4 µmol/ml
MgCl2: 8 µmol/ml
KCl: 33 µmol/ml
Na-phosphate-buffer, pH 7.4 100 µmol/ml
Glucose-6-phosphate: 5 µmol/ml

Solubilization of the test substance:
The test substance was dissolved in bi-distilled water at the concentrations of 50 mg/ml (toxicity test and original mutagenicity test) and 92.6 mg/ml (confirmatory mutagenicity test). Lower concentrations of the test material were obtained by appropriate dilution of the stock solutions with bi-distilled water. No precipitates or aggregates were noted.

Setting up of the test plates:
0.1 ml of the overnight cultures were mixed with 2 ml of top agar, either 0.5 ml of 100 mM sodium phosphate buffer (experiments without activation) or 0.5 ml of the activation mixture (experiments with activation) and 0.1 ml of a solution of the test substance, the substance for the positive control or the solvent for the negative control and poured on minimal agar in Petri dishes. Each Petri dish contained about 20 ml of minimal agar (1.5% agar supplemented with 2% salts of the Vogel-Bonner Medium E and 2% glucose). The top agar was composed of 0.6% agar and 0.6% NaCl. It was supplemented with 10% of 0.5 mM 1-histidine and 0.5 mM (+) biotin dissolved in water.

Preliminary Toxicity/Ranqe-Finding test:
A toxicity test (check for reduction in the number of revertant colonies) was carried out with strain TA 100 without and with microsomal activation at six concentrations of the test substance and one negative control according to Standard Operating Procedures of Genetic Toxicology. The highest concentration applied was 5000 µg/plate. The five lower concentrations decreased by a factor of 3. The plates were inverted and incubated for about 48 hours at 37 ± 1.5°C in darkness. Thereafter, they were evaluated by counting the colonies and determining the background lawn. One plate per test substance concentration, as well as each negative control was used.

Mutagenicity test:
The mutagenicity test was performed with strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 without and with microsomal activation according to Standard Operating Procedures of Genetic Toxicology. Each of the five concentrations of the test substance, a negative and a positive control were tested, using three plates per test substance concentration as well as each positive and negative control with each tester strain. The highest concentration applied in the first mutagenicity assay was 5000 µg/plate (because of lack of toxicity in the range finding test) and the four lower concentrations were each decreased by a factor of 3. The plates were inverted and incubated for about 48 hours at 37 ± 1.5°C in darkness. Thereafter, they were evaluated by counting the number of colonies and determining the background lawn.
Evaluation criteria:
The test substance is considered to be mutagenic in this test system if the following conditions are met:
At least a reproducible meaningful increase of the mean number of revertants per plate above that of the negative control at any concentration for one or more of the following strains: S. typhimurium TA 98, TA 100, TA 102, TA 1535 and TA 1537.
Generally a concentration-related effect should be demonstrable.
Statistics:
In deviation to the OECD guideline a statistical analysis was not performed. At present the use of statistical methods concerning this particular test system is not generally recommended. No appropriate statistical method is available.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
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
Additional information on results:
None
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Toxicity test/Range finding test:

Normal back-ground growth was observed at all concentrations. The test material exerted no toxic effect on the growth of the bacteria. From the results obtained, the highest concentration suitable for the first mutagenicity test was selected to be 5000 µg/plate without and with activation.

 

Mutagenicity test, original experiment: (concentration range: 61.7 to 5000 µg/plate)

In the experiment performed without and with metabolic activation, treatment with Cibacron Blau P-3R roh Lösung für Flüssighandelsform (FAT 41001/F) did not lead to an increase in the incidence of histidine-prototrophic mutants in comparison with the negative control.

 

Mutagenicity test, confirmatory experiment: (concentration range: 114.3 to 9259 µg/plate)

In the experiment carried out without and with metabolic activation, again none of the tested concentrations of Cibacron Blau P- 3R roh Lösung für Flüssighandelsform (FAT 41001/F) led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control. In the mutagenicity tests without and with metabolic activation, normal back-ground growth was observed. The numbers of revertant colonies were not reduced. The test material exerted no toxic effect on the growth of the bacteria. The various mutagens, pro-mutagens, sterility checks, sensitivity and resistance tests, etc., employed to ensure the test system was acceptable, all produced results within our established limits. There were no known circumstances or occurrences in this study that were considered to have affected the quality or integrity of the data.

Conclusions:
FAT 41001/F and its metabolites did not induce gene mutations in the strains of S. typhimurium used.
Executive summary:

The aim of this test was to evaluate the test compound for mutagenic activity in bacterial test systems (Salmonella typhimurium: TA 98, TA 100, TA 102, TA 1535 and TA 1537) in the absence and presence of a rat liver S9 activity system. This study was conducted in accordance with OECD test guideline 471, EU method B.13/14 and EPA OTS 798.5265 (Bacterial reverse mutation test). The concentration range of FAT 41001/F to be tested in the mutagenicity test was determined in a preliminary toxicity test. Thus, FAT 41001/F was tested for mutagenic effects without and with metabolic activation at five concentrations in the range of 61.7 to 5000 µg/plate. An independent repetition of the experiments was performed with the concentrations of 114.3 to 9259 µg/plate. The active ingredient is about 54%. 9259 µg/plate correspond to the concentration of about 5000 µg/plate of pure substance.

Toxicity test/Range finding test

In the experiment without and with metabolic activation no toxic effect of the test material on the growth of the bacteria was observed. Mutagenicity test, original experiment (concentration range: 61.7 to 5000 µg/plate). In the original experiment carried out without and with metabolic activation, none of the tested concentrations of FAT 41001/F led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control. Mutagenicity test, confirmatory experiment (concentration range: 114.3 to 9259 µg/plate) In the confirmatory experiment performed without and with metabolic activation, again, none of the tested concentrations of (FAT 41001/F) led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control. In the mutagenicity tests without and with metabolic activation, normal background growth was observed. The number of revertant colonies was not reduced. The test substance exerted no toxic effect on the growth of the bacteria. Based on the results of these experiments and on standard evaluation criteria, it is concluded that FAT 41001/F and its metabolites did not induce gene mutations in the strains of S. typhimurium used.

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
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
Specific details on test material used for the study:
Identification: FAT 41001/H TE
Purity: 77.3 %
Molecular Weight: 882.18 g/mol
Batch / Lot No.: BOP 01-12 (lot: BS-1106872)
Expiration Date: 31 January 2017
Description: Dark red-violet powder MSDA: Blue
Storage Conditions: -10 to -30 °C, protected from light
Receipt Date: 02 March 2015
Target gene:
Not applicable
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.
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:
- In the preliminary toxicity assay without metabolic activation (4-hours treatment and 16 hour recovery period): 0.2, 0.6, 2, 6, 20, 60, 200, 600 and 2000 μg/mL.
- In the preliminary toxicity assay without metabolic activation (20 hour continuous treatment): 0.2, 0.6, 2, 6, 20, 60, 200,600 and 2000 μg/mL.
- In the preliminary toxicity assay with metabolic activation (4-hours treatment and 16 hour recovery period): 0.2, 0.6, 2, 6, 20, 60, 200,600 and 2000 μg/mL.

- Chromosome aberration assay without metabolic activation (4-hours treatment and 16 hour recovery period): 50, 100, 250, 500, 750 and 1000 μg/mL.
- Chromosome aberration assay without metabolic activation (20 hour continuous treatment):50, 100, 200, 250, 300, 350, 400, 450, 500, 550, 600 and 750 μg/mL.
- Chromosome aberration assay with metabolic activation (4-hours treatment and 16 hour recovery period): 50, 100, 250, 500, 750 and 1000 μg/mL.

Analyses to determine the concentration, uniformity and stability of the test substance dose formulations were not performed.
Vehicle / solvent:
Water was used as the vehicle based on the solubility of the test substance and compatibility with the target cells.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Remarks:
dissolved ind diluted in distilled water.
Positive control substance:
mitomycin C
Remarks:
MMC used in the non-activated test system at 0.1 and 0.2 μg/mL
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Remarks:
dissolved and diluted in distilled water.
Positive control substance:
cyclophosphamide
Remarks:
Cyclophosphamide used in the S9 activated test system at 2.5, 5 and 7.5 μg/mL.
Details on test system and experimental conditions:
Preparation of target cells:
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.

NUMBER OF REPLICATIONS:
- In the preliminary toxicity test: single cultures.
- In the chromosome aberration assay: duplicate cultures.

DURATION (Exposure duration):
* For the chromosome aberration assay without metabolic activation:
- 4 hours (treatment time) and 16 hours (recovery time)
- 20 hours continuous treatment

* For the chromosome aberration assay with metabolic activation:
- 4 hours (treatment time) and 16 hours (recovery time)

METAPHASE-ARRESTING SUBSTANCE: 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.

TREATMENT OF TARGET CELLS (PRELIMINARY AND DEFINITIVE TEST):
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.

COLLECTION OF METAPHASE CELLS (PRELIMINARY AND DEFINITIVE TEST):
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. The cell counts and percent viability were used to determine cell growth inhibition relative to the vehicle control (% cytotoxicity). The cell growth in the treatment group relative to the vehicle control, was calculated based on the following formula:

RICC (%) = 100 - [(mean viable cellstreated - mean viable cellsbaseline) / (mean viable cellssolvent - mean viable cellsbaseline)]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.

NUMBER OF CELLS EVALUATED:
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. Slides were coded using random numbers by an individual not involved with the scoring process. Metaphase cells with 20 ± 2 centromeres were examined under oil immersion without prior knowledge of treatment groups. A minimum of 300 metaphase spreads from each dose level (150 per duplicate culture) were examined and scored for chromatid-type and chromosome-type aberrations (Scott et al., 1990). The number of metaphase spreads that were examined and scored per duplicate culture may be reduced if the percentage of aberrant cells reaches a significant level (at least 10% determined based on historical positive control data) before 150 cells are scored. Chromatid-type aberrations include chromatid and isochromatid breaks and exchange figures such as quadriradials (symmetrical and asymmetrical interchanges), triradials, and complex rearrangements. Chromosome-type aberrations include chromosome breaks and exchange figures such as dicentrics and rings. Fragments (chromatid or acentric) observed in the absence of any exchange figure were scored as a break (chromatid or chromosome). Fragments observed with an exchange figure will not be scored as an aberration but were considered part of the incomplete exchange. Pulverized cells and severely damaged cells (counted as 10 aberrations) were also recorded. Chromatid and isochromatid gaps were recorded but not included in the analysis. The XY vernier for each cell with a structural aberration was recorded. 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).
Evaluation criteria:
The test substance was considered to have induced a positive response if:
• at least one of the test concentrations exhibits a statistically significant increase when compared with the concurrent negative control (p ≤ 0.05), and
• the increase is concentration-related (p ≤ 0.05), and
• results are 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.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
Precipitate is visible at the beginning and at the end of treatment period at >= 750 μg/mL (exposure time 4 hours)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Toxicity of test item in CHO cells when treated for 4 hours in the absence of S9 activation was 37% at 750 μg/mL.Precipitate is visible at the beginning and at the end of treatment period at >= 750 μg/mL (exposure time 4 hours)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Toxicity of test item in CHO cells when treated for 20 hours in the absence of S9 activation was 52% at 300 μg/mL.Preciüitate is visible at 750 μg/mL at the beginning of treatment period and at >=550 μg/mL at the end of the treatment period.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
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, upon sonication for ten minutes at 27.0oC, the test substance formed workable suspensions in water at a concentration range of approximately 10 to 50 mg/mL.

PRELIMINARY TOXICITY ASSAY.

The preliminary toxicity assay was conducted to observe the cytotoxicity profile of the test substance and to select suitable dose levels for the definitive chromosome aberration assay. CHO cells were first exposed to nine dose levels, 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 ≥ 2 mg/mL, while concentrations ≤ 0.6 mg/mL were soluble in water. Visible precipitate (the culture was opaque dark blue) was observed in treatment medium at the following dose levels:

        Visible precipitate
   Treatment time  At the beginning of treatment period  At the conclusion of treatment period
 Non-activated  4 hr  ≥ 2000 μg/mL  ≥ 600 μg/mL
 Non-activated  20 hr  ≥ 2000 μg/mL  ≥ 600 μg/mL
 S9 -activated  4 hr  ≥ 2000 μg/mL  ≥ 600 μg/mL

The osmolality in treatment medium was measured as follows:

 Dose tested  Dose levels (μg/mL)  Osmolality (mmol/kg)
 Vehicle  0  270
 Highest  2000  302
 Lowest precipitating  600  273

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 7.44.

Cytotoxicity (≥ 50% reduction in cell growth index relative to the vehicle control) was observed at 2000 μg/mL in the non-activated 4-hour exposure group and at dose levels ≥ 600 μg/mL in the non-activated 20-hour exposure group. Cytotoxicity was not observed at any dose levels in the S9-activated 4-hour exposure group.

CHROMOSOME ABERRATION ASSAY:

In the chromosome aberration assay, the test substance formed workable suspensions in water at concentrations ≥ 1 mg/mL, while concentration 0.5 mg/mL was soluble in water. The pH of the highest dose level of test substance in treatment medium was 7.33. Visible precipitate (the culture was opaque dark blue) was observed in treatment medium at the following dose levels:

      Visible precipitate
   Treatment time  At the beginning of treatment period  At the conclusion of treatment period
 Non-activated  4 hr  ≥ 750 μg/mL  ≥ 750 μg/mL
 Non-activated  20 hr  7500 μg/mL  ≥ 550 μg/mL
 S9 -activated  4 hr  ≥ 750 μg/mL  ≥ 750 μg/mL

Toxicity of tht test item (cell growth inhibition relative to the vehicle control) in CHO cells when treated for 4 hours in the absence of S9 activation was 37% at 750 μg/mL, the highest test dose level evaluated for chromosome aberrations.The mitotic index at the highest dose level evaluated for chromosome aberrations, 750 μg/mL, was 18% reduced relative to the vehicle control. The dose levels selected for microscopic analysis were 100, 250, and 750 μg/mL. The percentage of cells with structural or numerical aberrations in the test substance-treated group was not significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher's Exact test).

Toxicity of the substance (cell growth inhibition relative to the vehicle control) in CHO cells when treated for 4 hours in the presence of S9 activation was not observed at 750 ìg/mL, the highest test dose level evaluated for chromosome aberrations. The mitotic index at the highest dose level evaluated for chromosome aberrations, 750 μg/mL, was 7% reduced relative to the vehicle control. The dose levels selected for microscopic analysis were 100, 250, and 750 μg/mL. The percentage of cells with structural or numerical aberrations in the test substance-treated group was not significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher's Exact test). The percentage of structurally aberrant cells in the CP (positive control) treatment group (26.0%) was statistically significant (p ≤ 0.01, Fisher's Exact test).

Toxicity of the test item (cell growth inhibition relative to the vehicle control) in CHO cells when treated for 20 hours in the absence of S9 activation was 52% at 300 μg/mL, the highest test dose level evaluated for chromosome aberrations. The mitotic index at the highest dose level evaluated for chromosome aberrations, 300 μg/mL, was 50% reduced relative to the vehicle control. The dose levels selected for microscopic analysis were 100, 200, and 300 μg/mL. The percentage of cells with structural or numerical aberrations in the test substance-treated group was not significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher's Exact test). The percentage of structurally aberrant cells in the MMC (positive control) treatment group (24.0%) was statistically significant (p ≤ 0.01, Fisher's Exact test).

The positive and vehicle controls fulfilled the requirements for a valid test.

Under the conditions of the assay described in this report, the test substance 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.

Conclusions:
FAT 41001/H 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:

The test substance, FAT 41001/H 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. This test was conducted based on test guideline OECD 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 the 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 (77.3%), using a correction factor of 1.29.

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, upon sonication for ten minutes at 27 °C, the test substance formed workable suspensions in water at a concentration range of approximately 10 to 50 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 2000 μg/mL in the non-activated 4-hour exposure group and at dose levels ≥ 600 μg/mL in the non-activated 20-hour exposure group. Cytotoxicity was not observed at any dose levels in the S9-activated 4-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed at dose levels ≥ 600 μg/mL in all three treatment groups. Based on these findings, the doses chosen for the chromosome aberration assay ranged from 50 to 1000 μg/mL for the non-activated and S9-activated 4-hour exposure groups, and from 50 to 750 μg/mL for the non-activated 20-hour exposure group.

In the chromosome aberration assay, 55 ± 5 % cytotoxicity (reduction in cell growth index relative to the vehicle control) was observed at dose levels ≥ 300 μg/mL in the non-activated 20-hour exposure group. Cytotoxicity was not observed at any dose level in the non-activated and S9-activated 4-hour exposure groups. At the conclusion of the treatment period, visible precipitate was observed at dose levels ≥ 750 μg/mL in the non-activated and S9-activated 4-hour exposure groups, and at dose levels ≥ 550 μg/mL in the non-activated 20-hour exposure group. The highest dose analyzed under each treatment condition exceeded the limit of solubility in treatment medium at the conclusion of the treatment period or produced 55 ± 5 % reduction in cell growth index relative to the vehicle control, which met the dose limit as recommended by testing guidelines for this assay.

No significant or dose-dependent increases in structural or numerical (polyploid or endoreduplicated cells) aberrations were observed in treatment groups with or without S9 (p > 0.05; Fisher’s Exact and Cochran-Armitage tests). 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 41001/H 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.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
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
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
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 41001/H TE
Chemical Name: Trisodium 1-amino-4-[[3-[[4-chloro-6-[(3-sulphonatophenyl)amino]-1,3,5-triazin-2-yl]amino]-2,4,6-trimethyl-5-sulphonatophenyl]amino]-9,10-dihydro-9,10-
dioxoanthracene-2-sulphonate
Commercial Name: Reactive Blue 049
EC No.: 276-481-8
CAS No.: 72214-18-7
Batch/Lot No.: BOP 01-12 (Lot: BS-1106872)
Purity: 77.3 % (provided by Sponsor)
Molecular Weight: 882.18 g/mol
Expiration Date: 31 January 2017
Description: Dark red-violet powder
Storage Conditions: -10 to -30 ºC, protected from light
Receipt Date: 02 March 2015
Target gene:
hypoxanthine-guanine phosphoribosyl transferase (HPRT)
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).
Frozen stock cultures were tested to confirm the absence of mycoplasma contamination and for karyotpye stability.
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:
- Preliminary toxicity assay: 9.77, 19.5, 39.1, 78.1, 156, 313, 625, 1250, 2500 and 5000 μg/mL with and without S9.
- Definitive mutagenoicity 1st assay: 237, 475, 949, 1270, 1690, 2250, 3000 and 4000 μg/mL with S9, and 29.7, 59.3, 119, 237, 475, 949, 1270 and 1690 μg/mL without S9.
- Definitive mutagenoicity 2nd assay: 237, 475, 949, 1270, 1690, 2250, 3000, 4000 and 5000 μg/mL with S9.
Test substance dilutions were prepared immediately before use and delivered to the test system.
Vehicle / solvent:
The vehicle used to prepare the test substance dose formulations, and also used as the vehicle control was distilled water, as indicated below:
- Vehicle: distilled water
- CAS number: 7732-18-5
- Supplier: Gibco
- Lot number: 1662088
- Purity/Grade: Sterile, distilled
- Expiration date: 30 December 2016
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Water
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
Cells were treated for 5 ± 0.5 hours in the presence and absence of S9, by addition of the test and control substance formulations to the treatment medium (with or without S9, as appropriate). This technique has been shown to be an effective method for detecting various chemical mutagens in this test system.

Preliminary Toxicity Test for Selection of Dose Levels
Cells were treated with 10 test substance concentrations, as well as the vehicle control, in the presence and absence of S9 using single cultures. The maximum concentration evaluated (5000 μg/mL). Lower concentrations were prepared by 2-fold dilutions. The pH of cultures at concentrations ≥2500 μg/mL were adjusted to pH 7 ± 0.5 using 1N hydrochloric acid (HCl, CAS No. 7647-01-1; Lot No. RNBD4214, Expiration 31 March 2018, Supplier: Sigma-Aldrich). No pH adjustment was made for the remaining concentrations. The pH of the cultures was determined using a pH meter due to the intense coloration of the test substance. Osmolality of the vehicle control and the highest concentration also was measured at the beginning of treatment. Concentrations evaluated in the definitive mutation assay were based on adjusted relative survival.


NUMBER OF REPLICATIONS: Duplicate



Evaluation criteria:
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:
Statistical analyses were performed using the method of Snee and Irr (1981), with significance established at the 0.05 level.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
No visible precipitate was observed at the beginning or end of treatment, and the test substance had no adverse impact on the osmolality of the cultures.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
SOLUBILITY TEST:
Water was selected as the solvent of choice based on the solubility of the test substance and compatibility with the target cells. After sonication at 27.0ºC for ten minutes, the test substance formed workable suspensions in sterile water at concentrations from ~10 to 50 mg/mL in the solubility test conducted at BioReliance.

Results:

Definitive mutagenicity assay:

Based on the results of the preliminary toxicity assay, FAT 41001/H TE was evaluated in the definitive mutagenicity assay at concentrations of 237, 475, 949, 1270, 1690, 2250, 3000 and 4000 μg/mL with S9, and 29.7, 59.3, 119, 237, 475, 949, 1270 and 1690 μg/mL without S9. No visible precipitate was observed at the beginning or end of treatment. pH adjustment with 1N HCl again was required at concentrations from 29.7 to 1690 μg/mL to maintain neutral pH. pH was measured at 4000 μg/mL after the addition of S9 and was within 7 ± 0.5; therefore, no pH adjustment was performed at concentrations ≥2250 μg/mL. The average adjusted relative survival was 11.10% at a concentration of 1690 μg/mL without S9. Cultures treated at concentrations of 237, 475, 949, 1270 and 1690 μg/mL without S9 were chosen for mutant selection (cultures treated at concentrations of 29.7, 59.3 and 119 μg/mL without S9 were discarded prior to selection because a sufficient number of higher concentrations was available). No statistically significant increases in mutant frequency were observed without S9 (p> 0.05). In contrast, the positive controls induced significant increases in mutant frequency (p< 0.01). However, no cultures with S9 exhibited 10 to 20 % adjusted relative survival, and this portion of the assay was repeated using adjusted concentrations. FAT 41001/H TE was evaluated in the mutagenicity assay retest at concentrations of 237, 475, 949, 1270, 1690, 2250, 3000, 4000 and 5000 μg/mL with S9. No visible precipitate was observed at the beginning or end of treatment. No pH adjustment was performed per the Study Director’s instructions and based on the pH results in the initial mutagenicity assay. The average adjusted relative survival was 14.73 % at a concentration of 4000 μg/mL with S9. Cultures treated at concentrations of 475, 949, 1690, 3000 and 4000 μg/mL with S9 were chosen for mutant selection (cultures treated at concentrations of 237, 1270 and 2250 μg/mL with S9 were discarded prior to selection because a sufficient number of other concentrations was available; cultures treated at a concentration of 5000 μg/mL with S9 were discarded prior to selection due to excessive cytotoxicity). No statistically significant increases in mutant frequency were observed with S9 (p> 0.05). In contrast, the positive controls induced significant increases in mutant frequency (p< 0.01).

All criteria for a valid assay ultimately were met.

CHO/HPRT Assay Historical Control Data (2011 -2013)

      Non-activated     S9 -activated
   Solvent control  0.2 microL/mL EMS  Solvent control  4.0 microL/mL B(a)P
 Mean MF  4.2 239.2  4.4  143.1 
 SD  3.6 144  3.9  76.2 
 Maximum  15.7 764.2  16.9  314.5 
 Minimum  0.0 12  0.0  5.8 

Solvent control (culture medium, distilled water, saline, DMSO, ethanol, acetone or vehicle supplied by Sponsor). It has been demonstrated that all of the above solvents exhibit the same mutant frequency range.

EMS Ethyl methanesulfonate

B(a)P Benzo(a)pyrene MF Mutant frequency per 106 clonable cells

SD Standard deviation

Conclusions:
These results indicate FAT 41001/H TE was negative in the In Vitro Mammalian Cell Forward Gene Mutation (CHO/HPRT) Assay with Duplicate Cultures in the presence and absence of S9, under the conditions and according to the criteria of the test protocol.
Executive summary:

The test substance, FAT 41001/H, 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). This assay was performed in accordance with the OECD test guideline 476 along with GLP compliance.

FAT 41001/H TE was prepared in distilled water and evaluated in a preliminary toxicity assay at concentrations of 9.77, 19.5, 39.1, 78.1, 156, 313, 625, 1250, 2500 and 5000 μg/mL with and without S9. No visible precipitate was observed at the beginning or end of treatment, and the test substance had no adverse impact on the osmolality of the cultures. pH adjustment with 1N HCl was required at concentrations ≥2500 μg/mL to maintain neutral pH. Adjusted relative survival was 22.37 and 11.39 % at concentrations of 2500 μg/mL with S9 and 1250 μg/mL without S9, respectively. Adjusted relative survival approximated 0 % at all higher concentrations.

Based on these results, FAT 41001/H TE was evaluated in the definitive mutagenicity assay at concentrations of 237, 475, 949, 1270, 1690, 2250, 3000 and 4000 μg/mL with S9, and 29.7, 59.3, 119, 237, 475, 949, 1270 and 1690 μg/mL without S9. No visible precipitate was observed at the beginning or end of treatment. pH adjustment with 1N HCl again was required at concentrations from 29.7 to 1690 μg/mL to maintain neutral pH. pH was measured at 4000 μg/mL after the addition of S9 and was within 7 ± 0.5; therefore, no pH adjustment was performed at concentrations ≥2250 μg/mL.

The average adjusted relative survival was 11.10% at a concentration of 1690 μg/mL without S9. Cultures treated at concentrations of 237, 475, 949, 1270 and 1690 μg/mL without S9 were chosen for mutant selection (cultures treated at concentrations of 29.7, 59.3 and 119 μg/mL without S9 were discarded prior to selection because a sufficient number of higher concentrations was available).

No statistically significant increases in mutant frequency were observed without S9 (p> 0.05). In contrast, the positive controls induced significant increases in mutant frequency (p< 0.01). However, no cultures with S9 exhibited 10 to 20 % adjusted relative survival, and this portion of the assay was repeated using adjusted concentrations.

FAT 41001/H TE was evaluated in the mutagenicity assay retest at concentrations of 237, 475, 949, 1270, 1690, 2250, 3000, 4000 and 5000 μg/mL with S9. No visible precipitate was observed at the beginning or end of treatment. No pH adjustment was performed per the Study Director’s instructions and based on the pH results in the initial mutagenicity assay. The average adjusted relative survival was 14.73 % at a concentration of 4000 μg/mL with S9. Cultures treated at concentrations of 475, 949, 1690, 3000 and 4000 μg/mL with S9 were chosen for mutant selection (cultures treated at concentrations of 237, 1270 and 2250 μg/mL with S9 were discarded prior to selection because a sufficient number of other concentrations was available; cultures treated at a concentration of 5000 μg/mL with S9 were discarded prior to selection due to excessive cytotoxicity). No statistically significant increases in mutant frequency were observed with S9 (p> 0.05). In contrast, the positive controls induced significant increases 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 41001/H TE was negative in the In Vitro Mammalian Cell Forward Gene Mutation (CHO/HPRT) Assay with Duplicate Cultures in the presence and absence of S9, under the conditions and according to the criteria of the test protocol.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

Based on the above mentioned results the substance does not need to be classified according to CLP Regulation (Regulation EC No.1272/2008).