Reaction mass ofDisodium [1-{[2-(hydroxy-kO)-3,5-dinitrophenyl]diazenyl-kN1}naphthalen-2-olato(2-)-kO][3-(hydroxy-kO)-4-{[2-(hydroxy-kO)naphthalen-1-yl]diazenyl-kN1}-7-nitronaphthalene-1-sulfonato(3-)]chromate(2-) andDisodium [1-{[2-(hydroxy-kO)-3,5-dinitrophenyl]diazenyl-kN2}naphthalen-2-olato(2-)-kO][3-(hydroxy-kO)-4-{[2-(hydroxy-kO)naphthalen-1-yl]diazenyl-kN1}-7-nitronaphthalene-1-sulfonato(3-)]chromate(2-)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

FAT 20037 has been tested for mutagenic effects in the Ames test according to OECD 471 involving Salmonella tester stains TA 98, TA 100, TA 102, TA 1535 and TA 1537 with and without metabolic activation. In addition, it has been investigated for mutagenic effects in eukaryotic cells using the HPRT test according to OECD guideline 476 and it has been investigated for clastogenic effects in mice in vivo according to OECD guideline 474.

 

In the bacterial reverse mutation assay, FAT 20037 was tested for mutagenic effects with and without metabolic activation at five concentrations in the range of 61.7 to 5000 µg/plate. An independent repetition of the experiment was performed using the same concentrations. In the original experiment performed with and without metabolic activation, the number of revertant counts increased strongly with strain TA 98, moderately with strains TA 100 and TA 1537 and slightly with strain TA 1535. In the confirmatory experiment performed without exogenous metabolic activation the number of revertant counts again increased strongly with TA 98 and moderately with strains TA 100 and TA 1537. A very weak increase in the number of back-mutants occurred on strain TA 102. The effect observed in the original experiment with strain TA 1535 could not be reproduced. In the confirmatory experiment carried out with metabolic activation, a strong increase in the number of back-mutants occurred with strain TA 98, a moderate increase with strains TA 100 and TA 1537 and very weak one with strain TA 1535. Based on the results of these experiments, it was concluded that Acid Black 107 exerted a clear-cut mutagenic action on strains S. typhimurium TA 98, TA 100 and TA 1537.

In addition to the bacterial Ames test, FAT 20037 was also investigated in a gene mutation assay with Chinese hamster cells V79 according to OECD guideline 476. Based on the results of a preliminary toxicity test, 500 µg/mL with and 400 µg/mL without metabolic activation were chosen as highest concentrations for the first mutagenicity assay. In the main test as well as in the confirmatory experiment, comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no relevant increase of the mutant frequencies as determined by the screening with positive controls. Hence, it was concluded that FAT 20037 and its metabolites did not show any mutagenic activity in this gene mutation assay with Chinese hamster cells V79.

Data to investigate potential clastogenic effects on the chromosomal levelin vitroare currently not available for the test substance, because a higher TIER testin vivois already available.

It is well-known for aromatic nitro-compounds to be positive in the Ames assay resulting from metabolism by the bacteria-specific enzyme nitro-reductase [Tweats et al. 2012]. However, it has been demonstrated in various publications that this is a bacteria-specific effect and that these Ames positive substances are not mutagenic in mammalian assays. The nitroreductase family comprises a group of flavin mononucleotide (FMN)- or flavin adenine dinucleotide (FAD) -dependent enzymes that are able to metabolize nitroaromatic and nitroheterocyclic derivatives (nitrosubstituted compounds) using the reducing power of nicotinamide adenine dinucleotide (NAD(P)H). These enzymes can be found in bacterial species and, to a lesser extent, in eukaryotes. The nitro-reductase proteins play a central role in the activation of nitro-compounds [de Oliveira et al. 2010]. For details please also refer to the expert statement provided in Chapter 13.2 of this dossier.

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: 08 March, 1993; Experiment start date - 19 March, 1993; Experiment end date - 04 May, 1993; Study completion date: 28 June, 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:

yes

Remarks:

a statistical analysis was not performed as no appropriate statistical method is available.

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:

SOURCE OF TEST MATERIAL
- Identification of the test material as used in the study report: Irgalan Schwarz BGL Roh Trocken (FAT 20037/C)
- Source and batch No.of test material: 9300001
- Expiration date of the lot/batch: February 1998

OTHER SPECIFICS:
- Purity: about 80 %

Target gene:

Histidine requiring genes of Salmonella strains

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: histidine-auxotrophic strains obtained from Prof. B. Ames, Berkeley, CA., U.S.A.

Metabolic activation:

with and without

Metabolic activation system:

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 29.4 and 30.6 mg/ml.

Test concentrations with justification for top dose:

Experiment without metabolic activation: 61.7, 185.2, 555.6, 1666.7 & 5000 µg/plate;
Experiment with metabolic activation: 61.7, 185.2, 555.6, 1666.7 & 5000 µg/plate;

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethylsulfoxide (Suspension)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without microsomal activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Without microsomal activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-nitrofluorene

Remarks:

Without microsomal activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2- aminoanthracene

Remarks:

With 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: 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 : increase of the mean number of revertants per plate above that of the negative control.

Evaluation criteria:

Assay acceptance criteria:
A test was considered acceptable if the mean colony counts of the control values of all strains were within the acceptable ranges and if the results of the positive controls met the criteria for a positive response. In either case the final decision was based on the scientific judgement of the Study Director.

Criteria for a positive response:
The test substance was 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 as no appropriate statistical method is available.

Key result

Species / strain:

other: Salmonella typhimurium TA 98, TA 100 and TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the mutagenicity tests without and with metabolic activation, due to toxicity of the test material, a slight decline in the number of revertant colonies was occasionally observed with all strains at the highest concentrations.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the mutagenicity tests without and with metabolic activation, due to toxicity of the test material, a slight decline in the number of revertant colonies was occasionally observed with all strains at the highest concentrations.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the mutagenicity tests without and with metabolic activation, due to toxicity of the test material, a slight decline in the number of revertant colonies was occasionally observed with all strains at the highest concentrations.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
Six concentrations of FAT 20037/C ranging from 20.6 to 5000 µg/plate were tested with strain S. typhimurium TA 100 to determine the highest concentration to be used in the mutagenicity assay. The experiments were performed with and without microsomal activation. The numbers of revertant colonies were markedly increased at the upper concentrations. From the results obtained, the highest concentration suitable for the mutagenicity test was selected to be 5000 µg/plate without and with activation.

Mutagenicity test, original experiment

In the original experiment performed without metabolic activation, treatment of strain TA 100 with FAT 20037/C led to a moderate, concentration dependent increase in the number of revertant counts at the concentrations of 185.2 to 1666.7 µg/plate. With strain TA 1535 a very weak increase in the number of back-mutants was observed at the concentrations of 1666.7 and 5000 µg/plate. With strain TA 98 a strong, concentration dependent increase in the number of backmutants was observed at all concentrations. With strain TA 1537 a moderate increase in the number of revertants was registered at the concentrations of 61.7 to 555.6 µg/plate. No effect was seen with strain TA 102.

In the original experiment performed with activation, treatment of strain TA 100 with FAT 20037/C led to a moderate, concentration dependent increase in the number of revertant counts at the concentrations of 185.2 to 1666.7 µg/plate. With strain TA 1535 a very weak increase in the number of back-mutants was observed at the concentration of 555.6 µg/plate only. With strain TA 98 a strong, concentration dependent increase in the number of back-mutants was observed at the concentrations of 61.7 to 1666.7 µg/plate. With strain TA 1537 a moderate increase in the number of revertants was registered at the concentrations of 61.7 to 1666.7 µg/plate. No effect was seen with strain TA 102. In the experiment with activation performed on strain TA 102 the mean number of colony counts in the negative control was somewhat above the acceptable range given on page 19. This, however, does not affect the test results.

Mutagenicity test, confirmatory experiment

In the confirmatory experiment performed without metabolic activation, treatment of strain TA 100 with FAT 20037/C again led to a moderate, concentration dependent increase in the number of revertant counts at the concentrations of 185.2 to 1666.7 µg/plate. In the confirmatory experiment performed without metabolic activation, treatment of strain TA 100 with FAT 20037/C again led to a moderate, concentration dependent increase in the number of revertant counts at the concentrations of 185.2 to 1666.7 µg/plate. The effect observed in the original experiment with strain TA 1535 could not be reproduced. With strain TA 102 a very weak increase in the number of revertants was registerd at the concentration of 555.6 µg/plate. With strain TA 98 a strong, concentration dependent increase in the number of back-mutants was observed at the concentrations of 61.7 to 1666.7 µg/plate . With strain TA 1537 a moderate increase in the number of revertants was registered at the same concentrations.

In the confirmatory experiment performed with activation, treatment of strain TA 100 with FAT 20037/C again led to a moderate, concentration dependent increase in the number of revertant counts at the concentrations of 185.2 to 1666.7 µg/plate. With strain TA 1535 a very weak increase in the number of back-mutants was observed at the same concentrations of 185.2 to 5000 µg/plate. With strain TA 98 a strong, concentration dependent increase in the number of back-mutants was observed at the concentrations of 61.7 to 1666.7 µg/plate. With strain TA 1537 a moderate increase in the number of revertants was registered at all concentrations. No effect was seen with strain TA 102. In the mutagenicity tests without and with metabolic activation, due to a growth-inhibiting effect of the test material, a slight decline in the number of revertant colonies was occasionally observed with all strains at the highest concentrations.

Conclusions:

FAT 20037/C exerted a clear-cut mutagenic action on strains S. typhimurium TA 98, TA 100 and TA 1537.

Executive summary:

The mutagenic potential of the test item was investigated in a bacterial reverse mutation assay according to OECD guideline 471 and EU method B.13/14 in compliance with GLP.

Based on the results of a preliminary toxicity test, FAT 20037/C 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 same concentrations.

In the original experiment performed without and with metabolic activation, after treatment of the bacterial cultures with FAT 20037/C, the number of revertant counts increased strongly with strain TA 98, moderately with strains TA 100 and TA 1537 and slightly with strain TA 1535. In the confimatory experiment performed without activation, after treatment of the bacterial cultures with FAT 20037/C the number of revertant counts again increased strongly with TA 98 and moderately with strains TA 100 and TA 1537. A very weak increase in the number of back-mutants occurred on strain TA 102. The effect observed in the original experiment with strain TA 1535 could not be reproduced. In the confirmatory experiment carried out with activation, a strong increase in the number of back-mutants occurred on strain TA 98, a moderate increase on strains TA 100 and TA 1537 and very weak one on strain TA 1535.

Based on the results of these experiments, it was concluded that FAT 20037/C exerted a clear-cut mutagenic action on strains S. typhimurium TA 98, TA 100 and TA 1537.

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: 14 July, 1994 Experimental start date - 15 August 1994; Experimental completion date - 22 December 1994; Study completion date - 23 January 1995.

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

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5300 (Detection of Gene Mutations in Somatic Cells in Culture)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EEC Directive 87/302, Annex (November 18, 1987) Part B; Mutagenicity testing and screening for carcinogenicity; In vitro mammalian cell gene mutation test. Official Journal of the European Communities, No L 133. Vol. 31, 61-63, May 30, 1988

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Identification of the test material as done in the study report: FAT 20037/C, (Irgalan schwarz BGL roh trocken)
- Source and batch No.of test material: 9300001
- Expiration date of the batch: February 28, 1998
- Purity: 69.2%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: stable
- Stability of the test substance in the solvent/vehicle: stable till expiry date

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (HPRT)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS AND MEDIA USED
V79 Chinese hamster cells were originally derived from embryonic lung tissue. The cells were cultured in Ham's F10 medium supplemented with 10 % pre-tested foetal calf serum, 100 U/ml penicillin and 100 µg/ml streptomycin in tissue culture (plastic) flasks. The humidity in the incubator was adjusted to >85 % rH, the air was enriched to 5 ± 2.0 Vol% CO2 and the temperature was 37 ± 1°C. Twice per week the growth medium was replaced by fresh one.

For cell lines:
- Absence of Mycoplasma contamination: Yes
- Number of passages if applicable: 5x10E4 cells per 175 sq.cm.
- Methods for maintenance in cell culture: Large stocks of the V79 cell line have been stored in liquid nitrogen allowing the repeated use of the same cell culture batch in experiments.
- Modal number of chromosomes: 22 ± 1
- Periodically checked for karyotype stability: yes
- Periodically ‘cleansed’ of spontaneous mutants: yes

Metabolic activation:

with and without

Metabolic activation system:

Rat-liver post mitochondrial supernatant (S9 fraction) was prepared in advance from male RAI rats, reared at the Animal Farm of CIBA-GEIGY, Sisseln, Switzerland. The animals 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. The homogenate was centrifuged at 9000x g for 15 minutes and the resulting supernatant (S9 fraction) was stored at approximately minus 80°C for no longer than one year. The protein content of the S9 fraction was 28.16 mg/ml. S9 fraction was thawed immediately before use. The S9 mixture was prepared just prior use in an activation experiment and kept on ice.

The S9 mixture consisted of:
• Rat liver S9 fraction 250.0 µl/ml
• Glucose-6-phosphate 10.0 µmol/ml
• NADP 8.0 µmol/ml
• CaCl2 20.0 µmol/ml
• MgCl2 20.0 µmol/ml
• Na2HPO4 1.0 µmol/ml
• FCS 30.0 µl/ml

Unused portions of S9 fraction and S9 mixture were discarded and not saved for another experiment. The S9 mixture was immediately filter-sterilised by passage through a 0.45 µm filter. The activation mixture was added to the medium at a concentration of 10 % in both the cytotoxicity test and the mutagenicity test and the final concentration of S9 fraction was 2.5% during the treatment.

Test concentrations with justification for top dose:

Mutagenicity test with metabolic activation (both original as well as confirmatory experiment): 18.52, 55.56, 166.67 and 500.0 µg/mL.
Mutagenicity test without metabolic activation (original experiment): 14.81, 44.44, 133.33 and 400.0 µg/mL.
Mutagenicity test without metabolic activation (confirmatory experiment): 12.96, 38.89, 116.67 and 350.0 µg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethylsulfoxide (DMSO)

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

Remarks:

With metabolic activation

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Duplicate
- Number of independent experiments : Two (one original, one confirmatory)

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 2.5-5.0x10E6

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 21 hours.
- Harvest time after the end of treatment (sampling/recovery times): The cultures were incubated at 37 °C for seven to eight days during which the cells could recover and divide to express the mutant phenotype.

FOR GENE MUTATION:
- Selection time (if incubation with a selective agent): 7 - 8 days.
- Selective agent: The selection medium was growth medium to which 6- thioguanine (6-TG) was added to a final concentration of 8 µg/mL.
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 2.5x10E5 V79 cells were seeded in 5 ml growth medium into a 25 cm² tissue culture flask and incubated overnight, After seven to eight days of growth the cultures were fixed and stained with Giemsa and the surviving colonies determined with the aid of an electronic colony counter (Artek Counter®, Fisher Scientific) or by the naked eye.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Cell number after treatement.

METHODS FOR MEASUREMENTS OF GENOTOXICIY : Mutant frequency.

Evaluation criteria:

- Assay acceptance criteria:
• The results of the experiments should not be influenced by a technical error, contamination or a recognized artifact.
• From each experiment, at least 3 concentrations of the test substance, one positive and one solvent control should be evaluated.
• The mutant frequency of the solvent controls (spontaneous mutant frequency) should not exceed 35x10E-6.
• The positive control should fulfil the criteria for a mutagenic substance.
• The highest concentration of the test substance applied in the mutagenicity test should either reduce the viable cells by about 50-90% or correspond to the test substance's solubility limit (precipitates in the culture). In case of non-toxic freely soluble compounds the highest tested concentration will be 5 mg/mL.

- Assay evaluation criteria:
All mutant frequencies are normalized to a virtual cloning efficiency of 100% at the end of the expression period. If the cloning efficiency of the viability cultures is lower than 15%, the corresponding mutant frequency is usually not calculated, owing to the high statistical insignificance of the result. For every concentration a mean mutant factor, which is defined as the ratio of the mean mutant frequencies of the treated cultures with the mean mutant frequencies of the solvent control cultures, will be calculated.

- Criteria for a positive response:
The test substance will be considered to be mutagenic if:
• The assay fulfills the validity criteria.
• The mutant frequency at one or more concentrations is significantly greater than that of the negative control and the number of normalized mutant clones in the treated and untreated cultures differs by more than 20.
• There is a significant dose-relationship as indicated by the linear trend analysis.
• The effects described above are reproducible.

Statistics:

Assessment of statistical significance of mutation frequency:
Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
A preliminary range finding test was run assessing cytotoxicity. FAT 20037/C was tested at concentrations up to 500 µg/mL. Higher concentration could not be applied due to solubility limitations in the vehicle. In the part with metabolic activation, at the highest concentration of 500 µg/mL an acute growth inhibiting effect of 55.45 % could be seen. Without metabolic activation treatment with FAT 20037/C at the highest concentration of 500 µg/mL caused an acute inhibition of growth of 99.48 %, while the next lower concentration of 250 µg/mL inhibited 66.35 %. Accordingly, 500 µg/mL with and 400.0 µg/mL without metabolic activation were chosen as highest concentrations for the first mutagenicity assay.

Mutagenicity test with metabolic activation:

The original experiment was performed at the following concentrations: 18.52, 55.56, 166.67 and 500.0 µg/mL. The mean growth inhibiting values found at the highest concentration after treatment and expression were 36.56 % and less than 3.06 % respectively. In the confirmatory experiment the concentrations applied were 18.52, 55.56, 166.67 and 500.0 µg/mL. The highest concentration revealed a mean acute growth inhibition of 10.75 %. The mean growth inhibitory effect after the expression period was 6.44 %. N-Nitrosodimethylamine (DMN, 1.0 µL/mL) was used as positive control. In both experiments comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no relevant increase of the mutant frequencies as determined by the screening with 6-Thioguanine (6-TG).

 

Mutagenicity test without metabolic activation:

The original experiment was performed at the following concentrations: 14.81, 44.44, 133.33 and 400.0 µg/mL. The mean growth inhibition value found at the highest concentration after treatment was 92.65 %. No toxicity was noted after expression. In the confirmatory experiment the concentrations applied were 12.96, 38.89, 116.67 and 350 µg/mL. The highest concentration revealed a mean acute, growth inhibitory effect of 76.50 %. The mean growth inhibition after the expression period was 16.93 %. Ethylmethansulfonate (EMS, 0.3 µL/mL) was used as positive control. In both experiments comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no relevant increase of the mutant frequencies as determined by the screening with 6-TG.

Conclusions:

FAT 20037/C and its metabolites did not show any mutagenic activity in this gene mutation assay with Chinese hamster cells V79.

Executive summary:

The mutagenic potential of the test item was investigated in a gene mutation test with Chinese hamster cells V79 according to OECD test guideline 476 and EPA OTS 798.5300, in compliance with GLP. Based on the results of a preliminary toxicity test, 500.0 µg/mL with and 400.0 µg/mL without metabolic activation were chosen as highest concentrations for the first mutagenicity assay. In the mutagenicity test with metabolic activation, the original experiment was performed at the following concentrations: 18.52, 55.56, 166.67 and 500.0 µg/mL. The mean growth inhibiting values found at the highest concentration after treatment and expression were 36.56 % and less than 3.06 % respectively. In the confirmatory experiment the concentrations applied were 18.52, 55.56, 166.67 and 500 µg/mL. The highest concentration revealed a mean acute growth inhibition of 10.75 %. The mean growth inhibitory effect after the expression period was 6.44%. N-Nitrosodimethylamine (DMN, 1.0 µL/mL) was used as positive control. In both experiments comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no relevant increase of the mutant frequencies as determined by the screening with 6-Thioguanine (6-TG). In the mutagenicity test without metabolic activation, the original experiment was performed at the following concentrations: 14.81, 44.44, 133.33 and 400.0 µg/mL. The mean growth inhibition value found at the highest concentration after treatment was 92.65 %. No toxicity was noted after expression. In the confirmatory experiment the concentrations applied were 12.96, 38.89, 116.67 and 350.0 µg/mL. The highest concentration revealed a mean acute, growth inhibitory effect of 76.50 %. The mean growth inhibition after the expression period was 16.93 %. Ethylmethansulfonate (EMS, 0.3 µL/mL) was used as positive control. In both experiments comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no relevant increase of the mutant frequencies as determined by the screening with positive control. Based on the results of above independently performed experiments and under the given experimental conditions, it was concluded that FAT 20037/C and its metabolites did not show any mutagenic activity in this gene mutation assay with Chinese hamster cells V79.

Reference 3

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

Type of assay:

in vitro mammalian chromosome aberration test

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

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 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 (Lot No. 3408) was obtained from Molecular Toxicology Inc. (Boone, NC). Each bulk preparation of S9 was assayed by the supplier for sterility and its ability to metabolize at least two pro-mutagens to forms mutagenic to Salmonella typhimurium TA100.
Immediately prior to use, the S9 was thawed and mixed with a cofactor pool to contain 2 mM magnesium chloride, 6 mM potassium chloride, 1 mM glucose-6-phosphate, 1 mM nicotinamide adenine dinucleotide phosphate (NADP) and 20 µL S9 per milliliter medium (McCoy's 5A serum-free medium supplemented with 100 units penicillin/mL, 100 µg streptomycin/mL, 1.5 mM L-glutamine and 2.5 µg/mL amphotericin B).

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

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:

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 Assays)
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.

Key result

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

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, 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

Conclusions:

FAT 20011/E 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 structurally similar substance FAT 20011/E was tested for genetic mutation in a 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 test Guideline 473, in a GLP certified laboratory. 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 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The clastogenic potential of the test substance was ruled out by way of negative outcome in an in vivo micronucleus assay. Based on the above results, the substance is determined to be devoid of any clastogenic potential.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiation date - 07 April, 1998; Experimental start date- 05 May 1998; End of Experiment - 23 June, 1998; Study Completion Date - 30 June 1998.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Identification of the test material as used in the study report: FAT 20037/C
- Source and batch No.of test material: 9300001
- Expiration date of the batch: December 1998
- Purity: approximately 80%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability of the test substance in the solvent/vehicle: at least 24 hours in water, saline, PEG 400, and CMC.

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL, CH-4414 Füllinsdorf
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 36.5 g (mean weight for males); 28.8 g (mean weight for females)
- Assigned to test groups randomly: yes
- Fasting period before study: 18 hours
- Housing: single in Makrolon Type I, with wire mesh top (EHRET GmbH, D-79302 Emmendingen)
- Diet: pelleted standard diet, ad libitum (ALTROMIN 1324, D-32791 Lage/Lippe)
- Water (e.g. ad libitum): tap water, ad libitum, (Gemeindewerke, D-64380 Roßdorf)
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 3 °C
- Humidity: 30-88 %
- Photoperiod: Artificial light 6.00 a.m. - 6.00 p.m.

IN-LIFE DATES: From: 05 May, 1998 To: 23 June, 1998

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Deionised water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: On the day of the experiment, the test article was formulated in deionised water. The vehicle was chosen to its non-toxicity for the animals. All animals received a single standard volume of 10 mL/kg body weight orally.

Duration of treatment / exposure:

once orally (gavage)

Frequency of treatment:

Single dose

Post exposure period:

Sampling of the bone marrow was done 24 and 48 hours after treatment, respectively.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control group

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

24 hour interval only

Dose / conc.:

670 mg/kg bw/day (nominal)

Remarks:

24 hour interval only

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

24 h and 48 h each.

No. of animals per sex per dose:

Six males and six females were assigned to each test group.

Control animals:

yes, concurrent vehicle

Positive control(s):

- Positive control used: Cyclophosphamide
- Route of administration: orally once
- Doses: 40 mg/kg bw

Tissues and cell types examined:

Bone marrow and polychromatic erythrocytes (PCE)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: In a pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 2000 mg/kg b.w. FAT 20037/C formulated in deionised water. The volume administered was 10 mL/kg bw. The dose was well tolerated, hence it was chosen to be the highest dose. Based on the above information, three adequate spaced dose levels extending over a single log range were applied at the central sampling interval 24 h after treatment. For the highest dose level an additional sample was taken at 48 h after treatment.

TREATMENT AND SAMPLING TIMES: Approximately 18 hours before treatment the animals received no food but water ad libitum. At the beginning of the treatment the animals (including the controls) were weighed and the individual volume to be administered was adjusted to the animals body weight. The animals received the test article, the vehicle or the positive control substance once orally (gavage). Twelve animals, six males and six females, were treated per dose group and sampling time. Sampling of the bone marrow was done 24 and 48 hours after treatment, respectively.

DETAILS OF SLIDE PREPARATION: The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald (MERCK, D-64293 Darmstadt)/Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, D-79110 Freiburg). At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS: Evaluation of the slides was performed using NIKON microscopes with 100 x oil immersion objectives. At least 2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in normochromatic erythrocytes per 2000 the PCEs. The analysis was performed with coded slides.
Five animals per sex and group were evaluated.

Evaluation criteria:

Acceptance Criteria:
The study was considered valid as the following criteria are met:
- the vehicle controls are in the range of our historical control data (0.03 - 0.26 % PCEs with micronuclei).
- the positive controls show substantially increased values
- more than 80 % of animals are évaluable

Evaluation of Results:
- A test article is classified as mutagenic if it induces either a dose-related increase in the number of micronucleated polychromatic erythrocytes or a statistically significant positive response for at least one of the test points.
- A test article producing neither a dose-related increase in the number of micronucleated polychromatic erythrocytes nor a statistically significant positive response at any of the test points is considered non-mutagenic in this system.
- This can be confirmed by means of the nonparametric Mann-Whitney test.
- However, both biological and statistical significance should be considered together.

Statistics:

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose: 2000 mg/kg bw
- Solubility: soulble in water
- Clinical signs of toxicity in test animals: reduction of spontaneous activity was the only sign observed at 1 and 6 hours post treatment
- High dose with and without activation: 2000 mg/kg bw

Summary of Micronucleus Test Results:

Test Group	Dose mg/kg b.w	sampling time (h)	PCEs with micronuclei (%)	Range	PCE/NCE
Vehicle	0	24	0.090	0-5	2000/1508
Test Article	200	24	0.060	0-3	2000/1533
Test Article	670	24	0.120	0-5	2000/1488
Test Article	2000	24	0.110	0-5	2000/1571
Cyclophosphamide	40	24	1.290	11-37	2000/2108
Test Article	2000	48	0.045	0-2	2000/2252

Biometry:

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Vehicle control versus test group	Significance	p
200 mg FAT 20037/C/kg bw; 24 h	n.t.	-
670 mg FAT 20037/C/kg bw; 24 h	-	0.2257
2000 mg FAT 20037/C/kg bw; 24 h	-	0.3341
40 mg CPA/kg bw; 24h	+	< 0.0001
2000 mg FAT 20037/C/kg bw; 48 h	n.t.	-

+ = significant; - not significant n.t. = not tested, as the mean micronucleus frequency was not above the vehicle control value

The mean number of normochromatic erythrocytes was increased after treatment with the test article as compared to the mean value of NCEs of the vehicle controls at preparation interval 48 hours, indicating that FAT 20037/C had cytotoxic properties in the bone marrow.

In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test article. The mean values of micronuclei observed after treatment with FAT 20037/C were near to the range of the vehicle control group.

40 mg/kg b.w. cyclophosphamide administered per os was used as positive control which showed a statistically significant increase of induced micronucleus frequency.

Conclusions:

FAT 20037/C was considered to be devoid of clastogenic potential in this micronucleus assay.

Executive summary:

A study was performed to investigate the potential of FAT 20037/C to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse according to OECD Guideline 474 and EU Method B.12, in compliance with GLP. The test article was formulated in deionised water. Deionised water was used as vehicle control. The volume administered orally (gavage) was 10 mL/kg bw. 24 h and 48 h after a single administration of the test article the bone marrow cells were collected for micronuclei analysis. 12 animals (6 males, 6 females) per test group were evaluated for the occurrence of micronuclei. At least 2000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 2000 PCE. The following dose levels of the test article were investigated: 24 h preparation interval: 200, 670, and 2000 mg/kg bw. 48 h preparation interval: 2000 mg/kg bw. The highest guideline-recommended dose (2000 mg/kg bw) was estimated by a pre-experiment to be suitable. The animals expressed slight toxic reactions. After treatment with the test article the mean number of NCEs at preparation interval 48 hours was increased as compared to the vehicle controls thus indicating that FAT 20037/C had cytotoxic effectiveness. In comparison to the corresponding vehicle controls there was no statistically significant or biological relevant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test article and with any dose level used. 40 mg/kg bw cyclophosphamide administered per os was used as positive control which showed a statistically significant increase of induced micronucleus frequency. Based on the findings of the study, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, FAT 20037/C was considered to be devoid of clastogenic potential in this micronucleus assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro:

Bacterial mutagenicity test

The mutagenic potential of the test item was investigated in a bacterial reverse mutation assay according to OECD guideline 471 and EU method B.13/14 in compliance with GLP. Based on the results of a preliminary toxicity test, FAT 20037/C 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 same concentrations. In the original experiment performed without and with metabolic activation, after treatment of the bacterial cultures with FAT 20037/C, the number of revertant counts increased strongly with strain TA 98, moderately with strains TA 100 and TA 1537 and slightly with strain TA 1535. In the confirmatory experiment performed without activation, after treatment of the bacterial cultures with FAT 20037/C the number of revertant counts again increased strongly with TA 98 and moderately with strains TA 100 and TA 1537. A very weak increase in the number of back-mutants occurred on strain TA 102. The effect observed in the original experiment with strain TA 1535 could not be reproduced. In the confirmatory experiment carried out with activation, a strong increase in the number of back-mutants occurred on strain TA 98, a moderate increase on strains TA 100 and TA 1537 and very weak one on strain TA 1535. Based on the results of these experiments, it was concluded that FAT 20037/C exerted a clear-cut mutagenic action on strains S. typhimurium TA 98, TA 100 and TA 1537.

Mammalian cell gene mutation test:

The mutagenic potential of the test item was investigated in a gene mutation test with Chinese hamster cells V79 according to OECD test guideline 476 and EPA OTS 798.5300, in compliance with GLP. Based on the results of a preliminary toxicity test,500.0µg/mL with and 400.0µg/mL without metabolic activation were chosen as highest concentrations for the first mutagenicity assay. In the mutagenicity test with metabolic activation, the original experiment was performed at the following concentrations: 18.52, 55.56, 166.67 and 500 µg/mL. The mean growth inhibiting values found at the highest concentration after treatment and expression were 36.56 % and less than 3.06 % respectively. In the confirmatory experiment the concentrations applied were 18.52, 55.56, 166.67 and 500.0µg/mL. The highest concentration revealed a mean acute growth inhibition of 10.75 %. The mean growth inhibitory effect after the expression period was 6.44 %. N-Nitrosodimethylamine (DMN, 1.0 µL/mL) was used as positive control. In both experiments comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no relevant increase of the mutant frequencies as determined by the screening with 6-Thioguanine (6-TG). In the mutagenicity test without metabolic activation, the original experiment was performed at the following concentrations: 14.81, 44.44, 133.33 and 400 µg/mL. The mean growth inhibition value found at the highest concentration after treatment was 92.65 %. No toxicity was noted after expression. In the confirmatory experiment the concentrations applied were 12.96, 38.89, 116.67 and 350.0 µg/mL. The highest concentration revealed a mean acute, growth inhibitory effect of 76.50 %. The mean growth inhibition after the expression period was 16.93 %. Ethylmethansulfonate (EMS, 0.3 µL/mL) was used as positive control. In both experiments, comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no relevant increase of the mutant frequencies as determined by the screening with positive control. Based on the results of above independently performed experiments and under the given experimental conditions, it was concluded that FAT 20037/C and its metabolites did not show any mutagenic activity in this gene mutation assay with Chinese hamster cells V79.

Chromosome aberration read-across study:

The read across substance FAT 20011/E was tested for genetic mutation in a 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 test Guideline 473, in a GLP certified laboratory. 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. Based on the above findings, FAT 20011/E 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.

In vivo Micronucleus test:

A study was performed to investigate the potential of FAT 20037/C to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse according to OECD Guideline 474 and EU Method B.12, in compliance with GLP. The test article was formulated in deionised water. Deionised water was used as vehicle control. The volume administered orally (gavage) was 10 mL/kg bw. 24 h and 48 h after a single administration of the test article the bone marrow cells were collected for micronuclei analysis. 12 animals (6 males, 6 females) per test group were evaluated for the occurrence of micronuclei. At least 2000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 2000 PCE. The following dose levels of the test article were investigated: 24 h preparation interval: 200, 670, and 2000 mg/kg bw. 48 h preparation interval: 2000 mg/kg bw. The highest guideline-recommended dose (2000 mg/kg bw) was estimated by a pre-experiment to be suitable. The animals expressed slight toxic reactions. After treatment with the test article the mean number of NCEs at preparation interval 48 hours was increased as compared to the vehicle controls thus indicating that FAT 20037/C had cytotoxic effectiveness. In comparison to the corresponding vehicle controls there was no statistically significant or biological relevant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test article and with any dose level used. 40 mg/kg bw cyclophosphamide administered per os was used as positive control which showed a statistically significant increase of induced micronucleus frequency. Based on the findings of the study, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, FAT 20037/C was considered to be devoid of clastogenic potential in this micronucleus assay. The test substance was determined to have mutagenic potential in bacterial cell reverse mutation assay, however this was negated by the negative results in a mammalian cell gene mutation assay. Further, the clastogenic potential of the test substance was ruled out by way of negative outcome in an in vivo micronucleus assay. Based on the above results, the substance is determined to be devoid of any genotoxic potential.

Justification for classification or non-classification

The substance was determined to have mutagenic potential in bacterial cell reverse mutation assay, however this was negated by the negative results in a mammalian cell gene mutation assay. Further, the clastogenic potential of the substance was ruled out by way of negative outcome in an in vivo micronucleus assay. Based on the above results, the substance is determined to be devoid of any genotoxic potential. Hence, the substance does not warrant classification for genetic toxicity according to the CLP (Regulation 1272/2008) criteria. Based on above findings in bacterial test systems and mammalian mutagenicity tests and the common mechanism between the reduction of nitro-compounds by bacterial nitro-reductases forming DNA-damaging radicals, which is widely explored in literature [de Oliveira et al. 2010], it is concluded, that the mutagenic effects observed in the Ames test with Acid Black 107 is a bacteria specific effect and not relevant to mammalians.