3-anilino-5-methyl-5-(4-phenoxyphenyl)-1,3-oxazolidine-2,4-dione;famoxadone (ISO)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Mutagenic effects - bacterial:

Ames study in S. typhimurium TA100, TA1535, TA97, TA98 and E. coli WP2uvrA. Negative. OECD 471, OPP 84-2. JMAFF 59 Nohsan 4200; Reliability = 1.

Ames study in S. typhimurium TA100, TA1535, TA97, TA98 and E. coli WP2uvrA. Negative. OECD 471, OECD 472, OPP 84-2. JMAFF 59 Nohsan 4200; Reliability = 2.

 

Clastogenic effects

Chromosome aberrations in human lymphocytes. Genotoxicity Negative; Positive in exhibiting clastogenic activity; OECD 473, OPP 84-2, EU Method B.10; Reliability = 1.

Chromosome aberrations in human lymphocytes Weakly clastogenic in human lymphocytes; OECD 473, OPP 84-2, EU Method B.10, JMAFF 59 Nohsan 4200; Reliability = 1.

 

Gene mutation in mammalian cells:

CHO/HPRT gene mutation assay. Negative. OECD 476, OPPTS 870.5300; Reliability = 1.

CHO/HPRT gene mutation assay. Negative. OECD 476, OPP 84-2; Reliability = 1.

 

UDS Assay

UDS in rat liver primary cell cultures. Negative. OECD 482, OPPTS 870.5550; Reliability = 1.

UDS in primary rat hepatocyte cultures. Positive. OECD 482, OPP 84-2; Reliability = 1.

UDS in primary rat hepatocyte cultures. Negative. OECD 482, OPP 84-2; Reliability = 1.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: MAFF Testing Guidelines for Mutagenicity Studies, 59 NohSan No. 4200

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Substance name: DPX-JE874
Lot #: DPX-JE874-133
Purity: 97.7%

Species / strain / cell type:

lymphocytes: human

Cytokinesis block (if used):

Colcemid® (0.1 µg/mL)

Metabolic activation:

with and without

Metabolic activation system:

Livers obtained from 8-9 week old male rats which received a single i.p. injection of 500 mg/kg Aroclor® 1254 five days prior to sacrifice. Livers were homogenized (1:3 w/v) in phosphate buffered saline (PBS) and the homogenate centrifuged at 9000 x g. The supernatant (S-9) was frozen, and the protein concentration was determined.

Test concentrations with justification for top dose:

In Trials 1 and 2: 0, 1, 5, 8, and 10 µg/mL
Trial 3: 0, 10, 15, and 18 µg/mL

Top dose was selected based on the limit of solubility in the culture medium

Vehicle / solvent:

Dimethyl sulfoxide (DMSO)

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Statistics:

For each trial, the proportion of abnormal cells and the proportion of cells with more than one aberration were evaluated using a Fisher Exact Test to compare each treatment level with the negative (solvent) control, where statistical significance was judged at the 5% level. A Cochran-Armitage test for linear trend (dose-response) was performed where appropriate; significance was judged at the 1% level. Chromatid and isochromatid gaps were excluded from the statistical evaluation.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with

Genotoxicity:

negative

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

other: At 10 µg/mL, slight to moderate cytotoxicity (as measured by a decrease in the MI), was observed under both treatment conditions, and the average AGT increased by approximately 20-27%

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

The test substance exhibited weak clastogenic activity in human lymphocytes without metabolic activation

Executive summary:

The study was conducted following OECD guideline 473, US EPA 84-2. The test substance was evaluated for clastogenci (chromosome-damaging) activity in human lymphocytes in vitro following 3-3.5 hour treatments with and without metabolic (S9) activation. Three independent trials were conducted. In Trails 1 and 2, concentrations of 0, 1, 5, 8, and 10 µg/mL were evaluated and in Trial 3, concentrations of 0, 10, 15, and 18 µg/mL were evaluated. A statistically significant increase in the number of chromosomally abnormal cells was observed  at 1 µg/mL in the first activated trial; however, this effect was not reproduced in activated Trial 2. Concentration-related increases in percent abnormal cells were evident in both of the first two non-activated trials and this effect was statistically significant in Trial 1. In addition, a statistically significant increase in the number of chromosomally abnormal cells was observed at 15 µg/mL in non-activated Trial 3.

The test substance exhibited weak clastogenic activity without metabolic activation, and is considered positive under the conditions of this study.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: EEC Commission Directive 67/548/EEC

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

Deviations:

no

GLP compliance:

yes

Type of assay:

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

Specific details on test material used for the study:

Substance name: DPX-JE874
Lot #: DPX-JE874-133
Purity: 97.7%

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

Livers obtained from 8-9 week old male rats which received a single i.p. injection of 500 mg/kg Aroclor® 1254 five days prior to sacrifice. The animals were fasted for 18-24 hours immediately preceding sacrifice. Liver homogenate was prepared using a ratio of 1 g liver (wet weight):3 mL phosphate buffered saline (PBS). The homogenate was centrifuged at 9000 x g. The supernatant (S-9) was frozen, and the protein concentration was determined.

Test concentrations with justification for top dose:

5, 10, 20 and 30 µg/mL

High dose was selected based on the solubility of the test substance in culture medium

Vehicle / solvent:

Dimethyl sulfoxide (DMSO)

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9,10-dimethylbenzanthracene

ethylmethanesulphonate

Statistics:

Mutant frequency data were transformed according to the formula Y = (mutant frequency + 1)^0.15. This power transformation provided data which satisfied the assumptions required for performing parametric statistical analyses for this assay. The average transformed frequency of each test concentration was compared to the solvent control by a Student’s t-test to determine whether there were any significant increases in the mutant frequency. The probability values determined on the transformed data were based on the analysis of Dunnett. Testing for dose-response relationships were performed by a two way (concentration and experiment) Analysis of Variance (AN0VA). Linear, quadratic and higher order effects were tested by an F-test for significance.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: No cytotoxicity was observed at any concentration tested under either treatment condition.

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Negative in the CHO/HPRT gene mutation assay

Executive summary:

The study was conducted following OECD guideline 476, US EPA 84-2. The test substance was tested for mutagenic activity in the CHO/HPRT assay with and without metabolic (rat liver S-9) activation. Two independent activated and non-activated trials were conducted using treatment concentrations of 5, 10, 20 and 30 µg/mL. No statistically significant increases in mutant frequency occurred at any test concentration under activated or nonactivated conditions, and no biologically significant dose-response relationships were present. Under the conditions of this study, the test substance is negative.

Reference 3

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: EEC Commission Directive 87/302/EEC, Part В (1988)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5550 - Unscheduled DNA Synthesis in Mammalian Cells in Culture

Deviations:

no

GLP compliance:

yes

Type of assay:

other: Unscheduled DNA synthesis

Specific details on test material used for the study:

Substance name: DPX-JE874
Lot #: DPX-JE874-221
Purity: 97.28%

Species / strain / cell type:

hepatocytes: rat

Metabolic activation:

not applicable

Test concentrations with justification for top dose:

5.00, 2.50, 1.0, 0.5, 0.25, 0.10 µg/mL.

The top dose was selected based on cytotoxicity. Cytotoxicity was determined by comparing the cell density of the treated plates to the cell density of the control plates 20 to 24 hours after initiation of treatment. The test substance was completely toxic at and above 25 µg/mL, and moderately toxic at 5 and 10 µg/mL. The cellular morphology was suitable for analysis at and below 5 µg/mL.

Vehicle / solvent:

Dimethyl sulfoxide (DMSO)

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

Key result

Species / strain:

hepatocytes: rat

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: The test substance was completely cytotoxic at 10 µg/mL, highly cytotoxic at 7.5 µg/mL, and slightly toxic at 5 µg/mL.

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Negative in UDS assay

Executive summary:

The study was conducted following OECD guideline 482 and OPPTS 870.5550.

The test substance was tested in the in vitro assay for unscheduled DNA synthesis in rat liver primary cell cultures. In the assay, rat hepatocytes were exposed to test substance prepared in DMSO and dosed into media. Fifteen concentrations were applied, ranging from 1000 to 0.0250 µg/mL, in the presence of 10 µCi/mL tritiated thymidine (3H-TdR). The test substance formed a transparent yellow solution in DMSO at a concentration of 100000 µg/mL, and a cloudy suspension with white precipitate at 1000 and 500 µg/mL in media, the maximum appropriate test concentration for the in vitro UDS assay. Cytotoxicity was determined by comparing the cell density of the treated plates to the cell density of the control plates 20 to 24 hours after initiation of treatment. In Trial 1, the test substance was completely toxic at and above 25 µg/mL, and moderately toxic at 5 and 10 µg/mL. The cellular morphology was suitable for analysis at and below 5 µg/mL. The test substance was nontoxic at and below 1 µg/mL. However, Trial 1 was unacceptable due to high background grains in the developed slides, but was used to select concentrations for Trial 2.

Based on toxicity results from Trial 1, eleven concentrations ranging from 25 µg/mL to 0.05 µg/mL were initiated in Trial 2. The test substance was completely toxic at dose levels at and above 7.5 µg/mL, moderately cytotoxic at 5 µg/mL, and slightly toxic at 2.5 µg/mL. The test substance was nontoxic at and below 1 µg/mL. Six treatments from 5 µg/mL to 0.1 µg/mL covered a range of toxicity from 49.6% to 115.5% survival and were selected for analysis of nuclear labeling. No dose levels showed an elevation in nuclei containing 5 or more net grains.

An independent repeat was performed to verify the results obtained in Trial 2. Trial 3 was unacceptable due to the lack of a response in the positive controls. A fourth assay was performed.

Nine concentrations ranging from 10 g/mL to 0.05 µg/mL were initiated in the confirmatory trial. The test substance was completely cytotoxic at 10 µg/mL, highly cytotoxic at 7.5 µg/mL, and slightly toxic at 5 µg/mL. The test substance was nontoxic at and below 2.5 µg/mL. Six treatments from 5 µg/mL to 0.1 µg/mL covered a range of toxicity from 80.0% to 111.6% survival and were selected for analysis of nuclear labeling. No dose levels showed an elevation in nuclei containing 5 or more net grains.

The test substance did not induce an increase in unscheduled DNA systhesis in either Trial 2 nor Trial 4. The test substance, was evaluated as negative in the in vitro assay for unscheduled DNA synthesis in rat liver primary cell cultures.

Reference 4

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: EEC Commission Directive 67/548/EEC

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

Deviations:

no

GLP compliance:

yes

Type of assay:

other: Unscheduled DNA synthesis

Specific details on test material used for the study:

Substance name: DPX-JE874
Lot #: DPX-JE874-133
Purity: 97.7%

Species / strain / cell type:

hepatocytes: rat

Metabolic activation:

not applicable

Test concentrations with justification for top dose:

Trials 1 and 2: 0.05, 0.1, 0.25, 0.5, 1.0, 2.5, 5.0, and 10 µg/mL. In Trial 3, an additional concentration of 7.5 µg/mL was included.

The top dose was selected based on the test substance solubility. The test substance appeared to precipitate at concentrations >10 µg/mL

Vehicle / solvent:

Dimethyl sulfoxide (DMSO)

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

Key result

Species / strain:

hepatocytes: rat

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

other: Upon visual inspection of the slides, signs of toxicity, (i.e., cells lacking cytoplasmic and nuclear grain counts), were evident at 10 µg/mL in all three trials.

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Positive in UDS assay

Executive summary:

The study was conducted following OECD guideline 482 and US EPA 84-2. The test substance was tested for its ability to induce UDS in primary rat hepatocyte cultures. Three independent trials were conducted. In Trials 1 and 2, concentrations of 0, 0.05, 0.25, 0.5, 1.0, 2.5, 5.0, and 10 µg/mL were tested. In Trial 3, an additional concentration of 7.5 µg/mL was included. UDS, as evidenced by mean net nuclear grain counts ≥+5, was observed in all trials at two or more concentrations. Under the conditions of this assay, the test substance is considered positive.

 

Reference 5

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: EEC Commission Directive 87/302/EEC

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

Deviations:

no

GLP compliance:

yes

Type of assay:

other: Unscheduled DNA synthesis

Specific details on test material used for the study:

Substance name: DPX-JE874
Lot #: DPX-JE874-221
Purity: 97.4%

Species / strain / cell type:

hepatocytes: rat

Metabolic activation:

not applicable

Test concentrations with justification for top dose:

Trial 1: 0.05, 0.1, 0.5, 1.0, 5.0, and 10 µg/mL
Trial 2: A concentration of 7.5 µg/mL was also included.

Vehicle / solvent:

Dimethyl sulfoxide (DMSO)

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

Species / strain:

hepatocytes: rat

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Upon visual inspection of the slides, signs of toxicity, (i.e., cells lacking cytoplasmic and nuclear grain counts), were evident at 10 µg/mL in both the trials.

Remarks:

The results of this study are inconsistent, some results are greater than +5NNGs and there is no clear concentration effect.

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Negative in UDS assay

Executive summary:

The study was conducted following OECD guideline 482 and US EPA 84-2. The test substance was tested for its ability to induce UDS in primary rat hepatocyte cultures. Two independent trials were conducted. In Trial 1, concentrations of 0, 0.05, 0.1, 0.5, 1, 5, and 10 µg/mL were tested. In trial 2，a concentration of 7.5 µg/mL was also included. Cytotoxicity precluded the evaluation of UDS at the concentration of 10 µg/mL. UDS was not observed in either trial at any  concentration evaluated. Under the conditions of this assay, the test substance is negative.

Reference 6

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japan Ministry of Agriculture, Forestry and Fisheries Testing Guidelines for Toxicology Studies, 59 NohSan No. 4200

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Substance name: DPX-JE874
Batch #: JE874-133
Purity: 97.7%

Target gene:

Genes coding for histidine and tryptophan biosynthetic enzymes

Species / strain / cell type:

other: S. typhimurium TA100, TA1535, TA97, TA98

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

Rat liver homogenate activation system (S9 Mix)

Test concentrations with justification for top dose:

Trial 1: 10, 50, 100, 500, 1000, 2500, 5000 µg/plate
Trial 2: 50, 100, 500, 1000, 2500, 5000 µg/plate

Vehicle / solvent:

Dimethyl sulfoxide (DMSO)

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene, ICR-191 acridine

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

other: S. typhimurium TA100, TA1535, TA97, TA98

Metabolic activation:

with and without

Genotoxicity:

negative

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Negative bacterial reverse mutation assay in S. typhimurium TA100, TA1535, TA97, TA98 and E. coli WP2uvrA

Executive summary:

The study was conducted according to OECD guideline 471, 472 and US EPA 84-2. The maximum concentration of test substance evaluated for mutagenicity in Salmonella typhimurium strains TA100, TA1535, TA97 and TA98 and E. coli strain WP2uvrA (pKM101) was 5000 µg/plate. No evidence of mutagenic activity was detected in two independent trials with or without metabolic activation. In this study, the test substance is negative.

Reference 7

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: U.S. EPA Health Effects Test Guidelines, Toxic Substances Control Act, article 799.9530 (1997)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EEC Commission Directive 87/302/EEC, Part В (1988)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes

Type of assay:

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

Specific details on test material used for the study:

Substance name: Famoxadone Technical
Lot #: DPX-JE874-221
Purity: 97.28%

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: The hypodiploid CHO cell line was originally derived from the ovary of the female Chinese hamster (Cricetulus griseus). The clone used in this assay was CHO-K1-BH4, obtained from Oak Ridge National Laboratories, Oak Ridge, Tennessee.

For cell lines:
- Absence of Mycoplasma contamination: Mycoplasma testing was performed by a commercial laboratory on stock cultures prior to preparing stocks for freezing and periodically on stock cultures used for assays. Both direct culturing methods and the indirect Hoechst staining method were used.
- Methods for maintenance in cell culture: Master stocks of CHO-K1-BH4 cells were maintained frozen in liquid nitrogen. Cells were maintained as monolayer cultures at 37 ± 1.5°C in a humidified atmosphere containing 5 ± 1.5% CO2.
- Doubling time: 11 to 14 hours
- Periodically checked for karyotype stability: yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: Cells used in this study were maintained in Ham's Nutrient Mixture F12 supplemented with L-glutamine, gentamicin, Fungizone, and fetal bovine serum (8% by volume), hereafter referred to as culture medium. Stock cultures were maintained in culture medium without the antibiotics. Cleansing medium used for reducing the frequency of HGPRT mutants prior to experimental studies consisted of culture medium with reduced serum content (5%) supplemented with 5.0 x 10^-6 M thymidine, 1.0 X 10^-5 M hypoxanthine, 1.0 x 10^-4 M glycine, and 3.2 x 10^-6 M methotrexate (HATG medium). Recovery medium is HATG medium with the methotrexate component removed and with the fetal bovine serum increased to 8% by volume. Selection medium for mutants was hypoxanthine-free F12 culture medium containing 4 µg/mL of TG (24 µM TG) with the fetal bovine serum component reduced to 5% by volume.

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 fraction induced with Aroclor™ 1254

Test concentrations with justification for top dose:

Without metabolic activation: Initial mutation test: 12.5, 25.0, 50.0, 75.0, 100, 150, 175, 200, and 250 µg/mL; confirmatory mutation assay: 25.0, 50.0, 100, 200, 250, 300, 350, 400 and 450 µg/mL.
With metabolic activation: Initial mutation test: 12.5, 25.0, 50.0, 75.0, 100, 150, 200, 250, 300 and
400 µg/mL; confirmatory mutation assay: 50.0, 100, 200, 300, 350, 400, 450, 500 and 600 µg/mL.

Vehicle / solvent:

Dimethyl sulfoxide (DMSO)

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 5-Bromo-2'-deoxyuridine (BrdU); 20-Methylcholanthrene (MCA)

Details on test system and experimental conditions:

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

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 4 x 10^6 cells
- Test substance added in medium

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 7 days
- Selection time (if incubation with a selective agent): 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 7-10 days
- Selective agent: 4 µg/mL 6-thioguanine

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Percentage of mean colony counts

Evaluation criteria:

Evaluation of a Positive Response: The test substance induces a positive response when:
The mutant frequency of a treated culture is significantly different from the mutant frequencies of the concurrent negative controls at the 95% or 99% confidence levels. This test compares variables distributed according to Poissonian expectations by summing up the probabilities in the tails of two binomial distributions. In addition, the mutant frequency must meet or exceed 15 X 10^-6 in order to compensate for random fluctuations that are typical for this assay.
A dose-related or toxicity-related increase in mutant frequency should be observed. The increase should be observed in both the initial and confirmatory assay, although this may not always be required, especially if different doses are used in the two trials.
If an increase in mutant frequency is observed near the highest testable toxic dose and the number of mutant colonies is more than twice the value needed to indicate a significant response, the test substance generally will be considered mutagenic. Smaller increases at a single dose near the highest testable toxic dose will be evaluated as equivocal and may require confirmation by a repeat assay. Significant mutagenic activity in one culture of a dose level should be confirmed in the replicate culture of the same dose level. This may not always be possible when differences in toxicity are observed or when there is a very weak response. Each assay is evaluated on a case by case basis.

Evaluation of a Negative Response: A test substance will be evaluated as non-mutagenic when:
The mutant frequency of none of the doses that allows greater than 10% survival is significantly greater than the mutant frequency of the vehicle control, where significance is determined at p <0.05.
A repeat assay does not confirm an earlier response and the same levels of toxic action have been reached in both trials.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Non-activation: At 125 µg/mL, the test substance was moderately cytotoxic and higher concentrations were lethal. In the presence of S9: Treatment at 250 µg/mL was highly cytotoxic and higher concentrations were lethal

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Dose Range Finding Assay: The test substance was tested in a preliminary dose range finding assay both with and without S9 metabolic activation. The test substance was soluble in dimethylsulfoxide (DMSO) at 503 mg/mL. In treatment medium, the test compound formed a precipitate at 2520 µg/mL and 5030 µg/mL; at 630 µg/mL, the test compound appeared translucent. The dose range finding assay was initiated with a high dose of 1000 µg/mL based on the solubility characteristics. Ten dose levels were used in each case that ranged from 1.97 µg/mL to 1000 µg/mL; vehicle controls were included under each activation condition. In the dose range finding assay, the test substance remained in solution in treatment medium from 1.97 µg/mL to 125 µg/mL, but had an opaque appearance at and above 250 µg/mL.

In the preliminary dose range finding assays, cells were exposed to the test substance from 1.97 µg/mL to 1000 µg/mL for about four hours in the presence and absence of metabolic activation. Under nonactivation conditions the test substance was noncytotoxic to weakly cytotoxic from 1.97 µg/mL to 62.5 µg/mL. At 125 µg/mL, the test substance was moderately cytotoxic and higher concentrations were lethal. In the presence of S9 metabolic activation, the test substance was noncytotoxic to weakly cytotoxic from 1.97 µg/mL to 125 µg/mL. Treatment at 250 µg/mL was highly cytotoxic and higher concentrations were lethal. The initial mutation assays were initiated based on the results of the preliminary dose range finding assays.

Table-1: Initial mutation assay without metabolic activation

Non-activation	Total mutant colonies	Absolute C.E. ± SD (%)	Mutant frequency in 10^-6 units
Vehicle control	8	116.5 ± 6.9	2.9
Vehicle control	9	113.2 ± 3.3	4.0
Positive control (50 µg/mL BrdU)	188	125.7 ± 11.8	68.0^a
Positive control (50 µg/mL BrdU)	146	104.2 ± 13.6	77.9^a
Test substance (µg/mL)
75	0	94.8 ± 20.1	[0.0]
75	0	105.5 ± 13.9	0.0
100	5	106.7 ± 2.6	[3.3]
100	1	107.0 ± 3.3	0.4
150	4	117.0 ± 6.1	1.4
150	1	143.3 ± 13.8	0.3
175	4	114.3 ± 3.8	1.6
175	1	114.3 ± 4.1	0.4
200	4	109.8 ± 3.8	1.7
200	5	92.2 ± 2.1	3.0
250	1	121.5 ± 10.1	0.5
250	1	120.8 ± 10.5	0.5

a Significant increase: Kastenbaum Bowman test p ≤0.01 and mutant frequency ≥15 x 10^-6

Table-2: Confirmatory mutation assay without metabolic activation

Non-activation	Total mutant colonies	Absolute C.E. ± SD (%)	Mutant frequency in 10^-6 units
Vehicle control	5	118.5 ± 4.8	1.8
Vehicle control	6	100.0 ± 7.0	2.5
Positive control (50 µg/mL BrdU)	162	122.5 ± 9.1	55.1^a
Positive control (50 µg/mL BrdU)	118	100.0 ± 6.0	59.0^a
Test substance (µg/mL)
200	0	88.7 ± 9.0	0.0
200	8	99.5 ± 0.7	3.4
250	5	101.8 ± 10.2	2.0
250	8	100.8 ± 4.0	3.3
300	5	67.0 ± 4.9	3.4
300	1	93.0 ± 10.8	0.4
350	7	84.3 ± 13.3	3.5
350	1	91.2 ± 5.8	0.5
400	14	108.0 ± 10.7	5.4^b
400	4	94.0 ± 3.1	1.8
450	5	97.0 ± 6.9	2.1
450	13	107.3 ± 2.8	5.0

a Significant increase: Kastenbaum Bowman test p ≤0.01 and mutant frequency ≥15 x 10^-6

b Significant increase: Kastenbaum Bowman test p ≤0.05 but mutant frequency <15 x 10^-6

Table-3: Initial mutation assay with metabolic activation

Activation	Total mutant colonies	Absolute C.E. ± SD (%)	Mutant frequency in 10^-6 units
Vehicle control	12	90.8 ± 3.2	5.5
Vehicle control	4	84.0 ± 0.9	2.0
Positive control (5 µg/mL MCA)	186	84.0 ± 9.9	92.3^a
Positive control (5 µg/mL MCA)	140	94.8 ± 3.2	67.1^a
Test substance (µg/mL)
100	6	101.2 ± 5.4	2.5
100	3	104.8 ± 4.5	1.2
150	12	95.8 ± 1.4	5.2
150	2	105.5 ± 10.1	0.8
200	14	100.0 ± 9.1	5.8
200	11	102.2 ± 5.8	4.5
250	15	108.2 ± 5.6	5.8
250	9	107.0 ± 4.8	3.5
300	21	95.0 ± 10.8	9.2^b
300	2	82.5 ± 2.8	1.0
400	4	82.7 ± 2.8	2.0
400	7	86.5 ± 8.5	3.4

a Significant increase: Kastenbaum Bowman test p ≤0.01 and mutant frequency ≥15 x 10^-6

b Significant increase: Kastenbaum Bowman test p ≤0.05 but mutant frequency <15 x 10^-6

Table-4: Confirmatory mutation assay with metabolic activation

Activation	Total mutant colonies	Absolute C.E. ± SD (%)	Mutant frequency in 10^-6 units
Vehicle control	5	100.7 ± 5.2	2.1
Vehicle control	4	82.0 ± 4.3	2.0
Positive control (5 µg/mL MCA)	245	91.8 ± 7.8	111.2^a
Positive control (5 µg/mL MCA)	230	91.8 ± 4.1	104.4^a
Test substance (µg/mL)
300	3	94.5 ± 2.1	1.3
300	6	91.5 ± 7.2	2.7
400	3	90.2 ± 6.8	1.4
400	2	85.2 ± 2.0	1.0
450	8	95.7 ± 4.9	3.5
450	16	89.8 ± 5.3	8.1^b
500	9	97.3 ± 2.6	3.9
500	3	86.7 ± 6.8	1.4
600	4	100.2 ± 8.8	1.8
600	1	84.2 ± 5.1	0.5

a Significant increase: Kastenbaum Bowman test p ≤0.01 and mutant frequency ≥15 x 10^-6

b Significant increase: Kastenbaum Bowman test p ≤0.01 but mutant frequency <15 x 10^-6

Conclusions:

Negative in the CHO/HPRT gene mutation assay

Executive summary:

The study was conducted following OECD guideline 476, OPPTS 870.5300. The objective of this in vitro assay was to evaluate the ability of famoxadone to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in Chinese hamster ovary cells under conditions with and without metabolic activation. The test substance was soluble in dimethylsulfoxide (DMSO) at 503 mg/mL. In treatment medium, the test substance formed a precipitate at 2520 µg/mL and 5030 µg/mL; at 630 µg/mL, the test substance appeared translucent. The dose range finding assay was initiated with a high dose of 1000 µg/mL based on the solubility characteristics. In the dose range finding assay, the test substance remained in solution in treatment medium from 1.97 µg/mL to 125 µg/mL, but the dosed treatment medium had an opaque appearance at and above 250 µg/mL.

In the preliminary dose range finding assay, cells were exposed to the test substance from 1.97 µg/mL to 1000 µg/mL for four hours in the presence and absence of metabolic activation (Aroclor-induced rat liver S9). Under nonactivation conditions, the test substance noncytotoxic to weakly cytotoxic from 1.97 µg/mL to 62.5 µg/mL. At 125 µg/mL, the test substance was moderately cytotoxic and higher concentrations were lethal. In the presence of S9 metabolic activation, the test substance was noncytotoxic to weakly cytotoxic from 1.97 µg/mL to 125 µg/mL. Treatment at 250 µg/mL was highly cytotoxic and higher concentrations were lethal. The initial mutation assays were initiated based on the results of the preliminary dose range finding assays.

In the initial trial of the nonactivation assay, six duplicate treatments from 75.0 µg/mL to 250 µg/mL were analyzed. One of the treatments at 75.0 µg/mL and one at 100 µg/mL were lost to contamination. In the remaining treatments, no cytotoxicity to weak cytotoxicity was observed based on relative survival (cloning efficiency after treatment) but high cytotoxicity was reached based on relative total growth (suspension growth during the expression period). No significant increases in the mutant frequency were observed at any of the assayed treatments. A confirmatory assay was performed.

In the confirmatory nonactivation assay, six duplicate treatments from 200 µg/mL to 450 µg/mL were analyzed. No cytotoxicity was observed based on relative survival, but moderate cytotoxicity was reached based on relative total growth. One treatment at 400 µg/mL had a mutant frequency that was significantly elevated but the mutant frequency was less than 15x10^-6 and a duplicate at the same concentration was not elevated. The test substance was therefore considered non-mutagenic without activation in this assay.

In the initial trial performed in the presence of metabolic activation, six duplicate treatments from 100 µg/mL to 400 µg/mL were analyzed. No cytotoxicity to moderate cytotoxicity was observed based on relative survival but cytotoxicity reached very high levels based on relative total growth. One treatment at 300 µg/mL had a mutant frequency that was significantly elevated but the mutant frequency was less than 15 x 10^-6 and a duplicate at the same concentration was not elevated. The test substance was considered non-mutagenic in the initial trial of the activation assay. A confirmatory assay was performed.

In the confirmatory activation assay, five duplicate treatments from 300 µg/mL to 600 µg/mL were analyzed. No cytotoxicity to weak cytotoxicity was observed based on relative survival but the higher concentrations were highly cytotoxic based on relative total growth. One treatment at 450 µg/mL had a mutant frequency that was significantly elevated but was less than 15 x 10^-6; in addition, a duplicate treatment at the same concentration was not elevated. The test substance was therefore evaluated as non-mutagenic with activation in this assay.

The test substance was evaluated as negative for inducing forward mutations at the HGPRT locus in CHO cells under the non-activation and S9 metabolic activation conditions used in this study.

Reference 8

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Japan Ministry of Agriculture, Forestry and Fisheries Testing Guidelines for Toxicology Studies, 59 NohSan No. 4200

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Substance name: DPX-JE874-221 Technical
Batch #: JE874-221
Purity: 97.4%

Target gene:

Genes coding for histidine and tryptophan biosynthetic enzymes

Species / strain / cell type:

other: S. typhimurium TA100, TA1535, TA97, TA98

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

Rat liver homogenate activation system (S9 Mix)

Test concentrations with justification for top dose:

0, 10, 50, 100, 500, 1000, 2500, and 5000 µg/plate

In accordance with EPA, OECD, and MAFF Japan test guidelines, the highest concentration evaluated in this study was 5000 µg/plate.

Vehicle / solvent:

Dimethyl sulfoxide (DMSO)

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene, ICR-191 acridine

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:
- Cell density: 1x10^8 bacteria
- Test substance added in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48 hours

Evaluation criteria:

A test substance was classified as positive when 1) the average number of revertants in any strain at any test substance concentration studied vas at least two times greater than the average number of revertants in the negative control and 2) there was a positive dose-response relationship in that same strain.

A test substance was classified as negative when either 1) there were no test substance concentrations with an average number of revertants which was at least two times greater than the average number of revertants in the negative control and 2) there was no positive dose-response relationship.

Results not meeting these criteria for positive or negative assessments will be evaluated on a case basis using scientific judgement and experience.

Statistics:

Trials were evaluated independently. For each tester strain, the average number of revertants and the standard deviation at each concentration with and without S9 activation were calculated.

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

other: S. typhimurium TA100, TA1535, TA97, TA98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Table 1: Summary of average revertants/plate without activation

Compound	Conc. mg/plate	TA100		TA1535		TA97		TA98		WP2uvrA
		Trial 1	Trial 2	Trial 1	Trial 2	Trial 1	Trial 2	Trial 1	Trial 2	Trial 1	Trial 2
Test substance	10	126 ± 6	132 ± 25	12 ± 3	9 ± 3	107 ± 22	139 ± 19	22 ± 4	18 ± 5	169 ± 11	168 ± 15
	50	80 ± 7	95 ± 4	13 ± 3	13 ± 3	105 ± 17	158 ± 6	19 ± 4	13 ± 3	185 ± 16	180 ± 17
	100	77 ± 12	95 ± 5	10 ± 1	7 ± 6	115 ± 21	173 ± 10	19 ± 8	16 ± 6	185 ± 8	169 ± 20
	500	99 ± 5	97 ± 24	7 ± 1	10 ± 7	115 ± 15	163 ± 17	16 ± 1	15 ± 3	178 ± 18	197 ± 12
	1000	86 ± 12	119 ± 26	10 ± 3	7 ± 3	113 ± 6	167 ± 4	18 ± 2	20 ± 3	168 ± 10	187 ± 6
	2500	100 ± 18	65 ± 18	11 ± 1	7 ± 1	113 ± 5	143 ± 23	17 ± 3	21 ± 8	161 ± 19	182 ± 11
	5000	95 ± 6	58 ± 19	10 ± 2	8 ± 4	123 ± 8	103 ± 15	22 ± 1	18 ± 3	158 ± 12	159 ± 13
DMSO	-	127 ± 10	133 ± 16	13 ± 1	14 ± 3	115 ± 10	150 ± 9	23 ± 8	21 ± 3	170 ± 10	169 ± 25
Sodium azide	2.0	554 ± 53	504 ± 18	1249 ± 40	607 ± 26	-	-	-	-	-	-
ICR-191	2.0	-	-	-	-	2193 ± 249	2602 ± 156	-	-	-	-
2-Nitrofluorene	25	-	-	-	-	-	-	1381 ± 76	1663 ± 34	-	-
Methylmethane sulfonate	1000	-	-	-	-	-	-	-	-	2211 ± 85	2019 ± 80

 

Table 2: Summary of average revertants/plate with activation

Compound	Conc. mg/plate	TA100		TA1535		TA97		TA98		WP2uvrA
		Trial 1	Trial 2	Trial 1	Trial 2	Trial 1	Trial 2	Trial 1	Trial 2	Trial 1	Trial 2
Test substance	10	178 ± 8	149 ± 12	13 ± 5	13 ± 6	127 ± 12	155 ± 12	28 ± 5	31 ± 4	182 ± 15	190 ± 12
	50	134 ± 8	110 ± 11	11 ± 1	9 ± 4	132 ± 7	157 ± 15	29 ± 3	25 ± 9	179 ± 2	188 ± 16
	100	127 ± 20	120 ± 4	11 ± 2	10 ± 2	133 ± 11	157 ± 8	24 ± 4	21 ± 2	170 ± 17	178 ± 23
	500	120 ± 20	99 ± 12	12 ± 2	11 ± 5	138 ± 8	155 ± 9	25 ± 3	23 ± 4	193 ± 8	175 ± 16
	1000	112 ± 11	105 ± 3	12 ± 6	8 ± 3	150 ± 12	148 ± 10	27 ± 2	31 ± 2	195 ± 18	173 ± 20
	2500	115 ± 10	95 ± 9	13 ± 3	9 ± 2	127 ± 20	150 ± 9	25 ± 6	22 ± 5	182 ± 15	162 ± 17
	5000	121 ± 9	109 ± 9	10 ± 1	6 ± 2	250 ± 2	134 ± 2	21 ± 5	18 ± 2	177 ± 9	152 ± 19
DMSO	-	134 ± 4	139 ± 3	13 ± 2	15 ± 4	101 ± 7	148 ± 10	26 ± 5	25 ± 4	190 ± 9	158 ± 8
2-aminoanthracene	1.0	1587 ± 217	827 ± 108	-	-	1327 ± 71	710 ± 129	-	-	-	-
2-aminoanthracene	2.0	-	-	384 ± 18	414 ± 61	-	-	1727 ± 80	1678 ± 333	-	-
2-aminoanthracene	25	-	-	-	-	-	-	-	-	2017 ± 79	1562 ± 118

Conclusions:

Negative bacterial reverse mutation assay in S. typhimurium TA100, TA1535, TA97, TA98 and E. coli WP2uvrA

Executive summary:

The test substance was evaluated for mutagenicity in Salmonella typhimurium strains TA100, TA1535, TA97, and TA98 and in Escherichia colі strain WP2uvrA (pKM101) with and without an exogenous metabolic activation system (S9). The study was conducted according to OECD guideline 471 and U. S. EPA 84-2. The maximum concentration tested was 5000 µg/plate. Additional concentrations tested included 0 (solvent control), 10, 50, 100, 500, 1000, and 2500 µg/plate. No evidence of mutagenic activity was detected in either of two independent trials; the test substance is negative.

Reference 9

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Substance name: DPX-JE874
Lot #: DPX-JE874-221
Purity: 97.4%

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Venous blood (supplied by the DuPont Clinical Specimen and Bioreagent Acquisition Group) was drawn aseptically from healthy donors

For lymphocytes:
- Sex and number of blood donors: One male and one female donor
- Whether whole blood or separated lymphocytes were used: Whole blood
- Whether blood from different donors were pooled or not: Not pooled. One replicate was from a male donor and the other from a female donor.

MEDIA USED
-The treatment medium for each culture consisted of 5 mL RPMI 1640 medium that had been supplemented with L-glutamine (2 mM), penicillin (50 IU/mL), streptomycin (50 µg/mL), and HEPES buffer (25 mM). In treatments with activation, S9 and cofactor components were included in the medium at final concentrations of: S9 (1 mg protein/mL), magnesium chloride (5 mM), glucose-6-phosphate (5 mM), and nicotinamide adenine dinucleotide phosphate (1.5 mM).

Cytokinesis block (if used):

Colcemid® (0.1 µg/mL)

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 fraction

Test concentrations with justification for top dose:

0, 5, 10, 15, 20, 25, and 30 µg/mL

Top dose was selected based on the limit of solubility in the culture medium

Vehicle / solvent:

Dimethyl sulfoxide (DMSO)

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

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

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3 hrs

FOR CHROMOSOME ABERRATION:
- Spindle inhibitor (cytogenetic assays): Colcemid® (01 µg/mL) present during the final 2 hrs to arrest cells in metaphase.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): Cultures were centrifuged, and the pelleted cells were resuspended and incubated for approximately 14-18 min in 0.075 M KCl at 37°C. Cells were fixed in at least three changes of methanol:glacial acetic acid (3:1, v/v) and dropped onto microscope slides which were then air-dried or dried on a slide warmer. Sister chromatid differentiation was achieved using a modified fluorescence plus-Giemsa method. Slides were stained in 0.05 mg/mL Hoechst 33258 in pH 7.0 phosphate buffer, rinsed in water, then exposed to 15W UV fluorescent light for 2 hrs while immersed in heated phosphate buffer. After rinsing in water, the slides were stained with 5% Giemsa in phosphate buffer for 20 min and rinsed again. After drying, the slides cleared in Hemo-De® clearing agent and coverslips mounted with Cytoseal 60®.
- Number of cells spread and analysed per concentration: 100 metaphase cells (50 from each replicate)
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): Only cells with 46 centromeres were scored


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Mitotic index (MI)

Rationale for test conditions:

Solubility testing indicated that the test substance was soluble in dimethyl sulfoxide (DMSO) and that the limit of solubility was approximately 30 µg/mL in culture medium. Therefore, the maximum concentration tested in the cytotoxicity assessment was 30 µg/mL. This was achieved when a 3 mg/mL solution was added to the culture medium in a volume of 1% (v/v)

Evaluation criteria:

The test substance was classified as clastogenic (positive) if both of the following effects were reproducible (i.e., evident in two or more trials under activated or nonactivated conditions):
1. The test substance produced a statistically significant increase in percent abnormal cells as compared to the negative (solvent) control at one or more test concentrations (p ≤0.05).
2. There was a statistically significant dose-related increase in percent abnormal cells (p ≤0.01).

The test substance was classified as non-clastogenic (negative) if the following criteria were met:
1. The test substance did not produce a statistically significant increase in percent abnormal ceils at any concentration tested.
2. There was no statistically significant dose-related increase in percent abnormal cells.

Statistics:

For each trial, the proportion of abnormal cells and the proportion of cells with more than one aberration were evaluated using a Fisher Exact Test to compare each treatment level with the negative (solvent) control, where statistical significance was judged at the 5% level. A Cochran-Armitage test for linear trend (dose-response) was performed where appropriate; significance was judged at the 1% level. Chromatid and isochromatid gaps were excluded from the statistical evaluation.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: With activation: Reductions in MI were not observed

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

other: Without activation: MI were decreased at all concentrations tested, with an 87% reduction in the MI observed at 30 µg/mL.

Vehicle controls validity:

valid

Positive controls validity:

valid

Table-1: Structural chromosome aberrations: 3-Hour treatment without activation

Test concentration (µg/mL)	cells scored	Harvest time (h)	Aberrations/cell	% Abn. Cells	%cells with >1 Aberr.	Mitotic index (%)
Trail-1
0 (DMSO)	100	18	0.05	5.0	0.0	6.2
10	100	21	0.07	4.0	1.0	5.8
15	100	21	0.07	6.0	1.0	3.3
20	100	21	0.16	14.0*	2.0	1.4
25	100	21	0.25	18.0*	3.0	1.4
MMC 0.35	100	18	0.61	43.0*	12.0*	1.0
Trail-2
0 (DMSO)	100	19	0.04	3.0	1.0	9.1
10	100	19	0.09	9.0	0.0	7.0
15	100	23	0.20	15.0*	4.0	5.6
20	100	23	0.17	14.0*	2.0	4.4
25	100	23	0.13	12.0*	1.0	2.6
MMC 0.35	100	19	0.52	42.0*	9.0*	6.9

Table-2: Structural chromosome aberrations: 3-Hour treatment with activation

Test concentration (µg/mL)	cells scored	Harvest time (h)	Aberrations/cell	% Abn. Cells	%cells with >1 Aberr.	Mitotic index (%)
Trail-1
0 (DMSO)	100	18	0.03	3.0	0.0	6.2
15	100	21	0.01	1.0	0.0	5.0
20	100	21	0.04	4.0	0.0	5.2
25	100	21	0.01	1.0	0.0	4.2
30	100	21	0.02	2.0	0.0	5.2
CP 10	100	18	0.50	37.0*	9.0*	1.2
Trail-2
0 (DMSO)	100	19	0.05	3.0	1.0	10.4
15	100	19	0.02	2.0	0.0	9.3
20	100	19	0.00	0.0	0.0	9.1
25	100	19	0.04	4.0	0.0	7.4
30	100	19	0.04	4.0	0.0	6.7
CP 10	100	19	0.64	39.0*	12.0*	5.8

*Statistically significant p <0.05

Conclusions:

The test substance exhibited clastogenic activity in human lymphocytes without metabolic activation

Executive summary:

The study was conducted following OECD guideline 473, US EPA 84-2.

The test substance was evaluated for clastogenic (chromosome-damaging) activity in human lymphocytes in vitro following 3-hr treatments with and without metabolic (S9) activation. Two independent trials were conducted at concentrations of 0, 5, 10, 15, 20, 25, and 30 µg/mL. For trials without S9 activation, chromosome aberrations evaluated at 0, 10, 15, 20, and 25 µg/mL; toxicity precluded evaluation of the 30 µg/mL level. For trials with S9 activation, metaphase cells from the 0, 15, 20, 25, and 30 µg/mL treatment levels were evaluated.

In the nonactivated trials, statistically significant increases in the percent of abnormal cells were observed at 20 and 25 µg/mL in Trial 1 and at 15, 20, and 25 µg/mL in Trial 2. Additionally, significant dose-related increases in the percent of abnormal cells were evident in both nonactivated trials. In trials with S9 activation, significant increases in the percent of abnormal cells were observed at any concentration evaluated.

The test substance exhibited clastogenic activity in human lymphocytes without S9 activation. Under the conditions of this assay, the test substance is positive.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Micronucleus Study

In vivo mouse micronucleus study in bone marrow cells of mice; Equivocal. OECD 474, OPP 84-2, EU Method B.10, JMAFF 59 Nohsan 4200; Reliability = 1

 

UDS Assay

UDS assay in hepatocytes of male rats. Negative. OPP 84-2; Reliability = 1.

UDS assay in hepatocytes isolated ex vivo. Negative. OECD 485, OPP 84-2; Reliability = 1.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

Deviations:

no

GLP compliance:

yes

Type of assay:

unscheduled DNA synthesis

Specific details on test material used for the study:

Substance name: DPX-JE874
Lot #: JE874-221
Purity: 97.4%

Species:

rat

Strain:

other: Crl:CD®(SD)BR

Sex:

male

Route of administration:

oral: gavage

Vehicle:

Corn oil

Duration of treatment / exposure:

Single dose

Frequency of treatment:

Single dose

Post exposure period:

4 and 18 hours after dosing

Dose / conc.:

1 000 mg/kg bw (total dose)

Dose / conc.:

2 500 mg/kg bw (total dose)

Dose / conc.:

5 000 mg/kg bw (total dose)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

DMN was prepared in phosphate buffered saline (PBS) and administered by oral intubation at a dose of 10 mg/kg. A fine suspension of 2AAF was made in 0.5% methyl cellulose using a Polytron® homogenizer and was administered orally by intubation at a dose of 50 mg/kg.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

The test substance did not induce UDS in hepatocytes of male rats following a single dose of up to 5000 mg/kg

Executive summary:

The study was conducted following US EPA 84-2. The test substance was evaluated for its ability to induce unscheduled DNA synthesis (UDS) in hepatocytes of male Crl:CD®(SD)BR rats. The animals were administered a single dose of 0 (corn oil control), 1000, 2500, or 5000 mg of the test substance/kg body weight by oral intubation. Liver cells were isolated at approximately 4 and 18 hours after dosing and cultured in the presence of [methyl-3H] thymidine. UDS was determined autoradiographically by counting silver grains. The 1000 mg/kg dose group was excluded from evaluation at the 4 hour time point due to presumed errors in slide labeling. At both sampling times, net nuclear grain counts in hepatocytes of animals were similar to concurrent negative control values. The test substance did not induce UDS in the liver of male rats treated in vivo.

Reference 2

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

Deviations:

no

GLP compliance:

yes

Type of assay:

unscheduled DNA synthesis

Specific details on test material used for the study:

Substance name: DPX-JE874
Lot #: JE874-221
Purity: 97.4%

Species:

rat

Strain:

other: Crl:CD®(SD)BR

Sex:

male

Route of administration:

oral: gavage

Vehicle:

Corn oil

Duration of treatment / exposure:

Single dose

Frequency of treatment:

Single dose

Dose / conc.:

800 mg/kg bw (total dose)

Dose / conc.:

2 000 mg/kg bw (total dose)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Positive control(s):

2-Acetamidofluorene (2-AAF) was freshly suspended in com oil using a Silverson homogeniser at 7.5 mg/mL to serve as the positive control for the 14-16 hour experiment. Dimethylnitrosamine (DMN) dissolved in purified water at 1.0 mg/mL was used as the positive control for the 2-4 hour experiment. Both positive control compounds were administered at a dose volume of 10 mL/kg.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

male rats showed no induction of UDS in hepatocytes isolated ex vivo approximately 14-16 or 2-4 hours after dosing.

Executive summary:

The test substance was tested for its ability to induce unscheduled DNA synthesis (UDS) in the livers of orally dosed male rats using an in vivo/in vitro procedure. The study was conducted following OECD guideline 486 and US PEA 84-2.

The Sponsor supplied information indicating that there was no substantial inter-sex difference in the toxicity of DPX-JE874-221 to rats. Accordingly male animals were used for this study.

The Sponsor supplied information indicating that the rat oral LD50 of test substance was >5000 mg/kg. In an initial confirmatory toxicity range-finder study, three male Sprague Dawley Crl:CD(R)BR rats were dosed once with 2000 mg/kg (the widely accepted limit dose for the UDS assay). During a 4 day post-dose observation period, no clinical signs were observed. Accordingly, 2000 mg/kg was selected as the top dose to be tested in the main study experiments, along with a lower dose of 800 mg/kg (40% of the top dose).

In the main study experiments, groups of five male rats were treated once with the solvent corn oil, the test substance (at 2000 or 800 mg/kg) or the required positive control, by oral gavage, at a dose volume of 10 mL/kg. The positive controls used were 75 mg/kg 2-acetamidofluorene (2-AAF) suspended in corn oil (14-16 hour experiment) and 10 mg/kg dimethylnitrosamine (DMN) dissolved in purified water (2-4 hour experiment).

No clinical signs were observed in any animal dosed in the main study experiments.

Approximately 14-16 hours (Experiment 1) or 2-4 hours (Experiment 2) after dosing, animals were killed and their livers perfused with collagenase to provide a primary culture of hepatocytes. Cultures were made from three animals in each dose group and were treated with [3H] thymidine. Six slides from each animal were prepared with fixed hepatocytes and of these, three were dipped in photographic emulsion to prepare autoradiograms. Slides were examined microscopically after development of the emulsion and staining, and the net grain count (NNG), the number of grains present in the nucleus minus the mean number of grains in three equivalent areas of cytoplasm, was determined for each of two of the three slides, each animal and dose group.

Negative (vehicle) control animals gave a group mean NNG value of less than zero with a maximum of 0.7% cells in repair. Group mean NNG values were increased by 2-AAF and DMN treatment to more than 8.6 and more than 50% cells were found to be in repair. In this study the vehicle control NNG value was consistent with both published and historical control data, and the system was shown to be sensitive to two known DNA damaging agents requiring metabolism for their action. The assay was therefore accepted as valid.

Treatment with 2000 or 800 mg/kg test substance did not produce a group mean NNG value greater than -1.7 nor were any more than 0.3% cells found in repair at either dose.

It was concluded that the test substance failed to induce UDS detectable under the experimental conditions employed.

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japan Ministry of Agriculture, Forestry and Fisheries Testing Guidelines for Toxicology Studies, 59 NohSan No. 4200

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

Substance name: DPX-JE874
Lot#: JE874-221
Purity: 97.4%

Species:

mouse

Strain:

other: Crl:CD®_1(ICR)BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Inc., Saint Constant, Quebec, Canada
- Age at study initiation: Approximately 53 days old
- Weight at study initiation: Male: 28.5-34.2 g; Female: 24.0-28.1 g
- Assigned to test groups randomly: Yes
- Housing: Individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2°C
- Humidity (%): 50 ± 10%
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

Corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Immediately prior to dosing, suspensions of the test substance in the vehicle were prepared at concentrations of 200, 100, and 50 mg/mL. Uniformity was maintained during dosing by constant stirring. Single acute doses of the appropriate suspensions were administered by oral intubation at 25 mL/kg, yielding treatments of 5000, 2500, and 1250 mg test substance/kg body weight.

Duration of treatment / exposure:

Single dose

Frequency of treatment:

Single dose

Post exposure period:

24, 48, 72 hours

Dose / conc.:

1 250 mg/kg bw (total dose)

Dose / conc.:

2 500 mg/kg bw (total dose)

Dose / conc.:

5 000 mg/kg bw (total dose)

No. of animals per sex per dose:

6 (high dose group); 5 (rest of the groups)

Control animals:

yes, concurrent vehicle

Positive control(s):

The CP was administered by intraperitoneal injection in a volume of 5 mL/kg. An 8.0 mg/mL CP solution was used, yielding a dose of 40 mg/kg.

Tissues and cell types examined:

Bone marrow cells

Details of tissue and slide preparation:

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Mice were treated with either the test substance, vehicle or positive indicator on the day following release from quarantine. Groups of 6 male and 6 female mice (high dose group) and 5 male and 5 female mice (negative control) were sacrificed approximately 24, 48, and 72 hrs post-dosing. For the intermediate and low dose groups, 5 males and 5 females were sacrificed 24 hrs post-dosing. A positive indicator group of 5 male and 5 female mice was concurrently treated and sacrificed approximately 24 hrs post-dosing.

DETAILS OF SLIDE PREPARATION: Immediately after sacrifice, marrow from both femurs of each animal was aspirated and flushed into approximately 3 mL prewarmed (37°C) fetal bovine serum. The marrow was collected by centrifugation (approximately 200 x g, 5 min). Most of the supernatant was removed and the cells were resuspended in the remaining 1-2 drops of serum. A Miniprep® automatic blood smearing instrument was used to prepare marrow smears. At least 3 slides per animal were prepared and fixed in absolute methanol for 8 min. Slides were stained for 3 min in 0.0125 mg/mL acridine orange in phosphate buffer (pH 7.4). Prior to scoring, a coverslip was floated on each slide using phosphate buffer.

Statistics:

Data for the proportion of PCEs and MNPCEs among 2000 erythrocytes (PCE frequency and MNPCE frequency, respectively) were transformed prior analysis using the arcsin square root function. This transformation appropriate for proportions since the distribution of the transformed data more closely approximates a normal distribution than do the nontransformed proportion. Transformed data for PCE or MNPCE frequency were analyzed separately for normality of distribution using the Shapiro-Wilkes test. If results indicated that the transformed values for PCE:MNPCE frequency normally distributed in both sexes, parametric methods (viz., Analysis of Variance (AN0VA) and Dunnett test) were used. If there was nonnormality in either sex, nonparametric methods (viz., Kruskal-Wallis test and Mann-Whitney U tests) were used for that variable using nontransformed proportions. Positive indicator data were not included in evaluating normality of distribution. Weight gain data were assumed to be normally distributed and were analyzed by ANOVA. Data from each sex and sacrifice time were analyzed separately, and individual comparisons to the control were made using each animal as the experimental unit. All analyses conducted at a significance level of 5%. Positive indicator data were analyzed separately.

Key result

Sex:

male/female

Genotoxicity:

other: equivocal

Remarks:

equivocal based on the incidence of soft stools and diarrhoea which are likely to be related to the corn oil dosing vehicle (possibly affecting absorption of the test material in the treated mice).

Toxicity:

yes

Remarks:

wet perineum and/or soft stools, diarrhea

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

The test substance did not induce micronuclei in bone marrow cells of mice; the material is negative in this in vivo assay.

Executive summary:

This study was conducted following US EPA 84-2 and OECD guideline 474. The test substance evaluated for its ability to induce micronuclei in bone marrow polychromatic erythrocytes (PCEs) of Crl:CD®-1(ICR)BR mice. Acute doses of 0, 1250, 2500, and 5000 mg/kg administered by oral intubation to male and female mice. In the control and high dose groups, bone marrow smears were prepared approximately 24, 48, and 72 hrs after dosing. In the intermediate and low dose groups, prepared approximately 24 hrs after dosing. Two thousand PCEs per animal were scored for micronuclei.

No statistically significant increases in the frequency of micronucleated PCEs were observed in test substance treated mice at any dose level or sampling time. In addition, no statistically significant depression in the proportion of PCEs among 1000 erythrocytes were observed. In this assay, the test substance is negative.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

The potential genotoxicity of famoxadone was assessed in nine in vitro assays and 3 in vivo assays. Famoxadone did not induce gene mutations in the bacterial or mammalian cell assays. A full battery of in vitro and in vivo genotoxicity studies were conducted with two lots of famoxadone (an early test lot, JE874-133, and a test lot upon which the composition of manufactured product was to be based upon, JE874-221).  The in vitro studies demonstrated no evidence of mutagenicity in bacteria (S. typhimurium and E. coli) or mammalian (CHO) cells.  Famoxadone was weakly clastogenic in two chromosome aberration studies without metabolic activation but was negative with activation.  An in vitro study for unscheduled DNA synthesis (UDS) with the initial test batch (JE874-133) in cultured rat hepatocytes was positive, but negative results were obtained in two subsequent in vitro UDS studies with batch JE874‑221.  Famoxadone failed to induce micronuclei in mouse bone marrow cells, and two in vivo/in vitro UDS studies in rats were negative when tested up to and including a limit dose of 5000 mg/kg bw.  Therefore, it can be concluded that famoxadone does not pose a mutagenic risk.

There was no clear evidence of carcinogenic or reproductive effects that were indicative or suggested famoxadone-induced genotoxicity. Overall, the weight of evidence indicated that famoxadone is not genotoxic in vivo. Based on an assessment of the robust genetic toxicity data for this substance, the substance does not need to be classified for mutagenicity according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.