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

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 4

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

Test material

Test material information

Constituent 1

Reference substance name:

5-methyl-5-(4-phenoxyphenyl)-3-(phenylamino)-1,3-oxazolidine-2,4-dione

EC Number:

603-520-1

Cas Number:

131807-57-3

Molecular formula:

C22H18N2O4

IUPAC Name:

5-methyl-5-(4-phenoxyphenyl)-3-(phenylamino)-1,3-oxazolidine-2,4-dione

Test material form:

solid

Specific details on test material used for the study:

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

Method

Target gene:

HPRT

Species / strain

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)

Controls

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.

Results and discussion

Test results

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.