Androsta-1,4-diene-17-carbothioic acid, 6,9-difluoro-11,17-dihydroxy-16-methyl-3-oxo-, (6.alpha.,11.beta.,16.alpha.,17.alpha.)-

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 May 2003 to 02 July 2003

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non-guideline and non-GLP study

Remarks:

Not intended for full compliance with full compliance with guidelines for mammalian cell mutagenicity testing as set forth by international regulatory authority

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

Compparable to OECD TG 476

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

other: ICH S2B guideline, "Genotoxicity: standard battery for genotoxicity testing of pharmaceuticals"

Principles of method if other than guideline:

The methodology used is comparable to that described in OECD guidance 476 and ICH S2B guidelines, but it was not intended to adhere to their specifications.

GLP compliance:

no

Type of assay:

other: L5178 TK+/- Mouse Lymphoma Forward Mutation Assay

Test material

Test material information

Constituent 1

Reference substance name:

(1R,2S,8S,10S,11S,13R,14R,15S,17S)-1,8-difluoro- 14,17dihydroxy-2,13,15-trimethyl-5-oxotetracyclo[8.7.0.0^{2,7}.0^{11,15}]heptadeca-3,6-diene-14-carbothioic S-acid

EC Number:

617-081-9

Cas Number:

80473-92-3

Molecular formula:

C21-H26-F2-O4-S

IUPAC Name:

(1R,2S,8S,10S,11S,13R,14R,15S,17S)-1,8-difluoro- 14,17dihydroxy-2,13,15-trimethyl-5-oxotetracyclo[8.7.0.0^{2,7}.0^{11,15}]heptadeca-3,6-diene-14-carbothioic S-acid

Test material form:

solid: particulate/powder

Remarks:

White powder

Specific details on test material used for the study:

Batch No: H03421

Method

Target gene:

Thymidine kinase (TK)

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Remarks:

mammalian cell line

Details on mammalian cell type (if applicable):

Type and identity of media
Growth Medium: The culture medium used for routine growth and subculture was RPMI 1640 (Amacher et al., 1980; Clive et al., 1987) supplemented with 10% (v/v) horse serum. Pluronic F68, L-glutamine, sodium pyruvate, penicillin and streptomycin.

Treatment Medium: Treatment was performed in Fischer's medium supplemented with 5% (v/v) horse serum. Pluronic F68, L-glutamine, sodium pyruvate, penicillin and streptomycin.

Cloning Medium: Cloning will be performed with RPMI 1640 culture medium supplemented with 20% (v/v) horse serum, L-glutamine. sodium pyruvate, penicillin and streptomycin and 0.24% (w/v) agar. Cloning medium for selection of tk-/-· mutants also will contain 3 µg/mL TFT (Clive et al., 1987).

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9: Commercial liver homogenate (9000x g supernatant) was purchased commercially and prepared from male Sprague-Dawley rats injected with Aroclor(TM) 1254 (200mg/ml in corn oil) at 500mg/kg.

Test concentrations with justification for top dose:

During mutagenicity screening, the test article was evaluated in single cultures at concentrations of 8.1, 16.2, 32.3, 64.5, 129, 258, 515, 1030 and 2050 μg/ml, with and without S9 (based upon the highest workable dose). The test article precipitated from solution upon addition to the aqueous treatment medium (and remained insoluble at the end of treatment) at concentrations >= 515 μg/ml with and without S9.
Cultures treated at concentrations >= 515 μg/ml with and without S9 were discarded due to cytotoxicity, along with the culture treated at 258 μg/ml without S9. Cultures treated at concentrations of 8.10 and 32.3 μg/ml with S9 and <= 16.2 μg/ml without S9 were excluded from evaluation because a sufficient number of higher concentrations was available.
Based upon the 2-day relative suspension growth, those cultures treated at concentrations of 16.2, 64.5, 129 and 258 μg/mL with S9, and 32.3, 64.5, 129 and 258 μg/mL without S9, were chosen for selection of TFTr mutants.

Vehicle / solvent:

Dimethyl sulfoxide (DMSO) was used as vehicle.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO was used as the vehicle control

True negative controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

other:

Remarks:

Positive control (MMS) was evaluated in the absence of S9, in duplicate cultures, at a concentration of 13.0 μg/ml

Details on test system and experimental conditions:

Test System
Test Cells
The heterozygous mouse lymphoma L5I78Y cell line (tk+/-) designated as clone 3.7.2C, will be used for this assay.

Source of Cells
Stock cultures were obtained from Dr. Donald Clive, Burroughs Wellcome Co.

Storage of Cells
For long-term storage, cells are stored frozen in liquid nitrogen. Working laboratory stock cultures are maintained in logarithmic growth by serial subculture. The working stocks typically are replaced by cells from the frozen stock after approximately four months.

Cell Culture Conditions
Unless otherwise noted, cultures will be grown under standard conditions (in an orbital shaker at 35-38 °C and 70 +/- 10 orbits per minute). A log will be kept to record growth and subculture operations for the working cell stocks. To reduce the frequency of spontaneous TK -/- mutants prior to use in a mutation assay, cell cultures will be exposed to conditions which select against the TK-/- phenotype, and then returned to normal growth medium for 3 to 8 days.

Tissue Culture Medias
Growth Medium
The culture medium used for routine growth and subculture will be RPMI 1640 (Amacher et al., 1980; Clive et al., 1987) supplemented with 10% (v/v) horse serum, Pluronic F68, L-glutamine, sodium pyruvate, penicillin and streptomycin.

Treatment Medium
Treatment will be performed in Fischer's medium supplemented with 5% (v/v) horse serum. Pluronic F68, L-glutamine, sodium pyruvate, penicillin and streptomycin.

Cloning Medium
Cloning will be performed with RPMI 1640 culture medium supplemented with 20% (v/v) horse serum, L-glutamine, sodium pyruvate, penicillin and streptomycin and 0.24% (w/v) agar. Cloning medium for selection of tk-/- mutants also will contain 3 µg/mL TFT (Clive et al., 1987).

Test for Mycoplasma Contamination
Mycoptasma tests are perfonned by an outside commercial laboratory on stock cultures prior to, and after, preparing frozen stocks. Working stocks also will be tested for mycoplasma at the end of their approximate 4- month "life span" to verify there was no contamination during use. Direct culture and indirect Hoechst staining methods will be used for mycoplasma detection.

Karyotype Stability
Karyotype analysis, including banding, is perfonned on each preparation of frozen stock cultures. Karyotypic stability, as measured by modal (mean) chromosome number, also will be performed on alI working stock cultures, prior to being discarded, to ensure that changes in the culture have not occurred.

Evaluation criteria:

Assay Evaluation Criteria
The test article will be evaluated as positive, negative, or equivocal. A positive evaluation indicates that the test article is mutagenic (induces gene/chromosomal mutations) in this test system, while a negative evaluation indicates the test article is non-mutagenic in this test system (causing no responses that can be interpreted as positive). In rare cases, however, conflicting results may be observed and the test article will be evaluated as equivocal in this test system. Separate evaluations will be made for the test article with and without S9.
Criteria for a Positive Response
A test article will be evaluated as positive in a given trial if a dosedependent, 2-fold increase in mutant frequency is observed (as compared to the appropriate concurrent negative control. This "2-fold" rule is based on extensive experience that indicates such responses are repeatable in additional trials.

Assay will be accepted if: a. absolute cloning efficiency of negative controls is between 60% and 130%, b. average 2-day suspension growth of negative controls is at least an 8-fold increase relative to original cell density, c. spontaneous mutant frequency should be in the range of 30-120 TFT mutants/10^6 clonable cells, d. at least one positive control in each trial should induce a mutant frequency >= 200 TFT mutants/10^6 clonable cells

Statistics:

Mutant frequencies will be included in the evaluation only if the cloning efficiency is >= 10 % (to avoid problems with the statistical distribution of scorable colonies among dishes). Likewise, treatments reducing the RTG to < 10 % may occur, but the results obtained at this high toxicity will be considered biologically irrelevant and will be excluded from evaluation.
For test articles ultimately judged to have no or weak mutagenic activity, a minimum of four acceptable concentrations is required. However, for test articles judged to be clearly mutagenic, fewer concentrations may be sufficient.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

mammalian cell line

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Test Article Handling
Thio acid was stored at room temperature, protected from light. Dimethylsulfoxide (DMSO; CAS No.67-68-5; Acros Organics, Lot No. A017325001) was used as the vehicle. At a concentration of 205 mg/mL, the highest prepared for treatment, the test article appeared to be a light-yellow suspension; it became freely soluble at <= 103 mg/mL and remained freely soluble at all subsequent lower dilutions.
Mutagenicity Screen
The test article was evaluated in single cultures at concentrations of 8.10, 16.2, 32.3, 64.5, 129, 258, 515, 1030 and 2050 μg/mL with and without S9 (based upon the highest workable dose). Vehicle controls were evaluated concurrently in triplicate cultures, while the concurrent positive controls were evaluated either at one concentration in duplicate cultures, or at two concentrations in single cultures. The test article precipitated from solution upon addition to the aqueous treatment medium (and remained insoluble at the end of treatment) at concentrations >= 515 μg/mL with and without S9). Based upon the 2-day relative suspension growth, those cultures treated at concentrations of 16.2, 64.5, 129 and 258 μg/mL with S9, and 32.3, 64.5, 129 and 258 μg/mL without S9, were chosen for selection of TFTr mutants. Those cultures treated at concentrations >= 515 μg/mL with and without S9 were discarded at the time of selection due to extreme cytotoxicity, and the culture treated at a concentration of258 μg/mL without S9 was excluded from evaluation due to excessive cytotoxicity. Cultures treated at concentrations of 8.10 and 32.3 μg/mL with S9, and <= l6.2 μg/mL without S9, were discarded because a sufficient number of higher concentrations was available. Relative total growth for the remaining cultures ranged from 16.7 to 76.3% with S9, and 16.1 to 69.2% without S9. The average mutant frequencies of the vehicle controls were 75.6 and 57.1 TFr mutants/10E6 clonable cells with and without S9, respectively. Mutant frequencies for those cultures treated with thio acid ranged from 50.5 to 71.9 TFTr mutants/10E6 clonable cells with S9, and 56.3 to 58.2 TFr mutants/10E6 clonable cells without S9. Thus, none of the cultures treated with thio acid exhibited a 2-fold increase in mutant frequency, relative to the concurrent vehicle controls, with or without S9. All positive and negative control values were within acceptable ranges, and all criteria for a valid study were met.
Sizing Analysis
The L5178Y TK+/- mutation assay produces a bimodal distribution of large and small mutant colonies. This bimodal distribution of mutant colony sizes is considered to reflect the scale of genetic damage, with the large colonies derived from cells with intragenic mutations that affect only the TK gene and the small colonies the result of larger mutations that affect cell growth as well as the TK gene. Colony sizing was performed on colonies from all TFT-selected cultures. Mutant colonies from all treated cultures, including the MMS and MCA positive controls, exhibited the expected bimodal distribution with large and small colonies. No change in the relative proportion of small and large colonies was apparent in the test article
treated cultures.

Remarks on result:

other: all strains/cell types tested Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation
These results indicate thio acid was negative in the L5178Y TK +/- Mouse Lymphoma Forward Mutation Screen, in the presence and absence of S9, under the conditions and according to the criteria of the test protocol.