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

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

Study period:

18th June 2003 - 2nd July 2003

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2203

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Principles of method if other than guideline:

The objective of this study was to evaluate the ability of thio acid propionate to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells, as assayed by colony growth in the presence of 5-trifluorothymidine (TFT). This assay was performed in the presence and absence of an exogenous mammalian metabolic activation system (S9) containing mammalian microsomal enzymes derived from AroclorTM-induced rat liver (S9). The mouse lymphoma L5178Y cell line, heterozygous at the TK locus and designated clone 3.7.2°C, was used for this assay.

GLP compliance:

no

Type of assay:

bacterial forward mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(6ALPHA,11BETA,16ALPHA,17ALPHA)-6,9-DIFLUORO-11-HYDRO XY-16-METHYL-3-OXO-17- (1-OXOPROPOXY)ANDROSTA-1,4-DIENE-CARBOTHIOIC ACID

EC Number:

617-084-5

Cas Number:

80474-45-9

Molecular formula:

C24H30F2O5S

IUPAC Name:

(6ALPHA,11BETA,16ALPHA,17ALPHA)-6,9-DIFLUORO-11-HYDRO XY-16-METHYL-3-OXO-17- (1-OXOPROPOXY)ANDROSTA-1,4-DIENE-CARBOTHIOIC ACID

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Test Article:
Sponsor Identification: Thio Acid Proionate, Batch No. H030714
Date Received: 28 April 2003
Physical Description: Light-yellowish-white powder with chucks
Storage Conditions: Room temperature, procted from light

Assay Information:
Type of Assay: L5178Y TK+/- Mouse Lymphoma Forward Mutation Screen
Protocol No.: 431SC5, Edition 3, for GlaxoSmithKline
Genetic Toxicology Assay no.: 25092-0-431SC

Method

Target gene:

Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Storage of cells:
For long-term storage, cells are stored from 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 oetherwise 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 steptomycin.

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 3µg/mL TFT (Clive et al., 1987).

Test concentrations with justification for top dose:

The test article was evaluated in single cultures at concentrations of 9.25, 18.5, 37.0, 74.0, 148, 295, 590, 1180, 2360 and 4720µg/mL with and without S9 (based upon a reported molecular weight of 468.53, the highest concentration evaluated approximated the limit dose of 10mM).

Vehicle / solvent:

Vehicle controls were evaluated concurrently in triplicate cultures, while the concurrent positive controls were evaluated 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 ≥148µg with and without S9.

Controls

Untreated negative controls:

yes

Remarks:

When the vehicle for the test article is medium, an untreated control will be included in each assay.

Positive controls:

yes

Remarks:

Known mutagens able to induce both small and large colonies will be evaluated concurrently.

Positive control substance:

methylmethanesulfonate

Details on test system and experimental conditions:

The test article, as well as the appropriate positive and negative controls, will be evaluated in the presence and absence of S9. Generally ten concentrations of test article, spanning approximately threee logs, will be evaluated in single cultures under each treatment condition.

The highest concentration of test article to be evaluated in the mutagenicity screen will be 5.00mg/mL (or 10.0mM, whichever is lower), or a concentration that is at least double the limit of solubility At least nine lower concentrations, generally will be prepared by two-fold serial dilutions, will be evaluated with and without S9.

Although ten or more concentrations may be used for treatment, only three are required for a valid assay. Treatment of the extra cultures compensates for normal varialtions in cellular toxicity and hels unsure the availability of at least three concentrations over a wide cytotixicity range. Test article-treated cultures may be elimated during the expression period due to excessive toxicity, or if a sufficient number of higher concentrations are available.

Unless otherwise specified by the Sponsor, the test article will be assayed as provided. Neutralization of acid or alkaline test articles (with HCL or NaOH) generally will be performed only after consultation with the Sponsor. Observations of test article solubility, pH and osmality (if warranted) will be noted and recorded. If the pH indicator in the medium has a color change, the pH will be taken using pH indicator strips.

Rationale for test conditions:

The mutagenicity screen will be performed using procedures based upon the methods described by Clive and Spector (1975), Clive et al., (1979), Amacher et al., (1980) and Clive et al., (1987). This methodology has been shown to detect a wide range of classes of chemical mutagens.

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Remarks:

All positive and negative control values were within acceptable ranges, and all criteria for a valid study were met

Positive controls validity:

valid

Remarks on result:

other: No change in the relative proportion of small and large colonies was apparent in the test article treated cultures.

Any other information on results incl. tables

Mutagenicity Screen:

The test article was evaluated in single cultures at concentrations of 9.25, 18.5, 37.0, 74.0, 148, 295, 590, 1180, 2360 and 4720µg/mL with and without S9 (based upon a reported molecular weight of 468.53, the highest concentration evaluated approximated the limit dose of 10mM). Vehicle controls were evaluated concurrently in triplicate cultures, while the concurrent positive controls were evaluated 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 ≥148µg with and without S9.

Based upon the 2 -day relative suspension growth, those cultures treated at concentrations of 590, 1180, 2350 and 4720µg with and without S9 were chosen for selection of TFT mutants. Cultures treated at concentrations ≤295µg/mL with and without S9 were discarded at the time of selection because a sufficient number of higher concentrations was available. Relative total growth for the remaining cultures ranged from 40.2 to 85.6% with S9, and 14.5 to 74.7% without S(. The arverage mutant frequencies of the vehicle controls were 75.6 and 57.1 TFT mutants/10⁶ clonable cells with and without S9, respectively. Mutant frequencies for those cultures treated with thio acid propionate ranged from 55.6 to 93.1 TFT mutants/10⁶ clobable cells with S9, and 55.4 to 92.6 TFT mutants/10⁶ clonable cells without S9. Thus, none of the cultures treated with thio acid propionate 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.

Applicant's summary and conclusion

Conclusions:

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.

All positive and negative control values were within acceptable ranges, and all criteria for a valid study were met.

Executive summary:

The objective of this study was to evaluate the ability of thio acid propionate to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells, as assayed by colony growth in the presence of 5-trifluorothymidine (TFT). This assay was performed in the presence and absence of an exogenous mammalian metabolic activation system (S9) containing mammalian microsomal enzymes derived from AroclorTM-induced rat liver (S9). The mouse lymphoma L5178Y cell line, heterozygous at the TK locus and designated clone 3.7.2°C, was used for this assay.

The test article was evaluated in single cultures at concentrations of 9.25, 18.5, 37.0, 74.0, 148, 295, 590, 1180, 2360 and 4720µg/mL with and without S9 (based upon a reported molecular weight of 468.53, the highest concentration evaluated approximated the limit dose of 10mM).

Vehicle controls were evaluated concurrently in triplicate cultures, while the concurrent positive controls were evaluated 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 ≥148µg with and without S9.

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.

All positive and negative control values were within acceptable ranges, and all criteria for a valid study were met.