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.)-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

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

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

Compparable to OECD TG 476

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

Specific details on test material used for the study:

Batch No: H03421

Target gene:

Thymidine kinase (TK)

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.

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.

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'.

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Batch number: 0000120575
Purity: 98.76%
Expiry date: 05 July 2016

Target gene:

Histidine or tryptophan gene loci in genetically modified strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and Escherichia coli WP2 uvrA (pKM101)

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital, 5-6 Benzoflavone-induced rat liver S9-mix containing 100 L S9 fraction per mL

Test concentrations with justification for top dose:

50, 150, 500, 1500, 2500 and 5000 μg/plate Thioacid. Testing also included negative control and positive controls (5 and 10 μg/plate benzo(a)pyrene, 5 μg/l 2-aminoanthracene)
Maximum concentration was 5000 μg/plate, which is the maximum concentration in accordance with current guidelines in the presence and absence of S9-mix respectively.

Vehicle / solvent:

For the test material and negative control acetone was used. For the positive controls, the vehicles used were DMSO and in one occasion (Sodium azide for TA100 and TA1535 strains in absence of S9-mix) sterile water.

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-aminoanthracene

Details on test system and experimental conditions:

One standard plate incorporation (Ames) test was conducted for each tester strain both in the presence and absence of rat liver S9-mix, together with appropriate vehicle and
positive controls with the exception of strain TA1537. In the initial test, the positive controls in the presence and absence of S9-mix failed for strain TA1537 therefore this data is was invalidated and is not reported. Accordingly, strain TA1537 was repeated in the presence and absence of S9-mix. Samples of vehicle control and the highest concentration of test article formulation were analysed to determine achieved concentration.
In the plate incorporation (Ames) test plates were treated with vehicle control, positive control or Thioacid at concentrations of 50, 150, 500, 1500, 2500 or 5000 g per plate both in the presence and absence of S9-mix. Plates treated with vehicle and positive controls were included for all strains in both the presence and absence of S9-mix.Untreated plates were also included to assess for vehicle effect of the non-standard vehicle (Acetone). For the vehicle and untreated controls 6 plates per group were used, for the test article treated groups 3 plates per group were used and for the positive controls 2 plates per group were used.

Measured concentration of thioacid concentration was 50.7 mg/ml (i.e. within 10% of nominal).
Incubation temperature was 37 C during the 3-day incubation period.

Evaluation criteria:

Criteria for assay acceptance:
The highest concentration tested is one that allows the maximum exposure up to 5000 g per plate, or the limit of solubility (see ‘a’ below) or toxicity (see ‘b’ below), whichever is the lower.
The values for vehicle control number of revertants for each strain must be within or comparable to the laboratory historical vehicle control ranges.
Positive controls must show clear unequivocal positive responses. In the event that positive control treatments in the absence of S9-mix do not meet the above acceptance criteria and need to be repeated, then treatments in the presence of S9-mix must also be included.
There should be an absence of confounding technical problems such as contamination, outliers, excessive toxicity, or unacceptable numbers of lost plates.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

5000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

5000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

5000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

5000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

5000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

There was clear evidence of bactericidal activity in the form of a diminution of the background bacterial lawn and/or a marked reduction in the number of revertant colonies for TA98 at 5000 g per plate in the absence of S9-mix only, and in the presence and absence of S9-mix at 5000 g per plate for strains TA100, TA1535 and TA1537. No test article-related increases in the numbers of revertant colonies were observed in strains TA100, TA1535, TA1537, TA98 or WP2uvrA(pKM101) in the presence or absence of S9-mix, at any of the concentrations tested in this study, indicating a negative result.

Conclusions:

Thioacid (MCL00U) was not mutagenic in the bacterial mutation assay, when tested in either the presence or absence of S9-mix. The maximum concentration analysed was 5000 μg per plate, the maximum concentration in accordance with current guidelines in the presence and absence of S9-mix respectively.

Executive summary:

The purpose of this study was to assess the potential of Thioacid (MCL00U) to induce gene mutations (base pair substitutions and frameshift mutations) in vitro in bacterial strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537) and Escherichia coli WP2 uvrA (pKM101). Thioacid (MCL00U) is a synthetic intermediate currently undergoing toxicological evaluation. In this study all formulations of the test article were prepared and all concentrations (including analyte concentrations) expressed in terms of parent compound. For the purposes of this report the test article is hereafter referred to as Thioacid.

Study Design

One standard plate incorporation (Ames) test was conducted for each tester strain both in the presence and absence of rat liver S9-mix, together with appropriate vehicle and positive controls with the exception of strain TA1537. In the initial test, the positive controls in the presence and absence of S9-mix failed for strain TA1537 therefore this data was invalidated and is not reported. Accordingly, strain TA1537 was repeated in the presence and absence of S9-mix. Acetone is a non standard vehicle with limited in house historical control data, therefore untreated controls were also included for all strains in the presence and absence of S9-mix for comparison, to ensure there was no vehicle effect on the test system. Samples of vehicle control and the highest concentration of test article formulation were analysed to determine achieved concentration.

Results

The data for the vehicle and untreated controls were within the laboratory historical vehicle (DMSO) control ranges and were also comparable to the acceptable ranges for spontaneous mutation frequency (Vehicle Controls) as detailed in the UKEMS recommended procedures [Gatehouse, 1990]. The data for the concurrent vehicle controls were similar to the untreated controls for the respective strain, thus indicating there was no effect of a non-standard vehicle (Acetone). The positive controls induced clear unequivocal increases in numbers of revertant colonies. Therefore the performance of the vehicle and positive controls were consistent with a valid assay. The maximum concentration tested and analysed was 5000 μg per plate, the maximum concentration in accordance with current guidelines. There was clear evidence of bactericidal activity in the form of a diminution of the background bacterial lawn and/or a marked reduction in the number of revertant colonies for TA98 at 5000 μg per plate in the absence of S9-mix only, and in the presence and absence of S9-mix at 5000 μg per plate for strains TA100, TA1535 and TA1537. No test article-related increases in the numbers of revertant colonies were observed in strains TA98, TA100, TA1535, TA1537 and WP2 uvrA (pKM101) in the presence or absence of S9-mix, at any of the concentrations tested in this study, therefore, indicating a negative result. The results of the formulation analysis showed that the achieved concentration of Thioacid was within 100% ±10% of nominal, therefore, formulations were acceptable for treatment.

Conclusion

Thioacid (MCL00U) was not mutagenic in the bacterial mutation assay, when tested in either the presence or absence of S9-mix. The maximum concentration analysed was 5000 μg per plate, the maximum concentration in accordance with current guidelines in the presence and absence of S9-mix respectively.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Based on the negative results of two in vitro tests, namely reverse bacterial mutation (Ames) and mammalian cell mutagenicity (Mouse Lymphoma Assay), carried out according to the respective OECD guidelines (OECD 471 and OECD 476 respectively) and adhering to GLP, the substance does not fulfil the criteria for classification as mutagenic, according to CLP.