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.)-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Mouse Lymphoma:

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.

Ames:

The purpose of this study was to assess the potential of Thioacid Propionate (MCM00U) 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 Propionate (MCM00U) 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 Propionate.

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 untreated, vehicle and positive controls. Samples of vehicle control and the highest concentration of test article formulation were analysed to determine achieved concentration.

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 (Dimethyl formamide). 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 in the presence and absence of S9-mix.

No test article-related increases in the numbers of revertant colonies were observed in strains TA98, TA100, TA1535, TA1537 or WP2 uvrA (pKM101) in the presence or absence of S9-mix, at any of the concentrations analysed in this study, therefore, indicating a negative result.

The results of the formulation analysis showed that the achieved concentration of Thioacid Propionate was within 100% ±10% of nominal, therefore, formulations were acceptable for treatment.

Thioacid Propionate (MCM00U) was not mutagenic in the bacterial mutation assay, when tested in either the presence or absence of S9-mix. The maximum concentration tested and analysed was 5000 µg per plate, the maximum concentration in accordance with current guidelines in the presence and absence of S9-mix.

Link to relevant study records

Reference

Reference 1

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

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

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

Target gene:

Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells

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.

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.

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.

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.

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19th November 2015 - 23rd November 2015

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:

Identity: Thioacid Propionate (MCM00U). Identity confirmed by IR
Salt form: Not applicable
Batch number & purity: 0000135240. 97.76% assigned purity
Molecular weight: 468.53 as Thioacid Propionate
Storage conditions: Approximately 4°C, protected from light
Expiry date: 26-July-2017

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

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:

In the plate incorporation (Ames) test plates were treated with vehicle control, positive control or Thioacid Propionate at concentrations of 50, 150, 500, 1500, 2500 or 5000 µg
per plate both in the presence and absence of S9-mix. Untreated control plates were included for all strains in both the presence and absence of S9-mix. For the vehicle controls 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.

The highest concentration tested was one that allowed maximum exposure up to 5000 µg/plate for freely soluble compounds, or the limit of solubility or toxicity, whichever was the lower.

Vehicle / solvent:

Vehicle substance: Dimethyl formamide

Samples of vehicle control and the highest concentration of test article formulation were analysed to determine achieved concentration.

Positive controls:

yes

Positive control substance:

other: Methyl Sulfoxide (DMSO) or Sterile Water

Details on test system and experimental conditions:

The bacterial strains were stored as frozen permanents and subsequently grown in nutrient broth to yield approximately 1 x 109 cells/mL. Bacteria inocula from vials of frozen cultures were used to prepare fresh day cultures in nutrient broth [containing ampicillin for the pKM101 plasmid-containing strains - S. typhimurium strains TA98 and
TA100 and E. coli WP2 uvrA (pKM101) to maintain the plasmid copy number]. Bacteria were cultured for 10 hours with shaking in an anhydric incubator at approximately
37ºC±1ºC. Treatments commenced within 3 hours of the end of the period of incubation.

Vögel Bonner plates, top agar (containing trace amounts of the amino acids required for auxotrophy and other nutrients), and S9-mix or buffer solutions were used for all strains. Vögel Bonner plates were used for sterility checks.

One plate incorporation (Ames) test was carried out for each tester strain both in the presence and absence of rat liver S9-mix together with appropriate untreated, vehicle and
positive controls.

Rationale for test conditions:

The plate incorporation test was carried out essentially as described by Maron and Ames [Maron, 1983] and according to current methodology.

The highest concentration tested was one that allowed maximum exposure up to 5000 µg/plate for freely soluble compounds, or the limit of solubility or toxicity, whichever was the lower.

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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

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

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on 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 (Dimethyl formamide). 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 in the presence and absence of S9-mix.

No test article-related increases in the numbers of revertant colonies were observed in strains TA98, TA100, TA1535, TA1537 or WP2 uvrA (pKM101) in the presence or absence of S9-mix, at any of the concentrations analysed in this study, therefore, indicating a negative result.

The results of the formulation analysis showed that the achieved concentration of Thioacid Propionate was within 100% ±10% of nominal, therefore, formulations were acceptable for treatment.

Analysis of Data:

Criteria for Assay Acceptance:

1. 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.

a) If test article solubility in the test system is a limiting factor, the maximum treatment concentration chosen for analysis (plate scoring) will be the lowest concentration at which compound precipitation is observed by eye on treatment plates at the end of the incubation period.

b) If toxicity is a limiting factor, the maximum treatment concentration selected for analysis will normally be the lowest concentration at which signs of significant bacterial toxicity are observed during plate scoring.

2. The values for vehicle control number of revertants for each strain must be within or comparable to the laboratory historical vehicle control ranges.

3. 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.

4. There should be an absence of confounding technical problems such as contamination, outliers, excessive toxicity, or unacceptable numbers of lost plates.

Tests that did not fulfil the required acceptable criteria were rejected and are not reported.

Table of Results:

Metabolic Activation	Test article	Concentration^1 (µg/plate)	Plate Incorporation Test Mean Number of Revertant Colonies per Plate
			TA100	TA1535	TA1537	TA98	WP2uvrA (pKM101)
With Activation	DMF	100 µL/plate	131.8	17.2	23.0	33.0	150.8
	Untreated	0	141.5	17.0	26.2	35.3	169.2
	Thioacid Propionate	50	121.7	18.3	19.3	36.3	165.3
	Thioacid Propionate	150	130.3	15.0	20.7	31.3	163.7
	Thioacid Propionate	500	120.0	15.7	22.0	40.7	168.3
	Thioacid Propionate	1500	130.3	16.0	19.3	34.3	173.0
	Thioacid Propionate	2500	126.3	14.3	19.7	23.3	166.3
	Thioacid Propionate	5000	126.0	9.3	19.0	35.0	151.7
	Benzo(a)pyrene	10	NT	NT	NT	557.5^2	NT
	2 -Aminoanthracene	5	4418.0^2	519.5^2	99.5^2	NT	NT
	2-Aminoanthracene	10	NT	NT	NT	NT	1714.0^2

1. Expressed in terms of parent compound

2. Positive control induced an unequivocal increase in revertant numbers

NT – Not tested

DMF– Dimethyl formamide

Conclusions:

Thioacid Propionate (MCM00U) was not mutagenic in the bacterial mutation assay, when tested in either the presence or absence of S9-mix. The maximum concentration tested and analysed was 5000 µg per plate, the maximum concentration in accordance with current guidelines in the presence and absence of S9-mix.

Executive summary:

The purpose of this study was to assess the potential of Thioacid Propionate (MCM00U) 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 Propionate (MCM00U) 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 Propionate.

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 untreated, vehicle and positive controls. Samples of vehicle control and the highest concentration of test article formulation were analysed to determine achieved concentration.

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 (Dimethyl formamide). 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 in the presence and absence of S9-mix.

No test article-related increases in the numbers of revertant colonies were observed in strains TA98, TA100, TA1535, TA1537 or WP2 uvrA (pKM101) in the presence or absence of S9-mix, at any of the concentrations analysed in this study, therefore, indicating a negative result.

The results of the formulation analysis showed that the achieved concentration of Thioacid Propionate was within 100% ±10% of nominal, therefore, formulations were acceptable for treatment.

Thioacid Propionate (MCM00U) was not mutagenic in the bacterial mutation assay, when tested in either the presence or absence of S9-mix. The maximum concentration tested and analysed was 5000 µg per plate, the maximum concentration in accordance with current guidelines in the presence and absence of S9-mix.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)