(R)-2-(2,4-Difluorphenyl)-1,1-difluor-3-(tetrazol-1-yl)-1-{5-[4-(2,2,2-trifluorethoxy)-phenyl]-2-pyridyl}-2-propanol-(R,R)-tartrate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The mutagenic potential of (R)-3-Amino-2-(2,4-difluorophenyl)-1,1-difluoro-1-(5-(4-(2,2,2-trifluoroethoxy)phenyl) pyridin-2-yl)propan-2-ol L-tartrate was investigated in a bacterial reverse gene mutation assay conducted according to OECD guideline 471 with and without metabolic activation. There were no biologically relevant increases of mutant colonies compared to the negative control observed in all strains. Consequently, the test item is considered to be non-mutagenic.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-02-21 to 2020-06-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21 July 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was suspended in DMSO and diluted prior to treatment. The solvent was compatible with the survival of the bacteria and the S9 activity.

Target gene:

Histidine locus

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells:
MOLTOX, INC., NC 28607, USA (TA98 and TA1535), Xenometrix AG, Switzerland (TA100 and TA1537)

MEDIA USED
- Type and identity of media: Nutrient medium: 8 g Nutrient Broth and 5 g NaCl per litre, plus 125 µL ampicillin (10 mg/mL) for TA98, TA100); Agar Plates: Vogel-Bonner Medium E agar plates contain per litre 15 g Agar Agar, 20 mL Vogel-Bonner salts and 50 mL glucose solution (40%); Overlay Agar: The overlay agar contains per litre: 7.0 g Agar Agar, 6.0 g NaCl, 10.5 mg L-histidine x HCl x H20 and 12.2 mg biotin.

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: MOLTOX, INC., NC 28607, USA

MEDIA USED
- Type and identity of media: Luria Bertani: 10 g tryptone 10 g NaCl and 5 g yeast extract per litre, plus 125 µL ampicillin (10 mg/mL); Agar Plates: Vogel-Bonner Medium E agar plates contain per litre 15 g Agar Agar, 20 mL Vogel-Bonner salts and 50 mL glucose solution (40%); Overlay Agar: The overlay agar contains per litre: 7.0 g Agar Agar, 6.0 g NaCl, 10.2 mg tryptophan.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Mammalian liver microsomal fraction S9 mix.

Test concentrations with justification for top dose:

The test item concentrations to be applied in the main experiments were chosen according to the results of the pre-experiment (see box "Any other information on materials and methods incl. tables"; Results: see box "Any other information on results incl. tables", Table 2). Two independent main experiments were performed with the following concentrations:
Experiment I:
with and without metabolic ativation: 0.316, 1.00, 3.16, 10.0, 31.6, 100, 316 and 1000 µg/plate (all tester strains except TA98 (with))
TA98, with metabolic activation: 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
Experiment II:
with and without metabolic ativation: 0.316, 1.00, 3.16, 10.0, 31.6, 100 and 316 µg/plate (all Salmonella strains)
E. coli WP2 uvrA, with and without metabolic activation: 1.00, 3.16, 10.0, 31.6, 100, 316 and 1000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (AppliChem Lot No. 0001760192, 0001731257, 0001603375)
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity.

Untreated negative controls:

yes

Remarks:

purified water

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

S. typhimurium: TA100, TA1535 (10 µg/plate),without S9

Untreated negative controls:

yes

Remarks:

purified water

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-NOPD; 4-nitro-o-phenylene-diamine

Remarks:

S. typhimurium: TA98, TA1537 (10 μg/plate for TA98, 40 μg/plate for TA1537), without S9

Untreated negative controls:

yes

Remarks:

purified water

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

E. coli WP2 uvrA (pKM 101) (1 μL/plate), without S9

Untreated negative controls:

yes

Remarks:

purified water

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-AA; 2-aminoanthracene

Remarks:

S. typhimurium: TA98, TA100, TA1535, TA1537 and E. coli WP2 uvrA (pKM 101) (2.5 μg/plate; 10 μg/plate for E. coli WP2 uvrA (pKM 101)), with metabolic activation

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): triplicate
- Number of independent experiments: two

METHOD OF TREATMENT/ EXPOSURE:
in agar (plate incorporation, Experiment I), pre-incubation (Experiment II)

EXPERIMENTAL PERFORMANCE
- Experiment I:
For the plate incorporation method the following materials were mixed in a test tube and poured over the surface of a minimal agar plate: 100 μL test solution at each dose level, solvent control, negative control or reference mutagen solution (positive control), 500 μL S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation), 100 μL Bacteria suspension, 2000 μL Overlay agar.
- Experiment II:
For the pre-incubation method 100 µL of the test solution was pre-incubated with the tester strains (100 µL) and sterile buffer or the metabolic activation system (500 µL) for 60 min at 37 °C prior to adding the overlay agar (2000 µL) and pouring onto the surface of a minimal agar plate.

DURATION
- Pre-incubation period (Experiment II): 60 min at 37 °C
- Exposure duration: 48 h in the dark at 37 °C

METHODS FOR MEASUREMENT OF CYTOTOXICITY
Cytotoxicity is considered either as a clearing or diminution of the background lawn (indicated as "N" or "B", respectively in the result tables) or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertant occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs.
A biologically relevant increase is described as follows:
- if in tester strains TA98, TA100 and and E. coli WP2 uvrA (pKM 101) the number of reversions is at least twice as high
- if in tester strains TA1535 and TA1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.

Evaluation criteria:

A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to ampicillin (TA98, TA100, E. coli WP2 uvrA (pKM 101))
- the negative control plates (A. dest.) with and without S9 mix are within the following ranges (mean values of the spontaneous reversion frequency are within the historical control data range (2017 - 2019, except for E. coli WP2 uvrA (pKM 101) for this tester strain the period was December 2019 to February 2020) (see box “Any other information on material and methods incl. tables”, Table 1)
- corresponding background growth on negative control, solvent control and test plates is observed
- the positive controls show a distinct enhancement of revertant rates over the control plate
- at least five different concentrations of each tester strain are analysable.

Statistics:

According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Key result

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535 and TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. No precipitation of the test item was observed in any tester strain used in experiments I and II (with and without metabolic activation), with the exception of a finding at a concentration of 5000 µg/plate, if tested.

Cytotoxic effects of the test item were noted in all tester strains evaluated in experiments I and II. In experiment I cytotoxic effects of the test item were observed in tester strains TA98 and TA1535 at concentrations of 31.6 µg/plate and higher (without metabolic activation) and at concentrations of 316 µg/plate and higher (with metabolic activation). In tester strain TA100 cytotoxic effects of the test item were noted at concentrations of 31.6 µg/plate and higher (without metabolic activation) and at concentrations of 100 µg/plate and higher (with metabolic activation). In tester strain TA1537 cytotoxic effects of the test item were observed at concentrations of 100 µg/plate and higher (without metabolic activation) and at concentrations of 316 µg/plate and higher (with metabolic activation). In tester strain WP2 uvrA (pKM 101) cytotoxic effects of the test item were noted at concentrations of 316 µg/plate and higher (with and without metabolic activation).
In experiment II cytotoxic effects of the test item were noted in tester strain TA98, at concentrations of 31.6 µg/plate and higher (without metabolic activation) and at a concentration of 316 µg/plate (with metabolic activation). In tester strains TA100, TA1535 and TA1537 cytotoxic effects of the test item were seen at concentrations of 31.6 µg/plate and higher (without metabolic activation) and at concentrations of 100 µg/plate and higher (with metabolic activation). In tester strain WP2 uvrA (pKM 101) cytotoxic effects of the test item were observed at concentrations of 100 µg/plate and higher (without metabolic activation) and at concentrations of 316 µg/plate and higher (with metabolic activation).

No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with (R)-3-Amino-2-(2,4-difluorophenyl)-1,1-difluoro-1-(5-(4-(2,2,2-trifluoroethoxy)phenyl)pyridin-2-yl)propan-2-ol L-tartrate at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.

Results of the pre-experiment:

Table 2: Results of the pre-experiment

Substance	Dose (µg/plate)	TA98				TA100
		Mutation Factor [toxicity]*				Mutation Factor [toxicity]*
		without S9		with S9		without S9		with S9
Solvent Control (DMSO)		1.0		1.0		1.0		1.0
4-NOPD	10.0	9.4		-		-		-
NaN3	10.0	-		-		4.1		-
2-AA	2.50	-		74.6		-		16.4
Test Item	3.16	1.3		1.7		1.2		1.0
	10.0	1.1		1.2		1.2		1.0
	31.6	0.9		1.1		0.9		1.1
	100	0.5	[B]	1.5		0.2	[B]	0.9
	316	0.0	[B, N]	0.4	[B]	0.0	[N]	0.0	[B]
	1000	0.0	[N]	0.0	[N]	0.0	[N]	0.0	[N]
	2500	0.0	[N]	0.0	[N]	0.0	[N]	0.0	[N]
	5000	0.0	[N]	0.0	[N]	0.0	[N]	0.0	[N]

B = Background lawn reduced; N = No background lawn

*Mutation factor= mean revertants (test item, negative or positive control)/ mean revertants (solvent control)

Conclusions:

Under the experimental conditions reported, (R)-3-Amino-2-(2,4-difluorophenyl)-1,1-difluoro-1-(5-(4-(2,2,2-trifluoroethoxy)phenyl) pyridin-2-yl)propan-2-ol L-tartrate did not cause gene mutations in an Ames Test conducted according to OECD 471. Therefore, the test item is considered to be non-mutagenic in this bacterial reverse gene mutation assay.

Executive summary:

In a bacterial reverse gene mutation assay conducted according to OECD guideline 471, S. typhimurium strains TA98, TA100, TA1535, TA1537 and tester strain E. coli WP2 uvrA (pKM 101) were exposed to (R)-3-Amino-2-(2,4-difluorophenyl)-1,1-difluoro-1-(5-(4-(2,2,2-trifluoroethoxy)phenyl) pyridin-2-yl)propan-2-ol L-tartrate (99.5% purity) in DMSO at the following concentrations in the presence and absence of mammalian metabolic activation:

Experiment I: 0.316, 1.00, 3.16, 10.0, 31.6, 100, 316 and 1000 µg/plate (all tester strains except TA98 (with metabolic activation)); 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (only tester strain TA98 (with metabolic activation))

Experiment II: 0.316, 1.00, 3.16, 10.0, 31.6, 100 and 316 µg/plate (all Salmonella strains); 1.00, 3.16, 10.0, 31.6, 100, 316 and 1000 µg/plate (E. coli WP2 uvrA (pKM 101))

No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation), with one exception: precipitation of the test item was observed in experiment I in tester strain TA98 at a concentration of 5000 µg/plate (with metabolic activation). The observed precipitation did not interfere with the scoring; thus, it did not impact the results. Cytotoxic effects of the test item were noted in all tester strains evaluated in experiment I and II. No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with the test item at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II. The positive controls induced the appropriate responses in the corresponding strains showing the validity of the results. Based on the results, the test item is considered to be non-mutagenic in the bacterial reverse gene mutation assay.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.5100; OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In a bacterial reverse gene mutation assay conducted according to OECD guideline 471, strains TA98, TA100, TA1535 and TA1537 of Salmonella typhimurium as well as E. coli WP2 uvrA (pKM 101) were exposed to the test item (R)-3-Amino-2-(2,4-difluorophenyl)-1,1-difluoro-1-(5-(4-(2,2,2-trifluoroethoxy)phenyl) pyridin-2-yl)propan-2-ol L-tartrate in DMSO in the presence and absence of mammalian metabolic activation. The positive controls induced the appropriate responses in the corresponding strains. No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation), with one exception: precipitation of the test item was observed in experiment I in tester strain TA98 at a concentration of 5000 µg/plate (with metabolic activation). The observed precipitation did not interfere with the scoring; thus it did not impact the results. Cytotoxic effects of the test item were noted in all tester strains evaluated in experiment I and II. No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with the test item at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II. Consequently, the test item was considered to be non-mutagenic and no classification for mutagenicity is warranted.

Justification for classification or non-classification

Based on the available data, the substance (R)-3-Amino-2-(2,4-difluorophenyl)-1,1-difluoro-1-(5-(4-(2,2,2-trifluoroethoxy)phenyl) pyridin-2-yl)propan-2-ol L-tartrate does not warrant classification for mutagenicity.