(2R)-2-(2,4-difluorophenyl)-1,1-difluoro-1-{5-[4-(2,2,2-trifluoroethoxy)phenyl]pyridin-2-yl}-3-(1H-tetrazol-1-yl)propan-2-ol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The genetic toxicity potential of the test item (2R)-2-(2,4-difluorophenyl)-1,1-difluoro-3-(tetrazol-1-yl)-1-[5-[4-(2,2,2-trifluoroethoxy)phenyl]pyridin-2-yl]propan-2-ol was assessed win a bacterial reverse mutation assay conducted in accordance with OECD test guideline 471. In this study, no indication of genetic toxicity potential of the test item was observed. The negative result is consolidated by the negative outcome of supporting studies, a non-GLP bacterial mutation assay and a GLP in vitro cytogenicity (mammalian chromosome aberration test).

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:

2011-01-28 to 2011-06-20

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:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: ZEN-B-18(1)
- Purity: 99.9% (HPLC)
- Purity test date: 2010-12-13

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: -60 °C or colder, stored in the dark without
desiccant
- Stability under storage conditions: 5 months

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Solubilized in DMSO
- Final dilution of a dissolved solid, stock liquid or gel: 500 mg/mL

Target gene:

S. typhimurium: Histidine locus
E. coli: Tryptophan locus

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from male Sprague-Dawley rats induced with a single intraperitoneal injection of Aroclor 1254, 500 mg/kg, five days prior to sacrifice. The S9 was prepared by and purchased from Moltox (Boone, NC). Upon arrival at BioReliance, the S9 was stored at -60 °C or colder until used. Each bulk preparation of S9 was assayed for its ability to metabolize at least two promutagens to forms mutagenic to Salmonella typhimurium TA100. The S9 mix was prepared immediately before its use and contained 10% S9, 5 mM glucose-6-phosphate, 4 mM ß-nicotinamide-adenine dinucleotide phosphate, 8 mM MgCl2 and 33 mM KCl in a 100 mM phosphate buffer at pH 7.4. The Sham S9 mixture (Sham mix), containing 100 mM phosphate buffer at pH 7.4, was prepared immediately before its use. To confirm the sterility of the S9 and Sham mixes, a 0.5 mL aliquot of each was plated on selective agar.

Test concentrations with justification for top dose:

Initial toxicity-mutation assay: 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate (with and without metabolic activation)
Confirmatory mutagenicity assay: 50, 150, 500, 1500, and 5000 μg per plate (with and without metabolic activation)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for percentage of solvent in the final culture medium: A solubility test was conducted using dimethyl sulfoxide (DMSO) to determine the highest soluble or workable stock concentration up to 500 mg/mL.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9, 75 µg/plate, strain TA1537

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without S9, 1.0 µg/plate, strain TA98

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9, 1.0 µg/plate, strains TA100 and TA1535

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9, 1000 µg/plate, strain WP2uvrA

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9, 1.0 µg/plate (TA98, TA1535, TA1537), 2.0 µg/plate (TA100), 15 µg/plate (WP2uvrA)

Details on test system and experimental conditions:

The tester strains used were the Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535 and TA1537 as described by Ames et al. (1975) and Escherichia coli WP2 uvrA as described by Green and Muriel (1976). Salmonella tester strains were from Dr. Bruce Ames’ Master cultures, E. coli tester strains were from the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland. Tester strains TA100, TA1535 and TA1537 were distributed by Moltox (Boone, NC), using cultures derived from the above sources.

Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester strain TA1535 is reverted by mutagens that cause basepair substitutions. Tester strain TA100 is reverted by mutagens that cause both frameshift and basepair substitution mutations. Specificity of the reversion mechanism in E. coli is sensitive to basepair substitution mutations, rather than frameshift mutations (Green and Muriel, 1976).

Overnight cultures were prepared by inoculating from the appropriate master plate, appropriate frozen permanent stock or with a lyophilized pellet into a vessel, containing ~30 to 50 mL of culture medium. To assure that cultures were harvested in late log phase, the length of incubation was controlled and monitored. Following inoculation, each flask was placed in a shaker/incubator programmed to begin shaking at approximately 125 to 175 rpm at 37 ± 2 °C approximately 12 to 14 hours before the anticipated time of harvest. Each culture was monitored spectrophotometrically for turbidity and was harvested at a percent transmittance yielding a titer of greater than or equal to 0.3x109 cells per milliliter. The actual titers were determined by viable count assays on nutrient agar plates.

Initial Toxicity-Mutation Assay
The initial toxicity-mutation assay was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. Vehicle control, positive controls and a minimum of eight dose levels of the test article were plated, two plates per dose, with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA on selective minimal agar in the presence and absence of Aroclor-induced rat liver S9.

Confirmatory Mutagenicity Assay
The confirmatory mutagenicity assay was used to evaluate and confirm the mutagenic potential of the test article. A minimum of five dose levels of test article along with appropriate vehicle control and positive controls were plated with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA on selective minimal agar in the presence and absence of Aroclor-induced rat liver S9. All dose levels of test article, vehicle control and positive controls were plated in triplicate.

Plating and Scoring Procedures
The test system was exposed to the test article via the plate incorporation methodologyoriginally described by Ames et al. (1975) and updated by Maron and Ames (1983). On the day of its use, minimal top agar, containing 0.8 % agar (W/V) and 0.5 % NaCl (W/V), was melted and supplemented with L-histidine, D-biotin and L-tryptophan solution to a final concentration of 50 μM each. Top agar not used with S9 or Sham mix was supplemented with 25 mL of water for each 100 mL of minimal top agar. For the preparation of media and reagents, all references to water imply sterile, deionized water. Bottom agar was Vogel-Bonner minimal medium E (Vogel and Bonner, 1956) containing 1.5 % (W/V) agar. Nutrient bottom agar was Vogel-Bonner minimal medium E containing 1.5 % (W/V) agar and supplemented with 2.5 % (W/V) Oxoid Nutrient Broth No. 2 (dry powder). Nutrient Broth was Vogel-Bonner salt solution supplemented with 2.5 % (W/V) Oxoid Nutrient Broth No. 2 (dry powder). Each plate was labeled with a code system that identified the test article, test phase, dose level, tester strain and activation, as described in detail in BioReliance's Standard Operating Procedures. One-half (0.5) milliliter of S9 or Sham mix, 100 μL of tester strain (cells seeded) and 50 μL of vehicle or test article dilution were added to 2.0 mL of molten selective top agar at 45±2°C. After vortexing, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. When plating the positive controls, the test article aliquot was replaced by a 50 μL aliquot of appropriate positive control. After the overlay had solidified, the plates were inverted and incubated for approximately 48 to 72 hours at 37 ± 2 °C. Plates that were not counted immediately following the incubation period were stored at 2-8°C until colony counting could be conducted.

Evaluation criteria:

The condition of the bacterial background lawn was evaluated for evidence of test article toxicity by using a dissecting microscope. Precipitate was evaluated after the incubation period by visual examination without magnification. Toxicity and degree of precipitation were scored relative to the vehicle control plate using the codes shown in table 1. Revertant colonies for a given tester strain and activation condition, except for positive controls, were counted either entirely by automated colony counter or entirely by hand unless the plate exhibited toxicity.

For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported. For the test article to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test article.

Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 3.0-times the mean vehicle control value. Data sets for tester strains TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 2.0-times the mean vehicle control value. An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response will be evaluated as negative, if it is neither positive nor equivocal.

Key result

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:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

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:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

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:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

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:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Solubility Test:
Dimethyl sulfoxide (DMSO) was selected as the solvent of choice based on information provided by the Sponsor, solubility of the test article and compatibility with the target cells. The test article formed a soluble and clear solution in DMSO at approximately 500 mg/mL, the maximum concentration tested in the solubility test.

Dosing Formulation Analysis:
Dosing formulations were sent to Calvert Laboratories (Scott Township, PA) for analysis. Concentration analysis indicates that the actual mean concentrations of the analyzed dose levels were between 96.9 and 115% of their respective targets. This indicates that the regulatory-required top dose level was achieved in each case and the results support the validity of the study conclusion. No test article was detected in the vehicle control samples. Stability analysis was conducted as part of the analytical method validation. The test item in DMSO at concentrations of 0.01 and 532 mg/mL was stable at ambient temperature (15 to 30 °C) for approximately 24 hours and frozen at -60 to -80 °C for 12 days. The 0.01 mg/mL formulated sample was also found to be stable when held frozen for 90 days; and the 532 mg/mL formulated sample was found to be stable when held frozen for 91 days.

Initial Toxicity-Mutation Assay:
No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.Precipitate was observed beginning at 500 μg per plate. No appreciable toxicity was observed.

Confirmatory Mutagenicity Assay:
No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.Precipitate was observed beginning at 500 μg per plate. No appreciable toxicity was observed.

Table 1: Summary of the results obtained with the test item (Initial Toxicity-Mutation Assay)

	Dose level (µg/plate)
Test strain	Control (50 µL)	1.5	5.0	15	50	150	500	1500	5000
Mean revertants per plate Without S9 activation
TA98	12	10	11	14	9	14	13	11	11
TA100	92	87	101	111	89	120	87	95	121
TA1535	13	6	16	10	14	13	9	12	16
TA1537	11	12	10	7	9	7	7	11	13
WP2uvrA	19	19	20	16	21	21	21	21	27
Mean revertant per plate With S9 activation
TA98	26	19	21	17	28	27	19	23	32
TA100	121	117	115	106	114	104	122	152	122
TA1535	11	9	13	7	14	11	8	11	14
TA1537	12	6	15	9	6	11	12	13	13
WP2uvrA	27	25	18	25	34	30	20	25	33

Table 2: Summary of the results obtained with the test item (Confirmatory Mutagenicity Assay)

Dose level (µg/plate)
Test strain	Control (50 µL)	50	150	500	1500	5000
Mean revertant per plate Without S9 activation
TA98	15	21	20	24	20	19
TA100	113	108	121	121	133	118
TA1535	6	12	10	8	8	8
TA1537	7	4	5	6	5	5
WP2uvrA	23	17	20	22	25	18
Mean revertants per plate With S9 activation
TA98	26	30	34	28	26	22
TA100	128	122	140	146	134	127
TA1535	11	9	10	9	9	12
TA1537	7	6	9	5	5	6
WP2uvrA	26	30	22	29	31	22

Conclusions:

Under the experimental conditions reported, the test item showed no evidence of mutagenic activity in a bacterial reverse mutation assay.

Executive summary:

In a bacterial reverse mutation assay conducted according to OECD 471, Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and one strain of Escherichia coli (WP2 uvrA) were exposed to (2R)-2-(2,4-difluorophenyl)-1,1-difluoro-3-(tetrazol-1-yl)-1-[5-[4-(2,2,2-trifluoroethoxy)phenyl]pyridin-2-yl]propan-2-ol (99.9% purity) in DMSO at concentrations of 0, 1.5, 5.0, 15, 150, 500, 1500 and 5000 µg per plate in the presence and absence of mammalian S9 metabolic activation. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies above background in all tester strains and both experiments (initial toxicity-mutation assay and confirmatory mutagenicity assay). 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 of OECD test guideline 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The negative result of the in vitro key study (OECD 471, Wagner 2011) is consolidated by the negative outcome of an in vivo micronucleus test condcuted according to OECD 474 under GLP conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In a bacterial reverse mutation assay conducted according to OECD 471, Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and one strain of Escherichia coli (WP2 uvrA) were exposed to (2R)-2-(2,4-difluorophenyl)-1,1-difluoro-3-(tetrazol-1-yl)-1-[5-[4-(2,2,2-trifluoroethoxy)phenyl]pyridin-2-yl]propan-2-ol in DMSO at concentrations of 0, 1.5, 5.0, 15, 150, 500, 1500 and 5000 µg per plate in the presence and absence of mammalian S9 metabolic activation. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies above background in all tester strains and both experiments (initial toxicity-mutation assay and confirmatory mutagenicity assay). Based on the results, the test item is considered to be non-mutagenic in the bacterial reverse gene mutation assay.

 

The negative result were consolidated by the negative outcomes of supporting studies, a non-GLP bacterial mutation assay and negative in vitro cytogenicity (mammalian chromosome aberration test) and by a negative in vivo rat bone marrow micronucleus assays.

Justification for classification or non-classification

Based on the available data, (2R)-2-(2,4-difluorophenyl)-1,1-difluoro-3-(tetrazol-1-yl)-1-[5-[4-(2,2,2-trifluoroethoxy)phenyl]pyridin-2-yl]propan-2-ol is considerd to be non-mutagenic and no classification for mutagenicity is warranted in accordance with CLP regulation 1272/2008.