2-(4-methylphenoxy)-N-(1H-pyrazol-3-yl)-N-[(thiophen-2-yl)methyl]acetamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames Test (OECD 471, GLP, K, rel. 1): non mutagenic up to the limit of solubility and /or up to cytotoxic concentrations in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 & E.coli WP2uvrA.

- Human lymphocytes chromosome aberration test (OECD 473, GLP, K, rel. 1): non clastogenic up to cytotoxic concentrations.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 February to 14 August 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD test Guideline No. 471 without any deviation.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine and tryptophan for S. typhimurium and E. coli, respectively

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

10% S9: S9-mix from the livers of male rats treated with 0.1 M NaH2PO4 / Na2HPO4, pH 7.4; 5 mM Glucose-6-phosphate; 4 mM NADP; 33 mM KCl; 8 mM MgCl2

Test concentrations with justification for top dose:

Test for Mutagenicity (Experiment 1) – Plate Incorporation Method: 63, 130, 250, 500, 1000 μg/plate (solubility limit), with and without S9-mix
Test for Mutagenicity (Experiment 2) – Pre-Incubation Method: 63, 130, 250, 500, 1000 μg/plate (solubility limit), with and without S9-mix

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: In solubility checks performed in house the test item was noted as immiscible in sterile distilled water at 50 mg/mL but fully miscile in DMSO at the same concentration. DMSO was therefore selected as the vehicle.
- Preparation of test formulation: The test item was dissolved in DMSO at 10.0 mg/mL and serially diluted to 5.0, 2.5, 1.3 and 0.63 mg/mL. All preparations were vortexed immediately before diluting.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

Remarks:

Without S9-mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

cyclophosphamide

other: 2-aminoanthracene

Remarks:

With S9-mix

Details on test system and experimental conditions:

SOURCE OF TEST SYSTEM
- Bacteria were purchased as dried discs from Molecular Toxicology Inc., Boone, NC, U.S.A. and stored in a refrigerator between 2 to 8°C. Frozen permanent stocks were prepared from fresh cultures of the discs and stored at -80°C ± 10°C in the presence of 9 % dimethyl sulfoxide (DMSO)

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Exposure duration: Plates were incubated at 37 °C ± 3 °C for 48 to 72 hours

NUMBER OF REPLICATIONS: Triplicate plates per dose level.

DETERMINATION OF CYTOTOXICITY
- Method: The plates were examinated via the naked eye and with the aid of a microscope for evidence of thinning (toxicity).

OTHERS:
After incubation, the plates were counted. The plates were also examined for the health status of the bacterial background lawn and possible precipitates of the test article at the end of the incubation period.

Rationale for test conditions:

Maximum concentration was 1000 μg/plate. Slight precipitate was visible with the naked eye only at 1.0 mg per plate. Therefore at this concentration, the test article was evaluated at the limit of solubility in the test system

Evaluation criteria:

Positive result will be considered positive when there is a significant increase in the number of colonies per plate in comparison to the concurrent negative control and a concentration-related increase over the exposure range tested. For the cases that there is historical data available, a positive result has to present an increase in the number of revertant colonies per plate in comparison to the historical data for at least one experimental condition. Biological relevance of the results will be considered first. A statistical method may be used as an aid in evaluating the test results but it will not be the only determining factor. A positive result indicates that the test article induces point mutations in S. typhimurium or E. coli.

Negative result (no evidence of genotoxicity) is concluded if there is no substantial increase in the number of colonies per plate; i.e. the results do not exceed the upper 98 percentile limit of the historical solvent/negative control range. A negative result indicates that the test article is non-mutagenic in S. typhimurium or E. coli.

Equivocal result: If no definite judgment can be made to fit the above criteria, even after repeated experiments, then the result will be described as equivocal. An equivocal result indicates that a definitive result cannot be made by performing the bacterial reverse mutation assay under the conditions described in this protocol.

Statistics:

Statistical analysis was applied to the numbers suspected to be abnormally high or to have a dose-related increase in revertant counts. The colony counts were transformed (square root) to normalize the data prior to using the one-sided Dunnett’s test (Mahon, G.A.T., et al., 1989).

Key result

Species / strain:

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

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

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:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Slight precipitate was observed with the naked eye starting at 250 µg/plate and increasing in a dose-dependent manner to a level of moderate precipitate at 1000 µg/plate. Precipitate was
not observed for any of the lower concentrations.
- Other confounding effects: None

HISTORICAL CONTROL DATA
All of the spontaneous reversion controls were within the acceptable range described in the protocol for spontaneous reversion. All the concurrent positive controls for each tester strain exceeded the minimum counts of the historical positive control data indicating that the sensitivity of the experiments was at historical levels.

MUTATION TEST
There were no statistically significant (p>0.01) increases in colony counts over the concurrent negative controls. The more sensitive preincubation test was performed to confirm the results of the plate incorporation
test as negative. There were five concentrations available for evaluation for mutagenicity for all conditions (OECD, 1997). There were two slight but statistically significant (p<0.01) increases in colony counts over the concurrent negative controls (TA1535 without S9 at 0.13 and 1.0 mg/plate). The results were within the Nucro-Technics historical data range (min-max) and a dose-response was not observed for this condition. Therefore the preincubation test results were concluded as clear negative which also confirmed the plate incorporation test results as negative.

OTHERS
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).

None

Conclusions:

Under the test condition, test item is not mutagenic with and without metabolic activation in S. typhimurium (strains TA1535, TA1537, TA98 and TA100) and E.coli WP2 uvrA- with or without metabolic activation.

Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coli strain WP2 uvrA- were exposed to test item diluted in DMSO both in the presence and absence of metabolic activation system (10% liver S9 in standard co-factors) using the Ames plate incorporation and pre‑incubation methods in Experiment 1 and Experiment 2, respectively.

Test for Mutagenicity (Experiment 1) – Plate Incorporation Method :

63, 130, 250, 500, 1000 μg/plate, with and without S9-mix

Test for Mutagenicity (Experiment 2) – Pre-Incubation Method :

63, 130, 250, 500, 1000 μg/plate, with and without S9-mix

Negative, vehicle (DMSO) and positive control groups were also included in mutagenicity tests.

At the end of the incubation period in the plate incorporation experiment, slight to moderate precipitate was visible between 0.25 and 1.0 mg per plate with the naked eye. Toxicity was not observed at any concentration evident by a normal background lawn and colony counts similar to the concurrent negative controls. Therefore, the test article was evaluated at the limit of solubility in the test system. For all strains and conditions, all 5 concentrations were analyzable for mutagenicity. The test item did not produced any statistically significant increases (p>0.01) in colony counts over the concurrent negative controls, with or without metabolic activation. A more sensitive preincubation test was designed to confirm the negative results of the plate incorporation test.

In the preincubation experiment, both in the presence and absence of S9, the concentrations of test item investigated were identical to the plate incorporation test. At the end of the incubation period, slight precipitate was visible with the naked eye only at 1.0 mg per plate. Therefore at this concentration, the test article was evaluated at the limit of solubility in the test system. Toxicity was similar to the plate incorporation test when compared to the concurrent negative controls with one exception. For TA1537 without S9, the colony counts were slightly reduced at the highest concentration of 1.0 mg/plate. Also, the background lawns were slightly reduced at the highest concentration of 1.0 mg/plate with and without S9. Despite this toxicity, all 5 concentrations were analyzable for mutagenicity. Therefore for these conditions, the test item was evaluated at the limit of test article toxicity. In the preincubation test with TA1535 without metabolic activation, there were two slight (1.25 and 1.44-fold), but statistically significant increases (p<0.01) in colony counts at 0.13 and 1.0 mg/plate over the concurrent negative control. Despite these increases, the results were within the historical control data range (min-max) and a dose-response was not observed. Therefore the preincubation test confirmed the negative results of the plate incorporation test.

 

For most strains and concentrations, the background lawn and the number of colony counts were observed to be normal when compared to the negative control with a few exceptions. For TA1537 with and without S9 at 1.0 mg/plate, the background lawn was slightly reduced when compared to the concurrent negative controls. Also, an obvious reduction in colony counts was observed for TA1537 without S9 at 1.0 mg/plate.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

 

Under the test condition, test item is not mutagenic with and without metabolic activation in S. typhimurium (strains TA1535, TA1537, TA98 and TA100) and E.coli WP2 uvrA- with or without metabolic activation.

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 June to 29 August 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted in compliance with OECD Guideline No. 473 (1997) without any deviation.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Qualifier:

according to guideline

Guideline:

other: 40 CFR 799.9537 TSCA in vitro mammalian chromosome aberration test (2011).

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction was obtained from a post mitochondrial 9,000 x g fraction of liver homogenate from Sprague-Dawley rats treated with Phenobarbital-5, 6-benzoflavone

Test concentrations with justification for top dose:

Preliminary experiment (cytotoxicity testt): The test item was dissolved in DMSO at 500 mg/mL and serially diluted 2-fold in DMSO. Cytotoxicity was observed at an exposure concentration range about 0.031 to 0.31 mg/mL Test article precipitate was observed in the test system starting at about 0.25 mg/mL up to the highest exposure concentration resulting in a range of slight to extreme precipitate. Based on these results, the dose designs described below were established for
the main study.

Main study:
- 35, 58, 97, 160 and 270 μg/mL yielded Relative Cell Growth (RCG) for a 3-hour exposure in the absence of S9 metabolic activation
- 1.3, 2.5, 5.0, 10, 20, 40, 80 and 160 μg/mL yielded RCGs for a 3-hour exposure in the presence of S9 and
- 23, 39, 65, 110 and 180 μg/mL, yielded RCGs for a 20-hour exposure in the absence of S9 activation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Formulation preparation: The test item was accurately weighed, dissolved in DMSO and serial dilutions prepared.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9 mix

Details on test system and experimental conditions:

TEST SYSTEM: Primary human lymphocytes were obtained from StemCell Technologies (Vancouver, BC, Canada) and were stored in liquid nitrogen until required. A typical cell contains 46 chromosomes. The health status of the cultures was observed under a microscope. The cultures were free of Mycoplasma by direct culture test

CELL CULTURE: Cells were cultured in RPMI 1640 Medium (Lonza, Walkersville, MD, U.S.A.) and supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine and antibiotic-antimycotic (100 units penicillin, 0.1 mg streptomycin and 0.25 μg amphotericin B) (Sigma Chemical Co., St. Louis, MO, U.S.A.) in Sterile Falcon tissue culture flasks (BD Biosciences, Bedford, MA, U.S.A.). Lymphocytes were stimulated to proliferate by the presence of Phytohemagglutinin (PHA) (Sigma Chemical Co., St. Louis, MO, U.S.A.) at 10 μg/mL. The cells were incubated in a humidified tissue culture incubator at 37 ± 2°C and 5 ± 2% CO2. Cells were collected by centrifugation and seeded in fresh medium.

DURATION
- Exposure duration: 3 hours (- S9), 20 hours (-S9) and 3 hours (+S9)
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours (± S9)

SPINDLE INHIBITOR (cytogenetic assays): Mitotic activity was arrested by addition of demecolcine (0.1 μg/mL), two to three hours before the harvest time.
STAIN (for cytogenetic assays): 10 % Giemsa

NUMBER OF REPLICATIONS:
Preliminary toxicity test: Single culture/dose
Main experiment: Single cultures/dose

NUMBER OF CELLS EVALUATED:
Well-spread metaphase cells with 44 - 48 chromosomes were analysed for chromosome aberrations as defined in Protocol SEN/265880 (Appendix I; Scott, et al., 1990; OECD, 1997). One hundred cells from each negative control and test item-treated culture or 50 cells from a positive control culture were examined (OECD, 1997).

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
The number of endoreduplicated and polyploidy cells, chromatid gaps and chromosome gaps were recorded when encountered, but were not included in the aforementioned calculations.

Rationale for test conditions:

The selection of the maximum dose level was based on results of the preliminary experiment (cytotoxicity and precipitate).

Evaluation criteria:

Negative control: The frequency of cells with chromosome aberrations (excluding gaps) in the vehicle control cultures will normally be within the laboratory historical control data range.
Positive control: All the positive control chemicals must induce positive responses (p≤0.01). Acceptable positive responses demonstrate the validity of the experiment and the integrity of the S9-mix.
Cytotoxicity: There must be at least three analyzable dose levels present for each experiment.

A test item can be classified as non-genotoxic if:
1. The number of induced chromosome aberrations in all evaluated dose groups is within the range of historical control data.
2. No toxicologically or statistically significant increase of the number of structural chromosome aberrations is observed following statistical analysis.
A test item can be classified as genotoxic if:
1. The number of induced structural chromosome aberrations is not in the range of historical control data.
2. Either a concentration-related or a statistically significant increase of the number of structural chromosome aberrations is observed. Marked increases only observed in one dose level will be assessed on a case by case basis.

Biological relevance of the results will be considered first. Statistical methods will be used to analyze the increases in aberration data as recommended in the OECD 473 guidelines. However, statistical significance will not be the only determining factor for a positive response.
A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations will be assessed on a case by case basis.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No significant change in pH when the test substance was added into media.
- Effects of osmolality: Osmolality did not increase by more than 50 mOsm.
- Precipitation: Yes; 0.25 mg/mL up to the highest exposure concentration resulting in a range of slight to extreme precipitate.

PRELIMINARY TOXICITY TEST:
Cytotoxicity, evaluated by Relative Cell Growth, was observed at an exposure concentration range about 0.031 to 0.31 mg/mL. This result varied depending on the condition. Test article precipitate was observed in the test system starting at about 0.25 mg/mL up to the highest exposure concentration resulting in a range of slight to extreme precipitate. The amount of precipitate and the concentration where it was observed varied slightly depending on the condition tested. Preliminary results were not included in this report as the results of the main study are selfsupporting.
Based on these results, the dose designs described below were established for the main study.

COMPARISON WITH HISTORICAL CONTROL DATA: All vehicle and solvent controls were in the range of historical laboratory control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the main study, 3 hours exposure without S9 to 35, 58, 97, 160 and 270 μg/mL test item resulted in Relative cell growth (RCG) of 108, 93, 98, 60 and 18%, respectively. At the four lowest concentrations, Relative mitotic index (RMI) of 102, 88, 110 and 48% were observed, respectively. Slight test article precipitate was observed only at the highest concentration of 270 μg/mL. Precipitate was not observed for any lower concentrations.
The concentrations of 0, 58, 97 and 160 μg/mL were analyzed for chromosome aberrations. At least one hundred metaphase cells from each culture were examined. A low level of cells with structural chromosome aberrations was encountered at all concentrations including the solvent control. At 0, 58, 97 and 160 μg/mL exposure to test item resulted in 3.5, 3.0, 1.5 and 3.0% of cells that were observed to have chromosome aberrations, respectively. A low level of polyploid cells was observed.
Cells exposed to test item for 3 hours in the presence of S9 at 1.3, 2.5, 5.0, 10, 20, 40, 80 and 160 μg/mL resulted in RCG of 109, 100, 121, 130, 97, 84, 60 and 11%, respectively. At the three lowest concentrations, RMI of 61, 55 and 45% were observed, respectively. Test article precipitate was not observed at any of the exposure concentrations. The concentrations 0, 1.3, 2.5 and 5.0 μg/mL were analyzed for chromosome aberrations. At least one hundred metaphase cells from each culture were examined. A low background level of cells with structural chromosome aberrations was
observed at all concentrations including the solvent control. At 0, 1.3, 2.5 and 5.0 μg/mL test item exposure, 1.5, 1.5, 2.5 and 2.0% of cells were observed to h ve chromosome aberrations, respectively. A low level of polyploid cells was observed.
The 20-hour exposure to test item in the absence of S9 demonstrated that doses of 23, 39, 65, 110 and 180 μg/mL resulted in RCG of 100, 97, 60, 56 and 19%, respectively. At the four lowest concentrations, RMI of 106, 82, 57 and 0% were observed, respectively. Test article precipitate was not visible at any of the exposure concentrations. The concentrations of 0, 23, 39 and 65 μg/mL were analyzed for chromosome aberrations. A low level of structural chromosome aberrations was encountered at all concentrations including the solvent controls. At exposures of 0, 23, 39 and 65 μg/mL of test item, 2.0, 2.0, 1.5 and 1.5% of cells were observed to have structural chromosome aberrations, respectively. A low level of polyploid cells was observed.

MAIN STUDY RESULTS:
The test item did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.
The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups. There was no indication of endoreduplication noted.

None

Conclusions:

The test item did not induce any statistically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolizing system. The test item was therefore considered to be nonclastogenic to human lymphocytes in vitro.

Executive summary:

In an in vitro chromosome aberration test performed according to OECD Guideline 473 and in compliance with GLP, cultured human lymphocytes were exposed to test item at the following concentrations:

Main study:

without S9: 0, 35, 58, 97, 160 and 270 μg/mL; 3 -hour treatment

without S9: 0, 23, 39, 65, 110 and 180 μg/mL; 20 -hour treatment

with S9: 0, 1.3, 2.5, 5.0, 10, 20, 40, 80 and 160 μg/mL 3 -hour treatment

The choice of doses was based on a preliminary experiment with observation of cytotoxicity and precipitates.

Mitotic activity was arrested by addition of colcemid at 0.1 µg/mL for each culture, two or three hours before the harvest. The cells were then treated with a hypotonic solution, fixed, stained and examined for mitotic indices and chromosomal aberrations. Vehicle and positive controls were also included in this test.

 

All vehicle (solvent) control groups had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated.

 

The test item did not induce any statistically significant increases in the frequency of cells with aberrations using a dose range that included a dose level that induced approximately 50% mitotic inhibition.

 

Under the test conditions, the test item was considered to be non-clastogenic to human lymphocytes in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

- In vivo micronucleus test (similar to OECD 474, GLP, K, rel. 2): non clastogenic, non aneugenic up to toxic concentrations

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Remarks:

no blood sample to show bone marrow exposure occurred and no systemic effects observed, 2000 PCE/animal instead of 4000 & historical mnPCE<0.1% in males, humidity <40%

Adequacy of study:

key study

Study period:

From February 21 to July 3, 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

no blood sample to show bone marrow exposure occurred & no effects observed & no systemic effects observed, 2000 PCE/animal instead of 4000 & historical MN PCE<0.1% in males, humidity <40%

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997 (old)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH (2011) EMA/CHMP/ICH/126642/2008. Guideline S2(R1): Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of assay:

micronucleus assay

Specific details on test material used for the study:

The Sponsor indicated that protection from light was not required for the bulk test article and for the test article and vehicle control dosing formulations.

Species:

mouse

Strain:

other: Crl:CD-1(ICR)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Portage, MI
- Age at study initiation: 40 days old
- weight: males are expected to weigh 24-34 grams and the females 18-28 grams
- Housing: Upon arrival: 3 per cage by sex for at least 3 days. After : all animals were housed individually in clean, stainless steel, wire-mesh cages suspended above cage-board. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals
- Diet: PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002 (meal), ad libitum
- Water: Reverse osmosis-treated (on-site) drinking water, delivered by an automatic watering system, ad libitum
- Acclimation period: 11-day and 18-day acclimation period for the range-finding and definitive phases, respectively.

ENVIRONMENTAL CONDITIONS
- Temperature: 21.3 to 21.5 °C
- Humidity: 35.4% to 39.0%
- Air changes: Air handling units were set to provide a minimum of 10 fresh air changes per hour.
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: From February 21 to April 17, 2013

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 1% methylcellulose [400 cps] in deionized water.
- Justification for choice of solvent/vehicle: not reported
- Concentration of test material in vehicle: 50; 100,150 and 200 mg/ml.
- Amount of vehicle (if gavage or dermal): 10 mL/kg (gavage)
- Lot/batch no. (if required): Methylcellulose, 400 cps (lot no. 2BF0758, retest date: 17 May 2013, manufactured by Spectrum Chemical Manufacturing Corporation, New Brunswick, NJ)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
An appropriate amount of test article was combined with a small amount of vehicle in a mortar and ground until a uniform mixture was obtained. The mixture was transferred to a glass container, the appropriate amount of vehicle was added to the container, and the mixture was stirred until the test article was wetted. The remaining vehicle was added, the mixture was homogenized using a Silverson L4RT or L4RT-A homogenizer until a uniform mixture was obtained, and the test article formulations were then stirred overnight. The test article formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures. Prior to dose administration, the test article formulations were visually inspected to ensure that the formulations were visibly homogenous and acceptable for dosing.
Dosing solutions were single formulations for each dosage level, divided into aliquots for daily dispensation, and stored at room temperature (18°C to 24°C).

Duration of treatment / exposure:

Three consecutive days (Positive control formulation administered to Group 5 animals once on study day 2 (third day of dosing)).

Frequency of treatment:

Once daily

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

1 500 mg/kg bw/day (nominal)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Range-finding test: 3 males and 3 females / dose
Main test: 6 Males and 6 Females / dose

Control animals:

yes, concurrent vehicle

Positive control(s):

- Positive control: Cyclophosphamide monohydrate
- Doses / concentrations: 60 mg/kg bw
- Route of administration: Oral

Tissues and cell types examined:

The objective of this study was to assess the potential of the test article to induce micronuclei in polychromatic erythrocytes (PCEs) in CD-1 mouse bone marrow.Bone marrow smears were prepared and the coded slides were counted for polychromatic, normochromatic, and micronucleated polychromatic erythrocytes following the final bone marrow sample collection on study day 3.

Details of tissue and slide preparation:

Bone marrow was aspirated or flushed 2 to 3 times from each femur into a centrifuge tube using a syringe containing heat inactivated fetal bovine serum (HI FBS). Animals not used for collection of bone marrow were euthanized by inhalation of carbon dioxide and discarded. The bone marrow was centrifuged and all but approximately 0.25 mL (or a volume approximately twice that of the cell pellet) of HI FBS was decanted, and the pellet was resuspended in the remaining HI FBS. Bone marrow smears were prepared by placing approximately 1 drop of cell suspension onto a minimum of 2 appropriately labeled, clean microscope slides. Each slide was coded so that the treatment group would not be revealed during subsequent analysis. The slides were air dried, fixed in 100% methanol for approximately 20 minutes, and allowed to air dry a second time. The slides were stored and shipped at ambient temperature to WIL Research Skokie for analysis.

CRITERIA FOR DOSE SELECTION:
Dosage levels for the range-finding phase were selected based on the recommendation of OECD Guideline 474, mammalian erythrocyte micronucleus test (July 1997), to dose up to the limit dosage of 2000 mg/kg/day. Additionally, the Sponsor has data which demonstrate that administration of the test item at 1000 mg/kg/day for 15 days did not result in maternal toxicity or fetal abnormalities (Charlap, 2013, WIL-884019), and a 28-day range finding study conducted in rats using an analog at doses of 10, 30, and 100 mg/kg did not result in any toxicity (Diehl, Draft, Study No. 20026750). In addition, no effects on survival, body weights, food consumption, or clinical observations were noted following administration of 500 to 2000 mg/kg/day for 3 consecutive days during the range-finding phase. Therefore, dosage levels of 1000, 1500, and 2000 mg/kg/day were selected for the definitive phase based on the lack of effects in the range-finding phase and the recommendation of the OECD Guideline 474.

TREATMENT AND SAMPLING TIMES:
For the range-finding and definitive phases, as appropriate, the vehicle and test article formulations were administered orally by gavage via an appropriately sized flexible Teflon®-shafted, stainless steel ball-tipped dosing cannula once daily for 3 consecutive days, through the day prior to the scheduled euthanasia. The positive control formulation for the definitive phase was administered in the same manner once on study day 2, the day prior to the scheduled euthanasia, to the positive control group animals. The dose volume for all groups was 10 mL/kg. Individual doses were based on the most recently recorded body weights to provide the correct mg/kg/day dosage. Adjusted doses became effective the day of collection of the body weights. The first day of dosing was study day 0.

DETAILS OF SLIDE PREPARATION:
Bone marrow was collected from the first 5 of 6 animals in each sex/group at the time of euthanasia (at least 18 to 24 hours following dose administration) from both femurs of animals euthanized by inhalation of carbon dioxide. Bone marrow was aspirated or flushed 2 to 3 times from each femur into a centrifuge tube using a syringe containing heat inactivated fetal bovine serum (HI FBS). Animals not used for collection of bone marrow were euthanized by inhalation of carbon dioxide and discarded. The bone marrow was centrifuged and all but approximately 0.25 mL (or a volume approximately twice that of the cell pellet) of HI FBS was decanted, and the pellet was resuspended in the remaining HI FBS. Bone marrow smears were prepared by placing approximately 1 drop of cell suspension onto a minimum of 2 appropriately labeled, clean microscope slides. Each slide was coded so that the treatment group would not be revealed during subsequent analysis. The slides were air dried, fixed in 100% methanol for approximately 20 minutes, and allowed to air dry a second time. The slides were stored and shipped at ambient temperature to WIL Research Skokie for analysis.

METHOD OF ANALYSIS:
Prior to analysis, the coded slides were stained with acridine orange (A/O) staining solution (Hayashi et al., 1983) and placed in numerical order using the animal numbers. Two separate evaluations were made for each slide: 1) a total of 1000 erythrocytes (both polychromatic erythrocytes [PCEs] and normochromatic erythrocytes [NCEs]) per animal were counted and the PCE:total erythrocytes [TE] ratio was determined; and 2) the number of micronucleated PCEs from a total of 2000 PCEs was scored per animal.

OTHER:

Evaluation criteria:

See section “Any other information on materials and methods incl. tables”

Statistics:

For the range-finding phase, data were not analyzed statistically due to the absence of a concurrent control group. For the definitive phase, analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test article-treated group to the vehicle control group by sex. Body weight, body weight change, and food consumption data were subjected to a parametric one-way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test article-treated groups to the vehicle control group. The positive control data were evaluated using the 2-sample t-test (Sokal and Rohlf, 1981) and compared to the vehicle control group.

For the definitive phase, the percentages of micronucleated cells in PCEs and in the ratio of PCEs to TEs for the test article-treated and vehicle control group (Group 1) were subjected to a parametric one-way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed statistically significant (p≤0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test article-treated groups to the vehicle control group (Group 1). In addition, the positive control and vehicle control groups were compared using a separate parametric one-way ANOVA. Statistical significance was assessed at a 95% confidence level (p≤0.05).

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 1000, 1500, and 2000 mg/kg/day
- Solubility: The analyzed dosing formulations met the protocol-specified acceptance criteria for suspensions (80% to 120% of target concentration) and were homogeneous. In addition, dosing formulations at concentrations of 100 and 200 mg/mL were demonstrated to be stable for 3 days at room temperature.
- Clinical signs of toxicity in test animals: All animals survived to the scheduled euthanasia. There were no test article-related clinical observations or effects on body weights or food consumption.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): Test item did not cause any statistically significant increases in the number of micronucleated polychromatic erythrocytes (MPCE) or micronucleated normochromatic erythrocytes (MNCE) in male or female rats. The coded positive control demonstrated the ability of the scorer to detect increases in micronucleated polychromatic erythrocytes.
- Ratio of PCE/NCE (for Micronucleus assay): Test item did not cause any statistically significant decreases in the proportion of polychromatic erythrocytes in male or female rats.
- Appropriateness of dose levels and route: All animals survived to the scheduled euthanasia. There were no test article-related clinical observations. All clinical findings in the test article-treated groups were noted with similar incidence in the vehicle control group, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory mice of this age and strain.

Table 7.6.2/1: Summary of results and statistical analysis

Treatment	Dosage	%MN PCEs	MN PCEs/2000 PCEs	PCE:TE Ratio
Male data
Vehicle	0	0.08 (0.09)	1.6 (1.8)	0.67 (0.04)
Test item	1000 mg/kg/day	0.03 (0.03)	0.6 (0.55)	0.63 (0.08)
	1500 mg/kg/day	0.04 (0.02)	0.8 (0.45)	0.63 (0.06)
	2000 mg/kg/day	0.04 (0.04)	0.8 (0.84)	0.66 (0.06)
Cyclophosphamide^a	60 mg/kg	1.15 (0.44*)	23 (8.8)	0.64 (0.19)
Female data
Vehicle	0	0.03 (0.03)	0.6 (0.55)	0.68 (0.13)
Test item	1000 mg/kg/day	0.00 (0.00)	0 (0)	0.64 (0.08)
	1500 mg/kg/day	0.03 (0.03)	0.6 (0.55)	0.62 (0.07)
	2000 mg/kg/day	0.01 (0.02)	0.2 (0.45)	0.66 (0.12)
Cyclophosphamide^a	60 mg/kg	0.82 (0.32*)	16.4 (6.3)	0.66 (0.08)

 

Vehicle: Basal diet

PCE: Polychromatic erythrocytes

TE: Total erythrocytes

MN: Micronucleated

Results of statistical analysis using the appropriate nonparametric method of analysis based on permutation (one-sided probabilities):

*p< 0.05 (significant)

Conclusions:

Under the test conditions, test item did not show any evidence of causing an increase in the induction of micronucleated polychromatic erythrocytes or bone marrow cell toxicity in male or female Crl:CD-1(ICR) mice.

Executive summary:

In an in vivo micronucleus test conducted according to OECD 474 guideline and in compliance with GLP, induction micronuclei by test item in bone marrow cells of male and female Crl:CD-1(ICR) mice was assessed. Mice (6 / sexe / dose) were exposed to the test substance (suspended in vehicle (1% methylcellulose [400 cps] in deionized water) by oral administration (gavage) at the dose levels of 500, 1000, 1500, and 2000 mg/kg/day for 3 days. A concurrent vehicle control group (Group 1) received the vehicle on a comparable regimen. A positive control group received a single oral dose of 60 mg/kg cyclophosphamide monohydrate (CPS) on study day 2, the day prior to the scheduled euthanasia. A dose range-finding study was performed in 3 animals / sexe / dose in order to determine the appropriate concentrations. All animals were observed for mortality and moribundity, clinical examinations, detailed physical examinations and body weights and individual food weights were recorded. Bone marrow collection for micronucleus evaluation was performed for 5 animals/sex/group. Bone marrow smears were prepared and the coded slides were counted for polychromatic, normochromatic, and micronucleated polychromatic erythrocytes. At least one smear from each animal was examined for the presence of micronuclei in 2000 polychromatic erythrocytes. The proportion of polychromatic erythrocytes was assessed by examination of at least 1000 erythrocytes from each animal. A record of the incidence of micronucleated normochromatic erythrocytes was also kept.

 

No statistically significant increases in the frequency of micronucleated polychromatic erythrocytes and no statistically significant decreases in the proportion of polychromatic erythrocytes were observed in mice at any dose level, compared to vehicle control values. The coded positive control slides demonstrated the ability of the scorer to detect increases in micronucleated polychromatic erythrocytes.

 

Under the test conditions, test item did not show any evidence of causing an increase in the induction of micronucleated polychromatic erythrocytes or bone marrow cell toxicity in male or female rats.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Table 7.6/1: Summary of genotoxicity tests

 

Test n°	Test / Guideline Reliability	Focus	Strains tested	Metabolic activation	Test concentration	Statement
1   Nucro-Technics, 2013	Ames Test (OECD 471) K, rel. 1	Gene mutation	TA 1535, TA 1537, TA 98, TA 100 E. coli WP2uvrA	-S9 +S9	Up to limit of solubility and /or up to cytotoxic concentrations	-S9 : non mutagenic +S9 : non mutagenic
2  Nucro-Technics, 2013	HL/CAT (OECD 473) K, rel. 1	Chromosomal aberration	Human Lymphocytes	-S9 +S9	Up to cytotoxic concentrations	-S9 : non clastogenic +S9 : non clastogenic
3 WIL research, 2013	In vivo MNT (OECD 474) K, rel. 2	Chromosomal aberration	Mice	NA	Up to toxic concentration	Non clastogenic, Non aneugenic

       

Gene mutation Assays (Tests n° 1):

A Bacterial Reverse mutation Assay (Ames test) was performed according to OECD guideline No. 471 with the substance (Test n°1, see Table 7.6/1). In the preincubation test with TA1535 without metabolic activation, there were two slight (1.25 and 1.44-fold), but statistically significant increases (p<0.01) in colony counts at 0.13 and 1.0 mg/plate over the concurrent negative control. Despite these increases, the results were within the historical control data range (min-max) and a dose-response was not observed. Therefore the preincubation test confirmed the negative results of the plate incorporation test. No other significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains under the test condition, with any dose of the substance, either in the presence or absence of metabolic activation. The substance does not induce gene mutations in bacteria whereas all positive control chemicals (with and without metabolic activation) induced significant increase of colonies. The substance is therefore considered as non-mutagenic according to the Ames test.

Chromosomal aberration (Test n°2)

The clastogenic potential of the substance was determined using an in vitro chromosome aberration test in human lymphocytes (OECD guideline No. 473, test n°2, see Table 7.6/1), which measures the potential of a substance to increase the incidence of structural chromosome aberrations in cultured human lymphocytes.

None of the dose levels up to the cytotoxicity limit with the substance, either in the presence or absence of metabolic activation, induced significant increases in the frequency of cells with aberrations in either of two separate experiments. The substance does not induce structural aberrations in the chromosomes of human lymphocytes under activation and non-activation conditions using a dose range that included a dose level that induced approximately 50% mitotic inhibition, whereas all the positive control items

induced significant increases in the frequency of aberrant cells indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated. The substance is therefore considered as negative for inducing chromosomal mutations in human lymphocyte cells under activation and non-activation conditions used in this assay.

Chromosomal aberration (Test n°3)

The clastogenic and aneugenic potential of the substance was determined using an in vivo mammalian erythrocytes micronucleus assay (OECD guideline No. 474, Test n°3, see Table 7.6/1), which identifies substances that cause micronuclei in erythroblasts. These micronuclei may originate from acentric fragments or whole chromosomes, and the test thus has the potential to identify both clastogenic and aneugenic chemicals. In this study erythroblasts were sampled from femur cells of mice (males and females) exposed to the test item by gavage. No statistically significant increases in the frequency of micronucleated polychromatic erythrocytes and no statistically significant decreases in the proportion of polychromatic erythrocytes were observed in mice at any dose level, compared to vehicle control values. The coded positive control slides demonstrated the ability of the scorer to detect increases in micronucleated polychromatic erythrocytes. The substance was therefore considered as negative for inducing chromosomal aberrations in mice femur erythrocytes under the conditions used in this study. The substance is therefore considered as non-clastogenic and non-aneugenic.

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification for human health according to the Regulation (EC) No. 1272/2008.

Self classification:

Based on the available data, no additional classification is proposed regarding genetic toxicity according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.