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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-08-22 to 2014-02-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
2010
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
(2E)-3-(2H-1,3-benzodioxol-5-yl)-N,N-diphenylprop-2-enamide
Cas Number:
1309389-73-8
Molecular formula:
C22H17NO3
IUPAC Name:
(2E)-3-(2H-1,3-benzodioxol-5-yl)-N,N-diphenylprop-2-enamide

Method

Species / strain
Species / strain / cell type:
lymphocytes: human peripheral blood lymphocytes
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: Human peripheral blood lymphocytes from two healthy, non-smoking female volunteers
- Suitability of cells: Yes, blood was received from donors who were not suspected of any virus infection or exposed to high levels of radiation or hazardous chemicals. All donors were non smokers and not heavy drinkers of alcohol. Donors were not taking any form of medication (contraceptive pill excluded).
- Normal cell cycle time: The measured cell cycle time of the donors falls within the range of 13±2 hours.

For lymphocytes:
- Sex, age and number of blood donors: Blood from 2 female donors was used in the range-finding and main study. The age of the donors was 28 and 31 in the range-finding study and 30 and 31 in the main experiment.
- Whether whole blood or separated lymphocytes were used: Whole blood cultures were used
- Whether blood from different donors were pooled or not: Blood was pooled using equal volumes from each donor prior to use.
- Mitogen used for lymphocytes: Phytohaemagglutinin (PHA, reagent grade)

MEDIA USED
- Type and composition of media: HEPES-buffered RPMI medium containing 10% (v/v) heat inactivated foetal calf serum and 0.52% penicillin / streptomycin
- Temperature: 37±1°C

Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- Source of S9: Obtained from Molecular Toxicology Incorporated, USA where it is prepared from male Sprague Dawley rats induced with Aroclor 1254.
- Method of preparation of S9 mix: Glucose-6-phosphate (180 mg/mL), β-Nicotinamide adenine dinucleotide phosphate (25 mg/mL), Potassium chloride (150 mM) and rat liver S-9 were mixed in the ratio 1:1:1:2.
- Concentration or volume of S9 mix and S9 in the final culture medium: The final concentration of the S9 mix in the test system was 5%.The final concentration of S9 in the test system was 2%.
- Quality controls of S9: Each batch was checked by the manufacturer for sterility, protein content, ability to convert known promutagens to bacterial mutagens and cytochrome P-450-catalyzed enzyme activities (alkoxyresorufin-O-dealkylase activities) as specified in the Certificate of Analysis.
Test concentrations with justification for top dose:
Range-finding study: 1.814 to 500 µg/mL (3-hour treatment +/- S9 and 20-hour treatment -S9);

Main study: 5 to 100 µg/mL (3-hour treatment -S9), 10 to 100 µg/mL (3-hour treatment +S9) and 2 to 40 µg/mL (20-hour treatment -S9)

Concentrations that were evaluated for micronuclei were: 10, 30 and 60 µg/mL (3-hour treatment -S9), 10, 30, 55 and 60 µg/mL (3-hour treatment +S9) and 8, 14 and 20 µg/mL (20-hour treatment -S9)
Vehicle / solvent:
- Vehicles used: DMSO (test substance, CPA), purified water (VIN, MMC)

- Justification for choice of solvent/vehicle: Solubility properties. DMSO is a commonly used solvent for this type of assay with a broad historical control database.

- Justification for percentage of solvent in the final culture medium: The maximum solvent concentration in the final culture medium was 1% which is known from historical control data to be not toxic and not mutagenic in this type of assay.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO (test substance, CPA), purified water (VIN, MMC)
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
vinblastine
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: For the range-finding study: Single (test substance) and duplicate (solvent control); for the main experiment: Duplicate (test substance and positive control) and quadruplicate (solvent control)
- Number of independent experiments: 1 range-finding assay and 1 main assay (each consisting of 3 tested exposure conditions)

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in: Medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3 hours (+/- S9) and 20 hours (- S9)
- Harvest time after the end of treatment (sampling/recovery times): 24 hours for all test conditions. For the 3-hour treatments, cytochalasin B was added after the 3-hour treatment period and incubated for further 21 hours. For the 20-hour treatment, cytochalasin B was added at the beginning od the treatment period and incubated, together with the test substance for 24 hours.

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Identity of cytokinesis blocking substance, concentration, and duration and period of cell exposure: Cytochalasin B was added (0.1 mL per culture) to the 24-hour treatment culture at the time of treatment. Cytochalasin B was added (0.1 mL per culture) to the 3-hour treatment cultures directly after the treatment period. The final concentration was 6 μg/mL per culture.
- Methods of slide preparation and staining technique used including the stain used: Fixed lymphocytes were centrifuged and resuspended in a minimal amount of fresh fixative. Several drops of cell suspension were gently spread onto multiple clean, dry microscope slides. Slides were air-dried then stored protected from light at room temperature prior to staining. Slides were stained by immersion in 125 μg/mL Acridine Orange in phosphate buffered saline (PBS), pH 6.8 for approximately 10 seconds, washed with PBS (with agitation) for a few seconds before transfer and immersion in a second container of PBS for approx. 10 minutes. Slides were air-dried and stored protected from light at room temperature prior to analysis.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): Slides from the cytotoxicity range-finder experiment were examined for
proportions of mono-, bi- and multinucleate cells, to a minimum of 200 cells per concentration. In the main experiment, slides were examined for RI to a minimum of 500 cells per culture.1000 binucleate cells from each culture (2000 per concentration) were analysed for micronuclei.
- Criteria for scoring micronucleated cells: See "Any other information on materials and methods incl. tables"

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Replication index

METHODS FOR MEASUREMENTS OF GENOTOXICIY
The proportion of binucleate cells with micronuclei (MNBN) as compared to the proportion in vehicle controls
Rationale for test conditions:
Test concentrations were chosen based on a dose-range finding test.

3-hour treatment (-S9): Precipitation occured at concentration of 38.88 µg/mL and above. At this concentration, 51% cytotoxicity was reached.

3-hour treatment (+S9): Precipitation occured at concentration of 38.88 µg/mL and above. At this concentration, 41% cytotoxicity was reached. At 64.80 µg/mL, 57% cytotoxicity was reached.

24-hour treatment (-S9): Marked cytotoxicity occured at concentrations of 8.398 µg/mL (36%), 14 µg/mL (79%) and 23.33 µg/mL (100%). Precipitation occured at concentrations of 38.88 µg/mL and above.

On this basis, test concentrations for the main assay were selected. The highest concentration selected for micronucleus analysis following all treatment conditions induced 55±5% cytotoxicity. Slides from the highest selected concentration and two or three lower concentrations were taken for microscopic analysis, such that a range of cytotoxicity from maximum to little was covered.
Evaluation criteria:
For valid data, the test article was considered to induce clastogenic and/or aneugenic events if:
1. A statistically significant increase in the frequency of MNBN cells at one or more concentrations was observed.
2. An incidence of MNBN cells at such a concentration that exceeded the normal range in both replicates was observed.
3. A concentration-related increase in the proportion of MNBN cells was observed. The test article was considered positive in this assay if all of the above criteria were met.

The test article was considered negative in this assay if none of the above criteria were met.

Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Evidence of a concentration-related effect was considered useful but not essential in the evaluation of a positive result
Statistics:
The proportions of MNBN cells in each replicate were used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test.

The proportion of MNBN cells for each treatment condition was compared with the proportion in vehicle controls by using Fisher's exact test.

Probability values of p≤0.05 were accepted as significant. Additionally, the number of micronuclei per binucleate cell were obtained and recorded.

Results and discussion

Test results
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
The limit cytotoxicity of 55±5% was reached at 60 µg/mL (3-hour treatment with and without S9) and at 20 µg/mL (24-hour treatment without S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: No marked changes in pH were observed at the highest concentration tested (500.0 μg/mL) in the Range Finder Experiment as compared to the concurrent vehicle controls.
- Data on osmolality: No marked changes in osmolality were observed at the highest concentration tested (500.0 μg/mL) in the Range Finder Experiment as compared to the concurrent vehicle controls (individual data not reported).
- Water solubility: The test item was soluble in DMSO up to a concentration of 175.0 mg/mL.
- Precipitation and time of the determination: Precipitation occured at the beginning of treatment from 38.88 µg/L onwards under all test conditions. Precipitation occured at the end of tretment from 108 and 180 µg/mL onwards for the 3-hour treatment times (-/+S9). Precipitation occured at harvest from 64.80 µg/mL (3-hours, -S9), from 108 µg/mL (3-hours, +S9) and from 38.88 µg/mL (24-hours, -S9) onwards under all test conditions.
- Definition of acceptable cells for analysis: See "Any other information on materials and methods incl. tables"

RANGE-FINDING/SCREENING STUDIES: Yes, cytotoxicity was tested in a range-finding study at concentrations between 1.814 to 500 µg/mL in duplicate (vehicle control) or single cultures (test item) under all three incubation conditions (3-hour treatment with and without S9 and 24-hour treatment without S9). Dose-dependent cytotoxic effects were observed in all cultures (see "Rationale for test conditions"). On this basis, test concentrations for the main experiment were selected.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: See "Attached background material"

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship: No concentration-response relationship was observed.
- Statistical analysis: No statistical significance was observed for any of the the test substance concentrations and negative controls. Positive control yielded the expected statistically significant responses.

Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements: See "Attached background material"
o In the case of the cytokinesis-block method: CBPI or RI; distribution of mono-, bi- and multi-nucleated cells: See "Attached background material"

- Genotoxicity results
o Number of cells with micronuclei separately for each treated and control culture and defining whether from binucleated or mononucleated cells, where appropriate: See "Attached background material"

HISTORICAL CONTROL DATA
- Positive historical control data: See "Attached background material"
- Negative (solvent/vehicle) historical control data: See "Attached background material"

Applicant's summary and conclusion

Conclusions:
In an in vitro Mammalian Cell Micronucleus Test according to OECD guideline 487, the test item did not induce micronuclei in cultured human peripheral blood lymphocytes following treatment in the absence and presence of a rat liver metabolic activation system (S-9), when tested up to cytotoxic concentrations.
Executive summary:

The test item was tested in an in vitro micronucleus assay according to OECD guideline 487 and GLP using duplicate human lymphocyte cultures prepared from the pooled blood of two female donors in a single experiment. Treatments covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S-9) from Aroclor 1254-induced rats. The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO) and the highest concentrations used in the Micronucleus Experiment, were determined following a preliminary cytotoxicity Range-Finder Experiment. Treatments were conducted (as detailed in the following summary table) 48 hours following mitogen stimulation by phytohaemagglutinin (PHA). The test article concentrations for micronucleus analysis were selected by evaluating the effect of the test item on the replication index (RI). Micronuclei were analysed at three or four concentrations and a summary of the data is presented in the following table:


 

































































































































Treatment



Concentration


(µg/mL)



Cytotoxicity


(%)



Mean


MNBN Cell


Frequency


(%)



Historical


Control


Range (%)



Statistical


Significance



3+21 hour -S9



Vehicle



-



0.80



0.10 – 1.10



-



10.00



11



1.05



 



NS



30.00



27



0.80



 



NS



60.00



55



0.65



 



NS



MMC, 0.80



ND



7.35



 



p ≤0.001



3+21 hour +S9



Vehicle



-



0.80



0.20 – 1.20



-



10.00



7



0.90



 



NS



30.00



27



0.85



 



NS



55.00



57



0.40



 



NS



60.00



50



0.45



 



NS



CPA, 6.25



ND



2.90



 



p ≤0.001



24+0 hour -S9



Vehicle



-



1.10



0.10 – 1.50



-



8.00



10



1.25



 



NS



14.00



24



1.00



 



NS



20.00



55



1.05



 



NS



VIN, 0.02



ND



16.92



 



p ≤0.001



 


Appropriate negative (vehicle) control cultures were included in the test system under each treatment condition. The proportion of micronucleated binucleate (MNBN) cells in the vehicle cultures fell within, or very close to, the 95th percentile of the current observed historical vehicle control (normal) ranges. Mitomycin C (MMC) and Vinblastine (VIN) were employed as clastogenic and aneugenic positive control chemicals, respectively, in the absence of rat liver S-9. Cyclophosphamide (CPA) was employed as a clastogenic positive control chemical in the presence of rat liver S-9.


 


Cells receiving these were sampled in the Micronucleus Experiment at 24 hours after the start of treatment; all compounds induced statistically significant increases in the proportion of cells with micronuclei. All acceptance criteria were considered met and the study was therefore accepted as valid. Treatment of cells with the test item in the absence and presence of S-9 resulted in frequencies of MNBN cells that were generally similar to and not significantly higher than those observed in concurrent vehicle controls for all concentrations analysed under all three treatment conditions. The MNBN cell frequency of all treated cultures fell within normal ranges with the exception of a single culture at 10 μg/mL, the lowest concentration analysed following 3+21 hours treatment in the presence of S-9. This isolated marginal increase of 1.3% compared to the normal range of 0.2-1.2% was not reproduced in the replicate culture at 10  g/mL or at any other concentration tested. In addition, as the mean MNBN cell frequency fell within the normal range and was not statistically significantly higher than the concurrent vehicle controls, it was considered of no biological relevance.


 


It is concluded that the test item did not induce micronuclei in cultured human peripheral blood lymphocytes following treatment in the absence and presence of a rat liver metabolic activation system (S-9), when tested up to cytotoxic concentrations.