1,4-dimethylpiperazine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2022-02-21 to 2022-06-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

1,4-dimethylpiperazine

EC Number:

203-412-0

EC Name:

1,4-dimethylpiperazine

Cas Number:

106-58-1

Molecular formula:

C6H14N2

IUPAC Name:

1,4-dimethylpiperazine

Details on test material:

- Name of test material (as cited in study report): Jeffcat DMP

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch number of test material: PFW210769
- Retest Date: 2023-11-25
- Purity: 99.91 A% . This substance is specified by total titratable amine content.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (+15 to +25°C), protect from light/humidity. At temperature no higher than 22ºC. Store under a dry inert gas blanket, such as nitrogen, to minimize contamination resulting from contact with air and water.
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: not indicated.
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: A solubility test was conducted at 114.2 mg/mL in sterile water to achieve a target test concentration of 1142 μg/mL in the final test solution.

OTHER SPECIFICS
- pH: 11.4
- Amine content: 17.39 meq/g.

Method

Species / strain

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Blood was collected from an individual male volunteers of 27 years of age for the preliminary cytotoxicity test and the micronucleus assay. The volunteer was non-smoker, not undergoing any drug treatment and not exposed to high levels of radiation or hazardous chemicals or any viral infection. The blood was sourced in accordance with the human ethical principles and regulations.
- Whole blood cultures were established in sterile disposable centrifuge tubes by placing 0.8 mL of heparinized blood into 8.55 mL RPMI FBS20 and 0.15 mL reagent grade PHA. Blood cultures were incubated inside a CO2 incubator at 37 ± 1°C in humidified atmosphere for approximately 48 hours.
- Approximately 48 hours before exposure to treatment, three sets of target blood cultures were established to represent 3-hour exposure in the presence (set 1) and absence (set 2) of metabolic activation and 24-hour exposure in the absence (set 3) of metabolic activation.

MEDIA USED
- RPMI 1640 medium supplemented with sodium bicarbonate, antibiotics and 20% fetal bovine serum (RPMI FBS20)
- Dulbecco’s Phosphate buffered saline (PBS)

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Aroclor 1254 induced rat liver S9 homogenate was used as the metabolic activation system.
- method of preparation of S9 mix: The S9 homogenate was prepared from male Wistar rats induced with a single intra-peritoneal injection of ready to use Aroclor 1254 at a dosage volume of 0.7 mL per rat, 5 days prior to sacrifice. The
S9 homogenate was prepared in batches and stored in a deep freezer maintained at -68 to -86 ºC. Each batch of S9 homogenate was characterized for its ability to metabolize the promutagens 2-Aminoanthracene and Benzo(a)pyrene to mutagens using Salmonella typhimurium TA100 strain, protein content using modified Lowry Assay (Sword and Thomson, 1980), and sterility. To check the sterility, the liver homogenate was streaked onto nutrient agar plates, in duplicate, and incubated for 72 hours at 37 ± 1 ºC. Blood cells were exposed to the treatment in the presence of S9 mix prepared as follows: NADP (25 mg/mL), Glucose-6-phosphate (180 mg/mL), 150 mM KCl and rat liver S9 mixed in the ratio of 1:1:1:2. For both the preliminary cytotoxicity test and micronucleus assays, the cofactor solutions were prepared by dissolving each of the following cofactors in 1.5 and 1.25 mL of RPMI 1640, for the preliminary toxicity assay and the micronucleus assay, respectively, and then sterilized by passing through a disposable 0.2 μm syringe filter. The S9 mix was prepared by mixing 3.0 mL and 2.5 mL of S9 homogenate with 4.5 and 3.75 mL of the co-factor solution, for the preliminary cytotoxicity test and micronucleus assays respectively. This mix was kept in an ice bath and used within one hour.

- concentration or volume of S9 mix and S9 in the final culture medium: 500 µL of S9 mix was added to the respective target blood cultures (set 1) to achieve a final concentration of 2% S9 (v/v) in the test solution

- quality controls of S9: For the initial pH and osmolality determination, 50 µL of either vehicle or the stock/dilution of the test item was transferred to two sets of tubes containing 4.75 mL of treatment medium pre-labeled for the presence and absence of metabolic activation. For the test in the presence of metabolic activation,
250 µL of S9 mix was added to each tube to achieve a concentration of 2% (v/v) of S9.
For the test in the absence of metabolic activation, 250 µL of 150 mM KCl was added to each tube. The tube contents were mixed and the pH (vehicle and all the test concentrations) and osmolality of the required test item treatment conditions in test solution were determined. The pH of the test solutions of 571 and 1142 µg/mL was adjusted with 1N HCl.
After 3-hour exposure, the first and second set of tubes were observed for any precipitation. The exposed test solution of the vehicle control and the test concentrations were centrifuged and transferred to pre-labeled tubes and the pH and osmolality were determined.
Target blood cultures in a single replicate were exposed to required concentrations of the test item and the sterile water control as follows:
Ten microliters (10 µL) of Cytochalasin B (6000 µg/mL) were added to all the tubes.

For the experiment in the presence of metabolic activation, 500 µL of S9 mix was added to the respective target blood cultures (set 1) to achieve a final concentration of 2% S9 (v/v) in the test solution. Similarly, for the experiments in the absence of metabolic activation, 500 µL of 150 mM KCl was added to the respective target blood cultures (set 2 and 3).

One hundred microliters (100 µL) of either vehicle or the required dilutions/stock of the test item were mixed with the medium in the respective blood cultures to achieve the required test concentrations as well as the vehicle control. The first and second set of blood cultures (with and without metabolic activation) were incubated for 3 hours, whereas, the third set of blood cultures were incubated for 24 hours to expose the cells to treatment.

After the 3-hour exposure, the cultures in the vehicle control and treatment groups (with and without metabolic activation) tubes were centrifuged and the supernatant discarded. The cell pellets were washed twice with PBS, resuspended in fresh RPMI FBS20, PHA and Cytochalasin B and incubated for approximately 24 hours from the start of treatment. Following the respective incubation periods, cells (sets 1, 2 and 3) were pelleted by centrifugation. The supernatant was removed and the cell pellet was suspended in KCl and further processed. Two slides were made from each culture, stained with acridine orange and scored for mitotic index.

Test concentrations with justification for top dose:

A preliminary cytotoxicity test was carried out to select test concentrations for the micronucleus assay at 17.84, 35.69, 71.38, 142.75, 285.5, 571 and 1142 µg/mL along with a sterile water control. The highest test concentration was chosen to correspond to 10 mM as per OECD TG.
The test item exposed to the blood cultures in the presence and absence of metabolic activation with the 3-hour exposure, did not exhibit the required level of toxicity (40 to 50 % CBPI) at any of the tested concentrations, both in the presence and absence of metabolic activation with the 3 hour exposure. The test item exposed to the blood cultures in the absence of metabolic activation with the 24-hour exposure, exhibited the required level of toxicity (40 to 50 % CBPI) at the highest tested concentration of 1142 μg/mL when compared to the respective sterile water control. Based on these observations, for the micronucleus assay, it was decided to test at the maximum concentration of 1142 μg/mL (10mM) in experiments 1, 2 and 3, because this is the highest concentration recommended by the OECD TG.

Doses tested in experiments 1, 2 and 3: 127, 381 and 1142 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: sterile water (100 µL)

- Justification for choice of solvent/vehicle: A solubility test was conducted at 114.2 mg/mL in sterile water to achieve a target test concentration of 1142 µg/mL in the final test solution.

- Justification for percentage of solvent in the final culture medium: not indicated

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

sterile water

Negative solvent / vehicle controls:

yes

Remarks:

sterile water

True negative controls:

no

Positive controls:

yes

Remarks:

dissolved in DMSO

Positive control substance:

cyclophosphamide

Remarks:

A stock solution of 300 µg/mL Cyclophosphamide (CPA) was prepared by mixing 1.2 mg CPA in 4 mL DMSO. From this stock solution, 100 microliters was dispensed into respective tubes to achieve a target concentration of 3 µg/mL cyclophosphamide (Group 5)

Controls 2

Untreated negative controls:

yes

Remarks:

sterile water

Negative solvent / vehicle controls:

yes

Remarks:

sterile water

True negative controls:

no

Positive controls:

yes

Remarks:

dissolved in DMSO

Positive control substance:

mitomycin C

Remarks:

A stock solution of 100 µg/mL of Mitomycin C (MMC) was diluted to 50 µg/mL by mixing 0.3 mL of the MMC stock in 0.3 mL DMSO. From this dilution 100 µL was dispensed into respective tubes to achieve a target concentration of 0.5 µg/mL (Group 5).

Controls 3

Untreated negative controls:

yes

Remarks:

sterile water

Negative solvent / vehicle controls:

yes

Remarks:

sterile water

True negative controls:

no

Positive controls:

yes

Remarks:

dissolved in DMSO

Positive control substance:

colchicine

Remarks:

A stock solution of 1000 µg/mL of Colchicine was prepared (2 mg Colchicine in 2 mL DMSOand diluted to 1 µg/mL (10 µL in 9.99 ml of DMSO). 100 µL was dispensed into respective tubes to achieve a target concentration of 0.01 μg/mL Colchicine (Group 6).

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 3, Experiment 1 (Presence of Metabolic Activation with 3-hour Exposure), Experiment 2 (Absence of Metabolic Activation with 3-hour Exposure) and Experiment 3 (Absence of Metabolic Activation with 24-hour Exposure).
- A stock solution of 114200 μg/mL was prepared by mixing 1142 mg of the test item in sterile water and making up the volume to 10 mL with sterile water in a volumetric flask. The above stock was further diluted in sterile water as follows to prepare the required concentrations of the test item and 100 μL was added to blood cultures in a 10 mL volume for the following final exposure amounts:
*Group 4; Test item stock/dilution: 100 μL stock; Volume of vehicle: 0 mL sterile water ; Test item conc/ mL of vehicle:114200 μg; Final test conc/culture: 11420 μg; Test item conc: 1142 μg/mL medium
*Group 3; Test item stock/dilution: 1 mL of stock; Volume of vehicle: 2 mL sterile water; Test item conc/ mL of vehicle: ~38100 μg; Final test conc/culture: 3810 μg; Test item conc: 381 μg/mL medium
*Group 2; Test item stock/dilution: 1 mL of dilution B; Volume of vehicle: 2mL sterile water; Test item conc/ mL of vehicle: ~12700 μg; Final test conc/culture: 1270 μg; Test item conc: 127 μg/mL medium

52 μL 1N HCl added to the tubes of G4 (the adjusted pH was 7.14, 7.19 and 7.21 for experiments 1, 2 and 3, respectively).

METHOD OF TREATMENT/ EXPOSURE:
- Initial pH and osmolality determination: 50 μL of either vehicle or the stock/dilution of the test item was transferred to two sets of tubes containing 4.75 mL of treatment medium pre-labeled for the presence and absence of metabolic activation.
- For the test in the presence of metabolic activation, 250 μL of S9 mix was added to each tube to achieve a concentration of 2% (v/v) of S9.
- For the test in the absence of metabolic activation, 250 μL of 150 mM KCl was added to each tube. The tube contents were mixed and the pH (vehicle and all the test concentrations) and osmolality of the required test item treatment conditions in test solution were determined. The pH of the test solutions of 571 and 1142 μg/mL was adjusted with 1N HCl. After 3-hour exposure, the first and second set of tubes were observed for any precipitation. The exposed test solution of the vehicle control and the test concentrations were centrifuged and transferred to pre-labeled tubes and the pH and osmolality were determined.
- Target blood cultures in a single replicate were exposed to required concentrations of the test item and the sterile water control as follows: Ten microliters (10 μL) of Cytochalasin B (6000 μg/mL) were added to all the tubes.
- For the experiment in the presence of metabolic activation: 500 μL of S9 mix was added to the respective target blood cultures (set 1) to achieve a final concentration of 2% S9 (v/v) in the test solution. Similarly, for the experiments in the absence of metabolic activation, 500 μL of 150 mM KCl was added to the respective target blood cultures (set 2 and 3). One hundred microliters (100 μL) of either vehicle or the required dilutions/stock of the test item were mixed with the medium in the respective blood cultures to achieve the required test concentrations as well as the vehicle control. The first and second set of blood cultures (with and without metabolic activation) were incubated for 3 hours, whereas, the third set of blood cultures were incubated for 24 hours to expose the cells to treatment. After the 3-hour exposure, the cultures in the vehicle control and treatment groups (with and without metabolic activation) tubes were centrifuged and the supernatant discarded. The cell pellets were washed twice with PBS, resuspended in fresh RPMI FBS20, PHA and Cytochalasin B and incubated for approximately 24 hours from the start of treatment. Following the respective incubation periods, cells (sets 1, 2 and 3) were pelleted by centrifugation. The supernatant was removed and the cell pellet was suspended in KCl and further processed. Two slides were made from each culture, stained with acridine orange and scored for mitotic index.
- Test substance added in medium; in suspension
- Exposure of Target Cells to Treatment; On the day of treatment, all test doses were prepared immediately before use and mixed with the test solution in the culture tubes. For the experiment in the presence of metabolic activation, 0.5 mL S9 mix was added to the respective culture tubes to achieve a final concentration of 2% S9 (v/v) in the test solution. Similarly, for experiments in the absence of metabolic activation, 0.5 mL of 150 mM KCl was added to the respective culture tubes. Cytochalasin B was added to all culture tubes to achieve a final concentration of 6 μg/mL. The target cells were exposed to the controls and the test item concentrations as follows:
* In Experiment 1, the target cells were exposed to three concentrations of the test item, the vehicle control and the positive control for 3-hours in the presence of metabolic activation.
* In Experiment 2, the target cells were exposed to three concentrations of the test item and the vehicle control for 3-hours in the absence of metabolic activation.
* In Experiments 3, the target cells were exposed to three concentrations of the test item, the vehicle control and the positive controls for 24-hours in the absence of metabolic activation.
One hundred microliters (100 μL) of the vehicle or corresponding stock solutions of the positive controls or the different dilutions of the test item were added separately to the respective pre-labelled tubes containing cell cultures to achieve the desired test concentrations and mixed well. These tubes were then placed on a tissue culture rotator inside a CO2 incubator at 37 ± 1 °C for 3 hours for experiments 1 and 2 and for 24 hours for experiment 3. After 3 hours exposure, the culture tubes from experiments 1 and 2 were centrifuged at 1000 rpm for 5 min. The supernatant was discarded and the cell pellets washed twice with PBS. The cell pellets were resuspended in fresh RPMI FBS20, PHA and Cytochalasin B and incubated for approximately 24 hours from the start of treatment.

TREATMENT AND HARVEST SCHEDULE:
- Harvest time after the end of treatment: Each culture from the controls and treatment groups was harvested at approximately 24 hours after the beginning of the treatment and processed separately. Cells were harvested by centrifugation at approximately 1000 rpm for 5 minutes. The supernatant was carefully removed and cell pellets were re-suspended in warm 0.56 % KCl for 15 minutes at room temperature to allow cell swelling to occur and then centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded and the cell pellets were gently resuspended in freshly prepared cold acetic acid: methanol fixative (1: 3) and incubated for 10 minutes at room temperature. The cell suspension was then centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded and the pellets were resuspended in a fresh aliquot of cold fixative. The tubes were then stored in the refrigerator overnight prior to slide preparation to ensure adequate fixation. At the end of the refrigeration period, the tubes were removed and the cell suspension was centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded and the cell pellet re-suspended in freshly prepared cold fixative at room temperature for 10 minutes. The above procedure was repeated and the cell pellet was re-suspended in minimum quantity of fresh cold fixative to produce a milky suspension and maintained at room temperature for 10 minutes, prior to preparing the slides. Several drops of the cell suspension were transferred onto clean glass slides and flame dried. Four slides per replicate were prepared and the slides were marked with the study number, respective code, presence or absence of metabolic activation, culture number, experiment number and replicate number. The slides were coded by an individual not involved in the scoring process. The slides were placed in the stain for approximately 7 minutes, rinsed briefly in PBS twice and then mounted with PBS and a coverslip for immediate viewing.


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- The frequency of lymphocytes containing one, two, or more than two nuclei in 1000 cells was assessed to determine the test item effect on cell cycle kinetics for all the treatment groups and the respective vehicle and positive controls.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- The micronuclei were counted in cells showing a clearly visible cytoplasm area. The frequency of micronuclei in 2000 bi-nucleated cells per concentration (1000 bi-nucleated cells per culture) were analyzed. After the completion of evaluation, a person other than the evaluator of that particular set decoded all the evaluation formats.

Rationale for test conditions:

Human blood cultures were established and used as the test system.

Testing approaches currently accepted under the OECD guidance for the assessment of mammalian cell clastogenicity include the use of human peripheral blood lymphocytes to assess the ability of a chemical substance to induce micronuclei under experimental conditions.
Human peripheral blood lymphocytes are useful in in vitro cytogenetics testing because of the easy availability of large numbers of human cells, their distribution throughout the body, circulation in all the tissues and a proportion of them are long-lived and they maintain a stable karyotype.

These cells are easy to culture and a proportion of the lymphocytes can be stimulated by mitogens to undergo mitosis in culture thus providing a population of dividing cells which is required for this test.

Evaluation criteria:

When all the validity criteria are fulfilled:

a. A test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
• At least one of the test concentrations exhibits a statistically significant increase in micronuclei compared with the concurrent vehicle control
• The increase is dose-dependent in at least one experimental condition when evaluated with an appropriate trend test
• Any of the results are outside the distribution of the historical vehicle control data
• When all of these criteria are met, the test chemical is then considered able to induce chromosome breaks and / or gain or loss in this test system.
b. A test chemical is considered to be clearly negative if, in all experimental conditions examined:
• None of the test concentrations exhibits a statistically significant increase in number of micronuclei compared with the concurrent vehicle control
• There is no concentration-related increase when evaluated with an appropriate trend test
• All results are inside the distribution of the historical vehicle control data
• The test chemical is then considered unable to induce chromosome breaks and/ or gain or loss in this test system.

Statistics:

Statistical analysis of the experimental data was carried out using validated SYSTAT Statistical package ver.12.0. Statistical significance was confirmed by Chi square test. All analysis and comparisons were evaluated at 5 % (p < 0.05) level.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

lymphocytes: human (3h exposure)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

34% cytostatis

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

lymphocytes: human (3h exposure)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

29% cytostatis

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

lymphocytes: human (24h exposure)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

38% cytostatis

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- S9 Characterization:
* Sterility Check: The S9 homogenate was found to be sterile.
* Metabolic Activation: The S9 homogenate was found to be active as evidenced by its ability to metabolize the promutagens, 2-Aminoanthacene and Benzo(a) pyrene to mutagens using S. typhimurium strain TA 100.
* Protein Content: The protein content of the S9 homogenate used for the preliminary study was 31.931 mg/mL and the protein content of the S9 homogenate used for the definitive assay was 32.384 mg/mL.

- Solubility of test item and justification for the selection of vehicle: The test item was soluble in sterile water (SW) at 114.2 mg/ mL. Sterile water was used as the vehicle. Sterile water is one of the solvents/vehicles compatible with this test system.

- Preliminary Cytotoxicity Test: The pH of the test solutions at 571 μg/mL and 1142 μg/mL were adjusted prior to the exposure. At the beginning and end of 3-hour exposure and in the presence and absence of metabolic activation, there was no precipitation of the test item at any of the tested concentrations. In the presence of metabolic activation, at the end of 3-hour exposure to treatment, the pH of the test medium was between 7.15 and 7.29 with 7.05 in the sterile water control, whereas in the absence of metabolic activation, the pH ranged between 7.14 and 7.29 with 7.09 in the sterile water control. At the end of 3-hour exposure to treatment, the osmolality of the test medium at the highest soluble test concentration, which was also the highest concentration (1142 μg/mL) was 329 and 327 mOSMOL/kg in the presence and absence of metabolic activation, respectively. The corresponding osmolality in the respective sterile water control was 314 and 312 mOSMOL/kg in the presence and absence of metabolic activation.

STUDY RESULTS
- Experiment I; Presence of Metabolic Activation with 3-hour Exposure:
* Cytotoxicity: In the assay carried out in the presence of metabolic activation with a 3-hour treatment, the highest concentration tested (1142 μg/mL) resulted in a reduction in CBPI compared with vehicle control values equivalent to 35 %.
* Micronucleus Analysis:
> The incidence of micronuclei in the binucleated sterile water control cells was comparable to the in-house historical control data.
> In the definitive test Experiment 1, the test item did not cause any statistically significant increase in the number of bi-nucleated cells containing micronuclei compared with the vehicle control at any of the test concentrations.
> The positive control, Cyclophosphamide caused a statistically significant (p < 0.05) increase in the number of bi-nucleate cells containing micronuclei, demonstrating the efficacy of the S9 mix and the sensitivity of the test system.

- Experiment 2; Absence of Metabolic Activation with 3-hour Exposure:
* Cytotoxicity: In this assay, carried out in the absence of metabolic activation with a 3-hour treatment, the highest concentration tested (1142 μg/mL) resulted in a reduction in CBPI compared with vehicle control values equivalent to 29 %.
* Micronucleus Analysis:
> The incidence of micronuclei in the binucleated sterile water control cells was comparable to the in-house historical control data.
> The test item did not cause any statistically significant increase in the number of bi-nucleate cells containing micronuclei compared with the vehicle control at any of the test concentrations.

- Experiment 3; Absence of Metabolic Activation with 24-hour Exposure:
* Cytotoxicity: In the assay carried out in the absence of metabolic activation with a 24-hour treatment, the highest concentration tested (1142 μg/mL) resulted in a reduction in CBPI compared with vehicle control values equivalent to 39 %.
* Micronucleus Analysis:
> The incidence of micronuclei in the binucleated sterile water control cells was comparable to the in-house historical control data
> The test item did not cause any statistically significant increase in the number of bi-nucleate cells containing micronuclei compared with the vehicle control at any of the test concentrations.
> The positive controls, Mitomycin C and colchicine caused a statistically significant (p < 0.05) increase in the number of bi-nucleate cells containing micronuclei, demonstrating the sensitivity of the test system.

No evidence of an increase in the number of binucleated cells containing micronuclei was obtained in any of the experiments at any test concentrations either in the presence or in the absence of metabolic activation. In each of these experiments, the respective positive controls produced a statistically significant increase in the number of binucleate cells containing micronuclei thereby demonstrating that the system was able to detect the effect of known clastogens.

Applicant's summary and conclusion

Conclusions:

It was concluded that the test item did not show any evidence of an increase in the number binucleated human lymphocyte cells containing micronuclei either in the presence or in the absence of a metabolic activation system at and up to the highest concentration of 1142 µg/mL and under the conditions of testing.