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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three in vitro reliable, key studies have been identified: one bacterial reverse mutation test, one micronucleus study and one mammalian cell gene mutation test, performed with the test substance dimethyl piperazine.



The bacterial reverse mutation assay was performed according to OECD Guideline 471 in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvrA (Divyashree, 2022). According to the results of the study, the test substance is not mutagenic in the Ames test with and without metabolic activation.



The mammalian cell gene mutation test was performed according to a method similar to OECD Guideline 476 in mouse lymphoma L5178Y cells (Myhr, 1980). The results of the test indicate that, under the conditions of this study, the test substance is negative with and without metabolic activation.


 


The micronucleus study was performed according to the OECD guideline 487 (Indrani, 2022). 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.

Link to relevant study records

Referenceopen allclose all

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
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
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.
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
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)
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).
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.
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
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
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.
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.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2022-01-27 to 2022-02-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
26 June 2020
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch number of test material: PFW210769
- CAS number: 106-58-1
- Chemical name: 1,4-dimethylpiperazine
- Physical Appearance : Liquid
- Retest Date : 2023-11-25
- Purity: 99.91 A%. The 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 specified
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: not specified
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: The test item was soluble in sterile water at 50 mg/mL

OTHER SPECIFICS
- pH : 11.4
- amine content: 17.36 meq/g
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9: male Wistar rats livers
- method of preparation of S9 mix: The S9 homogenate was prepared from male Wistar rats induced with a single intra-peritoneal injection of Aroclor 1254 (0.7 mL/rat ready to use solution), 5 days prior to sacrifice. The S9 homogenate was prepared in batches and stored in a deep freezer maintained at -68 to -86 ºC. S9 homogenate was thawed immediately before use and mixed with the cofactor solution containing 4 mM NADP, 5 mM Glucose-6-phosphate, 8 mM MgCl2 and 33 mM KCl in PBS. The co-factors solution was prepared by dissolving the following in 9 and 50 mL of cold PBS for the preliminary toxicity and mutation assay respectively. This solution was filter sterilized using a 0.2 μ disposable syringe filter:
*NADP (4 mM): 28.35 mg (Preliminary toxicity), 157.47 mg (Mutation assay)
*Glucose-6-phosphate (5 mM): 15.31mg (Preliminary toxicity), 85.03 mg (Mutation assay)
*Magnesium chloride (8 mM): 14.64mg (Preliminary toxicity), 81.32 mg (Mutation assay)
*Potassium chloride (33 mM): 22.14mg (Preliminary toxicity), 123.01 mg (Mutation assay)
S9 mix was prepared by mixing 1 and 5.5 mL of the S9 homogenate with 9 and 49.5 mL of the co-factors solution for the preliminary toxicity and mutation assay respectively, 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 (for the test in the presence of metabolic activation).
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): not specified
Test concentrations with justification for top dose:
Preliminary Toxicity Test for Selection of Test Doses:
- Tester Strain Culture: A loopful of the tester strain, TA 100 was inoculated into a tube containing Oxoid Nutrient broth No. 2 and the tube was incubated at 37 ± 1 ºC for 17 hours.
- Test Doses: (a) 50, (b) 100, (c) 200, (d) 400, (e) 800, (f) 1600, (g) 3200 and (h) 5000 μg/plate.
- Based on these observations, it was decided to test the OECD 471-recommended top dose of 5000 μg/plate in the mutation assay.

Mutation Assay
- Test Strain Cultures: Each of the tester strains from the master plates were inoculated into the respective tubes containing Oxoid Nutrient broth No. 2 and the tubes were incubated at 37 ± 1 °C for approximately 17 hours.
- Test Doses: Based on the absence of toxicity and precipitation in the preliminary toxicity test, the following five test doses were selected (with approximately half-log dose interval) for testing in the mutation assay: (a) 50, (b) 158, (c) 500, (d) 1581 and (e) 5000 μg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Sterile water (SW)
- Justification for choice of solvent/vehicle: SW is one of the vehicle compatible with this test system. Hence, based on the results of solubility test, SW is selected as the vehicle for the mutation assay.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
SW
True negative controls:
no
Positive controls:
yes
Remarks:
dissolved in DMSO
Positive control substance:
other: 2-Aminoanthracene
Remarks:
With S9; 4 µg/plate With TA98, TA100, TA1535, TA1537 and 30 µg/plate with WP2uvrA (pKM101)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
SW
True negative controls:
no
Positive controls:
yes
Remarks:
dissolved in DMSO
Positive control substance:
sodium azide
Remarks:
Without S9; 1 µg/plate With TA100 and TA1535
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
SW
True negative controls:
no
Positive controls:
yes
Remarks:
dissolved in DMSO
Positive control substance:
2-nitrofluorene
Remarks:
Without S9; 2 µg/plate With TA98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
SW
True negative controls:
no
Positive controls:
yes
Remarks:
dissolved in DMSO
Positive control substance:
9-aminoacridine
Remarks:
Without S9; 50 µg/plate With TA1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
SW
True negative controls:
no
Positive controls:
yes
Remarks:
dissolved in DMSO
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Without S9; 4 µg/plate With WP2uvrA (pKM101)
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Three replicate plates were maintained for the initial as well as the confirmatory mutation assays.
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation) for the initial mutation assay, pre-incubation for the confirmatory mutation assay
- Plating procedure for Preliminary Toxicity Test: One hundred microlitres (100 μL) each of the respective dilutions and the stock solution equivalent to above mentioned test doses was mixed with 2 mL of soft agar containing histidine and biotin, 500 μL of S9 mix (for the test in the presence of metabolic activation) or 500 μL of PBS (for the test in the absence of metabolic activation), 100 μL of overnight TA100 culture and overlaid onto pre-labeled VB agar plates in duplicate. A SW control, similarly treated, was maintained. After the agar had set, these plates were incubated at 37 ± 1 °C for approximately 68 hours.
- Test Item Stock and Dilutions: For both the initial and confirmatory mutation assays, a stock solution of 50000 μg/mL was prepared by mixing 500 mg test item in SW and making up the volume to 10 mL in a volumetric flask with SW. The above stock was further diluted in SW as follows to prepare 5 concentrations of the test item and 100 μL was added to cultures for the following final exposure amounts:
*Group NA: 1 mL stock (Test item stock or dilution), 4 mL (vehicle (SW)), 10000 μg (Test item concentration/mL of vehicle), 0 µg (Final test dose/plate)
*Group 2: 1 mL A (Test item stock or dilution), 19 mL (Volume of vehicle (SW)), 500 μg (Test item concentration/mL of vehicle), 50 μg (Final test dose/plate)
*Group 3: 0.79 mL A (Test item stock or dilution), 4.21 mL (vehicle (SW)), 1580 μg (Test item concentration/mL of vehicle), 158 μg (Final test dose/plate)
*Group 4: 2.5 mL A (Test item stock or dilution), 2.5 mL (vehicle (SW)), 5000 μg (Test item concentration/mL of vehicle), 500 μg (Final test dose/plate)
*Group 5: 1.581 mL stock (Test item stock or dilution), 3.419 mL (vehicle (SW)), 15810 μg (Test item concentration/mL of vehicle), 1581 μg (Final test dose/plate)
*Group 6: 100 μL stock (Test item stock or dilution), no vehicle (SW), 50000 μg (Test item concentration/mL of vehicle), 5000 μg (Final test dose/plate)
- Plating Procedure:
Initial mutation assay was conducted using the direct plate incorporation method as follows:
A. Presence of Metabolic Activation
a) 2.0 mL soft agar containing histidine-biotin/tryptophan
b) 100 μL test dose/vehicle/appropriate positive control
c) 100 μL bacterial culture
d) 500 μL S9 mix
B. Absence of Metabolic Activation
a) 2.0 mL soft agar containing histidine-biotin/tryptophan
b) 100 μL test dose /vehicle/appropriate positive control
c) 100 μL bacterial culture
d) 500 μL of PBS
These test constituents were transferred into sterile test tubes, mixed and overlaid onto VB agar plates and allowed to set. The plates were then incubated at 37 ± 1 °C for 67 hours. Revertant colonies were counted manually and the plates were examined for bacterial background lawn.

The confirmatory mutation assay was conducted using the pre-incubation method as follows:
A. Presence of Metabolic Activation
a) 100 μL test dose /vehicle/appropriate positive control
b) 100 μL bacterial culture
c) 500 μL S9 mix
B. Absence of Metabolic Activation
a) 100 μL test dose/vehicle/appropriate positive control
b) 100 μL bacterial culture
c) 500 μL PBS

These test constituents were transferred into sterile test tubes and kept in an incubator shaker for 20 minutes at 37ºC. After this period, 2 mL soft agar containing histidine-biotin / tryptophan were added to each of the tubes. The tube contents were mixed and overlaid onto VB agar plates and allowed to set. The plates were then incubated at 37 ± 1 °C for 67 hours.
Revertant colonies were counted manually and the plates were examined for bacterial background lawn.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition; decrease in number of revertants

METHODS FOR MEASUREMENTS OF GENOTOXICIY
The bacterial suspension of each tester strain was diluted up to 10-6 dilution in PBS. One hundred microlitres from the 10-6 dilution of each tester strain was mixed with 2mL of soft agar and plated onto nutrient agar plates in triplicate. The plates were incubated at 37 ± 1 °C for 67 hours for the initial as well as the confirmatory mutation assays. After incubation, the number of colonies in each plate were manually counted and expressed as the number of colony forming units per mL of the bacterial suspension.
Rationale for test conditions:
Each S. typhimurium tester strain contains, in addition to a mutation in the histidine operon, additional mutations that enhance sensitivity to some mutagens. The rfa mutation results in a cell wall deficiency that increases the permeability of the cell to certain classes of chemicals such as those containing large ring systems that would otherwise be excluded. The deletion in the uvrB gene results in a deficient DNA excision-repair system. Tester strains TA98 and TA100 also contain the pKM101 plasmid (carrying the R-factor). It has been suggested that the plasmid increases sensitivity to mutagens by modifying an existing bacterial DNA repair polymerase complex involved with the mismatch-repair process. TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. TA100 is reverted by both frameshift and base substitution mutagens and TA1535 is reverted only by mutagens that cause base substitutions. The E. coli tester strain has an AT base pair at the critical mutation site within the trpE gene (Wilcox et al., 1990). Tester strain WP2uvrA (pKM101) has a deletion in the uvrA gene resulting in a deficient DNA excision-repair system. Tryptophan revertants can arise due to a base change at the originally mutated site or by a base change elsewhere in the chromosome causing the original mutation to be suppressed. Thus, the specificity of the reversion mechanism is sensitive to base-pair substitution mutations (Green and Muriel, 1976).
Evaluation criteria:
To determine a positive result, there should be a dose related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of the test item either in the presence or absence of the metabolic activation system. The test will be judged positive, if the increase in mean revertants at the peak of the dose response is equal to or greater than 2 times the mean vehicle control value for strains TA98, TA100 and WP2uvrA (pKM101) or equal to or greater than 3 times the mean vehicle control value for strains TA1535 and TA1537. An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response will be evaluated as negative, if it is neither positive nor equivocal.
Statistics:
no statistics available
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
- The test item did not cause precipitation on the basal agar plates at any of the tested doses.
- The test item did not show toxicity to the tester strain at any of the tested doses as the intensity of the bacterial background lawn as well as the mean number of revertant colonies were comparable to the vehicle control plates, both in the presence and absence of metabolic activation.

STUDY RESULTS
Viable Counts of the Overnight Tester Strains:
- Viable counts of all the tester strains were within the required range of 1-2 x 10^9 CFU/mL for the initial toxicity-mutation as well as the confirmatory mutation assay.

Initial Mutation Assay – plate incorporation assay:
- The mean numbers of revertant colonies/plate in the sterile water control was comparable to the in-house spontaneous revertant counts for all the tester strains.
- The test item did not cause precipitation on the basal agar plates at any of the tested doses.
- The test item did not show toxicity to any of the tester strains up to the top dose of 5000 μg/plate, as the intensity of the bacterial background lawn and the mean number of revertant colonies was comparable to the vehicle control plates both in the presence and absence of metabolic activation. The tested doses showed no positive mutagenic increase in the mean number of revertant colonies for any of the tester strains when compared to the respective vehicle control plates, either in the presence or absence of metabolic activation.
- Positive control chemicals tested simultaneously produced a more than 3-fold increase in the mean numbers of revertant colonies for all the strains when compared to the respective vehicle control plates. No toxicity was observed in the positive controls as the intensity of the bacterial background lawn of all the tester strains was comparable to that of the respective vehicle control plates.

Confirmatory Mutation Assay– pre-incubation assay:
- The mean numbers of revertant colonies/plate in the sterile water control was comparable to the in-house spontaneous revertant counts for all the tester strains.
- The test item did not cause precipitation on the basal agar plates at any of the tested doses.
- The test item did not show toxicity to any of the tester strains up to the top dose of 5000 μg/plate, as the intensity of the bacterial background lawn and the mean number of revertant colonies was comparable to the vehicle control plates both in the presence and absence of metabolic activation. The tested doses showed no positive mutagenic increase in the mean number of revertant colonies for any of the tester strains when compared to the respective vehicle control plates, either in the presence or absence of metabolic activation.
- Positive control chemicals tested simultaneously produced a more than 3-fold increase in the mean numbers of revertant colonies for all the strains when compared to the respective vehicle control plates. No toxicity was observed in the positive controls as the intensity of the bacterial background lawn of all the tester strains was comparable to that of the respective vehicle control plates.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data:
- Negative (solvent/vehicle) historical control data:

DISCUSSION
Both the Salmonella typhimurium and Escherichia coli tester strains were found to be reliable and responsive to the different genotypic characterization tests like the amino acid requirement, rfa mutation, uvr mutation and the R-factor plasmids. Similarly, the spontaneous revertant counts of the vehicle control groups of these tester strains were in the ranges of the test facility’s
historical control data. The positive controls produced a more than 3-fold increase in the mean numbers of revertant colonies when compared to the respective vehicle controls, demonstrating the sensitivity of the assay procedure. The test item at doses up to 5000 μg/plate did not cause an increase in the mean numbers of revertant colonies in the strains TA98, TA100, WP2uvrA (pKM101), TA1535 and TA1537 either in the presence or absence of the metabolic activation system when compared to the respective vehicle control plates.

Genotypic Characterization
Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 demonstrated the requirement of histidine amino acid for their growth. Escherichia coli strain WP2uvrA (pKM101) demonstrated the requirement of tryptophan amino acid for its growth. Ampicillin resistance was demonstrated by the strains TA98, TA100 and WP2uvrA (pKM101) which carry R-factor plasmids. The presence of characteristic mutations like the rfa mutation was demonstrated by all the Salmonella typhimurium strains by their sensitivity to crystal violet. The uvrA mutation in the Escherichia coli strain and the uvrB mutation in the Salmonella typhimurium strains were demonstrated through their sensitivity to ultraviolet light. Finally, all these tester strains produced spontaneous revertant colonies which were within the frequency ranges of the test facility’s historical control data.

Conclusions:
It is concluded that the test item, was not mutagenic in this bacterial reverse mutation test up to the OECD 471-recommended top dose of 5000 μg/plate under the conditions of testing employed.
Endpoint:
in vitro transformation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1980-09-17 - 1980-11-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.21 (In Vitro Mammalian Cell Transformation Test)
Deviations:
no
GLP compliance:
not specified
Type of assay:
in vitro mammalian cell transformation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): 4236-30-10
- Physical state: liquid
Species / strain / cell type:
mammalian cell line, other: BALB/3T3 mouse cells
Details on mammalian cell type (if applicable):
- Type and identity of media: Eagle's Minimum Essential Medium (EMEM) supplemented with 10 % fetal bovine serum
- Periodically checked for Mycoplasma contamination: yes


Additional strain / cell type characteristics:
other: selected for low spontaneous frequencies of foci formation
Metabolic activation:
not specified
Test concentrations with justification for top dose:
0.01, 0.3, 3.0, 150.0, 300.0 nl/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: none (culture medium)
Untreated negative controls:
yes
Remarks:
culture medium
Negative solvent / vehicle controls:
other: not applicable
True negative controls:
no
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
Remarks:
5 µg/ml
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24h
- Exposure duration: 3d
- Expression time (cells in growth medium): 4 weeks (refeeding twice a week)
- Fixing the cell monolayers with methanol
- Staining with Giemsa

NUMBER OF REPLICATIONS: 15 dishes per dose, 15 for negative control, 15 for positive control

Examined by eye and by microscope to determine the number of foci of transformed cells.
Evaluation criteria:
- negative control flasks consist of a contiguous monolayer of cells which may or may not contain transformed foci. The lack of contiguous sheet of cells indicates growth conditions too poor to allow the reliable detection of weak transforming agents.
- the negative control transformation frequency does not exceed an average of 2-3 foci/flask. Attempts are made to isolate and maintain cell stocks (subclones of Balb/3T3 I13) with a very low spontaneous frequency of transformation.
- positive control yields an average number of foci/flask that is signicficantly different from the negative control at the 99% CL.
- a minimum of 8 flasks per test condition are available for analysis. At least 4 dose levels of test substance are assayed.
- the dose range of test substance assayed falls within the 50-100% survival range as determined by the preliminary toxicity test, which measures relative cloning efficiencies.
Statistics:
The statistical tables provided by Kastenbaum and Bowman are used to determine whether the results at each dose level are significantly different from the historical control at the 95% or 99% confidence levels (Kastenbaum and Bowman (1970) Tables for determining the statistical significance of mutation frequencies. Mut. Res. 9, 527-549).
The test compares variables distributed according to Poissonian expectations by summing the probabilities in the tails of two binomial distributions. The 95% confidence level must be met to consider the test substance active at a particular dose level.
Key result
Species / strain:
mammalian cell line, other: BALB/3T3 cells
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at concentrations of 1000 nl/ml and higher
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
Fifteen dose levels of the test compound are chosen (max 1000 nl/ml - min 0.061 nl/ml)(decreasing in two-fold dilution steps).
Each dose is applied to 3 culture dishes seeded 24h earlier with 200 cells per dish. After an exposure period of 3 days, the cells are washed and incubated in growth medium for an additional 4 days.
Surviving colonies are stained and counted. A relative survival for each dose is obtained by comparing the number of colonies surviving treatment to the colony counts in negative control dishes.
The highest dose chosen for subsequent transformation assays should normally cause no more than an 50% reduction in colony forming ability and is best located near 30 % reduction. Four lower doses (including 1 or 2 doses with low or no apparent toxicity) are also selected for the transformation assay.

COMPARISON WITH HISTORICAL CONTROL DATA:
The historical negative control for the subclone of 3T3 cells used in this assay consists of 147 flasks containing a total of 17 transformed foci for an average of 0.12 foci / flask. In this assay, one transformed focus was observed among the 15 negative control flasks for an average of 0.07 foci/flask. Sets of 15 flasks containing one focus are included in the historical database, so the effect of the test material could be evaluated by comparison with the historical spontaneous transformation frequency.
Remarks on result:
other: all strains/cell types tested
Conclusions:
Concentrations of the test material from 0.01 nl/ml to 300 nl/ml were not detectably transforming 3T3 cells. The positive control results showed that the sensitivity of the assay was normal. The test material is considered to be inactive in the Balb/3T3 in vitro transformation assay.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2006-11-1 - 2006-11-22
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
Although sufficient strains and doses are tested in presence and absence of metabolic activation and positive control substances were valid, it is difficult to judge the reliability based on a report in Japanese. Therefore a klimisch score of 4 was assigned.
Qualifier:
according to guideline
Guideline:
other: ISHL methods= CSCL methods (no OECD method)
Deviations:
no
GLP compliance:
no
Remarks:
CSCL & MHLW
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): N,N'-dimethylpiperazine
- Analytical purity: 100 wt%
- Lot/batch No.: PFW050197
Target gene:
Histidine locus (Salmonella)
Tryptophan locus (E. coli)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
- S9 mix: 313, 625, 1250, 2500, 5000 µg/plate
+ S9 mix: 313, 625, 1250, 2500, 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
Remarks:
0.01 µg/plate for TA100 and WP2uvrA, without S9; 0.1 µg/plate for TA98 without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
0.5 µg/plate for TA1535 without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ICR-191
Remarks:
1.0 µg/plate for TA1537 without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
5.0 µg/plate for TA100, TA98 and TA1537 with S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
2 µg/plate for TA1535 and 10 µg/plate for WP2uvrA with metabolic activation
Details on test system and experimental conditions:
No data
Evaluation criteria:
No data
Statistics:
No data
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No increase in colonies equal to or more than twice negative control at all doses with and without S9.
Conclusions:
The substance was not considered to be mugatenic in the Ames test under the conditions of the current test with and without metabolic activation.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1980-09-08 to 1980-09-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): 4236-30-10
- Substance type: Clear, light-yellow liquid
- Physical state: liquid
Target gene:
thymidine kinase locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media:
* maintained in Fischer's mouse leukemia medium suplemented with L-glutamine, sodium pyruvate and horse serum (10% by volume).
* cloning medium: preceding growth medium with the addition of agar to a final concentration of 0.35% to achieve a semisolid state.
* selection medium: cloning medium containing 100 µg/ml of BrdU or 3 µg/ml of TFT.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9
Test concentrations with justification for top dose:
93.8, 188.0, 375.0, 750.0, 1500.0 nl/ml
Vehicle / solvent:
vehicle(s)/solvent(s) used: water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
0.5 µl/ml without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-dimethylnitrosamine
Remarks:
0.3 µl/ml with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 2 or 3 days
- Selection time (if incubation with a selection agent): 10 days

SELECTION AGENT (mutation assays): 5-bromo-2'deoxyuridine or 5-trifluorothymidine

NUMBER OF REPLICATIONS: 3 selection plates per test concentration

NUMBER OF CELLS EVALUATED: mutant frequency is calculated as the ratio of mutant colonies to viable colonies times 1E-04.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
The minimum condition for mutagenesis is a mutant frequency that exceeds 150% of the concurrent background frequency by at least 10 x 1E-06. The background frequency is defined as the average mutant frequency of the solvent and untreated negative controls. The observation of a mutant frequency that meets the minimum criterion for a single treated culture within a range of assayed concentrations is not sufficient evidence to evaluate a test material as a mutagen. The following test results must be obtained to reach this conclusion for either activation/nonactivation conditions:
- Dose-related or toxicity-related increase in mutant frequency: it is desirable to obtain this relation for at least 3 doses.
- Increase in mutant frequency may be followed by only small or no further increases at higher concentrations or toxicities. However, a decrease in mutant frequency to values below the minimum criterion is not acceptable in a single assay. If the mutagenic activity at lower concentrations or toxicities was large, a repeat assay will be performed to confirm the mutagenic activity.
- If an increase of about 2 times the minimum criterion or greater is observed for a single dose near the highest testable toxicity, the test material will be considered mutagenic. Smaller increases at a single dose near the highest testable toxicity will require confirmation by a repeat assay.
- A negative correlation with dose is acceptable only if a positive correlation with toxicity exists. An apparent increase in mutagenic activity as a function of decreasing toxicity is not acceptable evidence for mutagenicity.

A test material will be evaluated as nonmutagenic in a single assay only if the minimum increase in mutant frequency is not observed for a range of applied concentrations that extends to toxicity causing 5% to 10% relative suspension growth.
If a repeat assay does not confirm an earlier, minimal response, the test material will be evaluated as nonmutagenic in this assay system.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Range of toxicities from moderate at 93.8 and 188 nL/mL (approximately 50% relative growth) to high at 1500 nl/mL (13.9% relative growth). Treatment with 3000 nL/mL was completely lethal to the cells
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Range of toxicities: no observable effect at 188 nL/mL to moderate toxicity at 1500 nL/mL. Treatment with 3000 nL/mL: completely lethal. Reduction in toxicity in 93.8 to 1500 nL/mL concentration range: possibility of interaction with activation system
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
In the preliminary cytotoxicity test, the 24-hour cell growth was reduced by exposures to concentrations of test material up to 1250 nl/ml. Treatment with 2500 nl/ml was almost completely lethal. Therefore, the mutation assay was initiated with a series of concentrations from 3000 nl/ml to 5.86 nl/ml in two-fold dilution steps in an effort to cover a wide range of toxic action.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Concentrations up to 1500 nL/mL became very toxic without metabolic activation and moderately toxic with rat liver S9 metabolic activation.
Treatment with 3000 nL/mL was lethal under both conditions.
Remarks on result:
other: all strains/cell types tested
Conclusions:
The test material did not induce significant changes in the mutant frequency at the TK locus in L5178Y mouse lymphoma cells.
Concentrations up to 1500 nl/ml were assayed and became very toxic without activation and moderately toxic with rat liver S9 metabolic activation. Treatment with 3000 nl/ml was lethal under both conditions. The test material was therefore considered to be inactive in the Mouse Lymphoma Forward Mutation Assay. According to the criteria laid down in the CLP Regulation, the test substance is not to be classified.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1996-01-19 - 1996-02-16
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Only two replicates per group were used, method for measuring toxicity was not specified, no historical control data provided, and the procedure (preincubation or plate incorporation) used for the preliminary reverse mutation test was not provided. However, all other parts of OECD guideline 471 were followed.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Only two replicates per group were used, method for measuring toxicity was not specified, no historical control data provided, and procedure (preincubation or plate incorporation) used for the preliminary reverse mutation test was not provided.
GLP compliance:
yes
Remarks:
Japanese Good Laboratory Practice
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): N,N'-dimethylpiperazine (DMP)
- Molecular weight (if other than submission substance): 114.19
- Physical state: colorless liquid
- Analytical purity: 99.0 % or more
- Impurities (identity and concentrations): water, 0.1 %
- Lot/batch No.: U3-1-1294
- Stability under test conditions: stable at room temperature; stability in solvent: stable in distilled water
- Storage condition of test material: at room temperature, shield under from light
Target gene:
Histidine locus (Salmonella strains) and tryptophan locus (E. coli strain)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: TA100 and TA1535 are base pair substitution detecting strains and TA98 and TA1537 are frameshift detecting strains
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
other: base pair substitution detecting strain
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and 5,6-benzoflavone induced rat liver S9
Test concentrations with justification for top dose:
313, 625, 1250, 2500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: the test substance was soluble in water at concentrations of 100 g/L or more. From these results, distilled water was used as the solvent for the test substance.
Untreated negative controls:
yes
Remarks:
distilled water
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide-used for strains TA100 and WP2 uvrA at 0.01 ug/plate and TA98 at 0.1 ug/plate
Remarks:
without activation
Untreated negative controls:
yes
Remarks:
distilled water
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without activation used for strain TA1535 at 0.5 ug/plate
Untreated negative controls:
yes
Remarks:
distilled water
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without activation used for strain TA1537 at 80 ug/ plate
Untreated negative controls:
yes
Remarks:
distilled water
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene-used for strain TA1535 at 2 ug/plate and WP2 uvrA at 10 ug/plate
Remarks:
with activation
Untreated negative controls:
yes
Remarks:
distilled water
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with activation used for strains TA100, TA98 and TA1537 at 5 ug/plate
Details on test system and experimental conditions:
METHOD OF APPLICATION: pre-incubation

DURATION
- Preincubation period: 30 minutes
- Exposure duration: 48 hours
- Selection time (if incubation with a selection agent): 48 hours (simultaneous with exposure)

SELECTION AGENT (mutation assays): L-histidine (Salmonella strains), L-tryptophan (E.coli strain)

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: cytotoxicity was determined but no information was given on how it was determined.
Evaluation criteria:
The number of revertant colonies at each dose was compared with that of the solvent control group for each bacterial strain with and without metabolic activation. The test substance was considered as positive when significant dose-related and more than two-fold increases in the number of revertant colonies were observed with sufficient reproducibility.
Statistics:
No statistical analysis was performed for evaluation of the test results.
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: A preliminary reverse mutation test was performed in each of the strains listed above with and without metabolic activation to determine the suitable dosage of the test substance to be applied in the reverse mutation test. The maximum dose level for the preliminary test was set at 5000 µg/plate and six dose levels (10, 50, 100, 500, 1000 and 5000 µg/plate) were employed.

Neither toxic effect nor more than two-fold increases in the number of revertant colonies were observed in any test substance treated groups with and without metabolic activation, while the dose related increase in the number of revertant colonies was observed in TA100 with and without metabolic activation. Considering these results, the maximum dose level for the reverse mutation test was set at 5000 µg/plate, and five different amounts of the test substance were prepared by serial dilution with a factor of 2 from the maximum dose.




Neither toxic nor more than two-fold increase in the number of revertant colonies were observed in any test substance treated groups with and without metabolic activation, while the dose related increase in the number of revertant colonies was observed in TA100 with and without metabolic activation. On the other hand, all of the positive controls produced more than two-fold increases in the number of the revertant colonies in comparison with that of the solvent control with all bacterial strains with and without metabolic activation. These results indicated that the test had been properly carried out.

Conclusions:
The test substance was evaluated for mutagenic potential in the reverse mutation test in the presence and absence of metabolic activation using S. typhimurium strains TA100, TA98, TA1535, TA1537 and E. coli strain WP2 uvrA. Based on the results of the assay, the test substance was considered to be negative under the test conditions employed up to a dose level of 5000 µg/plate. According to the criteria laid down in the CLP Regulation, the substance is considered not classified as mutagenic.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No in vivo genetic toxicity study has been conducted with the test substance. No positive result was obtained in the set of in vitro genotoxicity tests with this substance. Therefore, it is not necessary to perform an in vivo genetic toxicity test (column 2 adaptation, Annex IX, section 8.4).

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Genetic toxicity: in vitro


Three reliable in vitro studies were selected as key studies: a bacterial reverse mutation assay (Ames test), an in vitro micronucleus study, and an in vitro mammalian cell gene mutation test.


 


Bacterial reverse mutation assay (Ames test):


The results of a GLP compliant Ames Assay with the test substance conducted according to a method equivalent to OECD Guideline 471 (K1; Divyashree; 2022) were negative in strains TA1535, TA1537, TA98 and TA100 of Salmonella typhimurium and E. coli strain WP2 uvrA both with and without metabolic activation at following concentrations: 50, 158, 500, 1581 and 5000 μg/plate (plate incorporation for the initial mutation assay, and pre-incubation for the confirmatory assay). No cytotoxicity has been observed. The substance is considered not mutagenic.


 


In vitro mammalian cell gene mutation test:


In a study conducted according to a method equivalent to OECD Guideline 476, the test substance did not produce any statistically significant increases in the frequency of mutations of mouse lymphoma L5178Y cells at following concentrations: 93.8, 188.0, 375,0, 750.0 and 1500 nL/mL in tests with and without an S9 metabolic activation system (K1; Myhr, 1980). Concentrations up to 1500 nL/mL became very toxic without metabolic activation and moderately toxic with rat liver S9 metabolic activation. Treatment with 3000 nL/mL was lethal under both conditions. The test material was therefore considered to be inactive in the Mouse Lymphoma Forward Mutation Assay.


 


In vitro micronucleus test:


An GLP in vitro micronucleus test in human peripheral blood lymphocytes was conducted in accordance with OECD 487 (K1; Indrani; 2022). Cells were exposed to the following concentrations: 127, 381 and 1142 µg/mL, under the following conditions: 


- experiment 1: 3h- exposure with metabolic activation 


- experiment 2: 3h- exposure without metabolic activation 


- experiment 3: 24h- exposure without metabolic activation 


The frequencies of micronuclei from 2000 bi-nucleated cells per concentration (1000 bi-nucleated cells per culture) were analyzed. The data from the treatment groups were statistically compared with the vehicle control.
There was no evidence of statistically significant induction of micronuclei, either in the presence or in the absence of metabolic activation in any of the tested concentrations. In experiments 1 and 3, under identical conditions, the
respective positive control substances produced a large and statistically significant increase in micronuclei.
The study indicated that the test item does not have the potential to induce micronuclei in cultured human peripheral blood lymphocytes either in the presence or in the absence of metabolic activation at and up to the highest concentration tested, 1142 μg/mL (10mM) and under the conditions of testing. It can be concluded that the test item did not cause chromosomal damage in somatic cells.


 


 


A reliable, supporting cell transformation assay was also identified. This in vitro mammalian cell transformation assay in BALB/3T3 cells has been performed according to a method equivalent to EU Method B.21. Concentrations of the test material from 0.01 nL/mL to 300 nL/mL were not detectably transforming 3T3 cells. The positive control results showed that the sensitivity of the assay was normal. The test material is considered to be inactive in the Balb/3T3 in vitro transformation assay.


 


Genetic toxicity: in vivo


No in vivo genetic toxicity study has been conducted with the test substance. No positive result was obtained in the set of in vitro genotoxicity tests with this substance. Therefore, it is not necessary to perform an in vivo genetic toxicity test (column 2 adaptation, Annex IX, section 8.4).





Justification for classification or non-classification

Based on the available data from the test substance, and according to the criteria of the CLP Regulation (EC) 1272/2008, the test substance is considered not to be classified for mutagenicity.