N,N,4-trimethylpiperazine-1-ethylamine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-07-17 to 2018-02-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

26 September 2014

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

N,N,4-trimethylpiperazine-1-ethylamine

EC Number:

203-183-7

EC Name:

N,N,4-trimethylpiperazine-1-ethylamine

Cas Number:

104-19-8

Molecular formula:

C9H21N3

IUPAC Name:

dimethyl[2-(4-methylpiperazin-1-yl)ethyl]amine

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: PFW160141
- Expiration date of the lot/batch: no data
- Purity test date: no data
- Name of test material (as cited in study report): N,N,4-Trimethyl 1-Piperazineethanamine
- Lot/batch No.: PFW160141
- CAS No.: 4203-183-7
- Physical state: clear colorless liquid
- Analytical purity: 100% (per Protocol); 99.5% (per Certificate of Analysis)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room Temperature, protected from light
- Stability under test conditions: Not determined.
- Solubility and stability of the test substance in the solvent/vehicle: Not determined.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not determined.

Method

Target gene:

not applicable

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

mammalian cell line

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: American Type Culture Collection, Manassas, VA, repository number CCL 61.
- Suitability of cells: According to Guideline 473. In addtion, the use of CHO cells has been demonstrated to be an effective method of detection of chemical clastogens (Preston et al., 1981).
- Cell cycle length, doubling time or proliferation index: This cell line has an average cell cycle time of 10-14 hours.
- Number of passages if applicable: Working cell stocks were not used beyond passage 15
- Modal number of chromosomes: 20

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: McCoy's 5A medium
containing 10% fetal bovine serum, 1.5 mM L glutamine, 100 units/mL penicillin, 100 μg/mL s
treptomycin and 2.5 μg/mL Amphotericin B
- Periodically checked for Mycoplasma contamination: [yes/no] Yes, tested using the Hoechst staining procedure and found to be free of mycoplasma contamination
- Periodically checked for karyotype stability: [yes/no) No, however, in order to assure the karyotypic stability of the cell line, working cell stocks were not used beyond passage 15
- Periodically 'cleansed' against high spontaneous background: [yes/no] Not indicated

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

- Preliminary Toxicity Test A1: 0, 0.171, 0.513, 1.71, 5.13, 17.1, 51.3, 171, 513 and 1710 μg/mL (4-hour +/- S9 with 16 hour recovery period)
- Preliminary Toxicity Test A1: 0, 0.171, 0.513, 1.71, 5.13, 17.1, 51.3, 171, 513 and 1710 μg/mL (- S9 with 20 hour continuous treatment)

The results of a preliminary toxicity test were used to set the concentration range for the chromosome aberration test:
- Initial Group B1 (4-hour - S9 with 16 hour recovery time): 0, 100, 250, 500, 1000, 1650, 1710 μg/mL
- Initial Group B1 (4-hour + S9 with 16 hour recovery time): 0, 100, 250, 500, 1000, 1600, 1650, 1710 μg/mL
- Initial Group B1 (- S9 with 20 hour continuous treatment): 0, 100, 250, 500, 700, 800, 1000, 1200, 1400, 1600 μg/mL

- Repeat Group B2 (- S9 with 20 hour continuous) treatment): 0, 100, 250, 500, 700, 800, 1000, 1200, 1400, 1600 μg/mL

- Repeat Group B3 (4-hour - S9 with 16 hour recovery time): 0, 250, 500, 1000, 1650, 1710 μg/mL
- Repeat Group B3 (4-hour + S9 with 16 hour recovery time): 0, 250, 500, 1000, 1600 , 1650, 1710 μg/mL
- Repeat Group B3 (- S9 with 20 hour continuous treatment): 0, 100, 250, 500, 650, 700, 750, 800 μg/mL


For all groups, cells were collected 20 hours (± 30 minutes), 1.5 normal cell cycles, after initiation of treatment were selected for metaphase analysis.

Precipitation of the test substance dosing solution in the treatment medium was determined using unaided eye at the beginning and conclusion of treatment. The highest dose evaluated for the chromosome aberrations was selected based on the following:
- 4-hour without S9 : Limit dose for this assay (10 mM)
- 4-hour with S9 : Limit dose for this assay (10 mM)
- 20-hour without S9: 55 ± 5% cytotoxicity (cell growth inhibition relative to the vehicle control)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Water was the vehicle of choice based on the solubility of the test substance, and compatibility with the target cells. In a solubility test conducted at BioReliance, the test substance was soluble in water at a concentration of approximately 50 mg/mL, the maximum concentration tested for solubility.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation; at 10 and 20 μg/mL (final concentrations of 0.1 and 0.2 μg/mL respectively)

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation; at 0.25, 0.5 and 0.75 μg/mL (final concentrations of 2.5, 5 and 7.5 μg/mL respectively)

Details on test system and experimental conditions:

METHOD OF APPLICATION: no data
The pH of the highest dose of dosing solution in the treatment medium was measured using test tape.
When necessary, in order to maintain neutral pH in the treatment medium, pH was adjusted using 1N HCl . Treatment was carried out by refeeding the cultures as follows
Vehicle: Non-activated: 4.5 mL culture medium + 500 μL control substance dosing solution
Vehicle: S9-activated: 3.5 mL culture medium + 1 mL S9 mix + 500 μL control substance dosing solution
Test Substance: Non-activated: 4.5 mL culture medium + 500 μL test item dosing solution
Test Substance: S9-activated: 3.5 mL culture medium + 1 mL S9 mix + 500 μL test item dosing solution
Positive Control: Non-activated: 5 mL culture medium + 50 μL control substance dosing solution
Positive Control: S9-activated: 4 mL culture medium + 1 mL S9 mix + 50 μL control substance dosing solution
After the 4-hour treatment period in the non-activated and the S9-activated studies, the treatment medium was aspirated, the cells washed with calcium and magnesium free phosphate buffered saline (CMF-PBS), re-fed with complete medium and returned to the incubator under standard conditions.
For the definitive assay only, two hours prior to cell harvest, Colcemid was added to all cultures at a final concentration of 0.1 μg/mL.

DURATION
- Exposure duration: 4 hours (Groups 1 and 3); 20 hours (Groups 2)
- Expression time: 16 hours (Groups 1 and 3); 0 hours (Group 1)
- Fixation time: 20 hours (± 30 minutes)

SELECTION AGENT (mutation assays):
not applicable

SPINDLE INHIBITOR (cytogenetic assays):
Colcemid: For the definitive assay only, two hours prior to cell harvest, Colcemid was added to all cult
ures at a final concentration of 0.1 μg/mL

STAIN: Giemsa

NUMBER OF REPLICATIONS: Two cultures were tested per dose.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
For the chromosomal aberration assays only, cells were collected by centrifugation, treated with 0.075M KCl, washed
with fixative (methanol: glacial acetic acid, 3:1 v/v), capped and stored overnight or longer at 2 to 8°C.
To prepare slides, the cells were collected by centrifugation and the suspension of fixed cells was
applied to glass microscope slides and air-dried. The slides were stained with Giemsa, permanently
mounted, and identified by the laboratory study number, dose, treatment condition, harvest date, acti
vation system, test phase, and replicate tube design.

NUMBER OF CELLS EVALUATED: The mitotic index was recorded as the percentage of cells in mitosis per 500 cells counted.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in m
ammalian cells):
A minimum of 300 metaphase spreads containing 20 ± 2 centromeres from each dose (150 per
duplicate treatment) were examined and scored for chromatid-type and chromosome-type aberr
ations.
The number of metaphase spreads that were examined and scored per duplicate culture may be
reduced if the percentage of aberrant cells reaches a significant level (at least 10% determined based
on historical positive control data) before 150 cells are scored

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (percentage of cells in mitosis)

OTHER EXAMINATIONS:
- Determination of polyploidy: The percentage of cells with numerical aberrations (polyploid) was
evaluated for 150 cells per culture (a total of 300 per dose level)
- Determination of endoreplication: The percentage of cells with numerical aberrations
(endoreduplicated cells) was evaluated for 150 cells per culture (a total of 300 per dose level)
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole
or fragmented chromosomes (if applicable): not applicable

Evaluation criteria:

Vehicle Controls:
The frequency of cells with structural chromosomal aberrations should ideally be within the 95% control limits of the distribution of the historical negative control database. If the concurrent negative control data fall outside the 95% control limits, they may be acceptable as long as these data are note extreme outliers (indicative of experimental or human error).

Positive Controls:
The frequency of cells with structural chromosomal aberrations must be significantly greater than the concurrent vehicle control (p < 0.05). In addition, the cytotoxicity response must not exceed the upper limit for the assay (60%).

Cell Proliferation :
The average viable cell count in the vehicle control at harvest must be ≥ 1.5-fold the average viable cell baseline value.

Test Conditions:
The test substance must be tested using a 4-hr treatment with and without S9, as well as a 20 hr treatment without S9. However, all three treatment conditions need not be evaluated in the case of a positive test substance response under any treatment condition.

Analyzable Concentrations:
At least 300 metaphases must be analyzed from at least three appropriate test substance concentrations. The number of metaphases scored may be reduced when high numbers of cells with chromosomal aberrations (≥10% metaphases) are observed as with a positive test substance or the positive control substance.

The test substance was considered to have induced a positive response if
• at least one of the test concentrations exhibits a statistically significant increase when compared with the concurrent negative control (p ≤ 0.05), and
• the increase is concentration-related (p ≤ 0.05), and
• results are outside the 95% control limit of the historical negative control data.
The test substance was considered to have induced a clear negative response if none of the criteria for a positive response were met.

Statistics:

Statistical analysis was performed using the Fisher's exact test (p

Results and discussion

Test results

Test results 1

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

4h exposure

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

B1, B3

Cytotoxicity / choice of top concentrations:

other: see details in section "additional information on results"

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

4h exposure

Metabolic activation:

with

Genotoxicity:

ambiguous

Remarks:

B1 negative; B3 positive

Cytotoxicity / choice of top concentrations:

other: see details in section "additional information on results"

Vehicle controls validity:

valid

Untreated negative controls validity:

other:

Positive controls validity:

valid

Test results 4

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

20h exposure

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

B1 ambiguous; B2, B3 positive

Cytotoxicity / choice of top concentrations:

other: see details in section "additional information on results"

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH of the highest dose of test substance in treatment medium was measured and was adjusted using 1N HCl.
- Effects of osmolality: The osmolality of the test substance dose in treatment medium was considered acceptable.
- Water solubility: The test substance was soluble in the treatment medium at all doses tested at the beginning and conclusion of the treatment period.
- Precipitation: Precipitation of the test substance dosing solution in the treatment medium was determined using unaided eye at the beginning and conclusion of treatment.
- Definition of acceptable cells for analysis: Chromatid-type aberrations include chromatid and
isochromatid breaks and exchange figures such as quadriradials (symmetrical and asymmetrical
interchanges), triradials and complex rearrangements. Chromosome-type aberrations include chromosome breaks and exchange figures such as dicentrics and rings. Fragments (chromatid or
acentric) observed in the absence of any exchange figure were scored as a break (chromatid or chromosome). Fragments observed with an exchange figure were not scored as an aberration but were considered part of the incomplete exchange. Pulverized cells and severely damaged cells (counted as 10 aberrations) were also recorded.The XY vernier for each cell with a structural aberration was recorded. The percentage of cells with numerical aberrations (polyploid and endoreduplicated cells) was evaluated for 150 cells per culture (a total of 300 per dose level). The number and types of aberrations (structural and numerical) found, the percentage of structurally damaged cells in the total population of cells examined (percent aberrant cells), the percentage of numerically damaged cells in the total population of cells examined, and the average number of structural aberrations per cell (mean aberrations per cell) were calculated and reported for each treatment group. Chromatid and isochromatid gaps were presented in the data but were not included in the total percentage of cells with one or more aberrations or in the average number of aberrations per cell.

RANGE-FINDING/SCREENING STUDIES:
In the preliminary toxicity assay (A1), the doses tested ranged from 0.171 to 1710 µg/mL (10 mM), which was the limit dose for this assay. Cytotoxicity ( 50% reduction in cell growth index relative to the vehicle control) was observed at 1710 µg/mL in all three exposure groups. Based upon these results, the doses chosen for the chromosome aberration assay ranged from 100 to 1710 µg/mL for the non activated and S9-activated 4-hour exposure groups and from 100 to 1600 µg/mL for the non activated 20-hour exposure group.


Chromosome aberration test (CA) in mammalian cells:
- Results from cytotoxicity measurements:
o Relative Increase in cell count (RICC)

Cell Growth Inhibition (%) in Initial Chromosome Aberration Assay (B1)
- 4-hour without S9 : 35% at 1710 µg/mL
- 4-hour with S9 : 58% at 1710 µg/mL
- 20-hour without S9: 53% at 800 µg/mL
In the initial chromosome aberration assay (B1), cytotoxicity (≥ 50% reduction in cell growth index relative to the vehicle control) was not observed at any dose in the non-activated 4-hour exposure group. Cytotoxicity was observed at 1710 μg/mL in the S9-activated 4-hour exposure group and at doses ≥ 800 μg/mL in the non-activated 20-hour exposure group.

Cell Growth Inhibition (%) in Repeat Chromosome Aberration Assay (B2)
- 20-hour without S9: 59% at 700 µg/mL
In the repeat assay (B2), cytotoxicity (≥ 50% reduction in cell growth index relative to the vehicle control) was observed at doses ≥700 μg/mL in the non-activated 20-hour exposure group.

Cell Growth Inhibition (%) in Second Repeat Chromosome Aberration Assay (B3)
- 4-hour without S9 : 33% at 1710 µg/mL
- 4-hour with S9 : 13% at 1710 µg/mL
- 20-hour without S9: 52% at 700 µg/mL
In the second repeat assay (B3), cytotoxicity (≥ 50% reduction in cell growth index relative to the vehicle control) was not observed at any dose in the non-activated and S9-activated 4-hour exposure groups. Cytotoxicity was observed at doses ≥ 700 μg/mL in the non-activated 20-hour exposure group.

- Genotoxicity results
o Definition for chromosome aberrations, including gaps: Chromatid-type aberrations include chromatid and isochromatid breaks and exchange figures such as quadriradials (symmetrical and asymmetrical interchanges), triradials and complex rearrangements. Chromosome-type aberrations include chromosome breaks and exchange figures such as dicentrics and rings. Fragments (chromatid or acentric) observed in the absence of any exchange figure were scored as a break (chromatid or chromosome). Fragments observed with an exchange figure were not scored as an aberration but were considered part of the incomplete exchange. Chromatid and isochromatid gaps were presented in the data but were not included in the total percentage of cells with one or more aberrations or in the average number of aberrations per cell.
o Number of cells scored for each culture and concentration, number of cells with chromosomal aberrations and type given separately for each treated and control culture, including and excludling gaps: see summary tables for details

In the initial chromosome aberration assay (B1), cytotoxicity ( 50% reduction in cell growth index relative to the vehicle control) was not observed at any dose in the non activated 4-hour exposure group. Cytotoxicity was observed at 1710 µg/mL in the S9-activated 4-hour exposure group and at doses  800 µg/mL in the non-activated 20-hour exposure group. The doses selected for evaluation of chromosome aberrations were 500, 1000, and 1710 µg/mL for the non activated and S9-activated 4-hour exposure groups, and 100, 250, and 800 µg/mL for the non activated 20-hour exposure group.
Initial Chromosome Aberration Assay (B1)
In the non-activated 4-hour exposure group, no significant or dose dependent increases in structural aberrations were observed at any dose (p > 0.05; Fisher’s Exact and Cochran-Armitage tests). Statistically significant and dose-dependent increases in numerical (polyploid or endoreduplicated cells) aberrations (4.0% and 4.3%) were observed at 1000 and 1710 µg/mL, respectively (p < 0.05 or p < 0.01; Fisher’s Exact and Cochran-Armitage tests). Although the percentages of numerical aberrations were outside the historical control limit of 0.00 to 3.72%, they were within the historical control range of 0.00% to 5.00%. Therefore the statistically significant increase in numerical aberrations was considered biologically irrelevant.
In the S9-activated 4-hour exposure group, a statistically significant and dose-dependent increase in structural aberrations (8.0%) was observed at 1710 µg/mL (p < 0.01; Fisher’s Exact test and p < 0.05; Cochran-Armitage test). No significant or dose dependent increases in numerical aberrations were observed in the S9-activated 4-hour exposure group (p > 0.05; Fisher’s Exact and Cochran-Armitage tests).
In the non-activated 20-hour exposure group, statistically significant increases in structural aberrations (4.7%, 2.3%, and 1.7%) were observed at doses 100, 250, and 800 µg/mL, respectively (p < 0.05 or p < 0.01; Fisher’s Exact). However, the Cochran-Armitage test was negative for a dose response (p > 0.05). No significant or dose dependent increases in numerical aberrations were observed in the non-activated 20-hour exposure group (p > 0.05; Fisher’s Exact and Cochran-Armitage tests).

Repeat Chromosome Aberration Assay (B2)
In the repeat assay (B2), cytotoxicity ( 50% reduction in cell growth index relative to the vehicle control) was observed at doses  700 µg/mL in the non-activated 20-hour exposure group. The doses selected for evaluation of chromosome aberrations were 100, 250, and 700 µg/mL. Statistically significant and dose-dependent increases in structural aberrations (3.3%, and 6.0%) were observed at doses 250 and 700 µg/mL, respectively (p < 0.05 or p < 0.01; Fisher’s Exact and Cochran-Armitage tests). No significant or dose dependent increases in numerical aberrations were observed in the non-activated 20-hour exposure group (p > 0.05; Fisher’s Exact and Cochran-Armitage tests).

Second Repeat Chromosome Aberration Assay (B3)
In the second repeat assay (B3), cytotoxicity (>= 50% reduction in cell growth index relative to the vehicle control) was not observed at any dose in the non activated and S9-activated 4-hour exposure groups. Cytotoxicity was observed at doses  700 µg/mL in the non-activated 20-hour exposure group. The doses selected for evaluation of chromosome aberrations were 500, 1000, and 1710 µg/mL for the non activated and S9-activated 4-hour exposure groups, and 100, 250, and 700 µg/mL for the non activated 20-hour exposure group.
In the S9-activated 4-hour exposure group, a statistically significant increase in structural aberrations (2.3%) was observed at 1710 µg/mL (p < 0.05; Fisher’s Exact test). However, the Cochran-Armitage test was negative for a dose response (p > 0.05). No significant or dose dependent increases in numerical aberrations were observed in the S9-activated 4-hour exposure group (p > 0.05; Fisher’s Exact and Cochran-Armitage tests).
In the non-activated 4 and 20-hour exposure groups, due to significant variation in structural aberrations between the two scorers, the slides were evaluated by a different third scorer. Structural chromosomal aberration data from the initial scoring for these two exposure groups will not be reported, but maintained in the study file.
In the non-activated 4-hour exposure group, no significant or dose dependent increases in structural aberrations were observed at any dose (p > 0.05; Fisher’s Exact and Cochran-Armitage tests). Statistically significant and dose-dependent increases in numerical (polyploid or endoreduplicated cells) aberrations (2.0% and 2.3%) were observed at 1000 and 1710 µg/mL, respectively (p < 0.05 or p < 0.01; Fisher’s Exact and Cochran-Armitage tests). However, the percentage of numerical aberrations was within the historical control limit of 0.00 to 3.72%. Therefore the statistically significant increase in numerical aberrations was considered biologically irrelevant.
In the non-activated 20-hour exposure group, statistically significant and dose-dependent increases in structural aberrations (4.3%, and 8.0%) were observed at doses 250 and 700 µg/mL, respectively (p < 0.01; Fisher’s Exact and Cochran-Armitage tests). No significant or dose dependent increases in numerical aberrations were observed in the non-activated 20-hour exposure group (p > 0.05; Fisher’s Exact and Cochran-Armitage tests).

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data): see in table "Any other information including tables"

Remarks on result:

not determinable

Any other information on results incl. tables

Chromosome Aberration Assay

Based on the results of the preliminary toxicity test, the doses selected for testing in the chromosome aberration assay were as follows:

 

Treatment Condition	Treatment Time	Recovery Time	Doses (µg/mL)
Non-activated	4 hr	16 hr	100, 250, 500, 1000, 1650, 1710
	20 hr	0 hr	100, 250, 500, 700, 800, 1000, 1200, 1400, 1600
S9-activated	4 hr	16 hr	100, 250, 500, 1000, 1600, 1650, 1710

In order to confirm the results, the chromosomal aberration assay was repeated in the non-activated 20-hour exposure group at the following doses:

 

Treatment Condition	Treatment Time	Recovery Time	Doses (µg/mL)
Non-activated	20 hr	0 hr	100, 250, 500, 700, 800, 1000, 1200, 1400, 1600

In order to confirm the overall study conclusion, the chromosomal aberration assay was repeated again at the following doses:

Treatment Condition	Treatment Time	Recovery Time	Doses (µg/mL)
Non-activated	4 hr	16 hr	250, 500, 1000, 1650, 1710
	20 hr	0 hr	100, 250, 500, 650, 700, 750, 800
S9-activated	4 hr	16 hr	250, 500, 1000, 1600, 1650, 1710

 

Precipitation of the test substance dosing solution in the treatment medium was determined using unaided eye at the beginning and conclusion of treatment. The highest dose evaluated for the chromosome aberrations was selected based on the following:

 

4-hour (-S9)	4-hour (+S9)	20-hour (-S9)
Limit dose for this assay (10 mM)	Limit dose for this assay (10 mM)	55± 5% cytotoxicity (cell growth inhibition relative to the vehicle control)

Two additional doses were included in the evaluation.

 

 

Treatment of Target Cells (Preliminary Toxicity Test and Chromosomal Aberration Assays)

The pH of the highest dose of dosing solution in the treatment medium was measured using test tape. When necessary, in order to maintain neutral pH in the treatment medium, pH was adjusted using 1N HCl (Supplier: Sigma-Aldrich, Lot No. RNBF2514, Exp. Date Sep 2019). Treatment was carried out by refeeding the cultures as follows:

 

Treatment	Culture medium* (mL)	Volume of S9 mix (mL)	Volume of control / test substance dosing solution (µL)
Vehicle: Non-activated	4.5	-	500
Vehicle: S9-activated	3.5	1	500
Test Substance: Non-activated	4.5	-	500
Test Substance: S9-activated	3.5	1	500
Positive Control: Non-activated	5	-	50
Positive Control: S9-activated	4	1	50

Historical Control Values for Structural Aberrations

NON-ACTIVATED TEST SYSTEM

Historical Values	Structural Aberration (%)
	Negative Control^1			Positive Controls^2
	4-hour	20-hour		4-hour	20-hour
Mean	0.74	0.64		21.07	20.87
Standard Deviation	±0.83	±0.78		±5.41	±5.21
95% Control Limits	0.00 - 2.41	0.00 - 2.21		10.25 - 31.88	10.45 - 31.29
Range^4	0.00 - 3.00	0.00 - 3.50		7.50 - 33.00	7.00 - 34.00

 

  

S9-ACTIVATED TEST SYSTEM 

Historical Values	Structural Aberration (%)
	Negative Control^1	Positive Control^3
Mean	1.09	26.60
Standard Deviation	±1.39	±7.66
95% Control Limits	0.00 - 3.88	11.28 - 41.92
Range^4	0.00 - 9.50	12.50 - 52.00

 

^1.       Solvents include water, saline, DMSO, ethanol, acetone, and other non-standard and Sponsor supplied vehicles.

^2.       Positive control for non-activated studies, Mitomycin C (MMC).

^3.       Positive control for S9-activated studies, Cyclophosphamide (CP).

^4.       Range from minimum to maximum.

 

Historical Control Values for Numerical Aberrations

NON-ACTIVATED TEST SYSTEM

 

Historical Values	Numerical Aberration (%)
	Negative Control^1
	4-hour	20-hour
Mean	1.56	1.55
Standard Deviation	±1.08	±1.24
95% Control Limits	0.00 - 3.72	0.00 - 4.03
Range^2	0.00 -5.00	0.00 - 5.50

 

 

S9-ACTIVATED TEST SYSTEM

 

Historical Values	Numerical Aberration (%)
	Negative Control^1
Mean	2.68
Standard Deviation	±1.84
95% Control Limits	0.00 -6.35
Range^2	0.00 -10.00

 

^1.       Solvents include water, saline, DMSO, ethanol, acetone, and other non-standard and Sponsor supplied vehicles.

^2.       Range from minimum to maximum.

Applicant's summary and conclusion

Conclusions:

Under the conditions of the assay described in this report, the test substance was concluded to be positive for the induction of structural chromosomal aberrations and negative for the induction of numerical chromosomal aberrations in the non-activated and S9-activated test systems in the in vitro mammalian chromosome aberration test using CHO cells. Hence, based on the results of this study, the test substance is considered to be clastogenic in the in vitro mammalian chromosome aberration test using CHO cells.