N,N,4-trimethylpiperazine-1-ethylamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation study in mammalian cells

In a K1 Chinese Hamster Ovary assay (HPRT), performed according to the OECD Guideline 476, it was concluded that the test substance is not mutagenic in the Chinese Hamster Ovary test system under the experimental conditions described in the report.

Bacterial reverse mutation assay

In a K1 bacterial reverse mutation assay in Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and E. coli WP2 uvrA, performed according to OECD Guideline 471 (Ames), it was concluded that the test substance has no mutagenic properties towards any of the bacterial strains tested in the absence and in the presence of S9-mix under the test conditions described in the report.

 

In vitro chromosome aberration study in Chinese Hamster Ovary Cells 

In a K1 chromosome aberration study in Chinese hamster ovary cells, performed according to OECD Guideline 473, it was concluded that the test substance was clastogenic.

ToxTracker Test

In a K2 key study, the test substance was classified as not genotoxic in the ToxTracker assay when tested in the absence or presence of a metabolising system. The test substance did not induce DNA damage or activation of the unfolded protein response. However, the test substance induced a weak but significant level of oxidative stress.

 

Link to relevant study records

Reference

Reference 1

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

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

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.

Target gene:

not applicable

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.

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)

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

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

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

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

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

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.

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-06-21 to 2017-08-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Name of the test material: Jeffcat TAP, N,N,4-trimethyl 1-piperazineethanamine
- CAS no.: 104-19-8
- Purity: 100% (per protocol), 99.5% (per certificate of analysis)
- Molecular weight: 171.29 g/mol
- Description: clear colorless liquid
- Source and lot/batch No.of test material: PFW160141
- Expiration date of the lot/batch: no data
- Purity test date: 2016-07-15

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protected from light
- Stability under storage conditions: Not indicated
- Stability under test conditions: Not indicated
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: Not indicated

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Dr. Abraham W. Hsie, Biology Division, Oak Ridge National Laboratory, Oak Ridge, TN
- Suitability of cells: recommended by Guideline
- Modal number of chromosomes: 20
- Normal (negative control) cell cycle time: 12-14 hours

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
CHO cells were maintained in Ham's F12 medium supplemented with 3 mM L glutamine and 5% (v/v) heat-inactivated and dialyzed fetal bovine serum (Complete Ham’s F12) under standard conditions (37 ± 1C in a humidified atmosphere of 5 ± 1% CO2 in air). All media contained antimycotics and antibiotics.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes

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 Assay: 3.35, 6.69, 13.4, 26.8, 53.5, 107, 214, 428, 857 and 1713 µg/mL with and without S9;
Definitive Mutagenicity Assay: 107, 214, 428, 857 and 1713 µg/mL with and without S9;

The maximum concentration evaluated was based on solubility limitations of the test article in the vehicle. Based upon the results of the preliminary toxicity assay, the maximum concentration selected for the definitive mutagenicity assay was 1713 µg/mL

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 article and compatibility with the target cells. The test article formed a clear solution in water at a concentration of approximately 50 mg/mL in the solubility test conducted at the testing facility.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation; 0.200 µL/mL

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With metabolic activation; 4.00 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
Preliminary toxicity test
Cells were plated (on Day -1) in 25-cm2 cultures at a density of ~1 x 10^6 in 5 mL Complete Ham’s F12. Following an overnight incubation under standard conditions, the cultures were washed twice (on Day 0) with Hank’s Balanced Salt Solution (HBSS) and re fed with 4 mL treatment medium, or 3 mL treatment medium plus 1 mL S9 mix, as appropriate. Following addition of the test or control article dose formulations (500 µL) to the flasks, the cultures were incubated under standard conditions for 5 ± 0.5 hours.
Definitive mutagenicity assay
Cells were plated (on Day -1) in 75-cm2 cultures at a density of ~5 x 10^6 in 10 mL Complete Ham’s F12. Following an overnight incubation (on Day 0) at standard conditions, the cultures were washed twice with HBSS and re-fed with 10 mL treatment medium, or 8 mL treatment medium plus 2 mL S9 mix (adjusted for the test article dose volume if >1%, v/v), as appropriate. Following addition of the test or control article formulations (1.1 mL without S9 and 1.2 mL with S9) to the flasks, the cultures were incubated under standard conditions for 5 ± 0.5 hours (positive control articles were prepared in DMSO and added to the flasks using a 1% dose volume).

DURATION
- Exposure duration: 5h (+-0.5h)
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 7 days

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: single cultures in the preliminary toxicity assay; duplicate cultures in the mutagenicity assays

NUMBER OF CELLS EVALUATED:
For mutant frequencies: 1 x 10^6 cells
For number of clonable cells: 200 cells

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity was expressed as the adjusted relative survival (%; relative cloning efficiency x relative cell density at the time of cloning, as compared to the concurrent vehicle control)

Evaluation criteria:

The test substance was considered to have produced a positive response if it induced a dose-dependent increase in mutation frequency and an increase exceeding 95% historical vehicle control limits in at least one test dose level(s) as compared with concurrent vehicle control (p<0.01). If only one criterion was met (a statistically significant or dose-dependent increase or an increase exceeding the historical control 95% confidence interval), the result were considered equivocal. If none of these criteria were met, the results were considered to be negative.
Other criteria also may be used in reaching a conclusion about the study results (e.g., comparison to historical control values, biological significance, etc.).

Statistics:

Statistical analyses were performed using the method of Snee and Irr (1981), with significance established at the 0.05 level.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH of the cultures was adjusted at concentrations ≥107 µg/mL with and without S9 to maintain neutral pH.
- Effects of osmolality: The osmolality of the cultures was acceptable as it did not exceed the osmolality of the vehicle control by more than 120%.
- Water solubility: the solubility test showed that the substance is soluble in water at a conc of 50 mg/mL
- Precipitation: no visible precipitate was observed at the beginning or end of treatment.

RANGE-FINDING/SCREENING STUDIES:
Cells were treated with 10 test article concentrations, as well as the vehicle control, in the presence and absence of S9 using single cultures. The maximum concentration evaluated was selected based on test article solubility limitations in the vehicle. Lower concentrations were prepared by 2-fold dilutions. The pH of the treatment medium was measured and adjusted at concentrations ≥107 µg/mL using 1N hydrochloric acid (HCl). No pH adjustment was necessary to maintain neutral pH in the treatment medium at the remaining concentrations tested. The osmolality of the cultures was acceptable as it did not exceed the osmolality of the vehicle control by more than 120%. No visible precipitate was observed at the beginning or end of treatment. Adjusted relative survival was 81.67 and 54.75% at a concentration of 1713 µg/mL with and without S9, respectively.
Based upon the results of the preliminary toxicity assay, the concentrations selected for the definitive mutagenicity assay were 107, 214, 428, 857 and 1713 µg/mL with and without S9.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: The positive controls induced significant increases in mutant frequency (p < 0.01). All positive control values were within acceptable ranges.
- Negative (solvent/vehicle) historical control data: All vehicle control values were within acceptable ranges
All criteria for a valid assay were met.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The average adjusted relative survival was 99.51 and 81.84% at a concentration of 1713 µg/mL with and without S9, respectively.

Conclusions:

Under the conditions of the assay described in this report, the test item was concluded to be negative for the induction of forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system, in the in vitro mammalian cell forward gene mutation (CHO/HPRT) assay.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 June 2017 to 22 July 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

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

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protected 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:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

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:

Initial Toxicity-Mutation Assay: 1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate with and without S9-mix;
Confirmatory Mutagenicity Assay: 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate with and without S9-mix;

Since the test item formed a clear solution in sterile water for injection at approx. 50 mg/mL, 5000 µg/plate was selected as the maximum concentration in the initial toxicity-mutation assay. Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the confirmatory mutagenicity assay was 5000 μg per plate.

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 article and compatibility with the target cells. The test article formed a clear solution in water at a concentration of approximately 50 mg/mL in the solubility test conducted at the testing facility.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With S9: 1.0 μg/plate for TA98, TA1535 ; 2.0 μg/plate for TA100, TA1537; 15 μg/plate for WP2 uvrA

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Without S9: 1.0 μg/plate for TA98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without S9: 1.0 μg/plate for TA100, TA1535

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Without S9: 75 μg/plate for TA1537

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without S9: 1000 μg/plate for WP2 uvrA

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
One half (0.5) milliliter of S9 or Sham mix, 100 µL of tester strain (cells seeded) and 100 µL of vehicle or test article dilution were added to 2.0 mL of molten selective top agar at 45±2°C. When plating the positive controls, the test article aliquot was replaced by a 50.0 µL aliquot of appropriate positive control. After vortexing, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. After the overlay had solidified, the plates were inverted and incubated for 48 to 72 hours at 37±2°C. Plates that were not counted immediately following the incubation period were stored at 2 8C until colony counting could be conducted.

DURATION
- Exposure duration: 48 to 72 hours
- Selection time (if incubation with a selection agent): 48 to 72 hours (simultaneous with exposure)

SELECTION AGENT (mutation assays): Histidine (S. typhimurium) or Tryptophan (E. coli)

NUMBER OF REPLICATIONS:
Initial toxicity-mutation assay: duplicate
Confirmatory mutagenicity assay: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: A dose level is considered toxic if one or both of the following criteria are met: (1) A >50 % reduction in the mean number of revertants per plate as compared to the mean vehicle control value. This reduction must be accompanied by an abrupt dose dependent drop in the revertant count. (2) At least a moderate reduction in the background lawn

Evaluation criteria:

For the test article to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test article as specified below:
Strains TA1535 and TA1537
Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.
Strains TA98, TA100 and WP2 uvrA
Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.
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 was evaluated as negative if it was neither positive nor equivocal.

Statistics:

No formal hypothesis testing was done.
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Soluble at 50 mg/mL
- Precipitation: no precipitation has been observed
- Other: sterility results: No contaminant colonies were observed on the sterility plates for the vehicle control, the test article dilutions or the S9 and Sham mixes.

RANGE-FINDING/SCREENING STUDIES:
The initial toxicity-mutation assay was used to establish the dose range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. TA98, TA100, TA1535, TA1537 and WP2 uvrA were exposed to the vehicle alone, positive controls and eight dose levels of the test article, in duplicate, in the presence and absence of Aroclor induced rat liver S9. Neither precipitate nor toxicity was observed. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Based upon the results of the initial toxicity-mutation assay, the dose levels selected for the confirmatory mutagenicity assay were 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: all positive controls exhibited at least 3-fold increases in the number of revertants
- Negative (solvent/vehicle) historical control data: the number of revertants was within the characteristic ranges for all vehicle controls
All criteria for a valid study were met as described in the protocol.

Conclusions:

The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of the study, the test item did not cause a positive mutagenic response with any of the tester strains in either the presence or absence of Aroclor induced rat liver S9.

Reference 4

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

DNA damage, oxidative stress, protein damage and cellular stress were studied

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 21 May 2018 to 23 May 2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

ToxTracker is a panel of mammalian stem cell lines that contain different fluorescent reporters for induction of DNA damage, oxidative stress and protein damage. The differential induction of the GFP reporters as well as cytotoxicity of the tested compounds is determined by flow cytometry. The compound was analysed in the absence and presence of an S9 rat liver extract-based metabolising system. Exposure to control compounds are included in each test to determine technical performance and reproducibility of the ToxTracker assay. Quantitive data analysis is done using ToxPlot software.

GLP compliance:

no

Remarks:

non-GLP study, but general principles to conduct proper scientifically correct in vitro experiments and proper handling of test article (stock) solutions to prevent/minimise degradation. Good Cell Culture Practice guidelines from the OECD followed.

Type of assay:

other: ToxTracker is a mammalian stem cell-based assay that monitors activation of specific cellular signalling pathways for detection of the biological reactivity of compounds.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Huntsman (batch No.: no data)
- Expiration date of the lot/batch: no data
- Purity test date: no data
- Toxys reference: Txs000348

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
no data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
no data

Target gene:

Biomarker genes:
- DNA damage:
Bscl2 (ATR/Chk1 DNA damage signaling)
Rtkn (NF-kB signaling)

- Oxidative stress:
Srxn1 (Nrf2 antioxidant response)
Blvrb (Nrf2-independent)

- Protein damage:
Ddit3 (Unfolded protein response)

- Cellular stress:
Btg2 (p53 signaling)

Species / strain / cell type:

mammalian cell line, other: panel of six validated GFP-based mouse embryonic stem (mES) reporter cell lines

Details on mammalian cell type (if applicable):

CELLS USED
The ToxTracker assay is a panel of six validated GFP-based mouse embryonic stem (mES) reporter cell lines that can be used to identify the biological reactivity and potential carcinogenic properties of newly developed compounds in a single test.
ToxTracker consists of a panel of six different mES GFP reporter cell lines representing four distinct biological responses that are associated with carcinogenesis, i.e. general cellular stress, DNA damage,
oxidative stress and the unfolded protein response. The six independent mES reporter cell lines were seeded in gelatin-coated 96-well cell culture plates in 200 μl mES cell medium (50.000 cells per well).

MEDIA USED
The ToxTracker reporter cells are maintained by culturing them in gelatin-coated dishes in the presence of irradiated primary mouse embryonic fibroblasts (MEFs) in mES cell culture medium. During chemical exposures and reporter analysis the ToxTracker cells are cultured in the absence of fibroblasts in mES cell culture medium.

Metabolic activation:

with and without

Metabolic activation system:

S9 liver extract from aroclor1254- induced rats (Moltox)

Test concentrations with justification for top dose:

For each tested compound, ten concentrations were tested in 2-fold dilutions. The highest compound concentration that was tested was 10 mM, in line with the advised maximum concentration in the current guidelines for in vitro genotoxicity testing of chemical compounds.
concentrations tested: 0, 19.5, 39.0, 78.1, 156.3, 312.5, 625, 1250, 2500, 5000, 10000 μM

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: MQ
- Justification for choice of solvent/vehicle: no data

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Cisplatin (+/- S9; DNA damage), diethyl maleate (-S9; oxidative stress), tunicamycin (-S9; unfolded protein response), aflatoxin B1 (+S9; Aflatoxin B1)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 24 hours
- Expression time (cells in growth medium): Induction of the GFP reporters was determined after 24 h exposure using a Guava easyCyte 8HT flow cytometer (Millipore).

Regporter genes:
To allow easy assessment of the activation 1 status of these biomarker genes, we have generated green fluorescent (GFP) mES reporter cell lines. These reporters were created using bacterial artificial chromosomes (BAC) that contain the complete biomarker gene including promoter and regulatory elements ensuring physiological regulation of the GFP reporters following transfection into stem cells2.

NUMBER OF REPLICATIONS:
I. 3 independent replicates in absence of metabolic activation of the test article and the control compounds cisplatin, diethyl maleate and tunicamycin.
II. 3 independent replicates in presence of metabolic activation of the test article and the control compounds cisplatin and aflatoxin B1.

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity was estimated by cell count after 24 h exposure using a flow cytometer and is expressed as percentage of viable/intact cells after 24 h exposure compared to vehicle control exposed cells. For cytotoxicity assessment in the ToxTracker assay, the relative cell survival for the six different reporter cell lines was averaged.

Evaluation criteria:

The ToxTracker assay was considered to have a positive response when a compound induces at least a 2 fold increase in GFP expression in any of the reporters. Activation of the Bscl2-GFP or Rtkn-GFP reporters indicate induction of DNA damage, Srxn1-GFP and Blvrb-GFP indicate induction of cellular oxidative stress
and Ddit3-GFP activation is associated with the unfolded protein response. Only GFP inductions at compound concentrations that showed < 75% cytotoxicity are used for the ToxTracker analysis. Data from measurements > 75% cytotoxicity can not be interpreted in a meaningful way and are therefore discarded.
The No Observed Effect Level (NOEL) defines the highest concentrations where none of the ToxTracker reporters markers showed a >1.5-fold increase in fluorescence. The Lowest Observed Effect Level (LOEL) indicates the lowest concentration were at least one of the ToTracker reporters showed an induction of ≥2.0. The No Observed Genotoxicity Effect Level (NOGEL) is the highest concentration that did not activate the Bscl2-GFP and/or Rtkn-GFP reporters with a >1.5-fold increase in fluorescence. The Lowest Observed Genotoxicity Effect Level (LOGEL) is the lowest concentration that activated the Bscl2-GFP and/or Rtkn-GFP
genotoxicity reporters ≥2.0-fold.

Statistics:

Toxplot, a dedicated data analysis software package was developed. Toxplot imports raw GFP reporter data from the flow cytometer, calculates GFP induction levels and cytotoxicity, performs statistical analysis of the data and hierarchical clustering of the tested compounds, and visualises the data in a heatmap allowing convenient interpretation of obtained test results. To compare the induction of the six GFP reporters for a collection of compounds, each with different biological reactivities, dose-response relationships and kinetics, Toxplot calculates for each compound the level of GFP induction for every individual reporter at a specified level of cytotoxicity (typically 10%, 25% and 50%). GFP induction levels are calculated by linear regression between two data points around the specified cytotoxicity level. In case the specified level of cytotoxicity can not be reached at the highest tested compound concentration, Toxplot displays the GFP induction level at this top concentration. In the heatmap, Toxplot clearly marks the compounds that do not induce the selected level of cytotoxicity. Because the cytotoxicity for a compound can vary between the ToxTracker cell lines, calculations of the GFP induction levels of the individual reporters by Toxplot can slightly deviate from the GFP induction and cytotoxicity figures.

Key result

Species / strain:

mammalian cell line, other: panel of six validated GFP-based mouse embryonic stem (mES) reporter cell lines

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Significant cytotoxicity (>50%) was observed at a concentration of 1.7 mM in the absence of a metabolising system. No increased cytotoxicity was observed when tested in presence of a metabolising system.

Vehicle controls validity:

not examined

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
no data

Details on results:
• Significant cytotoxicity (>50%) was observed at a concentration of 1.7 mM in the absence of a metabolising system.
• No increased cytotoxicity was observed when tested in presence of a metabolising system.
• No autofluorescence was observed at the tested concentrations.
• No significant activation of the genotoxicity reporters was observed in absence and presence of a metabolising system.
• No significant activation of the p53 response in absence and presence of a metabolising system.
• A weak but significant induction of the Srxn1-GFP oxidative stress reporter was observed in the ToxTracker assay only in absence of a metabolising system, although >10-fold lower compared to the positive control diethyl maleate.
• No significant activation of the unfolded protein response was observed in absence and absence of a metabolising system.

The specificity of the ToxTracker reporter cell lines was extensively validated using different libraries of reference compounds as demanded by various regulatory agencies (Kirkland et al., 2008). Over 97% of all 4 tested chemicals were classified correctly, confirming the outstanding performance of the assay. Importantly, compounds that are often erroneously identified as genotoxic compounds by the conventional in vitro tests were identified by the assay as causing predominantly oxidative stress, explaining their positive results.

Reference: Kirkland, D., Kasper, P., Müller, L., Corvi, R., and Speit, G. (2008). Recommended lists of genotoxic and non-genotoxic chemicals for assessment of the performance of new or improved genotoxicity tests: a follow-up to an ECVAM workshop. Mutat Res 653, 99–108.

Conclusions:

The test substance was classified as not genotoxic in the ToxTracker assay when tested in the absence or presence of a metabolising system:
• No significant activation of the genotoxicity reporters was observed in absence and presence of a metabolising system.
• No significant activation of the p53 response in absence and presence of a metabolising system.
• A weak but significant induction of the Srxn1-GFP oxidative stress reporter was observed in the ToxTracker assay only in absence of a metabolising system, although >10-fold lower compared to the positive control diethyl maleate.
• No significant activation of the unfolded protein response was observed in absence and absence of a metabolising system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Published data with the structural analogue substance aminoethylpiperazine is included in the dossier to support the classification of the test substance as 'not mutagenic'.

In a K2 micronucleus test conducted with a structurally similar substance, aminoethylpiperazine, no induction in the frequency of micronuclei was noted in mice after intraperitoneal adminsitration of the test substance (Leung, 1993).

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

The assay (Heddle et al., 1983) was based on the procedures developed by Schmid et al. (1975) to screen chemicals for clastogenic potential (See section References)

GLP compliance:

no

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

Swiss Webster

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 6-8 weeks old

Route of administration:

intraperitoneal

Frequency of treatment:

single administration

Post exposure period:

Sampling times:
30h post administration
48h post administration
72h post administration

Dose / conc.:

175 mg/kg bw/day (nominal)

Dose / conc.:

350 mg/kg bw/day (nominal)

Dose / conc.:

560 mg/kg bw/day (nominal)

Control animals:

yes

Positive control(s):

Triethylenemelamine at 0.3 mg/kg bw

Tissues and cell types examined:

Micronuclei in peripheral, polychromatic erythrocytes from blood samples collected from the tail vein

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Test doses were selected from a preliminary range-finding test to determine approximate toxicity of the test materials.

DETAILS OF SLIDE PREPARATION: Micronuclei in peripheral, polychromatic erythrocytes were stained with Furr's R-66 Giemsa diluted in phosphate buffer (Gallupudi and Kamra, 1974). Slides were coded and read blindly to prevent bias. The polychromatic : normochromatic erythrocytes ratio for approximately 1000 total cells was calculated.

Evaluation criteria:

A positive result was concluded if at least one statistically significant increase above vehicle control was observed with an indication of a dose-related effect.

Statistics:

Data were compared for significant differences using the Fisher's Exact Test.

Key result

Sex:

male/female

Genotoxicity:

negative

Remarks:

at 30, 48 and 72h sampling times

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

With AEP, there was no increase at either the 48- or 72-h sample periods. Of the 3 dose levels, only the middle one (350 mg/kg), and only at the 30-h post-injection sample interval, produced a single statistically significant increase in the number of micronuclei.
 

 INDUCTION OF MICRONUCLEUS IN PERIPHERAL ERYTHROCYTES OF SWISS-WEBSTER MICE INJECTED INTRAPERITONEALLY WITH ALKYLENEAMINES

	Water	TEM	AEP
	10	0.3	175	350	560
	mg/kg	mg/kg	mg/kg
30 hours post dosing
Male	4.2±1.6	24.0±6.4^b	3.0±1.4	5.0±1.6	2.8±1.8
Female	1.4±1.7	17.0±5.3^b	2.0±1.6	4.0±1.9 a	1.8±1.3
48 hours post dosing
Male	2.4±0.9	-	3.8±3.4	2.0±1.4	1.2±1.6
Female	1.8±0.8	-	2.8±1.1	2.8±0.8	1.4±2.0
72 hours post dosing
Male	2.4±2.2	-	4.2±2.5	2.0±1.2	2.2±1.3
Female	1.2±1.3	-	1.8±1.6	1.6±0.9	2.2±1.8

TEM, triethylenemelamine.

Statistical analysis employed the Fisher's Exact text (1-tailed):ap < 0.01; b p < 0.001.

Conclusions:

In this micronucleus study with Swiss-Webster mice, no clastogenic activity was observed with AEP in male and female mice.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic toxicity: in vitro

In vitro gene mutation study in mammalian cells (HPRT) 

In a K1 study, Dutta, A (2018) investigated the effect of the test substance on the induction of forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of Chinese hamster ovary cells, according to the OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test). The test was performed in the presence of S9-mix (rat liver metabolic activation system induced by Aroclor 1254) with a 5-hour treatment period and in the absence of S9-mix with 7 days treatment period. The vehicle of the test item was water. 

Based upon the results of the preliminary toxicity assay, the test item was tested up to concentrations of 1713 µg/mL in the absence and presence of S9-mix for 5 hours, and no toxicity was observed. No visible precipitate was observed at the beginning or end of treatment. 

Positive control chemicals, ethylmethanesulphonate and benzo(a)pyrene, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control values were within acceptable ranges. 

Under the conditions of the assay described in this report, the test item was concluded to be negative for the induction of forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system, in the in vitro mammalian cell forward gene mutation (CHO/HPRT) assay.

Bacterial reverse mutation assay 

In K1 bacterial reverse mutation assay (Kamala, 2017), performed according to the OECD Guideline 471 (Bacterial Reverse Mutation Assay, Ames), the test substance was tested in four strains of Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and E. coli WP2 uvrA. The test substance, dissolved in water, was tested in the absence and in the presence of S9- mix: 1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 μg/plate in the initial toxicity-mutation study. Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the confirmatory mutagenicity assay was 5000 μg per plate. The test substance, dissolved in water, was used at six concentrations in the absence and in the presence of S9- mix: 15.0, 50.0, 150, 500, 1500 and 5000 μg/plate in the main study.

In the absence and in the presence of a metabolic activation system, the test substance did not cause any genotoxicity or cytotoxicity in any dose tested. No precipitation has been observed at any dose level. All positive controls exhibited at least 3-fold in the number of revertants. The number of revertants was within the characteristic ranges for all vehicle controls and all criteria for a valid study were met as described in the protocol.

It was concluded that the test substance has no mutagenic properties towards the various S. typhimurium strains and E. coli WP2 uvrA in either the presence or absence of Aroclor induced rat liver S9.

In vitro chromosome aberration study in Chinese Hamster Ovary Cells

The test substance was tested to evaluate the potential to induce structural chromosomal aberrations using Chinese hamster ovary (CHO) cells in both the absence and presence of an exogenous metabolic activation system (Roy S., 2018). CHO cells were treated for 4 hours in the absence and presence of S9, and for 20 hours in the absence of S9. Water was used as the vehicle.

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.

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.

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 95% 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). However, at this dose level significant cytotoxicity was observed (58% reduction in cell growth index relative to the vehicle control). 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).

In order to confirm the results, the chromosomal aberration assay was repeated in the non‑activated 20-hour exposure group at doses ranging from 100 to 1600 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. 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). However, at the highest dose level (700 µg/mL significant cytotoxicity was observed (59% reduction in cell growth index relative to the vehicle control). 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).

In order to confirm the overall study conclusion, the chromosomal aberration assay was repeated again at doses ranging from 250 to 1710 µg/mL for the non‑activated and S9-activated 4-hour exposure groups, and from 100 to 800 µg/mL for the non‑activated 20-hour exposure group.

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).

These results indicate that the test substance was positive for the induction of structural chromosomal aberrations and negative for the induction of numerical chromosomal aberrations in the presence and absence of the exogenous metabolic activation system.

As there were substantial differences among the repeat experiments in the chromosome aberration study in Chinese Hamster Ovary Cells, the test substance is considered equivocal and an additional ToxTracker Test test was conducted as a confirmatory test. 

Tox Tracker Test

ToxTracker is a panel of mammalian stem cell lines that contain different fluorescent reporters for induction of DNA damage, oxidative stress and protein damage. The differential induction of the GFP reporters as well as cytotoxicity of the tested compounds is determined by flow cytometry. The compound was analysed in the absence and presence of an S9 rat liver extract-based metabolising system. Exposure to control compounds are included in each test to determine technical performance and reproducibility of the ToxTracker assay. Quantitative data analysis is done using ToxPlot software. The validity of the ToxTracker assay results was confirmed by exposure to various reference compounds and assessing the specificity of the different reporter cell lines. Three test articles induced significant cytotoxicity (>50%), when tested in the absence and presence of S9 rat liver extract.

The test substance did not significantly activate the Bscl2-GFP and Rtkn-GFP genotoxicity reporters in ToxTracker when tested in the absence or presence of a metabolising system. In the presence of a metabolising system the test substance weakly activated (>1.5-fold) the Btg2-GFP reporter that is associated with the p53 response, but induction levels did not reach the 2 -fold threshold for a positive ToxTracker result. The test substance activated activated the Srxn1-GFP oxidative stress reporter in ToxTracker in absence of a metabolising system. The Ddit3-GFP reporter, that is associated with protein damage and the unfolded protein response, was not activated by the test compound in absence or presence of a metabolising system.

In conclusion, the test substance was classified as not genotoxic in the ToxTracker assay when tested in the absence or presence of a metabolising system. The test substance induced weak but significant levels of oxidative stress. No activation of the unfolded protein response was seen for the tested compound.

 

Genetic toxicity: in vivo

In vivo micronucleus test with analogue substance aminoethylpiperazine

No in vivo genetic toxicity study was available with the test substance. A key study conducted with the structurally related substance, Aminoethylpiperazine (CAS 140 -31 -8), is included as part of the genetic toxicity endpoint, to demonstrate the mutagenic potential of the test substance.

Aminoethylpiperazine is considered related to the test substance based on chemical structure and physico-chemical characteristics. Both substances are liquids with similar melting point, boiling point, relative density, vapour pressure, partition coefficient, water solubility and flash point. Both substances are considered not flammable, not explosive and not oxidising. Both aminoethylpiperazine and the test substance are considered acute toxic, skin corrosive and eye corrosive, but only aminoethylpiperazine is classified as sensitising to skin, reprotoxic cat 2, and STOT RE 1. Aminoethylpiperazine can be considered slightly more toxic than the test substance and inclusion of data with this analogue substance is thus a worst case approach.

In the key study performed with aminoethylpiperazine, mice were dosed by a single intraperitoneal injection. Triethylenemelamine (TEM) was used as the positive control agent. Blood samples were collected from the tail vein at 0, 48 and 72 h after dosing.

Micronuclei in peripheral, polychromatic erythrocytes were stained with Furr's R-66 Giemsa diluted in phosphate buffer (Gallupudi and Kamra, 1974). Slides were coded and read blindly to prevent bias. The polychromatic : normochromatic erythrocytes ratio for approximately 1000 total cells was calculated. Data were compared for significant differences using the Fisher's Exact Test. A positive result was concluded if at least one statistically significant increase above vehicle control was observed with an indication of a dose-related effect.

 

There was no increase at either the 48- or 72-h sample periods. Of the 3 dose levels, only the middle one (350 mg/kg), and only at the 30-h post-injection sample interval, produced a single statistically significant increase in the number of micronuclei. Since this induction was not dose-responsive, the increase is not considered biologically relevant and the test is concluded to be negative.

 

Justification for classification or non-classification

Based on the results and the criteria of the CLP Regulation (EC) 1272/2008, the test substance should not be classified as mutagenic.