2-[(3-aminopropyl)methylamino]ethanol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three reliable in vitro genetic toxicity studies are available. The test substance was demonstrated to be negative with or without metabolic activation in a bacterial mutation assay (Ames); negative with or without metabolic activation in a mammalian cell forward gene mutation (CHO/HPRT) assay;

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-06-06 to 2017-06-23

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

Qualifier:

according to guideline

Guideline:

other: ISO/IEC 17025:2005

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: PFW170183
- Expiration date of the lot/batch: no data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, protected from light

- purity: 98.83%
- molecular weight: 132.2 g/mol
- appearance: clear colorless liquid

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

Details on mammalian cell type (if applicable):

- TA98 and TA1537 are reverted from histidine dependence to histidine independence by framashift mutagens
- TA 1535 is reverted by mutagens that cause basepair substitutions
- TA100 is reverted by mutagens that cause both framashift and basepair substitutions mutations.
- E. coli is sensitive to basepair substitution mutationsrather than frameshift mutations

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.
- Test substance dilutions were prepared immediately before use and delivered to the test system at room temperature under filtered light.

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)
- Initial toxicity-mutation assay: 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 substance, in duplicate, in the presence and absence of metabolic activation. Dose levels for the confirmatory mutagenicity assay were based uppon post-treatment toxicity.
- Confirmatory mutagenicity assay: was used to evaluate and confirm the mutagenic potential of the test substance. TA98, TA100, TA1535, TA1537 and WP2 uvrA were exposed to the vehicle alone, positive controls and six dose levels of the test substance, in triplicate, in the presence and absence of metabolic activation.
-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 each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.
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. No precipitate was observed. Toxicity as reduction in revertant count was observed at 5000µg per plate with tester strain TA1535 in the presence of S9 activation.

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:

All criteria for a valid study were met. 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 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-06-05 - 2017-08-15

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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: PFW170183
- Expiration date of the lot/batch: 22 March 2019

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

OTHER SPECIFICS:
- Name of test material (as cited in study report): N-Aminopropyl Monomethylethanolamine
- Physical state: clear colorless liquid
- Analytical purity: 98.83%
- Molecular weight: 132.2 g/mol

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: American Type Culture Collection, Manassas, VA
- Suitability of cells: According to Guideline 473. In addition, 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: not indicated
- Sex, age and number of blood donors if applicable: not applicable
- Whether whole blood or separated lymphocytes were used if applicable: not applicable
- Number of passages if applicable: working cell stocks were not used beyond passage 15
- Methods for maintenance in cell culture if applicable: frozen
- Modal number of chromosomes: modal chromosome number of 20
- Normal (negative control) cell cycle time :average cell cycle time of 10-14 hours

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes, tested using the Hoechst staining procedure and found to be free of mycoplasma contamination
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: 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: 0.132, 0.396, 1.32, 3.96, 13.2, 39.6, 132, 396, 1320 µg/mL
Chromosome abberation assay- Non-activated (4h and 20h treatment): 10, 30, 100, 325, 650, 1320 µg/mL
Chromosome abberation assay- S9-activated (4h treatment): 10, 30, 100, 325, 650, 1320 µg/mL
The highest tested dose in the Preliminary toxicity test was 1320 µg/mL (=10 mM), which is the highest recommended dose in the OECD Guideline 473. Based on the results of the preliminary toxicity test, 1320 µg/mL was selected as the highest dose for the Chromosome aberration assay.

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 the laboratory, 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: in medium;
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 test item
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 control substance dosing solution
Test Substance: S9-activated: 3.5 mL culture medium + 1 mL S9 mix + 500 µL test substance 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: 4h (+/- S9) or 20h (-S9)
- Expression time (cells in growth medium): 16h (4h treatment) or 0h (20h treatment
- Fixation time (start of exposure up to fixation or harvest of cells): 24h

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 cultures at a final concentration of 0.1 µg/mL

STAIN (for cytogenetic assays):
Giemsa

NUMBER OF REPLICATIONS:
duplicate

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
For the definitive assay 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, activation 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 mammalian 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 aberrations.
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

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
Not applicable

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

- OTHER:

Evaluation criteria:

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 < or = 0.05) for a pairwise comparison of the frequency of aberrant cells in each treatment group with that of the vehicle control. The Cochran-Armitage trend test was used to assess dose-responsiveness.

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 initial pH of the highest dose of test substance in treatment medium was 9.5. The pH was adjusted using 1N HCl
- Effects of osmolality: The osmolality of the test substance dose in treatment medium was considered acceptable (<120% of vehicle): 285 mmol/kg at the highest dose level (vs 262 mmol/kg in vehicle control)
- Water solubility: soluble at 50 mg/mL
- Precipitation: no precipitation was observed
- 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.

RANGE-FINDING/SCREENING STUDIES:
CHO cells were exposed to vehicle alone and to nine concentrations of test substance with half-log dose spacing using single cultures. Precipitation of test substance dosing solution in the treatment medium was determined using unaided eye at the beginning and conclusion of treatment. The osmolality in treatment medium of the vehicle and that of the highest dose was measured. In the absence of cytotoxicity or visible precipitate in the treatment medium, the highest dose tested in the definitive chromosome aberration assay was 10 mM. Based on the results of the preliminary toxicity test, 1320 µg/mL was selected as the highest dose level in the Chromosome aberration assay.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: The results for the positive controls indicate that all criteria for a valid assay were met.
- Negative (solvent/vehicle) historical control data: The results for the vehicle controls indicate that all criteria for a valid assay were met.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxicity (>= 50% reduction in cell growth index relative to the vehicle control) was not observed at any dose in any of the three treatment groups in the preliminary toxicity test.
In the Chromosome aberration assay, mitotic inhibition of 1, 20 and 21% was observed for the 4h treatment without and with S9-activation, and for the 20h treatment, respectively.

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	10, 30, 100, 325, 650, 1320
	20 hr	0 hr	10, 30, 100, 325, 650, 1320
S9-activated	4 hr	16 hr	10, 30, 100, 325, 650, 1320

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)	Limit dose for this assay (10 mM)

Two additional doses were included in the evaluation.

Treatment of Target Cells (Preliminary Toxicity Test and Definitive Assay)

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

Conclusions:

Under the conditions of the assay described in this report, the test item was concluded to be negative for the induction of structural and numerical chromosome aberrations in the non-activated and S9-activated test systems in the in vitro mammalian chromosome aberration test using CHO cells.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-06-07 to 2017-08-02

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ISO/IEC 17025:2005

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
- Source and lot/batch No.of test material: PFW170183
- Expiration date of the lot/batch: no data


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, protected from light

- purity: 98.83%
- molecular weight: 132.2 g/mol
- description: clear colorless liquid

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: 2.58, 5.16, 10.3, 20.7, 41.3, 82.6, 165, 331, 661 and 1322 µg/mL with and without S9.
Definitive Mutagenicity Assay: 82.6, 165, 331, 661 and 1322 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. Based upon the results of the preliminary toxicity assay, the maximum concentration selected for the definitive mutagenicity assay was 1322 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 106 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 106 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 20 mL treatment medium, or 15 mL treatment medium plus5 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 (250 µL) 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 (eg comparison to historical control values).

Statistics:

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

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 ≥20.7 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 125.79 and 76.13% at a concentration of 1322 µg/mL with and without S9, respectively. Based upon these results, the concentrations chosen for the definitive mutagenicity assay were 82.6, 165, 331, 661 and 1322 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 96.30 and 90.32% at a concentration of 1322 µg/mL with and without S9, respectively. Cultures treated at all concentrations were chosen for mutant selection. No significant increases in mutant frequency , as compared to the concurrent vehicle controls, were observed at any concentration evaluated with or without S9 (p>0.01).

Conclusions:

Under the conditions of the assay described in this report, the test substance 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Genetic toxicity in vitro

Three in vitro toxicity studies are available.

Ames

The test substance was tested to evaluate its mutagenic potential by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system. Water was used as a vehicle. In the initial toxicity-mutation assay, the dose levels tested were 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate. No precipitate was observed. Toxicity as reduction in revertant count was observed at 5000 µg per plate with tester strain TA 1535 in the presence of S9 activation. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Based upon these results, the maximum dose tested in the confirmatory mutagenicity assay was 5000 µg per plate. In the confirmatory mutagenicity assay, the dose levels tested were 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate. 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. These results indicate that the test substance was negative for the ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system.

Chromosome aberration

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. 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, the doses tested ranged from 0.132 to 1320 µg/mL (10mM), which was the limit dose for this assay. Cytotoxicity (>=50% reduction in cell growth index relative to the vehicle control) was not observed at any dose in any of the three treatment groups. Based upon these results, the doses chosen for the chromosome aberration assay ranged from 10 to 1320 µg/mL for all three exposure groups. In the chromosome aberration assay, cytotoxicity (>=50% reduction in cell growth index relative to the vehicle control), was not observed at any dose in any of the three treatment groups. The doses selected for evaluation of chromosome aberrations were 325, 650, and 1320 µg/mL for all three exposure groups. No significant or dose-dependent increases in structural or numerical (polyploid or endoreduplicated cells) aberrations were observed in treatment groups with or without S9 (p>0.05; Fischer's Exact and Cochran-Armitage tests). These results indicate that the test substance was negative for the induction of structural and numerical chromosome aberrations in the presence and absence of the exogenous metabolic activation system.

Mammalian Cell forward Gene mutation (CHO/HPRT) assay

The test article was evaluated for its ability to induce 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, as assayed by colony growth in the presence of 6 -thioguanine (TG resistance). Water was used as the vehicle. In the preliminary toxicity assay, the concentrations tested were 2.58, 5.16, 10.3, 20.7, 41.3, 82.6, 165, 331, 661 and 1322 µg/mL. The maximuù concentration evaluated was based on solubility limitations of the test article in the vehicle. No visible precipitate was observed at the beginning or end of treatment. Adjusted relative survival was 125.79 and 76.13% at a concentration of 1322 µg/mL with and without S9, respectively. Based upon these results, the concentrations chosen for the definitive mutagenicity assay were 82.6, 165, 331, 661 and 1322 µg/mL with and without S9. In the definitive mutagenicity assay, no visible precipitate was observed at the beginning or end of treatment. The average adjusted relative survival was 96.30 and 90.32% at a concentration of 1322 µg/mL with and without S9, respectively. Cultures treated at all concentrations were chosen for mutant selection. No significant increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at any concentration evaluated with or without S9 (p>0.01). the positive controls induced significant increases in mutant frequency (p<0.01). These results indicate that the test substance was negative for the ability to induce 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.

Justification for classification or non-classification

Based on the results, the test substance is not classified as mutagenic, according to CLP regulation.