Reaction mass of 2-hydroxy-N-(2-(2-hydroxyethoxy)ethyl)-N-(2-hydroxyethyl)-N-methylethan-1-amininium hydroxide and 2-hydroxy-N,N-bis(2-hydroxyethyl)-N-methylethanaminium hydroxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Bacterial reverse mutation assay

In a K1 bacterial reverse mutation assay in Salmonella typhimurium strains TA98, TA100, TA1535,  TA1537 and Escherichia coli WP2 uvrA performed according to OECD Guideline 471, it was concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay in the absence and presence of metabolic activation.

- Chromosome aberration study

In a K1 in vitro chromosome aberration study in Chinese Hamster Ovary (CHO) Cells performed according to the OECD Guideline 473, the test item was considered to be negative for the induction of structural and numerical chromosome aberrations in the non-activated and S9-activated test systems.

-         In Vitro Mammalian Cell Forward Gene Mutation

In an in vitro mammalian cell forward gene mutation (CHO/HPRT) assay with duplicate cultures performed according to the OECD Guideline 476, 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.

Some of the data points both in the presence and absence of S9 were outside of the laboratory's historical range. Although according to the protocol the test item is considered equivocal, the registrant considers the test item to be negative for induction of forward mutations at the HPRT locus of CHO cells as no statistically significant increases compared to the solvent or a statistically significant dose related trend was observed.

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:

17 August 2017 - 31 August 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: no data
- Expiration date of the lot/batch: no data
- Purity test date: no data


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

OTHER SPECIFICS
Substance of Unknown or Variable Composition, Complex Reaction Products and Biological Materials

Target gene:

Histidine locus (Salmonella strains) and tryptophan locus (E. coli strain)

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

not specified

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
Confirmatory Mutagenicity Assay: 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate

In the Initial toxicity-mutation assay, the maximum recommended dose level of 5000 µg/plate was selected as the highest dose level. Based upon the results of the initial toxicity-mutation assay, the maximum dose level selected for the confirmatory mutagenicity assay was 5000 µg per plate.

Vehicle / solvent:

- Vehicle 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. The test substance formed a clear solution in water at a concentration of approximately 50 mg/mL in the solubility test conducted at the laboratory.

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 and TA1535; 2.0 µg/plate for TA100 and TA1537; 15.0 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 and 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 substance dilution were added to 2.0 mL of molten selective top agar at 45±2°C. When plating the positive controls, the test substance 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: 2 (initial toxicity-mutation assay) or 3 (confirmatory mutation assay)

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 substance 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 substance 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:

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
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- 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 substance 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 substance, 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: positive controls induced positive responses
- Negative (solvent/vehicle) historical control data: vehicle controls induced numbers of revertants within the historical control data

Conclusions:

All criteria for a valid study were met as described in the protocol. The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this 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:

07 August 2017 - 15 May 2019

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

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: D26-3-3-5

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

OTHER SPECIFICS:
Substance of Unknown or Variable Composition, Complex Reaction Products and Biological Materials

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 OECD Guideline
- Modal number of chromosomes: 20
- Normal (negative control) cell cycle time: 10-14 hours

MEDIA USED
- Type and identity of media:
Exponentially growing CHO K1 cells were seeded in complete medium (McCoy's 5A medium containing 10% fetal bovine serum, 1.5 mM L glutamine, 100 units/mL penicillin, 100 µg/mL streptomycin and 2.5 µg/mL Amphotericin B) for each treatment condition at a target of 5 x 10^5 cells/culture. The cultures were incubated under standard conditions (37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2 in air) for 16-24 hours.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes, using the Hoechst staining procedure
- Periodically checked for karyotype stability: no, 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: no data

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 was used as the metabolic activation system.

Test concentrations with justification for top dose:

Initial Toxicity-Mutation Assay: from 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/mL in both the absence and presence of S9-mix;
Confirmatory Mutagenicity Assay: 250, 1000, 2500, and 5000 µg/mL (4 hours in the absence and presence of S9, and for 20 hours in the absence of S9);

5000 µg/mL was selected as the highest dose level in the initial toxicity-mutation assay as this is the limit dose for the assay recommended in the guideline. Based upon these results, the maximum dose chosen for the chromosome aberration assay was 5000 µg/mL for all three exposure groups.

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:

mitomycin C

Remarks:

without S9-mix; at 0.1 and 0.2 µg/mL

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9-mix; at 2.5, 5, and 7.5 µg/mL

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 vehicle
Vehicle (S9-activated): 3.5 mL culture medium + 1 mL S9-mix + 500µL vehicle
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 positive control dosing solution
Positive Control (S9-activated): 4 mL culture medium + 1 mL S9-mix + 50 µL positive control 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 and 20 hours without S9 and for 4 hours with S9
- Expression time (cells in growth medium): 16 and 0 hours, after 4 and 20 hours exposure respectively
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours (+- 30 minutes)

SPINDLE INHIBITOR (cytogenetic assays): Colcemid

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicate

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
For the preliminary toxicity test and definitive assay, cells were collected 20 hours (± 30 minutes), 1.5 normal cell cycles, after initiation of treatment to ensure that the cells are analyzed in the first division metaphase. Just prior to harvest, the cell cultures was visually inspected for the degree of monolayer confluency relative to the vehicle control. The cells were trypsinized and counted and the cell viability was assessed using trypan blue dye exclusion.
The cell count was determined from a minimum of two cultures to determine the number of cells being treated (baseline). The data were presented as cell growth inhibition in the treatment group compared to vehicle control. Cell growth was determined by Relative Increase in Cell Counts (RICC) as a measure of cytotoxicity. The cell counts and percent viability were used to determine cell growth inhibition (CGI) relative to the vehicle control (% cytotoxicity = 100 – RICC).
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 BioReliance study number, dose, treatment condition, harvest date, activation system, test phase, and replicate tube design.

NUMBER OF CELLS EVALUATED: minimum 300 per dose

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

DETERMINATION OF CYTOTOXICITY
- Method: > or = 50% reduction in cell growth index relative to the vehicle control

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

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 =< 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.

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 initial pH of the highest dose of test substance in treatment medium was 10.0. The pH was adjusted using 1N HCl
- Effects of osmolality:
Preliminary toxicity-mutation assay: Since the osmolality of the highest dose in the treatment medium was slightly high (>120% of vehicle), the Study Director was consulted and per his directive, the slight increase in osmolality was considered acceptable for the purpose of the study.
- Water solubility: the test article was soluble in water at a concentration of 50 mg/mL, the maximum concentration tested
- Precipitation: no precipitation observed, the test substance was soluble in the treatment medium at all doses tested

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 test substance was soluble in the treatment medium at all doses tested at the beginning and conclusion of the treatment period. The osmolality was slightly increased, but was considered acceptable for the puropose of the study.
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 the results of the preliminary toxicity assay, the highest dose tested in the definitive chromosome aberration assay was 5000 µg/mL.


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.

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:

24 July 2017 - 14 September 2017

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
- Lot/batch No.of test material: D26-3-3-5
- Expiration date of the lot/batch: March 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protected from light
- Test substance dilutions were prepared immediately before use and delivered to the test system at room temperature under filtered light.

OTHER SPECIFICS:
- measurement of pH, osmolality, and precipitate in the culture medium to which the test chemical is added: Lower concentrations were prepared by 2-fold dilutions. The pH of the treatment medium was measuredadn adjusted when necessaryto maintain neutral pH in the treatment medium at the concentrations tested. The osmolality of the vehicle control and highest dose level in treatment medium also was measured at the beginning of treatment. Precipitation was assessed at the beginning and end of treatment.

Target gene:

The purpose of this study was to evaluate a test substance for its ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, as assayed by colony growth in the presence of 6-thioguanine (TG resistance, TGr).

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 in 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, frozen stock cultures were tested to confirm the absence of mycoplasma contamination
- Periodically checked for karyotype stability: yes; Cells used in the mutation assay did not exceed 15 passages from frozen stock
- Periodically 'cleansed' against high spontaneous background: yes, to reduce the frequency of spontaneous HPRT- mutants prior to use in an assay, the cells were cleansed in medium supplemented with hypoxanthine, aminopterin and thymidine (HAT).

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:

In the initial preliminary toxicity assay, the concentrations tested were 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 μg/mL. In order to achieve the maximum concentration for UVCB test substance per guidance, a second preliminary toxicity assay was performed to a maximum concentration of 5000 μg/mL.
In the second preliminary toxicity assay, the concentrations tested were 1000, 2000, 3000, 4000 and 5000 μg/mL. The maximum concentration evaluated approximated the limit dose for this assay.
Based upon second preliminary toxicity assay results, the top dose chosen for initial definitive mutagenicity assay was 5000 μg/mL. The concentrations chosen for the initial definitive mutagenicity assay were 313, 625, 1250, 2500 and 5000 μg/mL with and without S9.
In the confirmatory mutagenicity assay, the concentrations tested were 313, 625, 1250, 2500 and 5000 μg/mL with and without S9. The top dose was based on the results of the initial definitive mutagenicity assay.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionised 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. The test substance 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; at 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; at 4.00 µL/mL

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicates
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
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 4.5 mL treatment medium, or 3.5 mL treatment medium plus 1 mL S9 mix, as appropriate. Following addition of the test or control substance dose formulations (500 μL) to the flasks, the cultures were incubated under standard conditions for 5 ± 0.5 hours.

CHO/HPRT Mutagenicity Assays
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 10 mL treatment medium, or 8 mL treatment medium plus 2 mL S9 mix (adjusted for the test substance dose volume if >1%, v/v), as appropriate. Following addition of the test or control substance formulations (2.1 mL) to the flasks, the cultures were incubated under standard conditions for 5 ± 0.5 hours (positive control substances were prepared in DMSO and added to the flasks using a 1% dose volume).

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 5h (+-0.5h)
- Harvest time after the end of treatment (sampling/recovery times): 14 days

- Expression time (cells in growth medium between treatment and selection): 7 days
- Selection time (if incubation with a selective agent): 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 14 days
- Selective agent used: 6-thioguanine at 10 μM in 30 mL Complete Ham’s F12-Hx
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: selection and to determine cloning efficiency at the time of selection. At the end of the phenotypic expression period, the cultures were washed twice with CMF-HBSS and 1 x 10^6 cells from each culture were plated at a density of 6 x 10^5 cells/150-mm plate (4 plates total) in 30 mL Complete Ham’s F12-Hx containing 10 μM TG. Three 60-mm plates also were plated, at 200 cells/plate in 5 mL Complete Ham’s F12-Hx in triplicate, to determine the cloning efficiency at the time of selection. The plates were incubated under standard conditions for 7 days.
After the 7- day incubation period, the colonies were fixed with methanol, stained with crystal violet and counted. Mutant frequencies were expressed as the number of TGr mutants/10^6 clonable cells. The number of clonable cells was determined from the triplicate 60-mm plates.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
adjusted relative survival (RS, %; 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.). In such cases, the Study Director used sound scientific judgment and clearly reported and described any such considerations.

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:

not applicable

Positive controls validity:

not valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
Preliminary toxicity assay:
- Data on pH: The pH of the cultures was adjusted at concentrations ≥250 μg/mL with and without S9 to maintain neutral pH.
- Data on osmolality: The osmolality of the cultures was acceptable as it did not exceed the osmolality of the vehicle control by more than 120%.
- Precipitation and time of the determination: No visible precipitate was observed at the beginning or end of treatment.

Second preliminary toxicity assay:
- Data on pH: The pH of the cultures was adjusted at concentrations ≥1000 μg/mL with and without S9 to maintain neutral pH.
- Data on osmolality: The osmolality of the cultures was acceptable as it did not exceed the osmolality of the vehicle control by more than 120%.
- Precipitation and time of the determination: No visible precipitate was observed at the beginning or end of treatment.

Definitive Mutagenicity Assays
- Data on pH: The pH of the cultures was adjusted at concentrations ≥313 μg/mL with and without S9 to maintain neutral pH.
- Data on osmolality: The osmolality of the cultures was acceptable as it did not exceed the osmolality of the vehicle control by more than 120%.
- Precipitation and time of the determination: No visible precipitate was observed at the beginning or end of treatment.

RANGE-FINDING/SCREENING STUDIES:
Preliminary toxicity assay:
Cells were treated with 10 test substance concentrations in the initial preliminary toxicity assay and 5 test substance concentrations in the second preliminary toxicity assay, as well as the vehicle control, in the presence and absence of S9 using single cultures. Adjusted relative survival was 94.40 and 85.71% at a concentration of 2000 μg/mL with and without S9, respectively.
Second preliminary toxicity assay: Adjusted relative survival was 123.23 and 141.32% at a concentration of 5000 μg/mL with and without S9, respectively.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: The positive controls induced significant increases in mutant frequency (p < 0.01). All positive and vehicle control values were within acceptable ranges, and all criteria for a valid assay were met.
- Results from cytotoxicity measurements:
Definitive Mutagenicity Assay: The average adjusted relative survival was 68.39 and 104.8% at a concentration of 5000 μg/mL with and without S9, respectively.
Confirmatory Mutagenicity Assay: The average adjusted relative survival was 99.92 and 83.15% at a concentration of 5000 μg/mL with and without S9, respectively.
- Genotoxicity results:
Definitive Mutagenicity Assay: 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). While no statistically significant increases compared to the solvent or a statistically significant dose related trend was observed, some of the data points both in the presence and absence of S9 were outside of the laboratory historical range. The result is an equivocal per protocol.
Confirmatory Mutagenicity Assay: 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). No statistically significant increases compared to the solvent or a statistically significant dose related trend was observed, no data points both in the presence and absence of S9 were outside of the laboratory's historical range. The results were concluded to be negative per protocol criteria.

HISTORICAL CONTROL DATA (95% control limits, per million cells)
- Positive historical control data:
With S9: 7.8-301.0
Without S9: 0-529.9
- Negative (solvent/vehicle) historical control data:
With S9: 0.0-11.0
Without S9: 0.0-10.6

Conclusions:

The results were concluded to be negative per protocol criteria. 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Bacterial reverse mutation assay:

A key study (K1, Pant, 2017) was performed according to OECD Guideline 471 .

The test item was tested in the Salmonella typhimurium reverse mutation assay strains TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA. Water was used as the vehicle.

In the initial toxicity-mutation assay, the dose levels tested were 1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate. Neither precipitate nor toxicity were observed. 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 were 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 item 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.

In vitro Chromosome aberration test:

A key study (K1, Roy, 2017) performed an in vitro chromosome aberration study in Chinese Hamster Ovary (CHO) Cells according to OECD Guideline 473.

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 0.5 to 5000 µg/mL (the limit dose for this assay per OECD guidelines for UVCB test substances). 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 250 to 5000 µg/mL for all three exposure groups.

In the chromosome aberration assay, cytotoxicity was not observed at any dose in any of the three treatment groups. The doses selected for evaluation of chromosome aberrations were 1000, 2500, and 5000 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; Fisher’s Exact and Cochran-Armitage tests).

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

 In Vitro Mammalian Cell Forward Gene Mutation

A key study (K1, Pant, 2019) performed an in vitro mammalian cell forward gene mutation (CHO/HPRT) assay with duplicate cultures according to OECD Guideline 476.

Water was used as the vehicle. In the initial preliminary toxicity assay, the concentrations tested were 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 µg/mL. 

No visible precipitate was observed at the beginning or end of treatment. Adjusted relative survival was 94.40 and 85.71% at a concentration of 2000 µg/mL with and without S9, respectively.   

In order to achieve the maximum concentration for UVCB test substance, a second preliminary toxicity assay was performed. 

In the second preliminary toxicity assay, the concentrations tested were 1000, 2000, 3000, 4000 and 5000 µg/mL. The maximum concentration evaluated approximated the limit dose for this assay. No visible precipitate was observed at the beginning or end of treatment. Adjusted relative survival was 123.23 and 141.32% at a concentration of 5000 µg/mL with and without S9, respectively. Based upon these results, the concentrations chosen for the definitive mutagenicity assay were 313, 625, 1250, 2500 and 5000 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 68.39 and 104.8% at a concentration of 5000 µg/mL with and without S9, respectively. Cultures treated at all concentrations with and without S9 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). 

While no statistically significant increases compared to the solvent or a statistically significant dose related trend was observed, some of the data points both in the presence and absence of S9 were outside of the laboratory's historical range. Although according to the protocol the test item is considered equivocal, the registrant considers the test item to be negative for induction of forward mutations at the HPRT locus of CHO cells as no statistically significant increases compared to the solvent or a statistically significant dose related trend was observed

In the confirmatory mutagenicity assay, the concentrations tested were 313, 625, 1250, 2500 and 5000 μg/mL. No visible precipitate was observed at the beginning or end of treatment. The average adjusted relative survival was 99.92 and 83.15% at a concentration of 5000 μg/mL with and without S9, respectively. Cultures treated at all concentrations with and without S9 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). No statistically significant increases compared to the solvent or a statistically significant dose related trend was observed, no data points both in the presence and absence of S9 were outside of the laboratory historical range. The results were concluded to be negative per protocol criteria. 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 negative results observed in in vitro genetic toxicity tests and the criteria of the CLP Regulation (EC) 1272/2008, the test item should not be classified for mutagenicity.