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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 E. coli WP2 uvrA, performed according to OECD Guideline 471, it was concluded that the test item 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 Chromosomal 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 negative for the induction of structural and numerical chromosomal aberrations in the presence and absence of an exogenous metabolic activation system.

 

In vitro gene mutation study in mammalian cells: In a K1 in vitro Chinese Hamster Ovary Cell Forward Gene Mutation Assay, performed according to the OECD Guideline 476, it was concluded that the test substance was negative for the induction of forward mutations at the HPRT locus of CHO cells in the presence and the absence of an exogenous metabolic activation system. 

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Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Bacterial Reverse Mutation Assay
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 January 2011 - 26 January 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
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:
- Expiration date of the lot/batch: not specified

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, stored protected from light without desiccant
- Stability under test conditions: not specified
- Solubility and stability of the test substance in the solvent/vehicle: not specified

OTHER SPECIFICS:
- Purity: 100%
- Molecular weight : 178.2
- Appearance: clear colorless liquid
Target gene:
Histidine locus (histidine-dependent S. typhimurium strains); Tryptophan locus (tryptophan-dependent E. coli strains)
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 (B1): 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 µg per plate with and without S9-mix;
Confirmatory Mutagenicity Assay (B2): 5.0, 15, 50, 150, 500, 1500 and 5000 µg per plate with tester strain TA100 in the absence of S9 activation and 15, 50, 150, 500, 1500 and 5000 µg per plate with and without S9-mix;

The top dose of 5000 µg/plate in the Initial toxicity-mutation assay was selected based on the maximum recommended dose according to the Guideline. Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the confirmatory mutagenicity assay was 5000 µg/plate.
Vehicle / solvent:
- Vehicle used: - DMSO
- Justification for choice of solvent/vehicle: Dimethyl sulfoxide (DMSO) was selected as the solvent of choice based on the solubility of the test substance and compatibility with the target cells. The test substance formed a soluble and clear solution in DMSO at approximately 500 mg/mL, the maximum concentration tested in the solubility test.
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 and TA1537; 2.0 µg/plate for TA100; 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)
On the day of its use, minimal top agar, containing 0.8 % agar (W/V) and 0.5 % NaCl (W/V), was melted and supplemented with L-histidine, D-biotin and L-tryptophan solution to a final concentration of 50 μM each. Top agar not used with S9 or Sham mix was supplemented with 25 mL of water for each 100 mL of minimal top agar. For the preparation of media and reagents, all references to water imply sterile, deionized water. Bottom agar was Vogel-Bonner minimal medium E containing 1.5 % (W/V) agar. Nutrient bottom agar was Vogel-Bonner minimal medium E containing 1.5 % (W/V) agar and supplemented with 2.5 % (W/V) Oxoid Nutrient Broth No. 2 (dry powder). Nutrient Broth was Vogel-Bonner salt solution supplemented with 2.5 % (W/V) Oxoid Nutrient Broth No. 2 (dry powder).
Each plate was labeled with a code system that identified the test substance, test phase, dose level, tester strain and activation, as described in detail inthe laboratory's Standard Operating Procedures.
One-half (0.5) milliliter of S9 or Sham mix, 100 μL of tester strain (cells seeded) and 50 μL of vehicle or test substance dilution were added to 2.0 mL of molten selective top agar at 45±2°C. After vortexing, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. When plating the positive controls, the test substance aliquot was replaced by a 50 μL aliquot of appropriate positive control. After the overlay had solidified, the plates were inverted and incubated for approximately 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); Tryptophan (E. coli tryptophan-dependent strains)

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of revertants; reductino in the bacterial 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.
Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 3.0-times the mean vehicle control value. Data sets for tester strains TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 2.0-times the mean vehicle control value.
An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response will be evaluated as negative, if it is neither positive nor equivocal.
Statistics:
No formal hypothesis testing was done.
Species / strain:
other: TA98, TA100, TA1535, TA1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 500, 1500 or 5000 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 500, 1500 or 5000 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: not specified
- 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. Vehicle control, positive controls and a minimum of eight dose levels of the test substance were plated, two plates per dose, with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA on selective minimal agar in the presence and absence of Aroclor-induced rat liver S9.
No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. No precipitate was observed. Toxicity was observed beginning at 500 or 1500 μg per plate with most test conditions. Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the confirmatory mutagenicity assay was 5000 μg per plate..

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: the positive control chemicals induced clear positive responses
- Negative (solvent/vehicle) historical control data: vehicle control data were with the historical control data range


ADDITIONAL INFORMATION ON CYTOTOXICITY:
Initial Toxicity-Mutation Assay (B1): Toxicity was observed beginning at 500 or 1500 μg per plate with most test conditions.
Confirmatory Mutagenicity Assay (B2): Toxicity was observed beginning at 500, 1500 or at 5000 µg per plate with most test conditions.
Conclusions:
Interpretation of the results: negative with and without metabolic activation
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.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 February 2018 - 06 April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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 substance: 20141015
- Expiration date of the lot/batch: Jan 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test substance: 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): JEFFSOL® AG-1700
- Physical state: clear colorless liquid
- Analytical purity: 99.35%
- Molecular weight: 178.23 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: ranged from 0.178 to 1780 µg/mL.
Chromosome abberation assay- Non-activated (4h and 20h treatment): 28.05, 56.10, 66.00, 77.65, 91.35, 107.47, 126.44, 148.75, 175.00 µg/mL
Chromosome abberation assay- S9-activated (4h treatment): 32.06, 64.12, 75.43, 88.74, 104.40, 122.83, 144.50, 170.00, 200.00 µg/mL
The highest tested dose in the Preliminary toxicity test was 1780 µg/mL (=10 mM), which is the highest recommended dose in the OECD Guideline 473. Based on the results of the preliminary toxicity test, 200 µg/mL was selected as the highest dose for the Chromosome aberration assay.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO 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 DMSO at a concentration of approximately 500 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:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): duplicate
- Number of independent experiments: 3

METHOD OF TREATMENT/ EXPOSURE: 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: 5 mL culture medium + 50 µL test item
Vehicle: S9-activated: 4 mL culture medium + 1 mL S9 mix + 50 µL control substance dosing solution
Test Substance: Non-activated: 5 mL culture medium + 50 µL control substance dosing solution
Test Substance: S9-activated: 4 mL culture medium + 1 mL S9 mix + 50 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.

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4h (+/- S9) or 20h (-S9)
- Harvest time after the end of treatment (sampling/recovery times): 20h

FOR CHROMOSOME ABERRATION:
- Spindle inhibitor (cytogenetic assays): 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
- Methods of slide preparation and staining technique used including the stain 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 spread and analysed per concentration (number of replicate cultures and total number of cells scored): 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 scoring chromosome aberrations (selection of analysable cells and aberration identification):
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.
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.
- Determination of polyploidy: evaluated for 150 cells per culture (a total of 300 per dose level).
- Determination of endoreplication: evaluated for 150 cells per culture (a total of 300 per dose level).

METHODS FOR MEASUREMENT OF CYTOTOXICITY: The mitotic index was recorded as the percentage of cells in mitosis per 500 cells counted.


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.
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 7.5.
- Effects of osmolality: The osmolality of the test substance dose in treatment medium was considered acceptable: 429 mmol/kg at the highest dose level (vs 422 mmol/kg in vehicle control)
- Water solubility: no data
- Precipitation and time of the determination:
Preliminary Toxicity Assay: Visible precipitate was observed in treatment medium at ≥ 178 μg/mL in all three exposure groups
Main assay: Visible precipitate was observed in treatment medium at the end of the treatment period at 175 µg/ml in the non-activated groups (4h and 20h epxosure), and 200 µg/mL for the S9-activated 4h exposure period group.

- 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. Cytotoxicity (≥ 50% reduction in cell growth index relative to the vehicle control) and precipitate were observed at doses ≥ 178 μg/mL in all three exposure groups. Based on the results of the preliminary toxicity test, 200 µg/mL was selected as the highest dose level in the Chromosome aberration assay.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: The results for the positive and vehicle controls indicate that the following criteria for a valid assay were met.
- Results from cytotoxicity measurements: In the Chromosome aberration assay, mitotic inhibition of 36, 3 and 10% was observed for the 4h treatment without and with S9-activation, and for the 20h treatment, respectively.
- Genotoxicity results
No significant or dose-dependent increases in structural aberrations were observed at any dose in treatment groups with or without S9 (p > 0.05; Fisher’s Exact and Cochran-Armitage tests). No significant or dose-dependent increases in numerical (polyploid or endoreduplicated cells) aberrations were observed at any dose in the non-activated 4 and 20-hour exposure groups (p > 0.05; Fisher’s Exact and Cochran-Armitage tests).
In the S9-activated 4-hour exposure group, a statistically significant increase in numerical (polyploid or endoreduplicated cells) aberrations (5.7%) was observed at 144.5 μg/mL (p ≤ 0.05; Fisher’s Exact test). However, the Cochran-Armitage test was negative for a dose response (p > 0.05). In addition, the induction of numerical aberrations was within the historical 95% control limit of 0.00% to 6.35%. Therefore the statistically significant increase in numerical aberrations was considered biologically irrelevant.

HISTORICAL CONTROL DATA (95% control limits data range )
- Positive historical control data for structural aberrations:
Without S9, 4h exposure: 10.25 - 31.88 %
Without S9, 20h exposure: 10.45 - 31.29 %
With S9, 4h exposure: 11.28 - 41.92 %

- Negative (solvent/vehicle) historical control data for structural aberrations:
Without S9, 4h exposure: 0.00 - 2.41 %
Without S9, 20h exposure: 0.00 - 2.21%
With S9, 4h exposure: 0.00 - 3.88 %

- Negative (solvent/vehicle) historical control data for numerical aberrations:
Without S9, 4h exposure: 0.00 - 3.72 %
Without S9, 20h exposure: 0.00 - 4.03 %
With S9, 4h exposure: 0.00 -6.35 %

Conclusions:
Under the conditions of the assay described in this report, the test substance was concluded to be negative for the induction of structural and numerical chromosomal aberrations in the non-activated and S9-activated test systems in the in vitro mammalian chromosome aberration test using CHO cells.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 January 2018 to 09 May 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
updated and adopted 29 July 2016
Deviations:
no
Qualifier:
according to
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: AF18YR.783REACH.BTL
- Expiration date of the lot/batch: January 2019
- purity: 99.35%
- molecular weight: 178.23 g/mol
- description: clear colorless liquid

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at 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: The pH of the treatment medium was measured. The osmolality of the solvent control, highest dose level, lowest precipitating dose level and the highest soluble dose level in treatment medium also was measured at the beginning of treatment. Precipitation was assessed at the beginning and end of treatment. Concentrations evaluated in the definitive mutation assay were based on adjusted relative survival, calculated as described below.

Target gene:
hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Remarks:
CHO-K1-BH4
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: CHO-K1-BH4, obtained form Dr. Abraham W. Hsie, Biology Division, Oak Ridge National Laboratory, Oak Ridge, TN
- Suitability of cells: recommended by Guideline
- Absence of Mycoplasma contamination: yes
- Number of passages if applicable: cells did not exceed 15 passages from frozen stock.
- Methods for maintenance in cell culture: see relevant section below
- Cell cycle length, doubling time or proliferation index: population doubling time of 12-14 hours
- Modal number of chromosomes: 20
- Periodically checked for karyotype stability: yes
- Periodically ‘cleansed’ of spontaneous mutants: yes,cleansed in medium supplemented with hypoxanthine, aminopterin and thymidine

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, 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.
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:
The maximum concentration evaluated approximated the limit dose (10 mM) for this assay. Preliminary Toxicity Assay: 3.48, 6.96, 13.9, 27.8, 55.7, 111, 223, 446, 891 and 1782 µg/mL with and without S9.
The maximum concentration evaluated was based on solubility limitations (without S9) and cytotoxicity considerations (with S9)observed in the preliminary toxicity assay.
Definitive Mutagenicity Assay: 112.5, 300, 500, 1000 and 1782 µg/mL with S9 and 14.1, 28.1, 56.3, 112.5, 225 and 400 µg/mL without S9.

Based on the results obtained in the definitive mutagenicity assay, the condition without S9 was repeated at lower doses:
Confirmatory Mutagenicity Assay without S9: 12.5, 25, 50, 75, 100, 112.5, 150 and 300 µg/mL
Repeat Confirmatory Mutagenicity Assay without S9: 12.5, 25, 50, 55, 60, 65, 70, 75 and 100 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO 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 DMSO at a concentration of approximately 500 mg/mL in the solubility test conducted at BioReliance.
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:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): duplicates
- Number of independent experiments: 3

METHOD OF TREATMENT/ EXPOSURE:
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 (for +S9) and in 225-cm2 cultures at a density of ~2.67 x 106 in 30 mL Complete Ham’s F12 (for -S9). 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 [650 µL (for –S9) and 250 µL (for +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).

TREATMENT AND HARVEST SCHEDULE: - Exposure duration/duration of treatment: : 5h (+-0.5h)
- Harvest time after the end of treatment (sampling/recovery times): ): 14 days
FOR GENE MUTATION: - Expression time (cells in growth medium between treatment and selection): 7 days
- Selection time (if incubation with a selective agent): 7 day
- Fixation time (start of exposure up to fixation or harvest of cells): 14 days
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure. 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 mutant cells: Hypoxanthine-free Complete Ham’s F12 (Complete Ham’s F12-Hx) was used for mutant 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 2.4 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
No. of Cells Analyzed/Culture: 1 x 10^6

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: 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 article was considered to have produced a positive response if it induced a statistically significant and dose-dependent increase in mutant frequency (p≤ 0.01) that exceeded the 95% confidence limit of the historical vehicle control data from this laboratory. If only one criterion was met (a statistically significant or dose-dependent increase or an increase exceeding the historical control 95% confidence interval), the results 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.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Remarks:
Definitive Mutagenicity Assay
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
Chinese hamster Ovary (CHO)
Remarks:
Definitive Mutagenicity Assay
Metabolic activation:
without
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
Chinese hamster Ovary (CHO)
Remarks:
Confirmatory Mutagenicity Assay
Metabolic activation:
without
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Remarks:
Repeat of Confirmatory Mutagenicity
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The test substance did not have an adverse impact on the pH of the cultures (pH 7.5 at the top dose).- 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%.
- Precipitation: Precipitation was assessed at the beginning and end of treatment.
Preliminary Toxicity Assay: No precipitate was observed in the presence of metabolic activation. In absence of metabolic activation, visible precipitate was observed at 1782 μg/mL at the end of treatment period.
Definitive Mutagenicity Assay: Visible precipitate was observed at the end of treatment period at concentrations ≥ 112.5 μg/mL without S9 and ≥ 1000 μg/mL with S9.
Confirmatory Mutagenicity Assay without S9: Visible precipitate was observed at the end of treatment period at concentrations ≥75 μg/mL.
Repeat Confirmatory Mutagenicity Assay without S9: Visible precipitate was observed at the end of treatment period at concentrations ≥70 μg/mL.

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. Adjusted relative survival was 62.50 and 19.58% at a concentration of 1782 µg/mL with S9 and 223 µg/mL without S9, respectively. Adjusted relative survival was approximated 0% at all higher concentrations without S9.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: The positive controls induced significant increases in mutant frequency (p < 0.01) confirming the acceptable of the assay in the three independent assays. 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 94.32 and 15.18% at a concentration of 1782 μg/mL with S9 and 400 μg/mL without S9, respectively.
Confirmatory Mutagenicity Assay without S9: The average adjusted relative survival was 62.16% at a concentration of 75 μg/mL without S9.
Repeat Confirmatory Mutagenicity Assay without S9: The average adjusted relative survival was 38.42% at a concentration of 100 μg/mL without S9.

- Genotoxicity results:
Definitive Mutagenicity Assay: Cultures treated at concentrations of 112.5, 300, 500 and 1000 μg/mL with S9 and 14.1, 28.1, 56.3 and 112.5 μg/mL 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 S9 (p > 0.01). Significant increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at 112.5 μg/mL without S9 (p < 0.01). Although the response observed was outside the historical control range, there was no dose dependent trend observed. Therefore, this response did not meet all three criteria for a positive response without S9 and is considered to be equivocal. The positive controls induced significant increases in mutant frequency (p < 0.01) confirming the acceptable of the assay. The OECD Guideline 476 allows for the repeat of all or part of an assay, if the results are equivocal. The condition without S9 was considered equivocal; therefore, this portion of the assay was repeated at lower doses.

Confirmatory Mutagenicity Assay without S9: Cultures treated at concentrations of 12.5, 25, 50 and 75μg/mL was chosen for mutant selection. Although there was no significant increases in mutant frequency, as compared to the concurrent vehicle controls at any concentration evaluated without S9 (p > 0.01), there was a dose-related trend observed per statistical analysis. One of the replicates at the top dose (75 μg/mL) demonstrated a mutant frequency outside the 95% historical control. Therefore, this response did not meet all three criteria for a positive response without S9 and is considered to be equivocal. The positive controls induced significant increases in mutant frequency (p < 0.01), per the assay criteria. Again, the OECD Guideline 476 allows for the repeat of all or part of an assay, if the results are equivocal. The condition without S9 was considered equivocal; therefore, this portion of the assay was repeated at lower doses.
Repeat Confirmatory Mutagenicity Assay without S9: Cultures treated at concentrations of 12.5, 50, 55, 60, 65, 70 and 100 μg/mL was chosen for mutant selection. There was no significant increases in mutant frequency, as compared to the concurrent vehicle controls at any concentration evaluated without S9 (p > 0.01), no mutant frequency outside the 95% historical control, and no dose-related trend observed per statistical analysis. The positive controls induced significant increases in mutant frequency (p < 0.01), per the assay criteria. Therefore, this response did not meet all three criteria for a positive response without S9 and is considered to be negative.

HISTORICAL CONTROL DATA (95% control limits, per million cells)
- Positive historical control data:
With S9: 0-348
Without S9: 89-485
- Negative (solvent/vehicle) historical control data:
With S9: 0-13
Without S9: 0-13
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

Bacterial reverse mutation assay

In K1 bacterial reverse mutation assay (Wagner and VanDyke, 2011), performed according to the OECD Guideline 471, the test substance was tested in Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA in the presence and absence of Aroclor-induced rat liver S9. The assay was performed in two phases, using the plate incorporation method.

Dimethyl sulfoxide (DMSO) was selected as the solvent of choice based on the solubility of the test substance and compatibility with the target cells.

In the initial toxicity-mutation assay, the maximum dose tested was 5000 μg per plate; this dose was achieved using a concentration of 100 mg/mL and a 50 μL plating aliquot. The dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. No precipitate was observed. Toxicity was observed beginning at 500 or 1500 μg per plate with most test conditions. Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the confirmatory mutagenicity assay was 5000 μg per plate.

In the confirmatory mutagenicity assay, no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. The dose levels tested were 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate with tester strain TA100 in the absence of S9 activation and 15, 50, 150, 500, 1500 and 5000 μg per plate with the remaining test conditions. No precipitate was observed. Toxicity was observed beginning at 500, 1500 or at 5000 μg per plate with most test conditions.

Under the conditions of the study, the test substance was concluded to be negative in the Bacterial Reverse Mutation Assay.

 

In vitro chromosomal aberration assayin Chinese hamster ovary cells

In K1 chromosome aberration study performed according to OECD Guideline 473 (Roy, 2019), the test substance was tested to evaluate its potential to induce structural chromosomal aberrations using 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. DMSO was used as the vehicle.

In the preliminary toxicity assay, the doses tested ranged from 0.178 to 1780 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 doses ≥ 178 μg/mL in all three exposure groups. At the conclusion of the treatment period, visible precipitate was observed at doses ≥ 178 μg/mL in all three exposure groups. Based upon these results, the doses chosen for the chromosome aberration assay ranged from 28.05 to 175 μg/mL for the non-activated 4 and 20-hour exposure groups and from 32.06 to 200 μg/mL for the S9-activated 4-hour exposure group.

In the chromosome aberration assay, cytotoxicity was observed at doses of 107.47 µg/mL in the non activated 4 hour exposure group; at 200 µg/mL in the S9-activated 4-hour exposure group; and at doses 91.35 µg/mL in the non-activated 20-hour exposure group.  The doses selected for evaluation of chromosome aberrations were 66, 77.65, and 107.47 µg/mL for the non activated 4-hour exposure group; 144.50, 170.00, and 200 µg/mL for the S9-activated 4 hour exposure groups, and 28.05, 66.00, and 91.35 µg/mL for the non activated 20-hour exposure group.

No significant or dose dependent increases in structural aberrations were observed at any dose in treatment groups with or without S9 (p > 0.05; Fisher’s Exact and Cochran-Armitage tests). No significant or dose dependent increases in numerical (polyploid or endoreduplicated cells) aberrations were observed at any dose in the non-activated 4 and 20-hour exposure groups (p > 0.05; Fisher’s Exact and Cochran-Armitage tests).

In the S9-activated 4-hour exposure group, a statistically significant increase in numerical (polyploid or endoreduplicated cells) aberrations (5.7%) was observed at 144.5 µg/mL. However, the Cochran-Armitage test was negative for a dose response (p > 0.05). In addition, the induction of numerical aberrations was within the historical 95% control limit of 0.00% to 6.35%. Therefore the statistically significant increase in numerical aberrations was considered biologically irrelevant.

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

In vitro gene mutation study in Chinese hamster ovary cells

The test substance was evaluated in a K1 study (Pant, 2019) for its ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of 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, TGr). DMSO was used as the vehicle.

In the preliminary toxicity assay, the concentrations tested were 3.48, 6.96, 13.9, 27.8, 55.7, 111, 223, 446, 891 and 1782 μg/mL. The maximum concentration evaluated approximated the limit dose (10 mM) for this assay. Visible precipitate was observed at concentrations ≥223μg/mL at the beginning of treatment and at concentration 1782 μg/mL by the end of treatment without S9 activation. Adjusted relative survival was 62.50% and 19.58% at a concentration of 1782 μg/mL with S9 and 223 μg/mL without S9, respectively. Adjusted relative survival was approximated 0% at all higher concentrations without S9. Based upon these results, the concentrations chosen for the definitive mutagenicity assay were 112.5, 300, 500, 1000 and 1782 μg/mL with S9 and 14.1, 28.1, 56.3, 112.5, 225 and 400 μg/mL without S9.

In the definitive mutagenicity assay, visible precipitate was observed at concentrations ≥225 μg/mL at the beginning of treatment and at concentrations ≥112.5 μg/mL by the end of treatment without S9 and visible precipitate was observed at concentration 1782 μg/mL at the beginning of treatment and at concentrations ≥1000 μg/mL by the end of treatment with S9. The average adjusted relative survival was 94.32% and 15.18% at a concentration of 1782 μg/mL with S9 and 400 μg/mL without S9, respectively. Cultures treated at concentrations of 112.5, 300, 500 and 1000 μg/mL with S9 and 14.1, 28.1, 56.3 and 112.5 μg/mL 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 S9 (p > 0.01). However, significant increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at 112.5 μg/mL without S9 (p < 0.01). Although the response observed was outside the historical control range, there was no dose-dependent trend observed. Therefore, this response did not meet all three criteria for a positive response without S9 and is considered to be equivocal. The positive controls induced significant increases in mutant frequency (p < 0.01) confirming the acceptable of the assay. The OECD Guideline 476 allows for the repeat of all or part of an assay, if the results are equivocal. The condition without S9 was considered equivocal; therefore, this portion of the assay was repeated at lower doses. The concentrations chosen for confirmatory mutagenicity assay were 12.5, 25, 50, 75, 100, 112.5, 150 and 300 μg/mL without S9.

In the confirmatory mutagenicity assay, visible precipitate was observed at concentrations ≥75 μg/mL at the beginning of treatment and end of treatment. The average adjusted relative survival was 62.16% at a concentration of 75 μg/mL without S9. Cultures treated at concentrations of 12.5, 25, 50 and 75 μg/mL was chosen for mutant selection. Although there was no significant increases in mutant frequency, as compared to the concurrent vehicle controls at any concentration evaluated without S9 (p > 0.01), there was a dose-related trend observed per statistical analysis. One of the replicates at the top dose (75 μg/mL) demonstrated a mutant frequency outside the 95% historical control. Therefore, this response did not meet all three criteria for a positive response without S9 and is considered to be equivocal. The positive controls induced significant increases in mutant frequency (p < 0.01), per the assay criteria. Again, the OECD Guideline 476 allows for the repeat of all or part of an assay, if the results are equivocal. The condition without S9 was considered equivocal; therefore, this portion of the assay was repeated at lower doses. This equivocal response was retested in the retest of Confirmatory mutagenicity assay with dose levels 12.5, 25, 50, 55, 60, 65, 70, 75 and 100 μg/mL without S9.

In the retest of confirmatory mutagenicity assay, visible precipitate was observed at concentration 100 μg/mL at the beginning of treatment and at concentrations ≥70 μg/mL at the end of treatment. The average adjusted relative survival was 38.42% at a concentration of 100 μg/mL without S9. Cultures treated at concentrations of 12.5, 50, 55, 60, 65, 70 and 100 μg/mL were chosen for mutant selection. There was no significant increases in mutant frequency, as compared to the concurrent vehicle controls at any concentration evaluated without S9 (p > 0.01), no mutant frequency outside the 95% historical control, and no dose-related trend observed per statistical analysis. The positive controls induced significant increases in mutant frequency (p < 0.01), per the assay criteria. Therefore, this response did not meet all three criteria for a positive response without S9 and is considered to be negative.

These results indicate the test substance was negative for the ability to induce forward mutations at the HPRT locus of CHO cells, in the presence and absence of an exogenous metabolic activation system.

Justification for classification or non-classification

Based on the negative results in all in vitro genetic toxicity tests with the test item and the criteria laid out in the CLP Regulation (EC) 1272/2008, the substance should not be classified for mutagenicity.