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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Mutagenic effects - bacterial: Ames study. Negative. OECD 471 and OECD 472; Reliability = 1.

Clastogenic effects - mammalian: In vitro mammalian cell micronucleus test in CHO cells. Negative. OECD 487; Reliability = 1.

Mutagenic effects - mammalian: In vitro mammalian cell transformation assay. Negative. OECD 476; Reliability = 1

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
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)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell transformation assay
Target gene:
hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells
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
- Cell cycle length, doubling time or proliferation index: doubling time of 12-14 hours
-Cloning efficiency: greater than 80%

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Ham's F12 medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
Preliminary test concentrations: 1.33, 2.66, 5.32, 10.6, 21.3, 42.6, 85.1, 170, 341 and 681 μg/mL
Main test concentrations: 42.6, 85.1, 170, 341 and 681 μg/mL
-The maximum concentration evaluated approximated the 10 mM limit dose for this assay per OECD Guideline 476
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: No precipitate was seen before or after tests
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

NUMBER OF REPLICATIONS: dulpicate at each concentration level

- OTHER:METHOD OF APPLICATION: in medium, Complete Ham’s F12 medium
- Cell density at seeding (if applicable): ~5E6 in 10 mL

DURATION
- Preincubation period: overnight at standard conditions
- Exposure duration: 5 ± 0.5 hours
- Expression time (cells in growth medium): incubated under standard conditions for 7 days

SELECTION AGENT (mutation assays): Hypoxanthine-free Complete Ham’s F12 medium (Complete Ham’s F12 medium -Hx)

STAIN (for cytogenetic assays): crystal violet

NUMBER OF REPLICATIONS: tests done in duplicate

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Treatment
Cells were plated (on Day -1) in 75-cm2 cultures at a density of ~5 E6 in 10 mL Complete Ham’s F12 medium. Following an overnight incubation (on Day 0) at standard conditions, the cultures were washed twice with HBSS and re-fed with 5 mL treatment medium, or 4 mL treatment medium plus 1 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 (2.0 mL for vehicle control and test substance; 200 μL for positive control) 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).

Subculture for Phenotypic Expression and Initial Survival
After the 5-hour treatment, the treatment media were removed, the cultures were washed twice with CMF-HBSS and then were trypsinized and counted. Cells were subcultured at ~2.4 E6 cells/225-cm2 flask in 30 mL Complete Ham’s F12 medium in duplicate (or all available into 1 or 2 flasks) for phenotypic expression and incubated under standard conditions (larger numbers of cells may be subcultured for phenotypic expression where decreases in cloning efficiency are observed in the Preliminary Toxicity Test; i.e., there should be ~2.4E6 viable cells for phenotypic expression). An additional aliquot of cells was plated at 200 cells/60-mm plate in 5 mL Complete Ham’s F12 medium in triplicate for initial survival. The 60-mm plates were incubated under standard conditions for 7 days and the resulting colonies were fixed in methanol, stained with crystal violet, and counted.

The cultures were subcultured for 7 days, at 2- to 3-day intervals, to maintain logarithmic growth and permit expression of the mutant phenotype. At each subculture, the flasks were washed once (CMF-HBSS), trypsinized, counted and subcultured at ~2.4E6 cells/225-cm2 flask in 30 mL Complete Ham’s F12 in duplicate (or all available into 1 or 2 plates). Subculture was as follows based upon visual observation of the monolayer:
• ≥ 50% of the vehicle control, only one flask was subcultured (into duplicate flasks) and the back-up flask was discarded
• between 25% to 50% of the vehicle control, both flasks were subcultured
• ≤ 25% of the vehicle control), the culture(s) were re-fed with fresh medium and re-incubated for an additional 2 to 3 days

Mutant Selection
Hypoxanthine-free Complete Ham’s F12 medium (Complete Ham’s F12 medium -Hx) was used for mutant selection and to determine cloning efficiency at the time of selection. At the end of the phenotypic expression period, 2.4E6 cells from each culture were plated at a density of 6E5 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/E6 clonable cells. The number of clonable cells was determined from the triplicate 60-mm plates.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
The average absolute cloning efficiency of the vehicle controls must be >60% (at initial survival and selection). In addition, the average spontaneous mutant frequency of the vehicle controls should ideally be within the 95% control limits of the distribution of the historical negative control database. If the concurrent negative control data fell outside the 95% control limits, they may be acceptable as long as these data were not extreme outliers (indicative of experimental or human error). Spontaneous mutant frequencies were calculated separately for cultures with and without S9.

The positive controls must induce a significant increase in mutant frequency as compared to the concurrent vehicle controls (p≤ 0.01). A significant increase in the absence of S9 indicated the test system could identify a mutagen, while a significant increase in the presence of S9 was considered to have demonstrated the integrity of the S9 mix as well as the ability of the test system to detect a mutagen.

The highest concentration evaluated was the limit dose for this assay (2000 μg/mL or 10 mM), or must have induced 10 to 20% adjusted relative survival, or must be the highest concentration able to be prepared in the vehicle and administered (whichever is lowest). If increasing cytotoxicity was observed at precipitating concentrations, cytotoxicity was the determining factor. This latter requirement was waived if the highest concentration with acceptable cytotoxicity (>10% adjusted relative survival) was at least 75% of an excessively toxic concentration (cultures with adjusted relative survivals <10% were excluded from evaluation as excessively cytotoxic). There was no maximum concentration or toxicity requirement for test substances which clearly showed mutagenic activity.

A minimum of four acceptable concentrations was required for a valid assay. Fewer concentrations may be justified for test substances which clearly show mutagenic activity.
Statistics:
Statistical analyses were performed using the method of Snee and Irr, with significance established at the 0.05 level.

Once criteria for a valid assay were met, the responses observed in the assay were evaluated as follows.

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.
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
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no adverse impact on pH
- Effects of osmolality: no adverse impact on osmolality of cultures [280 mmol/kg for the vehicle control and 268 mmol/kg for the highest concentration (681 μg/mL)]
- Evaporation from medium: not specified
- Precipitation: No visible precipitate was observed at the beginning or end of treatment

RANGE-FINDING/SCREENING STUDIES: In the preliminary toxicity assay, the concentrations tested were 1.33, 2.66, 5.32, 10.6, 21.3, 42.6, 85.1, 170, 341 and 681 μg/mL. The maximum concentration evaluated approximated the 10 mM limit dose for this assay per OECD Guideline 476. The test substance formed clear solutions in water from 0.0133 to 6.81 mg/mL. No visible precipitate was observed at the beginning or end of treatment, and the test substance had no adverse impact on the pH or osmolality of the cultures [280 mmol/kg for the vehicle control and 268 mmol/kg for the highest concentration (681 μg/mL). Adjusted relative survival was 64.89 and 105.47% at a concentration of 681 μg/mL with and without S9, respectively. Based upon these results, the concentrations chosen for the definitive mutagenicity assay were 42.6, 85.1, 170, 341 and 681 μg/mL with and without S9.

HISTORICAL CONTROL DATA
- Positive historical control data:
0.2 μL/mL EMS (-S9 treatment) - mean: 259.7, st. dev: 135.1, 95% Control Limit: 0.0-529.9, Obs Range: 79.2-764.2
4.0 μL/mL B(a)P (+S9 treatment) - mean: 154.4, st. dev: 73.3, 95% Control Limit: 7.8-301.0, Obs Range: 34.9-314.5
- Negative (solvent/vehicle) historical control data: includes water and DMSO
-S9 treatment - mean: 4, st. dev: 3.3, 95% Control Limit: 0.0-10.6, Obs Range: 0.0-12.3
+S9 treatment - mean: 4.2, st. dev: 3.4, 95% Control Limit: 0.0-11.0, Obs Range: 0.0-14.0

The average adjusted relative survival was 100.47 and 94.52% at a concentration of 681 μg/mL with and without S9, respectively. Cultures treated at all concentrations with and without S9 were chosen for mutant selection. A statistically significant increase in mutant frequency, as compared to the concurrent vehicle control, was observed at a concentration of 85.1 μg/mL without S9 (p < 0.01). However, this increase was not dose-dependent, the mutant frequency for the vehicle control was on the lower end of the 95% control limit and the average mutant frequency at this test substance concentration was within the 95% control limit. Therefore, this increase was not biologically significant and considered spurious. No statistically significant or biologically relevant increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at the remaining concentrations evaluated with or without S9 (p > 0.01). 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.

Table 1: Summary of Results for Mutagenicity Assay -S9

Treatment Dose (μg/mL) S9 Cells (x 10^6) Cloning Efficiency  Relative Survival (adj., %) TGr Mutants/Plate Total Mutant Colonies Cloning Efficiency  Mutant Frequency (x 10^6)
(Colonies/Plate) % (Colonies/Plate) % Individual Average
Water 0 - 0.577 139 139 137 69.17 94.86 1 1 0 0 2 191 203 217 101.83 0.82 0.65
Water 0 - 0.609 158 153 149 76.67 105.14 0 0 0 1 1 166 190 167 87.17 0.48
Test Substance 42.6 - 0.715 131 138 132 66.83 91.66 3 2 4 1 10 140 163 163 77.67 5.36 6.94
42.6 - 0.688 171 160 146 79.50 109.03 7 3 5 2 17 182 162 155 83.17 8.52
85.1 - 0.716 169 192 153 85.67 117.49 3 6 4 6 19 154 162 137 75.50 10.49 9.99**
85.1 - 0.655 191 179 196 94.33 129.37 3 2 1 7 13 110 102 131 57.17 9.48
170 - 0.649 184 191 177 92.00 126.17 0 1 1 1 3 152 154 166 78.67 1.59 3.37
170 - 0.707 141 140 132 68.83 94.40 4 2 2 1 9 141 145 152 73.00 5.14
341 - 0.653 166 179 177 87.00 119.31 2 7 7 7 23 141 120 147 68.00 14.09 8.9
341 - 0.639 168 150 177 82.50 113.14 2 0 3 1 6 144 128 132 67.33 3.71
681 - 0.636 132 118 134 64.00 87.77 2 5 2 5 14 160 158 159 79.50 7.34 5.24
681 - 0.65 133 152 158 73.83 101.26 1 1 2 1 5 130 133 135 66.33 3.14
EMS 0.2a - 0.709 109 116 122 57.83 79.31 101 100 88 112 401 C 141 134 68.75 243.03 260.2**
EMS 0.2a - 0.679 103 103 97 50.50 69.26 134 102 104 116 456 C 134 140 68.50 277.37
a: μL/mL
Water aliquot volume: 100 μL/mL
C = Contaminated
** p<0.01 compared to the vehicle control (T-Test)

Table 2: Summary of Results for Mutagenicity Assay +S9

Treatment Dose (μg/mL) S9 Cells (x 10^6) Cloning Efficiency  Relative Survival (adj., %) TGr Mutants/Plate Total Mutant Colonies Cloning Efficiency  Mutant Frequency (x 10^6)
(Colonies/Plate) % (Colonies/Plate) % Individual Average
Water 0 + 0.514 169 182 187 89.67 101.13 0 0 0 0 0 179 196 176 91.83 0.00 3.66
Water 0 + 0.626 169 192 165 87.67 98.87 3 3 4 5 15 186 148 179 85.50 7.31
Test Substance 42.6 + 0.726 187 145 179 85.17 96.05 3 10 4 3 20 197 168 205 95.00 8.77 9.15
42.6 + 0.886 90 115 99 50.67 57.14 8 2 5 1 16 132 148 140 70.00 9.52
85.1 + 0.647 164 176 170 85.00 95.86 2 4 2 4 12 203 207 202 102.00 4.90 5.39
85.1 + 0.637 170 188 162 86.67 97.74 3 2 4 3 12 179 172 159 85.00 5.88
170 + 0.543 203 210 221 105.67 119.17 1 0 1 0 2 147 109 142 66.33 1.26 3.90
170 + 0.668 195 170 175 90.00 101.50 4 3 3 3 13 172 152 174 83.00 6.53
341 + 0.646 185 201 221 101.17 114.10 1 1 2 3 7 172 180 174 87.67 3.33 8.25
341 + 0.505 166 166 178 85.00 95.86 8 9 3 8 28 181 177 174 88.67 13.16
681 + 0.528 179 176 189 90.67 102.26 0 3 2 2 7 214 212 195 103.50 2.82 6.81
681 + 0.539 192 165 168 87.50 98.68 7 5 5 6 23 190 187 156 88.83 10.79
EMS 0.2a + 0.604 72 95 74 40.17 45.30 70 71 52 79 272 137 135 151 70.50 160.76 170.54**
EMS 0.2a + 0.579 77 77 90 40.67 45.86 38 72 92 93 295 146 135 128 68.17 180.32
a: μL/mL
Water aliquot volume: 100 μL/mL
** p<0.01 compared to the vehicle control (T-Test)
Conclusions:
The test substance was negative in the In Vitro Mammalian Cell Forward Gene Mutation (CHO/HPRT) Assay.
Executive summary:

The test substance was evaluated for its ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system (S9), as assayed by colony growth in the presence of 6-thioguanine (TG resistance, TGr) in accordance with OECD Guideline 476. The test substance was formulated in water and formed a clear solution at 6.81 mg/mL, the highest stock concentration used in the study.

In the preliminary toxicity assay, the concentrations tested were 1.33, 2.66, 5.32, 10.6, 21.3, 42.6, 85.1, 170, 341 and 681 μg/mL. The maximum concentration evaluated approximated the 10 mM limit dose for this assay per OECD Guideline 476. No visible precipitate was observed at the beginning or end of treatment, and the test substance had no adverse impact on the pH or osmolality of the cultures. Adjusted relative survival was 64.89 and 105.47% at a concentration of 681 μg/mL with and without S9, respectively. Based upon these results, the concentrations chosen for the definitive mutagenicity assay were 42.6, 85.1, 170, 341 and 681 μg/mL with and without S9.

In the definitive mutagenicity assay, no visible precipitate was observed at the beginning or end of treatment, and the test substance again had no adverse impact on the pH of the cultures. The average adjusted relative survival was 100.47 and 94.52% at a concentration of 681 μg/mL with and without S9, respectively. Cultures treated at all concentrations with and without S9 were chosen for mutant selection. A statistically significant increase in mutant frequency, as compared to the concurrent vehicle control, was observed at a concentration of 85.1 μg/mL without S9 (p < 0.01). However, this increase was not dose-dependent, the mutant frequency for the vehicle control was on the lower end of the 95% control limit and the average mutant frequency at this test substance concentration was within the 95% control limit. Therefore, this increase was not biologically significant and considered spurious. No statistically significant or biologically relevant increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at the remaining concentrations evaluated with or without S9 (p > 0.01). 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.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: EC Methods for the Determination of Toxicity and Other Health Effects Directive 640/2012/EC Method B.49 (2012)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
The CHO-K1 cell line is a proline auxotroph.
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: American Type Culture Collection (repository number CCL 61), Rockville, Maryland, U.S.A.
- Cell cycle length, doubling time or proliferation index: doubling time of 10-14 hours
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-1254-induced rat liver S9
Test concentrations with justification for top dose:
Preliminary Test: 1, 5, 10, 50, 75, 125, 250, 500, 680 µg/mL. Top dose was recommended limit does of OECD 487 of 10 mM.

Main Test: 75, 125, 250, 500, 680 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: sterile water
- Justification for choice of solvent/vehicle: The test substance formed a clear, colourless solution at 680 µg/mL (10 mM), the highest concentration used in the study.
Untreated negative controls:
yes
Remarks:
sterile water
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: The CHO cultures for the micronucleus assay were initiated in labeled sterile, tissue culture treated plates by seeding 0.5 mL of appropriately concentrated seeding stock to required wells. Plates were incubated at 37°C ± 2°C in a humidified atmosphere of 5 ± 2% CO2 in air. Approximately 24 hours after seeding, the culture medium was aspirated and replaced with treatment medium (with or without 10% S9 mixture) such that addition of the test substance volume (5 µL or 50 µL depending on the solvent) resulted in a total volume of 0.5 mL.
- Cell density at seeding: For 4-hour conditions: 5E4 cells/mL; For 24-hour conditions: 3E4 cell/mL

DURATION
- Preincubation period: 37°C ± 2°C for 24 hours
- Exposure duration: 4 hours with or without S9 mix and 24 hours without S9 mix
- Expression time (cells in growth medium): For 4-hour condidition, treatment medium was replaced with complete F12K culure medium and incubabted until cell harvest. Cells were harvested approximately 24 hours after exposure time.

STAIN (for cytogenetic assays): Cell harvest and staining were conducted following a modified version of Litron Laboratory’s In Vitro Micronucleus Analysis MicroFlow Kit®. Cell culture plates to be stained for micronuclei were placed on ice for approximately 20 minutes. Cells were then inoculated with nucleic acid dye A and exposed to cool white fluorescent light for approximately 30 minutes. Plates were removed from the light source and cells were washed with 1X buffer and inoculated with Complete Lysis Solution 1. Plates were incubated at 37 ± 2°C in a humidified atmosphere of 5 ± 2% CO2 in air for approximately 60 minutes. Plates were removed from incubator, and cells were inoculated with Complete Lysis Solution 2 and then incubated protected from light at room temperature for approximately 30 minutes. At the completion of the incubation, the plates were sealed with a plate cover, protected from light, and stored refrigerated overnight or up to 72 hours prior to analysis.

NUMBER OF REPLICATIONS: triplicate for each concentration

NUMBER OF CELLS EVALUATED: at least 20000 nucleated cells per sample

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: Cells were analyzed for induction of micronuclei and toxicity as indicated by the frequency of beads among the total nucleated cells counted.
Evaluation criteria:
The experimental unit is the cell; therefore the percentage of cells with micronuclei was used for the assessment. Evaluations were conducted on all samples. Whenever feasible, at least 20000 nucleated cells were analyzed per sample for induction of micronuclei, and toxicity as indicated by the frequency of beads among the total nucleated cells counted.
Statistics:
Statistical analysis was used as a guide to determine whether or not the test substance induced a positive response. Interpretation of the statistical analysis also relied on additional considerations including the magnitude of the observed test substance response relative to the vehicle control response and the presence of a dose-responsive trend.
All micronucleus statistical data analyses were evaluated one-tailed and at a significance level of 5%. Data was transformed prior to analysis using an arcsine square root transformation. This transformation was appropriate for statistical analyses of proportions since the distribution of the transformed data more closely approximates a normal distribution than does the non-transformed proportion of the data.
An ANOVA analysis of variance and a Dunnett’s test were performed on the transformed micornuclei frequencies. The frequency of micronuclei were evaluated only for an increasing response in relation to the concurrent vehicle control. A Williams test for dose-response trend was conducted.
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
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Measurements were taken from the two test substance concentration (500 and 680 µg/mL) and the vehicle control media. For the non-activated system, the pH was measured to be 8.20, 8.01, and 7.97 for the vehicle, 500, and 680 µg/mL, respectively. For the S-9 activated system, the pH was measured to be 7.88, 7.67, and 7.58 for the vehicle, 500, and 680 µg/mL, respectively.
- Effects of osmolality: Measurements were taken from the two test substance concentration (500 and 680 µg/mL) and the vehicle control media. For the non-activated system, the osmolality was measured to be 297, 303, and 306 mOsm/kg for the vehicle, 500, and 680 µg/mL, respectively. For the S-9 activated system, the osmolality was measured to be 303, 310, and 309 for the vehicle, 500, and 680 µg/mL, respectively. The observed changes in osmolality were <20% and were, therefore, not considered significant.
- Precipitation: No test substance precipitation was observed at either the beginning or end of treatment at any concentration.

RANGE-FINDING/SCREENING STUDIES: The preliminary toxicity assay tested 4-hour treatment (with and without activation) and 24-hour treatment (without activation) for the following concentrations: 1, 5, 10, 50, 75, 125, 250, 500, and 680 µg/mL. Test substance precipitation was not observed before or after treatment. Substantial toxicity (i.e., 55 ± 5% reduction of the cell survival relative to the vehicle control) was not observed in any test condition.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Negative (solvent/vehicle) historical control data: Sterile water: The 4-hour, Non-Activated (-S-9) micronuclei percentage range (95%) is 0.124-3.623%. The 4-hour, Activated (+S-9) micronuclei percentage range (95%) is 0.643-4.413%. The 24-hour, Non-Activated (-S-9) micronuclei percentage range (95%) is 0-5.383%.

In the initial micronucleus assay the cells usedexhibited an average cell cycle length of 0.95 normal cell cyclesduring the 24-hour total treatment time, which did not meet the OECD 487 (2016) requirement of 1.5-2.0 normal cell cycles. The initial micronucleus assay was considered invalid and was repeated using the initial concentrations. Data from the initial assay were not reported, but were archived with the study records.

In the 4-hour S9-activated and 24-hour non-activated test conditions no statistically significant increases in the percentage of micronuclei were observed at any of the test substance concentrations (p>0.05, Dunnett’s test). There was no evidence of an increasing dose responsive trend in the 4 hour S9-activated or 24-hour non-activated test conditions (p>0.05, William’s test).
In the 4-hour non-activated test condition a statistically significant increase in the frequency of micronuclei in relation to the vehicle control was observed at 500 µg/mL (p≤0.05, Dunnett’s test). In addition, a statistically significant dose responsive trend was observed at 500 and 680 µg/mL (p≤0.05, William’s test). This was considered to be due to large standard deviations in the frequency of micronuclei between replicates in the 500 and 680 µg/mL treatment groups, and not representative of a test substance–related mutagenic effect. However, a confirmatory assay was conducted for this testing condition using the same concentrations initially tested. 
In the confirmatory assay,no statistically significant increase in the frequency of micronuclei were observed at any test substance concentration in4-hour non-activated test condition(p>0.05, Dunnett’s test). There was no evidence of an increasing dose‑responsive trend (p>0.05, William’s test). No test substance precipitation or substantial toxicity was observed.
Cell proliferation calculations were conducted to determine the population doubling time. Cells used in the repeat micronucleus assay exhibited an average cell cycle length of 1.91 normal cell cyclesduring the 24‑hour total treatment time. Cells used in the repeat 4-hour non-activated micronucleus assay exhibited an average cell cycle length of 1.62 normal cell cyclesduring the 24‑hour total treatment time. The cell cycle length was within the OECD 487 guideline recommended cell cycle length from time of treatment through harvest of 1.5-2.0 normal cell cycle lengths.

Table 2: Micronucleus Assay Summary

Treatment S9 Treatment Time Average Relative Survival Average MN Frequency  Fold Increase in Micronuclei
µg/mL Activation (hours) (%) (%)  
0 (Sterile Water) -S9 4 100 1.55 NA
75  -S9 4 108.24 2.4 1.55
125  -S9 4 96.68 2.26 1.46
250  -S9 4 75.23 2.16 1.4
500  -S9 4 99.33 4.65a,b 3.01
680  -S9 4 92.14 4.06b 2.63
0.4MMC  -S9 4 59.67 4.70a 3.09
0.8MMC  -S9 4 52.71 4.90a 3.17
0 (Sterile Water)c  -S9 4 100 1.29 NA
75c  -S9 4 137.96 1.54 1.19
125c  -S9 4 93.21 1.06 0.82
250c  -S9 4 103.47 0.98 0.76
500c  -S9 4 112.91 0.94 0.73
680c  -S9 4 108.55 1 0.78
0.4MMCc  -S9 4 78.84 4.36a 3.38
0.8MMCc  -S9 4 76.03 6.01a 4.66
0 (Sterile Water) +S9 4 100 1.89 NA
75  +S9 4 106.42 2.86 1.51
125  +S9 4 114.12 1.83 0.96
250  +S9 4 109.49 2.33 1.23
500  +S9 4 123.5 2.56 1.36
680  +S9 4 96 2.5 1.32
0.4MMC  +S9 4 87.39 5.13a 2.71
0.8MMC  +S9 4 76.07 7.99a 4.22
0 (Sterile Water) -S9 24 100 0.83 NA
75  -S9 24 94.65 1.21 1.45
125  -S9 24 97.72 1.5 1.8
250  -S9 24 97.82 1.63 1.95
500  -S9 24 99.29 1.28 1.54
680  -S9 24 96.7 1.06 1.28
0.4MMC  -S9 24 46.26 5.21a 6.25
0.8MMC  -S9 24 35.47 6.50a 7.79
aStatistically significant (p≤0.05, Dunnett’s Test)
bStatistically significant dose responsive trend (p≤0.05, William’s Test)
cConfirmation Assay Data
Conclusions:
The test substance did not induce micronuclei in the in vitro mammalian micronucleus test in Chinese hamster ovary cells in the non-activated and S9-activated test systems. It was concluded that the test substance was negative in this in vitro test.
Executive summary:

The test substance was evaluated for its ability to induce micronuclei in Chinese Hamster Ovary (CHO) cells in vitro in the absence and presence of an exogenous metabolic activation system (Aroclor-induced rat liver S9) in accordance with OECD Guideline 487. The test substances formed a clear, colorless solution in sterile water at 6.8 mg/mL (1M), the highest concentration prepared for use on this study.

In the preliminary toxicity and micronucleus assays, the cells were treated for 4 and 24 hours in the non-activated test condition and for 4 hours in the S9-activated test condition. All cells were harvested 24 hours after treatment initiation. A vehicle and positive control group was included in each test condition. In the preliminary toxicity assay, the highest concentration tested was the OECD 487 (2016) recommended limit dose of 680 µg/mL (10 mM). The cells were exposed to nine concentrations of the test substance ranging from 1 to 680 µg/mL, as well as a vehicle control. Test substance precipitation was not observed at either the beginning or end of treatment. Relative cell survival was evaluated at the end of treatment via trypan blue staining. Substantial toxicity (i.e., 55 ± 5% reduction of the cell survival relative to the vehicle control) was not observed in any test condition.

The concentrations chosen for the micronucleus assay were 75, 125, 250, 500, and 680 µg/mL for all test conditions. Cells were stained using the Litron Laboratories In Vitro Micronucleus MicroFlow® Kit and analyzed on a flow cytometer. A total of 20000 nuclei were evaluated per sample. Relative cell survival was evaluated at the end of treatment via cytotoxicity ratio evaluation. In the initial micronucleus assay the cells did not meet the OECD 487 (2016) requirement of 1.52.0 normal cell cycles and was considered invalid. Data from the initial assay were not reported, but were archived with the study records.

In the main micronucleus assay test substance precipitation was not observed at either the beginning or end of treatment in any test condition. Substantial toxicity was not observed in any test condition. In the 4-hour S9-activated and 24-hour non-activated test conditions no statistically significant increases in the percentage of micronuclei were observed at any of the test substance concentrations (p>0.05, Dunnett’s test). There was no evidence of an increasing dose-responsive trend in the 4hour S9-activated or 24-hour non-activated test conditions (p>0.05, William’s test). In the 4-hour non-activated test condition a statistically significant increase in the frequency of micronuclei in relation to the vehicle control was observed at 500 µg/mL (p≤0.05, Dunnett’s test). In addition, a statistically significant dose responsive trend was observed at 500 and 680 µg/mL (p≤0.05, William’s test). This was considered to be due to large standard deviations in the frequency of micronuclei between replicates in the 500 and 680 µg/mL treatment groups, and not representative of a test substance–related mutagenic effect. However, a confirmatory assay was conducted for this testing condition using the same concentrations as initially tested.  In the confirmatory assay, no statistically significant increase in the frequency of micronuclei were observed at any test substance concentration in 4-hour non-activated test condition(p>0.05, Dunnett’s test). There was no evidence of an increasing doseresponsive trend (p>0.05, William’s test). No test substance precipitation or substantial toxicity was observed. All criteria for a valid study were met. Under the conditions of this study, the test substance did not induce micronuclei in the in vitro mammalian micronucleus test in Chinese hamster ovary cells in the non-activated and S9-activated test systems. It was concluded that the test substance was negative in this in vitro test.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
S. typhimurium: his-
E. coli: trp-
Species / strain / cell type:
other: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix from Aroclor 1254 induced rat liver homogenate
Test concentrations with justification for top dose:
20 µg - 5000 µg/plate (SPT and PIT; all strains)
20 µg - 8000 μg/plate (PIT; TA 1535)
Vehicle / solvent:
water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene, N-methyl-N'-nitro-N-nitrosoguanidine, 4-nitro-o-phenylendiamine, 9-aminoacridine, N-ethyl-N'-nitro-N-nitrosoguanidine
Details on test system and experimental conditions:
METHOD OF APPLICATION: 1st experiment: standard plate test (SPT); 2nd experiment: preincubation test (PIT); 3rd experiment: high dose preincubation test (PIT) only with TA 1535
Evaluation criteria:
In general, a substance to be characterized as positive in the bacterial tests has to fulfill the following requirements :
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results
Species / strain:
other: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA
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:
No precipitation was found. According to the results of the present study, the test substance is weakly mutagenic in TA1535 without metabolic activation system under the experimental conditions chosen here. An increase in the number of revertants (factor ≥ 2.0) was repeatable in two of three experiments, however, only in one of three experiments a dose-relationship was seen. Additionally, an at least doubled revertant factor was observed only in high doses ≥ 4000 µg/plate. Therefore, impurities rather than the test substance itself might be the cause for the effects observed.
Standard plate test (20 - 5000 µg/plate)
Strain Metabolic activation system mean revertants in Controls maximum revertant factor dose dependency Assessment
TA 98 no 27 1.0 no negative
  yes 41 1.0 no negative
TA 100 no 129 1.1 no negative
  yes 142 1.1 no negative
TA 1535 no 23 0.9 no negative
  yes 23 1.0 no negative
TA 1537 no 10 1.0 no negative
  yes 10 1.1 no negative
WP2 uvr A no 33 1.0 no negative
  yes 41 1.0 no negative
Preincubation test (20 - 5000 µg/plate)
Strain Metabolic activation system mean revertants in Controls maximum revertant factor dose dependency Assessment
TA 98 no 27 1.1 no negative
  yes 47 1.0 no negative
TA 100 no 122 1.2 no negative
  yes 112 1.2 no negative
TA 1535 no 18 2.0 yes weakly positive
  yes 19 1.2 no negative
TA 1537 no 12 1.0 no negative
  yes 12 1.4 no negative
WP2 uvr A no 32 1.0 no negative
  yes 39 1.2 no negative
           
Preincubation test (2000 - 8000 µg/plate)
Strain Metabolic activation system mean revertants in Controls maximum revertant factor dose dependency Assessment
TA 1535 no 19 2.6 no weakly positive
  yes 21 1.6 no negative

In the high dose PIT, the mean revertant factor in the 2000, 4000, 6000 and 8000 µg/plate treatments were 1.8, 2.6, 2.5 and 2.1, respectively. Therefore, no dose dependency was seen in this trial.

Conclusions:
The test substance is considered not to be mutagenic in bacteria. The weakly positive results in high doses were considered as caused by impurities.
Executive summary:

A test to examine the mutagenic potential of the test substance was conducted in accordance with OECD Guidelines 471 and 472. S. typhimurium strains TA98, TA100, TA1535, and TA1537, and E. coli WP2 urvA were exposed to the test substance at doses up to 8000 µg/plate with and without metabolic activation. No precipitation was found. According to the results of the present study, the test substance is weakly mutagenic in TA1535 without metabolic activation system under the experimental conditions chosen here. An increase in the number of revertants (factor ≥ 2.0) was repeatable in two of three experiments, however, only in one of three experiments a dose-relationship was seen. Additionally, an at least doubled revertant factor was observed only in high doses ≥ 4000 µg/plate. Therefore, impurities rather than the test substance itself might be the cause for the effects observed. Overall, the test substance was considered not mutagenic in any of the strains tested.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In an Ames test according to GLP and OECD Guideline 471 (and former OECD guideline 472), the test substance was tested for gene mutations in Salmonella typhimurium strains TA100, TA1535, TA98, TA1537 and E. coli WP2 uvrA with or without metabolic activation. A standard plate test (SPT) and a preincubation test (PIT) were performed at doses up to 5000 μg/plate in all strains; additionally, TA1535 was tested up to 8000 µg/plate in the PIT. The test substance was weakly mutagenic in TA1535 without metabolic activation system under the experimental conditions chosen, however, only in high doses ≥ 4000 µg/plate. Impurities rather than the test substance itself might be the cause for the effects observed. Therefore, the test substance is considered not mutagenic in the Ames test.

 

The test substance was evaluated for its ability to induce micronuclei in Chinese Hamster Ovary (CHO) cells in vitro in the absence and presence of an exogenous metabolic activation system (Aroclor-induced rat liver S9) in accordance with OECD Guideline 487. The concentrations chosen for the micronucleus assay were 75, 125, 250, 500, and 680 µg/mL for all test conditions. A total of 20000 nuclei were evaluated per sample. Under the conditions of this study, the test substance did not induce micronuclei in the in vitro mammalian micronucleus test in Chinese hamster ovary cells in the non-activated and S9-activated test systems. It was concluded that the test substance was negative in this in vitro test.

 

The test substance was evaluated for its ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system (S9), as assayed by colony growth in the presence of 6-thioguanine (TG resistance, TGr) in accordance with OECD Guideline 476. The concentrations chosen for the definitive mutagenicity assay were 42.6, 85.1, 170, 341 and 681 μg/mL with and without S9. A statistically significant increase in mutant frequency, as compared to the concurrent vehicle control, was observed at a concentration of 85.1 μg/mL without S9 (p < 0.01). However, this increase was not dose-dependent, the mutant frequency for the vehicle control was on the lower end of the 95% control limit and the average mutant frequency at this test substance concentration was within the 95% control limit. Therefore, this increase was not biologically significant and considered spurious. No statistically significant or biologically relevant increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at the remaining concentrations evaluated with or without S9 (p > 0.01). It was concluded that the test substance was negative in this in vitro test.

Justification for classification or non-classification

The test substance was negative for mutagenicity and clastogenicity in vitro in bacterial and mammalian cells, when tested in studies conducted according to OECD guidelines. The substance does not need to be classified for mutagenicity according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.