Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial Reverse Mutation Assay (OECD Guideline 471):

The substance was negative for the ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system (Pant, 2017).

Chromosome aberration assay in Chinese Hamster Ovary cells (OECD Guideline 473):

The substance was negative for the induction of structural and numerical chromosome aberrations in the presence and absence of the exogenous metabolic activation system (Roy, 2017).

In vitro mammalian cell forward gene mutation (CHO/HPRT) assay (OECD Guideline 476):

The substance was negative for the ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of Chinese hamster ovary cells, in the presence and absence of an exogenous metabolic activation system (Dutta, 2017).

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-07-24 to 2017-08-31
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: DR74271215
- Expiration date of the lot/batch: 04-05-2017
- Purity: 98.2% (per Certificate of Analysis)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protected from light
- Solubility and stability of the test substance in the solvent/vehicle: The test substance was soluble in water at a concentration of approximately 50 mg/mL, the maximum concentration tested for solubility.


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
Test substance dilutions were prepared immediately before use and delivered to the test system at room temperature under filtered light.
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
CELLS USED
- Source of cells: American Type Culture Collection, Manassas, VA
- The use of CHO cells has been demonstrated to be an effective method of detection of chemical clastogens
- In order to assure the karyotypic stability of the cell line, working cell stocks were not used beyond passage 15.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Exponentially growing CHO-K1 cells were seeded in complete medium (McCoy's 5A medium containing 10% fetal bovine serum, 1.5 mM L-glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin and 2.5 μg/mL Amphotericin B) for each treatment condition at a target of 5 x 1E05 cells/culture.
- Properly maintained: yes, the cultures were incubated under standard conditions (37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2 in air) for 16-24 hours.
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: In order to assure the karyotypic stability of the cell line, working cell stocks were not used beyond passage 15.
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
Colcemid at a final concentration of 0.1 µg/mL
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
In the preliminary toxicity assay, the doses tested were 0.2, 0.6, 2, 6, 20, 60, 200, 600 and 2000 µg/mL. The top dose tested was the limit dose for this assay, based on OECD guidelines. In the chromosomal aberration assay, the doses tested were 100, 250, 500, 1000, 1500, and 2000 µg/mL with and without metabolic activation. The top dose tested was the limit dose for this assay, based on OECD guidelines.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Water was the vehicle of choice based on the solubility of the test substance, and compatibility with the target cells. In a solubility test conducted at BioReliance, the test substance was soluble in water at a concentration of approximately 50 mg/mL, the maximum concentration tested for solubility.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: CHO cells were exposed to the test and control articles for 4 and 20 hours without S9 and for 4 hours with S9, and rinsed.
- Expression time (cells in growth medium): 20 hours (± 30 minutes), 1.5 normal cell cycles

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 1 in the preliminary toxicity assay; 2 in the chromosome aberration assay

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: To prepare slides, the cells were collected by centrifugation and the cells were resuspended in fresh fixative. The suspension of fixed cells was applied to glass microscope slides and air-dried. The slides were stained with Giemsa, permanently mounted.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 300 metaphase spreads containing 20 ± 2 centromeres from each dose (150 per duplicate treatment)

DETERMINATION OF CYTOTOXICITY- Method: RICC and mitotic index relative to solvent control

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- Cell density at seeding: 5 x 1E05 cells/culture
Evaluation criteria:
Evaluation of test results:
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.

Criteria for Determination of a Valid Test
Vehicle Controls
The frequency of cells with structural chromosomal aberrations should ideally be within the 95% control limits of the distribution of the historical negative control database. If the concurrent negative control data fall outside the 95% control limits, they may be acceptable as long as these data are note extreme outliers (indicative of experimental or human error).
Positive Controls
The frequency of cells with structural chromosomal aberrations must be significantly greater than the concurrent vehicle control (p ≤ 0.05). In addition, the cytotoxicity response must not exceed the upper limit for the assay (60%).
Cell Proliferation
The average viable cell count in the vehicle control at harvest must be ≥ 1.5-fold the average viable cell baseline value.
Test Conditions
The test substance must be tested using a 4-hr treatment with and without S9, as well as a 20-hr treatment without S9. However, all three treatment conditions need not be evaluated in the case of a positive test substance response under any treatment condition.
Analyzable Concentrations
At least 300 metaphases must be analyzed from at least three appropriate test substance concentrations. The number of metaphases scored may be reduced when high numbers of cells with chromosomal aberrations (≥10% metaphases) are observed as with a positive test substance or the positive control substance.
Statistics:
Statistical analysis was performed using the Fisher's exact test (p ≤ 0.05) for a pairwise comparison of the frequency of aberrant cells in each treatment group with that of the vehicle control. The Cochran-Armitage trend test was used to assess dose-responsiveness.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: pH values were considered acceptable
- Effects of osmolality: Osmolality values were considered acceptable
- Water solubility: Soluble in water at a concentration of approximately 50 mg/mL, the maximum concentration tested for solubility.
- Precipitation: No visible precipitate observed
- Definition of acceptable cells for analysis: Metaphase spreads containing 20 ± 2 centromeres

RANGE-FINDING/SCREENING STUDIES:
The test substance was soluble in the treatment medium at all doses tested at the beginning and conclusion of the treatment period.
Cytotoxicity (≥ 50% reduction in cell growth index relative to the vehicle control) was not observed at any dose in any of the three exposure groups.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Refer to main study report
- Negative (solvent/vehicle) historical control data: Refer to main study report

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: RICC and mitotic index relative to solvent control
- Other observations when applicable: Monolayer confluency
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 chromosome aberrations in the non-activated and S9-activated test systems in the in vitro mammalian chromosome aberration test using CHO cells.
Executive summary:

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

In the preliminary toxicity assay, the doses tested ranged from 0.2 to 2000 µg/mL, which was the limit dose for this assay. Cytotoxicity ( 50% reduction in cell growth index relative to the vehicle control) was not observed at any dose in any of the three exposure groups.  Based upon these results, the doses chosen for the chromosome aberration assay ranged from 100 to 2000 µg/mL for all three exposure groups.

In the chromosome aberration assay, cytotoxicity ( 50% reduction in cell growth index relative to the vehicle control) was not observed at any dose in any of the three exposure groups. The doses selected for evaluation of chromosome aberrations were 500, 1000, and 2000 µg/mL for all three exposure groups.

In the non-activated 4 and 20-hour exposure groups, no significant or dose‑dependent increases in structural aberrations were observed at any dose (p > 0.05; Fisher’s Exact and Cochran-Armitage tests).

In the S9-activated 4-hour exposure group, statistically significant increases in structural aberrations (3.3%) were observed at doses 1000, and 2000 µg/mL (p  0.05; Fisher’s Exact). However, the Cochran-Armitage test was negative for a dose response (p > 0.05). In addition, the increase was within the historical 95% control limit of 0.00% to 3.88%. Since the significant increases in structural aberrations did not meet any criteria for a positive response, the statistical significance can be attributed to the 0.0% induction of structural aberrations in the vehicle control. Therefore, the statistically significant increase in structural chromosomal aberrations was considered biologically irrelevant.

No significant or dose‑dependent increases in numerical (polyploid or endoreduplicated cells) aberrations were observed at any dose in any of the treatment groups (p > 0.05; Fisher’s Exact and Cochran-Armitage tests). 

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

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-05-30 to 2017-06-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 21 July 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ISO/IEC 17025:2005
Version / remarks:
ISO/IEC, 2005
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: DR74271215
- Expiration date of the lot/batch: 30-11-2017
- Purity: 98.2%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protected from light
- Solubility and stability of the test substance in the solvent/vehicle: Water was the vehicle of choice based on the solubility of the test substance and compatibility with the target cells. The test substance formed a clear solution in water at a concentration of approximately 50 mg/mL in the solubility test conducted at the testing laboratory.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
Test substance dilutions were prepared immediately before use and delivered to the test system at room temperature under filtered light.
Target gene:
Histidine locus (Salmonella strains) and tryptophan locus (E. coli strain): The Salmonella strains contain mutations in the histidine operon, thereby imposing a requirement for histidine in the growth medium. These strains contain the deep rough (rfa) mutation, which delete s the polysaccharide side chain from the lipopolysaccharides of the bacterial cell surface. This increases cell permeability of larger substances. The other mutation is a deletion of the uvrB gene, which codes for a protein of the DNA nucleotide excision repair system, resulting in an increased sensitivity in detecting many mutagens. This deletion also includes the nitrate reductase (chi) and biotin (bio) genes (bacteria require biotin for growth). Tester strains TA98 and TA100 contain the R-factor plasmid, pKM101. These strains are reverted by a number of mutagens that are detected weakly or not at all with the non-R-factor parent strains. pKM101 increases chemical and spontane ous mutagenesis by enhancing an error-prone DNA repair system, which is normally present in these organisms. The tester strain Escherichia coli WP2 uvrA carries the defect in one of the genes for tryptophan biosynthesis. Tryptophan-independent mutants (revertants) can arise either by a base change at the site of the original alteration or by a base change elsewhere in the chromosome so that the original defect is suppressed. This second possibility can occur in several different ways so that the system seems capable of detecting all types of mutagens, which substitute one base for another. Additionally, the strain is deficient in the DNA nucleotide excision repair system.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
Initial Toxicity-Mutation Assay: 1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate
Confirmatory Mutagenicity Assay: 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate
In the initial toxicity-mutation assay, neither precipitate nor toxicity was observed. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Based upon these results, the maximum dose tested in the confirmatory mutagenicity assay was 5000 µg per plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: Water was the vehicle of choice based on the solubility of the test substance and compatibility with the target cells. The test substance formed a clear solution in water at a concentration of approximately 50 mg/mL in the solubility test conducted at the testing laboratory.
Untreated negative controls:
yes
Remarks:
solvent control
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
With S9: 1.0 μg/plate for TA98, TA1535 ; 2.0 μg/plate for TA100, TA1537; 15 μg/plate for WP2 uvrA
Untreated negative controls:
yes
Remarks:
solvent control
Negative solvent / vehicle controls:
yes
Remarks:
water
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
Remarks:
solvent control
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Without S9: 1.0 μg/plate for TA100, TA1535
Untreated negative controls:
yes
Remarks:
solvent control
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Without S9: 75 μg/plate for TA1537
Untreated negative controls:
yes
Remarks:
solvent control
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Without S9: 1.0 μg/plate for WP2 uvrA
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
Tester strain: Each culture was monitored spectrophotometrically for turbidity and was harvested at a percent transmittance yielding a titer of greater than or equal to 0.3 x 1E09 cells per milliliter. The actual titers were determined by viable count assays on nutrient agar plates.

DURATION
- Exposure duration: 48 to 72 hours

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

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

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the growth of the bacterial background lawn
Rationale for test conditions:
see above in specific details on test material, test concentrations and details on test system and conditions
Evaluation criteria:
For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test substance as specified below:
- Strains TA1535 and TA1537:
Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.
- Strains TA98, TA100 and WP2 uvrA:
Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.
An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited.
A response was evaluated as negative if it was neither positive nor equivocal.
Statistics:
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.
Species / strain:
S. typhimurium, other: TA98, TA100, TA1535, TA1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: Soluble at 50 mg/mL
- Precipitation: no precipitation has been observed
- Other: sterility results: No contaminant colonies were observed on the sterility plates for the vehicle control, the test substance dilutions or the S9 and Sham mixes.

RANGE-FINDING/SCREENING STUDIES:
Based upon the results of the initial toxicity-mutation assay, the dose levels selected for the confirmatory mutagenicity assay were 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate. Neither precipitate nor toxicity was observed. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
Historical Negative and Positive Control Values (2015)
Revertants per plate:
Strain Control Activation
None Rat Liver
Mean SD Min Max 95% CL Mean SD Min Max 95% CL
TA98
Neg 16 5 6 43 6-26 23 7 5 53 9-37
Pos 190 191 42 2468 329 176 51 1786
TA100
Neg 90 12 62 233 66-114 98 15 63 157 68-128
Pos 697 172 239 1767 671 284 138 2692
TA1535
Neg 13 5 2 35 3-23 13 5 3 33 3-23
Pos 624 196 50 2509 137 110 24 1060
TA1537
Neg 7 3 1 20 1-13 9 3 2 23 3-15
Pos 392 292 24 2887 73 53 19 574
WP2 uvrA
Neg 25 8 7 73 9-41 28 8 10 96 12-44
Pos 336 112 89 1026 352 117 78 1409
SD=standard deviation; Min=minimum value; Max=maximum value; 95% CL = Mean ±2 SD (but not less than zero); Neg=negative control (including but not limited to deionized water, dimethyl sulfoxide, ethanol and acetone); Pos=positive control
Conclusions:
All criteria for a valid study were met. The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, the test substance 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 gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-06-01 to 2018-06-19
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- lot/batch No.of test material: DR74271215
- Expiration date of the lot/batch: 29 November 2018
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protected from light
- Stability under test conditions: While the Certificate of Analysis indicates an expiry date, it does not indicate the acceptable storage parameters for the test substance. Thus, the stability of the test substance has not been determined to cover the period of shipment to or storage at BioReliance.
- Solubility of the test substance in the solvent/vehicle: The test substance formed a clear solution in water at a concentration of approximately 50 mg/mL in the solubility test conducted at the test facility.
Target gene:
The purpose of this study was to evaluate a test article for its ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, as assayed by colony growth in the presence of 6-thioguanine (TG resistance, TGr).
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
In the preliminary toxicity assay, the concentrations tested were 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 μg/mL. The maximum concentration evaluated approximated the limit dose for this assay.Based upon the preliminary toxicity assay, the concentrations chosen for the definitive mutagenicity assay were 125, 250, 500, 1000 and 2000 μg/mL with and without S9.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Water was used as the vehicle for the test substance. Ethylmethanesulfonate (EMS) was diluted in DMSO (both obtained from Sigma-Aldrich) and used as the positive control for the non-activated test system. Benzo(a)pyrene [B(a)P] was also diluted in DMSO (both obtained from Sigma-Aldrich) and used as the positive control for the S9-activated system.
- Justification for choice of solvent/vehicle: Water was the vehicle of choice based on the solubility of the test substance and compatibility with the target cells.
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
DURATION
- Exposure duration: 5 +/- 0.5 hours
- Expression time (cells in growth medium): 7 to 8 days
- Selection time (if incubation with a selection agent): 7 to 10 days
SELECTION AGENT (mutation assays): 6-thioguanine
NUMBER OF REPLICATIONS: single cultures in the preliminary toxicity assay; duplicate cultures in the mutagenicity assay
NUMBER OF CELLS EVALUATED: 2.4 x 10e6 cells per culture
DETERMINATION OF CYTOTOXICITY
- Method: adjusted relative survival
Evaluation criteria:
The test substance was considered to have produced a positive response if it induced a dose-dependent increase in mutation frequency and an increase exceeding 95% historical vehicle control limits in at least one test dose level(s) as compared with concurrent vehicle control (p<0.01). If only one criterion was met (a statistically significant or dose-dependent increase or an increase exceeding the historical control 95% confidence interval), the result were considered equivocal. If none of these criteria were met, the results were considered to be negative.Other criteria also may be used in reaching a conclusion about the study results (e.g., comparison to historical control values, biological significance, etc.). In such cases, the Study Director used sound scientific judgment and clearly reported and described any such considerations.
Statistics:
Statistical analyses were performed using the method of Snee and Irr (1981), with significance established at the 0.05 level.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
The average adjusted relative survival was 119 and 85.66% at a concentration of 2000 μg/mL with and without S9, respectively for the definitive mutagenicity assay
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Preliminary toxicity assay: The pH of the cultures was adjusted at concentrations ≥31.3 μg/mL with and without S9 to maintain neutral pH.
Definitive mutagenicity assay: The pH of the cultures was adjusted at concentrations ≥125 μg/mL with and without S9 to maintain neutral pH.
- Effects of osmolality: The osmolality of the cultures was acceptable as it did not exceed the osmolality of the vehicle control by more than 120% in the preliminary toxicity assay
- Precipitation:No visible precipitate was observed at the beginning or end of treatment in both assays
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
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.
Executive summary:

All positive and vehicle control values were within acceptable ranges, and all criteria for a valid assay were met. The test substance as evaluated for its ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system, as assayed by colony growth in the presence of 6-thioguanine (TG resistance, TGr). Water was used as the vehicle.

In the preliminary toxicity assay, the concentrations tested were 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 μg/mL. The maximum concentration evaluated approximated the limit dose for this assay. No visible precipitate was observed at the beginning or end of treatment. Adjusted relative survival was 88.56 and 103.60% at a concentration of 2000 μg/mL with and without S9, respectively. Based upon these results, the concentrations chosen for the definitive mutagenicity assay were 125, 250, 500, 1000 and 2000 μg/mL with and without S9.

In the definitive mutagenicity assay, no visible precipitate was observed at the beginning or end of treatment. The average adjusted relative survival was 119 and 85.66% at a concentration of 2000 μg/mL with and without S9, respectively. Cultures treated at all concentrations with and without S9 were chosen for mutant selection. No significant increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at any concentration evaluated with or without S9 (p > 0.01). The positive controls induced significant increases in mutant frequency (p <0.01).

These results indicate that the test substance was negative for the ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system.

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

Genetic toxicity in vivo

Description of key information

No in vivo genetic toxicity study with the test substance is performed

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Genetic toxicity in vitro:

Bacterial Reverse Mutation Assay (OECD Guideline 471):

All criteria for a valid study were met as described in the protocol. The test substance was tested to evaluate its mutagenic potential by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system. Water was used as the vehicle.

In the initial toxicity-mutation assay, the dose levels tested were 1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate. Neither precipitate nor toxicity was observed. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Based upon these results, the maximum dose tested in the confirmatory mutagenicity assay was 5000 μg per plate.

In the confirmatory mutagenicity assay, the dose levels tested were 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate. Neither precipitate nor toxicity was observed. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

These results indicate the substance was negative for the ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system.

Chromosome aberration assay in Chinese Hamster Ovary cells (OECD Guideline 473):

Based upon the results of the preliminary toxicity assay, in which no cytotoxicity was observed at any dose in any of the three exposure groups, the doses chosen for the chromosome aberration asay ranged from 100 to 2000 µg/mL.

In the chromosome aberration assay, cytotoxicity (≥ 50% reduction in cell growth index relative to the vehicle control) was not observed at any dose in any of the three exposure groups. The doses selected for evaluation of chromosome aberrations were 500, 1000, and 2000 μg/mL for all three exposure groups.

In the non-activated 4 and 20-hour exposure groups, no significant or dose-dependent increases in structural aberrations were observed at any dose (p > 0.05; Fisher’s Exact and Cochran-Armitage tests).

In the S9-activated 4-hour exposure group, statistically significant increases in structural aberrations (3.3%) were observed at doses 1000, and 2000 μg/mL (p ≤ 0.05; Fisher’s Exact). However, the Cochran-Armitage test was negative for a dose response (p > 0.05). In addition, the increase was within the historical 95% control limit of 0.00% to 3.88%. Since the significant increases in structural aberrations did not meet any criteria for a positive response, the statistical significance can be attributed to the 0.0% induction of structural aberrations in the vehicle control. Therefore, the statistically significant increase in structural chromosomal aberrations was considered biologically irrelevant.

No significant or dose-dependent increases in numerical (polyploid or endoreduplicated cells) aberrations were observed at any dose in any of the treatment groups (p > 0.05; Fisher’s Exact and Cochran-Armitage tests).

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

In vitro mammalian cell forward gene mutation (CHO/HPRT) assay (OECD Guideline 476):

In the preliminary toxicity assay, the concentrations tested were 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 µg/mL. The maximum concentration evaluated approximated the limit dose for this assay. No visible precipitate was observed at the beginning or end of treatment. Adjusted relative survival was 88.56% and 103.60% at a concentration of 2000 µg/mL with and without S9, respectively.  Based upon these results, the concentrations chosen for the definitive mutagenicity assay were 125, 250, 500, 1000 and 2000 µg/mL with and without S9.

In the definitive mutagenicity assay, no visible precipitate was observed at the beginning or end of treatment. The average adjusted relative survival was 119% and 85.66% at a concentration of 2000 µg/mL with and without S9, respectively. Cultures treated at all concentrations with and without S9 were chosen for mutant selection. No significant increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at any concentration evaluated with or without S9 (p > 0.01). The positive controls induced significant increases in mutant frequency (p < 0.01).

These results indicate the substance was negative for the ability toinduce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system.


Justification for classification or non-classification

Based on the results of the available in vitro studies, the substance should not be classified for genetic toxicity according to the criteria laid down in the CLP Regulation.