Registration Dossier
Registration Dossier
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EC number: 219-006-1 | CAS number: 2312-35-8
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Propargite was determined to be negative overall for genotoxicity in vitro.
- Negative in the bacterial reverse mutation assay, equivalent or similar to EU Method B.13/14 (Brusick, 1977)
- Negative in the mammalian chromosome aberration test, equivalent or similar to EU Method B.10 (Kirkland, 1985)
- Negative in the DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells, EPA OPP 84-2 (Barfknecht, 1987)
- Negative in the CHO-HGPRT mutation assay (DMSO solvent), EPA OPP 84-2 (Bigger and Clarke, 1993a)
- Negative in the CHO-HGPRT mutation assay (acetone solvent), EPA OPP 84-2 (Bigger and Clarke, 1993b)
- Negative in the bacterial reverse mutation assay, OECD 471 and US EPA OPPTS 870.5100 (Wagner, 2014)
- Negative in the mammalian chromosome aberration test, OECD 473 and US EPA OPPTS 870.5375 (Roy, 2014)
- Positive in the presence of metabolic activation, mammalian cell gene mutation assay, OECD 476, US EPA OPPTS 870.5300 and JMAFF 59 Nousan Number 4200 (Stankowski, 2014)
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- Not stated
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- GLP compliance:
- no
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- S. typhimurium TA 1538
- Species / strain / cell type:
- Saccharomyces cerevisiae
- Details on mammalian cell type (if applicable):
- Strain: D4
- Metabolic activation:
- with and without
- Test concentrations with justification for top dose:
- 0, 0.001, 0.01, 0.1, 1.0 and 5.0 µL/plate
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: deionised water or DMSO
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 2-acetylaminofluorene
- 2-nitrofluorene
- other: methylnitrosoguanidine, quinacrine mustard, 2-anthramine, 8-aminoquinoline
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation). Direct to molten agar mixture containing biotin and histidine, plus 0.5 mL activated S-9 liver homogenate reaction mixture in the case of metabolic activation, and then final agar mixture poured into plates.
NUMBER OF REPLICATIONS: One
DURATION
- Exposure duration: 48 hours at 37 ºC - Key result
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Remarks:
- yes
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- Saccharomyces cerevisiae
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Conclusions:
- Interpretation of results: negative
Under the conditions of the test, the test material was determined to be negative for genotoxicity in a bacterial reverse mutation assay. - Executive summary:
In a non-GLP bacterial reverse mutation assay conducted in accordance with a procedure considered to be similar or equivalent to EU Method B.14, the genotoxicity of the test material was determined. Under the conditions of the test, the test substance was determined to be negative for genotoxicity.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 29th April 1985 to 6th June 1985
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- Methodology is based on in-house protocol of Microtest research Ltd which is based on studies that form the basis of EU Method B.10.
- GLP compliance:
- yes
- Type of assay:
- other: in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- rat liver S9 activation mix
- Test concentrations with justification for top dose:
- 25, 50 and 100 µg/mL (without metabolic activation)
50, 100 and 200 µg/mL (with metabolic activation) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- methylmethanesulfonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in suspension
DURATION
- Exposure duration: 2 hours
- Expression time (cells in growth medium): 22 hours
- Fixation time (start of exposure up to fixation or harvest of cells): at least 18 hours
SPINDLE INHIBITOR (cytogenetic assays): colchicine
STAIN (for cytogenetic assays): 4 % Gurr's Giemsa R66 in pH 6.8 buffer for five minutes
NUMBER OF REPLICATIONS: 3
NUMBER OF CELLS EVALUATED: 100 metaphases where possible - Evaluation criteria:
- N = normal
CHROMOSOME-TYPE ABERRATIONS
G = chromosome gap
C = chromosome deletion
C/C = interchange between chromosomes
C↑C - chromosome intrachange
D = dicentric
D+F = dicentric with accompanying fragment
AR = acentric ring
CR = centric ring
CR+F = centric ring with accompanying fragment
M = double minute
F = isolocus fragment
CHROMATID-TYPE ABERRATIONS
g = chromatid gap
c = chromatid deletion
SU = isochromatid deletion with sister union of broken ends
Nud = isochromatid deletion with non-union of broken ends distally
Nup = isochromatid deletion with non-union of broken ends proximally
c/c = interchange between chromatids of different chromosomes
c↑c = chromatid intrachange
i/c = isochromatid/chromatid interchange
m = single minute
OTHERS
Endo = endoreduplicated
Hyper = hyperdiploid
Poly = polypolid
Pulv = pulverised - Statistics:
- Number of aberrations were compared with appropriate controls using the following formula:
chi-squared = ((|O - E| - 0.5)2 / E) - Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- main test needed to be repeated with lower doses as initial test showed toxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- Cultures treated with the test material exhibited numbers of aberrations which were similar to or less than those seen in the solvent controls. Although one solvent control contained quite a large number of gaps, it was still within the normal range for a negative control. There was no significant increase in aberrations following treatment with the test material but some treatments, in the absence of metabolic activation, resulted in significant decreases in aberrations compared with the solvent controls.
RANGE-FINDING/SCREENING STUDIES:
It was seen that a dose of 1000 µg/mL induced approximately 90 % inhibition of mitosis in the absence and presence of metabolic activation. As a 50-80 % inhibition of mitosis is required in the aberration study, doses of 1000, 500, 250 and 125 µg/mL were chosen for the aberration assay. However, these does proved to be too toxic and a repeat assay was performed with 200 µg/mL as the top dose. - Conclusions:
- Interpretation of results: negative
Under the conditions of the test, it was concluded that the test material was not able to induce chromosome aberrations in CHO cells, either in the absence or presence of metabolic activation, when treated up to its limit of toxicity. - Executive summary:
D-014 was assayed in an in vitro cytogenetics assay using duplicate cultures of Chinese Hamster ovary (CHO) cells in the presence and absence of metabolic activation by a rat liver post-mitochondrial (S-9) fraction from Aroclor-1254 induced animals. Dose levels were determined from a preliminary toxicity experiment using single cultures which indicated that 100 µg/mL caused approximately 90 % inhibition of mitosis in the absence and presence of metabolic activation and therefore the doses chosen for the main study were 100, 500, 250 and 125 µg/mL. However, these doses provided to be too toxic in the main study and so it was repeated with lower doses of 200, 100, 50 and 25 µg/mL. Aberration analysis was carried out in the absence of metabolic activation at 100, 50 and 25 µg/mL and in the presence of metabolic activation at 200, 100 and 50 µg/mL. Appropriate solvent and positive controls were included.
Treatment of cells with D-014 in the absence of metabolic activation resulted in numbers of aberrations less than those seen in the solvent controls. Although one solvent control in the absence of metabolic activation contained quite a large number of gaps, it was still within the normal range for a negative control and significantly less than the positive controls. Treatment of cells with D-014 in the presence of metabolic activation resulted in numbers of aberrations which were similar to or less than those seen in the solvent controls. There were no significant increases in aberrations following treatment with D-014 although some of the decreases in the absence of metabolic activation were significant.
Under the conditions of the test, it was concluded that the test material was not able to induce chromosome aberrations in CHO cells, either in the absence or presence of metabolic activation, when treated up to its limit of toxicity.
- Endpoint:
- in vitro DNA damage and/or repair study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 7th May 1987 to 31st May 1987
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- EPA OPP 84-2
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- other: DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro
- Species / strain / cell type:
- hepatocytes: rat
- Metabolic activation:
- not applicable
- Test concentrations with justification for top dose:
- Nominal: 0.0167, 0.05, 0.167, 0.5, 1.67, 5.0, 16.7, 50, 167, 500, 1670 and 5000 µg/mL.
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-acetamidofluorene
- Details on test system and experimental conditions:
- CELL EXPOSURE
1 x 10E+5 viable hepatocytes were inoculated into 12 well cluster dishes containing 15 mm diameter Thermonax plastic coverslips in WME containing 10 % calf serum. The hepatocytes were allowed to attach for approximately 2 hours in a 37 ºC CO2 incubator. The cultures were rinsed and serum-free medium containing the test material and 10 µC/mL of 3H-thymidine (specific activity 50-80 Ci/mM) added to each culture. 18-20 hours after exposure, the cultures were washed three times with 3 mL volumes of phosphate buffered saline by aspiration.
CELL FIXATION
The cells on coverslips were swollen with 1 % sodium citrate for 10-15 minutes and fixed in three 30 minute changes of 100 % ethanol:glacial acetic acid (3:1). The coverslips were air dried and mounted cell surface up on glass slides with permount. Sides with dipped in NTB-2 photographic emulsion in the dark, allowed to dry overnight and stored at 4 ºC in light-proof slide boxes.
STAINING
After 7 days of exposure, autoradiographs were developed at approximately 15 ºC for 4 minutes, washed in distilled water with 5 mL glacial acetic acid for 30 seconds, immersed in Fixer for 10 minutes, washed in running tap water for 5 minutes, dried, stained in Harris Alum hematoxylin followed by a dip rinse in acid alcohol with rinsing in running tap water for 2-5 minutes and a dip rinse in ammonium water. The slides were then rinsed in running tap water for 2-5 minutes dipped in 70 % ethanol, followed by a 10-60 second dip in eosin solution. The slides were then rinsed in three separate baths of 95 % ethanol for 2 minute intervals. The slides were then air-dried and coverslipped in permount with excess emulsion scraped off. - Evaluation criteria:
- Unscheduled DNA 'repair' synthesis, evidence by a net increase in black silver grains over the nucleus, was quantified by determining nuclear and cytoplasmic grain counts using an Automated colony counter. A total of 150 cells/dose point were counted for autoradiographic UDS determinations. A correlation coefficient was calculated by the following method: an area/grain ratio was obtained by visually scoring an area of each slide containing 3-5 nuclear grains and then obtaining an object area count using the colony counter. Five such areas were scored, the differences summed and the mean obtained. This value served as a correction coefficient.
The cytoplasmic grain count was quantitated by randomly selecting the highest of three nuclear-sized areas adjacent to each nucleus. This value was then subtracted from the uncorrected nuclear grain count to determine the net nuclear grain count. Both nuclei of binucleated cells were recorded separately. Replicative DNA synthesis was evidenced by nuclei blackened with grains too numerous to count. The data of the HPC/DNA Repair Assay are reported as mean grains/nucleus from the triplicate wells. The solvent control should have a net nuclear grain count of 1 or less. In addition, the net nuclear grain count for the solvent control did not exceed the upper 95% confidence limits of mean historical data. Also the positive control should yield a mean net nuclear count that is within one standard deviation of the mean historical value.
The test material is reported to be positive when the minimum net grain count of 3 per nuclei is consistently observed in triplicate wells. - Key result
- Species / strain:
- hepatocytes: rat
- Metabolic activation:
- not applicable
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- The dose levels of 1.67 to 5000 µg/mL were determined to be cytotoxic and were not evaluated. The highest dose level evaluated was 0.5 µg/mL; additional levels evaluated were 0.0167, 0.05 and 0.167 µg/mL. None of the treated cultures produced mean net nuclear grain counts that were substantially greater than the solvent control. The negative and positive control values were within the acceptable range of mean historical data.
- Conclusions:
- Interpretation of results: negative
Under the conditions of the test, the test mateial was determined to be negative for genotoxicity. This conclusion is based upon the inability of the substance to produce a mean net nuclear gain count of plus five or greater at any level of concentration. - Executive summary:
Triplicate cultures were seeded with 1 x 105 viable cells, yielded from the liver of a male Fischer 344 rat, with 20 µL test material at 0.0167, 0.05, 0.167, 0.5, 1.67, 5.0, 16.7, 50, 167, 500, 1670 and 5000 µg/mL. In addition, acetone (as the vehicle) and 2-acetamidofluorene (2AAF) at a final concentration in the treatment medium of 1 x 10-7 M, serving as the positive control, were evaluated concurrently. The highest dose scored in the DNA Repair Test was 0.5 µg/mL due to excessive toxicity at the higher doses. Unscheduled DNA repair synthesis was quantified by a net nuclear increase of black silver grains for 50 cells/coverslip. This value was determined by subtracting the highest of three adjacent cytoplasmic counts from the nuclear counts. Three coverslips at each dose point were evaluated for a total of 150 cells/dose. The coverslips were evaluated at a magnification of approximately 1500X.
The results from the test material were negative in the Rat Hepatocyte Primary Culture/DNA Repair Test under the conditions of the assay. These findings are based upon the inability of the test material to produce a mean net nuclear grain count of five or greater at any level of concentration. The solvent and positive controls employed in the evaluation induced mean net nuclear grain counts that were within the acceptable limits of the historical data.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 27th January 1993 to 17th February 1993
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- EPA OPP 84-2
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- other: mammalian cell gene mutation assay
- Target gene:
- X-chromosome linked hypoxanthine-guanine phosphoribosyl trasferase (HGPRT) locus
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- - Periodically checked for Mycoplasma contamination: yes
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254-induced rat liver S-9
- Test concentrations with justification for top dose:
- 0, 1.0, 1.8, 2.6, 3.4, 4.2 and 5.0 µg/mL (initial assay; without metabolic activation)
0, 0.2, 1.0, 1.8, 2.6, 3.4 and 4.2 µg/mL (confirmatory assay; without metabolic activation)
0, 10, 25, 37.5, 50, 60 and 75 µg/mL (initial assay; with metabolic activation)
0, 10, 25, 37.5, 40, 45, 47.5 and 50 µg/mL (confirmatory assay; with metabolic activation) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- Exponentially growing CHO-K1-BH4 cells were seeded in F12FBS5-Hx at a density of 5 x 10E+5 cells/25 cm2 surface area of treatment flask and were incubated at 37 ± 1 ºC in a humidified atmosphere of 5 ± 1 % CO2 in air for 18-24 hours.
The time of initiation of treatment was designated as day 0. Treatment was carried out by refeeding the treatment flasks with 5 mL F12FBS-Hx/25 cm2 surface area of the treatment flask for the non-activated study and 4 mL F12FBS5-Hx and 1 mL S-9 mixture/25 cm2 surface area of treatment flask for the activated study, to which was added 50 µL dosing solution/25 cm2 surface area of treatment flask of test or control material in solvent or solvent alone. Cells were exposed, in duplicate, to five concentrations of the test material for 5 hours at 37 ± 1 ºC. After the treatment period, all media were aspirated, the cells washed with CMF-HBSS and cultured in F12FBS5-Hx for an additional 18-24 hours at 37 ± 1 ºC. At this time the cells were subcultured to assess cytotoxicity and to initiate the phenotypic expression period.
For evaluation of cytotoxicity, the replicates from each treatment condition were detached using trypsin and subcultured independently in F12DBS5-Hx, in triplicate, at a density of 100 cells/60 mm dish. After 7-10 days incubation, the colonies were rinsed with HBSS, fixed with methanol, stained with 10 % aqueous Giemsa, counted and cell survival determined.
For the expression of the mutant phenotype, the replicates from each treatment condition were trypsinized and subcultured independently, in duplicate, at a density no greater than 10E+6 cells/100 mm dish. Subculturing by trypsinizing at 2-3 day intervals was employed for the 7-9 day expression period. At this time, selection for the mutant phenotype was performed.
For selection of the TG-resistant phenotype, the replicates from each treatment condition were trypsinized, pooled and replated, in quintuplicate, at a density of 2 x 10E+5 cells/100 mm dish in F12FBS5-Hx containing 10 µm 6-thioguanine (TG, 2-amino-6-mercaptopurine). For cloning efficiency determinations at the time of selection, 100 cells/60 mm dish were plated in triplicate. After 7-10 days of incubation, the colonies were fixed, stained and counted for both cloning efficiency and mutant selection. - Evaluation criteria:
- The cytotoxic effects of each treatment were expressed relative to the vehicle control (relative cloning efficiency). The mutant frequency for each treatment was calculated by dividing the total number of mutant colonies by the number of cells selected (2 x 10E+6 cells: 10 plates at 2 x 10E+5 cells/plate), corrected for the cloning efficiency of cells prior to mutant selection, and is expressed as TG-resistant mutants per 10E+6 clonable cells. For experimental conditions in which no mutant colonies were observed, mutant frequencies were expressed as less than the frequency obtained with one mutant colony. Mutant frequencies generated from doses giving ≤ 10 % relative survival and presented but were not considered as valid data points.
For the CHO/HGPRT assay, spontaneous mutant frequencies range from 0 to 25 mutants per 10E+6 clonable cells. As a result, calculation of mutagenic responses in terms of fold increase in mutant frequency above the background rate does not provide a reliable indication of the significance of the observed response. The wide acceptable range in spontaneous mutant frequency also suggests the need to set a minimal mutant frequency for a response to be considered positive. A minimum level of >40 mutants per 10E+6 clonable cells was used.
The assay will be considered positive in the event of a dose-dependent increase in mutant frequencies with at least two consecutive doses showing mutant frequencies which are elevated above 40 mutants per 10E+6 clonable cells. In evaluating replicate treatments, the final determination of a positive point will be based on the mutant frequency derived from the mean of the replicate cultures. If a single point above 40 mutants per 10E+6 clonable cells is observed at the highest dose, the assay will be considered suspect. - Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Remarks:
- strain/cell type: HGPRT
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- RANGE-FINDING/SCREENING STUDIES:
Dose levels were selected following a preliminary toxicity test; toxicity was based upon cloning efficiency after treatment relative to the vehicle control. CHO cells were exposed to the vehicle alone and nine concentrations of test material ranging from 5000 to 0.5 µg/mL in the absence and presence of metabolic activation. Based on the results of the preliminary toxicity test, the doses chosen for the initial mutagenesis assay were 5.0, 4.2, 3.4, 2.6, 1.8 and 1.0 µg/mL in the absence of metabolic activation and 75, 60, 50, 37.5, 25 and 10 µg/mL in the presence of metabolic activation. The doses used in the confirmatory mutagenesis assay were 4.2, 3.4, 2.6, 1., 1.0 and 0.2 µg/mL in the absence of metabolic activation and 50, 47.5, 45, 40, 37.5, 25 and 10 µg/mL in the presence of metabolic activation.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the initial assay, the concurrent cytotoxicities, as measured by cloning efficiencies relative to vehicle controls, were 53, 39, 24, 10, 1 and 0 % without metabolic activation and 85, 77, 54, 3, 0 and 0 % with metabolic activation (lowest to highest dose). In the confirmatory assay, the concurrent cytotoxicities, as measured by cloning efficiencies relative to solvent controls, were 98, 43, 8, 6, 3 and 1 % without metabolic activation and 108, 62, 12, 3, 2, 4 and 0 % with metabolic activation (lowest to highest dose). - Conclusions:
- Interpretation of results: negative
Under the conditions of the test, the test material was found to be negative in the CHO-HGPRT mutation assay both in the absence and presence of metabolic activation. - Executive summary:
The test material was tested in the CHO/HGPRT mutation assay in the absence and presence of metabolic activation with Aroclor-induced rat liver S-9. The initial assay was conducted at 5.0, 4.2, 3.4, 2.6, 1.8 and 1.0 µg/mL in the non-activated study and at 75, 60, 37.5, 25 and 10 µg/mL in the activated study. The confirmatory assay was conducted at 4.2, 3.4, 2.6, 1.8, 1.0 and 0.2 µg/mL in the non-activated study and 50, 47.5, 45, 40, 37.5, 25 and 10 µg/mL in the activated study.
Under the conditions of these mutagenicity tests, the test material was determined to be negative in the absence and presence of metabolic activation.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 27th January 1993 to 14th June 1993
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- EPA OPP 84-2
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- other: mammalian cell gene mutation assay
- Target gene:
- X-chromosome linked hypoxanthine-guanine phosphoribosyl trasferase (HGPRT) locus
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- - Periodically checked for Mycoplasma contamination: yes
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254-induced rat liver S-9
- Test concentrations with justification for top dose:
- 0, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0 and 5.0 µg/mL (without metabolic activation; both assays)
0, 5, 10, 20, 30, 40 and 50 µg/mL (with metabolic activation; both assays) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- Exponentially growing CHO-K1-BH4 cells were seeded in F12FBS5-Hx at a density of 5 x 10E+5 cells/25 cm2 surface area of treatment flask and were incubated at 37 ± 1 ºC in a humidified atmosphere of 5 ± 1 % CO2 in air for 18-24 hours.
The time of initiation of treatment was designated as day 0. Treatment was carried out by refeeding the treatment flasks with 5 mL F12FBS5-Hx/25 cm2 surface area of the treatment flask for the non-activated study and 4 mL F12FBS5-Hx and 1 mL S-9 mixture/25 cm2 surface area of treatment flask for the activated study, to which was added 50 µL dosing solution/25 cm2 surface area of treatment flask of test or control substance in solvent or solvent alone. Cells were exposed, in duplicate, to five concentrations of the test material for 5 hours at 37 ± 1 ºC. After the treatment period, all media were aspirated, the cells washed with CMF-HBSS and cultured in F12FBS5-Hx for an additional 18-24 hours at 37 ± 1 ºC. At this time the cells were subcultured to assess cytotoxicity and to initiate the phenotypic expression period.
For evaluation of cytotoxicity, the replicates from each treatment condition were detached using trypsin and subcultured independently in F12FBS5-Hx, in triplicate, at a density of 100 cells/60 mm dish. After 7-10 days incubation, the colonies were rinsed with HBSS, fixed with methanol, stained with 10 % aqueous Giemsa, counted and cell survival determined.
For the expression of the mutant phenotype, the replicates from each treatment condition were trypsinized and subcultured independently in F12FBS5-Hx, in duplicate, at a density no greater than 10E+6 cells/100 mm dish. Subculturing by trypsinizing at 2-3 day intervals was employed for the 7-9 day expression period. At this time, selection for the mutant phenotype was performed.
For selection of the TG-resistant phenotype, the replicates from each treatment condition were trypsinized, pooled and replated, in quintuplicate, at a density of 2 x 10E+5 cells/100 mm dish in F12FBS5-Hx containing 10 µm 6-thioguanine (TG, 2-amino-6-mercaptopurine). For cloning efficiency determinations at the time of selection, 100 cells/60 mm dish were plated in triplicate. After 7-10 days of incubation, the colonies were fixed, stained and counted for both cloning efficiency and mutant selection. - Evaluation criteria:
- The cytotoxic effects of each treatment were expressed relative to the vehicle control (relative cloning efficiency). The mutant frequency for each treatment was calculated by dividing the total number of mutant colonies by the number of cells selected (2 x 10E+6 cells: 10 plates at 2 x 10E+5 cells/plate), corrected for the cloning efficiency of cells prior to mutant selection, and is expressed as TG-resistant mutants per 10E+6 clonable cells. For experimental conditions in which no mutant colonies were observed, mutant frequencies were expressed as less than the frequency obtained with one mutant colony. Mutant frequencies generated from doses giving ≤ 10 % relative survival and presented but were not considered as valid data points.
For the CHO/HGPRT assay, spontaneous mutant frequencies range from 0 to 25 mutants per 10E+6 clonable cells. As a result, calculation of mutagenic responses in terms of fold increase in mutant frequency above the background rate does not provide a reliable indication of the significance of the observed response. The wide acceptable range in spontaneous mutant frequency also suggests the need to set a minimal mutant frequency for a response to be considered positive. A minimum level of >40 mutants per 10E+6 clonable cells was used.
The assay will be considered positive in the event of a dose-dependent increase in mutant frequencies with at least two consecutive doses showing mutant frequencies which are elevated above 40 mutants per 10E+6 clonable cells. In evaluating replicate treatments, the final determination of a positive point will be based on the mutant frequency derived from the mean of the replicate cultures. If a single point above 40 mutants per 10E+6 clonable cells is observed at the highest dose, the assay will be considered suspect. - Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Remarks:
- strain/cell type: HGPRT
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- RANGE-FINDING/SCREENING STUDIES:
Dose levels were selected following a preliminary toxicity test; toxicity was based upon cloning efficiency after treatment relative to the vehicle control. CHO cells were exposed to the vehicle alone and nine concentrations of test material ranging from 5000 to 0.5 µg/mL in the absence and presence of metabolic activation. Based on the results of the preliminary toxicity test, the doses chosen for the initial mutagenesis assay were 0.5, 1.0, 1.5, 2.0, 3.0, 4.0 and 5.0 µg/mL in the absence of metabolic activation and 5.0, 10, 20, 30, 40 and 50 µg/mL in the presence of metabolic activation. The doses used in the confirmatory mutagenesis assay were the same as those used in the initial assay. The confirmatory assay was repeated due to lack of untreated controls both in the absence and presence of metabolic activation and low cloning efficiency of the nonactivated vehicle controls. Data from the first confirmatory assay are not reported.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the initial assay, the concurrent cytotoxicities, as measured by cloning efficiencies relative to vehicle controls, were 79, 23, 7, 11, 3 and 1 % without metabolic activation and 106, 85, 115, 16, 1 and 0 % with metabolic activation (lowest to highest dose). In the confirmatory assay, the concurrent cytotoxicities, as measured by cloning efficiencies relative to solvent controls, were 104, 75, 32, 17, 15, 8 and 7 % without metabolic activation and 152, 131, 119, 54, 12 and 5 % with metabolic activation (lowest to highest dose). - Conclusions:
- Interpretation of results: negative
Under the conditions of the test, the test material was found to be negative in the CHO-HGPRT mutation assay both in the absence and presence of metabolic activation. - Executive summary:
The test material was tested in the CHO/HGPRT mutation assay in the absence and presence of metabolic activation with Aroclor-induced rat liver S-9. The initial assay was conducted at 0.5, 1.0, 1.5, 2.0, 3.0, 4.0 and 5.0 µg/mL in the non-activated study and at 5.0, 10, 20, 30, 40 and 50 µg/mL in the activated study. The confirmatory assay was conducted at the same doses.
Under the conditions of these mutagenicity tests, the test material was determined to be negative in the absence and presence of metabolic activation.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 27 February 2014 to 17 March 2014
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- S typhimurium: Histidine locus
E. coli: Tryptophan locus - Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Details on mammalian cell type (if applicable):
- Before use in this study, strains were confirmed to express appropriate phenotypic characteristics (amino acid requirement, UV sensitivity, presence of rfa and R factors).
Overnight cultures were prepared by inoculating from the appropriate frozen permanent stock into a vessel containing 30 to 50 mL of culture medium. To assure that cultures were harvested in late log phase, the length of incubation was controlled and monitored. Following inoculation, each flask was placed in a shaker/incubator programmed to begin shaking at 125 to 175 rpm and incubating at 37 ± 2 °C for approximately 12 hours before the anticipated time of harvest. Each culture was monitored spectrophotometrically for turbidity and was harvested at a percent transmittance yielding a titre of greater than or equal to 0.3 x 10⁹ cells per mL. The actual titres were determined by viable count assays on nutrient agar plates. - Additional strain / cell type characteristics:
- not applicable
- Species / strain / cell type:
- E. coli WP2 uvr A
- Details on mammalian cell type (if applicable):
- Before use in this study, strains were confirmed to express appropriate phenotypic characteristics (amino acid requirement, UV sensitivity, presence of rfa and R factors).
Overnight cultures were prepared by inoculating from the appropriate frozen permanent stock into a vessel containing 30 to 50 mL of culture medium. To assure that cultures were harvested in late log phase, the length of incubation was controlled and monitored. Following inoculation, each flask was placed in a shaker/incubator programmed to begin shaking at 125 to 175 rpm and incubating at 37 ± 2 °C for approximately 12 hours before the anticipated time of harvest. Each culture was monitored spectrophotometrically for turbidity and was harvested at a percent transmittance yielding a titre of greater than or equal to 0.3 x 10⁹ cells per mL. The actual titres were determined by viable count assays on nutrient agar plates. - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- - Initial Toxicity-Mutation Assay: 0, 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate
- Confirmatory Mutagenicity Assay: 0, 15, 50, 150, 500, 1500 and 5000 μg per plate - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- methylmethanesulfonate
- other: 2-aminoanthracene
- 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).
One-half (0.5) millilitre of S9 or Sham mix, 100 μL of tester strain (cells seeded) and 50 μL of vehicle or test material 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 material aliquot was replaced by a 50 μL aliquot of appropriate positive control. After the overlay had solidified, the plates were inverted and incubated for 48 to 72 hours at 37 ± 2 °C. Plates that were not counted immediately following the incubation period were stored at 2 - 8 °C until colony counting could be conducted. The condition of the bacterial background lawn was evaluated for evidence of test material toxicity by using a dissecting microscope. Precipitate was evaluated after the incubation period by visual examination without magnification.
The solvent controls and the positive controls were run concurrently. The initial toxicity-mutation assay and confirmatory assay were conducted in triplicate. - Evaluation criteria:
- For the test material 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 material.
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 WP2uvrA 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:
- For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and reported.
- Key result
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- 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
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- 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
- Additional information on results:
- INITIAL TOXICITY-MUTATION ASSAY
No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Precipitate was observed beginning at 500 μg per plate. No toxicity was observed.
CONFIRMATORY ASSAY
No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Precipitate was observed beginning at 500 μg per plate. No toxicity was observed.
ANALYTICAL DETERMINATIONS
Test material stability was confirmed analytically in DMSO.
Concentration analysis indicates that the actual mean concentrations of the analysed dose levels were between 71.7 and 106.4 % of their respective targets with < 5.0 % relative standard deviation (RSD). To confirm the out-of-specification result observed at the high concentration in the initial toxicity-mutation assay, a repeat analysis was performed and the formulation was 79.7 % of target. The high dose formulation from the confirmatory mutagenicity assay was not reanalysed. Although the actual concentration of the high dose formulation in each assay was below the protocol-specified acceptable range of 85.0 to 115.0 % of target, precipitate was observed at the top three dose levels. This indicates that the test system was saturated with test substance and the test guideline recommended limit dose was achieved. Therefore, the Study Director concluded that the observed differences from nominal concentration had no adverse impact on the assessment of the test substance’s mutagenicity, and the results do not affect validity of the study conclusion. No test substance was detected in the vehicle control samples. - Conclusions:
- Interpretation of results: negative
Under the conditions of this study the test material is not mutagenic. - Executive summary:
A reverse mutation test was carried out in accordance with the standardised guidelines OECD 471 and US EPA OPPTS 870.5100 under GLP conditions.
The test material was evaluated for its mutagenic potential with four strains of Salmonella typhimurium (TA100, TA98, TA1535, and TA1537) and one strain of Escherichia coli (WP2uvrA). The test was conducted by the plate incorporation method in the presence and absence of metabolic activation (rat liver S9 mix).
In an 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. Precipitate was observed beginning at 500 μg per plate. No toxicity was observed. 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 15, 50, 150, 500, 1500 and 5000 μg per plate. Precipitate was observed beginning at 500 μg per plate. No toxicity was observed.
Based on these results, it is concluded that the test material is not mutagenic under the test conditions.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 04 February 2014 to 10 April 2014
- 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
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
- Deviations:
- no
- Principles of method if other than guideline:
- A repeat assay in the presence of S9 was omitted. Some regulators may interpret this as being necessary under the 1997 iteration of OECD 473, as only 200 metaphases were analysed.
- GLP compliance:
- yes
- Type of assay:
- other: in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: McCoy's 5A medium containing 10 % foetal bovine serum, 1.5 mM L-glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin and 2.5 μg/mL Amphotericin B
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes; prior to freezing, lot of cells was tested using the Hoechst staining procedure and found to be free of mycoplasma contamination.
Chinese hamster ovary (CHO-K1) cells (repository number CCL 61) were obtained from American Type Culture Collection, Manassas, VA. In order to assure the karyotypic stability of the cell line, working cell stocks were not used beyond passage 15. This cell line has an average cell cycle time of 10-14 hours with a modal chromosome number of 20. - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- Preliminary cytotoxicity assays:
- With and without S9 mix (4-hour treatment): 0.35, 1.05, 3.5, 10.5, 35, 105, 350, 1050 and 3500 µg/mL
- Without S9 mix (24-hour treatment): 0.35, 1.05, 3.5, 10.5, 35, 105, 350, 1050 and 3500 µg/mL
Chromosomal aberration assay:
- Without S9 mix (4-hour treatment): 0.1, 0.25, 0.5, 1, 1.5, 2, 2.5 and 3 µg/mL
- With S9 mix (4-hour treatment): 3, 5, 10, 12.5, 15, 17.5, 20, 22.5 and 25 µg/mL
- Without S9 mix (20-hour treatment): 0.025, 0.05, 0.1, 0.25, 0.35, 0.5, 0.75 and 1 µg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
A solubility test was conducted in order to determine the highest soluble or workable stock concentration up to 500 mg/mL in DMSO. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- Details on test system and experimental conditions:
- TEST PROCEDURE
Exponentially growing CHO-K1 cells were seeded in complete medium for each treatment condition at a target of 5 x 10^5 cells/culture. The cultures were incubated under standard conditions (37 ± 1 °C in a humidified atmosphere of 5 ± 1 % CO2 in air) for 16-24 hours.
Treatment of cells (Preliminary Toxicity Test and Definitive Assay) was carried out by re-feeding the cultures with 5 mL complete medium for the non-activated exposure or 5 mL S9 mix (4 mL serum-free culture medium + 1 mL of S9 cofactor pool) for the S9-activated exposure, to which was added 50 μL of test material dosing solution or vehicle alone. In the definitive assay, positive control cultures were re-suspended in either 5 mL of complete medium for the non-activated studies, or 5 mL of the S9 reaction mixture (4 mL serum free medium + 1 mL of S9 cofactor pool), to which was added 50 μL of positive control in solvent.
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. In the non-activated 20-hour treatment group, cultures with visible precipitate were washed with CMF-PBS to avoid precipitate interference with cell counts. Two hours prior to cell harvest, Colcemid® was added to all cultures at a final concentration of 0.1 μg/mL.
For the preliminary toxicity test and definitive assay, cells were collected at 20 hours (± 30 minutes), 1.5 normal cell cycles, after initiation of treatment to ensure that the cells are analysed in the first division metaphase. Just prior to harvest, the cell cultures was visually inspected for the degree of monolayer confluency relative to the vehicle control. The cells were trypsinized and counted and the cell viability was assessed using trypan blue dye exclusion. The cell count was determined from a minimum of two cultures to determine the number of cells being treated (baseline). The data was presented as cell growth inhibition in the treatment group compared to vehicle control. Cell growth was determined by Relative Increase in Cell Counts (RICC) as a measure of cytotoxicity (Fellows and O'Donovan 2007; Lorge et al., 2008). The cell counts and percent viability were used to determine cell growth inhibition relative to the vehicle control (% cytotoxicity).
For the definitive assay only, cells were collected by centrifugation, treated with 0.075 M 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 cells were re-suspended in fresh fixative. The suspension of fixed cells was applied to glass microscope slides and air-dried. The slides were stained with Giemsa and permanently mounted.
To ensure that a sufficient number of metaphase cells were present on the slides, the percentage of cells in mitosis per 500 cells scored (mitotic index) was determined and recorded for each coded culture selected for scoring chromosome aberrations. Metaphase cells with 20 ± 2 centromeres were examined under oil immersion without prior knowledge of treatment groups. Whenever possible, a minimum of 200 metaphase spreads from each concentration (100 per duplicate culture) were examined and scored for chromatid-type and chromosome-type aberrations. The number of metaphase spreads examined and scored per duplicate culture was reduced if the percentage of aberrant cells reached at least 10 %, determined based on historical positive control data, before 100 cells are scored. 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 aberrations but were considered part of the incomplete exchange. Pulverized cells and severely damaged cells (counted as 10 aberrations) were also recorded. Chromatid and isochromatid gaps were recorded but not included in the analysis. The XY vernier for each cell with a structural aberration was recorded. The percentage of cells with numerical aberrations (polyploid and endoreduplicated cells) was evaluated for 100 metaphases per culture (200 metaphases per concentration). - Evaluation criteria:
- CRITERIA FOR DETERMINATION OF A VALID TEST
The frequency of cells with structural chromosome aberrations in the vehicle control must be within the historical control range. The percentage of cells with aberrations must be statistically increased (p ≤ 0.05, Fisher's exact test) in the positive control treatment relative to the vehicle control.
EVALUATION OF TEST RESULTS
Toxicity induced by treatment was based upon cell growth inhibition relative to the vehicle control and was reported for the preliminary toxicity and chromosome aberration portions of the study. The number and types of aberrations (structural and numerical) found, the percentage of structurally damaged cells in the total population of cells examined (percent aberrant cells), the percentage of numerically damaged cells in the total population of cells examined, and the average number of structural aberrations per cell (mean aberrations per cell) were calculated and reported for each treatment group. Chromatid and isochromatid gaps were presented in the data but were not included in the total percentage of cells with one or more aberrations or in the average number of aberrations per cell.
The test substance was considered positive if it induced a statistically significant and dose-dependent increase the frequency of aberrant metaphases (p ≤ 0.05). If only Fisher's exact test is statistically significant without dose-dependent increase, the result was considered equivocal. If neither criterion was met, the results were considered to be negative. - Statistics:
- Statistical analysis of the induction in aberrant cells was performed using the Fisher's exact test. The Fisher's test was used to compare pairwise the percent aberrant cells of each treatment group with that of the vehicle control. The Cochran-Armitage test was used to evaluate dose-responsiveness.
- Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and 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:
- PRELIMINARY CYTOTOXICITY ASSAY
Substantial toxicity (i.e., ≥ 50 % cell growth inhibition, relative to the vehicle control) was observed at concentrations ≥ 3.5 μg/mL in the non-activated 4-hour exposure group, at concentrations ≥ 35 μg/mL in the S9-activated 4-hour exposure group, and at concentrations ≥ 1.05 μg/mL in the non-activated 20-hour continuous exposure group. The concentrations selected for testing in the chromosome aberration assay were based upon these results.
Visible precipitate was observed in treatment medium at concentrations ≥ 105 μg/mL, while concentrations ≤ 35 μg/mL were soluble in treatment medium at the beginning and conclusion of the treatment period. The osmolality in treatment medium of the highest concentration tested, 3500 μg/mL, was 368 mmol/kg. The osmolality in treatment medium of the lowest precipitating concentration, 105 μg/mL, was 418 mmol/kg. The osmolality in the treatment medium of the highest soluble concentration, 35 μg/mL, was 420 mmol/kg. The osmolality of the solvent (DMSO) in the treatment medium was 416 mmol/kg. The osmolality of the test substance concentrations in treatment medium is acceptable because it did not exceed the osmolality of the vehicle by more than 20 %. The pH of the highest concentration of test substance in treatment medium was 7.5.
CYTOGENICITY AND CHROMOSOME ANALYSIS
In the chromosome aberration assay, the test substance was soluble in DMSO and in the treatment medium at all concentrations tested at the beginning and conclusion of the treatment period. The pH of the highest concentration of test substance in treatment medium was 7.0.
Toxicity of the test material (cell growth inhibition relative to the vehicle control) in CHO cells when treated for 4 hours in the absence of S9 activation was 55 % at 2 μg/mL, the highest test concentration evaluated for chromosome aberrations. The mitotic index at the highest concentration evaluated for chromosome aberrations, 2 μg/mL, was reduced by 29 % relative to the vehicle control. The concentrations selected for microscopic analysis were 0.5, 1 and 2 μg/mL. The percentage of cells with structural or numerical aberrations in the test substance-treated group was not significantly increased relative to vehicle control at any concentration (p > 0.05, Fisher's Exact test). The percentage of structurally aberrant cells in the MMC (positive control) treatment group (23.0 %) was statistically significant (p ≤ 0.01, Fisher's Exact test).
Toxicity of the test material (cell growth inhibition relative to the vehicle control) in CHO cells when treated for 4 hours in the presence of S9 activation was 55 % at 10 μg/mL, the highest test concentration evaluated for chromosome aberrations. The mitotic index at the highest concentration evaluated for chromosome aberrations, 10 μg/mL, was reduced by 1 % relative to the vehicle control. The concentrations selected for microscopic analysis were 3, 5 and 10 μg/mL. The percentage of cells with structural or numerical aberrations in the test substance-treated group was not significantly increased relative to vehicle control at any concentration (p > 0.05, Fisher's Exact test). The percentage of structurally aberrant cells in the CP (positive control) treatment group (17.0 %) was statistically significant (p ≤ 0.01, Fisher's Exact test).
Toxicity of the test material (cell growth inhibition relative to the vehicle control) in CHO cells when treated for 20 hours in the absence of S9 activation was 68 % at 0.75 μg/mL, the highest test concentration evaluated for chromosome aberrations. The mitotic index at the highest concentration evaluated for chromosome aberrations, 0.75 μg/mL, was reduced by 41 % relative to the vehicle control. The concentrations selected for microscopic analysis were 0.1, 0.35 and 0.75 μg/mL. The percentage of cells with structural or numerical aberrations in the test substance-treated group was not significantly increased relative to vehicle control at any concentration (p > 0.05, Fisher's Exact test). The percentage of structurally aberrant cells in the MMC (positive control) treatment group (24.0 %) was statistically significant (p ≤ 0.01, Fisher's Exact test).
The results for the positive and negative controls indicate that all criteria for valid assay were met. Based on these criteria, results are justified and do not require a repeat of any portions of the study.
ANALYTICAL DETERMINATIONS
The test material in DMSO was stable at room temperature for at least 3.5 hours at concentrations of 0.0319 and 77.5 mg/mL. Analytical results indicated that the actual mean concentrations of the analysed samples (0.035 and 2.5 mg/mL) were 90.1 and 95.6 % of their respective nominal concentrations, with ≤ 5 % relative standard deviation (RSD). No test article was detected in the vehicle control sample. This indicates that the dose formulations were accurately prepared and were acceptable for use in the study. - Conclusions:
- Interpretation of results: negative
Under the conditions of this study the test material was concluded to be negative for the induction of structural and numerical chromosome aberrations in CHO cells in both the non-activated and the S9-activated test systems. - Executive summary:
The clastogenic potential of the test material was examined in an in vitro chromosomal aberration test using Chinese hamster ovary cells (CHO) conducted in accordance with the standardised guidelines OECD 473 and US EPA OPPTS 870.5375 under GLP conditions.
Based on the findings from a preliminary cytotoxicity assay, the doses chosen for the chromosome aberration assay ranged from 0.1 to 3 μg/mL for the non-activated 4-hour exposure group, from 3 to 25 μg/mL for the S9-activated 4-hour exposure group, and ranged from 0.025 to 1 μg/mL for the non-activated 20-hour continuous exposure group.
In the chromosome aberration assay, substantial toxicity was observed at concentrations ≥ 2 μg/mL in the non-activated 4-hour exposure group, at concentrations ≥ 10 μg/mL in the S9-activated 4-hour exposure group, and at concentrations ≥ 0.75 μg/mL in the non-activated 20- hour continuous exposure group. The highest dose analysed under each treatment condition produced an approximate 50 % cell growth inhibition relative to the vehicle control, which met the dose limit as recommended by testing guidelines for this assay.
No statistically significant or dose-dependent increases in structural or numerical (polyploid or endoreduplicated cells) aberrations were observed in treatment groups with or without S9 (p > 0.05; Fisher’s Exact and Cochran-Armitage tests). All vehicle control values were within historical ranges, and the positive controls induced significant increases in the percent of aberrant metaphases (p ≤ 0.01). Thus, all criteria for a valid study were met.
Under the conditions of this study the test material was concluded to be negative for the induction of structural and numerical chromosome aberrations in CHO cells in both the non-activated and the S9-activated test systems.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 03 February 2014 to 03 July 2014
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Version / remarks:
- MAFF Japan, 59 Nousan Number 4200
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- other: mammalian cell gene mutation assay
- Target gene:
- Thymidine kinase (TK) locus
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- - Type and identity of media: F0P supplemented with 10 % horse serum and 2 mM L-glutamine.
- Properly maintained: yes; L5178Y1/TK +/- cells, clone 3.7.2C. were received from Glaxo Wellcome Inc., Research Triangle Park, NC
- Periodically checked for Mycoplasma contamination: yes. Each batch of frozen cells was tested using the agar culture and Hoechst staining procedures and found to be free of mycoplasma contamination.
- Periodically "cleansed" against high spontaneous background: yes. Prior to use in the assay, L5178Y/TK+/- cells were cleansed to reduce the frequency of spontaneously occurring TK -/- cells. Using the procedure described by (Clive and Spector (1975), cells were cultured for 24 hours in the presence of thymidine, hypoxanthine, methotrexate and glycine to poison the TK -/-cells. L5178Y/TK +/-cells were prepared in 50 % conditioned F0P supplemented with 10 % horse serum and 2 mM L-glutamine (F10P) and 50 % Fischer’s Media for Leukemic Cells of Mice with 0.1 % Pluronics F-68 (FP). All media contained antibiotics. - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- Preliminary cytotoxicity (range finding) assay:
The highest concentration evaluated approximated the 10 mM limit dose:
- Without S9 mix (4-hour treatment): 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 3500 µg/mL
- Without S9 mix (4-hour treatment): 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 3500 µg/mL
- With S9 mix (24-hour treatment): 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 3500 µg/mL
Mutation assay I
- Without S9 mix (4-hour treatment): 0.5, 1.5, 5, 10, 15, 20, 30, 40 and 50 µg/mL
- Repeat with S9 mix (4-hour treatment): 0.5, 1.5, 5, 10, 15, 20, 30, 40 and 50 µg/mL
Mutation assay II
- Without S9 mix (24-hour treatment): 0.5, 1.5, 5, 10, 15, 20, 30, 40 and 50 µg/mL
- With S9 mix (4-hour treatment): 0.5, 1, 1.5, 2.5, 5, 7.5, 10, 15, 20, 25, 30, 40 and 50 µg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
A solubility test was conducted in order to determine the highest soluble or workable stock concentration up to 500 mg/mL in DMSO. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- methylmethanesulfonate
- Details on test system and experimental conditions:
- TEST PROCEDURE
The preparation and addition of the test material dosing solutions was carried out under yellow lighting and the cells were incubated, under standard conditions, in the dark during the exposure period. Treatment was carried out by combining 100 µL of dosing solution of test or control material in vehicle or vehicle alone, F0P medium or S9 mix with 6 x 10^6 L5178Y/TK+/- cells in a total volume of 10 mL. Each S9-activated 10 mL culture contained 4 mL S9 mix (final S9 concentration of 1.0 %). Cultures were capped tightly and incubated with mechanical mixing at 37 ± 1 °C for 4 or 24 hours.
For the preliminary toxicity assay only, after a 4-hour treatment in the presence and absence of S9 activation, cells were washed with culture medium and cultured in suspension for two days post-treatment, with cell concentration adjustment on the first day. After a 24 hour treatment in the absence of S9 activation, cells were washed with culture medium and immediately readjusted to 3 x 10^5 cells/mL. Cells were then cultured in suspension for an additional two days post-treatment with cell concentration adjustment on the first day.
For the definitive assay only, at the end of the exposure period, the cells were washed with culture medium and collected by centrifugation. The cells were re-suspended in 20 mL F10P on Day 1 and in 10 mL F10P on Day 2, and incubated under standard conditions for two days following treatment. Cell population adjustments to 3 x 10^5 cells/mL were made as follows:
- 4 hour treatment: 1 and 2 days after treatment
- 24 hour treatment: immediately after test material removal, and 2 and 3 days after treatment.
Cells from selected dose levels were cultured in triplicate with 2 to 4 µg TFT/mL at a density of 1 x 10^6 cells/100 mm plate in cloning medium containing 0.22 to 0.24 % agar. For estimation of cloning efficiency at the time of selection of those same cultures, 200 cells/100 mm plate were cultured in triplicate in cloning medium without TFT (viable cell (VC) plate). Cultures were incubated under standard conditions (37 ± 1 °C in a humidified atmosphere of 5 ± 1 % CO2 in air) for 10-14 days.
The total number of colonies per culture was determined for the VC plates and the total relative growth calculated. The total number of colonies per TFT plate was then determined for those cultures with ≥10 % total growth (including at least one concentration between 10 and 20 % total growth, if possible). Colonies were counted and the diameter of the TFT colonies from the positive control and vehicle control cultures and, in the case of a positive response, the test material-treated cultures were determined over a range from 0.2 to 1.1 mm. - Evaluation criteria:
- In evaluation of the data, increases in induced mutant frequency which occurred only at highly toxic concentrations (i.e., less than 10 % total growth) were not considered biologically relevant. All conclusions were based on scientific judgment; however, the following criteria are presented as a guide to interpretation of the data.
- A result was considered positive if a concentration-related increase in mutant frequency was observed in the treated cultures and one or more treatment conditions with 10 % or greater total growth exhibit induced mutant frequencies of >90 mutants per 10^6 clonable cells (based on the average mutant frequency of duplicate cultures). If the average vehicle control mutant frequency was >90 mutants per 10^6 clonable cells, a doubling of mutant frequency over the vehicle would also be required.
- A result was considered negative if the treated cultures exhibit induced mutant frequencies of less than 90 mutants per 10^6 clonable cells (based on the average mutant frequency of duplicate cultures) and there was no concentration-related increase in mutant frequency.
There are some situations in which a chemical would be considered negative when there was no culture showing between 10-20 % survival.
- There was no evidence of mutagenicity (e.g. no dose response or increase in induced mutant frequencies between 45 and 89 mutants per 10^6) in a series of data points within 100 to 20 % survival and there was at least one negative data point between 20 and 25 % survival.
-There was no evidence of mutagenicity (e.g. no dose response or increase in induced mutant frequencies between 45 and 89 mutants per 10^6) in a series of data points between 100 to 25 % survival and there was also a negative data point between 10 and 1 % survival. In this case, it would be acceptable to count the TFT colonies of cultures exhibiting <10 % total growth. - Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Remarks:
- in the presence of metabolic activation
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- PRELIMINARY CYTOTOXICITY ASSAY
Visible precipitate was observed at concentrations ≥50 µg/mL at the beginning of treatment and at concentrations ≥150 µg/mL by the end of treatment, and the test material did not have an adverse impact on the osmolality of the cultures (osmolality of the solvent and highest soluble test material concentration at the beginning of treatment, 15 µg/mL, were 455 and 451 mmol/kg, respectively). Relative suspension growth (RSG) was 19, 67 and 24 % at a concentration of 15 µg/mL using a 4-hour treatment with and without S9, and a 24-hour treatment without S9, respectively (RSG at all higher concentrations in all treatment groups was 0 %).
MUTATION ASSAYS
The results of the mutagenesis assays are presented in Tables 1 to 4.
No visible precipitate was observed at the beginning or end of treatment. Cultures treated at concentrations of 5, 10, 15, 20 and 30 µg/mL using a 4-hour treatment without S9 exhibited 14 to 80 % RSG and were cloned, while those cultures treated at concentrations of 0.5, 1.5, 5, 10 and 15 µg/mL using a 24 hour treatment without S9 exhibited 13 to 99 % RSG and were cloned (cultures treated at other lower or higher concentrations were discarded prior to cloning because a sufficient number of higher concentrations was available, or they were excluded from evaluation of mutagenicity due to excessive toxicity). Relative total growth (RTG) of the cloned cultures ranged from 7 to 84 % (4-hour treatment without S9) and from 10 to 93 % (24 hour treatment without S9). No increases in induced mutant frequency (IMF) were observed under either treatment condition. The average suspension growth and mutant frequency of the negative controls with S9 were unacceptable and that portion of the assay was repeated. A subsequent re-test with S9 also failed due to unacceptable average negative control mutant frequency.
A second re-test with S9 was conducted under identical conditions. No visible precipitate was observed at the beginning or end of treatment. Cultures treated at concentrations of 1.5, 5, 10, 20 and 40 µg/mL exhibited 26 to 104 % RSG and were cloned (cultures treated at the other concentrations were discarded prior to cloning because a sufficient number of higher concentrations was available, or were excluded from evaluation of mutagenicity due to excessive toxicity). RTG of the cloned cultures ranged from 13 to 101 %. IMFs ≥90 mutants per 10^6 clonable cells were observed in all cloned cultures treated at concentrations ≥5 µg/mL except for single replicates at concentrations of 10 and 20 µg/mL (IMFs were 69 and 88 mutants per 10^6 clonable cells, respectively). In addition, these increases were considered to be dose-dependent, and the increases observed at a concentration of 40 µg/mL also were >2-fold the concurrent vehicle control value.
A confirmatory assay was performed using test material concentrations of 1, 1.5, 2.5, 5, 7.5, 10, 15, 20, 25, 30, 40 and 50 µg/mL (4-hour treatment with S9). No visible precipitate was observed at the beginning or end of treatment. All cultures exhibited 21 to 96 % RSG and were cloned. RTG of the cloned cultures ranged from 21 to 103 %. IMFs ≥90 mutants per 10^6 clonable cells were observed in ten of the 14 cultures treated at concentrations of 5 – 30 µg/mL with S9, and seven of these increases also were >2 fold the concurrent vehicle control value. Although the IMFs decreased at concentrations of 40 and 50 µg/mL with S9, the response still appeared to be dose dependent (but with a plateau rather than linear). Except for the decrease in mutant frequencies at the two highest concentrations evaluated in this trial (40 and 50 µg/mL with S9), the results were remarkably similar to the previous trial.
The trifluorothymidine-resistant colonies for the cloned, test material-treated cultures with S9 and the positive and solvent control cultures with and without S9 were sized according to diameter over a range from approximately 0.2 to 1.1 mm. The data on colony size distributions showed both an increase in the frequency of small (≤0.63 mm) and large colonies (>0.63 mm) when the test material treated cultures were compared to the solvent control cultures. An increase in the frequency of small colonies is consistent with damage to multiple loci on chromosome 11 in addition to functional loss of the TK locus. An increase in large colony mutants is indicative of localized damage in the form of a point mutation or small deletion within the TK locus. The colony sizing for the MMS and DMBA positive controls yielded the expected increase in small colonies (verifying the adequacy of the methods used to detect small colony mutants) and large colonies.
All criteria for a valid study ultimately were met.
ANALYTICAL DETERMINATIONS
For the analysis of dosing formulation, the submitted formulations were found to be accurately prepared, except the low dose (0.05 mg/mL) formulation for Experiment B4 which was found to be below the acceptable range, but met ≤5.00 % RSD. The vehicle control samples were free of test article. - Conclusions:
- Interpretation of results: positive in the presence of metabolic activation
Under the conditions of this study, the test material was positive in the L5178Y/TK+/- Mouse Lymphoma Mutagenesis Assay, in the presence of S9 only, under the conditions and according to the criteria of the test protocol. The results were uniformly negative in the absence of S9. - Executive summary:
The test material was evaluated for its ability to produce gene mutations at the TK locus in Mouse Lymphoma cells in accordance with the standardised guidelines OECD 476, US EPA OPPTS 870.5300 and JMAFF 59 Nousan Number 4200 under GLP conditions.
The test material was prepared in DMSO and evaluated in a preliminary toxicity assay at concentrations from 0.5 to 3500 µg/mL using 4-hour treatments with and without S9, and a 24 hour treatment without S9 (the maximum concentration evaluated approximated the 10 mM limit dose for this assay). Visible precipitate was observed at concentrations ≥50 µg/mL at the beginning of treatment and at concentrations ≥150 µg/mL by the end of treatment, and the test material did not have an adverse impact on the osmolality of the cultures (osmolality of the solvent and highest soluble test material concentration at the beginning of treatment, 15 µg/mL, were 455 and 451 mmol/kg, respectively). Relative suspension growth (RSG) was 19, 67 and 24 % at a concentration of 15 µg/mL using a 4 hour treatment with and without S9, and a 24-hour treatment without S9, respectively (RSG at all higher concentrations in all treatment groups was 0 %).
Based on the results of the preliminary toxicity assay, cultures were treated in the mutagenicity assays at concentrations of 0.5, 1.5, 5, 10, 15, 20, 30, 40 and 50 µg/mL using all three treatment conditions (4 hour treatment with and without activation, 24 hour treatment without activation). No visible precipitate was observed at the beginning or end of treatment. Cultures treated at concentrations of 5, 10, 15, 20 and 30 µg/mL using a 4-hour treatment without S9 exhibited 14 to 80 % RSG and were cloned, while those cultures treated at concentrations of 0.5, 1.5, 5, 10 and 15 µg/mL using a 24 hour treatment without S9 exhibited 13 to 99 % RSG and were cloned (cultures treated at other lower or higher concentrations were discarded prior to cloning because a sufficient number of higher concentrations was available, or they were excluded from evaluation of mutagenicity due to excessive toxicity). Relative total growth (RTG) of the cloned cultures ranged from 7 to 84 % (4-hour treatment without S9) and from 10 to 93 % (24 hour treatment without S9). No increases in induced mutant frequency (IMF) were observed under either treatment condition. The average suspension growth and mutant frequency of the negative controls with S9 were unacceptable and that portion of the assay was repeated. A subsequent re-test with S9 also failed due to unacceptable average negative control mutant frequency.
A second re-test with S9 was conducted under identical conditions. No visible precipitate was observed at the beginning or end of treatment. Cultures treated at concentrations of 1.5, 5, 10, 20 and 40 µg/mL exhibited 26 to 104 % RSG and were cloned (cultures treated at the other concentrations were discarded prior to cloning because a sufficient number of higher concentrations was available, or were excluded from evaluation of mutagenicity due to excessive toxicity). RTG of the cloned cultures ranged from 13 to 101 %. IMFs ≥90 mutants per 10⁶ clonable cells were observed in all cloned cultures treated at concentrations ≥5 µg/mL except for single replicates at concentrations of 10 and 20 µg/mL (IMFs were 69 and 88 mutants per 10⁶ clonable cells, respectively). In addition, these increases were considered to be dose dependent, and the increases observed at a concentration of 40 µg/mL also were >2-fold the concurrent vehicle control value.
A confirmatory assay was performed using test material concentrations of 0.5, 1, 1.5, 2.5, 5, 7.5, 10, 15, 20, 25, 30, 40 and 50 µg/mL (4-hour treatment with S9). No visible precipitate was observed at the beginning or end of treatment. All cultures exhibited 21 to 96 % RSG and were cloned. RTG of the cloned cultures ranged from 21 to 103 %. IMFs ≥90 mutants per 10⁶ clonable cells were observed in ten of the 14 cultures treated at concentrations of 5 – 30 µg/mL with S9, and seven of these increases also were >2-fold the concurrent vehicle control value. Although the IMFs decreased at concentrations of 40 and 50 µg/mL with S9, the response still appeared to be dose dependent (but with a plateau rather than linear). Except for the decrease in mutant frequencies at the two highest concentrations evaluated in this trial (40 and 50 µg/mL with S9), the results were remarkably similar to the previous trial.
All criteria for a valid study ultimately were met.
Under the conditions of this study, the test material was positive in the L5178Y/TK+/- Mouse Lymphoma Mutagenesis Assay, in the presence of S9 only, under the conditions and according to the criteria of the test protocol. The results were uniformly negative in the absence of S9.
Referenceopen allclose all
Table 1: Summary of plate test results
| Revertants per plate | ||||||
| TA 1535 | TA 1537 | TA 1538 | TA 98 | TA 100 | D4 | |
| Non-activation | ||||||
| Solvent control | 21 | 10 | 16 | 27 | 154 | 29 |
| Positive control* | >1000 | 968 | 729 | >1000 | 467 | 288 |
| 0.001 UL test material | 22 | 16 | 11 | 22 | 171 | 23 |
| 0.01 UL test material | 15 | 14 | 12 | 36 | 149 | 19 |
| 0.1 UL test material | 28 | 3 | 9 | 25 | 136 | 22 |
| 1.0 UL test material | 18 | 11 | 8 | 36 | 171 | 17 |
| 5.0 UL test material | 13 | 5 | 17 | 45 | 140 | 26 |
| Activation | ||||||
| Solvent control | 22 | 13 | 20 | 46 | 141 | 20 |
| Positive control** | 254 | 299 | 682 | 436 | 465 | 20 |
| 0.001 UL test material | 18 | 9 | 15 | 48 | 68 | 16 |
| 0.01 UL test material | 14 | 15 | 18 | 32 | 153 | 16 |
| 0.1 UL test material | 8 | 6 | 10 | 36 | 92 | 17 |
| 1.0 UL test material | 17 | 13 | 3 | 38 | 102 | 15 |
| 5.0 UL test material | 25 | 17 | 6 | 22 | 146 | 16 |
*TA 1535: Methylnitrosoguanidine TA 1537: Quinaracene mustard TA 1538: 2-nitrofluorene TA 98: 2-nitrofluorene TA 100: Methylnitrosoguanidine D4: Methylnitrosoguanidine **TA 1535: 2-anthramine TA 1537: 8-aminoquinoline TA 1538: 2-acetylaminofluorene TA 98: 2-acetlyaminofluorene TA 100: 2-anthramine D4: 2-anthramine
Table 1: Results
| Treatment | Control -S-9 | 25 µg/mL -S-9 | 50 µg/mL -S-9 | 100 µg/mL -S-9 | 50 µg/mL MMS -S-9 | ||||||||||
| Culture | A | B | A+B | A | B | A+B | A | B | A+B | A | B | A+B | A | B | A+B |
| No. of cells scored | 100 | 100 | 200 | 100 | 95 | 195 | 57 | 75 | 132 | 100 | 86 | 186 | 25 | 25 | 50 |
| Gaps | 6 | 20 | 26 | 12 | 10 | 22 | 2 | 2 | 4 | 11 | 2 | 13 | 18 | 5 | 23 |
| Chromosome deletions | 1 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 1 | 5 | 4 | 9 |
| Chromosome exchanges | 0 | 1 | 1 | 0 | 0 | 0 | 1 | 0 | 1 | 0 | 1 | 1 | 0 | 2 | 2 |
| Chromatid deletions | 2 | 3 | 5 | 0 | 1 | 1 | 0 | 1 | 1 | 0 | 3 | 3 | 5 | 12 | 17 |
| Chromatid exchanges | 0 | 1 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 8 | 6 | 14 |
| Others | 1 | 0 | 1 | 0 | 2 | 2 | 0 | 0 | 0 | 1 | 4 | 5 | 1 | 0 | 1 |
| All abberations including gaps | 10 | 25 | 35 | 12 | 13 | 25 | 3 | 3 | 6 | 13 | 10 | 23 | 37 | 26 | 99 |
| All aberrations excluding gaps | 4 | 5 | 9 | 0 | 3 | 3 | 1 | 1 | 2 | 2 | 8 | 10 | 19 | 24 | 43 |
| Mitotic index | 8.2 | 0.4 | 0.4 | 0.8 | |||||||||||
| Frequency of aberrations/100 cells including gaps | 10 | 25 | 18 | 12 | 14 | 13 | 5 | 4 | 5 | 13 | 12 | 12 | 148 | 116 | 132 |
| Frequency of aberrations/100 cells excluding gaps | 4 | 5 | 5 | 0 | 3 | 2 | 2 | 1 | 2 | 2 | 9 | 5 | 76 | 96 | 86 |
| % of cells with aberrations including gaps | 10 | 17 | 14 | 10 | 12 | 11 | 5 | 4 | 5 | 12 | 11 | 11 | 60 | 48 | 54 |
| % of cells with aberrations excluding gaps | 4 | 5 | 5 | 0 | 3 | 2 | 2 | 1 | 2 | 2 | 8 | 5 | 44 | 48 | 46 |
| Treatment | Control +S-9 | 50 µg/mL +S-9 | 100 µg/mL +S-9 | 200 µg/mL +S-9 | 50 µg/mL CPA +S-9 | ||||||||||
| Culture | A | B | A+B | A | B | A+B | A | B | A+B | A | B | A+B | A | B | A+B |
| No. of cells scored | 100 | 100 | 200 | 100 | 100 | 200 | 100 |
100 |
200 |
100 |
100 |
200 |
25 |
25 |
50 |
Gaps |
2 |
2 |
4 |
4 |
3 |
7 |
8 |
8 |
16 |
3 |
2 |
5 |
18 |
5 |
23 |
Chromosome deletions |
0 |
2 |
2 |
0 |
4 |
4 |
1 |
0 |
1 |
1 |
0 |
1 |
7 |
3 |
10 |
Chromosome exchanges |
0 |
1 |
1 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
4 |
4 |
Chromatid deletions |
1 |
1 |
2 |
0 |
2 |
2 |
0 |
0 |
0 |
0 |
0 |
0 |
29 |
10 |
39 |
Chromatid exchanges |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
28 |
8 |
36 |
Others |
0 |
0 |
0 |
0 |
1 |
1 |
1 |
2 |
3 |
0 |
1 |
1 |
1 |
1 |
2 |
All abberations including gaps |
3 |
6 |
9 |
4 |
10 |
14 |
10 |
10 |
20 |
4 |
3 |
7 |
83 |
31 |
114 |
All aberrations excluding gaps |
1 |
4 |
5 |
0 |
7 |
7 |
2 |
2 |
4 |
1 |
1 |
2 |
65 |
26 |
91 |
Mitotic index |
4.2 |
1.8 |
0.8 |
0.4 |
|
||||||||||
Frequency of aberrations/100 cells including gaps |
3 |
6 |
5 |
4 |
10 |
7 |
10 |
10 |
10 |
4 |
3 |
4 |
332 |
124 |
228 |
Frequency of aberrations/100 cells excluding gaps |
1 |
4 |
3 |
0 |
7 |
4 |
2 |
2 |
2 |
1 |
1 |
1 |
260 |
104 |
182 |
% of cells with aberrations including gaps |
3 |
4 |
4 |
4 |
5 |
5 |
10 |
10 |
10 |
4 |
3 |
4 |
92 |
56 |
74 |
% of cells with aberrations excluding gaps |
1 |
2 |
2 |
0 |
3 |
2 |
2 |
2 |
2 |
1 |
1 |
1 |
92 |
56 |
74 |
Table 1: Autoradiographic analysis of DNA Repair
| Treatment | Concentration | Net nuclear grains (mean ± S.D.) |
| Acetone | 1% v/v | -14.8 ± 5.6 |
| 2AAF | 1 x 10-7 M* | 18.0 ± 7.3** |
| Test material | 0.0167 µg/mL | -14.4 ± 6.4 |
| Test material | 0.05 µg/mL | -13.3 ± 6.2 |
| Test material | 0.167 µg/mL | -14.4 ± 6.1 |
| Test material | 0.5 µg/mL | -12.0 ± 5.8 |
| Test material | 1.67 µg/mL | Cytotoxic |
| Test material | 5.0 µg/mL | Cytotoxic |
| Test material | 16.7 µg/mL | Cytotoxic |
| Test material | 50 µg/mL | Cytotoxic |
| Test material | 167 µg/mL | Cytotoxic |
| Test material | 500 µg/mL | Cytotoxic |
| Test material | 1670 µg/mL | Cytotoxic |
| Test material | 5000 µg/mL | Cytotoxic |
*final concentration in the treatment medium
**positive finding
Table 1: Initial non-activated study
| Cloning efficiency at selection | ||||||||||||
| Colonies per dish | Mutant colonies/selection dish | |||||||||||
| Treatment (µg/mL) | 1 | 2 | 3 | Total colonies | Cloning efficiency | 1 | 2 | 3 | 4 | 5 | Total mutant colonies | Mutants/106 clonable cells |
| Untreated A | 102 | 109 | 98 | 617 | 1.03 | 0 | 0 | 0 | 0 | 0 | 7 | 3.4 |
| Untreated B | 92 | 106 | 110 | 3 | 1 | 2 | 0 | 1 | ||||
| Vehicle A | 91 | 93 | 94 | 551 | 0.92 | 2 | 4 | 3 | 2 | 3 | 20 | 10.9 |
| Vehicle B | 94 | 91 | 88 | 1 | 2 | 2 | 1 | 0 | ||||
| Positive control A | 87 | 88 | 101 | 562 | 0.94 | 42 | 32 | 37 | 29 | 35 | 334 | 178.3 |
| Positive control B | 101 | 80 | 105 | 24 | 36 | 28 | 40 | 31 | ||||
| 1.0 A | 100 | 124 | 105 | 640 | 1.07 | 0 | 0 | 0 | 0 | 0 | 8 | 3.8 |
| 1.0 B | 103 | 123 | 85 | 2 | 3 | 0 | 2 | 1 | ||||
| 1.8 A | 110 | 96 | 94 | 587 | 0.98 | 0 | 2 | 0 | 0 | 0 | 2 | 1.0 |
| 1.8 B | 92 | 102 | 93 | 0 | 0 | 0 | 0 | 0 | ||||
| 2.6 A | 93 | 61 | 74 | 538 | 0.90 | 0 | 0 | 0 | * | 0 | 0 | <0.6** |
| 2.6 B | 113 | 93 | 104 | 0 | 0 | 0 | 0 | 0 | ||||
| 3.4 A | 102 | 77 | 92 | 578 | 0.96 | 0 | 0 | 0 | 0 | 0 | 0 | <0.5** |
| 3.4 B | 107 | 105 | 95 | 0 | 0 | 0 | 0 | 0 | ||||
| 4.2 A | 107 | 79 | 80 | 536 | 0.89 | 0 | 0 | 0 | 0 | 0 | 0 | <0.6** |
| 4.2 B | 87 | 102 | 81 | 0 | 0 | 0 | 0 | 0 | ||||
| 5.0 A | 65 | 76 | 64 | 456 | 0 | 0 | 0 | 0 | *** | *** | 0 | <1.3** |
| 5.0 B | 73 | 96 | 82 | 0 | 0 | *** | *** | *** | ||||
*plates lost due to contamination
**calculated on the basis of <1 mutant colony observed in the total number of dishes prepared
***plates not prepared due to lack of cells
Table 2: Initial activated study
| Cloning efficiency at selection | ||||||||||||
| Colonies per dish | Mutant colonies/selection dish | |||||||||||
| Treatment (µg/mL) | 1 | 2 | 3 | Total colonies | Cloning efficiency | 1 | 2 | 3 | 4 | 5 | Total mutant colonies | Mutants/106 clonable cells |
| Untreated A | 95 | 92 | 86 | 582 | 0.97 | 0 | 0 | 0 | 1 | 0 | 11 | 5.7 |
| Untreated B | 119 | 89 | 101 | 3 | 2 | 1 | 3 | 1 | ||||
| Vehicle A | 107 | 97 | 92 | 617 | 1.03 | 1 | 0 | 1 | 1 | 1 | 5 | 2.4 |
| Vehicle B | 85 | 117 | 119 | 1 | 0 | 0 | 0 | 0 | ||||
| Positive control (4 µg/mL) A | 78 | 79 | 88 | 509 | 0.85 | 32 | 38 | 41 | 30 | 47 | 202 | 119.1 |
| Positive control (4 µg/mL) B | 116 | 89 | 59 | 1 | 2 | 3 | 6 | 2 | ||||
| Positive control (5 µg/mL) A | 78 | 67 | 76 | 505 | 0.84 | 1 | 1 | 2 | 1 | 3 | 145 | 86.1 |
| Positive control (5 µg/mL) B | 106 | 84 | 94 | 34 | 28 | 29 | 26 | 20 | ||||
| 10 A | 99 | 90 | 92 | 581 | 0.97 | 4 | 3 | 3 | 1 | 2 | 18 | 9.3 |
| 10 B | 112 | 90 | 98 | 1 | 3 | 1 | 0 | 0 | ||||
| 25 A | 105 | 97 | 84 | 589 | 0.98 | 1 | 0 | 1 | 1 | 3 | 6 | 3.1 |
| 25 B | 106 | 109 | 88 | 0 | 0 | 0 | 0 | 0 | ||||
| 37.5 A | 85 | 71 | 82 | 478 | 0.80 | 1 | 0 | 0 | 0 | 0 | 3 | 1.9 |
| 37.5 B | 84 | 92 | 64 | 0 | 1 | 0 | 0 | 1 | ||||
| 50 A | 84 | 52 | 69 | 438 | 0.73 | 20 | 14 | 26 | 15 | 18 | 93 | 63.7 |
| 50 B | 81 | 71 | 81 | 0 | 0 | 0 | 0 | 0 | ||||
Table 3: Confirmatory non-activated study
| Cloning efficiency at selection | ||||||||||||
| Colonies per dish | Mutant colonies/selection dish | |||||||||||
| Treatment (µg/mL) | 1 | 2 | 3 | Total colonies | Cloning efficiency | 1 | 2 | 3 | 4 | 5 | Total mutant colonies | Mutants/106clonable cells |
| Untreated A | 128 | 123 | 102 | 689 | 1.16 | 0 | 0 | 0 | 0 | 0 | 3 | 1.3 |
| Untreated B | 119 | 119 | 107 | 1 | 0 | 0 | 0 | 2 | ||||
| Vehicle A | 108 | 103 | 96 | 562 | 0.94 | 1 | 0 | 0 | 0 | 0 | 4 | 2.1 |
| Vehicle B | 75 | 95 | 85 | 1 | 1 | 1 | 0 | 0 | ||||
| Positive control A | 82 | 91 | 94 | 571 | 0.95 | 41 | 45 | 44 | 52 | 41 | 468 | 245.9 |
| Positive control B | 95 | 108 | 101 | 39 | 57 | 44 | 56 | 49 | ||||
| 0.2 A | 105 | 69 | 91 | 545 | 0.91 | 0 | 0 | 0 | 0 | 0 | 6 | 3.3 |
| 0.2 B | 86 | 111 | 83 | 0 | 2 | 0 | 2 | 2 | ||||
| 1.0 A | 105 | 105 | 84 | 637 | 1.06 | 0 | 0 | 0 | 0 | 0 | 0 | <0.5* |
| 1.0 B | 122 | 125 | 96 | 0 | 0 | 0 | 0 | 0 | ||||
| 1.8 A | 86 | 82 | 71 | 531 | 0.89 | 0 | 0 | 0 | 0 | 0 | 1 | 0.6 |
| 1.8 B | 104 | 80 | 108 | 0 | 0 | 0 | 0 | 1 | ||||
| 2.6 A | 127 | 110 | 67 | 596 | 0.99 | 0 | 0 | 0 | 0 | 0 | 0 | <0.5* |
| 2.6 B | 95 | 101 | 96 | 0 | 0 | 0 | 0 | 0 | ||||
| 3.4 A | 108 | 78 | 94 | 595 | 0.99 | 0 | 0 | 0 | 1 | 0 | 1 | 0.5 |
| 3.4.B | 111 | 91 | 113 | 0 | 0 | 0 | 0 | 0 | ||||
| 4.2 A | 101 | 86 | 117 | 614 | 1.02 | 0 | 0 | 0 | 0 | 0 | 0 | <0.5* |
| 4.2 B | 99 | 111 | 100 | 0 | 0 | 0 | 0 | 0 | ||||
*calculated on the basis of <1 mutant colony observed in the total number of dished prepared
Table 4: Confirmatory activated study
| Cloning efficiency at selection | ||||||||||||
| Colonies per dish | Mutant colonies/selection dish | |||||||||||
| Treatment (µg/mL) | 1 | 2 | 3 | Total colonies | Cloning efficiency | 1 | 2 | 3 | 4 | 5 | Total mutant colonies | Mutants/106clonable cells |
| Untreated A | 126 | 90 | 89 | 620 | 1.03 | 1 | 0 | 0 | 0 | 0 | 6 | 2.9 |
| Untreated B | 91 | 117 | 107 | 0 | 1 | 0 | 2 | 2 | ||||
| Vehicle A | 96 | 91 | 59 | 511 | 0.85 | 2 | 5 | 2 | 2 | 2 | 25 | 14.7 |
| Vehicle B | 93 | 106 | 66 | 1 | 0 | 4 | 5 | 2 | ||||
| Positive control (4 µg/mL) A | 84 | 100 | 85 | 585 | 0.98 | 16 | 20 | 21 | 20 | 15 | 248 | 127.2 |
| Positive control (4 µg/mL) B | 108 | 94 | 114 | 28 | 26 | 35 | 37 | 30 | ||||
| Positive control (5 µg/mL) A | 107 | 88 | 77 | 560 | 0.93 | 18 | 24 | 14 | 18 | 19 | 238 | 127.5 |
| Positive control (5 µg/mL) B | 94 | 107 | 87 | 36 | 27 | 29 | 31 | 22 | ||||
| 10 A | 124 | 108 | 100 | 656 | 1.09 | 1 | 1 | 1 | 2 | 4 | 11 | 5.0 |
| 10 B | 117 | 95 | 112 | 0 | 1 | 0 | 0 | 1 | ||||
| 25 A | 98 | 109 | 102 | 580 | 0.97 | 0 | 0 | 1 | 0 | 0 | 3 | 1.6 |
| 25 B | 108 | 76 | 87 | 2 | 0 | 0 | 0 | 0 | ||||
| 37.5 A | 71 | 69 | 63 | 484 | 0.81 | 0 | 0 | 1 | 1 | 0 | 21 | 13.0 |
| 37.5 B | 107 | 92 | 82 | 0 | 0 | 1 | 1 | 0 | ||||
| 40 A | 84 | 95 | 97 | 513 | 0.86 | 0 | 0 | 0 | 0 | 0 | 0 | <0.6* |
| 40 B | 105 | 56 | 76 | 0 | 0 | 0 | 0 | 0 | ||||
| 45 A | 102 | 105 | 60 | 555 | 0.93 | 0 | 0 | 0 | 0 | 0 | 0 | <0.5* |
| 45 B | 134 | 64 | 90 | 0 | 0 | 0 | 0 | 0 | ||||
| 47.5 A | 102 | 60 | 77 | 489 | 0.82 | 0 | 2 | 0 | 0 | 0 | 2 | 1.2 |
| 47.5 B | 83 | 77 | 90 | 489 | 0.82 | 0 | 0 | 0 | 0 | 0 | ||
*calculated on the basis of <1 mutant colony observed in the total number of dishes prepared
Table 1: Initial non-activated study
| Cloning efficiency at selection | ||||||||||||
| Colonies per dish | Mutant colonies/selection dish | |||||||||||
| Treatment (µg/mL) | 1 | 2 | 3 | Total colonies | Cloning efficiency | 1 | 2 | 3 | 4 | 5 | Total mutant colonies | Mutants/106clonable cells |
| Untreated A | 93 | 126 | 100 | 580 | 0.97 | 0 | 3 | 0 | 0 | 0 | 14 | 7.2 |
| Untreated B | 86 | 79 | 96 | 3 | 0 | 3 | 2 | 3 | ||||
| Vehicle A | 108 | 129 | 105 | 620 | 1.03 | 1 | 1 | 1 | 2 | 0 | 16 | 7.7 |
| Vehicle B | 97 | 102 | 79 | 1 | 2 | 2 | 3 | 3 | ||||
| Positive control A | 105 | 88 | 114 | 598 | 1.00 | 49 | 51 | 57 | 41 | 47 | 424 | 212.7 |
| Positive control B | 98 | 95 | 98 | 39 | 38 | 40 | 28 | 34 | ||||
| 0.5 A | 128 | 144 | 126 | 689 | 1.15 | 0 | 0 | 0 | 0 | 01 | 9 | 3.9 |
| 0.5 B | 101 | 91 | 99 | 3 | 0 | 0 | 0 | 5 | ||||
| 1.0 A | 87 | 90 | 114 | 590 | 0.98 | 0 | 0 | 0 | 0 | 0 | 9 | 4.6 |
| 1.0 B | 94 | 98 | 107 | 2 | 1 | 1 | 3 | 2 | ||||
| 1.5 A | 112 | 88 | 117 | 633 | 1.06 | 0 | 0 | 0 | 0 | 0 | 0 | <0.5* |
| 1.5 B | 109 | 105 | 102 | 0 | 0 | 0 | 0 | 0 | ||||
| 2.0 A | 121 | 120 | 94 | 620 | 1.03 | 0 | 0 | 0 | 0 | 0 | 8 | 3.9 |
| 2.0 B | 92 | 85 | 108 | 1 | 3 | 1 | 2 | 1 | ||||
| 3.0 A | 89 | 103 | 97 | 572 | 0.95 | 0 | 0 | 0 | 0 | 0 | 0 | <0.5* |
| 3.0 B | 90 | 972 | 96 | 0 | 0 | 0 | 0 | 0 | ||||
| 4.0 A | 71 | 79 | 80 | 502 | 0.84 | 0 | 0 | ** | 0 | *** | 0 | <0.9** |
| 4.0 B | 98 | 90 | 84 | 0 | ** | 0 | 0 | 0 | ||||
| 5.0 A | 79 | 100 | 120 | 501 | 0.84 | 0 | 0 | 1 | *** | *** | 1 | 1.2 |
| 5.0 B | 63 | 65 | 74 | 0 | 0 | *** | *** | *** | ||||
*calculated on the basis of <1 mutant colony observed in the total number of dishes prepared
**plates lost due to contamination
***not plated due to toxicity
Table 2: Initial activated study
| Cloning efficiency at selection | ||||||||||||
| Colonies per dish | Mutant colonies/selection dish | |||||||||||
| Treatment (µg/mL) | 1 | 2 | 3 | Total colonies | Cloning efficiency | 1 | 2 | 3 | 4 | 5 | Total mutant colonies | Mutants/106clonable cells |
| Untreated A | 108 | 103 | 75 | 638 | 1.06 | 5 | 7 | 2 | 7 | 6 | 37 | 17.4 |
| Untreated B | 115 | 115 | 122 | 0 | 1 | 2 | 3 | 4 | ||||
| Vehicle A | 100 | 108 | 79 | 570 | 0.95 | 2 | 8 | 3 | 1 | 8 | 28 | 14.7 |
| Vehicle B | 91 | 104 | 88 | 0 | 1 | 1 | 1 | 3 | ||||
| Positive control (4 µg/mL) A | 94 | 100 | 96 | 544 | 0.91 | 37 | 42 | 53 | 44 | 40 | 385 | 212.3 |
| Positive control (4 µg/mL) B | 91 | 81 | 82 | 25 | 37 | 36 | 31 | 40 | ||||
| Positive control (5 µg/mL) A | 80 | 81 | 110 | 492 | 0.82 | 18 | 36 | 20 | 18 | 26 | 357 | 217.7 |
| Positive control (5 µg/mL) B | 113 | 103 | 97 | 42 | 49 | 52 | 49 | 47 | ||||
| 5.0 A | 129 | 81 | 110 | 639 | 1.07 | 1 | 2 | 1 | 0 | 2 | 17 | 8.0 |
| 5.0 B | 100 | 109 | 110 | 4 | 2 | 3 | 1 | 1 | ||||
| 10 A | 81 | 126 | 93 | 609 | 1.02 | 0 | 3 | 2 | 2 | 3 | 13 | 6.4 |
| 10 B | 97 | 109 | 103 | 0 | 1 | 1 | 1 | 0 | ||||
| 20 A | 77 | 72 | 94 | 500 | 0.83 | 1 | 0 | 2 | 3 | 1 | 16 | 9.6 |
| 20 B | 89 | 71 | 97 | 2 | 2 | 2 | 2 | 1 | ||||
| 30 A | 111 | 104 | 125 | 652 | 1.09 | 0 | 0 | 1 | 0 | 2 | 5 | 2.3 |
| 30 B | 105 | 105 | 102 | 0 | 2 | 0 | 0 | 0 | ||||
| 40 A | 83 | 103 | 86 | 554 | 0.92 | 0 | 0 | 0 | 0 | 0 | 0 | <0.5* |
| 40 B | 104 | 86 | 92 | 0 | 0 | 0 | 0 | 0 | ||||
| 50 A | too toxic to clone | |||||||||||
| 50 B | too toxic to clone | |||||||||||
*calculated on the basis of <1 mutant colony observed in the total number of dishes prepared
Table 3: Confirmatory non-activated study
| Cloning efficiency at selection | ||||||||||||
| Colonies per dish | Mutant colonies/selection dish | |||||||||||
| Treatment (µg/mL) | 1 | 2 | 3 | Total colonies | Cloning efficiency | 1 | 2 | 3 | 4 | 5 | Total mutant colonies | Mutants/106 clonable cells |
| Untreated A | 74 | 99 | 79 | 622 | 1.04 | 0 | 3 | 2 | 7 | 1 | 15 | 7.2 |
| Untreated B | 102 | 147 | 121 | 1 | 0 | 0 | 0 | 1 | ||||
| Vehicle A | 54 | 83 | 54 | 545 | 0.91 | 5 | 3 | 3 | 6 | 1 | 18 | 9.9 |
| Vehicle B | 89 | 131 | 134 | 0 | 0 | 0 | 0 | 0 | ||||
| Positive control A | 57 | 67 | 78 | 551 | 0.92 | 33 | 36 | 33 | 38 | 46 | 406 | 221.1 |
| Positive control B | 101 | 84 | 164 | 55 | 40 | 33 | 53 | 39 | ||||
| 0.5 A | 78 | 58 | 83 | 547 | 0.91 | 1 | 1 | 0 | 0 | 0 | 11 | 6.0 |
| 0.5 B | 108 | 113 | 107 | 2 | 2 | 2 | 2 | 1 | ||||
| 1.0 A | 80 | 73 | 84 | 503 | 0.84 | 0 | 0 | 0 | 0 | 0 | 0 | <0.6* |
| 1.0 B | 78 | 37 | 151 | 0 | 0 | 0 | 0 | 0 | ||||
| 1.5 A | 61 | 65 | 65 | 541 | 0.90 | 1 | 0 | 2 | 1 | 2 | 6 | 3.3 |
| 1.5 B | 131 | 113 | 106 | 0 | 0 | 0 | 0 | 0 | ||||
| 2.0 A | 75 | 83 | 66 | 564 | 0.94 | 2 | 1 | 2 | 2 | 2 | 10 | 5.3 |
| 2.0 B | 82 | 128 | 106 | 0 | 1 | 0 | 0 | 0 | ||||
| 3.0 A | 78 | 86 | 85 | 605 | 1.01 | 1 | 0 | 0 | 0 | 0 | 1 | 0.5 |
| 3.0 B | 108 | 101 | 147 | 0 | 0 | 0 | 0 | 0 | ||||
| 4.0 A | 71 | 85 | 88 | 628 | 1.05 | 0 | 0 | 1 | 0 | 0 | 1 | 0.5 |
| 4.0 B | 103 | 101 | 180 | 0 | 0 | 0 | 0 | 0 | ||||
| 5.0 A | 87 | 95 | 61 | 595 | 0.99 | 0 | 0 | 0 | 0 | 0 | 1 | 0.5 |
| 5.0 B | 117 | 116 | 119 | 0 | 1 | 0 | 0 | 0 | ||||
*calculated on the basis of <1 mutant colony observed in the total number of dishes prepared
Table 4: Confirmatory non-activated study
| Cloning efficiency at selection | ||||||||||||
| Colonies per dish | Mutant colonies/selection dish | |||||||||||
| Treatment (µg/mL) | 1 | 2 | 3 | Total colonies | Cloning efficiency | 1 | 2 | 3 | 4 | 5 | Total mutant colonies | Mutants/106clonable cells |
| Untreated A | 106 | 112 | 123 | 687 | 1.15 | 0 | 0 | 0 | 0 | 0 | 3 | 1.3 |
| Untreated B | 94 | 102 | 150 | 2 | 0 | 0 | 1 | 0 | ||||
| Vehicle A | 82 | 113 | 106 | 690 | 1.15 | 1 | 2 | 2 | 1 | 0 | 6 | 2.6 |
| Vehicle B | 118 | 116 | 155 | 0 | 0 | 0 | 0 | 0 | ||||
| Positive control (4 µg/mL) A | 57 | 80 | 83 | 554 | 0.92 | 25 | 38 | * | 36 | 31 | 228 | 154.3 |
| Positive control (4 µg/mL) B | 114 | 109 | 111 | 21 | * | 20 | 23 | 34 | ||||
| Positive control (5 µg/mL) A | 89 | 74 | 86 | 557 | 0.93 | 27 | 20 | 31 | 31 | 28 | 345 | 185.8 |
| Positive control (5 µg/mL) B | 81 | 98 | 129 | 49 | 41 | 43 | 40 | 35 | ||||
| 5.0 A | 103 | 88 | 109 | 641 | 1.07 | 0 | 0 | 0 | 1 | 0 | 14 | 6.6 |
| 5.0 B | 97 | 126 | 118 | 2 | 2 | 2 | 5 | 2 | ||||
| 10 A | 93 | 91 | 118 | 581 | 0.97 | 2 | 4 | 8 | 4 | 4 | 26 | 13.4 |
| 10 B | 94 | 86 | 99 | 0 | 1 | 2 | 0 | 1 | ||||
| 20 A | 104 | 113 | 91 | 643 | 1.07 | 1 | 3 | 0 | 1 | 1 | 11 | 5.7 |
| 20 B | 96 | 113 | 126 | 1 | 1 | 1 | * | 2 | ||||
| 30 A | 74 | 74 | 63 | 530 | 0.88 | 0 | 0 | 0 | 0 | 0 | 0 | <0.6** |
| 30 B | 78 | 107 | 134 | 0 | 0 | 0 | 0 | 0 | ||||
| 40 A | 73 | 79 | 75 | 560 | 0.93 | 0 | 0 | 0 | 0 | 0 | 0 | <0.5** |
| 40 B | 100 | 116 | 117 | 0 | 0 | 0 | 0 | 0 | ||||
| 50 A | 90 | 87 | 85 | 565 | 0.94 | 0 | 0 | 0 | 0 | 0 | 0 | <0.5** |
| 50 B | 81 | 117 | 105 | 0 | 0 | 0 | 0 | 0 | ||||
*plates lost due to contamination
**calculated on the basis of <1 mutant colony observed in the total number of dishes prepared
Table 1: Summary of Reverse Mutation Results (revertants/plate), Initial Toxicity and Mutation assay
Substance |
Dose level (µg/plate) |
TA98 |
TA100 |
TA1535 |
TA1537 |
WP2uvrA |
Without metabolic activation |
||||||
DMSO |
50 µL/plate |
20±4 |
86±6 |
11±0 |
8±2 |
30±7 |
Test material |
1.5 |
19±3 |
88±10 |
9±1 |
6±3 |
21±7 |
5.0 |
14±5 |
83±12 |
11±3 |
7±3 |
17±3 |
|
15 |
12±4 |
77±5 |
10±1 |
10±3 |
15±4** |
|
50 |
16±5 |
86±3 |
13±2 |
7±3 |
17±5 |
|
150 |
11±3 |
85±6 |
10±3 |
6±3 |
14±4 |
|
500* |
17±1 |
85±3 |
9±3 |
6±1 |
16±4 |
|
1500* |
13±5 |
80±2 |
9±2 |
7±3 |
16±2 |
|
5000* |
13±8 |
86±12 |
12±4 |
6±3 |
16±1 |
|
2NF |
1.0 |
134±21 |
|
|
|
|
SA |
1.0 |
|
543±15 |
530±50 |
|
|
9AAD |
75 |
|
|
|
218±33 |
|
MMS |
1000 |
|
|
|
|
255±2 |
With metabolic activation |
||||||
DMSO |
50 µL/plate |
25±5 |
83±4 |
9±1 |
7±3 |
21±6 |
Test material |
1.5 |
22±1 |
74±8 |
8±2 |
6±2 |
18±7 |
5.0 |
27±9 |
92±7 |
11±3 |
7±1 |
19±6 |
|
15 |
21±4 |
85±1 |
9±5 |
8±1 |
20±2 |
|
50 |
29±1 |
90±7 |
10±3 |
8±2 |
22±7 |
|
150 |
21±3 |
79±8 |
11±4 |
10±3 |
22±7 |
|
500* |
28±10 |
82±2 |
10±2 |
9±1 |
25±2 |
|
1500* |
23±3 |
73±10 |
7±2 |
13±2 |
21±3 |
|
5000* |
31±5 |
70±12 |
10±2 |
7±2 |
20±2 |
|
2AA |
1.0 |
334±26 |
|
63±3 |
|
|
2AA |
2.0 |
|
449±112 |
|
45±10 |
|
2AA |
15 |
|
|
|
|
251±29 |
SA = sodium azide
2AA = 2-aminoanthracene
9AAD = 9-Aminoacridine
2NF = 2-nitrofluorene
MMS = methyl methanesulfonate
*Precipitate was observed
**One replicate plate was lost due to contamination. Therefore, standard deviation was calculated where n = 2.
Table 2: Summary of Reverse Mutation Results (revertants/plate), Confirmatory assay
Substance |
Dose level (µg/plate) |
TA98 |
TA100 |
TA1535 |
TA1537 |
WP2uvrA |
Without metabolic activation |
||||||
DMSO |
50 µL/plate |
10±2 |
91±3 |
13±3 |
8±3 |
17±5 |
Test material |
15 |
9±1 |
79±4** |
7±1 |
6±1 |
23±4 |
50 |
10±4 |
88±7 |
9±4 |
4±3 |
20±2 |
|
150 |
9±2 |
85±8 |
9±3 |
4±0 |
14±1 |
|
500* |
9±1 |
92±10 |
11±2 |
6±2 |
22±1 |
|
1500* |
7±2 |
100±11 |
9±2 |
4±1 |
22±3 |
|
5000* |
9±2 |
103±18 |
11±4 |
4±3 |
16±1 |
|
2NF |
1.0 |
200±24 |
|
|
|
|
SA |
1.0 |
|
541±33 |
476±70 |
|
|
9AAD |
75 |
|
|
|
271±111 |
|
MMS |
1000 |
|
|
|
|
389±31 |
With metabolic activation |
||||||
DMSO |
50 µL/plate |
29±1 |
84±9 |
8±3 |
12±1 |
26±4 |
Test material |
15 |
27±4 |
85±2 |
9±1 |
9±0 |
28±3 |
50 |
22±4 |
81±6 |
13±2 |
11±4 |
27±1 |
|
150 |
30±6 |
79±16 |
13±1 |
8±2 |
25±7 |
|
500* |
28±6 |
86±5 |
12±2 |
12±2 |
23±2 |
|
1500* |
29±2 |
84±8 |
6±3 |
8±2 |
25±8 |
|
5000* |
22±4 |
94±9 |
12±4 |
8±3 |
19±2 |
|
2AA |
1.0 |
583±104 |
|
70±5 |
|
|
2AA |
2.0 |
|
596±141 |
|
65±10 |
|
2AA |
15 |
|
|
|
|
359±33 |
SA = sodium azide
2AA = 2-aminoanthracene
9AAD = 9-Aminoacridine
2NF = 2-nitrofluorene
MMS = methyl methanesulfonate
*Precipitate was observed
**No colonies were present on one replicate plate, and the plate could not be counted. Therefore, standard deviation was calculated where n = 2.
Table 1: Summary of In vitro Chromosome Aberration Results
Substance |
Concentration (µg/mL) |
Cytotoxicity¹(% of control) |
Aberrant cells |
Aberrations per Cell⁴ Mean ± SD |
Total Polyploid Cells (Mean %)⁵ |
|
Structural (Mean %)² |
Numerical (Mean %)³ |
|||||
20-hour continuous treatment –S9 |
||||||
DMSO |
NA |
NA |
0.5 |
1.5 |
0.005±0.071 |
1.5 |
Test material |
0.1 |
25 |
0.5 |
0.0 |
0.005±0.071 |
0.0 |
0.35 |
30 |
0.5 |
1.5 |
0.005±0.071 |
1.5 |
|
0.75 |
68 |
0.0 |
0.5 |
0.000±0.000 |
0.5 |
|
MMC |
0.1 |
38 |
24.0** |
0.5 |
0.290±0.556 |
0.5 |
4-hour continuous treatment with 16-hour recovery –S9 |
||||||
DMSO |
NA |
NA |
0.5 |
0.0 |
0.005±0.071 |
0.0 |
Test material |
0.5 |
2 |
1.0 |
0.0 |
0.010±0.100 |
0.0 |
1 |
23 |
1.5 |
1.0 |
0.015±0.122 |
1.0 |
|
2 |
55 |
0.5 |
1.5 |
0.005±0.071 |
1.5 |
|
MMC |
0.2 |
54 |
23.0** |
1.0 |
0.240±0.452 |
1.0 |
4-hour continuous treatment with 16-hour recovery +S9 |
||||||
DMSO |
NA |
NA |
1.0 |
2.5 |
0.010±0.100 |
1.5 |
Test material |
3 |
-15 |
1.5 |
1.0 |
0.015±0.122 |
0.5 |
5 |
-3 |
0.0 |
4.0 |
0.000±0.000 |
2.0 |
|
10 |
55 |
0.5 |
0.5 |
0.005±0.071 |
0.0 |
|
CP |
7.5 |
57 |
17.0** |
0.5 |
0.240±0.740 |
0.5 |
MMC = Mitomycin C
CP = Cyclophosphamide
Fisher’s Exact Test: * = p ≤ 0.05; ** = p ≤ 0.01.
¹Based on cell growth inhibition relative to solvent control.
²Does not include cells with only gaps.
³Includes polyploid and endoreduplicated cells.
⁴Does not include cells with only gaps. Severely damaged cells counted as 10 aberrations.
⁵Does not include endoreduplicated cell.
Table 1: Initial Mutagenesis Assay Results
Metabolic Activation and Exposure Time |
Test or Control Substance |
Concentration (µg/mL) |
Relative Total Growth (% of Control) |
Total Mutant Frequency (per 10⁶ cells) |
Induced Mutant Frequency (per 10⁶ cells) |
Without Activation 4 Hours |
DMSO |
NA |
100 |
53 |
NA |
NA |
51 |
||||
Test Material |
0.5 |
** |
** |
** |
|
0.5 |
** |
** |
** |
||
1.5 |
** |
** |
** |
||
1.5 |
** |
** |
** |
||
5 |
77 |
54 |
2 |
||
5 |
84 |
43 |
-9 |
||
10 |
57 |
56 |
4 |
||
10 |
52 |
69 |
17 |
||
15 |
42 |
39 |
-13 |
||
15 |
36 |
55 |
3 |
||
20 |
30 |
51 |
-1 |
||
20 |
35 |
56 |
4 |
||
30 |
7 |
90 |
38 |
||
30 |
7 |
77 |
25 |
||
40 |
*** |
*** |
*** |
||
40 |
2 |
**** |
**** |
||
50 |
*** |
*** |
*** |
||
50 |
*** |
*** |
*** |
||
MMS |
20 |
10 |
438 |
385 |
|
15 |
20 |
349 |
297 |
**Discarded prior to cloning because a sufficient number of higher concentrations was available
***Discarded prior to cloning due to excessive toxicity
****Excluded from evaluation of mutagenicity due to excessive toxicity
Table 2: Extended Treatment Assay Results
Metabolic Activation and Exposure Time |
Test or Control Substance |
Concentration (µg/mL) |
Relative Total Growth (% of Control) |
Total Mutant Frequency (per 10⁶ cells) |
Induced Mutant Frequency (per 10⁶ cells) |
Without Activation 24 Hours |
DMSO |
NA |
100 |
47 |
NA |
NA |
38 |
||||
Test Material |
0.5 |
80 |
52 |
9 |
|
0.5 |
93 |
39 |
-3 |
||
1.5 |
87 |
48 |
6 |
||
1.5 |
68 |
51 |
9 |
||
5 |
42 |
53 |
11 |
||
5 |
42 |
73 |
31 |
||
10 |
25 |
52 |
10 |
||
10 |
19 |
81 |
39 |
||
15 |
11 |
65 |
23 |
||
15 |
10 |
94 |
52 |
||
20 |
*** |
*** |
*** |
||
20 |
*** |
*** |
*** |
||
30 |
*** |
*** |
*** |
||
30 |
*** |
*** |
*** |
||
40 |
*** |
*** |
*** |
||
40 |
*** |
*** |
*** |
||
50 |
*** |
*** |
*** |
||
50 |
*** |
*** |
*** |
||
MMS |
7.5 |
21 |
684 |
641 |
|
5 |
30 |
465 |
423 |
***Discarded prior to cloning due to excessive toxicity
Table 3: Second Repeat Mutagenesis Assay Results
Metabolic Activation and Exposure Time |
Test or Control Substance |
Concentration (µg/mL) |
Relative Total Growth (% of Control) |
Total Mutant Frequency (per 10⁶ cells) |
Induced Mutant Frequency (per 10⁶ cells) |
With S9 Activation 4 Hours |
DMSO |
NA |
100 |
108 |
NA |
NA |
122 |
||||
Test Material |
0.5 |
** |
** |
** |
|
0.5 |
** |
** |
** |
||
1.5 |
73 |
170 |
55 |
||
1.5 |
101 |
141 |
26 |
||
5 |
59 |
220 |
105 |
||
5 |
55 |
221 |
106 |
||
10 |
40 |
219 |
104 |
||
10 |
49 |
184 |
69 |
||
15 |
** |
** |
** |
||
15 |
** |
** |
** |
||
20 |
33 |
221 |
106 |
||
20 |
33 |
204 |
88 |
||
30 |
** |
** |
** |
||
30 |
** |
** |
** |
||
40 |
17 |
238 |
123 |
||
40 |
13 |
296 |
181 |
||
50 |
8**** |
292 |
177 |
||
50 |
9**** |
249 |
134 |
||
DMBA |
1.5 |
17 |
555 |
440 |
|
1.0 |
36 |
469 |
354 |
**Discarded prior to cloning because a sufficient number of higher concentrations was available
****Excluded from evaluation of mutagenicity due to excessive toxicity
Table 4: Confirmatory Mutagenesis Assay Results
Metabolic Activation and Exposure Time |
Test or Control Substance |
Concentration (µg/mL) |
Relative Total Growth (% of Control) |
Total Mutant Frequency (per 10⁶ cells) |
Induced Mutant Frequency (per 10⁶ cells) |
With S9 Activation 4 Hours |
DMSO |
NA |
100 |
92 |
NA |
NA |
112 |
||||
Test Material |
0.5 |
76 |
132 |
30 |
|
0.5 |
87 |
123 |
22 |
||
1.0 |
91 |
151 |
49 |
||
1.0 |
82 |
152 |
50 |
||
1.5 |
95 |
123 |
21 |
||
1.5 |
103 |
132 |
30 |
||
2.5 |
74 |
177 |
75 |
||
2.5 |
92 |
152 |
50 |
||
5 |
68 |
206 |
104 |
||
5 |
55 |
213 |
111 |
||
7.5 |
61 |
199 |
97 |
||
7.5 |
70 |
175 |
74 |
||
10 |
51 |
196 |
94 |
||
10 |
49 |
241 |
139 |
||
15 |
44 |
224 |
122 |
||
15 |
53 |
160 |
59 |
||
20 |
38 |
237 |
135 |
||
20 |
50 |
168 |
66 |
||
25 |
38 |
184 |
83 |
||
25 |
42 |
197 |
95 |
||
30 |
30 |
248 |
146 |
||
30 |
30 |
230 |
128 |
||
40 |
35 |
177 |
75 |
||
40 |
29 |
155 |
53 |
||
50 |
22 |
185 |
83 |
||
50 |
21 |
135 |
33 |
||
DMBA |
1.5 |
4 |
1220 |
1118 |
|
1.0 |
22 |
585 |
483 |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
Propargite was determined to be negative for genotoxicity in vivo.
- Negative in the micronucleus assay, EPA OPP 84-2 (Putman and Young, 1994)
- Negative in the transgenic animal mutagenicity assay, OECD 488 and OECD 474 (McKeon and Huynh, 2015)
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 26th May 1994 to 16th September 1994
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- EPA OPP 84-2
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- other: micronucleus assay
- Species:
- mouse
- Strain:
- ICR
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Harlan Sprague-Dawley, Inc., Frederick, MD, USA
- Age at study initiation: 6-8 weeks old
- Weight at study initiation: 24.9-34.8 g (males); 22.8-30.3 g (females)
- Assigned to test groups randomly: yes
- Housing: up to five per cage in plastic autoclavable cages
- Diet: certified laboratory rodent chow ad libitum
- Water: tap water ad libitum
ENVIRONMENTAL CONDITIONS
- Temperature: 74 ± 6 ºF
- Humidity: 50 ± 20 %
- Photoperiod: 12 hours light/12 hours dark - Route of administration:
- intraperitoneal
- Vehicle:
- - Vehicle(s)/solvent(s) used: corn oil
- Duration of treatment / exposure:
- One IP injection at a constant volume of 10 mL/kg bw
- Dose / conc.:
- 37.5 mg/kg bw/day (nominal)
- Dose / conc.:
- 75 mg/kg bw/day (nominal)
- Dose / conc.:
- 150 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- Five
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- cyclophosphamide
- Tissues and cell types examined:
- Femur bone marrow
- Details of tissue and slide preparation:
- DETAILS OF SLIDE PREPARATION:
At the scheduled sacrifice time, five animals per sex per treatment were sacrificed and, immediately after sacrifice, the femurs exposed and the bone marrow aspirated into a syringe containing fetal bovine serum. The bone marrow cells were transferred into a centrifuge tube containing 1 mL fetal bovine serum and pelleted by centrifugation at approximately 100 x g for five minutes and the supernatant drawn off. The cells were resuspended by aspiration and a small drop of bone marrow suspension was spread onto a clean glass slide. Two to four slides were prepared from each animal. The slides were fixed in methanol, stained with May-Gruenwald-Giesma and permanently mounted. - Evaluation criteria:
- 1000 polychromatic erythrocytes were scored for the presence of micronuclei (defined as round, darkly staining nuclear fragments having a sharp contour with diameters usually from 1/20 to 1/5 of the erythrocyte). The number of micronucleated normocytes in the field of 1000 polychromatic erythrocytes was enumerated. The proportion of polychromatic erythrocytes to total erythrocytes counted was also recorded per 100 erythrocytes.
The incidence of micronucleated polychromatic erythrocytes per 1000 polychromatic erythrocytes was determined for each animal and treatment group. In order to quantify the proliferation state of the bone marrow as an indicator of bone marrow toxicity, the proportion of polychromatic erythrocytes to total erythrocytes was determined for each animal and treatment group.
The test material was considered negative if no statistically significant increase in micronucleated polychromatic erythrocytes above the concurrent vehicle control was observed at any sampling time. The test material was considered to be positive if a statistically significant treatment-related increase in micronucleated polychromatic erythrocytes was observed relate to the vehicle control. If a single treatment group was significantly elevated at one sacrifice time with no evidence of a dose-response, this assay was considered a suspect or unconfirmed positive. - Statistics:
- Statistical significance was determined using the Kastenbaum-Bowman table which are based on binomial distribution. All analyses were performed separately for each sex.
- Key result
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- yes
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): not statistically increased regardless of dose level or bone marrow collection time relative to respective vehicle controls (see Table 2).
- Ratio of PCE/NCE (for Micronucleus assay): 18-51 % reduction in male and female mice 48 and 72 hours following treatment with 75 and 150 mg/kg and in female mice 24 hours following treatment with 150 mg/kg relative to their respective vehicle controls (see Table 2). - Conclusions:
- Interpretation of results: negative
Under the conditions of the test, the test material did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow and was concluded to be negative in the micronucleus test using male and female ICR mice. - Executive summary:
Male and female ICR mice were exposed to 37.5, 75 or 150 mg/kg bw of the test material which was administered in a total volume of 10 mL/kg as a single IP injection. The high does level was calculated to be approximately 50 % of the LD50(3day). Corn oil was used as the vehicle. Mortality was observed in 2/20 males and 1/20 females receiving 150 mg/kg. Clinical signs following dose administration included lethargy and diarrhea in mice treated with 75 and 150 mg/kg. Bone marrow cells, collected 24, 48 and 72 hours after treatment, were examined microscopically for micronculeated polychromatic erythrocytes. Relative to their respective vehicle controls, an 18 to 51 % reduction in the frequency of polychromatic erythrocytes was observed in bone marrow from male and female mice following treatment with 75 and 150 mg/kg. This reduction demonstrates that the test material was transported to the bone marrow target tissue resulting in an inhibition of erythropoiesis. No significant increases in microncuelated polychromatic erythrocytes were observed in male or female ICR mice at 24, 48 or 72 hours after dose administration relative to their respective vehicle controls. The results of the assay indicate that under the conditions of the test, the test material did not induce a significant increase in micronucelated polychromatic erythrocytes in either male or female ICR mice. The test material was determined to be negative in the mouse micronculeus assay.
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 25 November 2014 to 16 March 2015
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- other: OECD 488
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- no
- Principles of method if other than guideline:
- The purpose of this study was to treat transgenic Fisher 344 male Rats (Big Blue®) with the test material in the feed and to determine both mutant frequency of the cII gene in liver and jejunum (Big Blue® Rat Assay) and formation of micronucleated reticulocytes in the peripheral blood. The Big Blue® Assay is a Transgenic Rodent (TGR) Mutation assay described in OECD Test Guideline 488 (OECD, 2013). The micronucleus test is an assay for cytogenetic damage in the bone marrow based upon OECD Test Guideline 474 (OECD, 2014).
- GLP compliance:
- yes
- Type of assay:
- transgenic rodent mutagenicity assay
- Species:
- rat
- Strain:
- Fischer 344
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: 8-10 weeks at the time of dosing
- Weight at study initiation: 191.3 – 255.5 g; the Study Director approved the use of three animals under 200 grams
- Assigned to test groups randomly: Yes. The animals were randomized by body weight, using a computer-generated randomization program.
- Fasting period before study: No
- Housing: Individually housed
- Diet: ad libitum diet in meal form
- Water: ad libitum drinking water via an automatic watering system
- Acclimation period: 11 days
ENVIRONMENTAL CONDITIONS
- Temperature: 20.5 – 24.0 °C
- Humidity: 30 to 70 %
- Air changes: At least 10 air changes per hour
- Photoperiod: 12 hour light / dark cycle
IN-LIFE DATES
From: 25 November 2014
To: 06 December 2014 - Route of administration:
- oral: feed
- Vehicle:
- - Vehicle(s)/solvent(s) used: None
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
DIET PREPARATION
- Rate of preparation of diet (frequency): The vehicle control and test material formulations were prepared four times during the study.
- Storage temperature of food: 2 - 8 °C
For Group 1, a blender was purged with approximately 1 kg of clean feed prior to use. The appropriate amount of clean feed was measured, mixed, then transferred to a double resealable polyethylene bag and stored at 2 - 8 °C until use.
For Groups 2-4, four portions of feed were designated for each formulation at each level. The premix feed was equal to 10 % of the total weight of the formulation batch, purge feed equal to 15 % of the total weight of each formulation batch, and two equal amounts of clean feed each at 37.5 % of the total weight of each formulation batch. Weighed quantities of the test material necessary to yield the final formulation concentration in % w/w were carefully spread on part of the premix feed in a mortar. The mixture was ground with a pestle and manually sieved through a Number 14 sieve, until no chemical/feed agglomerates remained. A roughly equal volume of the remaining premix feed was added and mixed in increments, each of which almost doubled the volume of the dosed premix feed, until all of the premix feed minus the weight of the test material was added.
One portion of the clean feed was layered evenly into a twin-shell blender. The dosed premix feed was added in roughly equal portions between the left and right wing of the blender and evenly spread over the clean feed. The premix mortar and pestle was rinsed with the purge feed, which was subsequently added to the blender. The other portion of the clean feed was then evenly spread over the purge feed. All blender ports were sealed and the blender was turned on and checked for leaks. The blender was operated for 5 minutes with the intensifier bar on and then for 10 minutes with the intensifier bar off. The formulations were then discharged from the blender and put into labelled double polyethylene bags and stored at 2 - 8 °C. All bulk formulated feed was stored at 2 - 8 °C and dispensed feed was stored at room temperature for up to 8 days. - Duration of treatment / exposure:
- 28 days
- Frequency of treatment:
- Continuous in diet
- Post exposure period:
- Animals were exposed to control and test agents for 28 consecutive days and left untreated for an additional 3 days to permit fixation of unrepaired DNA lesions into stable mutations.
- Dose / conc.:
- 80 ppm (nominal)
- Remarks:
- in diet
- Dose / conc.:
- 400 ppm (nominal)
- Remarks:
- in diet
- Dose / conc.:
- 800 ppm (nominal)
- Remarks:
- in diet
- No. of animals per sex per dose:
- 6 males per dose group
- Control animals:
- yes, plain diet
- Positive control(s):
- As permitted by OECD TG 488, DNA from the liver and jejunum of 5 male Positive Control animals from a previous study conducted at the testing facility was used as a concurrent packaging Positive Control during the post-life packaging and mutant analysis phase for each tissue. Positive Control DNA used was from a qualification study where male Big Blue® rats were treated via oral gavage at a dose volume of 10 mL/kg body weight on Days 1, 2 and 3 with 20 mg/kg/day N-ethyl- N-nitrosourea (Synonym; CAS number: 56-57-5) formulated in buffer solution (pH 6.0). Tissues were collected on study day 31, flash frozen for later DNA extraction and analysis.
- Tissues and cell types examined:
- Blood was collected for micronucleus assay.
Liver and jejunum were collected for mutant analysis. - Details of tissue and slide preparation:
- TEST PROCEDURE
At necropsy on Day 31, tissues were collected, flash frozen and held frozen for later mutant analysis. Genomic DNA was isolated from frozen tissues; lambda phage shuttle vectors were recovered from the genomic DNA and inserted into empty lambda bacteriophage capsids creating infectious phage. Phage were adsorbed onto permissive E. coli G1250 host cells and plated out for plaque formation under selective (24.0 ± 0.5 °C) and non-selective (37.0 ± 2.0 °C) temperature conditions. The mutant frequency of the cII gene can be determined for almost any tissue in the body using this technique.
MORTALITY CHECKS AND CAGE SIDE OBSERVATIONS
All animals were observed twice daily at least 6 hours apart for moribundity and mortality. Cage side observations were performed once daily. Detailed hands-on observations were performed on Day 1, weekly thereafter and on Day 31.
BODY WEIGHTS
Body weights of individual animals were recorded pre-dose on Day 1, weekly thereafter and prior to sacrifice on Day 31.
FOOD CONSUMPTION
Food consumption of individual animals was recorded weekly beginning on Day 1 and ending on Day 29.
BLOOD COLLECTION FOR MICRONUCLEUS ASSAY
On Day 26, surviving animals in each group were bled for evaluation of micronucleus. Two sets of blood samples/animal were fixed in extra cold methanol and stored at -80 ± 5 °C. Detection of micronucleated reticulocytes was made by Flow Cytometry using MicroFlow Kit as follows.
Peripheral blood samples were washed with ice cold MicroFlow buffer solution to remove the fixative. The cells were pelleted by centrifugation, and the supernatant was drawn off leaving a small amount of supernatant (50-100 μL) with the pellet. The cells were re-suspended and an aliquot of the suspension was added to the staining solution containing RNase, FITC-conjugated anti-CD 71 antibodies and PE-conjugated anti-CD 61 antibodies. Malaria BioStandard samples and CD71-negative and positive control standards were also processed simultaneously. All samples were incubated at 2 - 8 °C, protected from light, for approximately 30 minutes and re-suspended, followed by incubation at room temperature, protected from light, for an additional approximately 30 minutes. An appropriate amount of DNA staining solution was added then placed on wet ice for 5 minutes prior to flow cytometric analysis. The frequency of micronucleated reticulocytes in peripheral blood was analysed after flow cytometry calibration using Malaria infected biostandard and CD-71 negative control standards provided in the kit.
The proportion of reticulocytes to total erythrocytes (% RETs) was determined for each animal. The group value is calculated as a mean for the treatment group.
% RET = [(UL + UR) X 100] / [(UL + UR + LL + LR)] where UL: The number of events in the upper-left portion of the quadrant, UR: The number of events in the upper-right portion of the quadrant, LL: The number of events in the lower-left portion of the quadrant, LR: The number of events in the lower-right portion of the quadrant.
The % RETs served as a parameter of the test material cytotoxicity in bone marrow and, subsequently, in peripheral blood. A decrease in this ratio in the test material groups, as compared to the vehicle (negative) control, would indicate a toxic effect of the test material while an increase would represent a sign of recovery from earlier toxic insult. The animal with a % RET value of <5 % of the vehicle control was considered excessively cytotoxic and excluded from evaluation.
The quantitation of the MnRETs in peripheral blood was expressed as percentage of MnRETs per total number of cells evaluated. The % MnRETs for each animal was calculated as follows:
% MnRET = [(UR) X 100] / (UL + UR).
A positive control biological sample was supplied with the kit.
SACRIFICE, NECROPSY AND TISSUE COLLECTION
Surviving animals were sacrificed by CO2 overdose on Day 31, and necropsied. Prior to sacrifice, animals were weighed to the nearest 0.1 gram. To minimize the in situ degradation of the DNA, animals were euthanized and selected tissues were dissected in the general order listed below, weighed (except bone marrow), flash frozen, transferred to a box on dry ice then stored in a freezer set to ≤ -60 °C. A full necropsy with gross observations was performed; any gross lesions observed were identified and documented but not processed further.
Liver and jejunum were collected for mutant analysis. In addition, bone marrow (from the right femur, flushed with an appropriate amount of phosphate buffered saline), kidney, spleen, cauda epididymis, and testes were collected and held frozen but not analysed for mutants.
ORGAN WEIGHTS
Organs, listed above, except for bone marrow, were weighed for all animals that were necropsied at terminal sacrifice. Paired organs were weighed together. Organ weight data were collected to predict number of DNA extractions possible from a tissue.
EXTRACTION AND PROCESSING OF GENOMIC DNA
Liver and jejunum tissue samples were processed for DNA isolation from frozen tissues of the first five surviving animals in the control (Group 1) and the test material treated groups (Groups 2, 3, and 4). For Group 4, due to the loss of one liver, a total of five livers were analysed. Tissues were processed for DNA isolation using modifications to the standard Agilent RecoverEase™ digestion technique for somatic tissues. Isolated DNA was processed following methods based on Agilent instruction manual titled “λ Select-cII Mutation Detection System for Big Blue® Rodents” and Agilent instruction manual titled “Transpack Packaging Extract for Lambda Transgenic Shuttle Vector Recovery”. Isolated DNA samples were stored at 2 to 8 °C.
IN VITRO PACKAGING OF DNA AND PLATING OF PHAGE
Isolated DNA samples were processed using Agilent Transpack packaging extract, in order to recover lambda shuttle DNA vectors from the genomic DNA and to package the lambda shuttle vector DNA into empty phage capsids creating infectious phage particles. Packaged phage was adsorbed onto E. coli G1250 suspension cultures and the cells plated onto bottom agar plates. Plates with diluted phage were incubated overnight for determination calculation of phage titre. Plates with undiluted phage were incubated for determination of the number of mutants. After incubation, plates were scored visually for number of plaques per plate. - Evaluation criteria:
- MUTATION ASSAY
OECD 488 criteria are adopted. There are several criteria for determining a positive result, such as a dose-related increase in the mutant frequency, or a clear increase in the mutant frequency in a single dose group compared to the solvent/vehicle control group. At least three treated dose groups should be analysed in order to provide sufficient data for dose-response analysis. While biological relevance of the results should be the primary consideration, appropriate statistical methods may be used as an aid in evaluating the test results. Statistical tests used should consider the animal as the experimental unit. A test material for which the results do not meet the criteria in any tissue is considered non-mutagenic in this assay. For biological relevance of a negative result, tissue exposure should be confirmed.
MICRONUCLEUS TEST
The vehicle control group should be consistent with the historical vehicle control range of enucleated immature red blood cells or reticulocytes (RETs).
The historical vehicle control data are from Sprague-Dawley rats of 6-8 weeks of age, supplemented with the historical control data from BigBlue® rats of 8-12 weeks.
The %RET and %MnRET frequencies for the kit-supplied positive control samples should be within those values indicated for that particular lot by Litron laboratories.
Six animals/group were available for analysis. - Statistics:
- Dunnett’s test was conducted on body weight, body weight changes, and organ weight data (where appropriate). All required statistics were based on a two-sided significance value of p < 0.05.
TRANSGENIC MUTATION FREQUENCY
The individual animal is considered the experimental unit. The mutant frequency (MF) was calculated (number of mutant phage / number of total phage screened) for each tissue analysed from each animal. Since this ratio is extremely small and may not be normally distributed, a log10 transformation of the MF data was performed. The statistical analysis of MF was conducted in two parts. Initially, the Positive Control group was compared to the Vehicle Control. In the second part of the analysis all the groups (except the Positive Control) were compared to the Vehicle Control. In both cases log10 transformed MF data from Vehicle Control and treated groups were evaluated using a 1-Way Analysis of Variance (ANOVA). The suitability of using the parametric ANOVA was confirmed by testing parameters of the log10 transformed MF data for normality and equal variance.
MICRONUCLEUS TEST
Statistical analysis was performed on the micronucleus frequency (%MnRET) and %RET using animal as unit. The mean and standard deviation of %MnRET and %RET were presented for each treatment group.
Group variances of test substance group and concurrent vehicle control group in respective sampling time were tested (Levene’s test). With homogenous data distribution one-way ANOVA was performed followed by Dunnett’s post-hoc analysis to compare each dose group to concurrent vehicle control.
Statistical analysis was performed using Minitab® 16.1.0. - Key result
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- MUTATION ASSAY
All animals survived until scheduled sacrifice on Day 31. All animals were normal during cage side observations. There were no treatment-related findings during hands-on observation.
The test material at 800 ppm in the high dose group was associated with lower food consumption when compared to the control feed group. The differences were statistically significant in Weeks 1 and 2 (22.6 and 15.3 % lower, respectively, relative to Group 1) and overall for Days 1-29 (14.9 % lower). The mean food consumption of the 800 ppm rats during Weeks 3 and 4 was also lower by 10.0 and 11.7 %, respectively. The lower food consumption was associated with somewhat lower weekly body weight gains during Days 1-29, which were not statistically significant or toxicologically important. There were no food consumption values excluded from the data.
Mean daily consumption of the test material (mg/kg/day) ranged from 5.9 to 4.9, 27.1 to 22.7 and 47.6 to 42.8 in males given 80, 400 and 800 ppm, respectively. These are practically identical to the intakes achieved in the rat carcinogenicity bioassay evaluated in the most recent draft assessment report for the test material.
There were no treatment related effects on organ weights.
BIG BLUE® IN VIVO MUTATION ASSAY
The results are presented in Table 1.
Treatment with the test material did not cause statistically elevated mutant frequency at the cII gene in either liver or jejunum of male F344 Big Blue® rats.
Liver and jejunum tissues were processed for DNA isolation and analysis of cII mutants. Background cII mutation frequencies (x 10^-6, mean ± SD) of the control feed group 29.7 ± 8.2 in the liver and 45.4 ± 10.3 in the jejunum. These background incidences were consistent with the past experience of the laboratory.
Mutation frequencies in treated animals were similar to control values, at 33.5 ± 10.2, 38.8 ± 15.7 and 38.2 ± 11.4 (low, mid and high doses respectively) in liver, and likewise, 36.8 ± 12.3, 40.9 ± 11.0, and 27.7 ± 9.1 in the jejunum.
The positive controls demonstrated elevated mutation frequencies of 128.9 ± 35.0 in the liver and 209.8 ± 54.4 in the jejunum (4 – 5 times the control values, p<0.001) and were consistent with historical experience.
The study design and results obtained met protocol-specified assay acceptance criteria and were consistent with the study requirements of OECD Test Guideline 488 for transgenic rodent mutation assays giving confidence in the evaluation that mutation frequencies of the cII gene in liver and jejunum were not statistically increased in Big Blue® rats exposed to the test material under the conditions of the assay.
MICRONUCLEUS ASSAY
The results are presented in Table 2.
No increases in micronucleus formation were observed in peripheral blood samples collected from the treated Big Blue® transgenic F344 rats compared to the control animals, and the test material was negative in the micronucleus assay. The positive control sample supplied with the kit used gave a result within the specified range, demonstrating that the flow cytometry technique had been implemented successfully at the laboratory. Samples from the negative control animals also produced results within the expected range specified by the kit. Overall, the study design and results obtained met protocol-specified assay acceptance criteria.
ANALYTICAL DETERMINATIONS
The results of analysis demonstrated that the feed was accurately prepared, and homogenous. There was no test material measured in the control. Stability of the material in the diet was confirmed as satisfactory for the preparation interval. - Conclusions:
- Interpretation of results: negative
Mutation frequencies of the cII gene in liver and jejunum were not statistically increased in Big Blue® rats exposed to the test material up to and including doses provoking jejunal tumours in rat bioassays. There were similarly no increases in micronucleus formation in treated animals. The test material was not mutagenic or clastogenic in this in vivo assay. - Executive summary:
The purpose of this study was to determine both mutant frequency of the cII gene in liver and jejunum and the formation of micronucleated reticulocytes in the peripheral blood of transgenic Fisher 344 male Rats (Big Blue®) following dietary treatment with the test material for 28 days. The Big Blue® Assay is a Transgenic Rodent (TGR) Mutation assay described in OECD Test Guideline 488 (OECD, 2013). The micronucleus test is an assay for cytogenetic damage in the bone marrow based upon OECD Test Guideline 474 (OECD, 2014). The study was conducted under GLP conditions.
Male Big Blue® rats (6/group) were given either the vehicle alone (untreated feed, Group 1) or the test material in the feed for 28 consecutive days at dose levels of 80, 400 and 800 ppm (Groups 2-4). All animals were dosed by diet administration ad libitum, beginning on Day 1 and switched to normal, untreated feed on Day 29 after 28 consecutive days of feeding. Blood was collected on Day 26 for evaluation of micronucleated reticulocytes in peripheral blood by flow cytometry methods. Surviving animals were sacrificed by CO₂ overdose on Day 31, and subjected to necropsy (including organ weight data). Liver and jejunum were processed for DNA isolation, packaging, and determination of mutant frequency at the cII locus.
As permitted by OECD TG 488, DNA from Positive Control animals from a previous study (5 male Big Blue® rats, oral gavage, Days 1, 2 and 3 of 20 mg/kg/day N-ethyl-N nitrosourea (ENU), tissues collected on Day 31) was used as a concurrent packaging Positive Control.
End points evaluated during the study included mortality, clinical signs, body weights (and body weight changes), organ weights, food consumption, micronucleated reticulocytes in peripheral blood and mutant frequencies of the cII locus in liver and jejunum.
All animals survived until terminal sacrifice on Day 31 and there were no treatment-related signs noted during cage side and hands-on observations. There were no statistically significant differences in weekly mean body weight or body weight gain of animals given the test material in feed when compared to control feed. The absolute weight gain (from Day 1 to Day 31) of the animals fed 800 ppm was 19 % lower than the control mean, but it was not statistically significant. On Days 29 to 31, mean weekly body weight gain in the high dose group was statistically significantly higher (83 %) compared to control feed group. Weekly food consumption for Weeks 1 and 2 and total mean food consumption were statistically significantly lower in the 800 ppm group compared to control feed group.
Treatment of Big Blue® transgenic rats with the test material for 28 days in the diet did not cause statistically elevated mutant frequency at the cII gene in either liver or jejunum of F344 Big Blue® rats at any dose level under the conditions of the study. There were no toxicological effects observed that may have altered the outcome of the mutation study. There were also no significant increases in micronucleus formation in peripheral blood samples collected on Day 26.
In conclusion, Big Blue® transgenic F344 rats given standard rodent diet containing the test material ad libitum at levels of 80, 400 and 800 ppm for 28 consecutive days did not demonstrate statistically elevated mutant frequency at the cII gene in either liver or jejunum at any dose level under the conditions of the study. The mutation assay met all study objectives and assay acceptance criteria. The two tissues, liver and jejunum, gave background mutant frequency data consistent with past experience of the laboratory. The positive control treatment with ENU gave statistically increased mutant frequencies in both tissues demonstrating the utility of the test system to detect and quantify induced mutants following exposure to a known mutagen. The study design and results obtained were consistent with the study requirements of OECD Test Guideline 488 for transgenic rodent mutation assays. There was no evidence of toxicity that might affect these results in assessment of clinical observations, body weights, organ weights or food consumption. There were no significant increases in micronucleus formation in peripheral blood samples collected on Day 26. Small but statistically significant decreases in food consumption in rats receiving the test material at 800 ppm in feed that were associated with total mean body weight gain that was 19 % lower than control although not statistically significant supports the selection of 800 ppm as the high dose for this repeat-dose study.
Mutation frequencies of the cII gene in liver and jejunum were not statistically increased in Big Blue® rats exposed to the test material up to and including doses provoking jejunal tumours in rat bioassays. There were similarly no increases in micronucleus formation in treated animals. The study design and results obtained met protocol-specified assay acceptance criteria. The test material was not mutagenic or clastogenic in this in vivo assay.
Referenceopen allclose all
Pilot study
Mortality occurred within one to two days of dose administration as follows: all animals at 2500 mg/kg, 2/2 males at 1600 and 1000 mg/kg and 1/2 males at 500 mg/kg. Clinical signs included lethargy in mice treated with 100, 500, 1000 and 2500 mg/kg and crusty eyes in one 100 mg/kg male.
Toxicity study
Mortality occurred within two days of dose administration as follows: 4/5 males and 5/5 females at 800 mg/kg, 2/5 males and 3/5 females at 500 mg/kg, 3/5 males and 4/5 females at 300 mg/kg and 2/5 males and 1/5 females at 200 mg/kg. Clinical signs, which were noted on the day following dose administration included lethargy in both males and females at all dose levels, diarrhea in one female at 300 mg/kg and crusty eyes in one male at 500 mg/kg. The LD50/3 was calculated to be approximately 290 mg/kg. The high dose for the micronucleus test was set at 150 mg/kg which was approximately 50% of the LD50/3.
Table 1: Body weights and mortality from toxicity study
| Group mean body weights (g) | % change | ||||||
| Treatment | Sex | Pre-treatment | Day 1 | Day 3 | Day 1 | Day 3 | Mortality |
| 200 mg/kg | M | 27.3 ± 1.3 | 28.6 ± 1.8 | 25.7 ± 2.8 | 4.8 | -5.9 | 2/5 |
| F | 27.7 ± 0.6 | 26.1 ± 1.1 | 24.2 ± 0.7 | -5.8 | -12.6 | 1/5 | |
| 300 mg/kg | M | 30.8 ± 2.8 | 29.2 ± 2.1 | 26.7 ± 2.3 | -5.2 | -13.3 | 3/5 |
| F | 27.8 ± 0.4 | 26.8 ± 1.0 | 28.9* | -3.6 | -4.0 | 4/5 | |
| 500 mg/kg | M | 30.8 ± 1.8 | 30.4 ± 1.8 | 29.9 ± 2.9 | -1.3 | -2.9 | 2/5 |
| F | 27.7 ± 1.3 | 27.5 ± 0.8 | 26.9 ± 1.8 | -0.7 | -2.9 | 3/5 | |
| 800 mg/kg | M | 30.7 ± 1.2 | 30.0 ± 2.0 | 32.5* | -2.3 | 5.9 | 4/5 |
| F | 28.0 ± 1.3 | 27.4 ± 1.1 | ** | -2.1 | ** | 5/5 | |
| 2500 mg/kg | M | 25.7 ± 0.9 | 22.3* | ** | -13.2 | ** | 5/5 |
| F | 23.7 ± 0.6 | ** | ** | ** | ** | 5/5 | |
*standard deviation not available due to single surviving animal
**no data due to mortality
Table 2: Summary of results from micronucleus assay
| Micronculeated polychromatic erythrocytes | ||||||
| Treatment | Sex | Time (hr) | Number of mice | PCE/total erythrocytes | Number per 1000 PCEs (mean ± S.D.) | Number per PCE scored |
| Vehicle | M | 24 | 5 | 0.48 | 0.4 ± 0.55 | 2/5000 |
| 48 | 5 | 0.50 | 0.2 ± 0.45 | 1/5000 | ||
| 72 | 5 | 0.54 | 0.2 ± 0.45 | 1/5000 | ||
| F | 24 | 5 | 0.61 | 0.0 ± 0.00 | 0/5000 | |
| 48 | 5 | 0.50 | 0.4 ± 0.55 | 2/5000 | ||
| 72 | 5 | 0.63 | 0.0 ± 0.00 | 0/5000 | ||
| 37.5 mg/kg | M | 24 | 5 | 0.49 | 0.2 ± 0.45 | 1/5000 |
| 48 | 5 | 0.45 | 0.0 ± 0.00 | 0/5000 | ||
| 72 | 5 | 0.59 | 0.2 ± 0.45 | 1/5000 | ||
| F | 24 | 5 | 0.56 | 0.6 ± 0.55 | 3/5000 | |
| 48 | 5 | 0.51 | 0.2 ± 0.45 | 1/5000 | ||
| 72 | 5 | 0.67 | 0.2 ± 0.45 | 1/5000 | ||
| 75 mg/kg | M | 24 | 5 | 0.56 | 0.8 ± 1.30 | 4/5000 |
| 48 | 5 | 0.33 | 0.4 ± 0.55 | 2/5000 | ||
| 72 | 5 | 0.28 | 0.2 ± 0.45 | 1/5000 | ||
| F | 24 | 5 | 0.56 | 0.2 ± 0.45 | 1/5000 | |
| 48 | 5 | 0.40 | 0.2 ± 0.45 | 1/5000 | ||
| 72 | 5 | 0.46 | 0.0 ± 0.00 | 0/5000 | ||
| 150 mg/kg | M | 24 | 5 | 0.52 | 0.2 ± 0.45 | 1/5000 |
| 48 | 5 | 0.40 | 0.0 ± 0.00 | 0/5000 | ||
| 72 | 5 | 0.38 | 0.2 ± 0.45 | 1/5000 | ||
| F | 24 | 5 | 0.50 | 0.6 ± 0.89 | 3/5000 | |
| 48 | 5 | 0.37 | 0.2 ± 0.45 | 1/5000 | ||
| 72 | 5 | 0.31 | 0.0 ± 0.00 | 0/5000 | ||
| Positive control | M | 24 | 5 | 0.41 | 8.2 ± 6.61 | 41/5000 |
| F | 24 | 5 | 0.53 | 9.8 ± 4.32 | 49/5000 | |
Historical control data
Table 3: Negative control animals
| Ratio of PCE/total erythrocytes | MPCE/1000 PCE scored | |||
| Parameter | Males | Females | Males | Females |
| Mean | 0.57 | 0.60 | 0.41 | 0.47 |
| Standard Deviation | 0.09 | 0.08 | 0.76 | 0.75 |
| Range | 0.12 - 0.85 | 0.09 - 0.86 | 0 to 8 | 0 to 5 |
Table 4: Positive control animals
| Ratio of PCE/total erythrocytes | MPCE/1000 PCE scored | |||
| Parameter | Males | Females | Males | Females |
| Mean | 0.56 | 0.59 | 12.01 | 11.59 |
| Standard Deviation | 0.10 | 0.11 | 6.53 | 5.41 |
| Range | 0.12 - 0.85 | 0.04 - 0.80 | 1 to 51 | 2 to 40 |
Table 1: Mutation frequency at the cII gene in the liver and jejunum
Dose Group (ppm) |
Mutation frequency ± SD (n = 5; x10^-6) |
|
|
Liver |
Jejunum |
Control |
29.7 ± 8.2 |
45.4 ± 10.3 |
80 |
33.5 ± 10.2 |
36.8 ± 12.3 |
400 |
38.8 ± 15.7 |
40.9 ± 11.0 |
800 |
38.2 ± 11.4 |
27.7 ± 9.1 |
Positive control |
128.9* ± 35.0 |
209.8* ±54.4 |
* = statistically significant (ANOVA, P<0.001)
Table 2: Results of in vivo Micronucleus Test
Dose Group (ppm) |
Ratio of Reticulocytes:Total erythrocytes (mean ± SD, n = 6) |
% Micronucleated reticulocytes (mean ± SD, n = 6) |
Control |
2.7 ± 0.44 |
0.060 ± 0.03 |
80 |
2.2 ± 0.28 |
0.070 ± 0.04 |
400 |
2.2 ± 0.17 |
0.087 ± 0.03 |
800 |
2.1 ± 0.19 |
0.060 ± 0.02 |
Positive control (mean) |
1.5 |
1.510 |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
In studies conducted prior to 2014 propargite was not mutagenic as evidenced by the findings of five in vitro tests and one in vivo study. The results of these tests are all negative indicating that exposure to this substance does not lead to cellular mutations, damage to chromosomes/DNA or interference with cell mitosis. However, to ensure data was relevant to modern production batches, the mutagenicity testing was repeated in 2014 and 2015 with modern material of known technical specification; this included a transgenic rodent assay in the rat to confirm the non-genotoxic potential in vivo, particularly with respect to rat jejunal tissues where tumours had been observed in the rat carcinogenicity studies.
All assays employed were compliant with the contemporary OECD test guideline (TG) with perhaps the exception of the chromosome aberration assay, where a repeat assay in the presence of S9 was omitted. The 1997 iteration of the OECD 473 TG specifies that “negative results with metabolic activation need to be confirmed on a case-by-case basis” and in cases where “confirmation of negative results is not considered necessary, justification should be provided.” The justification provided by the laboratory in the study report is as follows: “the results for the positive and negative controls indicate that all criteria for a valid assay were met” and “based on these criteria, results are justified and do not require a repeat of any portions of the study.” Further, in the revised OECD 473 TG it states that there is no requirement for verification of a clearly positive or negative response, which clarifies the “case-by-case basis” provided in the 1997 iteration. Given that the results are clearly negative in the presence of S9, the omission is less of a concern, and is also covered by the negative result obtained in the in vivo micronucleus assay (OECD 474) conducted as part of the transgenic rodent assay.
Propargite technical produced a positive result in the mammalian cell mutagenicity assay using mouse lymphoma cells, but only in the presence of S9. If this were a genuine result, positive results might also have been expected in the Ames and in vitro cytogenetic assays. Nonetheless, to confirm the lack of genotoxicity following exposure to propargite technical, a Big Blue® Transgenic F344 Rat assay was conducted examining the liver (a metabolically active tissue) and the jejunum (the tissue downstream from the liver and in which tumours were observed in rat carcinogenicity assays). From the Big Blue® Transgenic assay, clear negative results were obtained, and the in vitro mutagenic result was not verified in vivo. Further, as an additional in vivo assay, micronuclei were assessed in the peripheral reticulocytes from the same Big Blue® rats. There was no increase in micronuclei at any dose level within this assay.
On review of the results, propargite technical is not mutagenic or clastogenic in vivo, including the jejunum which is a site of carcinogenesis in Sprague Dawley and Wistar rats. The negative Big Blue® F344 Rat assay result is fully transferable to the Sprague Dawley and Wistar rat strains, as the assay applied the same dose levels at which carcinogenicity occurred in previous studies, and the in vitro comparative ADME study examined both F344 and Sprague Dawley rat hepatocytes and found them to produce similar metabolic profiles.
Summary of the 2014 and 2015 genotoxicity studies
Test substance (batch and purity) |
Test system |
Concentrations/ dose |
Result |
Acceptability of the study |
Reference |
In vitro |
|||||
Propargite (Lot No.: LT3H20C569, 91.6 % purity) |
Ames test: Salmonella typhimurium (TA100, TA98, TA1535, and TA1537), and Escherichia coli (WP2uvrA). |
Up to 5000 µg/plate in both the presence and absence of metabolic activation (S9). |
Not mutagenic |
Fully acceptable |
Wagner, 2014 |
Chromosomal Aberration assay: Chinese hamster ovary |
Up to 3 µg/mL without metabolic activation (S9) mix (4 hour treatment). Up to 25 µg/mL with S9 mix (4 hour treatment). Up to 1 µg/mL without S9 mix (20 hour treatment). |
Not clastogenic or aneugenic |
Fully acceptable in combination with the OECD 474 assay conducted as part of the OECD 488 transgenic rodent assay |
Roy, 2014 |
|
Mammalian cell mutation assay: L5178Y Mouse Lymphoma cell line |
Up to 50 µg/mL in the presence and absence of metabolic activation |
Mutagenic in the presence of metabolic activation.
Not mutagenic in the absence of metabolic activation. |
Fully acceptable |
Stankowski, 2014 |
|
In vivo |
|||||
Propargite (Lot No.: LT3H20C569, 91.6 % purity) |
cII Locus and Peripheral Blood Micronucleus assay: Big Blue® Transgenic F344 Rat |
80, 400 and 800 ppm in the diet |
Not mutagenic in the liver or jejunum.
Not clastogenic in peripheral lymphocytes. |
Fully acceptable |
McKeon and Huynh, 2015 |
Justification for classification or non-classification
In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to genetic toxicity.
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