Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECDTG471): not mutagenic

Chromosome aberration (OECDTG473): clastogenic

Mouse lymphoma (OECDTG490): not mutagenic

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 July, 2000 - 25 August, 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
S. typhimurium: Histidine gene
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by Aroclor 1254
Test concentrations with justification for top dose:
- Experiment 1 and 2:
Due to cytotoxicity in an initial toxicity test, the following dose levels were used:
All 5 strains, without S9: 1.5, 5, 15, 50, 150 and 1500 μg/plate
All 5 strains, with S9: 5, 15, 50, 150, 500 and 1500 μg/plate
Vehicle / solvent:
- Solvent used: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
50 μL/plate DMSO
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
other: With S9 2-aminoanthracene: 0.7 μg/plate for TA 100, 1 μg/plate for TA 1535, 1.5 μg/plate for TA 98, TA 102 and TA 1537
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain.

DETERMINATION OF CYTOTOXICITY
- Method: the plates were examined for a decline in the number of spontaneous revertants, the existence of a normal background lawn and microscopically for microcolony growth.
Evaluation criteria:
No data.
Statistics:
X2-test (Mohn and Ellenberger, 1977)
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at concentrations of 150 µg/plate and upwards in the absence of S9-mix and at concentrations of 500 µg/plate and upwards in the presence of S9-mix.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at concentrations of 150 µg/plate and upwards in the absence of S9-mix and at concentrations of 500 µg/plate and upwards in the presence of S9-mix.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at concentrations of 150 µg/plate and upwards in the absence of S9-mix and at concentrations of 500 µg/plate and upwards in the presence of S9-mix.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at concentrations of 150 µg/plate and upwards in the absence of S9-mix and at concentrations of 500 µg/plate and upwards in the presence of S9-mix.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at concentrations of 150 µg/plate and upwards in the absence of S9-mix and at concentrations of 500 µg/plate and upwards in the presence of S9-mix.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed up to and including the top dose of 1500 μg/plate.

COMPARISON WITH HISTORICAL CONTROL DATA:
The number of spontaneous revertants observed using each of the five strains was close to those previously established in the laboratory and was within the range obtained by Ames et al. (1975) as well as reported by De Serres and Shelby (1979).
Similarly, the results with the positive control substances confirmed the known reversion properties and specificity of the tester strains as well as the full activity of the metabolizing system.
Conclusions:
The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay performed according to OECD 471 guideline and GLP principles.
Executive summary:

The mutagenic activity of the substance was evaluated in accordance with OECD 471 guideline and according to GLP principles. The test was performed in two independent direct plate experiments, both in the absence and presence of S9-mix up to and including 500 and 1500 μg/plate, respectively. Adequate negative and positive controls were included. Toxicity was observed in all Salmonella strains at concentrations of 150 µg/plate and upwards in the absence of S9-mix and at concentrations of 500 µg/plate and upwards in the presence of S9-mix. The substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the five S. typhimurium tester strains (TA1535, TA1537, TA98, TA100 and TA102), both in the absence and presence of S9-metabolic activation. These results were confirmed in independently repeated experiments. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 October, 2011 - 06 December, 2011
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)
Version / remarks:
21st July 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Methods of Testing New Chemical Substances (No. 0331-7 of Pharmaceutical and Food Safety Bureau dated March 31, 2011, 2011-03-29 Manufacturing Industries Bureau No. 5, No. 110331009 of Policy Planning Division, Environmental Health Department).
GLP compliance:
yes
Type of assay:
other: in vitro mammalian chromosome aberration test chromosome aberration
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Name of test material: 2-Heptan-1-ylcyclopentan-1-one
- Lot No.of test material: Masked
- Expiration date of the lot: No data.
- Purity: 99.3%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: To be stored being protected from light in a tight container in a cool place.
- Stability under test conditions: The investigational substance was confirmed to have been stable during the test period.
- Solubility and stability of the test substance in the solvent/vehicle: DMSO: No changes including generation of heat, bubbles, and/or smoke were seen until 3 hours after the preparation
Species / strain / cell type:
other: Chinese hamster lung (CHL/IU cells)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Distributed by National Institute of Hygienic Sciences (currently National Institute of Health Sciences, Japan) on November 15, 1984
- Number of passages if applicable: The cells after 29 and 7 subcultures were used in the cell proliferation inhibition test and the chromosomal aberration test, respectively.
- Methods for maintenance in cell culture if applicable:
- Modal number of chromosomes: No abnormality in chromosome number (84% of the cells had 25 chromosomes).
- Normal (negative control) cell cycle time: No abnormality in doubling time (15.2 hours).

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: MEM liquid medium was added with inactivated (56°C, 30 min) calf serum to the final concentration of 10%.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and 5,6-Benzoflavone.
Test concentrations with justification for top dose:
Dose range finding test:
With and without S9-mix, 6hr exposure; 24 hr fixation: 3.55, 7.11, 14.2, 28.4, 56.9, 114, 228, 455, 910 and 1820 μg/mL
Without S9-mix, 24hr exposure; 24 hr fixation: 3.55, 7.11, 14.2, 28.4, 56.9, 114, 228, 455, 910 and 1820 μg/mL
First cytogenetic test:
With and without S9-mix, 6hr exposure time, 24 hr fixation: 52.4, 65.5, 81.9, 102, 128, 160 and 200 μg/mL
Without S9-mix, 24hr exposure; 24 hr fixation: 41.9, 52.4, 65.5, 81.9, 102, 128 and 160 μg/mL
The following dose levels were selected for scoring of chromosome aberrations:
With S9-mix, 6hr exposure time, 24 hr fixation: 102, 128 and 160 μg/mL
Without S9-mix, 6hr exposure time, 24 hr fixation: 81.9, 102 and 128 μg/mL
Without S9-mix, 24hr exposure; 24 hr fixation: 81.9, 102 and 128 μg/mL
Vehicle / solvent:
- Solvent used: DMSO
- Justification for choice of solvent: Because the substance is hardly soluble in water and soluble in DMSO, the substance was dissolved in DMSO which was dehydrated by use of molecular sieves.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 8x10^3 cells/mL

DURATION
- Preincubation period: 72 hours
- Exposure duration: 6hr and 24hr
- Fixation time (start of exposure up to fixation or harvest of cells): 6hr and 24hr

SPINDLE INHIBITOR (cytogenetic assays): Colcemid

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: Duplicates in one experiment

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Two drops of the cell suspension were placed on slide glasses by the hand method to prepare two specimens for chromosome observation. The slide specimens were dried fully and then stained by use of a Giemsa solution for 12 min. The slides were dried after light washing with water.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 200 metaphase chromosome spreads per culture.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
Evaluation criteria:
a. The incidences of cells with structural aberration and polyploid cells in the negative control group are to be within the reference values calculated from the background data, AND the both incidences are to be lower than 5%.
b.The incidence of cells with structural aberration in the positive control group is to be within the reference value calculated from the background data, AND above 10%. In addition, the incidence of polyploid cells is to be lower than 5%.
When the conditions above were satisfied, the test was judged to become acceptable.

Less than 5% of incidence of aberrant cells was judged to be negative, whereas above 5% and less than 10% of the incidence with reproducibility was false positive. Above 10% of the incidence with reproducibility or dependence on the concentration of the investigational substance was judged to be positive. Cells with only a gap(s) were excluded from the number of aberrant cells in the judgment. No statistical test was performed.
Statistics:
D20 value, a concentration of the investigational substance required for inducing any aberration in 20% of metaphase images, was calculated by the least-squares method. TR value, a value for comparison representing incidence of exchange aberration (cte) per fixed concentration (mg/mL), was calculated by dividing the incidence (%) of chromatid exchange by concentration of the investigational substance (converted to mg/mL).
Key result
Species / strain:
other: hamster lung (CHL/IU cells)
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
other: hamster lung (CHL/IU cells)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In the cell proliferation inhibition test a concentration of 455 μg/mL and above showed precipitation in the culture medium at the time of completion of the treatment. In the chromosome aberration test a concentration of 160 μg/mL and above showed precipitation in the culture medium at the time of completion of the treatment.

RANGE-FINDING/SCREENING STUDIES: In all treatments, i.e., the S9-free short-time treatment (-S9 treatment), the short-time treatment with S9 treatment (+S9 treatment), and the continuous 24-hour treatment (24-hour treatment), cell proliferation inhibitory action was detected in a concentration-dependent manner. The 50% inhibitory concentration of cell proliferation in –S9 treatment, +S9 treatment, and 24-hour treatment was calculated to be 127, 129, and 113 μg/mL, respectively.

D20 AND TR VALUE:
The D20 values (mg/mL) and TR value (per mg/mL) calculated from the results of the chromosomal aberration test were as follows:

Treatment Type of aberration D20 value TR value (treatment concentration μg/mL)
Short-time treatment with S9 treatment Structural aberration 0.181 81.3 (160)

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
Short-term treatment (-S9): 56.5 ± 9.2 (29.0 - 83.9)
Short-term treatment (+S9): 37.3 ± 8.7 (11.2 - 63.4)
Continuous treatment (-S9): 43.0 ± 9.6 (14.3 - 71.7)
- Negative (solvent/vehicle) historical control data:
Short-term treatment (-S9): 0.6 ± 0.9 (0.0 - 3.1)
Short-term treatment (+S9): 0.2 ± 0.5 (0.0 - 1.6)
Continuous treatment (-S9): 0.4 ± 0.7 (0.0 - 2.5)
Both the incidences of chromosomal aberration in the negative and positive controls were within the reference values calculated from the background data, satisfying the forming conditions of the test. Therefore, it was judged that the test had been conducted in proper conditions.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Appropriate toxicity was reached at the dose levels selected for scoring in the absence and presence of S9.
Conclusions:
A chromosome aberration study with the substance was performed according to OECD 473 guideline and GLP principles, in cultured Chinese hamster lung fibroblast cell line (CHL/IU cells) in one experiment with and without S9-mix. It is concluded that the substance is clastogenic in cultured Chinese hamster lung fibroblast cell line (CHL/IU cells) in the presence of S9-mix.
Executive summary:

In a chromosome aberration study, cultured Chinese hamster lung fibroblast cell line (CHL/IU cells) were exposed to different concentrations (81.9 - 160 μg/mL) of the substance (dissolved in DMSO), in the presence and absence of S9-mix according to OECD 473 guideline and GLP principles.

In the cytogenetic assay, the substance was tested up to cytotoxic concentrations of 128 μg/mL for a 6 h and 24 h exposure time with a 24 h fixation time in the absence of S9 -mix.

In the presence of S9 -mix, the substance was tested up to the cytotoxic concentration of 160 μg/mL for a 6 h exposure time with a 24 h fixation time. The substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence of S9-mix. In the presence of S9 -mix, the substance did induce a statistically significant dose-dependent increase in the number of cells with chromosome aberrations. No effects of the substance on the number of polyploid cells were observed both in the absence and presence of S9-mix. Finally, it is concluded that the substance is clastogenic in cultured Chinese hamster lung fibroblast cell line (CHL/IU cells) in the presence of S9-mix.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 April, 2016 - 16 May, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Version / remarks:
28 July 2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
30 May 2008.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene
Target gene:
Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells.
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media:
Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 μg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 μg/mL) and 10% donor horse serum (giving R10 media) at with 5% CO2 in air. The cells have a generation time of approximately 12 hours and were subcultured accordingly. RPMI 1640 with 20% donor horse serum (R20), 10% donor horse serum (R10), and without serum (R0), are used during the course of the study.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone.
Test concentrations with justification for top dose:
Dose range finding test:
Without and with S9-mix, 4 hours treatment: 7.12, 14.24, 28.48, 56.97, 113.94, 227.88, 455.75, 911.5 and 1823 μg/mL
Without S9-mix, 24 hours treatment: 7.12, 14.24, 28.48, 56.97, 113.94, 227.88, 455.75, 911.5 and 1823 μg/mL
Main experiment:
Without S9-mix, 4 hours treatment: 2.5, 5, 10, 20, 30, 40, 50 and 60 μg/mL
With S9-mix, 4 hours treatment: 5, 10, 20, 30, 40, 50, 60 and 70 μg/mL
Without S9-mix, 24 hours treatment: 1.25, 2.5, 5, 10, 20, 30, 40 and 50 μg/mL
The following dose levels were selected to measure mutation frequencies at the TK-locus :
Without S9-mix, 4 hours treatment: 2.5, 5, 10, 20, 30 and 40 μg/mL
With S9-mix, 4 hours treatment: 20, 30, 40, 50, 60 and 70 μg/mL
Without S9-mix, 24 hours treatment: 5, 10, 20, 30, 40 and 50 μg/mL
Vehicle / solvent:
- Solvent used: DMSO.
- Justification for choice of solvent: The test item was dissolved in dimethyl sulfoxide. DMSO has been a ccepted and approved by authorities and international guidelines. No correction was made for the purity/composition of the test item.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Short-term treatment
With and without S9-mix: 4 hours
Prolonged treatment period
Without S9-mix: 24 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 10 to 12 days

SELECTION AGENT (mutation assays): 4 μg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS:
- Solvent controls: Duplicate cultures
- Treatment groups and positive control: Single cultures

NUMBER OF CELLS EVALUATED: 10E4 cells plated/concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (dose range finding test) and relative total growth (mutation experiment)
Evaluation criteria:
DATA EVALUATION
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the GEF and the increase is concentration related (e.g., using a trend test). The test chemical is then considered able to induce mutation in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test chemical is then considered unable to induce mutations in this test system.
Statistics:
In place of statistical analysis generally used for other tests, it relies on the use of a predefined induced mutant frequency (i.e. increase in MF above the concurrent control), designated the Global Evaluation Factor (GEF) of 126 x 10-6, which is based on the analysis of the distribution of the vehicle control MF data from participating laboratories.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and of osmolality:

µg/mL 0 7.12 14.24 28.48 56.97 113.94 227.88 455.75 911.5 1823
pH 7.28 7.32 7.34 7.34 7.35 7.36 7.36 7.36 7.37 7.36
mOsm 457 465 - 470 - 461 453 446 442 432
- = not determined

- Precipitation: Precipitation was observed at and above 227.88 μg/mL in all three exposure groups.

RANGE-FINDING/SCREENING STUDIES:
In the 4-hour exposures, both in the absence and presence of metabolic activation (S9), there was evidence of marked reductions in the relative suspension growth (%RSG) of 99% and 88% respectively at 56.97 μg/mLat cells treated with the test item when compared to the concurrent vehicle controls. The toxicity curve was particularly steep in the 4-hour exposure groups. In the 24-hour exposure a reduction of 70% was observed in the relative suspension growth (%RSG) at 56.97 μg/mL. In the subsequent mutagenicity experiments the maximum dose was limited by test item induced toxicity.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Negative (solvent/vehicle) and positive historical control data: See "Any other information on results incl. tables".

ADDITIONAL INFORMATION ON CYTOTOXICITY:
There was evidence of toxicity following exposure to the test item in all three of the exposure groups, as indicated by the %RSG and RTG values. There was evidence of reductions in viability (%V) in both 4-houre exposure groups, therefore indicating that residual toxicity occurred in these exposure groups. Optimum levels of toxicity were achieved in the 24-hour exposure group only. In both of the 4-hour exposures the toxicity curve was very steep and optimum toxicity could not be achieved for this reason.
However, the test item was tested and plated out for mutant expression beyond the toxic cut off point with no increase in mutant frequency being observed and, therefore, the results were considered to indicate that the test item had been adequately tested. Acceptable levels of toxicity were seen with both positive control substances. The test item dose levels 50 and 60 μg/mL in the 4-hour exposure in the absence of metabolic activation were not plated out for 5-TFT resistance and viability due to excessive toxicity. The dose levels 40 μg/mL in the 4-hour exposure in the absence of metabolic activation and 70 μg/mL in the presence of metabolic activation were plated out for 5-TFT resistance and viability but later discarded from analysis due to excessive toxicity.

Negative (solvent/vehicle) and positive historical control data:

Experiment -S9:

Vehicle control

Positive control

MF

MF

162.38

148.48

123.25

131.75

143.02

163.14

91.25

102.97

115.31

127.33

136.09

164.62

99.45

129.41

113.94

162.13

159.08

140.51

107.58

140.94

1439.27

725.42

784.41

715.32

1386.96

1381.05

795.75

891.24

1014.27

1736.47

873.64

860.09

1065.80

1382.02

978.98

1745.50

772.69

1028.72

905.61

1081.79

Mean             133.13

Min                  91.25

Max               164.62

SD                    22.95

Mean           1078.25

Min                715.32

Max             1745.50

SD                  322.38

Experiment +S9:

Vehicle control

Positive control

MF

MF

109.41

151.84

105.29

164.80

119.34

111.49

122.22

121.90

120.61

122.69

112.95

151.55

122.80

128.30

127.43

169.61

131.37

106.94

177.35

144.30

879.68

1219.61

779.42

533.15

726.20

1050.82

1081.26

713.66

1332.17

971.97

832.46

1177.21

978.95

704.07

1245.96

972.07

715.86

752.26

752.43

855.12

Mean             131.11

Min                105.29

Max               177.35

SD                    21.51

Mean             913.72

Min                533.15

Max             1332.17

SD                  216.53

Conclusions:
A mouse lymphoma assay with the substance was conducted according to OECD 490 guideline and GLP principles. It is concluded that the substance is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions of the study.
Executive summary:

The mouse lymphoma assay with the substance was conducted according to OECD 490 guideline and GLP principles.

In the 4 hours exposure period, the substance was tested up to and including concentrations of 40 and 70 μg/mL in the absence and presence of S9-mix, respectively. The incubation time was 4 hours. The relative suspension growth (RSG) was 13 and 10% in the absence and presence of S9-mix, respectively. In the 24 -hours exposure period in the absence of S9 -mix, the substance was tested up to and including of a concentration of 50 μg/mL. The RSG was reduced to 12%.

Positive control chemicals, ethylmethanesulphonate and cyclophosphamide induced appropriate responses.

In the absence and presence of S9-mix, the substance did not induce a significant increase in the mutation frequency. It is concluded that the substance is not mutagenic in the mouse lymphoma L5178Y test system.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Micronucleus study (OECDTG474): not mutagenic

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 September 2016 - 02 December 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
26 September 2014
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
30 may 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: Mammalian erythrocyte micronucleus test
Species:
mouse
Strain:
other: Hsd: ICR (CD-1®)
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo, B.V., Horst, The Netherlands
- Age at study initiation: approximately six to ten weeks
- Weight at study initiation: 23 to 30 g
- Assigned to test groups randomly: yes
- Fasting period before study: no data
- Housing: The animals were housed in groups of five in solid-floor polypropylene cages with wood-flake bedding.
- Diet: Free access to food (Envigo Teklad 2014C Global Certified Rodent Diet supplied by Envigo Laboratories UK Ltd., Oxon, UK)
- Water: Free access to mains drinking water
- Acclimation period: minimum of five days

ENVIRONMENTAL CONDITIONS (set to maintain)
- Temperature (°C): 19 to 25
- Humidity (%): 30 to 70
- Air changes (per hr): approximately fifteen
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
- Vehicle used: arachis oil
- Justification for choice of solvent/vehicle: no data
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was freshly prepared as required as a solution at the appropriate concentration in arachis oil. The test item was formulated within two hours of it being applied to the test system; it is assumed that the formulation was stable for this duration.

VOLUME: 10 mL/kg bw
Duration of treatment / exposure:
24 and 48 hours
Frequency of treatment:
Dosed once.
Dose / conc.:
0 mg/kg bw (total dose)
Remarks:
24 hours exposure time
Dose / conc.:
150 mg/kg bw (total dose)
Remarks:
24 hours exposure time
Dose / conc.:
300 mg/kg bw (total dose)
Remarks:
24 hours exposure time
Dose / conc.:
600 mg/kg bw (total dose)
Remarks:
24 and 48 hours exposure time
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide:
- Route of administration: orally at 50 mg/kg bw
- Doses / concentrations: Dissolved in sterile distilled water at a concentration of 5 mg/mL
Tissues and cell types examined:
The incidence of micronucleated cells per 4000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored. In addition, the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes was counted; these cells were also scored for incidence of micronuclei.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Based on the observed mortality and clinical signs in the toxicity assay, dose levels were selected for the micronucleus assay.

DETAILS OF SLIDE PREPARATION: Immediately following termination (i.e. 24 or 48 hours following dosing), both femurs were dissected from each animal, aspirated with foetal bovine serum and bone marrow smears prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol, stained in May-Grünwald / Giemsa, allowed to air-dry and a cover slip applied using mounting medium.

METHOD OF ANALYSIS: Stained bone marrow smears were coded and examined blind using light microscopy at x1000 magnification. The incidence of micronucleated cells per 4000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored. Micronuclei are normally circular in shape, although occasionally they may be oval or half-moon shaped, and have a sharp contour with even staining. In addition, the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes was counted; these cells were also scored for incidence of micronuclei.
The ratio of polychromatic to normochromatic erythrocytes was calculated together with appropriate group mean values and standard deviations.
Evaluation criteria:
Comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the test item groups and the number occurring in the vehicle control group.
A positive mutagenic response is demonstrated when a statistically significant, dose-responsive, toxicologically relevant increase in the number of micronucleated polychromatic erythrocytes is observed for either the 24 or 48-hour kill times when compared to the vehicle control group.
If these criteria were not fulfilled, then the test item was considered to be non-genotoxic under the conditions of the test.
A positive response for bone marrow toxicity was demonstrated when the dose group mean polychromatic to normochromatic ratio was shown to be statistically significantly lower than the vehicle control group.
Statistics:
All data were statistically analysed using appropriate statistical methods as recommended by the UKEMS Sub-committee on Guidelines for Mutagenicity Testing Report, Part III (1989).
The data was analysed following a transformation using Student's t-test (two tailed).
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Clinical signs of toxicity were observed at 600 mg/kg bw (24 and 48-hour) and included hunched posture and ptosis.
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: Groups of mice were dosed orally as follows: 1000 mg/kg bw (3 males and 1 female), 800 mg/kg bw (2 male) and 600 mg/kg bw (4 males and 2 females).
The clinical signs of hunched posture, lethargy, ataxia, ptosis, decreased respiration and labored respiration was observed at 1000 mg/kg bw. One of the animals dosed at 1000 mg/kg bw exceeded the severity band therefore was killed in extremis. The dose level was decreased to 800 mg/kg bw and the clinical signs of hunched posture, ptosis, ataxia and tiptoe gait were observed. The clinical signs were considered to be too severe and therefore the dose level was reduced to 600 mg/kg bw. The following clinical signs were observed, hunched posture, and ptosis. This was selected as the considered maximum tolerated dose level and the main test was initiated using this dose level. Acceptable bone marrow toxicity was observed at 600 mg/kg bw.

RESULTS OF DEFINITIVE STUDY
- Mortality Data and Clinical Observations:
There were no premature deaths in any of the dose groups. Clinical signs of toxicity were observed at 600 mg/kg bw (24 and 48-hour) and included hunched posture and ptosis. The clinical signs were considered to be indicative of systemic absorption.
- Evaluation of Bone Marrow Slides:
The test item Fleuramone did not show any indication of toxicity to bone marrow. The PCE/NCE ratio had no marked reductions when compared to the vehicle control. No statistically significant, decreases in the PCE/NCE ratio were observed in any of the exposure groups when compared to the vehicle control.
There was no evidence of any significant increases in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test item when compared to the vehicle control group.
The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.


Conclusions:
A micronucleus study with the substance was performed equivalent to OECD 474 guideline and GLP principles, in male mice. It is concluded that the substance is not mutagenic in the mouse micronucleus assay.
Executive summary:

In an in vivo micronucleus study, male mice were exposed to 150, 300 and 600 mg/kg bw of the substance, performed equivalent to OECD 474 guideline and GLP principles. Based on the results of the range finding test in which clinical signs of hunched posture, lethargy, ataxia, ptosis, decreased respiration and labored respiration were observed at 1000 mg/kg. One of the animals dosed at 1000mg/kg exceeded the severity band therefore was killed in extremis. The dose level was decreased to 800mg/kg and the clinical signs of hunched posture, ptosis, ataxia and tiptoe gait were observed. The clinical signs were considered to be too severe and therefore the dose level was reduced to 600 mg/kg. The following clinical signs were observed, hunched posture, and ptosis. This was selected as the considered maximum tolerated dose level and the main test was initiated using this dose level. At this dose levels and below there were no premature deaths in any of the dose groups. Clinical signs of toxicity were observed at 600 mg/kg bw (24 and 48-hour) and included hunched posture and ptosis. There was no evidence of any significant increases in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test substance when compared to the vehicle control group. The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test. It is concluded that the substance is not mutagenic in the mouse micronucleus assay.

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

Additional information

In vitro tests:

Ames test (OECD TG 471): The mutagenic activity of the substance was evaluated in accordance with OECD 471 guideline and according to GLP principles. The test was performed in two independent direct plate experiments, both in the absence and presence of S9-mix up to and including 500 and 1500 μg/plate, respectively. Adequate negative and positive controls were included. Toxicity was observed in all Salmonella strains at concentrations of 150 µg/plate and upwards in the absence of S9-mix and at concentrations of 500 µg/plate and upwards in the presence of S9-mix. The substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the five S. typhimurium tester strains (TA1535, TA1537, TA98, TA100 and TA102), both in the absence and presence of S9-metabolic activation. These results were confirmed in independently repeated experiments. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay.

Chromosome aberration (OECD TG 473): In a chromosome aberration study, cultured Chinese hamster lung fibroblast cell line (CHL/IU cells) were exposed to different concentrations (81.9 - 160 μg/mL) of the substance (dissolved in DMSO), in the presence and absence of S9-mix according to OECD 473 guideline and GLP principles.

In the cytogenetic assay, the substance was tested up to cytotoxic concentrations of 128 μg/mL for a 6 h and 24 h exposure time with a 24 h fixation time in the absence of S9 -mix.

In the absence of S9 -mix, the substance was tested up to the cytotoxic concentration of 160 μg/mL for a 6 h exposure time with a 24 h fixation time. The substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence of S9-mix. In the presence of S9 -mix, the substance did induce a statistically significant dose-dependent increase in the number of cells with chromosome aberrations. No effects of the substance on the number of polyploid cells were observed both in the absence and presence of S9-mix. Finally, it is concluded that the substance is clastogenic in cultured Chinese hamster lung fibroblast cell line (CHL/IU cells) in the presence of S9-mix.

Mouse lymphoma Assay (OECD TG 490):

The mouse lymphoma assay with the substance was conducted according to OECD 490 guideline and GLP principles.

In the 4 hours exposure period, the substance was tested up to and including concentrations of 40 and 70 μg/mL in the absence and presence of S9-mix, respectively. The incubation time was 4 hours. The relative suspension growth (RSG) was 13 and 10% in the absence and presence of S9-mix, respectively. In the 24 -hours exposure period in the absence of S9 -mix, the substance was tested up to and including of a concentration of 50 μg/mL. The RSG was reduced to 12%.

Positive control chemicals, ethylmethanesulphonate and cyclophosphamide induced appropriate responses.

In the absence and presence of S9-mix, the substance did not induce a significant increase in the mutation frequency. It is concluded that the substance is not mutagenic in the mouse lymphoma L5178Y test system.

In vivo test (OECD TG 474):

In an in vivo micronucleus study, male mice were exposed to 150, 300 and 600 mg/kg bw of the substance, performed equivalent to OECD 474 guideline and GLP principles. Based on the results of the range finding test in which clinical signs of hunched posture, lethargy, ataxia, ptosis, decreased respiration and labored respiration were observed at 1000 mg/kg. One of the animals dosed at 1000 mg/kg exceeded the severity band therefore was killed in extremis. The dose level was decreased to 800mg/kg and the clinical signs of hunched posture, ptosis, ataxia and tiptoe gait were observed. The clinical signs were considered to be too severe and therefore the dose level was reduced to 600 mg/kg. The following clinical signs were observed, hunched posture, and ptosis. This was selected as the considered maximum tolerated dose level and the main test was initiated using this dose level. At this dose levels and below there were no premature deaths in any of the dose groups. Clinical signs of toxicity were observed at 600 mg/kg bw (24 and 48-hour) and included hunched posture and ptosis. There was no evidence of any significant increases in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test substance when compared to the vehicle control group. The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test. It is concluded that the substance is not mutagenic in the mouse micronucleus assay.

Justification for classification or non-classification

Based on the results of the negative Ames test, negative Mouse lymphoma assay and the negative in vivo micronucleus study, the substance does not have to be classified for genotoxicity in accordance with Regulation (EC) No 1272/2008 and GHS.