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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test article was negative in an Ames, a chromosomal aberration and in a mammalian cell gene mutation assays. All studies were guideline and GLP compliant.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat-liver S9 mix
Test concentrations with justification for top dose:
preliminary toxicity test without metabolic activation: 20 - 5000 µg/plate
main test (with and without metabolic activation): 313, 625, 1250, 2500 and 5000 µg/plate
Vehicle / solvent:
Acetone
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see "Details on test system"
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS: 3METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48h

NUMBER OF REPLICATIONS: three Petri dishes are prepared per strain and per group

DETERMINATION OF CYTOTOXICITY
- Method: ; relative total growth

- POSITIVE CONTROLS
WITHOUT S9
1) for strain TA 98: 2-nitrofluorene, 10 µg/plate in dimethylsulfoxide
2) for strains TA 100 and TA 1535: sodium azide, 2.0 µg/plate in bidistilled water
3) for strain TA 1537: 9 (5)-aminoacridine hydrochloride monohydrate, 150 µg/plate in dimethylsulfoxide
4) for strain E. coli: 4-nitroquinoline-N-oxide, 1 µg/plate in dimethylsulfoxide.

WITH S9
1) for strains TA 98 and TA 100: 2-aminoanthracene, 2.5 µg/plate in dimethylsulfoxide
2) for strain TA 1535: cyclophosphamide monohydrate, 400 µg/plate in bidistilled water
3) for strain TA 1537: 2-aminoanthracene, 5µg/plate in dimethylsulfoxide;
4) for strain E. coli WP2uvrA: 2-aminoanthracene, 50 µg/plate in dimethylsulfoxide
.
Evaluation criteria:
Criteria for a positive response:
The test substance is considered to be positive in this test system if one or both of the following conditions are met:
- at least a reproducible doubling of the mean number of revertants per plate above that of the negative control at any concentration level for one or more of the following strains: TA 98, TA 1535, TA 1537 and E. coli
- a reproducible increase of the mean number of revertants per plate for any concentration above that of the negative control by at least a factor of 1.5 for strain TA 100.
Generally a concentration-related effect should be demonstrable.
Assay acceptance criteria:
A test is considered acceptable if the mean colony counts of the control values of all strains are within the acceptable ranges and if the results of the positive controls meet the criteria for a positive response.
In either case the final decision has to be based on scientific judgement.
Key result
Species / strain:
other: S.typhimurium TA 98, TA 100, TA 1535, TA 1537; Escherichia coli WP2uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
Nine concentrations of the test item ranging from 20 to 5000 µg/0.1 ml were tested to determine the highest concentration to be used in the mutagenicity assay. From the results obtained, the highest concentration suitable for the mutagenicity test was found to be 5000 µg/0.1 ml.

EXPERIMENTAL RESULT

Experiment 1

TA 1535 TA 100 TA 1537 TA 98 WP2uvrA
Dose (µg/plate) -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
0 20 13 116 130 12 11 29 35 26 33
313 13 16 108 121 8 13 23 52 22 31
625 13 17 114 124 9 12 18 43 13 33
1250 13 13 99 123 9 15 21 39 16 26
2500 17 15 114 115 10 13 18 43 26 37
5000 13 9 98 142 9 11 26 50 17 36
positive control 568 451 606 1781 1225 307 1225 815 443 1223

Experiment 2

TA 1535 TA 100 TA 1537 TA 98 WP2uvrA
Dose (µg/plate) -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
0 8 12 138 120 8 11 23 34 16 23
312.5 9 11 132 147 8 13 18 40 23 27
625 12 6 162 148 10 12 24 36 17 24
1250 9 12 133 129 9 13 16 34 27 17
2500 11 8 148 128 10 11 20 46 16 27
5000 11 10 140 134 7 11 20 48 26 19
positive control 455 477 667 1481 1585 126 1520 1184 379 546
Conclusions:
In the experiments performed without and with microsomal activation none of the tested concentrations of the test item led to an increase in the incidence of both histidine- or tryptophan-prototrophic mutants by comparison with the negative control.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
The CCL 61 cell line was derived as a subclone from the parental CHO cell line initiated from a biopsy of an ovary of an adult Chinese hamster and has a high proliferation rate (doubling time 12-14 hours). The cell line was maintained in Nutrient Mixture F-12 supplemented with 10% fetal calf serum + Penicillin/Streptomycin 100 units / ml / 100 µg / ml (Gibco AG, Basle, Switzerland) and saved in liquid nitrogen.
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced liver S9 mix of rats
Test concentrations with justification for top dose:
150.0 - 600.0 µg/ml
The highest concentration used (600 µg/ml) was based on the solubility properties of the test substance.
Vehicle / solvent:
Acetone
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
Two hours prior to harvesting, the cultures were treated with Colcemide 0.4 /µg/ml. The experiment was terminated by hypotonic treatment (0.075 M KCl solution) of the cells, followed by fixation (methanol:acetic acid, 3:1).
For the determination of the mitotic index the preparations from the various cultures were examined first, uncoded. The percentages of mitotic suppression in comparison with the controls were evaluated by counting at least 2000 cells per concentration. The determination of the mitotic coefficient was performed for the two experiments of the original study and for the third and fourth experiment of the confirmatory study separately. From the results of corresponding original run, five suitable concentrations were determined for the first and second experiment of the confirmatory study.
Two hundred metaphases were examined from the vehicle control and from the cultures treated with the various concentrations of the test substance. At least fifty metaphases each from the appropriate positive controls were analyzed.
Evaluation criteria:
Criteria for a positive response
- A test substance is considered to be active in this test system if in comparison to the negative control a marked increase in the number of specific chromosomal aberrations appears or if an increased number of exchange figures appears together with a high number of other specific chromosomal aberrations such as breaks and fragments.
A concentration-related response in the number of aberrations should be demonstrable.

Assay acceptance criteria
The (juality of the slides should allow, at least to a large extent, the chromosomes to be easily identifiable.
- In the negative controls the percentage of metaphases showing chromosomal aberrations should be in the range of that of the historical controls.
- The results of the positive control experiments should meet the criteria for a positive response.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
A weak increase in the number of cells with specific chromosomal aberrations (mainly due to chromatid breaks) was registered only in the second experiment (3 hours treatment, 15 hours recovery) performed with microsomal activation. However, these changes observed are within the range of spontaneous aberrations occasionally seen in control cultures (historical control data).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The highest concentration of 600.0 µg/ml available for analysis in the first experiment of the original study caused 69.1% suppression of mitotic activity. The highest concentration of 600.0 µg/ml available for analysis in the second experiment of the original study caused 17.1% suppression of mitotic activity. In the third experiment of the confirmatory study with a 42 hours treatment period the concentration'of 600.0 µg/ml available for analysis caused 52.8% suppression of mitotic activity. In the fourth experiment (3 hours treatment / 39 hours recovery) the concentration of 600.0 µg/ml available for analysis caused 14.4% suppression of mitotic activity.

EXPERIMENTAL RESULT

Experiment Metabolic Activation Treatment Treatment time (h) metaphases with specific aberrations (%) metaphases with unspecific aberrations (%)
Original Study Experiment 1 - Vehicle Control 18h 0.5 2
150 µg/ml 1 0.5
300 µg/ml 0.5 0.5
600 µg/ml 0.5 3.5
Positive control 20 16
Original Study Experiment 2 + Vehicle Control 3h (+15h recovery) 1 2
150 µg/ml 1 1
300 µg/ml 1 0
600 µg/ml 2 3
Positive control 27 8
Confirmatory Study Experiment 1 - Vehicle Control 18h 2 6.5
150 µg/ml 2 5
300 µg/ml 1.5 4
600 µg/ml 1.5 2
Positive control 22 22
Confirmatory Study Experiment 2 + Vehicle Control 3h (+15h recovery) 2.5 2
150 µg/ml 7 3
300 µg/ml 5.5 3
600 µg/ml 2 5.5
Positive control 24 6
Confirmatory Study Experiment 3 - Vehicle Control 42h 2.5 4
150 µg/ml 4 4
300 µg/ml 4 2.5
600 µg/ml 0.5 2
Confirmatory Study Experiment 4 + Vehicle Control 3h (+39h recovery) 2 4.5
150 µg/ml 2.5 3.5
300 µg/ml 1.5 4
600 µg/ml 3.5 6.5
Conclusions:
It is concluded that under the given experimental conditions no evidence of clastogenic effects was obtained in Chinese hamster ovary cells in vitro treated with the test substance.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 Jul 2018 - 23 Aug 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:
hgprt
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
For cell lines:
- Absence of Mycoplasma contamination: yes
- Cell cycle length, doubling time or proliferation index : doubling time of about 12 - 16 hours
- Modal number of chromosomes: 20

MEDIA USED
Ham's F12 medium containing stable glutamine and hypoxanthine (PAN Biotech; Cat. No. P04-15500) supplemented with 10% (v/v) fetal calf serum (FCS).
All media were supplemented with:
- 1% (v/v) penicillin/streptomycin (stock solution: 10000 IU / 10000 μg/mL)
- 1% (v/v) amphotericine B (stock solution: 250 μg/mL)
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system: phenobarbital and β-naphthoflavone induced rat S9 mix
- method of preparation of S9 mix : according to Ames et al
Test concentrations with justification for top dose:
0; 7.50; 10.00; 15.00; 20.00 µg/ml. Higher doeses were not analyzed since precipitation occured already at 20.00 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, acetone was selected as the vehicle, which had been demonstrated to be suitable in the CHO/HPRT assay and for which historical data are available.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9,10-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: one

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 20x10^6 cells in 40 mL
- Test substance added: in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 hours

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 7-9 days
- Selection time (if incubation with a selective agent): 6-7 days
- Fixation time (start of exposure up to fixation or harvest of cells): day 16
- Selective agent: 6-thioguanine (10 μg/mL)

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: cloning efficiency

METHODS FOR MEASUREMENTS OF GENOTOXICIY
The number of colonies in each flask was counted and recorded. The sum of the mutant colony counts within each test group was subsequently normalized per every 10^6 cells seeded. The uncorrected mutant frequency was corrected with the absolute cloning efficiency 2 for each test group to get the corrected mutant frequency (MFcorr.)
Evaluation criteria:
A test substance is considered to be clearly positive if all following criteria are met:
• A statistically significant increase in mutant frequencies is obtained.
• A dose-related increase in mutant frequencies is observed.
• The corrected mutation frequencies (MFcorr.) exceeds both the concurrent vehicle control value and the range of our laboratory’s historical negative control data (95% control limit)
Statistics:
An appropriate statistical trend test (MS EXCEL function RGP) was performed to assess a possible dose-related increase of mutant frequencies. The used model is one of the proposed models of the International Workshop on Genotoxicity Test procedures Workgroup Report. The dependent variable was the corrected mutant frequency and the independent variable was the concentration. The trend was judged as statistically significant whenever the one-sided p-value (probability value) was below 0.05 and the slope was greater than 0. In addition, a pair-wise comparison of each test group with the vehicle control group was carried out using one-sided Fisher's exact test with Bonferroni-Holm correction. The calculation was performed using R. If the results of these tests were statistically significant compared with the respective vehicle control, labels (s p ≤ 0.05) are printed in the tables. However, both, biological and statistical significance are considered together.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: No relevant test substance induced alterations of the pH was detected.
- Data on osmolality: No relevant test substance induced alterations of the osmolality was detected.
- Precipitation and time of the determination: In this study, in the absence and presence of S9 mix, test substance precipitation was observed macroscopically in culture medium at the end of treatment at 20.00 μg/mL and above.
- cell morphology: After 4 hours treatment either in the absence or presence of metabolic activation, the cell morphology and attachment of the cells was not adversely influenced (grade > 2) in any of the groups tested for gene mutations.

RANGE-FINDING/SCREENING STUDIES (if applicable):
Following the requirements of the current international guidelines and the ICPEMC Task Group a test substance should be tested up to a maximum concentration of 2 mg/mL, 2 μL/mL or 10 mM, whichever is the lowest. In case of toxicity, the top dose should result in approximately 10 - 20% relative survival (adjusted cloning efficiency), but not less than 10%. For relatively insoluble test substances at least one concentration should be scored showing no precipitation in culture medium at the end of the exposure period. The pH and osmolality of the test cultures were not significantly altered as compared to the
vehicle control. Precipitation of the test substance in the vehicle acetone was not observed in the stock solution (Test group: 5000.0 μg/mL). In culture medium, test substance precipitation occurred in all
applied concentrations (as low as 19.5 μg/mL) at the end of treatment in the absence and presence of S9 mix. After 4 hours treatment in the absence and presence of S9 mix, no cytotoxicity was observed.

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
There was no decrease in the number of colonies as described by the relative survival in the
presence and absence of S9 mix up to the highest evaluated concentrations for gene mutation.

STUDY RESULTS
In the 1st Experiment in the absence of S9 mix after 4 hours treatment the values for the corrected mutation frequencies (MFcorr.: 0.00 – 4.21 per 10^6 cells) were close to the respective vehicle control value (MFcorr.: 2.83 per 10^6 cells) and clearly within the range of the 95% control limit of our historical negative control data MFcorr.: 0.00 – 6.49 per 106 cells. After 4 hours treatment in the presence of S9 mix the values for the corrected mutation frequencies (MFcorr.: 2.45 – 5.09 per 10^6 cells) were below the respective vehicle control value (MFcorr.: 6.02 per 10^6 cells) and clearly within the range of the 95% control limit of our historical negative control data MFcorr.: 0.00 – 7.43 per 10^6 cells. The positive control substances EMS (without S9 mix; 400 μg/mL) and DMBA (with S9 mix; 1.25 μg/mL) induced a clear increase in mutation frequencies, as expected. The values of the corrected mutant frequencies (without S9 mix: MFcorr.: 288.44 per 10^6 cells; with S9 mix: MFcorr.: 62.06 per 10^6 cells) were within our historical positive control data range (without S9 mix: MFcorr.: 42.47 – 419.90 per 10^6 cells; with S9 mix: MFcorr.: 21.52 – 270.48 per 10^6 cells).

SUMMARY OF RESULTS

            Cytotoxicity***
Exp Exposure
period [h]
Test groups
[μg/mL]
S9
mix
Prec.* Genotoxicity**
MFcorr.
[per 106cells]
RS [%] CE2 [%]
1 4 Vehicle control1 - n.d. 2.83 100 100
0.63 - - n.c.1 101.9 n.c.1
1.25 - - n.c.1 94.2 n.c.1
2.5 - - n.c.1 106.8 n.c.1
5 - - n.c.1 109 n.c.1
7.5 - - 3.76 103 100.3
10 - - 0 102.7 88.1
15 - + 3.48 102.2 90.3
20   + 4.21 103.5 97.2
40 - + n.c.2 n.c.2 n.c.2
Positive control2   n.d. 288.44 85.5 62.6
1 4 Vehicle control1 + n.d. 6.02 100 100
0.63 + - n.c.1 98.5 n.c.1
1.25 + - n.c.1 130.3 n.c.1
2.5 + - n.c.1 109 n.c.1
5 + - n.c.1 107.4 n.c.1
7.5 + - 5.09 93.1 131.4
10 + - 2.45 86.7 109.4
15 + - 2.88 91.4 150.8
20 + + 3.39 103.8 78.9
40 + + n.c.2 n.c.2 n.c.2
Positive control3 + n.d. 62.06 131.9 94.3

* Microscopically or macroscopically visible precipitation in culture medium at the end of exposure period

** Mutant frequency MFcorr.: mutant colonies per 106 cells corrected with the CE2 value

*** Cloning efficiency related to the respective vehicle control

s Mutant frequency statistically significant higher than corresponding control values (p ≤ 0.05)

n.c.1 Culture was not continued since a minimum of only four analysable concentrations is required

n.c.2 Culture was not continued since only one concentration beyond the solubility limit is required

n.d. Not determined

1 Aceton 1% (v/v) 2 EMS 400 μg/mL 3 DMBA 1.25 μg/mL

Conclusions:
Under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.
Executive summary:

The test substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. One experiment with and without the addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation) was carried out. According to strong precipitation (from 19.5 μg/mL onward) of the test substance in culture medium in an initial range-finding cytotoxicity test, the maximum concentration to be used in this study was determined to be 40.0 μg/mL. The following concentrations were tested (test groups printed in bold type were evaluated for gene mutations):

1st Experiment, without S9 mix: 0; 0.63; 1.25; 2.50; 5.00; 7.50; 10.00; 15.00; 20.00; 40.00 μg/mL

1st Experiment, with S9 mix: 0; 0.63; 1.25; 2.50; 5.00; 7.50; 10.00; 15.00; 20.00; 40.00 μg/mL

Following attachment of the cells for 20 - 24 hours, cells were treated with the test substance for 4 hours in the absence and presence of metabolic activation. Subsequently, cells were cultured for 6 - 8 days and then selected in 6-thioguanine-containing medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted. The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, ethyl methanesulfonate (EMS) and 7,12-dimethylbenz[a]-anthracene (DMBA), led to the expected statistically significant increase in the frequencies of forward mutations. The highest tested concentrations in the main experiment showed clear test substance precipitates in culture medium macroscopically at the end of exposure period. No cytotoxicity was observed up to the highest concentrations evaluated for gene mutations. Based on the results of the present study, the test substance did not cause any biologically

relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing. Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

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

Genetic toxicity in vivo

Description of key information

Cytogenicity in vivo: micronucleus test, mouse: negative (GLP, OECD 474, 1990)

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
other: Tif: MAGf (SPF)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: animal farm CIBA-GEIGY
- Age at study initiation: Young adult mice
- Weight at study initiation: Females: 23-30 g; Males: 28-39 g
- Housing: group-caging
- Diet (e.g. ad libitum): standard diet of NAFAG No. 890 ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: at least 3 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-23
- Humidity (%): 50-65
- Photoperiod (hrs dark / hrs light): 12 hours/12 hours
Route of administration:
intraperitoneal
Vehicle:
Arachis oil
Details on exposure:
A solubility test was performed to determine the highest applicable dose level (solution/suspension) of the test substance for the tolerability test (up to a top dose level of 5000 mg/kg body weight). Arachis oil was found to be the best suited vehicle, yielding the highest applicable dose level of 5000 mg/kg.
A tolerability test was performed to determine the maximum tolerated dose level of the test substance, which is the highest causing no death in a group of four animals for the observation period of three consecutive days; that is the interval between administration and sacrifice of the animals in the micronucleus test, plus one day. The tolerability test was performed with three groups of four mice (two females and two males) receiving one single application. One group received the highest applicable dose (5000 mg/kg) and the other two groups received the doses of 1/5 and 1/25 of that amount respectively.
The animals were treated once with the highest tolerated dose of the test substance, 5000 mg/kg, and the appropriate treatment groups were sacrificed 16, 24 and 48 hours thereafter. Subsequently their femoral bone marrow erythrocytes were scored for micronuclei.
Duration of treatment / exposure:
one administration and then sacrificed after a period of 16, 24 and 48 hours
Frequency of treatment:
once
Post exposure period:
16, 24 and 48 hours
Dose / conc.:
5 000 mg/kg bw/day
No. of animals per sex per dose:
Five animals were evaluated out of 8 treated per group
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide (CPA, 64 mg/kg) was administered by oral gavage, at a volume of 20 ml/kg.
Tissues and cell types examined:
bone marrow: micronuclei in polychromatic erythrocytes
Details of tissue and slide preparation:
Preparation of bone marrow and preparation of the slides:
The animals were sacrificed by dislocation of the cervical vertebrae. Bone marrow was harvested from the shafts of both femurs with fetal calf serum and prepared on slides. After air-drying, the slides were stained with May-Gruenwald/Giemsa solution and mounted.
Evaluation criteria:
Criteria for scoring micronuclei:
Micronuclei are uniform, darkly stained, more or less round bodies in the cytoplasm of PCEs. Inclusions in PCEs which are reflective, improperly shaped or stained, or which are not in the focal plain of the cell are judged to be artifacts and are not scored as micronuclei. Cells containing more than one micronucleus are only counted once.
Prior to analysis the slides were coded. The slides of five animals/ sex/dose, showing good differentiation between mature and polychromatic erythrocytes, were selected for scoring. From each animal 1000 polychromatic erythrocytes were scored for micronuclei. The ratio of polychromatic to normochromatic erythrocytes was determined for each animal by counting a total of 1000 erythrocytes.

Assay evaluation criteria:
A test substance is considered to be positive in this test system if a statistically significant increase in the number of micronuclei in polychromatic erythrocytes occurs in comparison with the negative control at any dose and sampling time respectively. If ecjuivocal results are obtained, the final decision has to be based on scientific judgement.

Assay acceptance criteria:
1. The result of the experiments should not be influenced by a significant technical error or a recognized artifact.
2. The high dose of the test substance applied should be the maximum tolerated dose causing no death in a group of four animals in the range finding-test. In case of missing toxicity the high dose should be up to a maximum of 5000 mg/kg body weight or the highest applicable dose due to the limited solubility of the test substance.
3. The (juality of the slides must allow a clear differentiation between polychromatic and normochromatic erythrocytes.
4. At least five female and five male animals per dose and control group should be evaluated.
5. The positive control should fulfil the criteria for a positive substance.
Statistics:
The significance of differences was assessed by the Chi-Square-test (p < 0.05).
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
In the first step of the tolerability test the maximum dose of TK 13 282 (CGL 123) (5000 mg/kg) and the doses of 1000 and 200 mg/kg caused no death in a group of four animals.
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
The tolerability test was performed with three groups of four mice (two females and two males) receiving one single application. One group received the highest applicable dose (5000 mg/kg) and the other two groups received the doses of 1/5 and 1/25 of that amount respectively.
- Solubility: Arachis oil was found to be the best suited vehicle, yielding the highest applicable dose level of 5000 mg/kg.

EXPERIMENTAL RESULTS

Number of polychromatic erythrocytes with micronuclei and ratio of polychromatic to normochromatic (p/n) erythrocytes, arithmetic mean per sex and group.
Sacrifice Dose Sex polychromatic erythrocytes normochromatic
erythrocytes
ratio of
p / n erythrocytes
number of
polychromatic
erythrocytes
with micronuclei
% of
polychromatic
erythrocytes
with micronuclei
16h Control (Arachis oil) f 524 476 1.1 0.6 0.06
m 498 502 1 0.2 0.02
Test substance 5000 mg/kg f 541 459 1.2 1 0.1
m 505 495 1 0.4 0.04
24h Control (Arachis oil) f 514 486 1.1 1 0.1
m 504 496 1 0.6 0.06
Test substance 5000 mg/kg f 519 481 1.1 0.4 0.04
m 525 475 1.1 0.4 0.04
48h Control (Arachis oil) f 517 483 1.1 0.8 0.08
m 481 519 0.9 0.4 0.04
Test substance 5000 mg/kg f 506 494 1 0.4 0.04
m 486 514 0.9 0.4 0.04
CONTROLS
24h Control (Arachis oil) f 514 486 1.1 1 0.1
m 504 496 1 0.6 0.06
Cyclophosphamid (64 mg/kg) f 512 488 1 16.6 1.66
m 470 530 0.9 12.8 1.28
Conclusions:
Under the given experimental conditions no evidence for clastogenic or aneugenic effects was obtained in mice treated with the test item.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Gene mutation in bacteria:

In a GLP conform study according to OECD guideline 471, the substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 98 and Escherichia coli WP2 uvrA. The investigations were performed without and with (Aroclor 1254-induced rat liver S-9 mix) microsomal activation with the following concentrations of the trial substance: 313, 625, 1250, 2500 and 5000 µg/plate. In order to confirm the results, the experiments were repeated. In the experiments performed without and with microsomal activation, none of the tested concentrations of the test substance led to an increase in the incidence of both histidine- or tryptophan-prototrophic mutants by comparison with the negative control. No evidence of the induction of point mutations by the test substance or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium and E. coli used in these experiments

 According to the results of the present study, the test substance is not a mutagenic agent in the Salmonella typhimurium/ Escherichia coli reverse mutation assay under the experimental conditions chosen here.

Cytogenicity in mammalian cells:

In a GLP conform study according to OECD guideline 473, the substance was assessed in CHO-K1 cells in vitro for possible clastogenic activity both in the presence and in the absence of a metabolizing system. In the original study, the concentrations of 150.0, 300.0 and 600.0 µg/ml were selected for chromosome analysis from the first experiment (18 hours treatment without activation) and from the second experiment (3 hours treatment with activation and 15 hours recovery time) each. In the first and the second experiment of the confirmatory study, the same concentrations as mentioned above in the original study were selected for chromosome analysis. The concentrations of 150.0, 300.0 and 600.0 /ig/ml were selected for chromosome analysis in the third experiment (42 hours treatment without activation) and in the fourth experiment (3 hours treatment with activation and 39 hours recovery time) each. Two hundred metaphases were examined from the vehicle control and from the cultures treated with the various concentrations of the test substance. At least fifty metaphases each from the appropriate positive controls were analyzed. In the original study, in the first experiment (18 hours treatment, 0 hours recovery) performed without microsomal activation and in the second experiment (3 hours treatment, 15 hours recovery) performed with microsomal activation, the number of cells with specific chromosomal aberrations in the treatment groups showed no marked difference in comparison with the negative control. In the confirmatory study, in the first experiment (18 hours treatment, 0 hours recovery) performed without microsomal activation, in the third experiment (42 hours treatment, 0 hours recovery) performed without microsomal activation as well as in the fourth experiment (3 hours treatment, 39 hours recovery) performed with microsomal activation, the number of cells with specific chromosomal aberrations in all treatment groups showed no marked increase in comparison with the negative controls. In the second experiment (3 hours treatment, 15 hours recovery) performed with microsomal activation, a weak increase in the number of cells with specific chromosomal aberrations (mainly due to chromatid breaks) was registered. However, these changes observed are within the range of spontaneous aberrations occasionally seen in control cultures (historical control data).

It is concluded that under the given experimental conditions no evidence of clastogenic effects was obtained in Chinese hamster ovary cells in vitro treated with the test substance.

Mammalian cell gene muation assay:

In a GLP-cpmpliant HPRT assay following OECD guideline 476, the test article did not lead to a biologically relevant or dose-dependent increase in the number of mutant colonies, either without S9 mix or after the addition of a metabolizing system. The mutant frequencies at any concentration were within the 95% control limit of our historical negative control data. Additionally, no statistically significant dose-dependent increase in mutant colonies was observed after 4 hours treatment either in the absence or presence of metabolic activation. The mutation frequencies of the vehicle control groups were within our historical negative control data range (95% control limit) and, thus, fulfilled the acceptance criteria of this study. The proficiency of the laboratory to perform the HPRT assay in CHO cells was demonstrated by the laboratory’s historical control database on vehicle and positive controls and by X-bar chart to identify the variability of the vehicle control data. The increase in the frequencies of mutant colonies induced by the positive control substances EMS and DMBA clearly demonstrated the sensitivity of the test method and/or of the metabolic activity of the S9 mix employed. The values were within the range of the historical positive control data and, thus, fulfilled the acceptance criteria of this study. Thus, in the absence and the presence of metabolic activation, the test item is not a

mutagenic substance in the HPRT locus assay using CHO cells under the experimental conditions chosen.

Cytogenicity in vivo:

A micronucleus assay according to OECD guideline 474 and GLP requirements was performed with the test substanece. This test is performed to detect both chromosome breaking substances (clastogens) and aneuploidy inducing substances (aneugens). These effects are manifested by the formation of micronuclei in polychromatic erythrocytes (PCEs) from bone marrow cells in vivo. The animals were treated once with the highest tolerated dose of the test substance, 5000 mg/kg (as determined in the tolerability test), and the appropriate treatment groups were sacrificed 16, 24 and 48 hours thereafter. Subsequently their femoral bone marrow erythrocytes were scored for micronuclei. The evaluation of the bone marrow smears showed no statistically significant increase in the number of micronucleated polychromatic erythrocytes in comparison with the negative control animals at the respective sampling times. It is concluded that under the given experimental conditions no evidence for clastogenic or aneugenic effects was obtained in mice treated with the test substance.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is not considered to be classified as genotoxic under Regulation (EC) No 1272/2008.