Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Bacterial reverse mutation assay: Verspeek-Rip (2003) tested the substance in the Salmonella typhimurium reverse mutation assay with four histidine requiring strains of Salmonella typhimurium and in Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli WP2uvrA. Based on the results of this K1 study, performed according to OECD guideline 471 and EEC Directive 2000/23/EC, Part B: Methods for the Determination of Toxicity; B.13/14, it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
- Chromosome aberration assay: Buskens (2003) evaluated the substance for its ability to induce structural chromosome aberrations in cultured human lymphocytes. The study, performed according OECD Guideline 473 and EEC Directive 2000/32/EC, Part B, concluded that the substance is not clastogenic in human lymphocytes under the experimental conditions tested.

- Gene mutation study in mammalian cells: The substance was concluded to be negative for the induction of forward mutations at the hypoxanthine-guanine phosphoribosyl transfrase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system, in the in vitro mammalian cell forward gene mutation (CHO/HPRT) assay (Dutta, 2015; OECD 476).

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2003-03-19 - 2003-07-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
(1997)
Deviations:
yes
Remarks:
minor modifications cytogenetic assay as described by Evans, 1984
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
Directive 2000/32/EC, Part B
Deviations:
yes
Remarks:
minor modifications cytogenetic assay as described by Evans, 1984
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
- Name of test material (as cited in study report): XTJ 568
- Physical state: clear colourless liquid
- Analytical purity: 97.8%
- Impurities (identity and concentrations): no data
- Purity test date: no data
- Lot/batch No.: 8191-34
- Expiration date of the lot/batch: 01 January 2004
- Stability under test conditions: no data
- Storage condition of test material: at room temperature in the dark. Stable under storage conditions
- Other: specific gravity: > 0.94 g/cm3
Species / strain / cell type:
lymphocytes: human peripheral
Details on mammalian cell type (if applicable):
- Type and identity of media: F10 complete culture medium consisted of Ham's F10 medium with thymidine and hypoxanthine (Gibco), supplemented with 20% (v/v) heatinactivated (56°C; 30 min) foetal calf serium (Gibco), L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively), sodium bicarbonate (1.2 g/L) and 30 U/mL heparin
- Properly maintained: yes: all incubations were carried out in a humid atmosphere (80-100%) containing 5± 0.5% CO2 in air in the dark at 37 ± 1°C. The temperature, humidity and CO2- percentage were monitored throughout the experiment
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data
Metabolic activation:
with and without
Metabolic activation system:
S9-mix of liver rat induced with Aroclor-1254.
Test concentrations with justification for top dose:
Concentration range in the first main test (with metabolic activation): 333, 666 and 1000 µg/ml (3h exposure, 24h fixation)
Concentration range in the second main test (with metabolic activation): 100, 666 and 1250 µg/ml (3h exposure, 48h fixation)
Concentration range in the first main test (without metabolic activation): 333, 666 and 1000 µg/ml (3h exposure, 24h fixation)
Concentration range in the second main test (without metabolic activation): 100, 500 and 800 µg/ml (24h exposure, 24h fixation)
Concentration range in the second main test (without metabolic activation): 700, 850 and 875 µg/ml (48h exposure, 48h fixation)
Vehicle / solvent:
F10 medium buffered with 20 mM HEPES
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
F10 medium buffered with 20 mM HEPES
True negative controls:
no
Positive controls:
no
Positive control substance:
mitomycin C
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
F10 medium buffered with 20 mM HEPES
True negative controls:
no
Positive controls:
no
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation
Details on test system and experimental conditions:
Experiment 1: With/without S9: 3h exposure and 24h fixation time.
Experiment 2: With S9: 3h exposure and 48h fixation time. Without S9: 24h and 48h exposure time, 24h and 48h fixation time.

SPINDLE INHIBITOR (cytogenetic assays): colchicine

NUMBER OF REPLICATIONS: duplicates
NUMBER OF CELLS EVALUATED: at least 100 metaphase chromosome spreads per culture
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of each culture was determined by counting the number of metaphases per 1000 cells.
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) it induced a dose-related statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations
b) a statistically significant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship

A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, P< 0.05) increase in the number of cells with chromosome aberrations.
Statistics:
The incidence of aberrant cells (cells with one or more chromosome aberrations, inclusive or exclusive gaps) for each exposure group was compared to that of solvent control using Chi-square statistics:
X2=[(N-1) x (ad-bc)^2]/[(a+b) (c+d) (a+c) (b+d)]
b= total number of aberrant cells in control cultures
d = total number of non aberrant cells in control cultures
n0 = total number of cells scored in the control cultures
a = total number of aberrant cells in treated cultures to be compared with the control
c = total number of non aberrant cells in treated cultures to be compared with the control
n1 = the total number of cells scored in the treated cultures
N = sum of n0 and n1

If P [X2> [(N-1) x (ad-bc)^2]/[(a+b) (c+d) (a+c) (b+d)] (two-tailed) is small (P<0.05) the hypothesis that the incidence of cells with chromosome aberrations is the same for both the treated and the solvent control group is rejected and the number of aberrant cells in the test group is considered to be significantly different from the control group at the 95% confidence level.
Key result
Species / strain:
lymphocytes: human peripheral
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:
Observations: In the first cytogenetic assay the test substance increased the number of polyploid cells both in the absence and presence of S9-mix. This may indicate that the test substance has the potential to disturb mitotic processes and cell cycle progression, thereby inducing numerical chromosome aberrations.

The pH and osmolarity of a concentration of 100 µg/mL were 7.73 and 291 mOsm/kg respectively (compared to 7.51 and 290 mOsm/kg in the solvent control). The pH and osmolarity of this concentration was not measured in the first cytogenetic assay.

RANGE-FINDING/SCREENING STUDIES: The pH and osmolarity of a concentration of 5000 µg/mL were 10.01 and 305 mOsm/kg respectively (compared to 7.42 and 284 mOsm/kg in the solvent control). In the dose range finding test blood cultures were treated with 100, 333, 1000, 3330 and 5000 µg XTJ 568/mL culture medium with and without S9-mix.

COMPARISON WITH HISTORICAL CONTROL DATA: The number of cells with chromosome aberrations found in the solvent control culture were within the laboratory historical control data range {min=0, max=6 (mean=1.1, standard deviation: 1.1) aberrant cells per 100 metaphases in the absence of S9-mix; gaps excluded and min=0, max=5 (mean=0.9, standard deviation=1.0) aberrant cells per 100 metaphases in the presence of S9-mix; gaps excluded; for n=660 and 437 respectively}.
Remarks on result:
other: all strains/cell types tested
Conclusions:
The substance is not clastogenic in human lymphocytes under the experimental conditions tested. In the first cytogenetic assay, the substance increased the number of polyploid cells (with and without S9) indicating a potential to disturb mitotic processes and cell cycle progression, thereby inducing numerical chromosome aberrations.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-09-24 to 2015-11-11
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Well documented, GLP compliant report, according to OECD guideline 476. Some deviations were recorded on the washing frequency of pre-treated and post-treated cells, however without impact on the study. Dosing formulation analysis for concentrations and stability was not conducted (but, the solubility test showed that the substance is soluble in water at a conc of 50 mg/mL).
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
Minor deviations on washing protocol
GLP compliance:
yes
Type of assay:
in vitro mammalian cell transformation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report):XTJ-568
- Molecular weight (if other than submission substance): 220 g/mol
- Substance type: Clear colorless liquid
- Physical state: Liquid
- Analytical purity: 100%
- Lot/batch No.: 4H414
- Storage condition of test material: Room temperature, protected from light
- Other: CAS no: 897393-42-9
Target gene:
Hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, assayed by colony growth in the presence of 6-thioguanine (TG resistance).
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: cleansed in medium supplemented with hypoxanthine, aminopterin and thymidine (HAT)
- Properly maintained: no data
- Periodically checked for Mycoplasma contamination: yes, frozen stock cultures were tested to confirm the absence of mycoplasma contamination
- Periodically checked for karyotype stability: yes, frozen stock cultures were tested to confirm the karyotype stability
- Periodically "cleansed" against high spontaneous background: no data
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9 from male Sprague-Dawly rats
Test concentrations with justification for top dose:
Preliminary toxicity assay: 4.30, 8.59, 17.2, 34.4, 68.8, 138, 275, 550, 1100 and 2200 µg/mL
Definitive mutagenicity assay: 68.8, 138, 275, 550, 1100 and 2200 µg/mL
Mutagenicity assay repeat: 68.8, 138, 275, 550, 1100 and 2200 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Water was the vehicle of choice based on solubility of the test substance and compatibility with the target cells.
- Other: Test substance dilutions were prepared immediately before use and delivered to the test system at room temperature under filtered light.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Without metabolic activation, dissolved in DMSO, 0.200 µL/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
With metabolic activation, dissolved in DMSO, 4.00 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 1day
- Exposure duration: 5h (+-0.5h)
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 7 days

SELECTION AGENT (mutation assays): Hypoxanthine-free F12FCM5 (Hx F12FCM5) was used for mutant selection and to determine cloning efficiency
STAIN (for cytogenetic assays): after fixation, colonies were stained with crystal violet

NUMBER OF REPLICATIONS: duplicate cultures (triplicate for initial survival)

NUMBER OF CELLS EVALUATED: For determination of initial survival, the cultures were trypsinized, counted once for cell density, and then plated , in triplicate, at a density of 200 cells/60-mm dish in 5mL F12FCM5. For the mutant selection, 1 x 10^6 cells from each culture were plated at a density of 2 x 10^5 cells/100-mm plate, in quintuplicate, in 10 mL HX-F12FCM5 containing 10 µM TG.

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity was expressed as the adjusted relative survival (%; relative cloning efficiency x relative cell density at the time of cloning, as compared to the concurrent vehicle control)

Evaluation criteria:
Once criteria for a valid assay were met, the responses observed in the assay were evaluated as follows:
- The test substance was considered to have produced a positive response if it induced a statistically significant and dose-dependent increase in mutant frequency (p≤ 0.05) that exceeded the 95% confidence limit of the historical vehicle control data from this laboratory. If only one criterion was met (a statistically significant or dose-dependent increase or an increase exceeding the historical control 95% confidence interval), the results were considered equivocal. If none of these criteria were met, the results were considered to be negative.
- Other criteria also may be used in reaching a conclusion about the study results (e.g., comparison to historical control values, biological significance, etc.). In such cases, the Study Director used sound scientific judgment and clearly reported and described any such considerations.
- Other criteria also may be used in reaching a conclusion about the study results (e.g., comparison to historical control values, biological significance, etc.).
Statistics:
Statistical analyses were performed using the method of Snee and Irr (1981), with significance established at the 0.05 level.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
100.91% (with S9) and 83.67% (without S9) adjusted relative survival at 2200µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH of the cultures was adjusted at concentrations ≥34.4 μg/mL (Preliminary toxicity assay) or at concentrations ≥68.8 μg/mL (Definitive mutagenicity assays) using 1N hydrochloric acid to maintain neutral pH.
- Effects of osmolality: The osmolality of the cultures was acceptable as it did not exceed the osmolality of the vehicle control by more than 120%.
- Evaporation from medium: no data
- Water solubility: the solubility test showed that the substance is soluble in water at a conc of 50 mg/mL
- Precipitation: no visible precipitate was observed at the beginning or end of treatment.

RANGE-FINDING/SCREENING STUDIES:
Preliminary toxicity assay performed: cells were treated with 10 test substance concentrations, as well as the vehicle control, ranging from 4.30 to 2200 µg/mL in the absence and presence of S9 mix using single cultures. The cloning efficiency (%) was calculated for each culture. Based upon the results of the preliminary toxicity assay, the concentrations selected for the definitive mutagenicity assay were 68.8, 138, 275, 550, 1100 and 2200 μg/mL with and without S9.

COMPARISON WITH HISTORICAL CONTROL DATA: The test substance was considered to have produced a positive response if it induced a statistically significant and dose-dependent increase in mutant frequency (p≤ 0.05) that exceeded the 95% confidence limit of the historical vehicle control data from this laboratory.
No significant increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at any concentration evaluated with or without S9 (p > 0.05). The positive controls induced significant increases in mutant frequency (p < 0.01). All positive and vehicle control values were within acceptable ranges, and all criteria for a valid assay were met.

ADDITIONAL INFORMATION ON CYTOTOXICITY: The vehicle and positive control substances have been characterized as per the Certificates of Analysis on file with the Testing Facility. The stability of the vehicle and positive control substances and their mixtures was demonstrated by acceptable results that met the criteria for a valid test.
Remarks on result:
other: all strains/cell types tested
Conclusions:
Under the conditions of the assay described in this report, the substance was concluded to be negative for the induction of forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system, in the in vitro mammalian cell forward gene mutation (CHO/HPRT) assay.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2003-03-04 - 2003-03-13
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:
July 21, 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
Directive 2000/32/EC
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): XTJ 568
- Physical state: clear colourless liquid
- Analytical purity: 97.8% primary amines (% of total amine)
- Impurities (identity and concentrations): no data
- Purity test date: no data
- Lot/batch No.: 8191-34
- Expiration date of the lot/batch: 2004-01-01
- Stability under test conditions: no data
- Storage condition of test material: at room temperature in the dark; stable under storage conditions
- Other: specific gravity: > 0.94 g/cm3
Target gene:
Salmonella typhimurium: histidine
Escherichia coli: tryptophan
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Acrolor-1254 induced rat liver S9-mix.
Test concentrations with justification for top dose:
Concentration range in the main test (with metabolic activation): 100, 333, 1000, 3330 and 5000 µg/plate
Concentration range in the main test (without metabolic activation): 100, 333, 1000, 3330 and 5000 µg/plate
Vehicle / solvent:
Solvent: Milli-Q Water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Milli-Q Water
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Without metabolic activation; strain TA 1535 Migrated to IUCLID6: 5 µg/plate in saline
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Milli-Q Water
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Without metabolic activation; strain TA1537 Migrated to IUCLID6: 60 µg/plate in saline
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Milli-Q Water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: daunomycin, 4 µg/plate in saline
Remarks:
Without metabolic activation; strain TA98
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Milli-Q Water
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Without metabolic activation; strain TA100 Migrated to IUCLID6: 650 µg/plate in DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Milli-Q Water
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Without metabolic activation; strain WP2uvrA Migrated to IUCLID6: 10 µg/plate in DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Milli-Q Water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene in DMSO
Remarks:
With metabolic activation, strain TA1537 (2.5 µg/plate), TA1535, TA98 and TA100 (1 µg/plate) and WP2uvrA (5 µg/plate)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: not applicable (plate incorporation method)
- Exposure duration: 48 hours
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): no data

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: To determine the toxicity of XTJ 568, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined.

Evaluation criteria:
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) It induces a number of revertant colonies, dose related, greater than two-times the number of revertants induced by the solvent control in any of the tester strains, either with or without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment

A test substance is considered positive (mutagenic) in the test if:
a) It induces a number of revertant colonies, dose related, greater than two-times the number of revertants induced by the solvent control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 is considered to be not significant.
b) The positive response should be reproducible in at least one independently repeated experiment.
Statistics:
No statistics
Key result
Species / strain:
other: Salmonella typhimurium: TA1535, TA1537, TA100, TA98; 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:
TEST-SPECIFIC CONFOUNDING FACTORS
Precipitate: The substance did not precipitate in the top agar. Precipitation of the substance on the plates was not observed at the start or at the end of the incubation period in all tester strains.

RANGE-FINDING/SCREENING STUDIES: the substance was tested in the tester strains TA100 and WP2uvrA with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the absence and presence of S9-mix. Precipitate: The test substance did not precipitate in the top agar. Precipitation of the substance on the plates was not observed at the start or at the end of the incubation period in both tester strains. Toxicity: To determine the toxicity of the substance, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined. No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.

COMPARISON WITH HISTORICAL CONTROL DATA: The negative and strain-specific positive control values were within the laboratory background historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY: The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

Number of revertants: All bacterial strains showed negative responses over the entire dose range, i.e. no dose-related, two-fold, increase in the number of revertants in two independently repeated experiments.

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with and without metabolic activation.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

- Mouse bone marrow erythrocytes micronucleus test: Krsmanovic (2011) evaluated the substance for its genotoxic potential (clastogenicity/aneugenicity) as measured by its ability to increase the incidence of micronucleated polychromatic erythrocytes in bone marrow of male and female ICR mice. The study, performed according to OECD Guideline 474 and ICH S2A, ICH S2B, concluded that a single oral administration of the substance at doses up to and including a dose of 500 mg/kg did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow of male or female ICR mice. Therefore, the substance was concluded to be negative in the mouse micronucleus assay.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-08-29 - 2011-10-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH S2A (1996) and ICH S2B (1997)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): XTJ-568
- Substance type: clear colorless liquid
- Physical state: liquid
- Analytical purity: 100%
- Primary amine: 97%
- Lot/batch No.: 0G704
- Storage condition of test material: at room temperature (15 to 30°C), stored in the dark without desiccant
Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan, Frederick, MD
- Age at study initiation: dose range finding study: males and females, 7 weeks 1 or 2 days; definitive micronucleus study: males and females: 7 weeks 3 days
- Weight at study initiation: dose range finding study: male mice: 29.6 - 34.1 grams, female mice: 23.7-26.5 grams; definitive micronucleus study: male mice: 29.6-33.3 grams, female mice: 22.7-28.7 grams
- Assigned to test groups randomly: yes, under following basis: In each study, mice were assigned to the appropriate number of treatment groups using a randomization procedure based on equalization of group mean body weights. At the time of randomization, the weight variation of animals did not exceed ± 20% from their group mean. Following randomization, animals were identified by sequentially numbered ear tags assigned to each animal during randomization process. The cage card contained the following information: the animal number, sex, study number, test article identification, treatment group number, dose level and route of administration. Cage cards were color coded as a function of treatment group. Raw data records and specimens were also identified by the unique test animal number.

In the DRF study, mice were randomly assigned to four groups of five animals/sex, each.

In the definitive micronucleus study, mice were randomized into 8 groups (7+1) of 5 male and 5 female mice each. Animals in five of these groups were designated for 24 hour bone marrow collection, animals in the remaining two groups were designed for 48 hour bone marrow collection and animals in the 8th group were designated for the replacement in the event of mortality at the highest dose. Since mortality was not observed at the high dose, bone marrow was not collected from this group.
- Fasting period before study: no
- Housing: Animals were housed in an AAALAC-accredited facility with a controlled environment
- Diet (e.g. ad libitum): A certified laboratory rodent chow (Harlan 2018C Certified Global Rodent Diet) was provided ad libitum. The food was analyzed by the manufacturer for the concentrations of specified heavy metals, aflatoxin, chlorinated hydrocarbons, organophosphates and specified nutrients
- Water (e.g. ad libitum): Animals were allowed free access to tap water, which meets U.S. EPA drinking water standards (water source is Washington Suburban Sanitary Commission (WSSC) Potomac Plant. Drinking water was monitored at least annually for levels of specified microorganisms, pesticides, heavy metals, alkalinity and halogens
- Acclimation period: Virus antibody-free (VAF) mice were obtained from a supplier that monitored mice for evidence of ectoparasites, endoparasites, pathogenic bacteria, mycoplasmas, and appropriate murine viruses and were acclimatized for 8 or 9 days in the DRF study and for 10 days in the definitive study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): ± 22.2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): The animal rooms were supplied with at least 10 changes of fresh HEPA-filtered air every hour
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: deionized water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: All formulations were administered at a dose volume of 20 mL/kg body weight with the exception of one animal in the DRF study and one animal in the definitive study that were not dosed with the appropriate volumes, as per the records in the raw data.

The test article dose formulations were prepared fresh for each phase of study prior to dose administration. All formulations for the dose range finding study (100, 50, 37.5 and 25 mg/mL) and all formulations for the definitive study (25, 12.5 and 6.25 mg/mL) were prepared as follows:
- An appropriate amount of the test article was weighed into separate containers for each concentration
- Vehicle, ~80% of the final volume was added to the respective containers.
- Each formulation was vortexed briefly and remaining volume of the vehicle was added to achieve the final volume.

All formulations appeared as clear colorless solutions.
Duration of treatment / exposure:
Single exposure
Frequency of treatment:
Single exposure
Post exposure period:
Mice in 5 groups (either positive or vehicle control articles or 125, 250 or 500 mg/kg XTJ-568) were euthanized at 24 hours post-dose. Mice in two remaining groups (either dosed with vehicle control or 500 mg/kg substance) were euthanized at 48 hours post-dose. In addition, 5 animals/sex were dosed with the substance at 500 mg/kg to be used as replacement group, in the event of mortality at the high dose. As no mortality was observed, these animals were euthanized without bone marrow collection.
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
125 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide monohydrate
- Justification for choice of positive control(s): no data
- Route of administration: oral gavage
- Doses / concentrations: 50 mg/kg
Tissues and cell types examined:
Immediately following euthanasia, the femurs were exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing fetal bovine serum.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: A dose range finding study was performed. Five male and five female mice were exposed to XTJ-568 at 2000, 1000, 750 or 500 mg/kg. Mice were observed after dose administration and daily thereafter for 3 days for clinical signs of toxicity. Body weights were recorded before dose administration on Study Day 0 and on Study Days 1 and 3. Following the last observation on Study Day 3, animals were euthanized by exposure to CO2 followed by incision of the diaphragm and discarded without further examination.

DETAILS OF SLIDE PREPARATION: The bone marrow cells were transferred to a labeled centrifuge tube containing approximately 1 mL fetal bovine serum. The bone marrow cells were pelleted by centrifugation at approximately 100 x g for five minutes and the supernatant was drawn off, leaving a small amount of serum with the remaining cell pellet. The cells were re-suspended and a small drop of bone marrow suspension was spread onto a clean glass slide. Two slides were prepared from each mouse. The slides were air dried and fixed in methanol. One set of slides was stained with a nucleic acid-specific stain, acridine orange, and was used in the microscopic evaluation.

METHOD OF ANALYSIS: Scoring for micronuclei (bone marrow evaluation):
To control for bias, bone marrow slides were coded using a random number table by an individual not involved with the scoring process. Using a fluorescent microscope and medium magnification (400X; blue excitation filter in the rang of 440-490 nm and barrier filter combination at 520 nm), an area of acceptable quality was selected such that the cells were well spread and stained. Using oil immersion (1000 X), the following cell populations and cellular components were evaluated and enumerated:
- Polychromatic erythrocytes: PCE stain orange-red. PCEs are young erythrocytes and are the target cells for evaluation of the test article genotoxicity. 2000 PCEs per each animal were screened (scored) for the presence of micronuclei (MPCEs) resulting in evaluation of a total of 10000 PCEs per each treatment group.
- Normochromatic erythrocytes: NCEs appeared light green in color. NCEs are mature erythrocytes (red blood cels) and are the final cell population formed during erythropoiesis. The number of NCEs and micronucleated NCEs (MNCEs) in the field of 1000 total erythrocytes (ECs) is determined for each animal in order to determine the proportion of polychromatic erythrocytes to total erythrocytes (PCEs/ECs ratio).
- Micronuclei: Micronuclei are round, fluorescent green-stained nuclear (chromosome) fragments with a sharp contour and diameters usually from 1/20 to 1/5 of an erythrocyte. Micronuclei may occur in PCEs (MPCEs) or NCEs (MNCEs).
- PCEs/ECs ratio: The proportion of polychromatic erythrocytes to total erythrocytes would also be determined per total of 1000 erythrocytes (PCEs/ECs ratio) for each animal. In the event of high reduction and suppression in PCEs/EC ratio (80% or greater reduction in PCEs/ECs ratio relative to the negative control), it might not be possible to evaluate 2000 PCEs per animal for the presence of micronuclei.
Evaluation criteria:
The incidence of micronucleated polychromatic erythrocytes per 2000 PCEs for each mouse and per 10000 PCEs for each treatment group was determined. 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 mouse and treatment group (PCEs/ECs ratio). The proportion of polychromatic erythrocytes to total erythrocytes in test article animals should not be less than 20% of the control value.
As a guide to interpretation of the data, the following was considered:
- The test article would have been considered to induce a positive response if the incidence of micronucleated polychromatic erythrocytes at one or more doses is statistically elevated relative to the vehicle control (p < or = 0.05, binomial distribution, Kastenbaum-Bowmantables).
- In this study, the test article was judged negative because no statistically significant increase in the incidence of micronucleated polychromatic erythrocytes in the test substance groups relative to the concurrent negative (vehicle groups) is observed.
However, the results of the statistical analysis would not be the only criteria in determination of the test substance potential to increase the incidence of micronucleated PCEs. A dose-dependent increase in the incidence of micronucleated PCEs or biological relevance of generated data would have been taken in consideration during determination of test article positivity. in addition, if criteria for either a positive or negative genotoxic response were not met, the results would have been judged as equivocal.
Statistics:
Statistical significance was determined using the Kastenbaum-Bowman tables which are based on the binomial distribution (Kastenbaum and Bowman, 1970). All analyses were performed separately for each sex and sampling time.

Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
piloerection and lethargy were observed in both male and female mice at 500 mg/kg following dose administration. Piloerection persisted in male mice.
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
All male and female mice in 2000 mg/kg treatment group were found dead following dose administration. Mortality was also observed in 1/5 male and 1/5 females at 1000 mg/kg and in 2/5 male and 1/5 female mice at 750 mg/kg. Convulsions are noted following dose administration at 1000 and 750 mg/kg. Piloerection and lethargy were among the clinical signs observed in the surviving animals at these dose levels. All mice at the dose level 500 mg/kg were convulsing immediately after dosing and exhibited piloerection and lethargy following dose administration. Piloerection persisted on Study Day 1 in some of the mice. No appreciable changes in group mean body weights were observed.

RESULTS OF DEFINITIVE STUDY
Clinical signs:
No mortality was observed in any of the treatment groups. All mice in the control article (vehicle or positive) groups and mice in the test article groups at 125 and 250 mg/kg appeared normal during the study period. Piloerection and lethargy were observed in both male and female mice at 500 mg/kg following dose administration. Piloerection persisted in male mice.

Bone marrow evaluation:
- No appreciable reductions in the ratio of polychromatic erythrocytes to total erythrocytes (PCEs/ECs) in the test article groups relative to the respective vehicle control groups were observed suggesting that the test article did not inhibit erythropoiesis.
- No statistically significant increase in the incidence of micronucleated polychromatic erythrocytes in test article groups relative to the respective vehicle control groups was observed in the male or female mice at 24 or 48 hours after dose administration (p > 0.05, binominal distribution, Kastenbaum-Bowman tables).
- CP, the positive control, induced a statistically significant increase in the incidence of micronucleated PCEs (p < or = 0.05, binominal distribution, Kastenbaum tables) in both male and female mice. The number of micronucleated PCEs in the vehicle control groups did not exceed the historical vehicle control range. Based upon this, all criteria for a valid test were met as specified in the protocol.

Deviation:

The following deviation from the protocol occurred during the conducted of the dose range finding study, which was documented in the raw data with a deviation report:

As per written records, one male mouse in the 2000 mg/kg treatment group in the DRF study was dosed with the test article at a volume of 2 mL/kg instead of 20 mL/kg. As per Study Director's opinion, the volume recorded was a documentation error since the animal was found dead, which was considered to be a result of appropriate and correct administration and exposure. Another male mouse in the vehicle control group received the vehicle at a slightly higher volume of 28.4 mL/kg.

As per Study Director's opinion, these two events did not adversely impact the dose selection for the main study or the outcome of the study.

Conclusions:
Under the conditions of this study a single oral administration of the substance at doses up to and including 500 mg/kg did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow. Therefore, the substance was concluded to be negative in the micronucleus test using male or female ICR mice.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Genetic toxicity in vitro:

Bacterial reverse mutation assay:

The substance was tested in Salmonella typhimurium resverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA 1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli WP2uvrA. The test was performed in two independent experiments in the presence and absence of S9 -mix (Aroclor-1254 induced rat liver S9 -mix).

In the dose range finding test, the substance was tested up to concentrations of 5000 µg/plate in the absence and presence of S9 -mix in the strains TA100 and WP2uvrA. The substance did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. In the first and in the second mutation assay, the substance was tested up to concentrations of 5000 µg/plate in the absence and presence of S9 -mix. The bacterial background lawn was not reduced at any of the concentrations tested and no decrease in the number of revertants was observed. The presence of 5 and 10% (v/v) liver microsomal activation did not influence these findings. The substance did not induce a dose-related, two-fold increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9 -metabolic activation. These results were confirmed in an independently repeated experiment.

Based on the results of this study, it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Chromosome aberration assay:

Buskens (2003) investigated the effect of the substance on the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (Aroclor-1254 induced rat liver S9 -mix). The possible clastogenicity of the substance was tested in two independent experiments. In the first cytogenetic assay, the substance was tested up to 1000 µg/mL for a 3h exposure time with a 24h fixation time in the absence and presence of S9 -mix. Appropriate toxicity was reached at this dose level.

In the second cytogenetic assay, the substance was tested up to 800 µg/mL for a 24h continuous exposure time with a 24 h fixation time and up to 875 µg/mL for a 48 h continuous exposure time with a 48h fixation time in the absence of S9 -mix. In the presence of 1.8% (v/v) S9 -fraction the substance was tested up to 1250 µg/mL for a 3h exposure time with a 48h fixation time. Appropriate toxicity was reached at these dose levels.

Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9 -mix) functioned properly.

The substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and in the presence of S9 -mix, in two independently repeated experiments.

In the first cytogenetic assay the substance increased the number of polyploid cells both in the absence and presence of S9 -mix. This may indicate that the substance has the potential to disturb mitotic processes and cell cycle progression, thereby inducing numerical chromosome aberrations.

Finally, it is concluded that the substance is not clastogenic in human lymphocytes under the experimental conditions of this test.

In vitro gene mutation study in mammalian cells:

Dutta (2015) performed an in vitro gene mutation study in mammalian cells according to OECD guideline 476. The substance was concluded to be negative for the induction of forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system, in the in vitro mammalian cell forward gene mutation (CHO/HPRT) assay.

Genetic toxicity in vivo:

Mouse Bone Marrow Erythrocyte Micronucleus Test Following Oral Administration of XTJ-568:

Krsmanovic (2011) evaluated the substance for its genotoxic potential (clastogenicity/aneugenicity) as measured by its ability to increase the incidence of micronucleated polychromatic erythrocytes in bone marrow of male and female ICR mice. The micronucleus test was conducted in two phases: a dose range finding (DRF) study and a definitive micronucleus study/assay.

In order to evaluated toxicity of the test article and set the dose levels for the definitive micronucleus assay, the dose range finding study (DRF study) was conducted initially exposing five male and five female mice to the substance at 2000 mg/kg. As mortality was observed at this dose, 5 animals/sex were orally exposed to the substance at 1000 mg/kg. Since mortality was also observed at this dose, five animals/sex/group were exposed to the substance at 500 and 750 mg/kg.

Based on the mortality and clinical signs observed in the DRF study, a dose of 500 mg/kg was determined to be the maximum tolerated dose and was used as high dose in the definitive micronucleus study. Two lower doses, one fourth and one half of the highe dose, 125 and 150 mg/kg were also tested.

The definitive study consisted of 7 groups each containing 5 animals/sex. Mice in five of these groups were exposed to either the control articles (vehicle or positive) or to dose levels of 125, 250 or 500 mg/kg the substance and were euthanized at 24 hours post-dose. Mice in the remaining two groups were dosed either with vehicle control or the substance at a dose of 500 mg/kg and were euthanized at 48 hours post-dose. In addition, 5 animals/sex were dosed with the substance at 500 mg/kg to be used as replacement group, in the event of mortality at the high dose. As no mortality was observed, these animals were euthanized without bone marrow collection. Following scheduled euthanasia, bone marrow smears (slides) were prepared and stained with acridine orange stain (nucleic acid specific). Bone marrow cells (polychromatic erythrocytes (2000 PCEs/animal)] were examined microscopically for the presence of micronuclei (micronucleated PCEs; MPCEs). A statistical analysis of data was performed using the Kastenbaum-Bowman tables (binomial distribution, p< or = 0.05).

Under the conditions of the study a single oral administration of the substance at doses up to and including a dose of 500 mg/kg did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow of male or female ICR mice. Therefore, the substance was concluded to be negative in the mouse micronucleus assay.

Justification for classification or non-classification

Based on the results of the above mentioned studies, the substance does not need to be classified for genetic toxicity (germ cell mutagenicity).