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Genetic toxicity in vitro

Description of key information

In the preliminary toxicity test performed as part of the first experiment (plate incorporation assay) inhibition of growth of all the Salmonella typhimurium strains but not of Escherichia coliwas observed out of the 7 concentrations tested (5, 15, 50, 150, 500, 1500, 5000 µg/plate, spaced approximately half-log). A second experiment was therefore repeated on Salmonella typhimurium strains at lower concentrations (0.05,0.5, 1.5, 5 µg/plate). A third experiment concluded the study, using both the Salmonella typhimurium strains and Escherichia coli, following the pre-incubation method with metabolic activation and the plate test method without metabolic activation (concentrations of 0.15, 0.5, 1.5, 5 µg/plate).

In a preliminary cytotoxicity test IR5878 was tested at the dosage levels of 25, 50, 100, 250, 500, 1000, 2500 µg/mL (highest technically applicable concentration on the basis of IR5878 solubility in DMSO and maximum 1% solvent in the incubation mixture), with and without S9-mix, 3-hour pulse treatment and harvest at the 72ndhour (1.5 cell cycle lengths from the beginning of treatment). In a second test IR5878 was tested at the dosage levels of 25, 50, 100, 250, 500, 1000, 2500 µg/mL (highest technically applicable concentration), with a 24-hour continuous treatment in absence of metabolic activation and harvest at the 72ndhour (1.5 cell cycle lengths from the beginning of treatment). Finally, in a third test IR5878 was tested at the dosage level of 1000 µg/mL, with and without S9-mix, 3-hour pulse treatment but with a later harvesting time (i.e. at the 96thhour).

IR5878 up to 1000 μg/mL showed no evidence of either clastogenic or polyploidy-inducing activity in cultured human lymphocytes either in presence or absence of metabolic activation.

In a preliminary test IR5878 dissolved in culture medium at dosages of 50, 100, 250, 500, 1250 and 2500 µg/mL (highest technically applicable concentration on the basis of IR5878 solubility in DMSO and maximum 1% solvent in the incubation mixture), with and without S9-mix was tested with a 3-hour exposure. On the basis of the results of the preliminary test, 1000 µg/mL was chosen as the highest dose to be tested in the main experiments, both in the absence and in the presence of metabolic activation. Three additional doses (30, 100, 300 µg/mL) were also tested.

IR5878 was found to be not-mutagenic up to 1000 μg/mL in absence or in presence of metabolic activation when tested in Mouse Lymphoma L5178Y cells.

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:
1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Commission Directive 92/69/EEC (July 31, 1992) Council Directive 67/548/EEC, Method B.13 and B.14 MAFF 59 Nohsan No. 4200 (January 28, 1985)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial forward mutation assay
Specific details on test material used for the study:
Test material:
IR5878
Batch number: FCF/T/159-99 (ex 20274/71)
Purity: 93.72 ± 1.05 %
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Test concentrations with justification for top dose:
In the preliminary toxicity test performed as part of the first experiment (plate incorporation assay) inhibition of growth of all the Salmonella typhimurium strains but not of Escherichia coli was observed at 6 out of the 7 concentrations tested (5, 15, 50, 150, 500, 1500, 5000 µg/plate, spaced approximately half-log).
A second experiment was therefore repeated on Salmonella typhimurium strains at lower concentrations (0.05, 0.15, 0.5, 1.5, 5 µg/plate).
A third experiment concluded the study, using both the Salmonella typhimurium strains and Escherichia coli, following the pre-incubation method with metabolic activation and the plate test method without metabolic activation (concentrations of 0.05, 0.15, 0.5, 1.5, 5 µg/plate).
Vehicle / solvent:
Negative control (vehicle): dimethyl sulfoxide
Untreated negative controls:
yes
Positive controls:
yes
Positive control substance:
other:
Details on test system and experimental conditions:
Test material:
IR5878
Batch number: FCF/T/159-99 (ex 20274/71)
Purity: 93.72 ± 1.05 %

Test systems:
Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100
Escherichia coli strain WP2 uvrA

Control articles:
Negative control (vehicle): dimethyl sulfoxide
Positive controls in the test without metabolic activation:
Salmonella typhimurium strains TA 1535: hydrazine sulfate
Salmonella typhimurium strains TA 1537: 9-aminoacridine HCl monohydrate
Salmonella typhimurium strains TA 98 and TA 100: doxorubicine HCl
Escherichia coli strain WP2 uvrA: methyl methane-sulfonate
Positive controls in the test with metabolic activation:
Salmonella typhimurium strains TA 1535 and TA 1537: 2-aminoanthracene
Salmonella typhimurium strains TA 98 and TA 100: 2-aminofluorene
Escherichia coli strain WP2 uvrA-: 2-aminoanthracene

Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
positive
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:
positive
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:
positive
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:
positive
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In the preliminary toxicity test performed as part of the first experiment (plate incorporation assay) inhibition of growth of all the Salmonella typhimurium strains but not of Escherichia coli was observed at 6 out of the 7 concentrations tested (5, 15, 50, 150, 500, 1500, 5000 µg/plate, spaced approximately half-log). A second experiment was therefore repeated on Salmonella typhimurium strains at lower concentrations (0.05, 0.15, 0.5, 1.5, 5 µg/plate). A third experiment concluded the study, using both the Salmonella typhimurium strains and Escherichia coli, following the pre-incubation method with metabolic activation and the plate test method without metabolic activation (concentrations of 0.05, 0.15, 0.5, 1.5, 5 µg/plate).
Remarks on result:
mutagenic potential (based on QSAR/QSPR prediction)

In the preliminary toxicity test performed as part of the first experiment (plate incorporation assay) inhibition of growth of all theSalmonella typhimuriumstrains but not ofEscherichia coliwas observedout of the 7 concentrations tested (5, 15, 50, 150, 500, 1500, 5000 µg/plate, spaced approximately half-log). A second experiment was therefore repeated onSalmonella typhimuriumstrains at lower concentrations (0.05,0.5, 1.5, 5 µg/plate). A third experiment concluded the study, using both theSalmonella typhimuriumstrains andEscherichia coli, following the pre-incubation method with metabolic activation and the plate test method without metabolic activation (concentrations of0.15, 0.5, 1.5, 5 µg/plate).

Conclusions:
IR5878 was not mutagenic either in absence or in presence of metabolic activation, up to the concentration of 5 μg/plate when tested on Salmonella typhimurium TA 1535, TA 1537, TA 98, and TA 100 or up to the concentration of 5000 μg/plate when tested on Escherichia coli WP2 uvrA- strain according to Ames.

Executive summary:

In the preliminary toxicity test performed as part of the first experiment (plate incorporation assay) inhibition of growth of all the Salmonella typhimurium strains but not of Escherichia coli was observedout of the 7 concentrations tested (5, 15, 50, 150, 500, 1500, 5000 µg/plate, spaced approximately half-log). A second experiment was therefore repeated on Salmonella typhimurium strains at lower concentrations (0.05,0.5, 1.5, 5 µg/plate). A third experiment concluded the study, using both the Salmonella typhimurium strains and Escherichia coli, following the pre-incubation method with metabolic activation and the plate test method without metabolic activation (concentrations of0.15, 0.5, 1.5, 5 µg/plate).

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Commission Directive 92/69/EEC (July 31, 1992)
Principles of method if other than guideline:
Method:
First test: the preliminary cytotoxicity test was performed as part of the first core study. In this experiment IR5878 was tested at the dosage levels of 25, 50, 100, 250, 500, 1000, 2500 µg/mL (highest technically applicable concentration on the basis of IR5878 solubility in DMSO and maximum 1% solvent in the incubation mixture), with and without S9-mix, 3-hour pulse treatment and harvest at the 72nd hour (1.5 cell cycle lengths from the beginning of treatment). Metaphase analysis was carried out at 250, 500 and 1000 µg/mL.
Second test: IR5878 was tested at the dosage levels of 25, 50, 100, 250, 500, 1000, 2500 µg/mL (highest technically applicable concentration), with a 24-hour continuous treatment in absence of metabolic activation and harvest at the 72nd hour (1.5 cell cycle lengths from the beginning of treatment). Metaphase analysis was carried out at 250, 500 and 1000 µg/mL.
Third test: IR5878 was tested at the dosage level of 1000 µg/mL, with and without S9-mix, 3-hour pulse treatment but with a later harvesting time (i.e. at the 96th hour).
Human lymphocytes were obtained from peripheral blood of healthy male volunteers (1 donor/trial) just before starting of cultures.
Solvent and Positive control were used in each assay. Two slides were prepared from each culture and were examined at 1250 magnification for metaphase analysis (only metaphases containing 46±2 centromers were examined).
The test article did not affect pH and osmolarity of the treatment medium at any of the dosage levels assayed. At the doses of 1000 and 2500 µg/mL precipitation occurred.
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: in vitro gene mutation study in mammalian cells
Specific details on test material used for the study:
Test material:
IR5878
Batch number: FCF/T/168-00 (ex 20525/03/9)
Purity: 98.54 ± 0.51 %
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
Test system: cultured human peripheral lymphocytes
S9 fraction: from liver of Sprague Dawley rats pretreated with phenobarbital and -naphthoflavone
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction: from liver of Sprague Dawley rats pretreated with phenobarbital and -naphthoflavone
Test concentrations with justification for top dose:
First test: the preliminary cytotoxicity test was performed as part of the first core study. In this experiment IR5878 was tested at the dosage levels of 25, 50, 100, 250, 500, 1000, 2500 µg/mL (highest technically applicable concentration on the basis of IR5878 solubility in DMSO and maximum 1% solvent in the incubation mixture), with and without S9-mix, 3-hour pulse treatment and harvest at the 72nd hour (1.5 cell cycle lengths from the beginning of treatment). Metaphase analysis was carried out at 250, 500 and 1000 µg/mL.
Vehicle / solvent:
Negative control (vehicle): dimethyl sulfoxide
Untreated negative controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
Test system: cultured human peripheral lymphocytes

S9 fraction: from liver of Sprague Dawley rats pretreated with phenobarbital and -naphthoflavone

Control articles:
Negative control (vehicle): dimethyl sulfoxide
Positive standard in the absence of S9-mix: mitomycin C
Positive standard in the presence of S9-mix: cyclophosphamide
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:
valid
Positive controls validity:
valid
Additional information on results:
Treatments induced dose-related toxicity, with a reduction in mitotic index of approx. 30 – 50 % at the highest dose tested (1000 µg/mL) in different experiments, both with and without metabolic activation.
At neither sampling time, at none of IR5878 concentrations assayed there was an incidence of cells with chromosome aberrations, excluding gaps, statistically different from the control group, either in the presence or in the absence of metabolic activation.
At none of IR5878 concentrations assayed was observed an incidence of polyploid or endoreduplicated cells different from the control group observed, either in the presence or in the absence of metabolic activation.
As expected, the reference mutagens produced statistically significant increases in the percentage of cells with chromosome aberrations, both including and excluding gaps (p < 0.001).
Conclusions:
IR5878 up to 1000 μg/mL showed no evidence of either clastogenic or polyploidy-inducing activity in cultured human lymphocytes either in presence or absence of metabolic activation.
Executive summary:

In a preliminary cytotoxicity test IR5878 was tested at the dosage levels of 25, 50, 100, 250, 500, 1000, 2500 µg/mL (highest technically applicable concentration on the basis of IR5878 solubility in DMSO and maximum 1% solvent in the incubation mixture), with and without S9-mix, 3-hour pulse treatment and harvest at the 72ndhour (1.5 cell cycle lengths from the beginning of treatment). In a second test IR5878 was tested at the dosage levels of 25, 50, 100, 250, 500, 1000, 2500 µg/mL (highest technically applicable concentration), with a 24-hour continuous treatment in absence of metabolic activation and harvest at the 72ndhour (1.5 cell cycle lengths from the beginning of treatment). Finally, in a third test IR5878 was tested at the dosage level of 1000 µg/mL, with and without S9-mix, 3-hour pulse treatment but with a later harvesting time (i.e. at the 96thhour).

IR5878 up to 1000 μg/mL showed no evidence of either clastogenic or polyploidy-inducing activity in cultured human lymphocytes either in presence or absence of metabolic activation.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Commission Directive 96/54/EEC (July 30, 1996)
Deviations:
no
Principles of method if other than guideline:
Method:
A preliminary test was conducted with a 3-hour exposure in order to evaluate the citotoxicity of IR5878 on L5178Y cells and choose the dosage levels to be tested in the main experiments.
The tested concentrations of IR5878 dissolved in culture medium were of 50, 100, 250, 500, 1250 and 2500 µg/mL (highest technically applicable concentration on the basis of IR5878 solubility in DMSO and maximum 1% solvent in the incubation mixture), with and without S9-mix.
On the basis of the results of the preliminary test, 1000 µg/mL was chosen as the highest dose to be tested in the main experiments, both in the absence and in the presence of metabolic activation. Three additional doses (30, 100, 300 µg/mL) were also tested.
The cells were also exposed to the vehicle control (the solvent DMSO).
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: in vitro gene mutation study in mammalian cells
Specific details on test material used for the study:
Test material:
IR5878
Batch number: FCF/T/168-00 (ex 20525/03/9)
Purity: 98.54 ± 0.51 %
Test concentrations with justification for top dose:
A preliminary test was conducted with a 3-hour exposure in order to evaluate the citotoxicity of IR5878 on L5178Y cells and choose the dosage levels to be tested in the main experiments. The tested concentrations of IR5878 dissolved in culture medium were of 50, 100, 250, 500, 1250 and 2500 µg/mL (highest technically applicable concentration on the basis of IR5878 solubility in DMSO and maximum 1% solvent in the incubation mixture), with and without S9-mix.
Vehicle / solvent:
The cells were also exposed to the vehicle control (the solvent DMSO). Negative control (vehicle): dimethyl sulfoxide
Untreated negative controls:
yes
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
methylmethanesulfonate
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
IR5878 did not affect the pH and osmolality of the treatment medium at any of the concentrations assayed. IR5878 proved to be severely cytotoxic on L5178Y cells at 2500 µg/mL, both in the absence and in the presence of metabolic activation. At 1250 µg/mL the relative survivals in comparison with the solvent control were 25% and 14%, without and with metabolic activation, respectively.
In the two main experiments, at none of IR5878 concentrations assayed, there was an incidence of mutant frequency (M.F.) statistically different from the vehicle control group, either in the presence or in the absence of metabolic activation.
The reference mutagens induced an evident increase in mutant frequency in comparison to the vehicle control.
Conclusions:
IR5878 was found to be not-mutagenic up to 1000 μg/mL in absence or in presence of metabolic activation when tested in Mouse Lymphoma L5178Y cells.
Executive summary:

In a preliminary test IR5878 dissolved in culture medium at dosages of 50, 100, 250, 500, 1250 and 2500 µg/mL (highest technically applicable concentration on the basis of IR5878 solubility in DMSO and maximum 1% solvent in the incubation mixture), with and without S9-mix was tested with a 3-hour exposure. On the basis of the results of the preliminary test, 1000 µg/mL was chosen as the highest dose to be tested in the main experiments, both in the absence and in the presence of metabolic activation. Three additional doses (30, 100, 300 µg/mL) were also tested.

IR5878 was found to be not-mutagenic up to 1000 μg/mL in absence or in presence of metabolic activation when tested in Mouse Lymphoma L5178Y cells.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification