Registration Dossier
Registration Dossier
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EC number: 452-330-3 | CAS number: -
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Ames test, in vitro: negative with and without S9 mix, OECD TG 471, S. typhimurium and E. coli, Callander 2003 and Sokolowski 2016
Chromosome aberration, in vitro: negative with and without S9 mix, OECD TG 473, human lymphocytes, Bohnenberger 2008
Mammalian cell gene mutation, in vitro: negative with and without S9 mix, OECD TG 476, mouse lymphoma L5178Y cells, Wollny 2016
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 11 August 2003 to 21 August 2003
- 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:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: ICH Harmonised Tripartite Guidelines S2A: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals
- Qualifier:
- according to guideline
- Guideline:
- other: ICH Harmonised Tripartite Guidelines S2B: Genotoxicity. A Standard Battery for Genotoxicity Testing of Pharmaceuticals.
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- S. typhimurium: Histidine synthesis
E. coli: Tryptophan synthesis - Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Details on mammalian cell type (if applicable):
- - Type and identity of media: Top agar consisting of 0.6 % w/v agar and 0.5 % w/v sodium chloride in deionised water. Prior to testing, the molten top agar was prepared by adding sterile 0.5 mM histidine/0.5 mM biotin stock solution (10 mL solution: 100 mL) agar).
0.5 mL S9 mix (or S9 buffer) was then added to the number of aliquots of one strain required for one concentration, followed by 0.1 mL of the appropriate concentration of the test substance preparation. Finally 2.0 mL top agar was then added to each aliquot, and the resulting mixture poured onto the surface of a prepared pre-labelled Vogel Bonner plate (9 cm diameter vented Petri-dish prepared with 25 mL Vogel Bonner minimal medium and containing 1.5 % w/v agar and 2 % w/v glucose) and allowed to gel.
- Periodically checked for genotype stability: Yes. The overnight culture from each new frozen culture was screened for the deep-rough characters, DNA repair deficiency and Ampicillin resistance. The presence of the uvrB deletion was confirmed by testing the sensitivity of each culture to mitomycin C (10 µL of a 10 µg/mL solution) in the same manner as sensitivity to crystal violet was tested.
- Periodically "cleansed" against high spontaneous background: Yes. When fresh frozen stocks were prepared, the strains were tested for amino acid requirement. - Additional strain / cell type characteristics:
- not applicable
- Species / strain / cell type:
- E. coli, other: E. coli WP2P uvr A and E. coli WP2P
- Details on mammalian cell type (if applicable):
- - Type and identity of media: Top agar consisting of 0.6 % w/v agar and 0.5 % w/v sodium chloride in deionised water. Prior to testing, the molten top agar was prepared by adding sterile tryptophan solution (10 mL 0.5 mM stock: 100 mL agar).
0.5 mL S9 mix (or S9 buffer) was then added to the number of aliquots of one strain required for one concentration, followed by 0.1 mL of the appropriate concentration of the test substance preparation. Finally 2.0 mL top agar was then added to each aliquot, and the resulting mixture poured onto the surface of a prepared pre-labelled Vogel Bonner plate (9 cm diameter vented Petri-dish prepared with 25 mL Vogel Bonner minimal medium and containing 1.5 % w/v agar and 2 % w/v glucose) and allowed to gel.
- Periodically checked for genotype stability: Yes. The overnight culture from each new frozen culture was screened for the deep-rough characters, DNA repair deficiency and Ampicillin resistance. The presence of the uvrA mutation was confirmed by testing the sensitivity of each culture to mitomycin C (10 µL of a 10 µg/mL solution) in the same manner as sensitivity to crystal violet was tested.
- Periodically "cleansed" against high spontaneous background: Yes. When fresh frozen stocks were prepared, the strains were tested for amino acid requirement. - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9-mix
- Test concentrations with justification for top dose:
- 5000, 2500, 1000, 500, 200 and 100 µg/plate both in the presence and absence of metabolic activation
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: Details on positive control substance assignment is presented in table 1 in the field "Any other information on materials and methods incl. tables"
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Exposure duration: Plates were incubated inverted at 37 °C for three days in the dark.
NUMBER OF REPLICATIONS: Test concentrations were performed in triplicate
- EVALUATION PROCEDURE: Following the total incubation period the plates were examined for the lack of microbial contamination and evidence that the test was valid: i.e. there was a background lawn on the solvent control plates and on the plates for (at least) the lower concentrations of test substance, and that the positive controls had responded as expected. All plates were counted using an automated colony counter (Cardinal® automated counter linked to the Ames Study Manager system [Perceptive Instruments]) adjusted appropriately to permit the optimal counting of mutant colonies. - Evaluation criteria:
- Test data from individual experiments are considered valid if:
a) the concurrent solvent control data are acceptable
b) the positive control data show acceptable increases
Failure of one or more tester strain/S9 combinations does not invalidate the data for the remainder of a concurrent experiment.
A positive response in a (valid) individual experiment is acheived when one or both of the following are met:
a) a significant, dose related increase in the mean number of revertants is observed; or
b) a two-fold or greater increase in the mean number of revertant colonies (over that observed for the concurrent solvent control plates) is observed at one or more concentrations.
A negative result in a (valid) individual experiment is achieved when:
a) there is no significant dose-related increase in the mean number of revertant colonies per plate observed for the test substance; and
b) in the absence of any such dose response, no increase in colony numbers is observed (at any test concentration) which exceeds 2x the concurrent solvent control.
For a positive response in an individual experiment to be considered indicative of an unequivocal positive, i.e. mutagenic, result for that strain/S9 combination, then the observed effect(s) must be consistently reproducible. - Statistics:
- All derived calculations were carried out by the computerised data analysis system.
- Species / strain:
- other: S. typhimurium TA1535, TA1537, TA98 and TA100 and E. coli WP2P uvrA and WP2P
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not determined
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Species / strain:
- other: S. typhimurium TA1537 and TA98
- Metabolic activation:
- with
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- not determined
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Species / strain:
- other: S. typhimurium TA1535 and TA100 and E. coli WP2P uvrA and WP2P
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not determined
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- COMPARISON WITH HISTORICAL CONTROL DATA:
The results of the study were found to be comparable with the historical control. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative without metabolic activation All strains tested
negative with metabolic activation All strains tested except S. typhimurium TA1537 and TA98
positive with metabolic activation S. typhimurium TA1537 and TA98 strains
Under the specific conditions of this assay, the test material gave a negative (i.e. non-mutagenic), response in Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and E. coli strain WP2P and WP2P uvrA in the absence of metabolic activation and in all strains, except for TA1537 and TA98, in the presence of metabolic activation. The test material gave a positive (i.e. mutagenic) response in S. typhimurium strains TA1537 and TA98 in the presence of metabolic activation. The study is considered to be reliable, relevant and adequate for risk assessment and classification and labelling purposes. - Executive summary:
The potential of the test material to cause gene mutation in bacterial strains was determined in accordance with standardised guidelines OECD 471, EU Method B.13/14 and EPA OPPTS 870.1500. Four strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and two Escherichia coli strains (WP2P and WP2PuvrA) were treated in the presence and absence of at rat liver derived metabolic activation system (S9 mix). In two separate assays, the test material did not induce any significant, reproducible increases in the observed number of revertant colonies in any of the tester strains used in the absence of metabolic activation, nor in strains TA1535, TA100, WP2P and WP2PuvrA in the presence of metabolic activation. In both assays, the test material induced reproducible, dose related increases in revertant colony numbers in strains TA1537 and TA98 in the presence of metabolic activation. The test material gave a negative (i.e. non-mutagenic), response in S. typhimurium strains TA1535, TA1537, TA98 and TA100 and E. coli strain WP2P and WP2P uvrA in the absence of metabolic activation and in all strains, except for TA1537 and TA98, in the presence of metabolic activation. The test material gave a positive (i.e. mutagenic) response in S. typhimurium strains TA1537 and TA98 in the presence of metabolic activation.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 15 September 2015 to 28 September 2015
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Version / remarks:
- 1998
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- 2008
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- The S. typhimurium histidine (his) and the E. coli tryptophan (trp) reversion system measures his- → his+ and trp- → trp+ reversions, respectively. The S. typhimurium and Escherichia coli strains are constructed to differentiate between base pair (TA1535, TA100, WP2 uvrA pKM101, and WP2 pKM101) and frameshift (TA1537, TA98) mutations.
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2 uvr A pKM 101
- Species / strain / cell type:
- E. coli, other: WP2 pKM101
- Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbital induced rat liver S9
- Test concentrations with justification for top dose:
- Pre-experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate
In the pre-experiment, the concentration range of the test substance was 3 - 5000 μg/plate. The pre-experiment is reported as experiment I. Since no cytotoxic effects were observed in experiment I, 5000 μg/plate was chosen as the maximal concentration for experiment II. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubilisation properties, its relative nontoxicity to the bacteria. - Untreated negative controls:
- yes
- Remarks:
- untreated
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DSMO
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- Remarks:
- 10 µg/plate (in deionised water) for TA1535 and TA100 without S9
- Positive controls:
- yes
- Positive control substance:
- other: 4-nitro-o-phenylene-diamine
- Remarks:
- 10 µg/plate (in DMSO) for TA98 without S9; 50 µg/plate (in DMSO) for TA1537 without S9
- Positive controls:
- yes
- Positive control substance:
- methylmethanesulfonate
- Remarks:
- 2 µL/plate (in deionised water) for WP2 uvrA (pKM101) and WP2 (pKM101) without S9
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene
- Remarks:
- 2.5 µg/plate in DMSO for TA1535, TA1537, TA98, TA100 with S9; 10 µg/plate (WP2 uvra (pKM101), WP2 (pKM101) with S9
- Details on test system and experimental conditions:
- METHOD OF APPLICATION:in agar (plate incorporation - Expt. I); preincubation - Expt. II
DURATION
- Preincubation period: 60 mins
- Exposure duration: 72 hours
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth - Rationale for test conditions:
- The most widely used assays for detecting gene mutations are those using bacteria. They are relatively simple and rapid to perform, and give reliable data on the ability of an agent to interact with DNA and produce mutations.
Reverse mutation assays determine the frequency with which an agent reverses or suppresses the effect of the forward mutation. The genetic target presented to an agent is therefore small, specific and selective. Several bacterial strains, or a single strain with multiple markers are necessary to assure reliable detection of mutagens that may be specific to one tester strain or locus. The reversion of bacteria from growth-dependence on a particular amino acid to growth in the absence of that amino acid (reversion from auxotrophy to prototrophy) is the most widely used marker. - Evaluation criteria:
- A test substance is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice the colony count of the corresponding solvent control is observed.
A concentration dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A concentration dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls, such an increase is not considered biologically relevant. - Statistics:
- Not applicable.
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A pKM 101
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli, other: pKM101
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- The plates incubated with the test substance showed reduced background growth in experiment I in all strains with S9 mix, except strain WP2 pKM101.
- Conclusions:
- During the described mutagenicity tests and under the experimental conditions reported, the test substance did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. The test substance is considered to be non-mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay.
- Executive summary:
This study was performed to investigate the potential of the test substance dissolved in DMSO to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA1535, TA1537, TA98, and TA100, and the Escherichia coli strains WP2 uvrA pKM101 and WP2 pKM101.
The plates incubated with the test substance showed reduced background growth in experiment I in all strains with S9 mix, except strain WP2 pKM101. No cytotoxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed in all bacterial strains tested. No increase in revertant colony numbers of any of the six tester strains was observed following treatment with the test substance at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test substance did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test substance dissolved in DMSO is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
- 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
- Study period:
- 11 December 2007 to 25 February 2008
- 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:
- EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- lymphocytes: human
- Details on mammalian cell type (if applicable):
- - Blood collection and delivery: Blood samples were obtained from healthy donors not receiving medication. For this study, blood was collected from a female donor (30 years old) for the first experiment and from a 34 year-old female donor for the second experiment. Blood samples were drawn by venous puncture and collected in heparinized tubes. The tubes were sent to the testing facility within 24 hours of collection. Where necessary, blood was stored before use at 4 °C.
- Cell culture initiation: Blood cultures were set up in bulk within 24 hrs after collection in 75 cm² cell culture flasks. The culture medium was DMEM:F12 (Dulbecco's modified eagle medium/ Ham's F12 medium; mixture 1:1) containing 10 % FCS (foetal calf serum). The antibiotic solution contains 10,000 U/mL penicillin and 10,000 μg/mL streptomycin. Additionally, the medium was supplemented with Phytohemagglutinin (PHA, final concentration 3 μg/mL), the anticoagulant heparin (25,000 U.S.P.-U/mL), and HEPES (final concentration 10 mM).
The following volumes are added to the flasks (per 10 mL):
7.70 mL culture medium
1.00 mL fetal calf serum
0.10 mL L-glutamine
0.10 mL antibiotic solution
0.10 mL HEPES
0.05 mL phytohemagglutinin
0.05 mL heparin
0.90 mL whole blood
All incubations were done at 37° C in a humidified atmosphere with 5.5 % CO2 (94.5 % air). - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9-mix
- Test concentrations with justification for top dose:
- Experiment I:
- Without S9 mix 4 hour exposure: 15.3, 26.7, 46.8, 81.8, 143.2, 250.6, 438.5, 767.3, 1342.9 and 2350 µg/mL (approx. 9.5 mM)
- With S9 mix 4 hour exposure: 15.3, 26.7, 46.8, 81.8, 143.2, 250.6, 438.5, 767.3, 1342.9 and 2350 µg/mL (approx. 9.5 mM)
Experiment II:
- Without S9 mix 22 hour exposure: 46.8, 81.8, 143.2, 250.6, 438.5, 767.3, 1342.9 and 2350 µg/mL (approx. 9.5 mM)
- With S9 mix 22 hour exposure: 46.8, 81.8, 143.2, 250.6, 438.5, 767.3, 1342.9 and 2350 µg/mL (approx. 9.5 mM) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- without metabolic activation Migrated to IUCLID6: 4 hour exposure - 880 µg/mL; 22 hour exposure - 660 µg/mL
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- with metabolic activation Migrated to IUCLID6: 37.5 µg/mL
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
- Exposure time 4 hours with or without metabolic activation: About 50-80 hours after seeding for each test group, two blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks. The culture medium was replaced with serum-free medium (for treatment with S9 mix) or complete medium with 10 % FCS (v/v) (for treatment without S9 mix), containing the test material. For the treatment with metabolic activation, 50 μL S9 mix per mL medium were used. Concurrent solvent, and positive controls were performed. After 4 hours the cells were spun down by gentle centrifugation for 5 minutes. The supernatant with the dissolved test item was discarded and the cells were re-suspended in saline G. The washing procedure was repeated once.
Saline G solution (per litre):
NaCl 8000 mg
KCl 400 mg
glucose x H2O 1100 mg
Na2HPO4×7H2O 290 mg
KH2PO4 150 mg
pH was adjusted to 7.2
After washing the cells were re-suspended in complete culture medium and cultured until preparation.
- Exposure time 22 hours without metabolic activation: About 50-80 hours after seeding for each test group, two blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks. The culture medium was replaced with complete medium (with 10 % FCS) containing the test item without S9 mix. The culture medium at continuous treatment was not changed until preparation of the cells. Concurrent solvent and positive controls were performed. All cultures were incubated at 37° C in a humidified atmosphere with 5.5 % CO2 (94.5 % air).
DURATION
- Exposure duration: In Experiment I, the exposure period was 4 hours with and without metabolic activation. In Experiment II, the exposure periods were 4 hours with metabolic activation and 22 hours without metabolic activation.
- Fixation time (start of exposure up to fixation or harvest of cells): The chromosomes were prepared 22 hours after start of treatment with the test material.
SPINDLE INHIBITOR (cytogenetic assays): Three hours before harvesting, colcemid was added to the cultures (final concentration 0.2 μg/mL). The cultures were harvested by centrifugation 22 hrs after beginning of treatment. The supernatant was discarded and the cells were resuspended in approximately 5 mL hypotonic solution (0.0375 M KCl). The cell suspension was then allowed to stand at 37° C for 20 to 25 minutes.
STAIN (for cytogenetic assays): After removal of the hypotonic solution by centrifugation the cells were fixed with a mixture of methanol and glacial acetic acid (3 parts plus 1 part). At least two slides per experimental group were prepared by dropping the cell suspension onto a clean microscope slide. The cells for evaluation of cytogenetic damage were stained with Giemsa or according to the Fluorescent plus Giemsa technique respectively.
NUMBER OF REPLICATIONS: 2 blood cultures were set up in parallel for each concentration
NUMBER OF CELLS EVALUATED: 100 well spread metaphase cells.
The slides were evaluated using NIKON microscopes with 100 x oil immersion objectives. Breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates. 100 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides. Only metaphases with 46 ± 1 centromere regions were included in the analysis.
DETERMINATION OF CYTOTOXICITY
- Method: To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined.
OTHER EXAMINATIONS:
- Determination of polyploidy: The number of polyploid cells in 250 metaphase cells (% polyploid metaphases) was scored. If the chromosome (centromere) number was 46±1 then it is classified as a diploid cell and scored for aberrations. If less than 46±1 chromosomes are counted then the cell is ignored under the assumption that some chromosomes may have been lost for technical reasons. If multiple copies of the haploid chromosome number (other than diploid) are scored then the count is recorded and the cell classified as polyploid.
- Determination of endoreplication: If the chromosomes are arranged in closely opposed pairs, i.e. 4 chromatids instead of 2, the cell is scored as endoreduplicated.
RANGE FINDER:
A preliminary cytotoxicity test was performed to determine the concentrations to be used in the mutagenicity assay. Cytotoxicity was characterized by the percentages of mitotic suppression in comparison to the controls by counting 1000 cells per culture in duplicate. The experimental conditions in this pre-test phase were identical to those required and described below for the mutagenicity assay. The pre-test phase was performed with 10 concentrations of the test item and a solvent and positive control. All cell cultures were set up in duplicate. Exposure time was 4 hours (with and without S9 mix). The preparation interval was 22 hours after start of the exposure. Additional solvent control cultures (with and without S9 mix) were used in the presence of BrdU (5-bromodeoxyuridine; 6 μg/mL). - Evaluation criteria:
- The test material is classified as non-mutagenic if:
− the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of the historical control data of the laboratory (0.0 - 4.0 % aberrant cells, excluding gaps).
− no significant increase of the number of structural chromosome aberrations is observed.
The test material is classified as mutagenic if:
− the number of induced structural chromosome aberrations is not in the range of the historical control data of the laboratory (0.0 - 4.0 % aberrant cells, excluding gaps).
and
− either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.
Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications. The following criteria is valid:
A test item can be classified as aneugenic if:
- the number of induced numerical aberrations is not in the range of our historical control data (0.0 – 0.8 % polyploid cells). - Statistics:
- Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05). However, both biological and statistical significance should be considered together. If the criteria for the test material are not clearly met, the classification with regard to the historical data and the biological relevance is examined and/or a confirmatory experiment would be required.
- Species / strain:
- lymphocytes: human
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No relevant increase in the pH value was observed (e.g. Experiment I, solvent control pH 7.1 versus pH 7.18 at 2350 μg/mL).
- Effects of osmolality: No relevant increase in the osmolarity was observed (e.g. Experiment I, solvent control 329 mOsm versus 389 mOsm at 2350 μg/mL).
- Precipitation: In Experiment I, visible precipitation of the test material in the culture medium was observed at 1342.9 μg/mL and above in the absence and presence of S9 mix. In addition, precipitation occurred in Experiment II in the absence of S9 mix at 767.3 μg/mL and above and in the presence of S9 mix at 1342.9 μg/mL and above.
RANGE-FINDING/SCREENING STUDIES:
The highest concentration used in the pre-test was chosen with regard to the top concentration showing clear toxicity with reduced mitotic indices below 50 % of control, and/or the occurrence of precipitation. With respect to the solubility properties of the test material, 2350 μg/mL was applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations between 15.3 and 2350 μg/mL (with and without metabolic activation) were chosen for the evaluation of cytotoxicity. In the pre-test on toxicity, precipitation of the test material was observed before start of treatment at 1342.9 μg/mL and above in the absence and presence of metabolic activation. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I. Using reduced mitotic indices as an indicator for toxicity in Experiment I, no toxic effects were observed up to the highest applied concentration. Considering the toxicity data of Experiment I, 2350 μg/mL, in the absence and presence of metabolic activation, was chosen as top concentration in Experiment II.
COMPARISON WITH HISTORICAL CONTROL DATA: The study controls were comparable to the historical control data of the laboratory. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results: negative
Under the specific conditions of this assay, the did not induce structural chromosomal aberrations in human lymphocytes in vitro in the absence and presence of metabolic activation. The study is considered to be reliable, relevant and adequate for risk assessment and classification and labelling purposes. - Executive summary:
The potential of the test material to induce structural chromosomal aberrations was determined in a study performed in accordance with standardised guidelines OECD 473, EU Method B.10 and EPA OPPTS 870.5375. In each experimental group two parallel cultures were analysed, in the absence and presence of S9 mix, with 100 metaphase plates were scored for structural chromosomal aberrations per culture. The highest applied concentration in this study (2350 μg/mL) was chosen in line with the requirements of the guidelines followed. Dose selection of the cytogenetic experiments was performed considering the toxicity data and the occurrence of test material precipitation in accordance with the guidelines followed. In the absence and presence of metabolic activation, no cytotoxicity was observed up to the highest applied concentration being far in the range of test material precipitation. In both independent experiments, no biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test material. A dose-dependent increase in chromosomal aberrations was observed in Experiment I, in the presence of metabolic activation, but the values were within the range of the laboratory’s historical control data and considered as being biologically irrelevant. No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test material as compared to the frequencies of the controls. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations. Under the specific conditions of this assay, the test material did not induce structural chromosomal aberrations as determined by the chromosome aberration test in human lymphocytes in vitro. Therefore, the test material is considered to be non-clastogenic in this chromosome aberration test in the absence and presence of metabolic activation.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 26 November 2003 to 04 February 2004
- 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:
- EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- Thymidine kinase
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- - Type and identity of media: The tissue culture medium was prepared under sterile conditions by addition of donor horse serum (50 mL), L-glutamine (5 mL of 200 mM) and penicillin/streptomycin (5 mL of 5000 IU penicillin/mL; 5000 µg streptomycin/mL) to RPMI-1640 culture medium (500 mL, with Hepes). The volume of donor horse serum added was increased to 100 mL whenever the cells were dispensed into microwells and reduced to 25 mL for the treatment period.
- Properly maintained: Yes. A bank of L5178Y TK+/- cells were stored in a liquid nitrogen freezer. Following removal from liquid nitrogen, the cultures were kept at 37 °C under an atmosphere of 5 % CO2 in air in a gassing incubator or in a hot room in roller bottles rotated on a roller apparatus.
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
- Other: A fresh sample of cells was brought up from liquid nitrogen storage for each experiment. A minimum of 10^7 cells in exponential growth were required per treatment, therefore a bulk culture was prepared prior to each experiment and diluted with serum free medium to obtain a reduced serum content of 5 % at treatment time. Each 20 mL treatment culture (5 x 10^5 cells per mL) were taken from this culture. - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9-mix
- Test concentrations with justification for top dose:
- Experiment 1
With S9-mix
200, 500, 1000, 1500, 2000 and 2483 µg/mL (10 mM limit dose) test material and 1 µg/mL of BP as a positive control
DMSO as solvent control
Without S9-mix
125, 250, 500, 1000 and 2483 µg/mL (10 mM limit dose) test material and 500 µg/mL of EMS as a positive control.
DMSO as solvent control
Experiment 2
With S9-mix
200, 500, 1000, 1500, 2000 and 2483 µg/mL (10 mM limit dose) test material and 1 µg/mL of BP as a positive control
DMSO as solvent control
Without S9-mix
200, 500, 1000, 1500, 2000 and 2483 µg/mL (10 mM limit dose) test material and 500 µg/mL of EMS as a positive control.
DMSO as solvent control - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO 10 µL/mL
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- Remarks:
- With metabolic activation Migrated to IUCLID6: (BP) 1 µg/mL
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO 10 µL/mL
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- Without metabolic activation. Migrated to IUCLID6: (EMS) 500 µg/m
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
An individual stock of the test substance was prepared for each experiment in DMSO and dilutions were carried out as required in each case. All test and positive control substance during preparations were prepared as close to the time of culture treatment as possible and were dosed at 10 µL/mL culture.
Two series of exponentially growing suspension cultures of L5178Y cells were treated in duplicate with the solvent control, positive controls or a range of concentrations of the test material for 4 hours in the presence and absence of S9-mix. The cells were then cultured to allow any induced mutations to be expressed. During this expression time the growth rate was monitored and, where appropriate, the cells subcultured daily. At the end of the 48-hour expression time, samples were grown both in selective and non selective medium, and the results obtained used to determine the mutant frequency per viable cell.
DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): The post-treatment cultures were returned to the roller apparatus in the 37 °C hot room for a 48 hour expression period. To maintain exponential growth during the expression time, each culture was counted and, where appropriate, diluted daily to give approximately 2 x 10^5 cells per mL in 50 mL, thereby ensuring approximately 10^7 cells at each subculture.
- Selection time (if incubation with a selection agent): Not reported. For the assessment of mutants, a sample of each of the post-expression cultures was diluted to give 50 mL at 1 x 10^4 cells per mL. TFT was then added to the mutation cultures to give a final concentration of 4 µg/mL. Each TFT treated culture was then dispensed at 200 µL per well into 2 x 96 well microwell plates (2000 cells per well). These plates were then incubated (37 °C, 5 % CO2, 98 % relative humidity) to allow cell growth.
SELECTION AGENT (mutation assays): Trifluorothymidine (TFT).
NUMBER OF REPLICATIONS: Duplicate
DETERMINATION OF CYTOTOXICITY
- Method: Survival was measured by relative total growth (RTG). RTG is a measure of growth of test cultures both during the two-day expression and cloning phases of the assay, relative to the vehicle control.
DETERMINATION OF VIABILITY:
For the assessment of viability, a sample from each mutation culture (at 1 x 10^4/mL) was diluted to give 50 mL at 8 cells per mL. No TFT was added to these cultures. Each viability culture was then dispensed and incubated as for the mutation cultures.
DATA EVALUATION:
Cell growth in individual microwell plates was assessed after 10-13 days using a dissecting microscope. The survival plates and viability plates were scored for the number of wells containing no cell growth (negative wells). The mutation plates were scored so that each well contained either a small colony (considered to be associated with clastogenic effects), a large colony (considered to be associated with gene mutation effects) or no colony.
CALCUALTIONS:
Calculations were based on P(0), the proportion of wells in which a colony had not grown:
P(0) = number of negative wells/total wells plated
Cloning efficiency (CE) = -ln P(0)/number of cells per well
Relative suspension growth (RSG) is defined as the relative total two day suspension growth of the test culture compared to the total two-day suspension growth of the vehicle control. Relative total growth (RTG) is a measure of growth of test cultures both during the two-day expression and cloning phases of the assay, relative to the vehicle control. The RSG of each test culture was multiplied by the relative cloning efficiency of the test culture at the time of mutant selection and expressed relative to the cloning efficiency of the vehicle control. The highest concentration assayed was designed to reduce RTG to 10 %-20 % of the solvent control culture values unless limited by solubility, pH or osmolarity effects or a limit concentration of 5 µL/mL, 5000 µg/mL or 10 mM (whichever is the lowest). No mutation assay plates were quantified if survival fell below 10 %.
MUTANT FREQUENCY (M.F.)
The mutant frequency for each culture was then calculated:
M.F. = CE in selective medium (mutation)/CE in non-selective medium (viability)
The mutant frequency calculations were based on the total mutant colony counts. - Evaluation criteria:
- CRITERIA FOR A POSITIVE RESPONSE
A statistically significant dose-related increase in mutant frequency is required, but not only at concentrations eliciting high levels of toxicity. An associated absolute increase in mutant number above the solvent control values is a further requirement. Such a response must be reproducible in an independent experiment for the test substance to be described as positive in this assay.
CRITERIA FOR A NEGATIVE RESPONSE
A negative response is obtained when there is no reproducible statistically significant dose-related increase in mutant frequency. When reproducible significant increases in mutant frequency are seen only at high levels of toxicity, or when such increases are not accompanied by an increase in absolute numbers of mutants over solvent control values, consideration should be given to such factors as statistical significance of the difference between treated and control cultures, and dose response relationships in order to clarify the response. Failing this, results from an independent experiment should be obtained to clarify the result.
CRITERIA FOR SCORING MUTATION PLATES
Each well of mutation plates was scored as containing either a small colony, a large colony or no colony.
Small colony: an average diameter less than 25 % of the diameter of the well, usually around 15 % of the diameter of the well. A small colony should also have a dense clonal morphology
Large colony: Average diameter greater than 25 % of the diameter of the well. A large colony should also have shown less densely packed cells, especially around the edges of the colony.
Any well which contained more than one small colony was scored as a small colony. Any well which contained more than one large colony was scored as a large colony. Wells containing a combination of large and small colonies was scored as a large colony. An empty well was one which contained no cell growth. - Statistics:
- The data were considered by logit regression, using a complimentary log-log link function. The dependent variable was the number of empty wells. This procedure provided maximum likelihood estimates of log mutant frequencies. Variances were inflated by the between duplicate heterogeneity factor. Intergroup comparisons of log mutant frequency comparing each treated group with the solvent control were performed within each experiment. All tests were one-sided. Similar analyses were carried out separately for the positive controls.
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- the increases observed were small and not reproducible and therefore considered not to be of biological importance
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of osmolality: Treatment of the culture medium with concentrations of the test material used in this study had no significant effect on osmolarity
- Effects of pH:Treatment of the culture medium with concentrations of the test material used in this study had no significant effect on pH
COMPARISON WITH HISTORICAL CONTROL DATA: The results were found to concur with the historical control data included as part of the evaluation.
ADDITIONAL INFORMATION ON CYTOTOXICITY: The maximum concentration of the test material selected for testing was 2483 µg/mL; this concentration is approximately equivalent to 10 mM and as such is the limit concentration for this assay. This concentration resulted in survival levels relative to the solvent control of 16 % and 10 % in the presence of metabolic activation in Experiment 1 and in the absence of metabolic activation in Experiment 2 respectively. The maximum concentration evaluated for mutant frequency in the absence of metabolic activation in Experiment 1 and in the presence of metabolic activation in Experiment 2 was 2000 µg/mL resulting in 10 % and 22 % survival respectively.
The test material was found to be biologically active in the test system causing concentration related reductions in survival down to values of 16 % and 10 % with and without metabolic activation respectively. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative both in the presence and absence of S9-mix
Under the conditions of the assay, the test material was determined not to be mutagenic in L5178Y TK+/- cells treated in vitro in either the presence or absence of metabolic activation. The study is considered to be reliable, relevant and adequate for risk assessment and classification and labelling purposes. - Executive summary:
The potential of the test material to cause gene mutation or clastogenic effects in mammalian cells was determined in accordance with standardised guidelines OECD 476, EU Method B.17 and EPA OPPTS 870.5300. L51788Y TK+/- mouse lymphoma cells were treated in vitro both in the presence and absence of a rat liver derived auxillary metabolic system (S9 mix) in two independent experiments. Large and small mutant colonies were scored for all cultures in each experiment.
The test material was tested up to a maximum concentration of 2483 µg/mL in the presence and absence of metabolic activation. This concentration is approximately equivalent to 10 mM and as such is the limit concentration for this assay. Minimum survival levels, compared to the solvent cultures of 16 % and 10 % were observed in cultures treated with the maximum concentration of the test material in the presence and absence of metabolic activation respectively. No significant increased in mutant frequency were observed in cultures treated with the test material in either the presence or absence of metabolic activation in either of the independent experiments. The positive controls induced appropriate increases in mutant frequency in all mutation experiments thus demonstrating the activity of the S9 mix and that the assay was performing satisfactorily in being capable of detecting known mutagens. Under the conditions of this study, the test material was determined not to be mutagenic in L5178Y TK+/-cells treated in vitro either in the presence or absence of metabolic activation.
Referenceopen allclose all
Table 2: Results with test material
Strain |
Compound |
Dose µg/plate |
Mean revertants/plate |
Treated/solvent |
Individual revertant colony counts |
Mean revertants/plate |
Treated/solvent |
Individual revertant colony counts |
|||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
||||
Experiment 1 |
Experiment 2 |
||||||||||||||
TA 100 |
Test material |
5000 |
95 |
194.7 |
1.1 |
1.5 |
97,97,91 |
219,223,142 |
94 |
259.7 |
1.0 |
1.3 |
88,93,101 |
276,278,225 |
|
2500 |
91.3 |
165 |
1.0 |
1.3 |
84,91,99 |
163,164,168 |
85.7 |
245 |
0.9 |
1.2 |
83,84,90 |
237,273,225 |
|||
1000 |
78.7 |
138.3 |
0.9 |
1.0 |
77,51,108 |
152,135,128 |
95.3 |
214.3 |
1.0 |
1.0 |
72,106,108 |
219,224,200 |
|||
500 |
82 |
124.7 |
0.9 |
0.9 |
86,77,83 |
138,112,124 |
101.3 |
219.7 |
1.1 |
1.1 |
101,114,89 |
201,257,201 |
|||
200 |
103.7 |
125 |
1.2 |
0.9 |
101,110,100 |
110,125,140 |
96.3 |
212.3 |
1.0 |
1.0 |
100,101,88 |
226,219,192 |
|||
100 |
101 |
112 |
1.2 |
0.8 |
95,117,91 |
130,106,100 |
94.3 |
220 |
1.0 |
1.1 |
96,105,82 |
212,235,213 |
|||
DMSO |
87.4 |
131.8 |
91,84,90,83,89 |
136,133,151,133,106 |
95.8 |
207.6 |
97,99,94,101,88 |
184,239,184,229,202 |
|||||||
TA 1535 |
Test material |
5000 |
4.0 |
8.7 |
0.8 |
1.5 |
4,4,M |
10,6,10 |
3.3 |
8.7 |
0.6 |
1.0 |
4,2,4 |
9,11,6 |
|
2500 |
7.0 |
11.3 |
1.3 |
2.0 |
7,4,10 |
10,12,12 |
6.0 |
8.7 |
1.0 |
1.0 |
7,4,7 |
9,12,5 |
|||
1000 |
4.0 |
9.7 |
0.8 |
1.7 |
6,1,5 |
4,23,2 |
5.7 |
7.0 |
1.0 |
0.8 |
7,6,4 |
4,11,6 |
|||
500 |
4.7 |
7.0 |
0.9 |
1.2 |
1,10,3 |
9,5,7 |
5.3 |
5.0 |
0.9 |
0.6 |
2,7,7 |
6,5,4 |
|||
200 |
7.0 |
10.7 |
1.3 |
1.8 |
10,7,4 |
15,11,6 |
6.0 |
8.3 |
1.0 |
1.0 |
10,7,1 |
10,9,6 |
|||
100 |
5.7 |
13.7 |
1.1 |
2.4 |
5,6,6 |
9,5,27 |
4.3 |
9.3 |
0.7 |
1.1 |
5,4,4 |
7,10,11 |
|||
DMSO |
5.2 |
5.8 |
2,5,7,1,11 |
4,5,5,6,9 |
5.8 |
8.4 |
4,4,4,6,11 |
6,9,9,9,9 |
|||||||
TA 1537 |
Test material |
5000 |
8.3 |
27 |
0.8 |
3.5 |
6,12,7 |
21,32,28 |
4.0 |
31.3 |
0.4 |
4.2 |
2,6,4 |
27,29,38 |
|
2500 |
3.3 |
19.7 |
0.3 |
2.5 |
4,4,2 |
18,26,15 |
6.3 |
17.7 |
0.6 |
2.4 |
5,9,5 |
9,17,27 |
|||
1000 |
8.0 |
11.7 |
0.7 |
1.5 |
6,6,12 |
18,13,4 |
8.7 |
9.7 |
0.9 |
1.3 |
9,11,6 |
13,4,12 |
|||
500 |
6.0 |
15 |
0.5 |
1.9 |
7,6,5 |
11,15,19 |
6.3 |
8.0 |
0.6 |
1.1 |
6,6,7 |
7,6,11 |
|||
200 |
8.7 |
9.3 |
0.8 |
1.2 |
13,6,7 |
5,6,17 |
8.7 |
6.7 |
0.9 |
0.9 |
10,1,15 |
11, 5, 4 |
|||
100 |
10.3 |
11.3 |
0.9 |
1.5 |
9,12,10 |
15,10,9 |
8 |
7.0 |
0.8 |
0.9 |
10,9,5 |
10,5,6 |
|||
DMSO |
11 |
7.8 |
18,5,11,10,11 |
7,7,9,9,7 |
10 |
7.4 |
7,11,11,6,15 |
7,7,7,6,10 |
|||||||
TA 98 |
Test material |
5000 |
13.3 |
618.7 |
0.9 |
24.6 |
11,16,13 |
715,609,532 |
24.3 |
635.3 |
1.9 |
27.6 |
17,21,35 |
461,629,819 |
|
2500 |
17.7 |
252.7 |
1.2 |
10 |
19,18,16 |
267,252,239 |
14.7 |
206 |
1.1 |
9.0 |
17,11,16 |
223,134,261 |
|||
1000 |
11 |
75 |
0.8 |
3.0 |
10,11,12 |
84,55,86 |
11.3 |
64.3 |
0.9 |
2.8 |
12,13,9 |
60,65,68 |
|||
500 |
10.7 |
51.7 |
0.7 |
2.1 |
12,9,11 |
44,60,51 |
18.7 |
48.3 |
1.4 |
2.1 |
12,27,17 |
49,47,49 |
|||
200 |
19.7 |
31.7 |
1.3 |
1.3 |
23,17,19 |
38,29,28 |
14.3 |
31.3 |
1.1 |
1.4 |
11,16,16 |
35,21,38 |
|||
100 |
15 |
25.3 |
1.0 |
1.0 |
16,13,16 |
24,26,26 |
17.7 |
28.7 |
1.4 |
1.2 |
26,9,18 |
39,28,19 |
|||
DMSO |
14.6 |
25.2 |
16,17,10,19,11 |
23,17,23,28,35 |
13 |
23 |
10,13,13,12,17 |
28,27,17,15,28 |
|||||||
WP2 (pKM101) |
Test material |
5000 |
53.7 |
55.7 |
0.8 |
0.6 |
39,55,67 |
49,56,62 |
39.7 |
56 |
1.0 |
0.9 |
47,46,26 |
56,57,55 |
|
2500 |
63.3 |
78.7 |
0.9 |
0.9 |
66,73,51 |
66,95,75 |
37.7 |
57.3 |
0.9 |
0.9 |
30,49,34 |
39,86,47 |
|||
1000 |
64.3 |
75 |
0.9 |
0.8 |
72,47,74 |
65,82,78 |
46 |
71.3 |
1.1 |
1.1 |
43,50,45 |
73,79,62 |
|||
500 |
80.7 |
106 |
1.2 |
1.2 |
83,97,62 |
105,116,97 |
37 |
65.3 |
0.9 |
1.0 |
39,29,43 |
65,57,74 |
|||
200 |
104 |
92.3 |
1.5 |
1.0 |
101,116,95 |
89,105,83 |
39.7 |
64.3 |
1.0 |
1.0 |
41,35,43 |
73,62,58 |
|||
100 |
118.3 |
94.3 |
1.7 |
1.0 |
83,154,118 |
99,83,101 |
35.7 |
57 |
0.9 |
0.9 |
39,45,23 |
58,47,66 |
|||
DMSO |
69.6 |
90 |
51,66,82,69,80 |
61,75,114,106,94 |
40.6 |
63.6 |
39,44,41,32,47 |
58,56,82,61,61 |
|||||||
WP2 uvrA (pKM101) |
Test material |
5000 |
158.3 |
160.3 |
1.0 |
0.6 |
141,149,185 |
118,158,205 |
154.7 |
258 |
1.1 |
1.4 |
129,160,175 |
235,263,276 |
|
2500 |
186 |
294 |
1.2 |
1.1 |
169,188,201 |
274,296,312 |
139.7 |
199.3 |
1.0 |
1.1 |
130,145,144 |
202,213,183 |
|||
1000 |
198.7 |
227 |
1.3 |
0.9 |
185,214,197 |
206,223,252 |
147.7 |
202.7 |
1.1 |
1.1 |
139,166,138 |
186,225,197 |
|||
500 |
217 |
270 |
1.4 |
1.0 |
256,218,177 |
242,283,285 |
137.7 |
174.3 |
1.0 |
0.9 |
140,117,156 |
166,183,174 |
|||
200 |
232 |
269 |
1.5 |
1.0 |
233,246,217 |
265,284,258 |
130 |
177 |
0.9 |
1.0 |
101,119,170 |
144,206,181 |
|||
100 |
238.3 |
248.3 |
1.5 |
0.9 |
272,237,206 |
261,216,268 |
138.7 |
185.7 |
1.0 |
1.0 |
140,146,130 |
198,173,186 |
|||
DMSO |
156.8 |
263.4 |
173,149,147,135,180 |
259,219,304,267,268 |
137.8 |
184.2 |
128,125,151,161,124 |
168,157,190,209,197 |
|||||||
M = Plate missing |
|||||||||||||||
In both experiments, in the absence and presence of metabolic activation, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. A dose dependent increase was observed in Experiment I in the presence of metabolic activation (0.0, 1.0, 2.0 % aberrant cells, excluding gaps). The values were within the range of the laboratory’s historical control data (0.0 -4.0 % aberrant cells, excluding gaps) and considered as biologically irrelevant.
In both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (0.0–0.2 %) as compared to the rates of the solvent controls (0.0–0.2 %).
In both experiments, either EMS (880.0 or 660.0 μg/mL) or CPA (37.5 μg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.
The proliferation index of the lymphocytes in solvent control cultures in Experiment I with and without metabolic activation (1.13 and 1.20, respectively), in Experiment II with and without metabolic activation (1.17 and 1.71, respectively), was checked by analysing the proportion of mitotic cells in the 1st, 2nd and 3rd metaphase (M1, M1+, M2 and M3) indicating that the lymphocytes divided about 1.5 times within the early preparation interval. This is also proven by the occurrence of sufficient numbers of mitotic cells and by a clear clastogenicity observed after treatment with the positive control substances.
Table 1: Summary of results
Experiment |
Preparation interval |
Test item concentration (µg/mL) |
Polyploid cells (%) |
Mitotic indices (% of control) |
Aberrant cells (%) |
||
Incl. gaps* |
Excl. gaps* |
With exchanges |
|||||
Exposure period 4 hours without S9 mix |
|||||||
I |
22 hours |
Solvent control1 |
0.2 |
100.0 |
0.5 |
0.0 |
0.0 |
Positive control2 |
0.0 |
76.3 |
9.5 |
8.5S |
3.5 |
||
438.5 |
0.2 |
75.8 |
2.0 |
1.5 |
0.0 |
||
767.3 |
0.2 |
80.5 |
1.5 |
1.0 |
0.0 |
||
1342.9P |
0.2 |
85.8 |
2.5 |
1.5 |
0.0 |
||
Exposure period 22 hours with S9 mix |
|||||||
I |
22 hours |
Solvent control1 |
0.0 |
100.0 |
1.5 |
1.0 |
0.0 |
Positive control3 |
0.0 |
60.9 |
11.0 |
11.0S |
3.0 |
||
250.6 |
0.0 |
100.0 |
1.5 |
1.5 |
0.0 |
||
438.5 |
0.0 |
82.8 |
1.5 |
1.5 |
0.0 |
||
767.3P |
0.0 |
73.8 |
2.0 |
2.0 |
0.0 |
||
Exposure period 4 hours without S9 mix |
|||||||
I |
22 hours |
Solvent control1 |
0.2 |
100.0 |
0.5 |
0.5 |
0.0 |
Positive control4 |
0.0 |
39.0 |
15.0 |
14.5S |
3.5 |
||
438.5 |
0.0 |
112.0 |
0.0 |
0.0 |
0.0 |
||
767.3 |
0.0 |
103.5 |
1.0 |
1.0 |
0.0 |
||
1342.9P |
0.2 |
116.0 |
2.0 |
2.0 |
0.0 |
||
II |
22 hours |
Solvent control1 |
0.2 |
100.0 |
0.5 |
0.5 |
0.0 |
Positive control4 |
0.0 |
42.0 |
9.5 |
9.5S |
0.0 |
||
438.5 |
0.0 |
101.6 |
2.0 |
2.0 |
1.0 |
||
767.3 |
0.2 |
118.7 |
1.0 |
1.0 |
0.0 |
||
1342.9P |
0.0 |
98.2 |
3.0 |
2.5 |
0.0 |
||
*Inclusive cells carrying exchanges
PPrecipitation occurred
SAberration frequency statistically significant higher than corresponding control values
1DMSO 0.5% (v/v)
2EMS 88/0 µg/mL
3EMS 660.0 µg/mL
4CPA 37.5 µg/mL
Mutation data
Statistically significant increases in mutant frequency compared to the solvent control cultures were observed in certain cultures treated with the test material in Experiment 1 in the absence of metabolic activation and in Experiment 2 in the presence of metabolic activation. These increases were all small (less than twice the concurrent background values), not reproducible and therefore considered not to be of biological importance. The positive controls, EMS and BP induced induced appropriate increases in mutant frequency in all mutation experiments, demonstrating the activity of the S9 mix and that the assay was performing satisfactorily in being capable of detecting known mutagens.
Table 1: Summary of results from Experiment 1
Without S9 mix |
With S9 mix |
||||
Concentration (µg/mL) |
Mean % Relative Total Growth |
Mean Mutant Frequency (x 10-4) |
Concentration (µg/mL) |
Mean % Relative Total Growth |
Mean Mutant Frequency (x 10-4) |
Test material |
|||||
2483 |
0 |
b |
2483 |
16 |
2.9 |
2000 |
10 |
1.8 |
1000 |
63 |
2.2 |
1500 |
21 |
2.7* |
500 |
122 |
2.0 |
1000 |
33 |
2.8* |
250 |
137 |
2.7 |
500 |
91 |
2.6* |
125 |
118 |
2.7 |
200 |
84 |
2.6* |
|||
Solvent control |
|||||
DMSO (10 µL/mL) |
100 |
1.7 |
DMSO (10 µL/mL) |
100 |
2.0 |
Positive Control |
|||||
EMS 500 |
3.5 |
11.5** |
BP 1 |
40 |
5.9** |
b = not reported due to excessive toxicity
* = p<0.05
** p<0.01
Table 2: Summary of results from Experiment 2
Without S9 mix |
With S9 mix |
||||
Concentration (µg/mL) |
Mean % Relative Total Growth |
Mean Mutant Frequency (x 10-4) |
Concentration (µg/mL) |
Mean % Relative Total Growth |
Mean Mutant Frequency (x 10-4) |
Test material |
|||||
2483 |
10 |
0.9 |
2483 |
6 |
b |
2000 |
28 |
1.4 |
2000 |
22 |
1.7* |
1500 |
43 |
1.3 |
1500 |
34 |
1.6 |
1000 |
45 |
1.3 |
1000 |
54 |
1.4 |
500 |
117 |
1.0 |
500 |
80 |
1.2 |
200 |
114 |
0.9 |
200 |
90 |
1.0 |
Solvent control |
|||||
DMSO (10 µL/mL) |
100 |
0.9 |
DMSO (10 µL/mL) |
100 |
1.1 |
Positive Control |
|||||
EMS 500 |
45 |
12.4** |
BP 1 |
50 |
9.2** |
b = not reported due to excessive toxicity
* = p<0.05
** p<0.01
Table 3: pH and osmolality data
Treatment |
pH |
Osmolality (mmol/kg) |
Solvent Control 10 µL/mL |
7.21 |
471 |
Test material |
||
125 µg/mL |
7.19 |
426 |
250 µg/mL |
7.19 |
449 |
500 µg/mL |
7.20 |
437 |
1000 µg/mL |
7.18 |
446 |
2483 µg/mL |
7.18 |
412 |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
Micronucleus test, in vivo: negative, OECD TG 474, rat, Dunton 2016
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Remarks:
- The in vivo micronucleus study was conducted solely to comply with a non-EU national registration requirement, and has been provided here in accordance with REACH, Article 22(1)e.
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- Experimental start date: 17 September 2015, Experimental end date: 04 December 2015
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Version / remarks:
- 1997
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian erythrocyte micronucleus test
- Species:
- rat
- Strain:
- other: Crl: WI(Han)
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River (K) Ltd, Margate, Kent, CT9 4LT, United Kingdom
- Age at study initiation: four to five weeks (dose sighting and range finding), five to six weeks (main study)
- Weight at study initiation: 82 to 123 g for males and 77 to 111 g for females (dose sighting and range finding) and 127 to 170 g (males only, main study)
- Assigned to test groups randomly: yes
- Fasting period before study: not specified
- Housing: in groups of up to three, by sex, in grid-floor cages suspended over paper-lined trays
- Diet (ad libitum): pelleted rodent diet, LabDiet 5l0S EURodent Diet, by PMI Nutrition International
- Water (ad libitum): mains tap water
- Acclimation period: 11 days for the dose sighting and range finding phase and five days for the main study
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 22 °C
- Humidity (%): 45 to 70%
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours darkness - Route of administration:
- oral: gavage
- Vehicle:
- The vehicle used in the study was 0.5% w/v carboxymethylcellulose with 0.1% v/v Tween 80, whihc is a common vehicle for oral gavage dosing to rodents.
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
A quantity of test item was weighed into a mortar, wetted with a small quantity of vehicle and made into a smooth paste using a pestle. After further addition of vehicle and mixing, the resultant suspension was transferred into a stoppered measuring cylinder. The mortar was thoroughly rinsed out with vehicle and these rinsings were added to the suspension which was then made up to final volume with vehicle and homogenised. Formulations were dispensed into amber glass bottles for dosing.
Formulations were stirred for a minimum of five minutes before the start and until the completion of dosing, to ensure thorough re-suspension and homogeneity. - Duration of treatment / exposure:
- Animals were dosed twice, depending on the severity of the clinical signs, approximately 24 hours apart, by oral gavage.
- Frequency of treatment:
- Two times, approximately 24 hours apart
- Post exposure period:
- 24 hours after first (all groups) and second dose (groups 1 to 4)
- Dose / conc.:
- 312.5 mg/kg bw/day (nominal)
- Dose / conc.:
- 625 mg/kg bw/day (nominal)
- Dose / conc.:
- 1 250 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- Six animals per dose group, negative and positive control
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- Cyclophosphamide monohydrate (CPA), the positive Control item, was a 3 mg/mL solution of CPA in UHP water.
- Tissues and cell types examined:
- One femur from each animal was exposed by dissection of the surrounding muscle and connective tissues and the shank of the bone removed. The bone marrow cells from each femur were aspirated using a syringe into labelled centrifuge tubes containing foetal bovine serum.
- Details of tissue and slide preparation:
- The bone marrow cells were centrifuged, the supernatant withdrawn, and the cells re-suspended in a minimal volume of foetal bovine serum. One drop of cell suspension was placed on each of two slides and spread by drawing an edge of a clean glass microscope slide along from the drop to the end of the slide. All slides were left to air dry and age overnight before fixing for 5 minutes in methanol. Fixed slides were stained for 20 to 30 minutes in 11.5 % (v/v) Giemsa in Sorensen’s buffer pH 6.0, based on the method of Gollapudi and Kamra
- Evaluation criteria:
- The negative and positive Controls from every experiment were compared with historical ranges of these parameters. The micronucleus test was considered valid because the following criteria were met:
• The incidence of MN-PCE and the PCE/NCE ratio in the negative Control group fell within the historical negative Control range.
• The positive Control item induced a significant increase in the frequency of MN-PCE, and the incidence of MN-PCE and the PCE/NCE ratio in the positive Control group fell within the historical positive Control range.
• At least five animals out of each group were available for analysis.
The test substane was concluded to be neither clastogenic nor aneugenic because the study met the acceptance criteria and no statistically significant increases in the frequency of MN-PCE were seen, relative to negative Controls, in any group given the test substance.
For the test to have been considered positive i.e. the test substance was considered to induce clastogenic/aneugenic damage, the following criteria would have had to be met:
• A statistically significant increase in the frequency of MN-PCE occurred at one or more dose levels.
• The incidence and distribution of MN-PCE in individual animals at the dose level(s) showing statistical significance exceeded Sequani’s historical negative Control data.
• A dose-related increase in the frequency of MN-PCE (where more than two dose levels are analysed) was observed.
Results which only partially satisfy the above criteria would be dealt with on a case-by-case basis. Evidence of a dose-related effect was considered useful but not essential in the evaluation of a positive result.
Biological relevance was taken into account, for example consistency of response within and between dose levels.
A test was considered to be negative if there was neither a dose-response curve nor any group showed statistically significant increases in the frequency of micronucleated PCEs compared to the negative Controls. - Statistics:
- The data were analysed in accordance with the UKEMS guidelines. The data analysed were the proportion of MN-PCE and the ratio of polychromatic to normochromatic cells (PCE/NCE).
The proportion of micronucleated PCEs (with respect to the total number of PCEs counted) is considered to be a measure of chromosomal or cell division apparatus damage. The ratio PCE/NCE is considered to provide an estimate of general toxicity of the test item to bone marrow. In this case testing is concerned with a reduction from the negative Control value. The preferred approach for the MN-PCE data is to combine the data within each group and construct a 2x2 contingency table for each treated group with the negative Control. The groups are then compared using a one tailed Fisher Exact test. However, this approach must first be validated by carrying out a test for between animal heterogeneity using a chi square test. If the heterogeneity test is significant at the 1% level then an exact Wilcoxon Rank Sum test is used instead of the Fisher Exact test. The same method is used to compare the positive and negative Controls.
For the PCE/NCE data, the groups given the test substance were compared with the negative Control using one tailed exact Wilcoxon Rank Sum tests. - Key result
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- The only effect observed was yellow stained bedding 24 hours after the second dosing (low dose) or 24 hours after the first and second dosing (mid and high dose).
- Conclusions:
- There was no evidence of clastogenicity or aneugenicity following oral (gavage) administration of the test substance up to the maximum tolerated dose of 1250 mg/kg/day in male rats, as tested in an in vivo micronucleus assay in accordance with OECD TG 474.
- Executive summary:
The potential of the test substance to produce clastogenicity or aneugenicity following oral administration by gavage in the male rat was studied under GLP in accordance with OECD TG 474. Female and male rats of the Crl:WI(Han) strain, approximately 4 to 5 weeks old and weighing 77 to 111 g (females) and 82 to 123 g (males), were used in a dose sighting and range finder study conducted with doses of 500, 1250 and 2000 mg/kg bw/day to establish the maximum tolerable dose (MTD). The test substance was administered by oral gavage in a volume of 10 mL/kg bw of a suspension, using 0.5% w/v carboxymethylcellulose with 0.1% v/v Tween 80 as the vehicle. The MTD was determined to be 1250 mg/kg bw/day in female and male rats, and the main study was conducted with male rats only. In the main test, thirty male rats, five to six weeks old and weighing between 160 to 210 g, were arbitrarily assigned to one of three treatment groups, a vehicle control group or a positive control group, so that each group consisted of six individual animals.
In the main study, the test substance was administered at doses of 312.5, 625 or 1250 mg/kg bw/day in 10 mL suspension with the vehicle, animals in the negative control group received the vehicle only, and the positive control substance cyclophosphamide monohydrate was dosed at 15 mg/kg bw/day in 3 mL water. Animals were dosed twice, depending on the severity of the clinical signs, about 24 hours apart, while animals in the positive control group were dosed only once. Animals were killed about 24 hours after the final dose and the bone marrow cells were obtained from one femur taken from each animal. Slides of bone marrow cells were prepared, coded and score following the test guidance.
There were no adverse clinical effects following oral exposure to the test substance, the negative or positive controls in the main test. The micronucleus frequency in male rats exposed to the test substance was not statistically significantly increased. There was no statistically significant reduction in the PCE/NCE ratio in male rats treated with the test substance and there was no evidence of clastogenicity or aneugenicity. Since proof of exposure to the bone marrow was demonstrated in the range finding study, this indicated a lack of toxicity of the test substances to the bone marrow. The animals treated with the positive control substance had statistically significant increases in the number of micronucleated cells compared with the concurrent negative control group, which demonstrated that the test system was capable of detecting a known clastogen.
Reference
Table 1: Individual Micronucleus data
Animal number |
Group |
Dose (mg/kg bw/day) |
Total PCE |
NCE/1000 cells |
MN-PCE |
MN-NCE |
PCE/NCE |
1 |
1 |
0 |
2000 |
537 |
3 |
1 |
0.86 |
2 |
1 |
0 |
2000 |
523 |
1 |
0 |
0.91 |
3 |
1 |
0 |
2000 |
450 |
2 |
0 |
1.22 |
4 |
1 |
0 |
2000 |
519 |
3 |
0 |
0.93 |
5 |
1 |
0 |
2000 |
593 |
0 |
0 |
0.69 |
6 |
1 |
0 |
2000 |
620 |
0 |
0 |
0.61 |
7 |
2 |
312.5 |
2000 |
536 |
3 |
0 |
0.87 |
8 |
2 |
312.5 |
2000 |
528 |
0 |
0 |
0.89 |
9 |
2 |
312.5 |
2000 |
579 |
0 |
0 |
0.73 |
10 |
2 |
312.5 |
2000 |
506 |
3 |
0 |
0.98 |
11 |
2 |
312.5 |
2000 |
529 |
2 |
1 |
0.89 |
12 |
2 |
312.5 |
2000 |
640 |
1 |
0 |
0.56 |
13 |
3 |
625 |
2000 |
593 |
1 |
0 |
0.69 |
14 |
3 |
625 |
2000 |
515 |
1 |
0 |
0.94 |
15 |
3 |
625 |
2000 |
497 |
0 |
0 |
1.01 |
16 |
3 |
625 |
2000 |
492 |
0 |
0 |
1.03 |
17 |
3 |
625 |
2000 |
502 |
0 |
1 |
0.99 |
18 |
3 |
625 |
2000 |
518 |
0 |
0 |
0.93 |
19 |
4 |
1250 |
2000 |
476 |
1 |
0 |
1.10 |
20 |
4 |
1250 |
2000 |
559 |
1 |
0 |
0.79 |
21 |
4 |
1250 |
2000 |
473 |
1 |
0 |
1.11 |
22 |
4 |
1250 |
2000 |
611 |
0 |
0 |
0.64 |
23 |
4 |
1250 |
2000 |
604 |
1 |
0 |
0.66 |
24 |
4 |
1250 |
2000 |
605 |
2 |
0 |
0.65 |
25 |
5 |
CPA 15 mg/kg |
2000 |
601 |
21 |
2 |
0.66 |
26 |
5 |
CPA 15 mg/kg |
2000 |
567 |
17 |
0 |
0.76 |
27 |
5 |
CPA 15 mg/kg |
2000 |
560 |
19 |
0 |
0.79 |
28 |
5 |
CPA 15 mg/kg |
2000 |
625 |
23 |
0 |
0.60 |
29 |
5 |
CPA 15 mg/kg |
2000 |
538 |
25 |
0 |
0.86 |
30 |
5 |
CPA 15 mg/kg |
2000 |
649 |
15 |
0 |
0.54 |
Table 2: Historical control data on rats
Male negative control |
|||||||
|
N |
Mean |
SD |
Range (mean ± SD) |
Range (min / max) |
||
PCE |
143 |
2000.0 |
0.1 |
1999.9 |
2000.1 |
2000 |
2001 |
NCE/1000 cells |
143 |
538.0 |
92.1 |
445.8 |
630.1 |
344 |
952 |
MN-PCE/2000 PCE |
143 |
0.9 |
1.0 |
-0.2 |
1.9 |
0 |
6 |
MN-NCE/1000 cells |
143 |
0.1 |
0.3 |
-0.2 |
0.4 |
0 |
2 |
PEC/NCE ratio |
143 |
0.9 |
0.3 |
0.6 |
1.2 |
0.1 |
1.9 |
Male positive control |
|||||||
|
N |
Mean |
SD |
Range (mean ± SD) |
Range (min / max) |
||
PCE |
123 |
2000.0 |
0.5 |
1999.6 |
2000.5 |
2000 |
2005 |
NCE/1000 cells |
123 |
594.2 |
71.6 |
522.6 |
665.7 |
406 |
809 |
MN-PCE/2000 PCE |
123 |
24.6 |
11.2 |
13.4 |
35.8 |
3 |
70 |
MN-NCE/1000 cells |
123 |
0.2 |
0.5 |
-0.3 |
0.7 |
0 |
2 |
PEC/NCE ratio |
123 |
0.7 |
0.2 |
0.5 |
0.9 |
0.2 |
1.5 |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
In vitro data
Gene mutation in mammalian cells:
The potential of the test material to cause gene mutation or clastogenic effects in mammalian cells was determined in accordance with standardised guidelines OECD 476, EU Method B.17 and EPA OPPTS 870.5300. L51788Y TK+/-mouse lymphoma cells were treated in vitro both in the presence and absence of a rat liver derived auxillary metabolic system (S9 mix) in two independent experiments. Large and small mutant colonies were scored for all cultures in each experiment.
The test material was tested up to a maximum concentration of 2483 µg/mL in the presence and absence of metabolic activation. This concentration is approximately equivalent to 10 mM and as such is the limit concentration for this assay. Minimum survival levels, compared to the solvent cultures of 16 % and 10 % were observed in cultures treated with the maximum concentration of the test material in the presence and absence of metabolic activation respectively. No significant increase in mutant frequency were observed in cultures treated with the test material in either the presence or absence of metabolic activation in either of the independent experiments. The positive controls induced appropriate increases in mutant frequency in all mutation experiments thus demonstrating the activity of the S9 mix and that the assay was performing satisfactorily in being capable of detecting known mutagens. Under the conditions of this study, the test material was determined not to be mutagenic in L5178Y TK+/-cells treated in vitro either in the presence or absence of metabolic activation.
Bacterial reverse mutation
The potential of the test material to cause gene mutation in bacterial strains was determined in accordance with standardised guidelines OECD 471, EU method B.13/14 and EPA OPPTS 870.1500. Four strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and two Escherichia coli strains (WP2P and WP2PuvrA) were treated in the presence and absence of a rat liver derived metabolic activation system (S9 mix). In two separate assays, the test material did not induce any significant, reproducible increases in the observed number of revertant colonies in any of the tester strains used in the absence of metabolic activation, nor in strains TA1535, TA100, WP2P and WP2PuvrA in the presence of metabolic activation. In both assays, the test material induced reproducible, dose related increases in revertant colony numbers in strains TA1537 and TA98 in the presence of metabolic activation.
Further bacterial reverse mutagenicity studies were available for assessment:
An Ames test performed on Salmonella typhimurium TA1537 and TA98 with the purest substance tested (test material 2) in the presence of metabolic activation (Calandar, 2003b) gave negative results, thus suggesting that the impurity profile is responsible for the observed specific result.
Chromosome aberration test in vitro
The potential of the test material to induce structural chromosomal aberrations was determined in a study performed in accordance with standardised guidelines OECD 473, EU Method B.10 and EPA OPPTS 870.5375, using human lymphocytes. In each experimental group two parallel cultures were analysed, in the absence and presence of metabolic activation (S9 mix), with 100 metaphase plates scored for structural chromosomal aberrations per culture. The highest applied concentration in this study (2350 μg/mL, approximately 9.5 mM) was chosen in line with the requirements of the guideline indications. Dose selection of the cytogenetic experiments was performed considering the toxicity data and the occurrence of test material precipitation in accordance with the guidelines followed. In the absence and presence of metabolic activation, no cytotoxicity was observed up to the highest applied concentration being far in the range of test material precipitation. In both independent experiments, no biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test material. A dose-dependent increase in chromosomal aberrations was observed in Experiment I, in the presence of metabolic activation, but the values were within the range of the laboratory’s historical control data and considered as being biologically irrelevant. No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test material as compared to the frequencies of the controls. Appropriate mutagens used as positive controls induced statistically significant increases in cells with structural chromosome aberrations thus confirming sensitivity of the test system and that appropriate methods were used. Under the specific conditions of this assay, the test material did not induce structural chromosomal aberrations as determined by the chromosome aberration test in human lymphocytes in vitro. Therefore, the test material is considered to be non-clastogenic in this chromosome aberration test in the absence and presence of metabolic activation.
The available data is considered to be complete and the conclusion, non-mutagenic, was taken forward for risk assessment.
Justification for selection of genetic toxicity endpoint
No selection made as three key in vitro studies are available.
Short description of key information:
IN VITRO DATA
Gene mutation in mammalian cells: Negative, L5178Y TK+/-, OECD 476, EU
Method B.17, EPA OPPTS 870.5300, Clay 2004
Reverse mutation in bacteria: Positive (S. typhimurium strains TA 1537
and TA98 in the presence of S9-mix), OECD 471, EU method B.13/14, EPA
OPPTS 870.5100, ICH S2A & S2B - Callander 2003
In vitro chromosome aberration: Negative, human lymphocytes with and
without metabolic activation, OECD 473, EU method B.10, EPA OPPTS
870.5375 - Bohnenberger 2008
Endpoint Conclusion: No adverse effect observed (negative)
Justification for classification or non-classification
In accordance with criteria for classification as defined in Regulation (EC) No. 1272/2008, Annex I, 3.5.2.3, the test material does not require classification for genetic toxicity based on the overall negative response noted in the available genetic toxicity studies.
In accordance with the criteria for classification set out by Directive 2001/59/EC, Annex 6, 3.2.3, the test material does not require classification for genetic toxicity based on the overall negative response noted in the available genetic toxicity studies.
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