Registration Dossier
Registration Dossier
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EC number: 807-621-3 | CAS number: 1428450-95-6
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
In order to determine the genotoxic and mutagenic potential of the testsubastance W630 an Ames test, an in vitro micro nucleus and a mutation assay with mouse lymphoma cells were perfornmed.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- adopted in 1997
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his operon
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9) prepared from the livers of Aroclor 1254-induced rats
- Test concentrations with justification for top dose:
- Experiment 1: 5, 15.81, 50, 158.1, 500, 1581 and 5000 µg/plate with and without metabolic activation
Experiment 2: for strains TA98 and TA1535: 20.48, 51.2, 128, 320, 800 and 2000 µg/plate with and without metabolic activation; for strains TA100, TA102 and TA1537: 8.192, 20.48, 51.2, 128, 320 and 800 µg/plate with and without metabolic activation - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that the test substance was soluble in DMSO at concentrations of at least 100 mg/mL. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- benzo(a)pyrene
- mitomycin C
- other: 2-aminoanthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION:
Experiment 1: in agar (plate incorporation)
Experiment 2: preincubation
DURATION
- Preincubation period: 20 min
- Exposure duration: 72 h
NUMBER OF REPLICATIONS: two separate experiments using triplicate plates with and without metabolic activation
DETERMINATION OF CYTOTOXICITY
- Method: clearing of background lawn - Evaluation criteria:
- The test substance was considered positive in this assay if all of the following criteria were met:
- When assessed using Dunnett's test, an increase in revertant numbers gave a significant response (p ≤ 0.01) which was concentration related.
- The positive trend/effects described above were reproducible. - Statistics:
- Dunnett's test was used to compare the counts at each concentration with the control.
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test substance was observed in both experiments at 2000 µg/plate and above.
COMPARISON WITH HISTORICAL CONTROL DATA: All negative controls were in the range of the historical control data.
ADDITIONAL INFORMATION ON CYTOTOXICITY: Cytotoxic effects of the test substance were observed in all tester strains, starting for TA102 and TA1537 at 320 µg/plate with metabolic activation and 500 µg/plate without metabolic activation, for TA100 at 500 µg/plate with and without metabolic activation and for TA98 and TA1535 at 800 µg/plate with metabolic activation and 1581 µg/plate without metabolic activation. The cytotoxic effects ranged from slight diminution of the back ground lawn to complete extermination of the test bacteria at higher concentrations. - Conclusions:
- It was concluded that W630 (Sample ID: 26480) did not induce mutation in five
histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of
Salmonella typhimurium when tested under the conditions of this study. These
conditions included treatments at concentrations up to 5000 μg/plate (the maximum
recommended concentration according to current regulatory guidelines) or toxic
concentrations, in the absence and in the presence of a rat liver metabolic activation
system (S-9). - Executive summary:
W630 (Sample ID: 26480) was assayed for mutation in five histidine-requiring strains
(TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, both in
the absence and in the presence of metabolic activation by an Aroclor 1254-induced
rat liver post-mitochondrial fraction (S-9), in two separate experiments.
All W630 (Sample ID: 26480) treatments in this study were performed using
formulations prepared in anhydrous analytical grade dimethyl sulphoxide (DMSO).
Experiment 1 treatments of all the tester strains were performed in the absence and in
the presence of S-9, using final concentrations of W630 (Sample ID: 26480) at 5,
15.81, 50, 158.1, 500, 1581 and 5000 μg/plate, plus negative (vehicle) and positive
controls. Following these treatments, evidence of toxicity was observed at 500 and/or
1581 μg/plate and above in all strains in the absence and presence of S-9.
Experiment 2 treatments of all the tester strains were performed in the absence and in
the presence of S-9. For strains TA98 and TA1535, the maximum test concentration
was reduced to 2000 μg/plate and for strains TA100, TA1537 and TA102 the
maximum test concentration was reduced to 800 μg/plate, based on toxicity observed
in Experiment 1. Narrowed concentration intervals were employed covering the
ranges 20.48 – 2000 μg/plate or 8.192 – 800 μg/plate respectively, in order to
examine more closely those concentrations of W630 (Sample ID: 26480) approaching
the maximum test concentration. In addition, all treatments in the presence of S-9
were further modified by the inclusion of a pre-incubation step. Following these
treatments, evidence of toxicity was observed at 320 μg/plate and above in strain
TA1537 and TA102 in the presence of S-9; 800 μg/plate and above in strain TA98 in
the absence and presence of S-9 and strain TA1535 in the presence of S-9; and 800
and/or 2000 μg/plate in strain TA100 in the absence and presence of S-9, and strains
TA1535, TA1537 and TA102 in the absence of S-9.
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 15 Aug - 18 Oct 2012
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- other: OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test) 2010
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian cell micronucleus test
- Target gene:
- not applicable
- Species / strain / cell type:
- lymphocytes: cultured peripheral human lymphocytes
- Details on mammalian cell type (if applicable):
- - Cell proliferation: Blood was collected from healthy, non-smoking volunteers. No donor was suspected of any virus infection or exposed to high levels of radiation or hazardous chemicals. All donors are non-smokers and are not heavy drinkers of alcohol. Donors were not taking any form of medication (contraceptive pill excluded). The measured cell cycle time of the donors falls within the range 13 +/- 2 h.
- Type and identity of media: HEPES-buffered RPMI medium containing 10% (v/v) heat inactivated foetal calf serum and 0.52% penicillin / streptomycin. The mitogen Phytohaemagglutinin (PHA, reagent grade) was included in the culture medium at a concentration of approx. 2% of culture to stimulate the lymphocytes to divide.
- Properly maintained: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254
- Test concentrations with justification for top dose:
- Range finding experiment:
3 h treatment: 3.6, 6.1, 10.1, 16.8, 28.0, 46.7, 77.8, 129.6, 216, 360, 600 and 1000 µg/mL with and without metabolic activation
24 h treatment: 3.6, 6.1, 10.1, 16.8, 28.0, 46.7, 77.8, 129.6, 216, 360, 600 and 1000 µg/mL without metabolic activation
Experiment 1:
3 h treatment: 25, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150 and 200 µg/mL without metabolic activation
3 h treatment: 25, 50, 100, 110, 120, 130, 140, 150, 160, 180, 200 and 250 µg/mL with metabolic activation
Experiment 2:
24 h treatment: 2, 4, 8, 10, 12, 14, 16, 18, 20, 22, 25, 30 and 40 µg/mL without metabolic activation - Vehicle / solvent:
- - Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that the test substance was soluble in anhydrous analytical grade DMSO at concentrations up to at least 280.1 mg/mL. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- vinblastine
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 3 and 24 h
- Fixation time (start of exposure up to fixation or harvest of cells): 3 and 24 h treatment: 24 h
ACTIN POLYMERISATION INHIBITOR (cytogenetic assays): cytochalasin B, 6 µg/mL
STAIN (for cytogenetic assays): Acridine Orange in PBS (pH 6.8; 125 μg/mL)
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: 2000 binucleated cells per concentration
DETERMINATION OF CYTOTOXICITY
- Method: Replication Index (RI); Cytotoxicity (%) is expressed as (100 – Relative RI)
OTHER: Duplicate samples (1 mL) of test article formulations (highest and lowest concentrations) and vehicle control from the micronucleus experiment were retained for analysis of achieved concentration. - Evaluation criteria:
- For valid data, the test article was considered to induce clastogenic and/or aneugenic events if:
1. A statistically significant increase in the frequency of micronucleated binucleate cells at one or more concentrations was observed.
2. An incidence of micronucleated binucleate cells at such a concentration that exceeded the normal range in both replicates was observed.
3. A concentration-related increase in the proportion of micronucleated binucleate cells was observed.
The test substance was considered positive in this assay if all of the above criteria were met.
The test substance was considered negative in this assay if none of the above criteria were met.
Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Evidence of a concentration-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 concentrations, or effects occurring only at high or very toxic concentrations. - Statistics:
- The proportions of micronucleated binucleate cells in each replicate were used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test. The proportion of micronucleated binucleate cells for each treatment condition was compared with the proportion in vehicle controls by using Fisher's exact test. Probability values of p ≤ 0.05 were accepted as significant.
- Key result
- Species / strain:
- lymphocytes: cultured peripheral human lymphocytes
- Metabolic activation:
- with and without
- Genotoxicity:
- other: with S9 mix: negative; without S9 mix: ambiguous
- 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 pH and osmolality: No significant changes in osmolality or pH were observed at the highest concentration tested (1000 μg/mL) as compared to the concurrent vehicle controls.
- Precipitation: The solubility limit in culture medium was in the range of 175.1 to 350.2 μg/mL, as indicated by precipitation at the higher concentration which persisted for at least 24 h after test article addition.
RANGE-FINDING/SCREENING STUDIES: A range finding experiment was conducted to select the concentrations for the micronucleus experiment. A maximum concentration of 1000 μg/mL was selected for the cytotoxicity range finding experiment, in order that treatments were performed in excess of the limit of test substance solubility in culture medium. Slides from the experiment were examined, uncoded, for proportions of mono-, bi- and multinucleate cells, to a minimum of 200 cells per concentration.
COMPARISON WITH HISTORICAL CONTROL DATA: The proportion of micronucleated binucleate cells in the vehicle cultures fell within current historical control ranges.
ADDITIONAL INFORMATION ON CYTOTOXICITY: The highest concentration selected for micronucleus analysis following 24 h treatment in the absence of S9 mix induced 55 ± 5% cytotoxicity. Following 3 h treatments in the absence and presence of S9 mix, the highest concentrations for micronucleus analysis were considered sufficiently close to 50% cytotoxicity to be accepted (the highest concentrations induced 49 and 44% cytotoxicity in the absence and presence of S9 mix, respectively). In addition, analysis of slides from highly cytotoxic concentrations was avoided. - Conclusions:
- It is concluded that W630 (Sample ID: 26480) induced micronuclei in cultured human
peripheral blood lymphocytes following 3+21 hour treatments in the absence of a rat
liver metabolic activation system (S-9) at the highest clearly cytotoxic
(49% cytotoxicity) concentration analysed.
In the same test system, W630 (Sample ID: 26480) did not induce biologically
relevant increases in micronuclei following 3+21 hour treatments in the presence of
S-9 and 24+0 hour treatments in the absence of S-9, when tested up to cytotoxic
concentrations. - Executive summary:
W630 (Sample ID: 26480) was tested in an in vitro micronucleus assay using
duplicate human lymphocyte cultures prepared from the pooled blood of two female
donors in a single experiment. Treatments covering a broad range of concentrations,
separated by narrow intervals, were performed both in the absence and presence of
metabolic activation (S-9) from Aroclor 1254 induced animals. The test article was
formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO) and the
highest concentrations used in the Micronucleus Experiment, limited by toxicity, were
determined following a preliminary cytotoxicity Range-Finder Experiment.
Treatments were conducted (as detailed in the following summary table) 48 hours
following mitogen stimulation by phytohaemagglutinin (PHA). The test article
concentrations for micronucleus analysis were selected by evaluating the effect of
W630 (Sample ID: 26480) on the replication index (RI). In the Micronucleus
Experiment, micronuclei were analysed at three concentrations.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 10 May - 22 June 2012
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- yes
- Remarks:
- only 200 cells per concentration scored; limited documentation; no short-term treatment without S9 mix
- GLP compliance:
- no
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- not applicable
- Species / strain / cell type:
- lymphocytes: cultured peripheral human lymphocytes
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix prepared from the livers of rats treated with Aroclor 1254
- Test concentrations with justification for top dose:
- Experiment 1:
3 h treatment: 50, 100, 120, 140, 150, 160, 170, 180, 190, 200, 225 and 250 µg/mL with metabolic activation
Experiment 2:
20 h treatment: 2.5, 5, 10, 20, 30, 40, 45, 50, 55, 60, 70, 80 and 100 µg/mL without metabolic activation - Vehicle / solvent:
- - Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that the test substance was soluble in anhydrous analytical grade DMSO at a concentration of at least 280.3 mg/mL. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- cyclophosphamide
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 3 and 20 h
- Fixation time (start of exposure up to fixation or harvest of cells): 3 and 20 h treatment: 20 h
NUMBER OF CELLS EVALUATED: 200 cells per concentration (except for the treatment with NQO and CPA: 113 and 61 cells, respectively)
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes - Species / strain:
- lymphocytes: cultured peripheral human lymphocytes
- Metabolic activation:
- with
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- The highest concentrations analysed induced mitotic inhibition: 59% at 170 µg/mL with S9-mix.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not specified
- Positive controls validity:
- valid
- Species / strain:
- lymphocytes: cultured peripheral human lymphocytes
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- The highest concentrations analysed induced mitotic inhibition: 47% at 30 µg/mL without S9-mix
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- True negative controls validity:
- not specified
- Positive controls validity:
- valid
- Additional information on results:
- RANGE-FINDING/SCREENING STUDIES: A maximum concentration of 2784 μg/mL was selected for the cytotoxicity range finding experiment in order tha treatment was performed up a maximum concentration equivalent to 10 mM. Concentrations selected for the chromosome aberration experiment were based on the results of this cytotoxicity range finding experiment.
COMPARISON WITH HISTORICAL CONTROL DATA: Numbers of aberrant cells (excluding gaps) in all cultures treated for 20 h in the absence of S9 mix fell within the current historical control range. Both replicates of the positive controls in the absence and presence of S9 mix exceeded the historical control range and were statistically significantly higher than the concurrent vehicle controls. - Conclusions:
- It is concluded that W630 (Sample ID: 25130) did induce structural chromosome
aberrations in cultured human peripheral blood lymphocytes following treatment for
3+17 hours in the presence of a rat liver metabolic activation system (S-9). In the same
test system, W630 (Sample ID: 25130) did not induce chromosome aberrations
following treatment for 20+0 hours in the absence of S-9, when tested up to cytotoxic
concentrations. - Executive summary:
It is concluded that W630 (Sample ID: 25130) did induce structural chromosome
aberrations in cultured human peripheral blood lymphocytes following treatment for
3+17 hours in the presence of a rat liver metabolic activation system (S-9). In the same
test system, W630 (Sample ID: 25130) did not induce chromosome aberrations
following treatment for 20+0 hours in the absence of S-9, when tested up to cytotoxic
concentrations.
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 10 May - 14 June 2012
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- other: OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test) 2010
- Deviations:
- yes
- Remarks:
- limited documentation, no short-term treatment without S9 mix
- GLP compliance:
- no
- Type of assay:
- in vitro mammalian cell micronucleus test
- Target gene:
- not applicable
- Species / strain / cell type:
- lymphocytes: cultured peripheral human lymphocytes
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix prepared from the livers of rats treated with Aroclor 1254
- Test concentrations with justification for top dose:
- Experiment 1:
3 h treatment: 50, 100, 120, 140, 150, 160, 170, 180, 190, 200, 225 and 250 µg/mL with metabolic activation
Experiment 2:
24 h treatment: 2, 4, 8, 10, 12, 14, 16, 18, 20, 22, 26, 30 and 40 µg/mL without metabolic activation - Vehicle / solvent:
- - Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that the test substance was soluble in anhydrous analytical grade DMSO at a concentration of at least 280.3 mg/mL. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- other: vinblastine
- Remarks:
- mitomycin C (MMC): 0.8 µg/mL (24 h, -S9); vinblastine (VIN): 0.02 µg/mL (24 h, -S9); cyclophosphamide (CPA): 12.5 µg/mL (3 h, +S9)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 3 and 24 h
- Fixation time (start of exposure up to fixation or harvest of cells): 3 and 24 h treatment: 24 h
NUMBER OF CELLS EVALUATED: 2000 binucleated cells per concentration (except for the treatment with vinblastine: 1285 binucleated cells)
DETERMINATION OF CYTOTOXICITY
- Method: Replication Index (RI) - Species / strain:
- lymphocytes: cultured peripheral human lymphocytes
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- RANGE-FINDING/SCREENING STUDIES: A maximum concentration of 2784 μg/mL was selected for the cytotoxicity range finding experiment in order that treatment was performed up a maximum concentration equivalent to 10 mM. Concentrations selected for the micronucleus experiment were based on the results of this cytotoxicity range finding experiment.
COMPARISON WITH HISTORICAL CONTROL DATA: The micronucleated binucleate cell frequency of all treated cultures fell within historical control ranges. Both replicates of the positive controls in the absence of S9 mix exceeded the normal historical control range and were statistically significantly higher than the concurrent vehicle control. - Conclusions:
- It is concluded that W630 (Sample ID: 25130) did not induce micronuclei in cultured
human peripheral blood lymphocytes following treatment 1sv ]+2I hours in the
presence of a rat liver metabolic activation system (S-9) and for 24+0 hours in the
absence of S-9, when tested up to cytotoxic concentrations. - Executive summary:
It is concluded that W630 (Sample ID: 25130) did not induce micronuclei in cultured
human peripheral blood lymphocytes following treatment 1sv ]+2I hours in the
presence of a rat liver metabolic activation system (S-9) and for 24+0 hours in the
absence of S-9, when tested up to cytotoxic concentrations.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2012-08-10 - 2013-01-18
- 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)
- Version / remarks:
- 1997
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian cell gene mutation tests using the thymidine kinase gene
- Target gene:
- thymidine kinase (tk) locus
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Metabolic activation system:
- The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was obtained from Molecular Toxicology Incorporated, USA, where it was prepared from male Sprague Dawley rats induced with Aroclor 1254. The batches of MolToxTM S-9 were stored frozen in aliquots at <-50°C prior to use. Each batch was checked by the manufacturer for sterility, protein content, ability to convert known promutagens to bacterial mutagens and cytochrome P-450-catalyzed enzyme activities (alkoxyresorufin-O-dealkylase activities).
- Test concentrations with justification for top dose:
- Range-Finder 3 h: 21.88 - 700 µg/mL (limited solubility in culture medium)
Range-Finder 24 h: 2.735 - 700 µg/mL (limited solubility in culture medium)
Experiment 1 -S9: 10 - 150 µg/mL (135 µg/mL, cytotoxicity)
Experiment 1 +S9: 10 - 175 µg/mL (135 µg/mL, cytotoxicity)
Experiment 2 -S9, 24 h: 2 - 40 µg/mL (30 µg/mL, cytotoxicity)
Experiment 2 +S9, 3 h: 20 - 180 µg/mL (cytotoxicity) - Vehicle / solvent:
- DMSO
- Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- methylmethanesulfonate
- Details on test system and experimental conditions:
- The master stock of L5178Y tk+/- (3.7.2C) mouse lymphoma cells originated from Dr Donald Clive, Burroughs Wellcome Co. Cells supplied to Covance Laboratories Ltd. were stored as frozen stocks in liquid nitrogen. Full details of the supplier are documented in central records. Each batch of frozen cells was purged of tk- mutants, checked for spontaneous mutant frequency and that it was mycoplasma free. For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and incubated in a humidified atmosphere of 5±1% (v/v) CO2 in air. When the cells were growing well, subcultures were established in an appropriate number of flasks.
- Statistics:
- Test for linear trend: χ2 (one-sided), significant at 5%, 1% and 0.1%
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with
- Genotoxicity:
- ambiguous
- Remarks:
- 3 h treatment
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- True negative controls validity:
- not specified
- Positive controls validity:
- valid
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Remarks:
- 3 h and 24 h treatment
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- True negative controls validity:
- not specified
- Positive controls validity:
- valid
- Conclusions:
- It is concluded that W630 (Sample ID: 26480) showed evidence of inducing mutation at the tk locus of L5178Y mouse lymphoma cells when tested for 3 hours in the presence of a rat liver metabolic activation system (S-9) in two independent experiments. Concentration-related increases in mutant frequency were observed in both experiments, but the induced mutant frequency exceeded the global evaluation factor only in Experiment 2 at the highest toxic concentration analysed, which was close to the limit of toxicity for the assay. The magnitude of response was therefore not well reproduced between experiments and the observation may be considered of uncertain biological relevance.
In the same test system, W630 (Sample ID: 26480) did not induce mutation following treatment for 3 and 24 hours in the absence of S-9 when tested up to toxic concentrations. - Executive summary:
W630 (Sample ID: 26480) was assayed for the ability to induce mutation at the tk locus (5-trifluorothymidine [TFT] resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity Range-Finder Experiment followed by two independent experiments, each conducted in the absence and presence of metabolic activation by an Aroclor 1254 induced male Sprague-Dawley rat liver post-mitochondrial fraction (S-9). The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO).
A 3 hour treatment incubation period was used for all experiments performed in the presence of S-9. In the absence of S-9, the Range-Finder Experiment was performed using 3 and 24 hour treatment incubation periods, Experiment 1 was performed using a 3 hour treatment incubation and Experiment 2 was performed using a 24 hour treatment incubation.
Referenceopen allclose all
Table 1. Test results of experiment 1 (plate incorporation).
Bacterial Reverse Mutation Assay, mean revertants colonies/plate ± standard deviation |
|||||
EXPERIMENT 1 (plate incorporation) |
|||||
S9-Mix |
Without
|
||||
Test item (µg/plate) |
T98 |
TA100 |
TA1535 |
TA1537 |
TA102 |
DMSO |
23.8 ± 6.1 |
94.8 ± 3.6 |
14.0 ± 4.1 |
6.0 ± 2.5 |
275.4 ± 33.5 |
5 |
24.0 ± 7.0 |
103.7 ± 7.6 |
13.3 ± 3.1 |
6.7 ± 2.5 |
276.7 ± 11.7 |
15.81 |
20.3 ± 4.5 |
102.3 ± 4.0 |
16.3 ± 2.5 |
6.3 ± 2.5 |
280.3 ± 34.3 |
50 |
19.7 ± 1.2 |
80.0 ± 6.0 |
22.0 ± 5.0 * |
7.3 ± 1.5 |
275.3 ± 10.3 |
158.1 |
19.3 ± 3.8 |
84.7 ± 8.5 |
16.0 ± 4.6 |
7.3 ± 2.9 |
270.7 ± 11.0 |
500 |
16.3 ± 4.5 |
80.3 ± 5.5 S |
15.7 ± 4.5 |
4.7 ± 0.6 S |
217.7 ± 35.2 S |
1581 |
5.3 ± 3.5 S |
T |
5.0 ± 1.0 V |
T |
T |
5000 |
P, T |
P, T |
P, T |
P, T |
P, T |
2NF |
905.0 ± 37.0 |
--- |
--- |
--- |
--- |
NaN3 |
--- |
614.3 ± 58.6 |
549.7 ± 82.0 |
--- |
--- |
AAC |
--- |
--- |
--- |
158.7 ± 31.2 |
--- |
MMC |
--- |
--- |
--- |
--- |
850.3 ± 133.3 |
S9-Mix
|
With |
||||
Test item (µg/plate) |
TA98 |
TA100 |
TA1535 |
TA1537 |
TA102 |
DMSO |
30.4 ± 7.6 |
98.6 ± 9.0 |
18.0 ± 5.0 |
13.2 ± 5.0 |
251.6 ± 10.1 |
5 |
34.7 ± 5.1 |
110.3 ± 10.8 |
19.3 ± 4.9 |
18.7 ± 4.7 |
237.7 ± 14.4 |
15.81 |
43.0 ± 11.3 |
113.7 ± 5.5 |
16.7 ± 3.1 |
17.0 ± 6.1 |
254.0 ± 14.7 |
50 |
36.7 ± 2.5 |
106.7 ± 11.7 |
21.0 ± 9.5 |
16.0 ± 1.0 |
247.0 ± 13.1 |
158.1 |
39.0 ± 9.2 |
93.7 ± 12.4 |
19.0 ± 2.0 |
13.0 ± 2.6 |
228.7 ± 21.1 |
500 |
26.0 ± 5.3 |
73.7 ± 12.7 S |
11.7 ± 2.9 |
8.3 ± 4.0 S |
181.3 ± 23.2 S |
1581 |
15.0 ± 2.6 S |
T |
5.3 ± 4.0 V |
T |
T |
5000 |
P, T |
P, T |
P, T |
P, T |
P, T |
BaP |
297.7 ± 110.2 |
--- |
--- |
--- |
--- |
AAN |
--- |
1081.7 ± 192.8 |
265.7 ± 3.8 |
198.3 ± 24.4 |
1387.3 ± 79.5 |
P: precipitation of the test substance observed S: slight thinning of background bacterial lawn V: very thin background bacterial lawn T: toxic, no revertant colonies
Positive controls: 2NF = 2-nitrofluorene NaN3 = sodium azide AAC = 9-aminoacridine MMC = Mitomycin C BaP = benzo[a]pyrene AAN = 2-aminoanthracene |
|||||
* p<0.05 |
|||||
Table 2. Test results of experiment 2 (pre-incubation).
Bacterial Reverse Mutation Assay, mean revertants colonies/plate ± standard deviation |
|||||
EXPERIMENT 2 (pre-incubation) |
|||||
S9-Mix |
Without
|
||||
Test item (µg/plate) |
T98 |
TA100 |
TA1535 |
TA1537 |
TA102 |
DMSO |
22.2 ± 5.2 |
94.8 ± 14.6 |
17.6 ± 2.9 |
11.2 ± 4.2 |
233.4 ± 12.6 |
8.192 |
--- |
94.7 ± 14.0 |
--- |
10.0 ± 4.0 |
243.3 ± 8.1 |
20.48 |
24.3 ± 8.1 |
70.7 ± 8.7 |
22.7 ± 6.8 |
9.7 ± 3.5 |
236.7 ± 4.6 |
51.2 |
16.7 ± 4.9 |
87.7 ± 5.8 |
14.7 ± 5.8 |
9.0 ± 2.6 |
221.3 ± 0.6 |
128 |
20.3 ± 2.3 |
96.3 ± 17.2 |
16.0 ± 2.6 |
8.0 ± 3.6 |
201.0 ± 22.1 |
320 |
20.3 ± 3.2 |
87.0 ± 9.8 |
11.7 ± 3.8 |
10.3 ± 4.5 |
180.7 ± 15.0 |
800 |
10.3 ± 2.5 |
47.3 ± 6.7 |
11.3 ± 4.7 |
4.7 ± 1.5 |
102.7 ± 5.7 V |
2000 |
5.3 ± 2.1 P, V |
--- |
2.3 ± 0.6 P, V |
--- |
--- |
2NF |
735.7 ± 87.9 |
--- |
--- |
--- |
--- |
NaN3 |
--- |
671.7 ± 94.0 |
686.7 ± 24.1 |
--- |
--- |
AAC |
--- |
--- |
--- |
100.3 ± 34.8 |
--- |
MMC |
--- |
--- |
--- |
--- |
715.0 ± 99.3 |
S9-Mix
|
With |
||||
Test item (µg/plate) |
TA98 |
TA100 |
TA1535 |
TA1537 |
TA102 |
DMSO |
33.8 ± 6.1 |
120.0 ± 9.9 |
20.6 ± 4.5 |
17.0 ± 4.0 |
225.2 ± 23.6 |
8.192 |
--- |
114.0 ± 18.5 |
--- |
19.3 ± 1.5 |
261.3 ± 11.6 * |
20.48 |
39.3 ± 4.7 |
112.7 ± 7.5 |
15.7 ± 0.6 |
18.7 ± 2.5 |
249.0 ± 15.6 |
51.2 |
39.0 ± 7.5 |
126.7 ± 14.0 |
17.7 ± 5.5 |
16.7 ± 1.2 |
259.0 ± 15.6 * |
128 |
32.3 ± 6.7 |
125.7 ± 7.4 |
16.3 ± 3.5 |
21.0 ± 3.0 |
232.7 ± 13.7 |
320 |
28.3 ± 5.9 |
119.0 ± 5.6 |
12.7 ± 2.5 |
19.3 ± 0.6 S |
229.7 ± 2.3 S |
800 |
15.0 ± 3.5 S |
T |
2.3 ± 0.6 V |
T |
T |
2000 |
P, T |
--- |
P, T |
--- |
--- |
BaP |
339.0 ± 28.2 |
--- |
--- |
--- |
--- |
AAN |
--- |
1414.7 ± 33.5 |
257.7 ± 10.4 |
260.0 ± 13.9 |
942.3 ± 199.4 |
P: precipitation of the test substance observed S: slight thinning of background bacterial lawn V: very thin background bacterial lawn T: toxic, no revertant colonies
Positive controls: 2NF = 2-nitrofluorene NaN3 = sodium azide AAC = 9-aminoacridine MMC = Mitomycin C BaP = benzo[a]pyrene AAN = 2-aminoanthracene |
|||||
* p<0.05 |
|||||
Table 1: Results of Experiment 1 and 2.
Test item |
Concentration in µg/mL |
Cytotoxicity (%) |
Number of cells with MN in % |
Exposure period 3 h, fixation time 24 h, without S9 mix |
|||
DMSO |
- |
- |
0.50 |
MMC |
0.8 |
ND |
11.70** |
Test substance |
50 |
7 |
0.40 |
90 |
19 |
0.70 |
|
110 |
49 |
1.95** |
|
Exposure period 3 h, fixation time 24 h, with S9 mix |
|||
DMSO |
- |
- |
0.5 |
CPA |
12.5 |
3.4 |
3.40* |
Test substance |
50 |
0 |
1.05* |
120 |
18 |
0.80 |
|
140 |
44 |
1.10** |
|
Exposure period 24 h, fixation time 24 h, without S9 mix |
|||
DMSO |
- |
- |
0.70 |
VIN |
0.02 |
ND |
16.01** |
Test substance |
4 |
10 |
0.90 |
14 |
28 |
0.40 |
|
18 |
55 |
0.80 |
|
CPA: Cyclophosphamide
DMSO: Dimethylsulfoxide
MMC: Mitomycin C
ND: Not determined
VIN: Vinblastine
*: Statistical significant (p ≤ 0.05)
**: Statistical significant (p ≤ 0.001)
Treatment of cells with test substance for 3 h in the absence of S-9 resulted in frequencies of micronucleated binucleate cells that were significantly higher (p ≤ 0.001) than those observed in concurrent vehicle controls at the highest concentration analysed only (110 μg/mL inducing 49% cytotoxicity which was concentration-limiting toxicity according to current test guidelines). The micronucleated binucleate cell frequency of both treated cultures at 110 μg/mL exceeded the historical control range.
Treatment of cells for 3 h with metabolic activation resulted in frequencies of micronucleated binucleate cells that were significantly higher (p ≤ 0.05) than those observed in concurrent vehicle controls at the lowest and highest concentrations analysed (50 and 140 μg/mL). The micronucleated binucleate cell frequency of only single cultures at 50 and 140 μg/mL exceeded the historical control range. These increases were not reproduced in the replicate cultures, showed no concentration-related increases and the mean micronucleated binucleate cell frequencies fell within the historical control range, therefore, they were considered to be negative
Analysis of achieved concentration:
Results of formulation analyses demonstrated achieved concentrations within 100 ± 10% of the nominal test substance concentrations and were therefore considered acceptable. No test article was detected in the vehicle sample.
Validity of study:
1) The binomial dispersion test demonstrated acceptable heterogeneity (in terms of micronucleated binucleate cell frequency) between replicate cultures.
2) The frequency of micronucleated binucleate cells in vehicle controls fell within the normal range.
3) The positive control chemicals induced statistically significant increases in the proportion of cells with micronuclei. Both replicate cultures at the positive control concentration analysed under each treatment condition demonstrated micronucleated binucleate cell frequencies that clearly exceeded the current historical vehicle control ranges.
4) A minimum of 50% of cells had gone through at least one cell division (as measured by binucleate + multinucleate cell counts) in vehicle control cultures at the time of harvest.
Table 1. Results of the chromosomal aberration test.
Test item |
Concentration [µg/mL] |
Scored cells |
Mitotic Inhibition (%) |
No. of cells with aberrations |
No. of cells with aberrations |
Exposure period 20 h, fixation time 20 h, without S9 mix |
|||||
DMSO |
- |
200 |
- |
1 |
0 |
NQO |
2.5 |
113 |
- |
45*** |
40*** |
Test substance |
10 |
200 |
2 |
1 |
1 |
20 |
200 |
43 |
0 |
0 |
|
30 |
200 |
47 |
5 |
5 |
|
Exposure period 3 h, fixation time 20 h, with S9 mix |
|||||
DMSO |
- |
200 |
- |
2 |
0 |
CPA |
12.5 |
61 |
- |
42*** |
40*** |
Test substance |
50 |
200 |
20 |
2 |
1 |
120 |
200 |
31 |
6* |
5 |
|
170 |
202 |
59 |
15*** |
14*** |
|
CPA: Cyclophosphamide
DMSO:Dimethylsulfoxide
NQO: 4-nitroquinoline-N-oxide
*: Statistical significant (p ≤ 0.05)
***: Statistical significant (p ≤ 0.001)
Treatment of cells for 3 h in the presence of S9 mix resulted in frequencies of cells with structural aberrations that were significantly higher (p ≤ 0.05) than those observed in concurrent vehicle control cultures for the highest two concentrations analysed (120 and 170 μg/mL). The number of aberrant cells (excluding gaps) exceeded the vehicel control in both cultures at 170 μg/mL. Thus, a concentration-related increase in structural chromosome aberrations was observed under this treatment condition.
Incidental increases in the frequency of cells with numerical aberrations, which exceeded the concurrent vehicle controls and the historical control range, were observed in single cultures at 20 μg/mL following 20 h treatment in the absence of S9 mix and at 120 and 170 μg/mL following 3 h treatment in the presence of S9 mix. These increases were not reproduced in the replicate culture or consistently at any other concentration tested. Thus, these incidental observations were considered to be negative. Furthermore, the assay is not specifically designed to evaluate the potential to induce numerical aberrations and they were not assessed quantitatively as part of this study.
Table 1: Results of the micronucleus test.
Test item |
Concentration in µg/mL |
Cytotoxicity (%) |
Number of cells with MN in % |
Exposure period 3 h, fixation time 24 h, with S9 mix |
|||
DMSO |
- |
- |
0.45 |
CPA |
12.50 |
- |
1.6*** |
Test substance |
50 |
19 |
0.35 |
100 |
37 |
0.65 |
|
120 |
45 |
0.30 |
|
140 |
61 |
0.55 |
|
Exposure period 24 h, fixation time 24 h, without S9 mix |
|||
DMSO |
- |
- |
0.30 |
MMC |
0.80 |
- |
7.00*** |
VIN |
0.02 |
- |
11.52*** |
Test substance |
2 |
7 |
0.35 |
10 |
26 |
0.45 |
|
20 |
54 |
0.55 |
|
CPA: Cyclophosphamide
DMSO:Dimethylsulfoxide
MMC: Mitomycin C
VIN: Vinblastine
***: Statistical significant (p ≤ 0.001)
The micronucleated binucleate cell frequency of replicate culture B of the positive control in the presence of S9 mix did not exceed the historical control range but equalled the upper limit of the historical control range (1.10%). However, the mean micronucleated binucleate cell frequency exceeded the historical control range and was statistically significantly higher than the concurrent vehicle control.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
no adverse effect observed: negative
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: see 'Remark'
- Remarks:
- Basic data given: This micronucleus assay is part of a 90-day repeated dose toxicity study performed according to OECD 408. The test item was assessed for its cumulative toxicity when administered daily to rats by gavage for a period of 90 days and for its potential to induce the formation of micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of Wistar rats. No positive control was used.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- yes
- Remarks:
- In this study, the animals were treated orally by gavage for period of at least 90 days; no positive control
- GLP compliance:
- yes
- Type of assay:
- micronucleus assay
- Species:
- rat
- Strain:
- Wistar
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Harlan Laboratories B.V., Horst, The Netherlands
- Age at study initiation: 7 weeks
- Weight at study initiation: 212 - 256 g (males), 140 - 160 g (females)
- Housing: Animals were housed in groups of 3 or 4 in Makrolon type-4 cages with wire mesh tops and standard softwood bedding including paper enrichment.
- Diet: Pelleted standard HarlanTeklad 2914C rat / mouse maintenance diet, ad libitum
- Water: Community tap water, ad libitum
- Acclimation period: 7 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES:
From: 29 Nov 2012
To: 28 Feb 2013 - Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle(s)/solvent(s) used: PEG 300
- Batch no.: BCBJ2013V - Details on exposure:
- PREPARATION OF DOSING SOLUTIONS: The dose formulations were prepared weekly. The test substance was weighed into a glass beaker on a tared balance and the vehicle added. The mixtures were stirred using a magnetic stirrer. Homogeneity of the test substance in the vehicle was maintained during the daily administration period using a magnetic stirrer.
- Duration of treatment / exposure:
- 90 days
- Frequency of treatment:
- daily
- Remarks:
- Doses / Concentrations:
50, 200, 800 mg/kg bw/day
Basis:
actual ingested - No. of animals per sex per dose:
- 7
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- none
- Tissues and cell types examined:
- Tissue: bone marrow
Cell type: bone marrow cells - Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION: This micronucleus assay is part of 90 day repeated dose toxicity study. The dose levels were selected based on a previous dose range finding toxicity study.
TREATMENT AND SAMPLING TIMES: 24 hours after the last treatment of the animals, animals were sacrificed and the right femurs were removed.
DETAILS OF SLIDE PREPARATION: The epiphyses were cut off and the marrow was flushed out with warmed fetal calf serum (FCS), using a syringe. The nucleated cells were separated from the erythrocytes using the method of Romagna. Briefly, the cell suspensions were passed through a column consisting of α-Cellulose (Sigma) and Cellulose (Sigmacell type 50). The columns were then washed with Hank´s buffered saline. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. The pellet was resuspended in a small drop of FCS and spread on slides. The smears were air-dried, fixed in methanol and stained with May-Grünwald /Giemsa. Cover slips were mounted with EUKITT. At least one slide was made from each bone marrow sample.
METHOD OF ANALYSIS: Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. At least 4000 immature erythrocytes per animal should be scored for the incidence of micronucleated immature erythrocytes. At least 2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides. - Evaluation criteria:
- A test item can be classified as mutagenic if it induces either a dose-related increase in the number of micronucleated polychromatic erythrocytes or a statistically significant positive response for at least one of the test points.
A test item producing neither a dose-related increase in the number of micronucleated polychromatic erythrocytes nor a statistically significant positive response at any of the test points, is considered non-mutagenic in this system.
This can be confirmed by means of the nonparametric Mann-Whitney test, if necessary. However, both biological and statistical significance should be considered together.
Acceptance Criteria
The study is considered valid if the following criteria show that:
- at least 5 animals per group and sex are evaluable
- PCE to erythrocyte ratio should not be less than 20% of the negative control. - Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- not examined
- Additional information on results:
- RESULTS
- Induction of micronuclei: In comparison to the corresponding vehicle controls, there was no statistically significant or biologically relevant enhancement of the frequency of the detected micronuclei in males and females at any dose level. The mean values of micronuclei observed after treatment with the test item were below or near the value of the respective vehicle control group (see Table 1+2) and within the historical control data (see Table 3).
- Ratio of PCE/NCE: The ratio between polychromatic and normochromatic erythrocytes was determined in the same sample to evaluate any cytotoxic effect due to treatment with the test substance and reported as the number of PCEs per 2000 erythrocytes. After treatment with the test substance, the mean number of PCEs per 2000 erythrocytes was not decreased as compared to the mean value of PCEs per 2000 erythrocytes of the vehicle control, thus indicating that the test substance did not induce cytotoxic effects in the bone marrow of exposed rats. - Conclusions:
- Interpretation of results: negative
Under the experimental conditions reported, the test substance did not induce micronuclei in the bone marrow of the rats exposed to the test substance for 90 days as part of a 90-day repeated dose toxicity study. Therefore, test substance is considered to be non-mutagenic in this micronucleus assay.
Reference
Table 1: Males
Test group |
Dose [mg/kg bw/day] |
PCEs with micronuclei [%] |
Range |
PCE per 2000 erythrocytes |
Vehicle (PEG 300) |
0 |
0.314 |
1-11 |
948 |
Test item |
50 |
0.350 |
1-17 |
965 |
Test item |
200 |
0.357 |
0-11 |
954 |
Test item |
800 |
0.257 |
3-9 |
1008 |
Table 2: Females
Test group |
Dose [mg/kg bw/day] |
PCEs with micronuclei [%] |
Range |
PCE per 2000 erythrocytes |
Vehicle (PEG 300) |
0 |
0.414 |
0-21 |
1039 |
Test item |
50 |
0.364 |
4-11 |
1144 |
Test item |
200 |
0.250 |
1-7 |
1095 |
Test item |
800 |
0.393 |
2-23 |
1135 |
Table 3: Historical controls 2006-2011
Micronuclated cells |
Males |
Females |
Total |
Males |
Females |
Total |
Mean ± SD [%] |
0.184± 0.054 |
0.160± 0.053 |
0.175± 0.055 |
2.154± 0.820 |
1.034± 0.432 |
1.723± 0.884 |
Range of mean group [%] |
0.085- 0.320 |
0.058- 0.283 |
0.058- 0.320 |
0.670- 4.058 |
0.470– 2.875 |
0.470– 4.058 |
Range (individual animal data) |
0-12 |
0-12 |
0-12 |
8-136 |
5-74 |
5-136 |
No. of Experiments |
51 |
35 |
51 |
49 |
32 |
49 |
The values included in the historical control data base were obtained from single time application using differentroutes(e.g. orally, intravenously, intraperitoneally).
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
The genotoxic potential of W630 has been investigated in several in vitro studies and in one in vivo study.
In vitro studies
An Ames test was performed according to OECD 471 and under GLP conditions to investigate the potential of W630 to induce gene mutations the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102 (Mc Garry, 2012). W630 was investigated in the plate incorporation method in test concentrations from 5 to 5000 µg/plate with and without metabolic activation. An independent repeat was performed as a pre-incubation test. There was no evidence of mutagenic activity following incubation with W630 in the presence and the absence of metabolic activation. Precipitation was observed on the test plates at concentrations of 2000 µg/plate and above. Cytotoxic effects of the test substance were observed in all tester strains, starting for TA102 and TA1537 at 320 µg/plate with metabolic activation and 500 µg/plate without metabolic activation, for TA100 at 500 µg/plate with and without metabolic activation and for TA98 and TA1535 at 800 µg/plate with metabolic activation and 1581 µg/plate without metabolic activation. The cytotoxic effects ranged from slight reduction of the background lawn to complete extermination of the test bacteria at higher concentrations. The positive controls were shown to be valid.Under the experimental conditions of this study, W630 did not induce gene mutations in the tested strains up to 5000 µg/plate with and without metabolic activation.
W630 was tested in an in vitro micronucleus assay using human blood lymphocytes according to OECD 487 and under GLP conditions (Watters, 2013). Treatments covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence and presence of metabolic activation. Cells were exposed to W630 for 3 h with and without S9-mix and continuously for 24 h in the absence of S9-mix.
Mitomycin C and Vinblastine were used as clastogenic and aneugenic positive control chemicals respectively, in the absence of S9-mix. Cyclophosphamide was used as a clastogenic positive control chemical in the presence of S9-mix. All positive control compounds induced statistically significant increases in the proportion of cells with micronuclei. The acceptance criteria were met and the study was therefore considered valid.
Blood lymphocytes were analysed for micronuclei in the presence of metabolic activation at 50, 120 and 140 µg/mL. In the absence of metabolic activation, cells were analysed for micronuclei at 50, 90 and 110 µg/mL after short-term treatment and at 4, 14 and 18 µg/mL after long-term treatment.
Treatment of cells with test substance for 3 h without S9-mix resulted in frequencies of micronucleated binucleate (MNBN) cells that were significantly higher (p ≤ 0.001) than those observed in concurrent vehicle controls (DMSO) at the highest concentration analysed (110 μg/mL inducing 49% cytotoxicity which was concentration-limiting toxicity according to current test guidelines). The MNBN cell frequency of both treated cultures at 110 µg/mL exceeded the historical control range. However, long-term treatment (24 h) of cells with W630 in the absence of metabolic activation resulted in frequencies of MNBN cells that were similar to and not significantly higher than those observed in concurrent vehicle controls (DMSO) for all concentrations analysed. The MNBN cell frequency of all treated cultures fell within the historical control range.
Treatment of cells for 3 h with S9-mix resulted in frequencies of MNBN cells that were significantly higher (p ≤ 0.05) than those observed in concurrent vehicle controls (DMSO) at the lowest and highest concentrations analysed (50 and 140 μg/mL). The MNBN cell frequency of only single cultures at 50 and 140 µg/mL exceed the historical control range. These increases were not reproduced in the replicate cultures, showed no concentration-related increases and the mean MNBN cell frequencies fell within the historical control range, therefore, the test result in the presence of metabolic activation was considered to be negative.
In summary, W630 induced micronuclei in cultured human peripheral blood lymphocytes following 3 h treatment in the absence of metabolic activation at the highest clearly cytotoxic (49% cytotoxicity) concentration analysed. However, in the same test system, W630 did not induce biologically relevant increases in the frequencies of micronuclei after long-term treatment (24 h) without S9-mix, when tested up to cytotoxic concentrations. Therefore, under the experimental conditions of this test, the result obtained in the absence of metabolic activation is considered to be ambiguous.
The result observed in the presence of metabolic activation was considered to be incidental as no concentration-related increase and no reproducible results in both cultures were found and the mean MNBN cell frequency was in the range of the historical control data. Thus, the overall result of the micronucleus assay in the presence of metabolic activation is negative.
In addition, a non-GLP screening micronucleus assay is available which was performed similar to OECD 487 (Watters, 2012).Cells were exposed to W630 for 3 h with S9-mix and continuously for 24 h in the absence of S9-mix.
Mitomycin C and Vinblastine were used as the clastogenic and aneugenic positive control chemicals respectively in the absence of S9-mix. Cyclophosphamide was used as a clastogenic positive control chemical in the presence of S9-mix.
The MNBN cell frequency of replicate culture B of the positive control with S9-mix (Cyclophosphamide) did not exceed the historical control range but equaled the upper limit of the historical control range (1.10%). However, the mean MNBN cell frequency exceeded the historical control range and was statistically significantly higher than the concurrent vehicle control. Both replicates of the positive controls in the absence of S9-mix (Mitomycin C and Vinblastine) exceeded the normal historical control range and were statistically significantly higher than the concurrent vehicle control. Therefore the test system showed sufficient sensitivity and the data can be considered as valid.
Cells were analysed for micronuclei in cultures treated at 50, 100, 120 and 140 µg/mL with S9-mix and at 2, 10 and 20 µg/mL without S9-mix.The highest concentrations analysed for micronuclei induced cytotoxicity: 61% at 140 µg/mL and 54% at 20 µg/mL.
Treatment of cells with W630 for 3 h in the presence of S9-mix and for 24 h in the absence of S9-mix resulted in frequencies of micronucleated binucleate (MNBN) cells that were similar to and not significantly higher than those observed in concurrent vehicle controls for all test concentrations analysed. The MNBN cell frequency of all treated cultures fell within the historical control range.
Under the experimental conditions of this test, W630 did not induce micronuclei in cultured human peripheral blood lymphocytes following 3 h treatment in the presence of metabolic activation and following 24 h in the absence of metabolic activation when tested up to cytotoxic concentrations.
The clastogenic potential of W630 was evaluated in a non-GLP screening chromosome aberration test in cultured peripheral human lymphocytes conducted similar to OECD 473 (Watters, 2012). Human lymphocytes were exposed for 20 h to W630 in the absence of metabolic activation and for 3 h in the presence of metabolic activation. Metaphase cells were analysed for chromosome aberration in cultures treated at 10, 20 and 30 µg/mL in the absence of S9 -mix and at 50, 120 and 170 µg/mL in the presence of S9-mix.
The highest concentrations analysed for aberrations induced mitotic inhibition: 47% at 30 µg/mL and 59% at 170 µg/mL. The positive controls 4-nitroquinoline-N-oxide and cyclophosphamide induced clastogenic effects and demonstrated the sensitivity of the test system and (in the case of cyclophosphamide) the activity of the used S9-mix.
Treatment of cells for 3 h in the presence of S9-mix resulted in frequencies of cells with structural aberrations that were significantly higher (p ≤ 0.05) than those observed in concurrent vehicle control cultures (DMSO) for the highest two concentrations analysed (120 and 170 µg/mL). The number of aberrant cells (excluding gaps) exceeded the historical control range in both cultures at 170 µg/mL. Thus, a concentration-related increase in structural chromosome aberrations was observed under this treatment condition.
Incidental increases in the frequency of cells with numerical aberrations, which exceeded the concurrent vehicle control (DMSO) and the historical control ranges, were observed in single cultures at 20 µg/mL following 20 h treatment in the absence of S9-mix and at 120 and 170 µg/mL following 3 h treatment in the presence of S9-mix. These increases were not reproduced in the replicate culture or at any other concentration tested. Thus, these incidental observations can be considered to be of no biological relevance. Furthermore, the assay is not specifically designed to evaluate the potential to induce numerical aberrations and they were not assessed quantitatively as part of this study.
It is concluded that W630 did induce structural chromosome aberrations in cultured human peripheral blood lymphocytes following treatment for 3 h in the presence of metabolic activation. In the same test system, W630 did not induce chromosome aberrations following treatment for 20 h without metabolic activation, when tested up to cytotoxic concentrations.
In vivo studies
The micronucleus (MN) test was applied to investigate W630 for clastogenic effects on the chromosomes of bone marrow erythroblasts in a study conducted similar to OECD 474 (Braun, 2013). The MN test was performed within a 90-day repeated dose toxicity study carried out according to OECD 408.
W630 was assessed for its cumulative toxicity when administered daily to rats by gavage for a period of 90 days, and its potential to induce the formation of micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of Wistar rats. The following dose levels of the test substance were investigated: 50, 200, and 800 mg/kg bw/day. From seven males and females per dose group the right femurs were collected at necropsy and bone marrow samples were evaluated for the occurrence of micronuclei. For each animal at least 2000 polychromatic erythrocytes were scored for micronuclei. The ratio between polychromatic and normochromatic erythrocytes was determined in the same sample to evaluate any cytotoxic effect due to treatment with the test substance and reported as the number of PCEs per 2000 erythrocytes. After treatment with the test substance, the mean number of PCEs per 2000 erythrocytes was not decreased as compared with the mean value of PCEs per 2000 erythrocytes of the vehicle control, thus indicating that W630 did not induce cytotoxic effects in the bone marrow. In comparison to the corresponding vehicle controls, there was no statistically significant or biologically relevant increase in the frequency of the detected micronuclei in males and females at any dose level. The mean values of micronuclei observed after treatment with W630 were below or near the value of the respective vehicle control group and within the historical control range. Under the experimental conditions reported, W630 did not induce a clastogenic effect on the micronuclei of bone marrow in rats exposed to the test substance at doses of up to 800 mg/kg bw/day for 90 days as part of a 90-day repeated dose toxicity study.
Taking into account all available data on genetic toxicity for W630, there was only one positive chromosome aberration test in human lymphocytes. This non-GLP screening chromosome aberration test performedsimilar to OECD 473 showed structural chromosome aberrations following the 3 h treatment with W630 in the presence of metabolic activation (Watters, 2012). In the same test system, W630 did not induce chromosome aberrations following treatment for 20 h without metabolic activation, when tested up to cytotoxic concentrations.
In contrast to this, W630 in the presence of metabolic activation was tested negative in an in vitro micronucleus assay according to OECD 487 and under GLP conditions (Watters, 2013). In the absence of metabolic activation, the outcome of the study was considered to be ambiguous based on a positive result at cytotoxic concentration for the 3 h treatment and a negative result for the 24 h treatment.
In a non-GLP screening micronucleus assay, W630did not induce micronuclei in human lymphocytes following the 3 h treatment with metabolic activation and following 24 h without metabolic activation (Watters, 2012).
The available study on gene mutation in bacteria (Ames test) was negative (Mc Garry, 2012).
In order to further investigate W630 for clastogenic effects, an in vivo micronucleus assay was performed within a 90-day repeated dose toxicity study in rats (Braun, 2013).Under the experimental conditions reported, W630 did not induce clastogenic effects on bone marrow in rats exposed to W630 at doses of up to 800 mg/kg bw/day for 90 days.
Therefore, it can be concluded that W630 is not considered to have a genotoxic potential.
Justification for selection of genetic toxicity endpoint
There was one study available showing a positive result for in vitro
chromosome aberration in human lymphocytes in the presence of metabolic
activation. The available studies on in vitro gene mutation in bacteria
and in vivo micronucleus test were negative. Two in vitro micronucleus
assays were negative or ambiguous.
Short description of key information:
In vitro:
Gene mutation (Bacterial reverse mutation assay / Ames test): S.
typhimurium TA 1535, TA 1537, TA 98, TA 100, and TA 102: negative with
and without metabolic activation (according to OECD 471)
Chromosome aberration in cultured peripheral human lymphocytes
(micronucleus assay): negative with metabolic activation and ambiguous
without metabolic activation (according to OECD 487; -S9 mix:
concentrations of 50, 90 and 110 µg/mL (3 h treatment) and 4, 14 and 18
µg/mL (24 h treatment); +S9 mix: concentrations of 50, 120 and 140 µg/mL
(3 h treatment))
Chromosome aberration in cultured peripheral human lymphocytes
(micronucleus assay): negative with and without metabolic activation
(similar to OECD 487; -S9 mix: concentrations of 2, 10 and 20 µg/mL (24
h treatment); +S9 mix: concentrations of 50, 100, 120 and 140 µg/mL (3 h
treatment))
Chromosome aberration in cultured peripheral human lymphocytes
(chromosome aberration test): negative with metabolic activation and
positive with metabolic activation (similar to OECD 473; -S9 mix:
concentrations of 10, 20, 30 µg/mL (20 h treatment); +S9 mix:
concentrations of 50, 120, 170 µg/mL (3 h treatment))
In vivo:
Chromosome aberration (micronucleus assay): negative (similar to OECD
474; 3 dose levels (50, 200, 800 mg/kg bw/day; rats were treated orally
by gavage for a period of at least 90 d; preparation 24 h after the last
treatment))
Endpoint Conclusion: No adverse effect observed (negative)
Justification for classification or non-classification
The available data on genetic toxicity of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.
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