Registration Dossier
Registration Dossier
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EC number: 308-766-0 | CAS number: 98283-67-1
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Genetic toxicity in vitro:
Bacterial mutagenicity (Ames / OECD 471): negative with and without metabolic activation
Mammalian cytogenicity (CA / OECD 473): negative with and without metabolic activation (RA from CAS 110615-47-9)
Mammalian mutagenicity (MLA / OECD 476): negative with and without metabolic activation (RA from CAS 68515-73-1)
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 04 Jan - 22 Feb 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP - Guideline study. Due to cytotoxicity more marked with pre-incubation, of the six tested doses, only four instead of 5 doses were analysable in the second assay in tester strain TA100 (+S9) as recommended by OECD guideline 471.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- (1997)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Groupe interministeriel des produits chimiques, Paris, France
- 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
- Additional strain / cell type characteristics:
- other: TA 1535, TA 1537, TA 98 and TA 100 carry a mutation of the uvr B gene and the deep rough mutation (rfa), TA100 and TA98 contain the R-factor plasmid (pkM101). TA 102 carries the deep rough mutation (rfa) and contains the R-factor plasmid (pAQ1)
- 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:
- Preliminary toxicity test:
- 50, 150, 500, 1500, 5000 µg/plate (with and without metabolic activation)
Experiment I:
- 5, 15, 50, 150, 500, 1000 µg/plate (without metabolic activation; TA 1535, TA 1537, TA 100 and TA 102)
- 5, 15, 50, 150, 500, 1500 µg/plate (without metabolic activation, TA 98)
- 15, 50, 150, 500, 1500, 3000 µg/plate (with metabolic activation, all tester strains)
Experiment II:
- 5, 15, 50, 150, 500, 1000 µg/plate (without metabolic activation, TA 1535)
- 1.5, 5, 15, 50, 150, 500 µg/plate (without metabolic activation, TA 1537)
- 5, 15, 50, 150, 500, 750 µg/plate (without metabolic activation, TA 98, TA 100 and TA 102)
- 5, 15, 50, 150, 500, 1500 µg/plate (with metabolic activation, TA 98, TA 102 and TA 1535, pre-incubation method)
- 5, 15, 50, 150, 500, 1000 µg/plate (with metabolic activation, TA 100 and TA 1537, pre-incubation method) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: sterile water
- Justification for choice of solvent/vehicle: The vehicle was chosen due to the solubility properties of the test item. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: -S9-mix: sodium azide (TA 1535, TA 100), 9-amino-acridine (TA 1537), 2-nitro fluorene (TA 98), mitomycin c (TA 102); +S9-mix: 2-anthramine (TA 1535, TA 1537, TA 98, TA 100), benzo[a]pyrene (TA 102)
- Remarks:
- sodium azide (1 µg/plate), 9-amino-acridine (50 µg/plate), 2-nitro fluorene (2 µg/plate), mitomycin c (0.125 µg/plate), 2-anthramine (2/1 µg/plate without/with pre-incubation), benzo[a]pyrene (2 µg/plate without/with pre-incubation)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: plate incorporation; pre-incubation (experiment II with metabolic activation)
DURATION
- Pre-incubation period: 60 minutes
NUMBER OF REPLICATIONS: 3 plates for each test concentration
DETERMINATION OF CYTOTOXICITY
- Method: In order to choose the range of doses for the test, the toxic activity of the test item was determined. The toxicity assay was carried out in all the strains to be tested under the same conditions as the mutagenicity test with and without metabolic activation but using only one plate per dose instead of 3. The plates were incubated for approximately 44 hours at ca. 37°C, and the revertants were counted. The maximum dose in the preliminary toxicity assay was the maximum dose according to OECD Guideline 471 e.g. 5000 μg/plate. Toxicity was checked by microscopic examination of the background growth. - Evaluation criteria:
- A test system is considered as mutagenic if
- a test item causing a positive response proportional to the dose for at least 3 doses with, for the highest increase, a value greater than or equal to 3 times the value for the solvent control, is considered positive in the assay (TA 1535 and TA 1537)
- a test item causing a positive response proportional to the dose for at least 3 doses with, for the highest increase, a value greater than or equal to 2 times the value for the solvent control, is considered positive in the assay (TA 98, TA 100 and TA 102)
In some borderline cases, an additional criterion to be considered is the comparison between the number of revertants induced by the test item and the laboratory historical control data. Indeed, an increase in each individual value that is above the highest value of corresponding historical control data can help supporting a conclusion such as “equivocal” or “weak” mutagen. If a test item causes a positive response during a single assay and that result cannot be reproduced in at least 1 independent assay, the initial positive result may be considered as not significant. If, in all experimental conditions examined, none of the above criteria are fulfilled, a test item is considered clearly negative and unable to induce mutations in this test system. - Statistics:
- For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated. In addition, data was analysed by means of Dunnett's method allowing the comparison of the mean value for each dose to the mean value for the corresponding solvent control.
- Key result
- Species / strain:
- other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Exp I: -S9: 1000 µg/plate: TA1535, TA1537, TA100; 1500 µg/plate: TA98; +S9: 1500 µg/plate: TA100; 3000 µg/plate: TA1535, TA1537, TA98; Exp II: -S9: 750 µg/plate: TA100; 1000 µg/plate: TA1535; +S9: 500 µg/plate: TA1535, TA1537, TA100; 1500 µg/plate: TA98
- Vehicle controls validity:
- valid
- Untreated 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
- Remarks:
- Exp I: -S9: 1000 µg/plate: TA102; +S9: 3000 µg/plate: TA102; Exp II: -S9: 750 µg/plate: TA102; +S9: 150 µg/plate: TA102
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test item was fully soluble in the vehicle (sterile water).
- Precipitation: No precipitation was observed in the main mutagenicity assay (experiment I and II) neither in the presence nor in the absence of metabolic activation.
RANGE-FINDING/SCREENING STUDIES
Cytotoxicity of the test item was assessed in a preliminary experiment resulting in:
- an important toxicity with the presence of microcolonies at the two highest tested doses of 5000 and 1500μg/plate without metabolic activation in strains TA1535, TA1537, TA100 and TA102
- an important toxicity with the presence of microcolonies at the highest tested dose of 5000μg/plate in all strains with metabolic activation and in strain TA98 without metabolic activation
- a moderate toxicity at the tested dose of 1500μg/plate in strain TA98 without metabolic activation and in strain TA100 with metabolic activation
- a slight toxicity at the tested dose of 500μg/plate in strains TA1535 ad TA1537 without metabolic activation and at the tested dose of 1500μg/plate in strains TA1535, TA1537, TA98 and TA102 with metabolic activation.
Therefore, the maximum doses retained for the first mutagenicity assay in the absence of metabolic activation were 1000 µg/plate (TA1535, TA1537, TA100 and TA102) and 1500μg/plate (TA 98) and 3000 µg/plate in all strains in the presence of metabolic activation.
COMPARISON WITH HISTORICAL CONTROL DATA
The results were within the range of the historical control data.
ADDITIONAL INFORMATION ON CYTOTOXICITY
Cytotoxicity was recorded at 1000 µg/plate (TA 1535, TA 1537, TA 100, TA 102) and 1500 µg/plate (TA 98) in the absence of metabolic activation and at 1500 µg/plate (TA 100) and 3000 µg/plate (TA 1535, TA 1537, TA 98, TA 102) in the presence of metabolic activation (experiment I). In the second experiment cytotoxicity was present at 750 µg/plate (TA 100, TA 102) and at 1000 µg/plate /TA 1535) in the absence of metabolic activation and at 150 µg/plate (TA 102), 500 µg/plate (TA 1535, TA 1537, TA 100) and 1500 µg/plate (TA 98) in the presence of metabolic activation. - Conclusions:
- A bacterial gene mutation assay (Ames test) was performed with the test material according to OECD TG 471 and in compliance with GLP. The test material was considered not to be mutagenic in the presence and absence of metabolic activation in the tester strains used.
- 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:
- 16 Nov - 02 Dec 1993
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: GLP - Guideline study with acceptable restrictions. Positive controls were only used in the 20-h treatment.
- Justification for type of information:
- refer to category justification provided in IUCLID section 13
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- yes
- Remarks:
- positive controls were only used in the 20-h treatment
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- yes
- Remarks:
- positive controls were only used in the 20-h treatment
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- Not applicable
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: Flow Laboratories, UK
- Cell cycle length, doubling time or proliferation index: 12 - 18 h
MEDIA USED
- Type and identity of media including CO2 concentration: Stock cultures: Eagle’s Minimal Essential Medium (EMEM) containing 10% (v/v) fetal calf serum, 1% L-glutamine, 1% non-essential amino acids and 2% Penicillin/Streptomycin; Treatment cultures: FCS-free MEM; 37 °C at 6% CO2
- Properly maintained: yes
- Periodically checked for karyotype stability: yes - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9-mix) (CCR, Rossdorf, Germany), prepared from livers of male Wistar rats induced with Arochlor 1254 (500 mg/kg bw, single i.p. injection)
- Test concentrations with justification for top dose:
- Preliminary toxicity test: 0.5, 1, 2.5, 5, 10, 25, 50, 100, 250, 500, 1000, 2500 and 5000 µg/mL (±S9)
7 h and 28 h culture period: 2, 4, 8 and 16 µg/mL (-S9); 20, 40, 80 and 160 µg/mL (+S9)
20 h culture period: 0.5, 1, 2, 4, 8 and 16 µg/mL (-S9); 5, 10, 20, 40, 80 and 160 µg/mL (+S9) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: medium and DMSO
- Untreated negative controls:
- yes
- Remarks:
- medium
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 20 h experiment: -S9: ethylmethanesulfonate (EMS), 0.75 mg/mL, +S9; cyclophosphamide (CPA), 1 µg/mL
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Pre-incubation period (main experiment): 15 - 30 h
- Exposure duration: 4 h
- Fixation time (start of exposure up to fixation or harvest of cells): 7, 20, 28 h
SPINDLE INHIBITOR: colcemide 0.4 µg/mL medium (2-2.5 h before harvesting)
STAIN: Giemsa solution
NUMBER OF REPLICATIONS: duplicate cultures
NUMBER OF CELLS EVALUATED: 100 metaphases per culture
DETERMINATION OF CYTOTOXICITY
- Method: relative plating efficiency (preliminary toxicity test); mitotic index of 500 cells (main experiment)
OTHER EXAMINATIONS:
- Determination of polyploidy: yes
ACCEPTABILITY OF THE ASSAY
The test is considered acceptable if the results comply with the following criteria: the number of cells with structural aberrations excluding gaps of the negative and/or solvent control should fall within the range of approx. 0.0 - 4.0%, which can usually be found in negative control preparations of this cell type; the positive controls should induce a significant increase versus negative controls in the proportion of cells with structural chromosomal aberrations. - Evaluation criteria:
- If the mitotic indices are reduced, the highest concentrations leading to a reduction of the mitotic index to approx. 20 - 50% are analysed. If no toxicity by reduction of the mitotic index is found, the highest tested concentration should be investigated unless other indications of toxicity were observed. In this case, the highest obviously non-toxic concentration was chosen for chromosomal analysis. The test substance was classified as mutagenic if a significant, concentration-related increase in the proportion of structural aberrations was induced or a significant positive response for at least one test concentration was found. The test substance was classified as non-mutagenic in this test system, if neither a significant concentration-related increase in the proportion of structural chromosomal aberrations nor a significant positive response at any of the analysed test substance concentrations was detected.
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Preliminary cytotoxicity test: ≥ 5 µg/mL (-S9) and ≥ 100 µg/mL (+S9); main experiment: at 160 µg/mL (+S9, all 3 fixation times)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- RANGE-FINDING/SCREENING STUDIES:
In the experiment on plating efficiency, strong toxic effects were noticed at ≥ 5 µg/mL without metabolic activation and ≥100 µg/mL with S9 mix.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the cytogenetic experiments, the test substance was applied up to 16 µg/mL without and 160 µg/mL with metabolic activation. In the experiments without S9 mix, no substantial reduction of the Mitotic Index (MI) was observed at the highest concentration. With metabolic activation, there was no cellular growth to be detected at 160 µg/mL at all three fixation times, and nearly no mitoses to be detected at 80 µg/mL 7 h after treatment. No substantial reduction of the MI was determined at 80 µg/mL after incubation periods of 20 and 28 h. Treatment concentrations for chromosome analysis were selected by evaluating the effect of the test substance on the mitotic index. The highest concentrations for chromosome analysis from the 7, 20 and 28 h harvesting time points were those that resulted in 50-80% reduction of mitotic index or the highest test concentration if no cytotoxicity was observed. - Conclusions:
- In a chromosomal aberration assay according to OECD 473 and GLP, no clastogenic effect was observed for the test material, either in the presence or absence 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:
- key study
- Study period:
- 20 Dec 1990 - 27 Mar 1991
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: GLP - Guideline study with acceptable restrictions. Colony sizing procedures were not described, and colony size was not reported for the negative and positive controls. No mutant frequency values were provided.
- Justification for type of information:
- refer to category justification provided in IUCLID section 13
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- yes
- Remarks:
- Colony sizing procedures were not described, and colony size was not reported for the negative and positive controls. No mutant frequency values were provided.
- GLP compliance:
- yes
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- TK locus
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- - Type and identity of media: Fischer's medium for leukemic cells of mice with 0.1 % Pluronics (treatment medium); supplemented with 10 % horse serum and 4 mM L-glutamine (expression period medium). Cloning medium was treatment medium with 20 % horse serum and 0.25 % agar.
- Properly maintained: yes
- Periodically "cleansed" against high spontaneous background: yes - Additional strain / cell type characteristics:
- other: heterozygous at the thymidine kinase locus (TK +/-); clone 3.7.2C
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1242 and Aroclor 1254 (2:1 mixture) induced rat liver S9-mix
- Test concentrations with justification for top dose:
- Experiment I:
With S9 mix: 13-1004 µg/mL - data only presented for concentrations up to 179 µg/mL due to higher doses prohibiting cloning
Without S9 mix: 7.5-565 µg/mL - data only presented for concentrations up to 101 µg/mL due to higher doses prohibiting cloning
Experiment II:
With S9-mix: 103-260 µg/mL - data only presented for concentrations ranging from 161 to 234 µg/mL due to higher doses prohibiting cloning - Vehicle / solvent:
- - Vehicle/solvent used: sterile, distilled water
- Justification for choice of solvent/vehicle: solubility - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- sterile distilled water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: ethylmethanesulphonate (-S9; 0.5 and 0.25 µL/mL); 7,12-dimethylbenzanthracene (+S9; 5 and 2.5 µg/mL)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 10-12 days
- Fixation time (start of exposure up to fixation or harvest of cells): 12-14 days
SELECTION AGENT: trifluorothymidine
NUMBER OF REPLICATIONS: 1 tube was treated per test substance group and positive control group; 2 tubes were treated for the solvent control group. 3 plates with selection agent and 3 plates without selection agent were plated per treatment tube.
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
OTHER: for expression of the mutant phenotype, the cultures were counted and adjusted to 3.0E+5 cells/mL (if the cell population exceeded 3.0E+5 cells/mL) at approximately 24 and 48 hours after treatment in 20 and 10 mL total volume, respectively. - Evaluation criteria:
- The following criteria were used as guidelines in judging the significance of the activity of the test substance in the system. In evaluating the results, it was considered that increases in mutant frequencies, which occurred only at highly toxic concentrations, may have been due to epigenetic events. However, it was impossible to formulate criteria which could apply to all types of data which may have been generated and therefore the scientist's evaluation was the final endpoint.
1. Positive: if there was a positive dose response and one or more of the three highest doses in the 0% or greater total growth range exhibited a mutant frequency which was two-fold greater than the background level. All data including that from cultures with less than 10% total growth would be used to establish the dose response relationship.
2. Equivocal: if there was no dose response but any one or more of the three highest doses with 10% or greater total growth exhibited a two-fold increase in mutant frequency over background, or if there was a dose response but no culture exhibited a two-fold increase in mutant frequency over background.
3. Negative: if there was no dose response in cultures with 10% or greater total growth and none of these test cultures exhibited a two-fold or greater increase in mutant frequency over background. - Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- yes, at 1003 µg/mL (with/without S9)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- RANGE-FINDING/SCREENING STUDIES:
The optimal dose levels for the mutagenicity assays were selected following a preliminary toxicity test based on cell population growth relative to the solvent controls. L5178Y cells were exposed to solvent alone and seven concentrations of the test substance ranging from 5013 to 0.01 ug/ml for 4 hours in the absence and presence metabolic activation. Each tube was gassed with 5 +/- 1 % CO2 in air and placed on a Bellco roller drum apparatus rotating at approximately 25 rpm. The final solvent concentration in the culture medium was 1 % by volume. The test solutions were prepared under amber light s and kept in darkness during the entire exposure period. After approximately 4 hours, the test substance in solution was removed by centrifuging the cells at 1000 rpm for 10 minutes and decanting the supernatant. The cells were washed twice in 10 ml of medium (containing serum), resuspended in 20 ml of medium (containing serum), gassed with 5 +/- 1 % CO2 in air, and replaced on the roller drum apparatus. Cell population density was determined 24 and 48 hours after the initial exposure to the test substance by removing a sample from each treatment tube, diluting in 0.1 % trypsin, incubating at 37 +/- 1 degrees C for 10 minutes, and counting the samples with an electronic cell counter. The cultures were adjusted to 2.0 E5 cells/ml (if the cell population exceeded 3.0 E5 cells/ml) after 24 hours, only.
The toxicity test indicted 100 % toxicity at 1003 ug/ml for both the non-activated cultures and for the activated cultures.
ADDITIONAL INFORMATION ON CYTOTOXICITY: See below - Conclusions:
- The test material was evaluated in the L5178Y TK+/-Mouse Lymphoma Mutagenesis Assay performed equivalent to OECD 476 and under GLP conditions in the absence and presence of metabolic activation. Under the conditions of the assay, the test substance was found to be negative for mutagenic activity.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Justification for type of information:
- refer to category justification provided in IUCLID section 13
- Reason / purpose for cross-reference:
- read-across source
- Target gene:
- Not applicable
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: Flow Laboratories, UK
- Cell cycle length, doubling time or proliferation index: 12 - 18 h
MEDIA USED
- Type and identity of media including CO2 concentration: Stock cultures: Eagle’s Minimal Essential Medium (EMEM) containing 10% (v/v) fetal calf serum, 1% L-glutamine, 1% non-essential amino acids and 2% Penicillin/Streptomycin; Treatment cultures: FCS-free MEM; 37 °C at 6% CO2
- Properly maintained: yes
- Periodically checked for karyotype stability: yes - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9-mix) (CCR, Rossdorf, Germany), prepared from livers of male Wistar rats induced with Arochlor 1254 (500 mg/kg bw, single i.p. injection)
- Test concentrations with justification for top dose:
- Preliminary toxicity test: 0.5, 1, 2.5, 5, 10, 25, 50, 100, 250, 500, 1000, 2500 and 5000 µg/mL (±S9)
7 h and 28 h culture period: 2, 4, 8 and 16 µg/mL (-S9); 20, 40, 80 and 160 µg/mL (+S9)
20 h culture period: 0.5, 1, 2, 4, 8 and 16 µg/mL (-S9); 5, 10, 20, 40, 80 and 160 µg/mL (+S9) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: medium and DMSO
- Untreated negative controls:
- yes
- Remarks:
- medium
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 20 h experiment: -S9: ethylmethanesulfonate (EMS), 0.75 mg/mL, +S9; cyclophosphamide (CPA), 1 µg/mL
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Pre-incubation period (main experiment): 15 - 30 h
- Exposure duration: 4 h
- Fixation time (start of exposure up to fixation or harvest of cells): 7, 20, 28 h
SPINDLE INHIBITOR: colcemide 0.4 µg/mL medium (2-2.5 h before harvesting)
STAIN: Giemsa solution
NUMBER OF REPLICATIONS: duplicate cultures
NUMBER OF CELLS EVALUATED: 100 metaphases per culture
DETERMINATION OF CYTOTOXICITY
- Method: relative plating efficiency (preliminary toxicity test); mitotic index of 500 cells (main experiment)
OTHER EXAMINATIONS:
- Determination of polyploidy: yes
ACCEPTABILITY OF THE ASSAY
The test is considered acceptable if the results comply with the following criteria: the number of cells with structural aberrations excluding gaps of the negative and/or solvent control should fall within the range of approx. 0.0 - 4.0%, which can usually be found in negative control preparations of this cell type; the positive controls should induce a significant increase versus negative controls in the proportion of cells with structural chromosomal aberrations. - Evaluation criteria:
- If the mitotic indices are reduced, the highest concentrations leading to a reduction of the mitotic index to approx. 20 - 50% are analysed. If no toxicity by reduction of the mitotic index is found, the highest tested concentration should be investigated unless other indications of toxicity were observed. In this case, the highest obviously non-toxic concentration was chosen for chromosomal analysis. The test substance was classified as mutagenic if a significant, concentration-related increase in the proportion of structural aberrations was induced or a significant positive response for at least one test concentration was found. The test substance was classified as non-mutagenic in this test system, if neither a significant concentration-related increase in the proportion of structural chromosomal aberrations nor a significant positive response at any of the analysed test substance concentrations was detected.
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Preliminary cytotoxicity test: ≥ 5 µg/mL (-S9) and ≥ 100 µg/mL (+S9); main experiment: at 160 µg/mL (+S9, all 3 fixation times)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- RANGE-FINDING/SCREENING STUDIES:
In the experiment on plating efficiency, strong toxic effects were noticed at ≥ 5 µg/mL without metabolic activation and ≥100 µg/mL with S9 mix.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the cytogenetic experiments, the test substance was applied up to 16 µg/mL without and 160 µg/mL with metabolic activation. In the experiments without S9 mix, no substantial reduction of the Mitotic Index (MI) was observed at the highest concentration. With metabolic activation, there was no cellular growth to be detected at 160 µg/mL at all three fixation times, and nearly no mitoses to be detected at 80 µg/mL 7 h after treatment. No substantial reduction of the MI was determined at 80 µg/mL after incubation periods of 20 and 28 h. Treatment concentrations for chromosome analysis were selected by evaluating the effect of the test substance on the mitotic index. The highest concentrations for chromosome analysis from the 7, 20 and 28 h harvesting time points were those that resulted in 50-80% reduction of mitotic index or the highest test concentration if no cytotoxicity was observed. - Conclusions:
- In a chromosomal aberration assay according to OECD 473 and GLP, no clastogenic effect was observed for the test material, either in the presence or absence of metabolic activation.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Justification for type of information:
- refer to category justification provided in IUCLID section 13
- Reason / purpose for cross-reference:
- read-across source
- Target gene:
- TK locus
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- - Type and identity of media: Fischer's medium for leukemic cells of mice with 0.1 % Pluronics (treatment medium); supplemented with 10 % horse serum and 4 mM L-glutamine (expression period medium). Cloning medium was treatment medium with 20 % horse serum and 0.25 % agar.
- Properly maintained: yes
- Periodically "cleansed" against high spontaneous background: yes - Additional strain / cell type characteristics:
- other: heterozygous at the thymidine kinase locus (TK +/-); clone 3.7.2C
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1242 and Aroclor 1254 (2:1 mixture) induced rat liver S9-mix
- Test concentrations with justification for top dose:
- Experiment I:
With S9 mix: 13-1004 µg/mL - data only presented for concentrations up to 179 µg/mL due to higher doses prohibiting cloning
Without S9 mix: 7.5-565 µg/mL - data only presented for concentrations up to 101 µg/mL due to higher doses prohibiting cloning
Experiment II:
With S9-mix: 103-260 µg/mL - data only presented for concentrations ranging from 161 to 234 µg/mL due to higher doses prohibiting cloning - Vehicle / solvent:
- - Vehicle/solvent used: sterile, distilled water
- Justification for choice of solvent/vehicle: solubility - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- sterile distilled water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: ethylmethanesulphonate (-S9; 0.5 and 0.25 µL/mL); 7,12-dimethylbenzanthracene (+S9; 5 and 2.5 µg/mL)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 10-12 days
- Fixation time (start of exposure up to fixation or harvest of cells): 12-14 days
SELECTION AGENT: trifluorothymidine
NUMBER OF REPLICATIONS: 1 tube was treated per test substance group and positive control group; 2 tubes were treated for the solvent control group. 3 plates with selection agent and 3 plates without selection agent were plated per treatment tube.
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
OTHER: for expression of the mutant phenotype, the cultures were counted and adjusted to 3.0E+5 cells/mL (if the cell population exceeded 3.0E+5 cells/mL) at approximately 24 and 48 hours after treatment in 20 and 10 mL total volume, respectively. - Evaluation criteria:
- The following criteria were used as guidelines in judging the significance of the activity of the test substance in the system. In evaluating the results, it was considered that increases in mutant frequencies, which occurred only at highly toxic concentrations, may have been due to epigenetic events. However, it was impossible to formulate criteria which could apply to all types of data which may have been generated and therefore the scientist's evaluation was the final endpoint.
1. Positive: if there was a positive dose response and one or more of the three highest doses in the 0% or greater total growth range exhibited a mutant frequency which was two-fold greater than the background level. All data including that from cultures with less than 10% total growth would be used to establish the dose response relationship.
2. Equivocal: if there was no dose response but any one or more of the three highest doses with 10% or greater total growth exhibited a two-fold increase in mutant frequency over background, or if there was a dose response but no culture exhibited a two-fold increase in mutant frequency over background.
3. Negative: if there was no dose response in cultures with 10% or greater total growth and none of these test cultures exhibited a two-fold or greater increase in mutant frequency over background. - Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- yes, at 1003 µg/mL (with/without S9)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- RANGE-FINDING/SCREENING STUDIES:
The optimal dose levels for the mutagenicity assays were selected following a preliminary toxicity test based on cell population growth relative to the solvent controls. L5178Y cells were exposed to solvent alone and seven concentrations of the test substance ranging from 5013 to 0.01 ug/ml for 4 hours in the absence and presence metabolic activation. Each tube was gassed with 5 +/- 1 % CO2 in air and placed on a Bellco roller drum apparatus rotating at approximately 25 rpm. The final solvent concentration in the culture medium was 1 % by volume. The test solutions were prepared under amber light s and kept in darkness during the entire exposure period. After approximately 4 hours, the test substance in solution was removed by centrifuging the cells at 1000 rpm for 10 minutes and decanting the supernatant. The cells were washed twice in 10 ml of medium (containing serum), resuspended in 20 ml of medium (containing serum), gassed with 5 +/- 1 % CO2 in air, and replaced on the roller drum apparatus. Cell population density was determined 24 and 48 hours after the initial exposure to the test substance by removing a sample from each treatment tube, diluting in 0.1 % trypsin, incubating at 37 +/- 1 degrees C for 10 minutes, and counting the samples with an electronic cell counter. The cultures were adjusted to 2.0 E5 cells/ml (if the cell population exceeded 3.0 E5 cells/ml) after 24 hours, only.
The toxicity test indicted 100 % toxicity at 1003 ug/ml for both the non-activated cultures and for the activated cultures.
ADDITIONAL INFORMATION ON CYTOTOXICITY: See below - Conclusions:
- The test material was evaluated in the L5178Y TK+/-Mouse Lymphoma Mutagenesis Assay performed equivalent to OECD 476 and under GLP conditions in the absence and presence of metabolic activation. Under the conditions of the assay, the test substance was found to be negative for mutagenic activity.
Referenceopen allclose all
Table 1: Results of the preliminary cytotoxicity test
Toxicity Assay |
Dose in µg/plate |
TA 1535 |
TA 1537 |
TA 98 |
TA 100 |
TA 102 |
|||||
T |
P |
T |
P |
T |
P |
T |
P |
T |
P |
||
Test item without S9-mix |
0 50 150 500 1500 5000 |
- - - + +++ +++ |
- - - - - - |
- - - + +++ +++ |
- - - - - - |
- - - - ++ +++ |
- - - - - - |
- - - - +++ +++ |
- - - - - - |
- - - - +++ +++ |
- - - - - - |
Top Dose in first mutagenicity assay |
|
1000 |
1000 |
1500 |
1000 |
1000 |
|||||
Test item with S9-mix |
0 50 150 500 1500 5000 |
- - - - + +++ |
- - - - - - |
- - - - + +++ |
- - - - - - |
- - - - + +++ |
- - - - - - |
- - - - ++ +++ |
- - - - - - |
- - - - + +++ |
- - - - - - |
Top dose in first mutagenicity assay |
|
3000 |
3000 |
3000 |
3000 |
3000 |
|||||
T: toxicity (- non toxic: + slightly toxic: ++ moderately toxic: +++ strongly toxic)
P: precipitation (- absence: + slight precipitate: ++ moderate precipitate: +++ important precipitate)
Table 2: Results of mutagenicity assay (experiment I)
With or without S9-Mix |
Test substance concentration (μg/plate) |
Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation) |
||||
Base-pair substitution type |
Frameshift type |
|||||
TA 100 |
TA1535 |
TA 102 |
TA98 |
TA1537 |
||
– |
0 |
93.8 ± 6.1 |
14.0 ± 2.8 |
107.7 ± 23.9 |
16.2 ± 1.9 |
6.8 ± 3.4 |
– |
5 |
85.7 ± 7.1 |
14.3 ± 3.5 |
105.3 ± 15.5 |
10.3 ± 1.2 |
6.0 ± 3.0 |
– |
15 |
97.3 ± 3.5 |
17.3 ± 5.9 |
112.7 ± 9.2 |
14.3 ± 2.1 |
4.7 ± 0.6 |
– |
50 |
95.0 ± 11.3 |
16. 7 ± 1.5 |
127.3 ± 8.1 |
15.7 ± 4.2 |
4.0 ± 1.0 |
– |
150 |
85.3 ± 6.5 |
13.0 ± 2.6 |
114.7 ± 1.2 |
10.7 ± 6.4 |
3.7 ± 3.8 |
– |
500 |
82.7 ± 29.1 |
15.3 ± 4.5 |
100.7 ± 32.9 |
12.0 ± 4.0 |
4.0 ± 2.6 |
– |
1000 |
18.7 ± 1.5 |
9.3 ± 2.1 |
50.0 ± 2.0 |
- |
** |
– |
1500 |
- |
- |
- |
4.3 ± 2.1 |
- |
Positive controls, –S9 |
Name |
NaN |
NaN |
MM-c |
2-NF |
9-AA |
Concentrations (μg/plate) |
1 |
1 |
0.125 |
2 |
50 |
|
Mean No. of colonies/plate (average of 3 ± SD) |
624.0 ± 97.3 |
477.3 ± 258.8 |
480.0 ± 142.2 |
230.7 ± 31.9 |
450.7 ± 117.1 |
|
+ |
0 |
110.5 ± 16.1 |
12.0 ± 3.7 |
179.3 ± 27.1 |
21.7 ± 4.1 |
6.7 ± 1.9 |
+ |
15 |
102.7 ± 5.5 |
8.7 ± 2.9 |
210.0 ± 28.8 |
24.0 ± 4.0 |
6.0 ± 1.0 |
+ |
50 |
104.0 ± 5.0 |
12.3 ± 2.5 |
193.3 ± 17.5 |
19.3 ± 8.1 |
6.7 ± 3.2 |
+ |
150 |
84.3 ± 2.5 |
9.0 ± 2.0 |
155.3 ± 25.0 |
19.0 ± 5.2 |
8.0 ± 2.6 |
+ |
500 |
94.7 ± 5.7 |
7.7 ± 0.6 |
189.3 ± 12.2 |
27.0 ± 6.6 |
5.0 ± 4.6 |
+ |
1500 |
31.3 ± 4.9 |
11.0 ± 2.6 |
126.0 ± 30.0 |
24.0 ± 7.9 |
3.0 ± 2.6 |
+ |
3000 |
** |
** |
22.7 ± 6.7 |
** |
** |
Positive controls, +S9 |
Name |
2-AA |
2-AA |
BaP |
2-AA |
2-AA |
Concentrations (μg/plate) |
2 |
2 |
2 |
2 |
2 |
|
Mean No. of colonies/plate (average of 3 ± SD) |
1738.7 ± 268.8 |
418.7 ± 66.2 |
912.0 ± 224.0 |
1888.0 ± 279.0 |
154.7 ± 24.1 |
|
**: not analyzable due to toxicity
NaN: sodium azide
MM-c: mitomycin c
2 -NF: 2 -nitrofluorene
9 -AA: 9 -aminoacridine
2 -AA: 2 -anthramine
BaP: benz[a]pyrene
-: concentration not assessed
Table 3: Results of mutagenicity assay (experiment II)
With or without S9-Mix |
Test substance concentration (μg/plate) |
Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation) |
||||
Base-pair substitution type |
Frameshift type |
|||||
TA 100 |
TA1535 |
TA 102 |
TA98 |
TA1537 |
||
– |
0 |
114.3 ± 19.7 |
12.5 ± 3.2 |
165.3 ± 20.1 |
14.8 ± 2.0 |
5.7 ± 1.6 |
|
1.5 |
- |
- |
- |
- |
9.3 ± 2.1 |
– |
5 |
109.7 ± 10.1 |
12.0 ± 3.5 |
196.0 ± 7.2 |
18.0 ± 5.2 |
9.0 ± 2.6 |
– |
15 |
104.0 ± 6.0 |
11.0 ± 2.6 |
188.7 ± 26.6 |
14.0 ± 1.0 |
10.0 ± 5.0 |
– |
50 |
91.0 ± 8.5 |
14.3 ± 6.4 |
173.3 ± 3.1 |
12.0 ± 1.0 |
7.7 ± 3.8 |
– |
150 |
107.7 ± 12.2 |
15.3 ± 4.2 |
139.3 ± 8.1 |
14.0 ± 1.7 |
4.7 ± 0.6 |
– |
500 |
93.3 ± 11.9 |
10.3 ± 4.0 |
141.3 ± 16.0 |
15.3 ± 4.2 |
6.7 ± 0.6 |
|
750 |
65.0 ± 2.8 |
- |
95.3 ± 21.6 |
13.3 ± 0.6 |
- |
– |
1000 |
- |
8.0 ± 2.6 |
- |
- |
- |
Positive controls, –S9 |
Name |
NaN |
NaN |
MM-c |
2-NF |
9-AA |
Concentrations (μg/plate) |
1 |
1 |
0.125 |
2 |
50 |
|
Mean No. of colonies/plate (average of 3 ± SD) |
544.0 ± 48.0 |
421.3 ± 21.2 |
490.7 ± 33.3 |
244.7 ± 36.7 |
580.0 ± 102.1 |
|
+ |
0 |
71.7 ± 5.9 |
11.2 ± 3.3 |
202.0 ± 29.4 |
20.2 ± 6.5 |
5.7 ± 1.2 |
+ |
5 |
74.0 ± 12.1 |
9.7 ± 2.1 |
178.0 ± 32.7 |
23.7 ± 7.5 |
5.7 ± 0.6 |
+ |
15 |
78.3 ± 6.7 |
10.0 ± 2.0 |
164.7 ± 9.5 |
21.3 ± 5.0 |
9.7 ± 2.3 |
+ |
50 |
81.7 ± 7.2 |
9.7 ± 1.5 |
146.0 ± 9.2 |
22.3 ± 2.5 |
8.3 ± 8.4 |
+ |
150 |
94.0 ± 6.9 |
10.3 ± 1.5 |
116.0 ± 5.3 |
18.3 ± 4.7 |
6.7 ± 3.1 |
+ |
500 |
** |
6.0 ± 2.0 |
117.3 ± 11.4 |
16.3 ± 6.7 |
2.7 ± 1.5 |
|
1000 |
** |
- |
- |
- |
** |
+ |
1500 |
- |
** |
72.0 ± 22.6 |
** |
- |
Positive controls, +S9 |
Name |
2-AA |
2-AA |
BaP |
2-AA |
2-AA |
Concentrations (μg/plate) |
1 |
1 |
2 |
1 |
1 |
|
Mean No. of colonies/plate (average of 3 ± SD) |
1280.7 ± 287.0 |
232.7 ± 20.2 |
536.0 ± 60.4 |
1152.0 ± 180.3 |
83.3 ± 12.7 |
|
**: not analyzable due to toxicity
NaN: sodium azide
MM-c: mitomycin c
2 -NF: 2 -nitrofluorene
9 -AA: 9 -aminoacridine
2 -AA: 2 -anthramine
BaP: benz[a]pyrene
-: concentration not assessed
Table 1: Test results of experiment without S9 mix
Test item
|
Concentration |
Mitotic Index |
Aberrant cells in % |
|
in µg/mL |
in % |
with gaps |
without gaps |
|
Exposure period 4 h, fixation time 7 h |
||||
Medium |
0 |
100.0 |
2.5 |
1.5 |
Test substance |
16 |
93.2 |
2.5 |
2.0 |
Exposure period 4 h, fixation time 20 h |
||||
Medium |
0 |
100.0 |
1.0 |
0.5 |
PC (EMS) |
1.5 |
52.2 |
28.5 |
22 |
Test substance |
2 |
n.d. |
2.0 |
2.0 |
8 |
79.6 |
2.5 |
2.0 |
|
16 |
84.6 |
5.0 |
2.0 |
|
Exposure period 4 h, fixation time 28 h |
||||
Medium |
0 |
100.0 |
2.5 |
1.0 |
|
16 |
97.7 |
1.5 |
1.5 |
PC (positive control): EMS (ethylmethanesulfonate)
n.d.: not determined: no reduction at higher concentrations
Table 2. Test results of experiment with S9 mix
Test item
|
Concentration |
Mitotic Index |
Aberrant cells in % |
|
in µg/mL |
in % |
with gaps |
without gaps |
|
Exposure period 4 h, fixation time 7 h |
||||
Medium |
0 |
100.0 |
5.0 |
3.0 |
Test substance |
40 |
113.1 |
3.5 |
2.5 |
Exposure period 4 h, fixation time 20 h |
||||
Medium |
0 |
100 |
3.5 |
2.5 |
PC (CP) |
1 |
25.4 |
34.5 |
33.5 |
Test substance |
10 |
n.d. |
1.5 |
0.0 |
40 |
n.d. |
3.5 |
1.5 |
|
80 |
86.3 |
5.0 |
2.0 |
|
Exposure period 4 h, fixation time 28 h |
||||
Medium |
0 |
100 |
4.0 |
2.5 |
|
80 |
92.9 |
3.0 |
3.0 |
PC (positive control): CP (cyclophosphamid)
n.d.: not determined: no reduction at higher concentrations
After a two day expression period, ten doses of the non-activated and the activated cultures were selected for cloning. The non-activated cultures that were cloned were treated with 101, 75, 57, 42, 32, 24, 18, 13, 10 and 7.5 µg/ml, which produced a range in suspension growth of 39% to 100%. The activated cultures that were cloned were treated with 179, 134, 101, 75, 57, 42, 32, 24, 18 and 13 µg/mL. These concentrations produced a range in suspension growth of 61% to 106%.
None of the non-activated cultures that were cloned exhibited a mutant frequency which was at least two times the mean mutant frequency of the solvent controls. The total growths of these cultures ranged from 39% to 104%. A dose-dependent response was not noted in the treated cultures.
None of the activated cultures that were cloned exhibited a mutant frequency which was at least two times the mean mutant frequency of the solvent control. The total growths of these cultures ranged from 68% to 117%. A dose-dependent response was not noted in the treated cultures.
In the second experiment, the activated cultures that were cloned were treated with 234, 229, 225, 221, 210, 199, 189, 180, 171 and 161 µg/mL. These concentrations produced a range in suspension growth of 12% to 71%. None of these cultures exhibited a mutant frequency which was at least two times the mean mutant frequency of the solvent control. The total growths of these cultures ranged from 11% to 73%. A significant dose-dependent response was not noted in the treated cultures.
The solvent and positive controls fulfilled the requirements for a valid test.
Table 1: Test results of experiment without S9 mix
Test item
|
Concentration |
Mitotic Index |
Aberrant cells in % |
|
in µg/mL |
in % |
with gaps |
without gaps |
|
Exposure period 4 h, fixation time 7 h |
||||
Medium |
0 |
100.0 |
2.5 |
1.5 |
Test substance |
16 |
93.2 |
2.5 |
2.0 |
Exposure period 4 h, fixation time 20 h |
||||
Medium |
0 |
100.0 |
1.0 |
0.5 |
PC (EMS) |
1.5 |
52.2 |
28.5 |
22 |
Test substance |
2 |
n.d. |
2.0 |
2.0 |
8 |
79.6 |
2.5 |
2.0 |
|
16 |
84.6 |
5.0 |
2.0 |
|
Exposure period 4 h, fixation time 28 h |
||||
Medium |
0 |
100.0 |
2.5 |
1.0 |
|
16 |
97.7 |
1.5 |
1.5 |
PC (positive control): EMS (ethylmethanesulfonate)
n.d.: not determined: no reduction at higher concentrations
Table 2. Test results of experiment with S9 mix
Test item
|
Concentration |
Mitotic Index |
Aberrant cells in % |
|
in µg/mL |
in % |
with gaps |
without gaps |
|
Exposure period 4 h, fixation time 7 h |
||||
Medium |
0 |
100.0 |
5.0 |
3.0 |
Test substance |
40 |
113.1 |
3.5 |
2.5 |
Exposure period 4 h, fixation time 20 h |
||||
Medium |
0 |
100 |
3.5 |
2.5 |
PC (CP) |
1 |
25.4 |
34.5 |
33.5 |
Test substance |
10 |
n.d. |
1.5 |
0.0 |
40 |
n.d. |
3.5 |
1.5 |
|
80 |
86.3 |
5.0 |
2.0 |
|
Exposure period 4 h, fixation time 28 h |
||||
Medium |
0 |
100 |
4.0 |
2.5 |
|
80 |
92.9 |
3.0 |
3.0 |
PC (positive control): CP (cyclophosphamid)
n.d.: not determined: no reduction at higher concentrations
After a two day expression period, ten doses of the non-activated and the activated cultures were selected for cloning. The non-activated cultures that were cloned were treated with 101, 75, 57, 42, 32, 24, 18, 13, 10 and 7.5 µg/ml, which produced a range in suspension growth of 39% to 100%. The activated cultures that were cloned were treated with 179, 134, 101, 75, 57, 42, 32, 24, 18 and 13 µg/mL. These concentrations produced a range in suspension growth of 61% to 106%.
None of the non-activated cultures that were cloned exhibited a mutant frequency which was at least two times the mean mutant frequency of the solvent controls. The total growths of these cultures ranged from 39% to 104%. A dose-dependent response was not noted in the treated cultures.
None of the activated cultures that were cloned exhibited a mutant frequency which was at least two times the mean mutant frequency of the solvent control. The total growths of these cultures ranged from 68% to 117%. A dose-dependent response was not noted in the treated cultures.
In the second experiment, the activated cultures that were cloned were treated with 234, 229, 225, 221, 210, 199, 189, 180, 171 and 161 µg/mL. These concentrations produced a range in suspension growth of 12% to 71%. None of these cultures exhibited a mutant frequency which was at least two times the mean mutant frequency of the solvent control. The total growths of these cultures ranged from 11% to 73%. A significant dose-dependent response was not noted in the treated cultures.
The solvent and positive controls fulfilled the requirements for a valid test.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
Genetic toxicity in vivo:
Mammalian cytogenicity (MNT / OECD 476): negative with and without metabolic activation (RA from CAS 110615-47-9)
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Justification for type of information:
- refer to category justification provided in IUCLID section 13
- Reason / purpose for cross-reference:
- read-across source
- Species:
- mouse
- Strain:
- other: Crl:CD-1TM(ICR)BR
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Limited, Margate, UK
- Age at study initiation: approx. 5-8 weeks
- Weight at study initiation: 25-30 g
- Housing: in groups of up to seven in solid-floor polypropylene cages with woodflakes bedding
- Diet: Rat and Mouse Expanded Diet No. 1 (Special Diets Services Limited, Witham, UK), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hour): 15
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- intraperitoneal
- Vehicle:
- - Vehicle/solvent used: sterile, distilled water
- Amount of vehicle (gavage): 10 mL/kg - Details on exposure:
- PREPARATION OF DOSING SOLUTIONS: dosing solutions were prepared freshly by dissolving the test substance in sterile water yielding final concentrations of 25, 12.5 and 6.25 mg/mL (corresponding to 250, 125 and 6.25 mg/kg bw, respectively).
- Duration of treatment / exposure:
- not applicable
- Frequency of treatment:
- single treatment
- Post exposure period:
- 24 and 48 h (vehicle and high dose group)
24 h (low, mid dose and positive control group) - Dose / conc.:
- 62.5 mg/kg bw/day (nominal)
- Remarks:
- low dose
- Dose / conc.:
- 125 mg/kg bw/day (nominal)
- Remarks:
- mid dose
- Dose / conc.:
- 250 mg/kg bw/day (nominal)
- Remarks:
- high dose
- No. of animals per sex per dose:
- 7 (vehicle and dose groups), 5 (positive control)
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- Cyclophosphamide
- Route of administration: oral
- Doses / concentrations: 5 mg/mL in sterile water (corresponding to 50 mg/kg bw ) - Tissues and cell types examined:
- Femur bone marrow smears
- Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION:
A range finding study in 2 male and 2 female mice was performed to find the maximum tolerated dose level, a suitable route of administration and to assess gender-specific differences in toxicity.
TREATMENT AND SAMPLING TIMES:
24 h sampling time: 0, 62.5, 125 and 250 mg/kg bw (test substance); 50 mg/kg bw (positive control)
48 h sampling time: 0 and 250 mg/kg bw (test substance)
All animals were observed for signs of overt toxicity and death one hour after dosing and then once daily as applicable and immediately prior to sacrifice.
DETAILS OF SLIDE PREPARATION:
Immediately following sacrifice (i.e. 24 or 48 h following dosing), both femurs were dissected from each animal, aspirated with fetal calf serum and bone marrow smears were prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol and stained with May-Grünwald/Giemsa, allowed to air-dry and coverslipped using mounting medium.
METHOD OF ANALYSIS:
Stained bone marrow smears were coded and examined blind using light microscopy at x1000 magnification. The incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored. Micronuclei are normally circular in shape, although occasionally they may be oval or half-moon shaped, and have a sharp contour with even staining. In addition, the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes was counted; these cells were also scored for incidence of micronuclei. The ratio of polychromatic to normochromatic erythrocytes was calculated together with appropriate group mean values and standard deviations. - Evaluation criteria:
- A comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the test material groups and the number occurring in the corresponding vehicle control group.
A positive mutagenic response was demonstrated when a biologically relevant statistically significant, dose-related increase in the number of micronucleated polychromatic erythrocytes was observed for either the 24 or 48-hour time points, when compared to their corresponding control group.
If these criteria were not demonstrated, then the test material was considered to be non-genotoxic under the conditions of the test.
A positive response for bone marrow toxicity was demonstrated when the dose group mean polychromatic to normochromatic ratio was shown to be statistically significantly lower than the concurrent vehicle control group. - Statistics:
- All data were statistically analysed using appropriate statistical methods as recommended by the UKEMS Sub-committee on Guidelines for Mutagenicity Testing Report, Part III (1989). The data were analysed following a transformation using Student's t-test (two tailed) and any significant results were confirmed using the one way analysis of variance. Statistical significance was indicated at *p < 0.05, **p < 0.01 and ***p < 0.001.
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- yes
- Remarks:
- ≥500 mg/kg bw (range finding toxicity study): all animals dosed i.p. died within the study period of 48 h; 250 mg/kg bw (main study): 2/7 animals died in the 48-h harvest time group
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS OF RANGE-FINDING STUDY
- Dose range: 250, 500, 1000, 2000 mg/kg bw (intraperitoneal) and 2000 mg/kg bw (oral)
- Clinical signs of toxicity in test animals: in animals dosed with the test material via the intraperitoneal route premature deaths occurred at ≥ 500 mg/kg bw. Clinical signs were observed at ≥ 250 mg/kg bw (and increased in severity with increasing doses) and included: hunched posture, lethargy, pilo-erection, decreased respiratory rate, ptosis, ataxia, splayed gait, prostration, laboured respiration and pallor of the extremities.
- Sacrifice time: 48 h
- Dose selection: based on the results, the maximum tolerated dose of the test material was 250 mg/kg bw and thus selected as high dose for the main study.
- Other: no marked differences in the toxicity of the test substance were noted between male and female mice. Therefore, it was considered acceptable to use males only for the main study.
RESULTS OF DEFINITIVE STUDY
- Clinical signs: there were two premature deaths seen in the 48-h 250 mg/kg bw test material dose group. Clinical signs were observed in animals dosed with the test material at 250 mg/kg bw in both the 24 and 48 h groups. These included hunched posture, lethargy, pilo-erection, decreased respiratory rate, ptosis and ataxia.
- Induction of micronuclei: there was a small, statistically significant increase in the frequency of micronucleated PCEs in the 24-h 125 mg/kg bw test material dose group when compared to the respective control group. However, the response was within the current historical range for vehicle controls, the 24-h vehicle control PCE+MN value was very low and no individual animal values for micronucleated PCEs were greater than it would be considered acceptable for vehicle control animals. Therefore, the response was considered to be of no biological relevance. The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes, hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.
- Ratio of PCE/NCE: there was a statistically significant decrease in the PCE/NCE ratio of the 24-h 250 mg/kg bw test material group when compared to the concurrent vehicle control group. The response was part of a dose-related reduction in PCE/NCE ratios and was taken to indicate that cytotoxicity and exposure of the bone marrow had been achieved.
- Appropriateness of dose levels and route: it was considered that the loss of animals due to premature death did not affect the integrity of the study, because at least five analysable animals were available in each group, as recommended in the OECD test guideline No. 474. - Conclusions:
- The test material has been tested according to OECD 474 and under GLP conditions. No significant increase in the incidence of micronucleated polychromatic erythrocytes was observed in the bone marrow of male mice 24 or 48 hours after a single oral dose of up to and including 250 mg/kg bw. A reduction in the PCE/total erythrocyte ratio was only observed in the 24 hour positive control group. It is concluded that the test substance is negative for the induction of micronuclei under the conditions of the study.
- 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
- Study period:
- 03 Feb - 07 Mar 2000
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: see 'Remark'
- Remarks:
- GLP - Guideline study. According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (Dec 2012)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.
- Justification for type of information:
- refer to category justification provided in IUCLID section 13
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM
- Type of assay:
- micronucleus assay
- Species:
- mouse
- Strain:
- other: Crl:CD-1TM(ICR)BR
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Limited, Margate, UK
- Age at study initiation: approx. 5-8 weeks
- Weight at study initiation: 25-30 g
- Housing: in groups of up to seven in solid-floor polypropylene cages with woodflakes bedding
- Diet: Rat and Mouse Expanded Diet No. 1 (Special Diets Services Limited, Witham, UK), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hour): 15
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- intraperitoneal
- Vehicle:
- - Vehicle/solvent used: sterile, distilled water
- Amount of vehicle (gavage): 10 mL/kg - Details on exposure:
- PREPARATION OF DOSING SOLUTIONS: dosing solutions were prepared freshly by dissolving the test substance in sterile water yielding final concentrations of 25, 12.5 and 6.25 mg/mL (corresponding to 250, 125 and 6.25 mg/kg bw, respectively).
- Duration of treatment / exposure:
- not applicable
- Frequency of treatment:
- single treatment
- Post exposure period:
- 24 and 48 h (vehicle and high dose group)
24 h (low, mid dose and positive control group) - Dose / conc.:
- 62.5 mg/kg bw/day (nominal)
- Remarks:
- low dose
- Dose / conc.:
- 125 mg/kg bw/day (nominal)
- Remarks:
- mid dose
- Dose / conc.:
- 250 mg/kg bw/day (nominal)
- Remarks:
- high dose
- No. of animals per sex per dose:
- 7 (vehicle and dose groups), 5 (positive control)
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- Cyclophosphamide
- Route of administration: oral
- Doses / concentrations: 5 mg/mL in sterile water (corresponding to 50 mg/kg bw ) - Tissues and cell types examined:
- Femur bone marrow smears
- Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION:
A range finding study in 2 male and 2 female mice was performed to find the maximum tolerated dose level, a suitable route of administration and to assess gender-specific differences in toxicity.
TREATMENT AND SAMPLING TIMES:
24 h sampling time: 0, 62.5, 125 and 250 mg/kg bw (test substance); 50 mg/kg bw (positive control)
48 h sampling time: 0 and 250 mg/kg bw (test substance)
All animals were observed for signs of overt toxicity and death one hour after dosing and then once daily as applicable and immediately prior to sacrifice.
DETAILS OF SLIDE PREPARATION:
Immediately following sacrifice (i.e. 24 or 48 h following dosing), both femurs were dissected from each animal, aspirated with fetal calf serum and bone marrow smears were prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol and stained with May-Grünwald/Giemsa, allowed to air-dry and coverslipped using mounting medium.
METHOD OF ANALYSIS:
Stained bone marrow smears were coded and examined blind using light microscopy at x1000 magnification. The incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored. Micronuclei are normally circular in shape, although occasionally they may be oval or half-moon shaped, and have a sharp contour with even staining. In addition, the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes was counted; these cells were also scored for incidence of micronuclei. The ratio of polychromatic to normochromatic erythrocytes was calculated together with appropriate group mean values and standard deviations. - Evaluation criteria:
- A comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the test material groups and the number occurring in the corresponding vehicle control group.
A positive mutagenic response was demonstrated when a biologically relevant statistically significant, dose-related increase in the number of micronucleated polychromatic erythrocytes was observed for either the 24 or 48-hour time points, when compared to their corresponding control group.
If these criteria were not demonstrated, then the test material was considered to be non-genotoxic under the conditions of the test.
A positive response for bone marrow toxicity was demonstrated when the dose group mean polychromatic to normochromatic ratio was shown to be statistically significantly lower than the concurrent vehicle control group. - Statistics:
- All data were statistically analysed using appropriate statistical methods as recommended by the UKEMS Sub-committee on Guidelines for Mutagenicity Testing Report, Part III (1989). The data were analysed following a transformation using Student's t-test (two tailed) and any significant results were confirmed using the one way analysis of variance. Statistical significance was indicated at *p < 0.05, **p < 0.01 and ***p < 0.001.
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- yes
- Remarks:
- ≥500 mg/kg bw (range finding toxicity study): all animals dosed i.p. died within the study period of 48 h; 250 mg/kg bw (main study): 2/7 animals died in the 48-h harvest time group
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS OF RANGE-FINDING STUDY
- Dose range: 250, 500, 1000, 2000 mg/kg bw (intraperitoneal) and 2000 mg/kg bw (oral)
- Clinical signs of toxicity in test animals: in animals dosed with the test material via the intraperitoneal route premature deaths occurred at ≥ 500 mg/kg bw. Clinical signs were observed at ≥ 250 mg/kg bw (and increased in severity with increasing doses) and included: hunched posture, lethargy, pilo-erection, decreased respiratory rate, ptosis, ataxia, splayed gait, prostration, laboured respiration and pallor of the extremities.
- Sacrifice time: 48 h
- Dose selection: based on the results, the maximum tolerated dose of the test material was 250 mg/kg bw and thus selected as high dose for the main study.
- Other: no marked differences in the toxicity of the test substance were noted between male and female mice. Therefore, it was considered acceptable to use males only for the main study.
RESULTS OF DEFINITIVE STUDY
- Clinical signs: there were two premature deaths seen in the 48-h 250 mg/kg bw test material dose group. Clinical signs were observed in animals dosed with the test material at 250 mg/kg bw in both the 24 and 48 h groups. These included hunched posture, lethargy, pilo-erection, decreased respiratory rate, ptosis and ataxia.
- Induction of micronuclei: there was a small, statistically significant increase in the frequency of micronucleated PCEs in the 24-h 125 mg/kg bw test material dose group when compared to the respective control group. However, the response was within the current historical range for vehicle controls, the 24-h vehicle control PCE+MN value was very low and no individual animal values for micronucleated PCEs were greater than it would be considered acceptable for vehicle control animals. Therefore, the response was considered to be of no biological relevance. The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes, hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.
- Ratio of PCE/NCE: there was a statistically significant decrease in the PCE/NCE ratio of the 24-h 250 mg/kg bw test material group when compared to the concurrent vehicle control group. The response was part of a dose-related reduction in PCE/NCE ratios and was taken to indicate that cytotoxicity and exposure of the bone marrow had been achieved.
- Appropriateness of dose levels and route: it was considered that the loss of animals due to premature death did not affect the integrity of the study, because at least five analysable animals were available in each group, as recommended in the OECD test guideline No. 474. - Conclusions:
- The test material has been tested according to OECD 474 and under GLP conditions. No significant increase in the incidence of micronucleated polychromatic erythrocytes was observed in the bone marrow of male mice 24 or 48 hours after a single oral dose of up to and including 250 mg/kg bw. A reduction in the PCE/total erythrocyte ratio was only observed in the 24 hour positive control group. It is concluded that the test substance is negative for the induction of micronuclei under the conditions of the study.
Referenceopen allclose all
Table 1. Results of the in vivo micronucleus assay
Treatment group |
Dose (Conc) [mg/kg] |
Sampling time [h] |
Number of PCE with MN [per 2000 PCEs] |
PCE/NCE ratio |
||
Mean value |
SD |
Mean value |
SD |
|||
Vehicle control |
0 |
48 |
1.0 |
1.3 |
1.87 |
0.29 |
|
0 |
24 |
0.7 |
0.8 |
1.72 |
0.65 |
Positive control |
50 |
24 |
54.8*** |
15.9 |
1.62 |
0.41 |
Test substance |
250 (a) |
48 |
2.2 |
1.6 |
1.25 |
0.80 |
|
250 |
24 |
1.3 |
1.4 |
0.98* |
0.28 |
|
125 |
24 |
2.1** |
0.9 |
1.42 |
0.26 |
|
62.5 |
24 |
0.9 |
0.9 |
2.12 |
1.25 |
Positive control: cyclophosphamide
*p<0.05, **p<0.01, ***p < 0.001, Student's t-test (two tailed) and one way analysis of variance
(a): data from 5 animals
PE: polychromatic erythrocytes
NCE: normochromatic erythrocytes
SD: standard deviation
Table 1. Results of the in vivo micronucleus assay
Treatment group |
Dose (Conc) [mg/kg] |
Sampling time [h] |
Number of PCE with MN [per 2000 PCEs] |
PCE/NCE ratio |
||
Mean value |
SD |
Mean value |
SD |
|||
Vehicle control |
0 |
48 |
1.0 |
1.3 |
1.87 |
0.29 |
|
0 |
24 |
0.7 |
0.8 |
1.72 |
0.65 |
Positive control |
50 |
24 |
54.8*** |
15.9 |
1.62 |
0.41 |
Test substance |
250 (a) |
48 |
2.2 |
1.6 |
1.25 |
0.80 |
|
250 |
24 |
1.3 |
1.4 |
0.98* |
0.28 |
|
125 |
24 |
2.1** |
0.9 |
1.42 |
0.26 |
|
62.5 |
24 |
0.9 |
0.9 |
2.12 |
1.25 |
Positive control: cyclophosphamide
*p<0.05, **p<0.01, ***p < 0.001, Student's t-test (two tailed) and one way analysis of variance
(a): data from 5 animals
PE: polychromatic erythrocytes
NCE: normochromatic erythrocytes
SD: standard deviation
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
There are only limited data (Ames Test / OECD 471) available on genetic toxicity of D-Glucopyranose, oligomeric, undecyl glycoside. Therefore, in order to fulfil the standard information requirements set out in Annex VIII, 8.4, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006, read-across from the structurally related substances D-Glucopyranose, oligomeric, C10-16-alkyl glycosides (CAS 110615-47-9) and D-Glucopyranose, oligomers, decyl octyl glycosides (CAS 68515-73-1) is conducted following a category approach.
Genetic toxicity (mutagenicity) in bacteria in vitro
A bacterial gene mutation study (Ames test) performed according to OECD TG 471 and in compliance with GLP with D-Glucopyranose, oligomeric, undecyl glycoside is available (Institut Pasteur De Lille, 2016). The strains Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 were tested according to the plate incorporation (experiment I -/+ S9-mix, experiment II -S9-mix) and pre-incubation (experiment II +S9-mix) procedure in the absence and presence of a metabolic activation system (Aroclor 1254-induced rat liver S9-mix). Both experiments were conducted in three repetitions at concentrations ranging from 1.5 to 3000 µg/plate (vehicle: sterile water). No significant increase in the number of revertants was observed in any of the tester strains with and without metabolic activation. Cytotoxicity was recorded at 1000 µg/plate (TA 1535, TA 1537, TA 100, TA 102) and 1500 µg/plate (TA 98) in the absence of metabolic activation and at 1500 µg/plate (TA 100) and 3000 µg/plate (TA 1535, TA 1537, TA 98, TA 102) in the presence of metabolic activation (experiment I). In the second experiment cytotoxicity was present at 750 µg/plate (TA 100, TA 102) and at 1000 µg/plate /TA 1535) in the absence of metabolic activation and at 150 µg/plate (TA 102), 500 µg/plate (TA 1535, TA 1537, TA 100) and 1500 µg/plate (TA 98) in the presence of metabolic activation. Appropriate solvent and positive controls were included into the test and gave the expected results. Hence, the test item was considered to be not mutagenic to bacteria under the conditions of the test.
Genetic toxicity (cytogenicity) in mammalian cells in vitro
In a chromosome aberration study (Henkel, 1995) performed according to OECD TG 473 and in compliance with GLP Chinese hamster lung fibroblasts (V79) were treated with the category member D-Glucopyranose, oligomeric, C10-16-alkyl glycosides (CAS 110615-47-9) or vehicle (medium) in duplicates each in the absence or presence of a metabolic activation system (Aroclor-induced rat liver S9-mix) at concentrations of 2, 4, 8 and 16 µg/mL (7 and 28 h fixation time, without S9-mix), 0.5, 1, 2, 4, 8 and 16 µg/mL (20 h fixation time, without S9-mix), 20, 40, 80 and 160 µg/mL (7 and 28 h fixation time, with S9-mix) and 5, 10, 20, 40, 80 and 160 µg/mL (20 h fixation time, with S9-mix) for 4 h. Fixation of the cells was performed at the appropriate time points after start of exposure with the test material. Appropriate solvent (DMSO), negative (medium) and positive controls (EMS and CPA) were included in the test and gave the expected results. Cytotoxicity (mitotic index) of the test material was assessed in a preliminary cytotoxicity test indicating cytotoxic effects at concentrations ≥5 µg/mL (without S9-mix) and ≥100 µg/mL (with S9-mix). In the main experiment cytotoxicity was only recorded in the presence of S9-mix at 160 µg/mL and all fixation time points (7, 20 and 28 h) and at 80 µg/mL at 7h. The test substance did not cause a statistically significant, dose-related increase in chromosome aberrations under the tested conditions. Moreover, no indication of an increase in the frequency of polyploid metaphases after treatment with the test material compared to the negative controls was observed. Based on the results of the study the test material is considered not to be clastogenic in this chromosome aberration test in vitro. Therefore, D-Glucopyranose, oligomeric, undecyl glycoside is not considered to induce chromosome aberrations, either.
Genetic toxicity (mutagenicity) in mammalian cells in vitro
An in vitro gene mutation test in Mouse lymphoma L5178Y cells was performed with the category member D-Glucopyranose, oligomers, decyl octyl glycosides (CAS 68515-73-1) equivalent or similar to OECD TG 476 and in compliance with GLP (Microbiological Associates, 1991). Mouse lymphoma cells were treated with the test material or vehicle (distilled water) alone in the absence or presence of a metabolic activation system (Aroclor 1242 and Aroclor 1254 (2:1 mixture) induced rat liver S9-mix) in triplicates at concentrations from 7.5 to 565 µg/mL (-S9-mix, experiment I), 13 to 1004 µg/mL (+S9-mix, experiment I) and 103 to 260 µg/mL (+S9-mix, experiment II) for 4 hours followed by 2 days of culture for expression of the relevant genes. Thereafter, the mouse lymphoma cells were cultured in microtiter plates containing trifluorothymidine selective medium for additional 10 - 12 days. Fixation of the cells was performed 12 to 14 days after start of exposure with the test material. Appropriate solvent (distilled water) and positive controls (EMS and DMBA) were included in the test and gave the expected result. Cytotoxicity was observed in a preliminary toxicity test with L5178Y mouse lymphoma cells treated with concentrations from 0.01 to 5013 µg/mL for 4 h in the absence and presence of metabolic activation. Severe cytotoxicity was recorded at 1003 µg/mL in both, the presence and absence of metabolic activation. Based on the results of the dose range finding test an appropriate range of dose levels was chosen for the mutagenicity assays considering the highest dose level was the recommended 1004 µg/mL. The test substance did not cause a statistically significant, dose-related increase in mutant frequency either in the presence or absence of metabolic activation. Based on the results of the study the test material is considered not to be mutagenic in this L5178Y TK +/- Mouse Lymphoma Mutagenesis Assay in vitro. Therefore, D-Glucopyranose, oligomeric, undecyl glycoside is not considered to be mutagenic in mammalian cells, either.
Genetic toxicity (cytogenicity) in mammalian cells in vivo
An in vivo chromosome aberration test (Micronucleus Test) w ith the category member D-Glucopyranose, oligomeric, C10-16-alkyl glycosides was performed according to OECD TG 474 and in compliance with GLP (Safepharm Laboratories, 2000). The test material was once administered intraperitoneal to 7 male Crl:CD mice per dose group at concentrations of 62.5, 125 and 250 mg/kg bw. Appropriate control animals were administered a single dose of vehicle alone (sterile water, 7 animals) or the positive control substance (50 mg/kg bw cyclophosphamide, 5 animals). Bone marrow cells were freshly isolated from animals of the vehicle and high dose group 24 and 48 hours post-application and of the low, medium and positive dose group 24 hours post-application. The ratio of polychromatic to normochromatic erythrocytes (PCE/NCE) and the number of polychromatic erythrocytes (PCE) with micronuclei were analyzed to evaluate bone marrow cytotoxicity. A range finding study in two mice of each sex per dose group was performed to find the maximum tolerated dose level (up to 2000 mg/kg bw), a suitable route of administration (oral vs intraperitoneal) and to assess gender-specific differences in toxicity. Briefly, all animals treated with ≥500 mg/kg bw of the test material died within 24 hours post-application. Therefore, the highest concentration used in the main study was selected to be 250 mg/kg bw. No marked difference in toxicity between the sexes was observed, therefore, the main study was performed with male mice only. In the main micronucleus study, 2/7 animals of the high dose (250 mg/kg bw) 48 hour post-exposure group died. Clinical signs of toxicity were observed in the high dose group in both the 24 and 48 hour post-exposure group. Animals showed hunched posture, lethargy, piloerection, decreased respiratory rate, ptosis and ataxia. A small, statistically significant increase in the incidence of micronucleated polychromatic erythrocytes was recorded in the medium dose group (125 mg/kg bw) 24 hours post-administration of the test material when compared to the vehicle control group. Nevertheless, the response was within the current historical control range and therefore considered not to be of biological relevance. A statistically significant decrease in the PCE/NCE ratio was observed in the highest dose group (250 mg/kg bw) 24 hours post administration of the test material when compared to the vehicle control group. The response was part of a dose-related effect and sufficient evidence for exposure of the bone marrow with the test material. In summary, the test material was found not to produce a toxicologically significant increase in the frequency of micronuclei in polychromatic erythrocytes of mice under the conditions of the test. Positive and negative controls were included in the study and showed the expected result indicating that the test system was valid under the experimental conditions. Based on the results of the study the test substance is considered to be non-genotoxic under the conditions of the test. In conclusion, D-Glucopyranose, oligomeric, undecyl glycoside is not considered to be genotoxic in vivo, either.
Taken together, the available data on genetic toxicity of D-Glucopyranose, oligomeric, undecyl glycoside and the category members indicate that the target substance D-Glucopyranose, oligomeric, undecyl glycoside is neither mutagenic in bacterial and mammalian cells in vitro nor clastogenic in mammalian cells in vitro and mammals in vivo.
Justification for classification or non-classification
The available data on genetic toxicity of D-Glucopyranose, oligomeric, undecyl glycoside and the structurally related substances according to Regulation (EC) No 1907/2006, Annex XI, 1.5 do not meet the criteria for classification according to Regulation (EC) No 1272/2008; therefore, D-Glucopyranose, oligomeric, undecyl glycoside does not meet the criteria for classification, either, and the data are thus conclusive but not sufficient for classification.
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