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EC number: 240-344-0 | CAS number: 16215-49-9
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- Endpoint summary
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- Ecotoxicological Summary
- Aquatic toxicity
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- Short-term toxicity to fish
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- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
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Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
The substance dibutyl peroxydicarbonate was tested in an in vitro genotoxicity testing battery (OECD 471, 476, 487 and 490, GLP) as required by Annex VIII of the REACH regulation (EC) 1907/2006.
Dibutyl peroxydicarbonate was tested negative in a bacterial reverse gene mutation test conducted according to OECD 471 and in an in vitro micronucleus assay conducted according to OECD 487.
In a mammalian cell HPRT mutation assay conducted according to OECD 476, the substance showed equivocal results for inducing mutagenic effects. However, in a follow up mammalian cell gene mutation assay according to OECD 490, dibutyl peroxydicarbonate did not induce mutagenic effects in mouse lymphoma L5178Y cells. Based on the weight of evidence, the test item was considered to not induce genetic toxicity.
Link to relevant study records
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 2017-03-27 to 2017-11-30
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
- Version / remarks:
- adopted 29 July 2016
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell micronucleus test
- Specific details on test material used for the study:
- TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: A solubility test was performed with different solvents and vehicles up to the maximum recommended concentration of 2 mg/mL. Due to the nature of the test item it was not possible to prepare a solution of the test item with cell culture medium. Therefore the test item was dissolved in ethanol at a 200fold increased concentration and rediluted in cell culture medium at a ratio 1:200 to reach the final test item concentrations and a final concentration of 0.5% v/v ethanol in the samples. The test item solution were used within 1 hour. The solvent was compatible with the survival of the cells and the S9 activity. - Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- CELLS USED
V79 cells (ATCC, CCL-93) were stored over liquid nitrogen (vapour phase) in the cell bank of Eurofins Munich, as large stock cultures allowing the repeated use of the same cell culture batch in experiments. Routine checking of mycoplasma infections were carried out before freezing.
For the experiments thawed cultures were set up in 75 cm² cell culture plastic flasks at 37 °C in a 5% carbon dioxide atmosphere (95% air). 5 x 10^5 cells per flask were seeded in 15 mL of MEM (minimum essential medium) supplemented with 10% FBS (fetal bovine serum) and subcultures were made every 3-4 days.
MEDIA USED:
MEM medium supplemented with:
- 10% fetal bovine serum (FBS)
- 100 U/100 µg/mL penicillin/streptomycin solution
- 2 mM L-glutamine
- 2.5 µg/mL amphotericin
- 25 mM HEPES
Treatment Medium (short-term exposure):
- Complete culture medium with 0% FBS.
After Treatment Medium / Treatment Medium (long-term exposure):
- Complete culture medium with 10% FBS and 1.5 µg/mL cytochalasin B. - Additional strain / cell type characteristics:
- not applicable
- Cytokinesis block (if used):
- Cytochalasin B
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- The concentrations evaluated in the main experiment were based on the results obtained in the pre-experiment:
Experiment 1:
- Without metabolic activation: 0.10, 0.25, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 7.5, 10, 25 and 50 µg/mL; 0.50, 1.0, 2.0 and 4.0 µg/mL was selected as highest concentration for the microscopic analysis of micronuclei.
- With metabolic activation: 50, 100, 200, 300, 400, 500, 750, 1000, 1250, 1500 and 2000 µg/mL; 50, 100 and 300 µg/mL was selected as highest concentration for the microscopic analysis of micronuclei.
Experiment 2:
- Without metabolic activation: 0.5, 1.0, 2.5, 5.0, 7.5, 10, 12.5, 15, 17.5, 20, 25, 50, 75 and 100 µg/mL; 12.5, 17.5 and 20 µg/mL was selected as highest concentration for the microscopic analysis of micronuclei. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: ethanol (0.5% v/v in culture medium)
- Untreated negative controls:
- yes
- Remarks:
- Cell culture medium
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Cell culture medium with 0.5% ethanol
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- methylmethanesulfonate
- Remarks:
- clastogenic control; without metabolic activation, final concentration: 20 µg/mL
- Untreated negative controls:
- yes
- Remarks:
- Cell culture medium
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Cell culture medium with 0.5% ethanol
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- clastogenic control, with metabolic activation, final concentration: 2.5 µg/mL
- Untreated negative controls:
- yes
- Remarks:
- Cell culture medium
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Cell culture medium with 0.5% ethanol
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: Colchicine
- Remarks:
- aneugenic control, without metabolic activation, final concentration: 1.5 and 0.08 µg/mL
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 50000 cells were seeded per cell culture flask, containing 5 mL complete culture medium (minimum essential medium supplemented with 10% FBS).
Seeding of the cultures: Three or four-day-old stock cultures (in exponential growth), more than 50% confluent, were rinsed with Ca-Mg-free PBS solution prior to the trypsin treatment. Cells subsequently were trypsinised with a solution of 0.2% trypsin in Ca-Mg-free PBS at 37 °C for 5 min. By adding complete culture medium the detachment was stopped and a single cell suspension was prepared.
Experiment 1:
Exponentially growing V79 cells were seeded into 25 cm² cell culture flasks (two flasks per test group). Approx. 50000 cells were seeded per cell culture flask, containing 5 mL complete culture medium (minimum essential medium supplemented with 10% FBS). After an attachment period of approx. 48 h, the complete culture medium was removed and subsequently the test item was added to the treatment medium in appropriate concentrations. The cells were incubated with the test item for 4 h in presence or absence of metabolic activation. At the end of the incubation, the treatment medium was removed and the cells were washed twice with PBS. Subsequently, the cells were incubated in complete culture medium + 1.5 µg/mL cytochalasin B for 20 h at 37 °C.
Experiment 2:
If negative or equivocal results are obtained, they should be confirmed using continuous treatment (long-term treatment) without metabolic activation. Approx. 50 000 exponentially growing V79 cells were seeded in 25 cm² cell culture flasks in absence of metabolic activation. After an attachment period of approx. 48 h the test item was added in complete culture medium. 1 h later 1.5 µg/mL cytochalasin B were added and the cells were incubated for 23 h at 37 °C. At the end of the treatment the cell culture medium was removed and the cells were prepared for microscopic analysis.
Number of Cultures:
Duplicate cultures were performed at each concentration level except for the pre-experiment.
Preparation of the Cultures:
At the end of the cultivation, the complete culture medium was removed. Subsequently, cells were trypsinated and resuspended in about 9 mL complete culture medium. The cultures were transferred into tubes and incubated with hypotonic solution (0.4% KCl) for some minutes at room temperature. Prior to this an aliquot of each culture was removed to determine the cell count by a cell counter (AL-Systems). After the treatment with the hypotonic solution the cells were fixed with methanol + glacial acetic acid (3+1). The cells were resuspended gently and the suspension was dropped onto clean glass slides. Consecutively, the cells were dried on a heating plate. Finally, the cells were stained with acridine orange solution.
Analysis of Micronuclei:
At least 2000 binucleated cells per concentration (1000 binucleated cells per slide) were analysed for micronuclei according to criteria of Fenech, i.e. clearly surrounded by a nuclear membrane, having an area of less than one-third of that of the main nucleus, being located within the cytoplasm of the cell and not linked to the main nucleus via nucleoplasmatic bridges. Mononucleated and multinucleated cells and cells with more than six micronuclei were not considered.
As an assessment of the cytotoxicity, a cytokinesis block proliferation index (CBPI) was determined from 500 cells according to the following formula:
CBPI= (c1 x 1) + (c2 x 2) + (cx x 3)/n
c1: mononucleate cells
c2: binucleate cells
cx: multinucleate cells
n: total number of cells
The CBPI can be used to calculate the % cytostasis, which indicates the inhibition of cell growth of treated cultures in comparison to control cultures: % Cytostasis= 100 – 100 x ((CBPIT – 1) / (CBPIC – 1))
CBPIT: Cytokinesis Block proliferation index of treated cultures
CBPIC: Cytokinesis Block proliferation index of control cultures - Evaluation criteria:
- A mutation assay is considered acceptable if it meets the following criteria:
- The concurrent negative/solvent control is considered acceptable for addition to the laboratory historical negative/solvent control database.
- Concurrent positive controls should induce responses that are compatible with those generated in the laboratory’s historical positive control data base and produce a statistically significant increase compared with the concurrent negative/solvent control.
- Cell proliferation criteria in the negative/solvent control according to OECD 487 [4] should be fulfilled.
- All experimental conditions are tested unless one resulted in positive results.
- Adequate number of cells and concentrations are analysable.
- Criteria for the selection of top concentration are fulfilled.
A test item is considered to be clearly positive if, in any of the experimental conditions examined:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
- the increase is concentration-related in at least one experimental condition when evaluated with an appropriate trend test
- any of the results are outside the distribution of the historical negative/solvent control data (e.g. Poisson-based 95% control limits).
When all of these criteria are met, the test item is considered able to induce chromosome breaks and/or gain or loss in this test system. A test item is considered to be clearly negative if in all experimental conditions examined none of the criteria mentioned above are met. - Statistics:
- The nonparametric Chi-Quadrat Test was performed to verify the results of the experiment. No statistically significant enhancement (p< 0.05) of cells with micronuclei was noted in the concentration groups of the test item evaluated in experiment I and II.
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:The pH-value detected with the test item was within the physiological range (pH 7.4).
- Precipitation: No precipitate of the test item was noted in any concentration group evaluated in experiment I and II.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
In experiment I without metabolic activation no increase of the cytostasis above 30% was noted up to a concentration of 1.0 µg/mL. At a concentration of 2.0 µg/mL a cytostasis of 51% and at a concentration of 4.0 µg/mL a cytostasis of 65% was noted.
In experiment I with metabolic activation no increase of the cytostasis above 30% was noted up to a concentration of 50 µg/mL. At a concentration of 100 µg/mL a cytostasis of 33% and at a concentration of 300 µg/mL a cytostasis of 61% was noted.
In experiment II without metabolic activation no increase of the cytostasis above 30% was noted up to a concentration of 17.5 µg/mL. At a concentration 20 µg/mL a cytostasis of 57% was noted.
Micronuclei Analysis:
In experiment I without metabolic activation the micronucleated cell frequencies of the negative control (0.60%) and the solvent control (1.00%) were within the historical control limits of the negative control (0.39% – 1.40%,) and the solvent control (0.45% - 1.55%), respectively. The mean values of micronucleated cell frequencies found after treatment with the test item were 0.70% (0.5 µg/mL), 0.70% (1.0 µg/mL), 0.90% (2.0 µg/mL) and 0.85% (4.0 µg/mL). The number of micronucleated cells found in the groups treated with the test item was within the historical control limits of the negative and solvent control and did not show a biologically relevant increase compared to the concurrent solvent control.
In experiment I with metabolic activation the micronucleated cell frequencies of the negative control (1.55%) and the solvent control (1.28%) were within the historical control limits of the negative control (0.37% – 1.68%) and the solvent control (0.23%-1.88%), respectively. The mean values of micronucleated cell frequencies found after treatment with the test item were 0.85% (50 µg/mL), 1.55% (100 µg/mL) and 1.20% (300 µg/mL). The number of micronucleated cells found in the groups treated with the test item was within the historical control limits of the negative and solvent control and did not show a biologically relevant increase compared to the concurrent solvent control. In experiment II without metabolic activation the micronucleated cell frequencies of the negative control (0.95%) and the solvent control (0.70%) were within the historical control limits of the negative control (0.39% – 1.40 %) and the solvent control (0.45% - 1.55%), respectively. The mean values of micronucleated cell frequencies found after treatment with the test item were 0.65% (12.5 µg/mL), 0.80% (17.5 µg/mL) and 1.05% (20 µg/mL). The number of micronucleated cells found in the groups treated with the test item was within the historical control limits of the negative and solvent control and did not show a biologically relevant increase compared to the concurrent solvent control.
The nonparametric x² Test was performed to verify the results in both experiments. No statistically significant enhancement (p< 0.05) of cells with micronuclei was noted in the concentration groups of the test item evaluated in experiment I and II
For individual results please refer to Table 3 to 5 in box "Any other information on results incl. tables". - Remarks on result:
- other:
- Remarks:
- Experiment 1
- Conclusions:
- In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural and/or numerical chromosomal damage in Chinese hamster V79 cells. Therefore, the test item is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in this in vitro Mammalian Cell Micronucleus Test.
- Executive summary:
In an in vitro mammalian micronucleus assay (OECD 487), V79 cells cultured in vitro were exposed to Dibutyl peroxydicarbonate (50.6% peroxide) in cell culture medium with 0.5% ethanol in experiment 1 (short-term exposure, 4 h) at concentrations of 0.50, 1.0, 2.0 and 4.0 µg/mL (without metabolic activation) and at 50, 100 and 300 µg/mL (with metabolic activation). In experiment 2 (long-term exposure 24 h, without metabolic activation), concentration of 12.5, 17.5 and 20 µg/mL were used.
In experiment 1 without metabolic activation the mean values of micronucleated cell frequencies found after treatment with the test item were 0.70% (0.5 µg/mL), 0.70% (1.0 µg/mL), 0.90% (2.0 µg/mL) and 0.85% (4.0 µg/mL). In experiment I with metabolic activation the mean values of micronucleated cell frequencies found after treatment with the test item were 0.85% (50 µg/mL), 1.55% (100 µg/mL) and 1.20% (300 µg/mL). In experiment 2 without metabolic activation the mean values of micronucleated cell frequencies found after treatment with the test item were 0.65% (12.5 µg/mL), 0.80% (17.5 µg/mL) and 1.05% (20 µg/mL). No statistically significant enhancement (p< 0.05) of cells with micronuclei was noted in the concentration groups of the test item evaluated in experiment 1 and 2.
The positive controls did induce the appropriate response. In conclusion, it can be stated that during the study described and under the experimental conditions reported, Dibutyl peroxydicarbonate did not induce structural and/or numerical chromosomal damage in Chinese hamster V79 cells. Therefore, Dibutyl peroxydicarbonate is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in this in vitro Mammalian Cell Micronucleus Test.
This study is classified as acceptable and satisfies the requirements for Test Guideline OECD 487 for in vitro mutagenicity data.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 2017-04-03 to 2017-05-22
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- - Name: Dibutyl peroxydicarbonate
- Common name: Peroxan NBC-50
- CAS No: 13215-49-9
- Batch No: 1230518-02
- Molecular weight: not provided by the sponsor
- Physical state: liquid
- Colour: clear
- Density: 0.88 g/cm³
- Active components: 50.6% Peroxide
- Expiry date: not provided by the sponsor
- Storage conditions: =< -20°C
- Safety precautions: routine hygienic procedures will be sufficient to assure personnel helath and safety - Target gene:
- Histidine
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Details on mammalian cell type (if applicable):
- CELLS USED:
Sources of cells:
TA98, TA1535 and TA102: MOLTOX, INC., NC 28607, USA
TA100 and TA1537: Xenometrix AG, Switzerland
MEDIA USED:
Nutrient medium (per litre):
- 8 g Nutrient Borth
- 5 g NaCl
(solution of 125 µL ampicillin (10 mg/mL) (TA98, TA100, TA102) was added to retain phenotypic characteristics of strain)
Vogel-Bonner-salts (per litre)
- 10 g MgSO4 x 7 H2O
- 100 g citric acid
- 175 g NaNH4HPO4 x 4 H2O
- 500 g K2HPO4
Vogel-Bonner Medium E agar plates (per litre)
- 15 g Agar Agar
- 20 mL Vogel-Bonner salta
- 50 mL glucose-solution (40%)
Overlay agar (per litre)
- 7 g Agar Agar
- 6 g NaCl
- 10.5 mg L-histidine x HCl x H2O
- 12.2 mg biotin - Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- The test item concentrations in the main experiments were chosen according to the results of the pre-experiment:
Experiment 1: 0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 µL/plate
Experiment 2: 0.00158, 0.0050, 0.0158, 0.050, 0.158, 0.50 and 1.58 µL/plate, all tester strains (with metabolic activation) and TA102 (with and without metabolic activation); 0.05, 0.158, 0.50, 1.58, 5.0, 15.8 and 50 nL/plate, all tester strains (without metabolic activation), except TA102 - Vehicle / solvent:
- - Vehicle: ethanol
- Untreated negative controls:
- yes
- Remarks:
- A. dest.
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Ethanol
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- Untreated negative controls:
- yes
- Remarks:
- A. dest.
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Ethanol
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 4-nitro-o-phenylene-diamine
- Untreated negative controls:
- yes
- Remarks:
- A. dest.
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Ethanol
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- methylmethanesulfonate
- Untreated negative controls:
- yes
- Remarks:
- A. dest.
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Ethanol
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION
- in agar (plate incorporation)
EXPERIMENTAL PERFORMANCE
For the plate incorporation method the following materials were mixed in a test tube and poured over the surface of a minimal agar plate:
- 100 µL Test solution at each dose level, solvent control, negative control or reference mutagen solution (positive control),
- 500 µL S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation),
- 100 µL Bacteria suspension (cf. Preparation of bacteria, pre-culture of the strain),
- 2000 µL Overlay agar.
After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY
- Method: clearing or diminution of background lawn or reduction in number of revertants down to mutation factor of approximately < 0.5 in relation to solvent control - Evaluation criteria:
- The mutation factor is calculated by dividing the mean value of the revertant counts through the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA98, TA100 and TA102 the number of reversions is at least twice as high
- if in tester strains TA1535 and TA1537 the number of reversions is at least three times higher than the reversion rate of solvent control. - Statistics:
- not applicable
- Key result
- Species / strain:
- S. typhimurium, other: TA98, TA102, TA1535, TA1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Precipitation of the test item was observed in all tester strains used in experiment I at concentrations of 2.5 µL/plate and higher (with and without metabolic activation).
Toxic effects of the test item were noted in most tester strains evaluated in experiment I and II. In experiment I, toxic effects were observed in tester strain TA98 at concentrations of 1.0 µL/plate and higher (without S9 mix). In tester strains TA1535 and TA102 toxic effects of the test item were noted at concentrations of 1.0 µL/plate and higher (without S9 mix) and at concentrations of 2.5 µL/plate and higher (with S9 mix). In tester strain TA1537 toxic effects were observed at concentrations of 1.0 µL/plate and higher (with and without S9 mix). In experiment II toxic effects of the test item were noted in tester strain TA98 at concentrations at 15.8 nL/plate and higher (without metabolic activation) and at a concentration of 1.58 µL/plate (with S9 mix). In tester strains TA100, TA1535 and TA1537 toxic effects of the test item were observed at concentrations of 15.8 nL/plate and higher (without metabolic activation) and at concentrations of 0.50 µL/plate and higher (with metabolic activation). In tester strain TA102 toxic effects of the test item were noted at concentrations of 0.050 µL/plate and higher (without S9 mix) and at a concentration of 1.58 µL/plate (with S9 mix). - Remarks on result:
- other:
- Remarks:
- Experiment 1
- Conclusions:
- In conclusion, the test item is not genotoxic in the bacterial reverse gene mutation assay in the presence and absence of mammalian metabolic activation.
- Executive summary:
In a reverse gene mutation assay in bacteria (OECD 471) strains of S. typhimurium (TA1537, TA1535, TA102, TA100 and TA98) were exposed to Dibutyl peroxydicarbonate in ethanol at concentrations of 0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 µl/plate (experiment I) in the presence and absence of metabolic activation. In experiment II all tester strains (with metabolic activation) and TA 102 (with and without metabolic activation) were exposed to concentrations of 0.00158, 0.0050, 0.0158, 0.050, 0.158, 0.50 and 1.58 µL/plate. Tester strain TA102 (without metabolic activation) was tested at concentrations of 0.050, 0.158, 0.50, 1.58, 5.0, 15.8 and 50 nL/plate. The positive controls did induce the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.
This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 2017-04-24 to 2017-04-18
- 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:
- adopted July 29 2016
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell gene mutation test using the Hprt and xprt genes
- Target gene:
- HPRT (hypoxanthine-guanine-phosphoribosyl-transferase)
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: Eurofins BioPharma Product Testing Munich GmbH
- Doubling time: 12 - 14 h
- Cloning efficiency: > 50%
- Modal number of chromosomes: Yes
MEDIA USED
- Type and identity of media: Complete culture media - Metabolic activation:
- with and without
- Metabolic activation system:
- Mammalian Microsomal Fraction S9 Mix
- Test concentrations with justification for top dose:
- Pre-test for toxicity:
0.025, 0.05, 0.1, 0.25, 0.5, 1, 1.5 and 2 mg/mL without/with S9 mix
Main test:
Experiment I: 0.005, 0.01, 0.03, 0.04, 0.06, 0.08, 0.1, 0.3 and 0.5 mg/mL without S9 mix and 0.025, 0.05, 0.1, 0.25, 0.5, 0.75, 1, 1.5, 2 mg/mL with S9 mix
Experiment II: 0.0002, 0.0005, 0.001, 0.002, 0.005, 0.007, 0.01, 0.02, 0.03 and 0.04 mg/mL without S9 mix
The test item was investigated at the following concentrations:
Experiment I: 0.005, 0.01, 0.03, 0.04, 0.06 mg/mL without S9 mix and 0.025, 0.05, 0.1, 0.25, 0.5 mg/mL with S9 mix
Experiment II: 0.007, 0.01, 0.02, 0.03 and 0.04 mg/mL without S9 mix - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Ethanol
- Untreated negative controls:
- yes
- Remarks:
- MEM medium
- Negative solvent / vehicle controls:
- yes
- Remarks:
- ethanol (1 % v/v)
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- 300 µg/mL, without S9
- Untreated negative controls:
- yes
- Remarks:
- MEM medium
- Negative solvent / vehicle controls:
- yes
- Remarks:
- ethanol (1 % v/v)
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- Remarks:
- 1.0 µg/mL, with S9
- Details on test system and experimental conditions:
- Method of application: in medium
DURATION
- Preincubation period: 24 hours
- Exposure duration: 4 hours (short time exposure, Experiment I with and without metabolic activation)
20 hours (long time exposure, Experiment II without metabolic activation)
- Expression time (cells in growth medium): 7 - 9 days
- Selection time (if incubation with a selection agent): 7-12 days
SELECTION AGENT (mutation assays): 11 µg/mL thioguanine (TG)
NUMBER OF REPLICATIONS: one culture per test group (expression period)
DETERMINATION OF CYTOTOXICITY: Cytotoxicity (relative survival) was calculated based on the cloning efficiency of cells plated immediately after treatment adjusted by any loss of cells during treatment - Evaluation criteria:
- A test chemical is considered to be clearly negative if, in all experimental conditions examined
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend-test
- all results are inside the distribution of the historical negative control data
A test chemical is considered to be clearly positive if, in any of the experimental conditions examined
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control, and
- the increase is concentration-related when evaluated with an appropriate trend test, and
- any of the results are outside the distribution of the historical negative control data.
- if there is by chance a low spontaneous mutation rate in the corresponding negative and solvent controls a concentration related increase of the mutations within their range has to be discussed.
According to the OECD guideline, the biological relevance is considered first for the interpretation of results. - Statistics:
- The non-parametric Mann-Whitney test was applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative/solvent controls. Mutant frequencies of the negative/solvent controls were used as reference.
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- ambiguous
- Cytotoxicity / choice of top concentrations:
- not determined
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Precipitation:
No precipitation of the test item was noted in any of the experiments.
Toxicity:
A biologically relevant growth inhibition (reduction of relative survival below 70%) was observed after the treatment with the test item in experiment I and II with and without metabolic activation. In experiment I without metabolic activation, the relative survival was 1% for the highest concentration (0.3 mg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 2.0 mg/mL with a relative survival of 0%. In experiment II without metabolic activation, the relative survival was 15% for the highest concentration (0.04 mg/mL) evaluated.
Mutagenicity:
In the experiment without and with metabolic activation all validity criteria were met. The mutant values of the negative and solvent controls were within or even lower than the historical data range of the test facility and the cloning efficiencies of the negative and solvent controls were > 50%. The positive controls, DMBA (1 µg/mL) and EMS (300 µg/mL) showed statistically significant increases in mutant frequency, thereby demonstrating both the sensitivity and validity of the test systems. In the experiment I without metabolic activation, the mutant values of the negative controls, the solvent controls and two of five mutant values of the test item concentrations found were within the historical control data of the test facility Eurofins Munich (about 7.8-39.7 mutants per 10^6 cells). The positive control EMS induced a distinct increase in mutant frequency with 273.2 mutants/10^6 cells.
The mutant frequencies of the negative controls were 39.5 and 39.2 mutants per 10^6 cells, of the solvent controls 35.2 and 23.9 mutants per 10^6 cells, respectively. - Remarks on result:
- other: equivocal results obtained in short-term and long-term experiments
- Conclusions:
- In this study, under the given conditions, the results of the mutagenic potential of the test item dibutyl peroxydicarbonate, were found to be equivocal.
- Executive summary:
In a mammalian cell HPRT gene mutation assay conducted in accordance with OECD 476, V79 cells cultured in vitro were exposed to dibutyl peroxydicarbonate (50.6% peroxide) in 1% ethanol investigated at concentrations of 0.005, 0.01, 0.03, 0.04, 0.06, 0.08, 0.1 and 0.3 mg/mL in the absence of mammalian metabolic activation (experiment I, 4-hour exposure) and at concentrations of 0.025, 0.05, 0.1, 0.25, 0.5, 0.75, 1, 1.5 and 2 mg/mL in the presence of mammalian metabolic activation (experiment I, 4-hour exposure). For experiment II (20-hour exposure), concentrations tested were 0.0002, 0.0005, 0.001, 0.002, 0.005, 0.007, 0.01, 0.02, 0.03 and 0.04 mg/mL in the absence of metabolic activation. An experiment II with metabolic activation could not be run for technical reasons. DMBA and EMS were used as positive controls and showed distinct and biologically relevant effects in mutation frequency, showing the validity of the experiments.
In experiment I without metabolic activation, a biologically relevant increase of mutants was found after treatment with the test item at cytotoxic concentrations. The concentration 0.04 mg/mL induced a slight increase above the historic control range. A statistical analysis displayed that these mutant frequencies were significantly increased over those of the solvent controls, but no dose-response relationship was determined. Therefore, no clearly positive response is observed.
In experiment II without metabolic activation, the mutant values were all within or even lower than the historical control data. The highest mutant frequency was observed at the highest evaluated concentration of 0.04 mg/mL with a relative survival of 15%. This increase was also statistically significant compared to the solvent control and a dose-response relationship was determined. However, as the mutant values of the test item were within the historical control data, these significances were considered as not biologically relevant. Thus, the 20 h exposure without S9 was clearly negative.
In experiment I with metabolic activation, a dose related increase in both cytotoxicity and mutant frequency was observed. The top two concentrations induced appropriate maximum levels of cytotoxicity and very slight, but significantly increased mutant frequencies compared to the control, which minimally meet the criteria for a positive response.
In summary, as the short-term and long-term treatments without metabolic activation were contrary to each other and the short-term incubation with metabolic activation was slightly positive, the all over result was regarded as equivocal.
This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS OECD 476 for in vitro mutagenicity data.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 2018-07-19 to 2018-12-03
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
- Version / remarks:
- adopted July 29, 2016
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell gene mutation tests using the thymidine kinase gene
- Specific details on test material used for the study:
- Name: Dibutyl peroxydicarbonate
Common name: Peroxan NBC-50
CAS No.: 16215-49-9
Physical state: liquid
Colour: clear
Density: 0.88 g/cm3
Active components: 50.5% peroxide
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Source: Pergan GmbH (Sponsor), batch no.: 1262684-01
- Purity test date: 2018-06-26
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ≤ -20 °C
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test material was diluted in ethanol (0.5% v/v) - Target gene:
- Thymidine kinase
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: Eurofins Munich stock
- Cell cycle length, doubling time or proliferation index: 10-12 hours doubling time, cloning efficiency >50%
- Methods for maintenance in cell culture if applicable: Thawed stock cultures are maintained in plastic culture flasks in RPMI 1640 complete medium and subcultured three times per week
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Roswell Park Memorial Institute (RPMI) 1640, supplemented with 9.0 µg/mL hypoxanthine; 15.0 µg/mL thymidine, 22.5 µg/mL glycine and 0.1 µg/mL methotrexate
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: not specified
- Periodically 'cleansed' against high spontaneous background: yes, by culturing in RPMI 1640 supplemented with 9.0 µg/mL hypoxanthine, 15.0 µg/mL thymidine, 22.5 µg/mL glycine, 0.1 µg/mL methotrexate - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9
- Test concentrations with justification for top dose:
- without metabolic activation: 50, 60, 70, 72, 74, 76, 78, and 80 µg/mL
with metabolic activation: 25, 50, 75, 100, 150, 300, 500, 750 µg/mL
The selection of the concentrations used in the main experiment was based on data from the pre-experiment. Due to the unusual toxicity 76 µg/mL (without metabolic activation) and 300 µg/mL (with metabolic activation) were selected as the highest concentrations. Since there was first an increase of toxicity followed by a decrease, two upper concentrations (78 and 80 µg/mL, without metabolic activation; 500 and 750 µg/mL, with metabolic activation) were evaluated in the main experiment. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: 0.5% v/v ethanol
- Justification for choice of solvent/vehicle: A solubility test was performed with different solvents and vehicles up to the maximum recommended concentration of 2 mg/mL.
Based on the results of the solubility test ethanol (0.5% v/v; Supplier: AppliChem; Lot: 7A012351) was used as solvent. - Untreated negative controls:
- yes
- Remarks:
- treatment medium
- Negative solvent / vehicle controls:
- yes
- Remarks:
- 0.5% v/v ethanol
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Untreated negative controls:
- yes
- Remarks:
- treatment medium
- Negative solvent / vehicle controls:
- yes
- Remarks:
- 0.5% v/v ethanol
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- methylmethanesulfonate
- Untreated negative controls:
- yes
- Remarks:
- treatment medium
- Negative solvent / vehicle controls:
- yes
- Remarks:
- 0.5% v/v ethanol
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 1x10^7 cells suspended in 11 mL RPMI medium with 5% horse serum
DURATION
- Preincubation period: none
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 12 days
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency and relative total growth
SELECTION AGENT (mutation assays): Trifluorothymidine (TFT) - Rationale for test conditions:
- The mutation assay is considered acceptable if it meets the criteria mentioned in current international guidelines and the current recommendations of the IWGT:
- At least three out of four 96-well plates from the TFT resistance-testing portion of the experiment are scorable
- The cloning efficiency of the negative and/or solvent controls is in the range 65% - 120%.
- The spontaneous mutant frequency in the negative and/or solvent controls is in the range 50 - 170 mutants per 10^6 cells
- The cell number of the negative/solvent controls should undergo 8 - 32-fold increase during a 2-day growth period (short-term treatment)
- The clastogenic positive controls (MMS and B[a]P) have to produce an induced mutant frequency (total mutant frequency minus concurrent negative control mutant frequency) of at least 300 mutants per 10^6 cells with at least 40% of the colonies being small colonies or with an induced small colony mutant frequency of at least 150 mutants per 10^6 cells
- The relative total growth must be greater than 10%. - Evaluation criteria:
- Test item is considered mutagenic if the following criteria are met:
- the induced mutant frequency meets or exceeds the Global Evaluation factor of 126 mutants per 1x10^6 cells
- a dose-dependent increase in mutant frequency is detected
Besides, combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (≥40% of total colonies) is an indication for potential clastogenic effects and/or chromosomal aberrations. - Statistics:
- The non-parametric Mann-Whitney test was applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative/solvent controls. Mutant frequencies of the solvent/negative controls were used as reference. Statistical significance at the 5% level (p < 0.05) was evaluated.
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- The relative total growth was 13.1% at 76 µg/mL without S9 and 13.7% at 300 µg/mL with S9
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH-value detected with the test item was within the physiological range.
- Effects of osmolality: The osmolality was within the physiological range.
- Precipitation: In the main experiment no precipitation was observed.
RANGE-FINDING/SCREENING STUDIES: The selection of the concentrations used in the main experiment was based on data from the pre-experiment according to the OECD guideline 490. Due to the nature of the test item an explicit determination of the toxicity in the pre-experiment was not clearly evaluable. Thus for the relative suspension growth (RSG) values a first increase in toxicity could be observed, followed by a further decrease. Therefore the selection of appropriate concentration was adapted to cover the toxicity range, containing the first time toxicity occurs and two upper evaluated concentrations.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%): All mutant frequencies for negative, solvent and positive controls were found within the historical range of the test facility Eurofins Munich.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: relative suspension growth
For individual results see tables 1 to 10 in box "Any other information on results incl. tables". - Conclusions:
- The test item dibutyl peroxydicarbonate is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.
- Executive summary:
In a mammalian cell gene mutation assay using the Thymidine Kinase Gene conducted according to OECD Guideline 490, mouse lymphoma L5178Y cells cultured in vitro were exposed to dibutyl peroxydicarbonate (50.5% active peroxide) at concentrations of 50, 60, 70, 72, 74, 76, 78, and 80 µg/mL without S9 metabolic activation and at concentrations 25, 50, 75, 100, 150, 300, 500, and 750 µg/mL with S9 metabolic activation. The selection of the concentrations used in the main experiment was based on data from the pre-experiment. Due to the unusual toxicity 76 µg/mL (without metabolic activation) and 300 µg/mL (with metabolic activation) were selected as the highest concentrations. Since there was first an increase of toxicity followed by a decrease, two upper concentrations (78 and 80 µg/mL, without metabolic activation; 500 and 750 µg/mL, with metabolic activation) were evaluated in the main experiment. The experiment without and with metabolic activation was performed as 4 h short-term exposure assay.
The relative total growth (RTG) was 13.1% (at concentration 76 µg/mL without metabolic activation) and 13.7% (at concentration 300 µg/mL with metabolic activation) at time of first toxicity. In the experiment with metabolic activation for the highest evaluated concentrations 500 and 750 µg/mL, an increase in toxicity was observed. A relative total growth value of 18.2% at 500 µg/mL and 36.1% at 750 µg/mL was observed. In the experiment without metabolic activation, the increase in toxicity at concentrations 78 and 80 µg/mL had RTG values of 26.3% and 8.6%, respectively.
The positive controls EMS, MMS and B(a)P showed distinct effects in mutation frequency, thus proving the ability of the test system to detect potential mutagenic effects. In the main experiment without and with metabolic activation, all validity criteria were met. Furthermore, the negative and vehicle controls had valid mutant frequencies. In experiments with the test item, no biologically relevant increase was observed. In the experiment without metabolic activation a statistically significant concentration-response relationship was observed. Since the Global Evaluation Factor was not exceeded in any evaluated concentrations and a strong toxicity was observed in the highest tested concentrations, this effect was considered as not biologically relevant. In the experiment with metabolic activation no evidence for a concentration-response relationship was detected. Based on the non-mutagenic effects of the test item, an assessment of clastogenicity was not feasible. The positive controls proved the test system valid in indicating potential clastogenic effects.
This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 490 for in vitro mutagenicity data.
Referenceopen allclose all
Table 3: Test for cytotoxicity
Dose Group | Concentration [µg/mL] |
CBPI | Relative Cell Growth [%] |
Cytostasis [%] |
Precipitate +/- |
without metabolic activation | |||||
C | 0 | 1.53 | 101 | 0 | - |
S | 0 | 1.53 | 100 | 0 | - |
1 | 7.8 | 1.31 | 60 | 40 | - |
2 | 15.6 | 1.23 | 43 | 57 | - |
3 | 31.3 | 1.36 | 68 | 32 | - |
4 | 62.5 | 1.31 | 60 | 40 | - |
5 | 125 | 1.26 | 50 | 50 | - |
6 | 250 | 1.03 | 5 | 95 | - |
7 | 500 | 1.02 | 5 | 95 | + |
8 | 1000 | 1.01 | 2 | 98 | + |
with metabolic activation | |||||
C | 0 | 1.44 | 123 | 0 | - |
S | 0 | 1.36 | 100 | 0 | - |
1 | 7.8 | 1.49 | 134 | 0 | - |
2 | 15.6 | 1.37 | 102 | 0 | - |
3 | 31.3 | 1.38 | 105 | 0 | - |
4 | 62.5 | 1.40 | 110 | 0 | - |
5 | 125 | 1.36 | 98 | 2 | - |
6 | 250 | 1.35 | 97 | 3 | - |
7 | 500 | 1.25 | 70 | 30 | - |
8 | 1000 | 1.18 | 50 | 50 | + |
9 | 1500 | 1.21 | 57 | 43 | + |
10 | 2000 | 1.16 | 45 | 55 | + |
C: Negative Control (Culture medium)
CBPI: Cytokinesis Block Proliferation Index, CBPI = ((c1x 1) + (c2x 2) + (cxx 3))/n
Relative Cell Growth:100 x ((CBPITest conc– 1) / (CBPIcontrol-1))
c1:mononucleate cells
c2:binucleate cells
cx:multinucleate cells
n:total number of cells
Cytostasis [%] = 100- Relative Cell Growth [%]
the cytostasis is defined 0, when the relative cell growth exceeds 100%
Table 4: Experiment 1 and 2, without metabolic activation
|
Dose Group |
Concentration [µg/mL] |
Number of cells evaluated |
Cytostasis [%] |
Relative Cell Growth |
Micro-nucleated |
Historical Control Limits Negative Control |
Precipitation |
Statistical Significant Increasea |
Exp. I 4 h treatment, 24 h fixation interval |
C |
0 |
2000 |
2 |
98 |
0.60 |
0.39% - 1.40% |
/ |
/ |
S |
0 |
2000 |
0 |
100 |
1.00 |
/ |
/ |
||
3 |
0.50 |
2000 |
12 |
88 |
0.70 |
- |
- |
||
4 |
1.0 |
2000 |
20 |
80 |
0.70 |
- |
- |
||
5 |
2.0 |
2000 |
51 |
49 |
0.90 |
- |
- |
||
7 |
4.0 |
2000 |
65 |
35 |
0.85 |
- |
- |
||
MMS |
20 |
2000 |
18 |
82 |
2.85 |
- |
+ |
||
Colc |
1.5 |
2000 |
18 |
82 |
2.55 |
- |
+ |
||
|
|
|
|
||||||
Exp. II 24 h treatment, 24 h fixation interval |
C |
0 |
2000 |
0* |
128 |
0.95 |
0.39% - 1.40% |
/ |
/ |
S |
0 |
2000 |
0 |
100 |
0.70 |
/ |
( |
||
7 |
12.5 |
2000 |
30 |
70 |
0.65 |
- |
- |
||
9 |
17.5 |
2000 |
16 |
84 |
0.80 |
- |
- |
||
10 |
20 |
2000 |
57 |
43 |
1.05 |
- |
- |
||
MMS |
20 |
2000 |
0* |
108 |
5.85 |
- |
+ |
||
Colc |
0.08 |
1906 |
1 |
99 |
8.07 |
- |
+ |
Table 5: Experiment 1, with metabolic activation
|
Dose Group |
Concentration [µg/mL] |
Number |
Cytostasis [%] |
Relative Cell Growth |
Micro- Nucleated |
Historical Control Limits Negative Control |
Precipitation |
Statistical Significant Increasea |
Exp. I 4 h treatment, 24 h fixation interval |
C |
0 |
2000 |
5 |
95 |
1.55 |
0.37% - 1.68% |
/ |
/ |
S |
0 |
4000 |
0 |
100 |
1.28 |
/ |
/ |
||
1 |
50 |
2000 |
21 |
79 |
0.85 |
- |
- |
||
2 |
100 |
4000 |
33 |
67 |
1.55 |
- |
- |
||
4 |
300 |
4000 |
61 |
39 |
1.20 |
- |
- |
||
CPA |
2.5 |
2000 |
30 |
70 |
5.75 |
- |
+ |
Pre-experiment for toxicity and Main experiments I and II:
Result tables are attched in box "Attached background material"
Table1: Experiment I – Toxicity, without metabolic activation
Dose Group | Concen-tration | Number of cells at the | Number of colonies per flask | CE [%] | Adjusted CE [%] | Relative Survival (RS) [%] | |||
[mg/mL] | beginning of treatment | end of treatment | I | II | mean | ||||
NC1 | 0 | 10000000 | 12019000 | 151 | 142 | 147 | 73 | 88 | 96 |
NC2 | 10000000 | 13804000 | 176 | 104 | 140 | 70 | 97 | 106 | |
S1 | 0 | 10000000 | 11815000 | 158 | 158 | 158 | 79 | 93 | 100 |
S2 | 10000000 | 11237000 | 160 | 159 | 160 | 80 | 90 | ||
1 | 0.005 | 10000000 | 8925000 | 128 | 111 | 120 | 60 | 53 | 58 |
2 | 0.01 | 10000000 | 13821000 | 107 | 110 | 109 | 54 | 75 | 82 |
3 | 0.03 | 10000000 | 7786000 | 52 | 75 | 64 | 32 | 25 | 27 |
4 | 0.04 | 20000000 | 17000000 | 46 | 37 | 42 | 21 | 18 | 19 |
5 | 0.06 | 20000000 | 10778000 | 24 | 22 | 23 | 12 | 6 | 7 |
6 | 0.08 | 20000000 | 7174000 | 21 | 19 | 20 | 10 | 4 | 4 |
7 | 0.1 | 20000000 | 5168000 | 25 | 31 | 28 | 14 | 4 | 4 |
8 | 0.3 | 20000000 | 2815200 | 12 | 5 | 9 | 4 | 1 | 1 |
EMS | 300 µg/mL | 10000000 | 11645000 | 178 | 145 | 162 | 81 | 94 | 103 |
NC: negative control
S: solvent control (0.5% v/v ethanol)
CE: cloning efficiency
EMS: Ethylmethanesulfonate [300 µg/mL]
Table 2: Experiment I – Mutagenicity, without metabolic activation
CE in non-selective medium | CE in selective medium | |||||||||||||
Dose Group | Concen-tration | Number of colonies per flask | CE [%] | Number of colonies per flask | CE [%] | Mutant Frequency per 106 cells | ||||||||
[mg/mL] | I | II | mean | I | II | III | IV | V | mean | SD | ||||
NC1 | 0 | 148 | 171 | 160 | 80 | 8 | 12 | 18 | 11 | 14 | 12.6 | 3.3 | 0.0032 | 39.5 |
NC2 | 155 | 146 | 151 | 75 | 15 | 12 | 8 | 8 | 16 | 11.8 | 3.4 | 0.0030 | 39.2 | |
S1 | 0 | 154 | 153 | 154 | 77 | 7 | 16 | 9 | 10 | 12 | 10.8 | 3.1 | 0.0027 | 35.2 |
S2 | 180 | 163 | 172 | 86 | 9 | 9 | 5 | 9 | 9 | 8.2 | 1.6 | 0.0021 | 23.9 | |
1 | 0.005 | 147 | 148 | 148 | 74 | 11 | 11 | 12 | 9 | 8 | 10.2 | 1.5 | 0.0026 | 34.6 |
2 | 0.01 | 160 | 140 | 150 | 75 | 19 | 15 | 20 | 17 | 16 | 17.4 | 1.9 | 0.0044 | 58.0 |
3 | 0.03 | 135 | 135 | 135 | 68 | 9 | 9 | 9 | 7 | 6 | 8.0 | 1.3 | 0.0020 | 29.6 |
4 | 0.04 | 141 | 142 | 142 | 71 | 11 | 13 | 11 | 12 | 12 | 11.8 | 0.7 | 0.0030 | 41.7 |
5 | 0.06 | 151 | 148 | 150 | 75 | 8 | 18 | 14 | 16 | 15 | 14.2 | 3.4 | 0.0036 | 47.5 |
EMS | 300 µg/mL | 176 | 141 | 159 | 79 | 81 | 88 | 90 | 82 | 92 | 86.6 | 4.4 | 0.0217 | 273.2 |
NC: negative control
S: solvent control (0.5% v/v ethanol)
CE: cloning efficiency
EMS: Ethylmethanesulfonate
Table 3: Experiment I – Toxicity, with metabolic activation
Dose Group | Concen-tration | Number of cells at the | Number of colonies per flask | CE [%] | Adjusted CE [%] | Relative Sur-vival (RS) [%] | |||
[mg/mL] | beginning of treatment | end of treatment | I | II | mean | ||||
NC1 | 0 | 10000000 | 13464000 | 128 | 157 | 143 | 71 | 96 | 88 |
NC2 | 10000000 | 12274000 | 135 | 151 | 143 | 72 | 88 | 81 | |
S1 | 0 | 10000000 | 13362000 | 149 | 175 | 162 | 81 | 108 | 100 |
S2 | 10000000 | 12648000 | 177 | 168 | 173 | 86 | 109 | ||
1 | 0.025 | 10000000 | 12274000 | 122 | 128 | 125 | 63 | 77 | 71 |
2 | 0.05 | 10000000 | 11373000 | 111 | 81 | 96 | 48 | 55 | 50 |
3 | 0.1 | 10000000 | 10047000 | 88 | 126 | 107 | 54 | 54 | 49 |
4 | 0.25 | 10000000 | 7854000 | 44 | 39 | 42 | 21 | 16 | 15 |
5 | 0.5 | 20000000 | 14110000 | 36 | 38 | 37 | 19 | 13 | 12 |
6 | 0.75 | 20000000 | 8602000 | 43 | 50 | 47 | 23 | 10 | 9 |
7 | 1 | 20000000 | 8806000 | 14 | 25 | 20 | 10 | 4 | 4 |
8 | 1.5 | 20000000 | 6528000 | 14 | 24 | 19 | 10 | 3 | 3 |
9 | 2 | 20000000 | 3434000 | 6 | 6 | 6 | 3 | 1 | 0 |
DMBA | 1 µg/mL | 10000000 | 13413000 | 151 | 158 | 155 | 77 | 104 | 95 |
NC: negative control
S: solvent control (0.5% v/v ethanol)
CE: cloning efficiency
DMBA: 7,12-dimethylbenz(a)anthracene
Table 4: Experiment I – Mutagenicity, with metabolic activation
CE in non-selective medium | CE in selective medium | |||||||||||||
Dose Group | Concentration | Number of colonies per flask | CE [%] | Number of colonies per flask | CE [%] | Mutant Frequency per 106 cells | ||||||||
[mg/mL] | I | II | mean | I | II | III | IV | V | mean | SD | ||||
NC1 | 0 | 145 | 149 | 147 | 74 | 10 | 11 | 12 | 6 | 9 | 9.6 | 2.1 | 0.0024 | 32.7 |
NC2 | 164 | 165 | 165 | 82 | 9 | 2 | 2 | 6 | 4 | 4.6 | 2.7 | 0.0012 | 14.0 | |
S1 | 0 | 143 | 142 | 143 | 71 | 7 | 7 | 6 | 11 | 9 | 8.0 | 1.8 | 0.0020 | 28.1 |
S2 | 141 | 132 | 137 | 68 | 10 | 10 | 6 | 6 | 9 | 8.2 | 1.8 | 0.0021 | 30.0 | |
1 | 0.03 | 155 | 154 | 155 | 77 | 8 | 7 | 4 | 7 | 5 | 6.2 | 1.5 | 0.0016 | 20.1 |
2 | 0.05 | 145 | 131 | 138 | 69 | 5 | 6 | 8 | 4 | 5 | 5.6 | 1.4 | 0.0014 | 20.3 |
3 | 0.1 | 169 | 160 | 165 | 82 | 10 | 13 | 4 | 6 | 6 | 7.8 | 3.2 | 0.0020 | 23.7 |
4 | 0.25 | 142 | 124 | 133 | 67 | 6 | 13 | 15 | 13 | 10 | 11.4 | 3.1 | 0.0029 | 42.9 |
5 | 0.5 | 154 | 148 | 151 | 76 | 13 | 14 | 13 | 11 | 16 | 13.4 | 1.6 | 0.0034 | 44.4 |
DMBA | 1 µg/mL | 147 | 171 | 159 | 80 | 81 | 60 | 68 | 66 | 54 | 65.8 | 9.0 | 0.0165 | 206.9 |
NC: negative control
S: solvent control (0.5% v/v ethanol)
CE: cloning efficiency
DMBA: 7,12-dimethylbenz(a)anthracene
Table 5: Experiment II – Toxicity, without metabolic activation
Dose Group | Concen-tration | Number of cells at the | Number of colonies per flask | CE [%] | Adjusted CE [%] | Relative Survival (RS) [%] | |||
[mg/mL] | beginning of treatment | end of treatment | I | II | mean | ||||
NC1 | 0 | 10000000 | 38250000 | 182 | 209 | 196 | 98 | 374 | 107 |
NC2 | 10000000 | 39610000 | 207 | 240 | 224 | 112 | 443 | 126 | |
S1 | 0 | 10000000 | 36040000 | 188 | 196 | 192 | 96 | 346 | 100 |
S2 | 10000000 | 35870000 | 201 | 196 | 199 | 99 | 356 | ||
1 | 0.0002 | 10000000 | 34170000 | 198 | 221 | 210 | 105 | 358 | 102 |
2 | 0.0005 | 10000000 | 33830000 | 202 | 206 | 204 | 102 | 345 | 98 |
3 | 0.001 | 10000000 | 34680000 | 230 | 189 | 210 | 105 | 363 | 103 |
4 | 0.002 | 10000000 | 33830000 | 184 | 193 | 189 | 94 | 319 | 91 |
5 | 0.005 | 10000000 | 36550000 | 204 | 183 | 194 | 97 | 354 | 101 |
6 | 0.007 | 10000000 | 37230000 | 187 | 180 | 184 | 92 | 342 | 97 |
7 | 0.01 | 20000000 | 64260000 | 232 | 259 | 246 | 123 | 394 | 112 |
8 | 0.02 | 20000000 | 71060000 | 257 | 269 | 263 | 132 | 467 | 133 |
9 | 0.03 | 20000000 | 48960000 | 223 | 214 | 219 | 109 | 267 | 76 |
10 | 0.04 | 20000000 | 13906000 | 166 | 141 | 154 | 77 | 53 | 15 |
EMS | 300 µg/mL | 10000000 | 32130000 | 176 | 172 | 174 | 87 | 280 | 80 |
NC: negative control
S: solvent control (0.5% v/v ethanol)
CE: cloning efficiency
EMS: Ethylmethanesulfonate
Table 6: Experiment II – Mutagenicity, without metabolic activation
CE in non-selective medium | CE in selective medium | |||||||||||||
Dose Group | Concen-tration | Number of colonies per flask | CE [%] | Number of colonies per flask | CE [%] | Mutant Frequency per 106 cells | ||||||||
[mg/mL] | I | II | mean | I | II | III | IV | V | mean | SD | ||||
NC1 | 0 | 175 | 189 | 182 | 91 | 2 | 8 | 0 | 5 | 3 | 3.6 | 2.7 | 0.0009 | 9.9 |
NC2 | 196 | 181 | 189 | 94 | 4 | 7 | 9 | 12 | 7 | 7.8 | 2.6 | 0.0020 | 20.7 | |
S1 | 0 | 161 | 169 | 165 | 83 | 9 | 7 | 7 | 9 | 10 | 8.4 | 1.2 | 0.0021 | 25.5 |
S2 | 186 | 202 | 194 | 97 | 7 | 5 | 5 | 9 | 4 | 6.0 | 1.8 | 0.0015 | 15.5 | |
6 | 0.007 | 183 | 194 | 189 | 94 | 7 | 3 | 6 | 5 | 5 | 5.2 | 1.3 | 0.0013 | 13.8 |
7 | 0.01 | 183 | 210 | 197 | 98 | 7 | 6 | 5 | 4 | 10 | 6.4 | 2.1 | 0.0016 | 16.3 |
8 | 0.02 | 213 | 218 | 216 | 108 | 14 | 6 | 8 | 9 | 9 | 9.2 | 2.6 | 0.0023 | 21.3 |
9 | 0.03 | 191 | 220 | 206 | 103 | 5 | 5 | 7 | 15 | 9 | 8.2 | 3.7 | 0.0021 | 20.0 |
10 | 0.04 | 178 | 205 | 192 | 96 | 14 | 11 | 14 | 12 | 11 | 12.4 | 1.4 | 0.0031 | 32.4 |
EMS | 300 µg/mL | 156 | 168 | 162 | 81 | 202 | 203 | 210 | 240 | 211 | 213.2 | 13.9 | 0.0533 | 658.0 |
NC: negative control
S: solvent control (0.5% v/v ethanol)
CE: cloning
efficiency
EMS: Ethylmethanesulfonate
Table 1: Pre-Experiment for Toxicity, without metabolic activation
Test Group |
Concentration [µg/mL] |
Number of Cells 4 h after Treatment |
Number of Cells 24 h after Treatment |
Number of Cells 48 h after Treatment |
SGa |
RSGb[%] |
C1 |
0 |
339000 |
963000 |
1320000 |
12.7 |
98.8 |
C2 |
363000 |
996000 |
1280000 |
12.7 |
99.1 |
|
S1 |
0 |
319000 |
870000 |
1390000 |
12.1 |
100.0 |
S2 |
325000 |
927000 |
1470000 |
13.6 |
||
1 |
25 |
325000 |
983000 |
1130000 |
11.1 |
86.4 |
2 |
50 |
356000 |
1020000 |
1380000 |
14.1 |
109.5 |
3 |
150 |
77500 |
44200 |
54800 |
0.2 |
1.3 |
4 |
500 |
98700 |
78700 |
269000 |
0.8 |
6.3 |
5 P |
1000 |
51700 |
21200 |
24900 |
0.1 |
0.6 |
6 P |
2000 |
61900 |
17400 |
13300 |
0.0 |
0.3 |
C: Negative control
S: Solvent control (0.5% ethanol; v/v)
P: Precipitation
a: Suspension Growth, SG = [((value 24 h x 30) / 1x107) x ((value 48 h x 20) / (value 24 h*x20))]
* : for value 24 h > 3x105 then value 24 h = 3x105
b: Relative Suspension Growth, RSG = [(value SG / value SG of corresponding controls) x 100]
Table 2: Pre-Experiment for Toxicity, with metabolic activation
Test Group |
Concentration [µg/mL] |
Number of Cells 4 h after Treatment |
Number of Cells 24 h after Treatment |
Number of Cells 48 h after Treatment |
SGa |
RSGb[%] |
C1 |
0 |
321000 |
873000 |
1360000 |
11.9 |
103.7 |
C2 |
313000 |
936000 |
1270000 |
11.9 |
103.8 |
|
S1 |
0 |
307000 |
879000 |
1190000 |
10.5 |
100.0 |
S2 |
325000 |
882000 |
1410000 |
12.4 |
||
1 |
25 |
311000 |
816000 |
1420000 |
11.6 |
101.2 |
2 |
50 |
327000 |
894000 |
1330000 |
11.9 |
103.9 |
3 |
150 |
258000 |
232000 |
737000 |
2.2 |
19.3 |
4 P |
500 |
263000 |
416000 |
1430000 |
5.9 |
52.0 |
5 P |
1000 |
175000 |
350000 |
1430000 |
5.0 |
43.7 |
6 P |
2000 |
200000 |
219000 |
879000 |
2.6 |
23.0 |
C: Negative control
S: Solvent control (0.5% ethanol; v/v)
P: Precipitation
a: Suspension Growth, SG = [((value 24 h x 30) / 1x107) x ((value 48 h x 20) / (value 24 h*x20))]
*: for value 24 h > 3x105 then value 24 h = 3x105
b: Relative Suspension Growth, RSG = [(value SG / value SG of corresponding controls) x 100]
Table 3: Main Experiment - Toxicity data, without metabolic activation
Test Group |
Concentration [µg/mL] |
Number of Cells 4 h after Treatment |
Number of Cells 24 h after Treatment |
Number of Cells 48 h after Treatment |
SGa |
RSGb[%] |
RCEc[%] |
RTGd[%] |
C1 |
0 |
365000 |
773000 |
1600000 |
12.4 |
107.0 |
107.4 |
114.9 |
C2 |
397000 |
891000 |
1420000 |
12.7 |
109.5 |
100.4 |
109.9 |
|
S1 |
0 |
371000 |
726000 |
1510000 |
11.0 |
100.0 |
100.0 |
100.0 |
S2 |
374000 |
799000 |
1520000 |
12.1 |
||||
1 |
50 |
358000 |
917000 |
1430000 |
13.1 |
113.5 |
89.8 |
101.9 |
3 |
60 |
325000 |
631000 |
1440000 |
9.1 |
78.6 |
92.6 |
72.9 |
5 |
70 |
304000 |
465000 |
1420000 |
6.6 |
57.2 |
111.2 |
63.5 |
6 |
72 |
302000 |
329000 |
970000 |
3.2 |
27.6 |
109.2 |
30.2 |
7 |
74 |
275000 |
303000 |
833000 |
2.5 |
21.8 |
85.8 |
18.7 |
8 |
76 |
266000 |
274000 |
553000 |
1.7 |
14.4 |
91.2 |
13.1 |
9 |
78 |
253000 |
310000 |
992000 |
3.1 |
26.6 |
98.8 |
26.3 |
10 |
80 |
192000 |
186000 |
352000 |
1.1 |
9.1 |
94.1 |
8.6 |
EMS |
300 |
364000 |
693000 |
1500000 |
10.4 |
90.0 |
75.1 |
67.6 |
MMS |
10 |
369000 |
729000 |
1260000 |
9.2 |
79.5 |
59.0 |
46.9 |
C: Negative control
S: Solvent control (0.5% ethanol; v/v)
P: Precipitation
a: Suspension Growth, SG = [((value 24h x 30) / 1x107) x ((value 48 h x 20) / (value 24 h*x20))]
*: for value 24 h > 3x105 then value 24 h = 3x105
b: Relative Suspension Growth, RSG = [(value SG / value SG of corresponding controls) x 100]
Table 4: Main Experiment - Mutagenicity data, without metabolic activation
Cloning Efficiency (CE) |
Mutagenicity Data |
||||||||||
Test Group |
Concen-tration [µg/mL] |
Plate 1e |
Plate 2e |
CEf[%] |
Number of cultures / 96 wells |
MFg [mutants / 106cells] |
IMFh [mutants / 106cells] |
||||
Plate 1e |
Plate 2e |
Plate 3e |
Plate 4e |
Mean |
|||||||
C1 |
0 |
76 |
83 |
110.1 |
25 |
26 |
26 |
32 |
27.3 |
152.1 |
/ |
C2 |
73 |
82 |
102.9 |
24 |
26 |
19 |
30 |
24.8 |
145.6 |
/ |
|
S1 |
0 |
70 |
80 |
95.0 |
16 |
27 |
17 |
23 |
20.8 |
129.1 |
/ |
S2 |
82 |
77 |
110.1 |
28 |
24 |
21 |
21 |
23.5 |
127.9 |
/ |
|
1 |
50 |
67 |
81 |
92.1 |
26 |
16 |
11 |
21 |
18.5 |
117.7 |
-10.9 |
3 |
60 |
73 |
77 |
95.0 |
21 |
25 |
23 |
25 |
23.5 |
147.9 |
19.4 |
5 |
70 |
78 |
83 |
114.0 |
22 |
22 |
31 |
23 |
24.5 |
129.9 |
1.4 |
6 |
72 |
82 |
78 |
112.0 |
36 |
36 |
31 |
19 |
30.5 |
173.1 |
44.5 |
7 |
74 |
72 |
73 |
88.0 |
31 |
33 |
39 |
33 |
34.0 |
249.2 |
120.7 |
8 |
76 |
80 |
69 |
93.5 |
31 |
30 |
29 |
36 |
31.5 |
213.1 |
84.6 |
9 |
78 |
78 |
76 |
101.2 |
27 |
35 |
31 |
25 |
29.5 |
182.2 |
53.6 |
10 |
80 |
78 |
73 |
96.5 |
39 |
32 |
35 |
26 |
33.0 |
219.7 |
91.2 |
EMS |
300 |
68 |
68 |
77.0 |
75 |
63 |
77 |
70 |
71.3 |
895.0 |
766.5 |
MMS |
10 |
63 |
56 |
60.4 |
68 |
66 |
68 |
68 |
67.5 |
1005.0 |
876.5 |
C: Negative control
S: Solvent control (0.5% ethanol; v/v)
a: Suspension Growth, SG = [((value 24hx30)/1x107) x ((value 48 h x 20)/(value 24 h*x20))]
* : for value 24h > 3x105then value 24h = 3x105
b: Relative Suspension Growth, RSG = [(value SG / value SG of corresponding controls) x 100]
c: Relative Cloning Efficiency, RCE = [(CEdose group/ CEof corresponding controls) x 100]
Cloning Efficiency, CE = ((-LN (((96 - (mean P1,P2)) / 96)) / 1.6) x 100)
d: Relative Total Growth, RTG = (RSG x RCE)/100
EMS: Ethylmethanesulfonate
MMS: Methylmethanesulfonate
Table 5: Main Experiment - Colony sizing, without metabolic activation
Test Group* |
Concentration [µg/mL] |
Wells with at least 1 colony |
Large colonies |
Small colonies |
% small colonies |
C1 |
0 |
109 |
87 |
22 |
20.2 |
C2 |
99 |
75 |
24 |
24.2 |
|
S1 |
0 |
83 |
56 |
27 |
32.5 |
S2 |
94 |
74 |
20 |
21.3 |
|
EMS |
300 |
285 |
241 |
44 |
15.4 |
MMS |
10 |
270 |
138 |
132 |
48.9 |
C: Negative control
S: Solvent control (0.5% ethanol; v/v)
e: Number of cultures with cell growth.
f: Cloning Efficiency, CE = ((-LN (((96 - (mean P1,P2)) / 96)) / 1.6) x 100)
g: Mutant frequency, MF = {-ln [negative cultures/total wells (selective medium)] / -ln [negative cultures/total wells (non selective medium)]}x800
h: Induced mutant frequency, IMF = mutant frequency sample – mean value mutant frequency corresponding controls
EMS: Ethylmethanesulfonate
MMS: Methylmethanesulfonate
Table 6: Main Experiment - Toxicity data, with metabolic activation
Test Group |
Concen-tration [µg/mL] |
Number of Cells 4 h after Treatment |
Number of Cells 24 h after Treatment |
Number of Cells 48 h after Treatment |
SGa |
RSGb[%] |
RCEc[%] |
RTGd[%] |
C1 |
0 |
351000 |
926000 |
1410000 |
13.1 |
92.9 |
103.2 |
95.9 |
C2 |
377000 |
1010000 |
1430000 |
14.4 |
102.8 |
96.9 |
99.6 |
|
S1 |
0 |
350000 |
954000 |
1420000 |
13.5 |
100.0 |
100.0 |
100.0 |
S2 |
369000 |
983000 |
1480000 |
14.5 |
||||
1 |
25 |
342000 |
927000 |
1450000 |
13.4 |
95.7 |
94.0 |
89.9 |
2 |
50 |
364000 |
975000 |
1390000 |
13.6 |
96.5 |
89.8 |
86.7 |
3 |
75 |
336000 |
826000 |
1420000 |
11.7 |
83.5 |
100.0 |
83.5 |
4 |
100 |
364000 |
782000 |
1460000 |
11.4 |
81.3 |
98.4 |
80.0 |
5 |
150 |
266000 |
338000 |
885000 |
3.0 |
21.3 |
96.9 |
20.6 |
6 |
300 |
276000 |
269000 |
643000 |
1.9 |
13.7 |
100.0 |
13.7 |
7 |
500 |
256000 |
277000 |
879000 |
2.6 |
18.8 |
96.9 |
18.2 |
8 |
750 |
282000 |
366000 |
1320000 |
4.8 |
34.4 |
104.9 |
36.1 |
B[a]P |
2.5 |
329000 |
514000 |
1380000 |
7.1 |
50.5 |
73.3 |
37.0 |
C: Negative control
S: Solvent control (0.5% ethanol; v/v)
EMS: Ethylmethanesulfonate
MMS: Methylmethanesulfonate
*: Based on the non-mutagenic effects of the test item, an assessment of clastogenicity was not feasible.
Table 7: Main Experiment - Mutagenicity data, with metabolic activation
Cloning Efficiency (CE) |
Mutagenicity Data |
||||||||||
Test Group |
Concen-tration [µg/mL] |
Plate 1e |
Plate 2e |
CEf[%] |
Number of cultures / 96 wells |
MFg [mutants / 106cells] |
IMFh [mutants / 106cells] |
||||
Plate 1e |
Plate 2e |
Plate 3e |
Plate 4e |
Mean |
|||||||
C1 |
0 |
78 |
74 |
98.0 |
11 |
11 |
16 |
10 |
12.0 |
68.3 |
/ |
C2 |
75 |
73 |
92.1 |
17 |
10 |
10 |
12 |
12.3 |
74.4 |
/ |
|
S1 |
0 |
76 |
75 |
96.5 |
16 |
14 |
24 |
14 |
17.0 |
101.7 |
/ |
S2 |
73 |
76 |
93.5 |
15 |
12 |
13 |
8 |
12.0 |
71.6 |
/ |
|
1 |
25 |
71 |
75 |
89.3 |
17 |
12 |
22 |
17 |
17.0 |
109.7 |
23.0 |
2 |
50 |
73 |
70 |
85.4 |
16 |
15 |
21 |
13 |
16.3 |
109.0 |
22.4 |
3 |
75 |
78 |
72 |
95.0 |
18 |
12 |
14 |
14 |
14.5 |
86.4 |
-0.3 |
4 |
100 |
76 |
73 |
93.5 |
13 |
15 |
10 |
16 |
13.5 |
81.2 |
-5.4 |
5 |
150 |
73 |
75 |
92.1 |
10 |
12 |
21 |
14 |
14.3 |
88.0 |
1.3 |
6 |
300 |
77 |
73 |
95.0 |
19 |
16 |
20 |
22 |
19.3 |
118.0 |
31.3 |
7 |
500 |
73 |
75 |
92.1 |
21 |
18 |
19 |
16 |
18.5 |
116.4 |
29.7 |
B[a]P |
2.5 |
68 |
61 |
69.6 |
40 |
47 |
41 |
43 |
42.8 |
424.0 |
337.3 |
C: Negative control
S: Solvent control (0.5% ethanol; v/v)
a: Suspension Growth, SG = [((value 24hx30)/1x107) x ((value 48 h x 20)/(value 24 h*x20))]
* : for value 24h > 3x105then value 24h = 3x105
b: Relative Suspension Growth, RSG = [(value SG / value SG of corresponding controls) x 100]
c: Relative Cloning Efficiency, RCE = [(CEdose group/ CEof corresponding controls) x 100]
Cloning Efficiency, CE = ((-LN (((96 - (mean P1,P2)) / 96)) / 1.6) x 100)
d: Relative Total Growth, RTG = (RSG x RCE)/100
B[a]P: Benzo[a]pyrene
Table 8: Main Experiment - Colony sizing, with metabolic activation
Test Group* |
Concentration [µg/mL] |
Wells with at least 1 colony |
Large colonies |
Small colonies |
% small colonies |
C1 |
0 |
48 |
38 |
10 |
20.8 |
C2 |
49 |
43 |
6 |
12.2 |
|
S1 |
0 |
68 |
53 |
15 |
22.1 |
S2 |
48 |
38 |
10 |
20.8 |
|
B[a]P |
2.5 |
171 |
95 |
76 |
44.4 |
C: Negative control
S: Solvent control (0.5% ethanol; v/v)
e: Number of cultures with cell growth.
f: Cloning Efficiency, CE = ((-LN (((96 - (mean P1,P2)) / 96)) / 1.6) x 100)
g: Mutant frequency,MF = {-ln [negative cultures/total wells (selective medium)] / -ln [negative cultures/total wells (non selective medium)]}x800
h: Induced mutant frequency, IMF = mutant frequency sample – mean value mutant frequency corresponding controls
B[a]P: Benzo[a]pyrene
Table 9: Main Experiment - Biometry, without metabolic activation
Test Group |
Concentration [µg/mL] |
mean Mutant Frequency |
mean induced Mutant Frequency |
p-value |
statistical Significance |
C1 |
0 |
152.1 |
/ |
0.2236 |
- |
C2 |
145.6 |
/ |
|||
S1 |
0 |
129.1 |
/ |
/ |
/ |
S2 |
127.9 |
/ |
/ |
/ |
|
1 |
50 |
117.7 |
-10.9 |
0.6545 |
- |
3 |
60 |
147.9 |
19.4 |
0.2404 |
- |
5 |
70 |
129.9 |
1.4 |
0.6505 |
- |
6 |
72 |
173.1 |
44.5 |
0.1394 |
- |
7 |
74 |
249.2 |
120.7 |
0.0040 |
+ |
8 |
76 |
213.1 |
84.6 |
0.0040 |
+ |
9 |
78 |
182.2 |
53.6 |
0.0263 |
+ |
10 |
80 |
219.7 |
91.2 |
0.0061 |
+ |
EMS |
300 |
895.0 |
766.5 |
0.0040 |
+ |
MMS |
10 |
1005.0 |
876.5 |
0.0040 |
+ |
C: Negative control
S: Solvent control (0.5% ethanol; v/v)
B[a]P: Benzo[a]pyrene
*: Based on the non-mutagenic effects of the test item, an assessment of clastogenicity was not feasible.
Table 10: Main Experiment - Biometry, with metabolic activation
Test Group |
Concentration [µg/mL] |
mean Mutant Frequency |
mean induced Mutant Frequency |
p-value |
statistical Significance |
C1 |
0 |
68.3 |
/ |
0.2236 |
- |
C2 |
74.4 |
/ |
|||
S1 |
0 |
101.7 |
/ |
/ |
/ |
S2 |
71.6 |
/ |
/ |
/ |
|
1 |
25 |
109.7 |
23.0 |
0.2606 |
- |
2 |
50 |
109.0 |
22.4 |
0.0990 |
- |
3 |
75 |
86.4 |
-0.3 |
0.7798 |
- |
4 |
100 |
81.2 |
-5.4 |
> 0.9999 |
- |
5 |
150 |
88.0 |
1.3 |
0.9091 |
- |
6 |
300 |
118.0 |
31.3 |
0.0424 |
+ |
7 |
500 |
116.4 |
29.7 |
0.0424 |
+ |
B[a]P |
2.5 |
424.0 |
337.3 |
0.0020 |
+ |
C: Negative control
S: Solvent control (0.5% ethanol; v/v)
EMS: Ethylmethanesulfonate
MMS: Methylmethanesulfonate
+: significant
-: not significant
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Dibutyl peroxydicarbonate was evaluated for genotoxicity/mutagenicity in four of the standard genetic toxicology tests. It was evaluated in the Ames test, the in vitro mammalian HPRT gene mutation assay, the in vitro mammalian mouse lymphoma gene mutation assay using the thymidine kinase locus and in the in vitro mammalian micronucleus test. Negative results were obtained in the bacterial Ames test, the mouse lymphoma assay and the micronucleus test. The in vitro mammalian HPRT assay gave borderline results. The study director concluded that dibutyl peroxydicarbonate is considered to be mutagenic at cytotoxic concentrations in the first experiment without and with metabolic activation in the HPRT locus using V79 cells of the Chinese Hamster. As the mutagenic potential of the test item could not be verified in the long-term experiment without metabolic activation, the response was concluded to be equivocal in the HPRT mutagenicity test.
Because the results were different, it is important to compare the results observed in the HPRT and the mouse lymphoma assays. Three different test conditions were used for the HPRT assay, 4 h and 20 h exposure without exogeneous metabolic activation (S9) and a 4 h exposure with S9. The 4 h exposure without S9 had background mutant frequencies that were close to the maximum values shown in the laboratory historical controls. While there was an overall dose response both for cytotoxicity and for mutant frequency, there was substantial variability and the highest mutant frequency occurred at one of the lower concentrations (a concentration that demonstrated only a minor amount of cytotoxicity). Thus, the response without S9, while suggestive of a positive response, was not clearly positive. The 4 h exposure with S9 showed substantial variability in the concurrent negative controls. There was a dose related increase in both cytotoxicity and mutant frequency. The top two concentrations induced appropriate maximum levels of cytotoxicity and very small increases in mutant frequency. These increases were sufficient to minimally meet the criteria for a positive response. The 20 h exposure without S9 was clearly negative. A 20 h exposure with S9 cannot be run for technical reasons. Thus, the overall response in the HPRT assay was a weak positive (observed with S9).
For the mouse lymphoma assay, two test conditions, 4 h exposure with and without S9, were used. Without S9, there was a dose related increase in cytotoxicity. Ten concentrations were used to cover the dose range and several concentrations induced the appropriate maximum levels of cytotoxicity. Because none of these cultures showed an increase in mutant frequency that met the criteria for a positive response, the result was clearly negative. With S9 there was also a dose related increase in cytotoxicity. Three of the eight concentrations induced the appropriate maximum level of cytotoxicity. There was no dose related increase in the mutant frequency. The result with S9 was also clearly negative. Thus, the overall response for the mouse lymphoma assay was a clear negative.
When comparing the results obtained in the three gene mutation assays (Ames test, HPRT and the mouse lymphoma assay) that were conducted for dibutyl peroxydicarbonate, it is important to bear in mind that while all three assays respond to test materials that induce point mutations, the mouse lymphoma assays detects a much broader range of genetic damage (OECD TG490). If dibutyl peroxydicarbonate was capable of inducing point mutations, it would be expected that positive results would have been obtained in all three assays. Because the results seen in the HPRT assay were not robustly positive, it can be concluded that the clear negative results for the Ames test and the mouse lymphoma assay outweigh the results and that dibutyl peroxydicarbonate is not mutagenic. Because the mouse lymphoma assay can detect viable chromosome damage, the negative results in both that assay, and the in vitro micronucleus test indicate that dibutyl peroxydicarbonate does not induce chromosomal effects. Therefore, it can be concluded that dibutyl peroxydicarbonate is not genotoxic/mutagenic.
Justification for classification or non-classification
Based on the available data for mutagenicity used in a weight of evidence approach, dibutyl peroxydicarbonate is not considered to be genotoxic and no classification is warranted in accordance with CLP regulation (EC) 1272/2008.
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