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EC number: 208-021-9 | CAS number: 505-84-0
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
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- Nanomaterial pour density
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- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
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- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
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- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- Experimental start date 28 July 2017 Experimental completion date 17 November 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 018
- Report date:
- 2018
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell micronucleus test
Test material
- Reference substance name:
- Diethoxymethane
- EC Number:
- 207-330-6
- EC Name:
- Diethoxymethane
- Cas Number:
- 462-95-3
- Molecular formula:
- C5H12O2
- IUPAC Name:
- Diethoxymethane
- Test material form:
- liquid
- Details on test material:
- Identification: Diethoxymethane
Synonym: Ethylal
CAS Number: 462-95-3
Physical State/Appearance: Clear colourless liquid
Storage Conditions: Approximately 4 °C, in the dark, under nitrogen
No correction for purity was required.
Constituent 1
- Specific details on test material used for the study:
- Identification: Ethylal
Chemical name: Diethoxymethane
CAS Number: 462-95-3
Physical state/Appearance: Clear colorless liquid
Storage Conditions: Approximately 4 °C in the dark under nitrogen
Intended use/Application: Not supplied
No correction for purity was made.
Method
- Target gene:
- not applicable
Species / strain
- Species / strain / cell type:
- primary culture, other: whole blood
- Details on mammalian cell type (if applicable):
- Cells
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non-smoking volunteer (18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. Based on over 20 years in-house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 hours. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 hours.
The details of the donors used are:
Preliminary Toxicity Test: male, aged 29 years
Main Experiment: female, aged 25 years
Cell Culture
Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% fetal bovine serum (FBS), at approximately 37 ºC with 5% CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).
- Cytokinesis block (if used):
- Cytochalasin B
- Metabolic activation:
- with and without
- Metabolic activation system:
- standard metabolizing system (S9)
- Test concentrations with justification for top dose:
- Preliminary Toxicity Test
The dose range of test item used was 0, 4.07, 8.14, 16.28, 32.56, 65.13, 130.25, 260.5, 521 and 1042 μg/mL.
Main Experiment
The dose range of test item used for all three exposures was 0, 65.13, 130.25, 260.5, 521, 781.5, 1042 μg/mL. - Vehicle / solvent:
- The test item was miscible in aqueous media (MEM) at 10.42 mg/mL by mixing on a vortex for approximately 10 seconds in a solubility check performed in-house. The highest concentration of the test item tested in this study was 10.42 mg/mL.
Controlsopen allclose all
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Minimal Essential Medium
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- 0.2 μg/mL for 4-hour exposure
- Positive control substance:
- mitomycin C
- Remarks:
- Absence of S9-mix
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Minimal Essential Medium
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- 0.075 μg/mL for 24-hour continuous exposure
- Positive control substance:
- other: Demecolcine
- Remarks:
- Absence of S9-mix
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Minimal Essential Medium
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- 5 μg/mL for 4-hour exposure
- Positive control substance:
- cyclophosphamide
- Remarks:
- Presence of S9-mix
- Details on test system and experimental conditions:
- Culture conditions
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:
8.05-9.05 mL MEM, 10% (FBS)
0.1 mL Li-heparin
0.1 mL phytohaemagglutinin
0.75 mL heparinized whole blood
4-Hour Exposure With Metabolic Activation (S9)
After approximately 48 hours incubation at approximately 37 ºC, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 1.0 mL of the appropriate solution of vehicle control or test item was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1.0 mL of 20% S9-mix (i.e. 2% final concentration of S9 in standard co-factors) was added to the cultures of the Preliminary Toxicity Test and the Main Experiment. All cultures were then returned to the incubator. The nominal total volume of each culture was 10 mL.
After 4 hours at approximately 37 ºC, the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium, supplemented with Cytochalasin B at a final concentration of 4.5 μg/mL, and then incubated for a further 24 hours.
4-Hour Exposure Without Metabolic Activation (S9)
After approximately 48 hours incubation at approximately 37 ºC with 5% CO2 in humidified air, the cultures were decanted into tubes and centrifuged. Approximately 9 mL of the culture medium was removed and reserved. The cells were then resuspended in the required volume of fresh MEM (including serum) and dosed with 1.0 mL of the appropriate vehicle control, test item solution or 0.1 mL of positive control solution. The nominal total volume for each culture was 10 mL.
After 4 hours at approximately 37 ºC, the cultures were centrifuged, the treatment medium was removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium, supplemented with Cytochalasin B, at a final concentration of 4.5 μg/mL, and then incubated for a further 24 hours.
24-Hour Exposure Without Metabolic Activation (S9)
The exposure was continuous for 24 hours in the absence of metabolic activation. Therefore, when the cultures were established the culture volume was a nominal 9 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 1.0 mL of vehicle control, test item dose solution or 0.1 mL of positive control solution. The nominal total volume of each culture was 10 mL. The cultures were then incubated for 24 hours, the tubes and the cells washed in MEM before resuspension in fresh MEM with serum. At this point Cytochalasin B was added at a final concentration of 4.5 μg/mL, and then the cells were incubated for a further 24 hours.
The extended exposure detailed above does not follow the suggested cell treatment schedule in the Guideline. This is because it avoids any potential interaction between Cytochalasin B and the test item during exposure to the cells and any effect this may have on the activity or response. Additionally, as the stability or reactivity of the test item is unknown prior to the start of the study this modification of the schedule is considered more effective and reproducible due to the in-house observations on human lymphocytes and their particular growth characteristics in this study type and also the significant laboratory historical control data using the above format.
The preliminary toxicity test was performed using the exposure conditions as described for the Main Experiment but using single cultures only, whereas the Main Experiment used replicate cultures.
Preliminary Toxicity Test
Three exposure groups were used:
i) 4-hour exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
iii) 24-hour continuous exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
The dose range of test item used was 0, 4.07, 8.14, 16.28, 32.56, 65.13, 130.25, 260.5, 521 and 1042 μg/mL.
Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.
Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for the evaluation of the frequency of binucleate cells and to calculate the cytokinesis block proliferation index (CBPI). Coded slides were evaluated for the CBPI. The CBPI data were used to estimate test item toxicity and for selection of the dose levels for the experiments of the main test.
Main Experiment
Three exposure groups were used for Main Experiment:
i) 4-hour exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
iii) 24-hour continuous exposure to the test item without S9-mix, followed by a 24-hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
The dose range of test item used for all three exposures was 0, 65.13, 130.25, 260.5, 521, 781.5, 1042 μg/mL.
Cell Harvest
At the end of the Cytochalasin B treatment period the cells were centrifuged, the culture medium was drawn off and discarded, and the cells resuspended in MEM. The cells were then treated with a mild hypotonic solution (0.0375M KCl) before being fixed with fresh methanol/glacial acetic acid (19:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC prior to slide making.
Preparation of Microscope Slides
The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labelled with the appropriate identification data.
Staining
When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium. - Evaluation criteria:
- Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly negative if, in most/all of the experimental conditions examined:
1. None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is no dose-related increase.
3. The results in all evaluated dose groups should be within the range of the laboratory historical control data.
Providing that all of the acceptability criteria are fulfilled, a test item may be considered to be clearly positive, if in any of the experimental conditions examined, there is one or more of the following applicable:
1. At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is an increase which can be considered to be dose-related.
3. The results are substantially outside the range of the laboratory historical negative control data.
When all the criteria are met, the test item is considered able to induce chromosome breaks and/or gain or loss in this test system.
There is no requirement for verification of a clear positive or negative response.
In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations.
Test items that induce micronuclei in the MNvit test may do so because they induce chromosome breakage, chromosome loss, or a combination of the two. Further analysis using anti-kinetechore antibodies, centromere specific in situ probes, or other methods can be used to determine whether the mechanism of micronucleus induction is due to clastogenic and/or aneugenic activity. - Statistics:
- The frequency of binucleate cells with micronuclei was compared, where necessary, with the concurrent vehicle control value using the Chi-squared Test on observed numbers of cells with micronuclei. Other statistical analyses may be used if appropriate (Hoffman et al., 2003). A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the frequency of binucleate cells with micronuclei was less than 0.05 and there was a dose-related increase in the frequency of binucleate cells with micronuclei which was reproducible.
Results and discussion
Test results
- Species / strain:
- primary culture, other: whole blood
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- Preliminary Toxicity Test (Table 1)
The dose range for the Preliminary Toxicity Test was 4.07 to 1042 μg/mL. The molecular weight of the test item was given as 104.15, therefore, the maximum dose level was 1042 μg/mL, which was calculated to be equivalent to the 10mM concentration (the maximum recommended dose level).
No precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at any dose level tested in any of the exposure groups.
Microscopic assessment of the slides prepared from the exposed cultures showed that binucleate cells were present at up to the maximum concentration in all three exposure groups. The test item induced no evidence of toxicity in any of the exposure groups.
The selection of the maximum dose level for the Main Experiment was, therefore, based on the maximum recommended dose level (1042 μg/mL) for all three exposure group.
Micronucleus Test – Main Experiment
As during the Preliminary Toxicity Test, there were binucleate cells suitable for scoring at the maximum dose level of test item, 1042 μg/mL, for all three exposure groups.
No precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at any dose level tested in any of the exposure groups.
The CBPI data confirm the qualitative observations in that no dose-related inhibition of CBPI was observed Tables 2 to 6).
The maximum dose level selected for analysis of binucleate cells was the maximum recommended dose level (1042 μg/mL) for all three exposure groups.
The vehicle control cultures had frequencies of cells with micronuclei within the expected range. The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The test item did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei, either in the absence or presence of metabolic activation.
Any other information on results incl. tables
The dose levels of the controls and the test item are given in the table below:
Group | Final concentration of test item Ethylal (µg/mL) |
4-hour without S9 | 0*,65.13, 130.25, 260.5, 521*, 781.5*, 1042*,MMC0.2* |
4-hour with S9 (2%) | 0*, 65.13, 130.25, 260.5, 521*, 781.5*, 1042*,CP5* |
24-hour without S9 | 0*, 65.13, 130.25, 260.5, 521*, 781.5*, 1042*,DC0.075* |
* = Dose levels selected for analysis of micronucleus frequency in binucleate cells
MMC = MitomycinC
CP = Cyclophosphamide
DC =Demecolcine
Table 1: CBPI - Preliminary Toxicity test
4-hour exposure without S9 |
4-hour exposure with S9 |
24-hour exposure without S9 | |||||||||||||||
Dose Level μg/mL | Nucleate Cells/500 Cells |
CBPI |
% Cytostasis |
Dose Level μg/mL | Nucleate Cells/500 Cells |
CBPI |
% Cytostasis |
Dose Level μg/mL | Nucleate Cells/500 Cells |
CBPI |
% Cytostasis | ||||||
Mono |
Bi |
Multi |
Mono |
Bi |
Multi |
Mono |
Bi |
Multi | |||||||||
0 |
248 |
228 |
24 |
1.55 |
0 |
0 |
229 |
244 |
27 |
1.60 |
0 |
0 |
83 |
313 |
104 |
2.04 |
0 |
4.07 |
- |
- |
- |
- |
- |
4.07 |
- |
- |
- |
- |
- |
4.07 |
- |
- |
- |
- |
- |
8.14 |
- |
- |
- |
- |
- |
8.14 |
- |
- |
- |
- |
- |
8.14 |
- |
- |
- |
- |
- |
16.28 |
- |
- |
- |
- |
- |
16.28 |
- |
- |
- |
- |
- |
16.28 |
- |
- |
- |
- |
- |
32.56 |
- |
- |
- |
- |
- |
32.56 |
- |
- |
- |
- |
- |
32.56 |
- |
- |
- |
- |
- |
65.13 |
- |
- |
- |
- |
- |
65.13 |
- |
- |
- |
- |
- |
65.13 |
- |
- |
- |
- |
- |
130.25 |
- |
- |
- |
- |
- |
130.25 |
- |
- |
- |
- |
- |
130.25 |
- |
- |
- |
- |
- |
260.5 |
244 |
229 |
27 |
1.57 |
0‡ |
260.5 |
270 |
210 |
20 |
1.50 |
17 |
260.5 |
101 |
306 |
93 |
1.98 |
6 |
521 |
296 |
193 |
11 |
1.43 |
22 |
521 |
201 |
282 |
17 |
1.63 |
0‡ |
521 |
54 |
354 |
92 |
2.08 |
0‡ |
1042 |
228 |
255 |
17 |
1.58 |
0‡ |
1042 |
259 |
224 |
17 |
1.52 |
13 |
1042 |
66 |
345 |
89 |
2.05 |
0‡ |
- = Not selected for scoring
‡ = Cytosis reported as 0 as the CBPI value is equal to or higher than the solvent control
Table 2: CBPI Data - Main Experiment - 4HOUR Exposure With and Without Metabolic Activation (S9)
4-Hour exposure without S9 | 4-hour exposure with S9 | ||||||||||||||
Dose level (µg/mL) |
Replicate |
Nucleate cells /500 cells |
CBPI |
Mean CBPI |
% Cytostasis |
Dose level (µg/mL) |
Replicate |
Nucleate cells /500 cells |
CBPI |
Mean CBPI |
% Cytostasis | ||||
M ono | Bi | Multi | M ono | Bi | Multi | ||||||||||
0 | A | 125 | 316 | 59 | 1.87 |
1.89 |
0 |
0 | A | 227 | 241 | 32 | 1.61 |
1.65 |
0 |
B | 109 | 333 | 58 | 1.90 | B | 203 | 251 | 46 | 1.69 | ||||||
65.13 | A | - | - | - | - |
- |
- |
65.13 | A | - | - | - | - |
- |
- |
B | - | - | - | - | B | - | - | - | - | ||||||
130.25 | A | - | - | - | - |
- |
- |
130.25 | A | - | - | - | - |
- |
- |
B | - | - | - | - | B | - | - | - | - | ||||||
260.5 | A | - | - | - | - |
- |
- |
260.5 | A | - | - | - | - |
- |
- |
B | - | - | - | - | B | - | - | - | - | ||||||
521 | A | 150 | 306 | 44 | 1.79 |
1.80 |
10 |
521 | A | 231 | 244 | 25 | 1.59 |
1.59 |
10 |
B | 150 | 302 | 48 | 1.80 | B | 233 | 244 | 23 | 1.58 | ||||||
781.5 | A | 125 | 331 | 44 | 1.84 |
1.84 |
5 |
781.5 | A | 217 | 253 | 30 | 1.63 |
1.59 |
9 |
B | 113 | 353 | 34 | 1.84 | B | 258 | 209 | 33 | 1.55 | ||||||
1042 | A | 144 | 325 | 31 | 1.77 |
1.76 |
15 |
1042 | A | 265 | 209 | 26 | 1.52 |
1.58 |
12 |
B | 172 | 286 | 42 | 1.74 | B | 218 | 247 | 35 | 1.63 | ||||||
MMC 0.2 | A | 268 | 227 | 5 | 1.47 |
1.48 |
46 |
CP 5 | A | 356 | 142 | 2 | 1.29 |
1.31 |
53 |
B | 258 | 238 | 4 | 1.49 | B | 346 | 146 | 8 | 1.32 |
MMC = Mitomycin C
CP = Cyclophosphamide
- = No selected for scoring
Table 3: CBPI Data - Main Experiment - 24-Hour Exposure Without Metabolic Activation (S9)
24-Hour exposure without S9 | |||||||
Dose level (µg/mL) |
Replicate |
Nucleate cells /500 cells |
CBPI |
Mean CBPI |
% Cytostasis | ||
Mono | Bi | Multi | |||||
0 | A | 82 | 383 | 65 | 1.97 |
1.96 |
0 |
B | 96 | 340 | 64 | 1.94 | |||
65.13 | A | - | - | - | - |
- |
- |
B | - | - | - | - | |||
130.25 | A | - | - | - | - |
- |
- |
B | - | - | - | - | |||
260.5 | A | - | - | - | - |
- |
- |
B | - | - | - | - | |||
521 | A | 91 | 346 | 63 | 1.94 |
1.96 |
0‡ |
B | 89 | 336 | 75 | 1.97 | |||
781.5 | A | 75 | 366 | 59 | 1.97 |
1.99 |
0‡ |
B | 74 | 345 | 81 | 2.01 | |||
1042 | A | 69 | 366 | 65 | 1.99 |
1.97 |
0‡ |
B | 88 | 346 | 57 | 1.94 | |||
DC 0.075 | A | 196 | 259 | 45 | 1.70 |
1.66 |
31 |
B | 237 | 218 | 45 | 1.62 |
DC = Demecolcine
- = Not selected for scoring
‡ = Cytosis reported as 0 as the CBPI value is equal to or higher than the solvent control
Table 4: CBPI and Micronucleus Data - Main Experiment - 4-Hour Exposure Without Metabolic Activation (S9)
Exposure Time +/-S9 |
Dose Level (μg/mL) |
Replicate |
Nucleate cells /500 cells |
CBPI |
% Cytostasis | Micronuclei (MN) Per 1000 Binucleate cells | % Binucleate Cells with MN | Mean % Binucleate Cells with MN | ||||
Mono |
Bi |
Multi |
1 MN |
2 MN |
>2 MN | |||||||
4Hr-S9 |
0 | A | 125 | 316 | 59 | 1.87 |
0 | 5 | 0 | 0 | 0.50 |
0.65 |
B | 109 | 333 | 58 | 1.90 | 7 | 1 | 0 | 0.80 | ||||
521 | A | 150 | 306 | 44 | 1.79 |
10 | 3 | 0 | 0 | 0.30 |
0.40 | |
B | 150 | 302 | 48 | 1.80 | 5 | 0 | 0 | 0.50 | ||||
781.5 | A | 125 | 331 | 44 | 1.84 |
5 | 6 | 0 | 0 | 0.60 |
0.50 | |
B | 113 | 353 | 34 | 1.84 | 4 | 0 | 0 | 0.40 | ||||
1042 | A | 144 | 325 | 31 | 1.77 |
15 | 4 | 1 | 0 | 0.50 |
0.45 | |
B | 172 | 286 | 42 | 1.74 | 3 | 1 | 0 | 0.40 | ||||
MMC 0.2 | A | 268 | 227 | 5 | 1.47 |
46 | 62 | 5 | 0 | 6.70 |
9.7*** | |
B | 258 | 238 | 4 | 1.49 | 116 | 8 | 3 | 12.70 |
MMC = Mitomycin C
*** = P<0.001
Table 5: CBPI and Micronucleus Data - Experiment - 4-Hour Exposure With Metabolic Activation (S9)
Exposure Time +/-S9 |
Dose Level (μg/mL) |
Replicate |
Nucleate cells /500 cells |
CBPI |
% Cytostasis | Micronuclei (MN) Per 1000 Binucleate cells | % Binucleate Cells with MN | Mean % Binucleate Cells with MN | ||||
Mono |
Bi |
Multi |
1 MN |
2 MN |
>2 MN | |||||||
4Hr+S9 |
0 | A | 227 | 241 | 32 | 1.61 |
0 | 5 | 0 | 0 | 0.50 |
0.45 |
B | 203 | 251 | 46 | 1.69 | 4 | 0 | 0 | 0.40 | ||||
521 | A | 231 | 244 | 25 | 1.59 |
10 | 1 | 1 | 0 | 0.20 |
0.25 | |
B | 233 | 244 | 23 | 1.58 | 3 | 0 | 0 | 0.30 | ||||
781.5 | A | 217 | 253 | 30 | 1.63 |
9 | 5 | 1 | 1 | 0.70 |
0.55 | |
B | 258 | 209 | 33 | 1.55 | 4 | 0 | 0 | 0.40 | ||||
1042 | A | 265 | 209 | 26 | 1.52 |
12 | 4 | 0 | 0 | 0.40 |
0.50 | |
B | 218 | 247 | 35 | 1.63 | 6 | 0 | 0 | 0.60 | ||||
CP 5 | A | 356 | 142 | 2 | 1.29 |
53 | 35 | 2 | 0 | 3.70 |
4.15*** | |
B | 346 | 146 | 8 | 1.32 | 44 | 2 | 0 | 4.60 |
CP = Cyclophosphamide
*** = P<0.001
Table 6: CBPI and Micronucleus Data - Main Experiment - 24-Hour Exposure Without Metabolic Activation (S9)
Exposure Time +/- S9 |
Dose Level (μg/mL) |
Replicate |
Nucleate cells /500 cells |
CBPI |
% Cytostasis | Micronuclei (MN) Per 1000 Binucleate cells | % Binucleate Cells with MN | Mean % Binucleate Cells with MN | ||||
Mono |
Bi |
Multi |
1 MN |
2 MN |
>2 MN | |||||||
24Hr-S9 |
0 | A | 82 | 383 | 65 | 1.97 |
0 | 6 | 0 | 1 | 0.70 |
0.55 |
B | 96 | 340 | 64 | 1.94 | 3 | 0 | 1 | 0.40 | ||||
521 | A | 91 | 346 | 63 | 1.94 |
0‡ | 5 | 0 | 0 | 0.50 |
0.65 | |
B | 89 | 336 | 75 | 1.97 | 6 | 0 | 2 | 0.80 | ||||
781.5 | A | 75 | 366 | 59 | 1.97 |
0‡ | 7 | 0 | 0 | 0.70 |
0.90 | |
B | 74 | 345 | 81 | 2.01 | 11 | 0 | 0 | 1.10 | ||||
1042 | A | 69 | 366 | 65 | 1.99 |
0‡ | 0 | 0 | 0 | 0.00 |
0.40 | |
B | 88 | 346 | 57 | 1.94 | 2 | 0 | 6 | 0.80 | ||||
DC 0.075 | A | 196 | 259 | 45 | 1.70 |
31 | 40 | 9 | 5 | 5.40 |
5.75*** | |
B | 237 | 218 | 45 | 1.62 | 36 | 19 | 6 | 6.10 |
DC = Demecolcine
*** = P<0.001
‡ = Cytosis reported as 0 as the CBPI value is equal to or higher than the solvent control
Applicant's summary and conclusion
- Conclusions:
- Under the conditions of this test, the test item, Ethylal, did not induce a statistically significant increase in the frequency of binucleate cells with micronuclei in either the absence or presence of a metabolizing system. The test item was therefore considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.
- Executive summary:
Introduction
This report describes the results of an in vitro study for the detection of the clastogenic and aneugenic potential of the test item on the nuclei of normal human lymphocytes.
Methods
Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells at three dose levels, together with vehicle and positive controls. Three exposure conditions in a single experiment were used for the study using a 4-hour exposure in the presence and absence of a standard metabolizing system (S9) at a 2%
final concentration and a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B.
The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be the maximum recommended limit (1042 µg/mL). The dose levels selected for the Main Test were as follows:
Group
Final concentration of test item Ethylal (µg/mL)
4-hour without S9
0, 65.13, 130.25, 260.5, 521, 781.5, 1042
4-hour with S9 (2%)
24-hour without S9
Results
All vehicle (Eagle's minimal essential medium with HEPES buffer (MEM)) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes.
The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The test item was non-toxic to human lymphocytes and did not induce any statistically significant increases in the frequency of cells with micronuclei, using a dose range that included a dose level that was the maximum recommended dose level.
Conclusion
Under the conditions of this study, the test item, Ethylal, was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.
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