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Genetic toxicity: in vivo

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in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
GLP-study according to OECD guideline 474.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of divinylbenzene and ethylstyrene
EC Number:
Cas Number:
Molecular formula:
Divinylbenzene: C10H10 Ethylstyrene: C10H12
Reaction mass of divinylbenzene and ethylstyrene
Details on test material:
Test Material Name: Divinylbenzene 55
Chemical Name: reaction mass of divinylbenzene and ethylstyrene
Synonyms: DVB-55
Supplier, City, State (Lot, Reference Number): The Dow Chemical Company, Midland, Michigan (lot # XF30012V44)
Purity/Characterization (Method of Analysis and Reference): The purity of the test material was determined to be 56.1% Diethenylbenzene by gas chromatography with identification by gas chromatography mass spectrometry (Butler, 2009).

Test animals

Details on test animals or test system and environmental conditions:
- Source: Charles River Laboratories (Raleigh, North Carolina)
- Age at study initiation: Approximately 8 weeks
- Weight at study initiation: 25.8 g (mean)
- Assigned to test groups randomly: yes
- Fasting period before study: not applicable
- Housing: animals were housed one per cage in stainless steel cages. Cages had wire mesh floors and were suspended above absorbent paper. Non-woven gauze was placed in the cages to provide a cushion from the flooring for rodent feet. The gauze provided environmental enrichment. Cages contained a hanging feeder and a pressure activated lixit valve-type watering system.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least one week prior to the start of the study

- Temperature (°C): 22°C with a tolerance of ± 1°C (and a maximum permissible excursion of ± 3°C)
- Humidity (%): 40-70%
- Air changes (per hr): 12-15 times/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/dark (on at 6:00 a.m. and off at 6:00 p.m.)

Administration / exposure

Route of administration:
inhalation: vapour
not applicable
Details on exposure:
Chambers: the animals were exposed to filtered air or DVB-55 vapors in 4 cubic meter stainless steel and glass Rochester-type whole-body exposure chambers [1.5 meters (m) x 1.5 m wide x 1.3 m deep with a pyramidal top and bottom]. Chamber airflow was maintained at approximately 900 liters per minute. This flow rate was sufficient to provide the normal concentration of oxygen to the animals and 12-15 calculated air changes per hour. The chambers were operated at a slightly negative pressure, relative to the surrounding area. The airflow through the exposure chamber was calibrated with a gas meter (Singer Aluminum Diaphragm Meter, Model AL-1400, American Meter Division, Philadelphia, Pennsylvania) prior to the start of the study. Chamber temperature and relative humidity data were recorded from a thermometer (Control Company, Friendswood, Texas) and hygrometer (Brooklyn Thermometer Co., Inc., Farmingdale, New York) stationed in the interior of each chamber. The chamber temperature and relative humidity was controlled by a system designed to maintain values of approximately 22 ± 3 °C and 30% – 70%, respectively. Chamber temperature, relative humidity, and airflow data were recorded once per hour during each exposure period.
Generation System: The various concentrations of DVB-55 were generated using a glass J-tube method. Liquid test material was pumped into the glass J-tube assembly and vaporized by the flow of compressed nitrogen gas passing through the bead bed of the glass J-tube. The nitrogen was heated with a flameless heat torch (FHT-4, Master Appliance Corporation, Racine, Wisconsin) to the minimum extent necessary to vaporize the test material. All chambers, including the 0 ppm (negative control) chamber received the same volume percent of supplemental nitrogen (carrier gas). The minimum amount of nitrogen necessary to reach the desired chamber concentrations were used. The generation system was electrically grounded and the J-tubes were changed as needed. The vaporized test material and carrier gas was mixed and diluted with supply air to achieve a total flow of 900 liters per minute at the desired test chamber concentration.
Duration of treatment / exposure:
3 consecutive days
Frequency of treatment:
6 hours/day
Post exposure period:
2 and 48 hours after the termination of the third exposure
Doses / concentrationsopen allclose all
Dose / conc.:
37.5 ppm (nominal)
35.0 ± 3.0 ppm (analytical)
Dose / conc.:
75 ppm (nominal)
74.1 ± 4.3 ppm (analytical)
Dose / conc.:
150 ppm (nominal)
142.9 ± 7.9 ppm (analytical)
No. of animals per sex per dose:
5/exposure/timepoint, except for highest exposure where 6/timepoint were used
Control animals:
yes, sham-exposed
Positive control(s):
Cyclophosphamide monohydrate (CP), CAS Number 6055-19-2, Source: Sigma, St. Louis, Missouri.

CP was administered by oral gavage in an aliquot of 10 mL/kg body weight (bw). CP when administered by oral gavage has been shown to induce micronuclei in polychromatic erythrocytes. The dose level for CP was 40 mg/kg and was based upon experience which indicated a pronounced micronuclei induction in B6C3F1/Crl mice at this dose level. CP was administered only once, approximately 48 hours (±1 hour) before sacrifice. A frozen stock solution of CP dissolved in distilled water (thawed and brought to room temperature prior to use) was used.


Tissues and cell types examined:
Micronucleus formation in peripheral blood reticulocytes was determined by flow cytometry with traditional blood smears prepared as a backup. Approximately 5000 RETs were analyzed per blood sample. The number of normochromatic erythrocytes (NCE), MN-NCE, RET, and MN-RET were recorded for each sample and the frequency of MN-RET was determined to provide an indication of genotoxic potential. The frequency of RETs relative to total erythrocytes was determined to provide an indication of perturbations in hematopoetic activity indicative of cell toxicity. For each of the treatment groups, a mean and standard deviation was calculated to describe the frequency of RET and MN-RET observed. The analyses were conducted utilizing a Coulter EPICS XL-MCL flow cytometer (Beckman Coulter).
Details of tissue and slide preparation:
Micronucleus formation in peripheral blood reticulocytes was determined by flow cytometry with traditional blood smears prepared as a backup. Samples were prepared and analyzed per instructions in the mouse MicroFlow Micronucleus Analysis Kit Manual (Litron Laboratories, Rochester, New York). At the end of the specified interval following treatment, a peripheral blood sample was collected from the orbital sinus of all surviving animals into anticoagulant solution following anesthesia with isoflurane. The blood samples were fixed in ultracold (-70 to -80°C) methanol within five hours of collection. All fixed blood samples were stored at -80°C. Fixed blood samples were washed with a cold, buffered salt solution and isolated by centrifugation. The resulting cell pellets were stored at 4°C until staining. Blood samples were ultimately incubated with RNAse to degrade the high levels of RNA present in the reticulocytes (RET) and a fluorescently labeled antibody to the transferrin receptor (anti-CD71-FITC) to specifically identify the RET. A propidium iodide solution was added to each sample immediately before flow cytometry (FCM) analysis to stain the DNA, including that of micronuclei. Blood samples were analyzed by high-speed FCM. In this system, the sample was moved at a high velocity past a laser set to provide 488 nm excitation. The fluorescent wavelengths emitted by each cell were collected by photomultiplier tubes. Using the previously described staining procedure, the propidium iodide-stained DNA of the micronuclei emitted a red fluorescence and the anti-CD71-FITC antibody emitted a high green fluorescent signal permitting differentiation between cells with and without micronuclei. In addition to obtaining fluorescent profiles, FCM simultaneously provided cell size information by determining the light scatter properties of each cell or combination of cells.
Evaluation criteria:
A test was considered valid if all of the following conditions were met: the range of MN-RET values in the negative controls were within reasonable limits of the recent laboratory background range. There was a significant increase in the incidence of MN-RET in the positive control treatment as compared to the concurrent negative controls. The mean for percent RET value in one or more of the test material treated groups was greater than or equal to 20% of the control value indicating no undue effect on erythropoiesis (toxicity). A test material was considered positive in this assay if the following criterion was met: statistically significant increase in MN-RET frequency at one or more dose levels accompanied by a dose response. A test material was considered negative in this assay if the following criterion was met: no statistically significant dose-related increase in MN-RET when compared to the negative control. A test result not meeting the criteria for either the positive or the negative response was considered to be equivocal.
Descriptive statistics only (means and standard deviations) were reported for chamber concentration, temperature, humidity, airflow, and body weights. MN-RET and percent RET were tested for equality of variance using Bartlett's test (alpha = 0.01). If the results from Bartlett's test were significant, then the data for the parameter may have been subjected to a transformation to obtain equality of the variances. The transformations that were examined are the common log, the inverse, and the square root in that order. The data was reviewed and an appropriate form of the data selected and subjected to the following analysis. The MN-RET data and the data on percent RET were analyzed by a one-way analysis of variance. Pairwise comparisons of treated vs. control groups were done, if the dose effect was significant, by Dunnett’s t-test, one-sided (upper) for MN-RET and two-sided for the percent RET. Linear dose-related trend tests were performed if any of the pairwise comparisons yield significant differences. The alpha level at which all tests were conducted was 0.05. The MN-NCE was not analyzed statistically and was only used as an adjunct end point to evaluate the biological significance of the MN-RET results. The final interpretation of biological significance of the responses is based on both statistical outcome and scientific judgment.

Results and discussion

Test results
Vehicle controls validity:
Negative controls validity:
not applicable
Positive controls validity:
Additional information on results:
Mean chamber concentration values for the 0, 37.5, 75 and 150 ppm chambers were 0, 35.0 ± 3.0, 74.1 ± 4.3, or 142.9 ± 7.9 ppm DVB-55 (study mean), respectively. The actual exposure concentrations ranged from approximately 93 to 98% of the targeted values. Animals that were sacrificed two hours after the final exposure had body weight decreases of more than 10% in all exposure levels. Body weights of animals sacrificed 48 hours after the final exposure were minimally affected. On the initial day of exposure animals in the 37.5 and 75 ppm exposure groups exhibited decreased activity. These same mice had decreased faeces on the second day of exposure and no remarkable clinical observations by the third day. In the highest exposure group (150 ppm), 7 out of 12 mice displayed decreased activity on the initial day of exposure. By the second day, all mice at the highest exposure level had a decrease in the quantity of faeces, however, these mice had no remarkable clinical observations by the third day. Three hours after the initial exposure decreases in body temperature were observed in all test material exposure groups (up to 9.2°C). The body temperature of the animals was minimally affected following subsequent exposures after this initial body temperature decrease. There were no significant differences in MN-RET frequencies between the groups exposed with the test material and the negative controls. The adequacy of the experimental conditions for the detection of induced micronuclei was ascertained from the observation of a significant increase in the frequencies of micronucleated RETs in the positive control group. The percent RET values observed in the test material-exposed animals were significantly decreased from the negative control values in all exposure groups. The percent RET values of the positive control animals were found to be significantly lower than those of the negative control animals. Treatment-related toxicity was observed in male mice administered three consecutive targeted daily exposures of DVB-55 up to 150 ppm (actual exposure was142.9 ± 7.9 ppm). All exposure groups had a significant decrease in the percent RET indicating that DVB-55 was systemically available to the target tissue. Based upon the results of the study reported herein, it was concluded that the test material, DVB-55, did not induce a significant increase in the frequencies of micronucleated reticulocytes in the peripheral blood when given as a six hour inhalation exposure on three consecutive days to male B6C3F1/Crl mice. Hence, DVB-55 is considered negative in this test system under the experimental conditions used.

Applicant's summary and conclusion

DVB-55, did not induce a significant increase in the frequencies of micronucleated reticulocytes in the peripheral blood when given as a six hour inhalation exposure on three consecutive days to male B6C3F1/Crl mice. Hence, DVB-55 is considered negative in this test system under the experimental conditions used.
Executive summary:

The in vivo genotoxic potential of DVB-55 (a mixture of diethenylbenzene and ethenylethylbenzene) was evaluated by examining the incidence of micronucleated reticulocytes (MN-RET) in the peripheral blood. In a previously published study, Kligerman et al. (1996, Mutat. Res, 370:107-113) showed a weak positive effect for the induction of micronuclei following inhalation exposure of mice to DVB-55. In the current study, male B6C3F1/Crl mice were whole-body exposed to actual concentrations of 0, 35.0 ± 3.0, 74.1 ± 4.3, or 142.9 ± 7.9 ppm DVB-55 six hours/day for three days. The exposure concentrations were selected to overlap those employed by Kligerman et al. with the highest concentration representing the maximum tolerated exposure concentration. Peripheral blood from exposed mice was sampled 2 and 48 hours after the final exposure. The first sampling time was selected to replicate the timing of the Kligerman et al. study and the second time was consistent with the OECD test guidelines. All animals were observed for clinical signs prior to the exposure, immediately following the exposure, approximately three hours following each exposure, and daily after the final exposure. Groups of mice, five/exposure/timepoint, except the highest exposure where six/timepoint, were sacrificed approximately 2 and 48 hours after the final exposure for the collection of peripheral blood and evaluation of RET (approximately 5000/animal) for MN by flow cytometry. The proportion of RET was determined based upon 5000 RET per animal and the results expressed as a percentage. Mice treated with 40 mg/kg bw cyclophosphamide monohydrate by a single oral gavage dose and sacrificed 48 hours later served as positive controls. All animals survived to the end of the observation period. Treatment-related clinical signs including decreased activity, decreased faeces, and decreased body temperatures were observed in all test material exposure groups. There were no statistically significant increases in the frequencies of MN-RET in groups exposed to the test material and sampled at approximately 2 and 48 hours after the final exposure. There were statistically significant decreases in the percent RET in all test material exposure groups. There was a significant increase in the frequency of MN-RET and a decrease in the percentage of RET in the positive control chemical group as compared to the negative control group. Under the experimental conditions used, DVB-55 was considered to be negative in the mouse peripheral blood micronucleus test via inhalation exposure.