Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 30, 2015 to December 3, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
other: Mouse peripheral blood micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
2-butoxyethyl benzoate
EC Number:
226-685-8
EC Name:
2-butoxyethyl benzoate
Cas Number:
5451-76-3
Molecular formula:
C13H18O3
IUPAC Name:
2-butoxyethyl benzoate
Test material form:
other: liquid
Details on test material:
2-Butoxyethyl benzoate, Lot # 201303443-19; Purity 99.2 %
Specific details on test material used for the study:
Test Material Name: 2-Butoxyethyl benzoate
Chemical Name: 2-Butoxyethanol benzoate
Supplier, City, State (Lot, Reference Number): The Dow Chemical Company, Midland, Michigan (Lot # 201303443-19).
Purity/Characterization (Method of Analysis and Reference): The purity of the test material was determined to be 99.2% area (corrected for water) by gas chromatography with identification by nuclear magnetic resonance and gas chromatography mass spectrometry (Gobbi, 2014).
Test Material Stability Under Storage Conditions: The test material was determined to have two years of stability under ambient storage conditions (Wachowicz, et al., 2015).

Test animals

Species:
mouse
Strain:
other: Outbred Crl:CD1(ICR)
Details on species / strain selection:
Criteria for Selecting the Strain:
1. Availability of historical negative control data
2. Suitability for utilization in the MNT
3. General suitability for toxicity testing
Sex:
male/female
Details on test animals or test system and environmental conditions:
Species and Sex: Male and female mice were used for the RFT. Only males were used for the MNT due to the results of the RFT, which showed no apparent difference in the toxicity between the sexes.
Strain: Outbred Crl:CD1(ICR) mice
Criteria for Selecting the Strain:
1. Availability of historical negative control data
2. Suitability for utilization in the MNT
3. General suitability for toxicity testing
Supplier and Location: Charles River (Kingston, New York)
Age at Study Start: Approximately 8-10 weeks

Physical Acclimation:
During the acclimation period each animal was evaluated by a veterinarian trained in the field of Laboratory Animal Medicine, or a trained animal/toxicology technician, to determine the general health status and acceptability for study purposes. The Toxicology and Environmental Research and Consulting Laboratory is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International). The animals were housed one per cage in stainless steel cages, in rooms designed to maintain adequate conditions (temperature, humidity, and photocycle), and acclimated to the laboratory for at least one week prior to the start of the study.

Housing:
After assignment, 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 the rodents' feet. The gauze also provided environmental enrichment. Cages contained a hanging feeder and a pressure activated lixit valve-type watering system. The following environmental conditions were maintained in the animal room.
Temperature: 22°C with a range of 20°C-26°C
Humidity: 40-70%
Air Changes: 10-15 times/hour (average)
Photoperiod: 12-hour light/dark (on at 6:00 a.m. and off at 6:00 p.m.)

Randomization and Identification:
Before administration of test material began, animals were stratified by body weight and then randomly assigned to treatment groups using a computer program designed to increase the probability of uniform group mean weights and standard deviations at the start of the study. Animals placed on study were uniquely identified via subcutaneously implanted transponders (BioMedic Data Systems, Seaford, Delaware) that were correlated to unique alphanumeric identification numbers.

Feed and Water:
Feed and municipal water were provided ad libitum. Animals were provided LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in pelleted form. Analyses of the feed were performed by PMI Nutrition International to confirm the diet provides adequate nutrition and to quantify the levels of selected contaminants. Drinking water obtained from the municipal water source was periodically analyzed for chemical parameters and biological contaminants by the municipal water department. In addition, specific analyses for chemical contaminants were conducted at periodic intervals by an independent testing facility. Copies of these analyses are maintained in the study file.

Animal Welfare:
In accordance with the U.S. Department of Agriculture Animal Welfare Regulations, 9 CFR, Subchapter A, Parts 1-4, the animal care and use activities required for conduct of this study were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC). The IACUC has determined that the proposed Activities were in full accordance with these Final Rules. The IACUC-approved Animal Care and Use Activities to be used for this study were Genetic Tox 01, Blood Collection 01, Humane Endpoints 01, and Animal ID 01.


Administration / exposure

Route of administration:
oral: gavage
Vehicle:
The vehicle used to mix the test material (corn oil; CAS number 8001-30-7), served as the negative control.
Details on exposure:
Dose Levels:
The dosing solutions of the test material were prepared and used fresh on each of the two consecutive days of administration. A frozen stock solution of CP dissolved in distilled water (thawed and brought to room temperature prior to use) served as the positive control. The vehicle used to mix the test material (corn oil; CAS number 8001-30-7), served as the negative control. The concentrations of the test material in the dosing solutions used for the first day of dosing were verified using high performance liquid chromatography with ultraviolet detection (HPLC/UV).

Animal Dosing:
The test material, the vehicle (negative control), and CP (positive control) were administered by oral gavage. Oral gavage is one of the generally accepted routes of administering the test material in this assay system. CP when administered by oral gavage has been shown to induce micronuclei in polychromatic erythrocytes of Crl:CD1(ICR) mice (De Boeck et al., 2005; Gollapudi et al., 1986). Dosing solutions were administered in aliquots of 10 ml/kg bodyweight (bw).
Duration of treatment / exposure:
In the Micronucleus Test (MNT), groups of male mice were administered 0, 375, 750, or 1500 mg/kg bw/day of the test material on two consecutive days. CP was administered only once at a dose level of 40 mg/kg bw. The dose levels were based on the results of the RFT.
Frequency of treatment:
Once per day.
Post exposure period:
48 hours
Doses / concentrationsopen allclose all
Dose / conc.:
375 mg/kg bw/day
Dose / conc.:
750 mg/kg bw/day
Dose / conc.:
1 500 mg/kg bw/day
No. of animals per sex per dose:
There were six mice/dose except in the 1500 mg/kg bw/day group where an additional group of two mice were dosed as possible replacements in the event of deaths occurring among the treated animals of this group.
Control animals:
yes, concurrent vehicle
Positive control(s):
Positive Control Chemical:
Chemical Name: Cyclophosphamide monohydrate (CP)
CAS Number: 6055-19-2
Source: Sigma, St. Louis, Missouri

CP was administered only once at a dose level of 40 mg/kg bw.

Examinations

Tissues and cell types examined:
Cells Examined:
Approximately 5,000 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 hematopoietic 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 flow cytometer (Beckman Coulter Gallios flow cytometer).
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
The dose levels were based on the results of the RFT.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Rationale for Selection of Sampling Intervals for Micronucleus Test (MNT):
It takes approximately 48 hours for the treated erythroblasts to undergo one posttreatment division and mature into reticulocytes and reach a peak incidence in the peripheral blood (CSGMT, 1992). However, if the treatment induces cell cycle delay, or if there is a delayed absorption and metabolism of the test material, a longer sampling time (such as 72 hours) may be needed to recover the damage induced in the erythroblasts. The sampling time used in this study provided an opportunity to recover any delayed effect (i.e., effect induced by the first treatment) as well as a more immediate effect (i.e., effect induced by the second treatment) in the same treated animals.

Peripheral Blood:
Micronucleus formation in peripheral blood reticulocytes was determined by flow cytometry (FCM; Beckman Coulter Gallios) 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. Briefly, the blood samples were fixed in ultracold (-70 to -80°C) methanol within five hours of collection. All fixed blood samples were stored at approximately -80°C until the time of analysis. 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 FCM analysis to stain the DNA, including that of micronuclei. 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.

DETAILS OF SLIDE PREPARATION:
Duplicate cell smears were prepared and stored to serve as backups in the event that the flow cytometric analysis was not possible. Blood was collected into a microtainer tube coated with EDTA (Becton Dickinson, Franklin Lakes, New Jersey). Wedge smears were prepared, fixed in methanol, and stored at room temperature.

METHOD OF ANALYSIS:
Micronucleus formation in peripheral blood reticulocytes was determined by flow cytometry (FCM; Beckman Coulter Gallios) 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).
Evaluation criteria:
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 ≥ 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.
• The statistically significant increase is out of historical range.

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.
Statistics:
MN-RET and percent RET was tested for equality of variance using Bartlett's test (alpha = 0.01; Winer, 1971). The raw data and the following transformations were examined; the common log, the inverse, and the square root in that order. The results from Bartlett's test were significant for MN-RET and percent RET in the positive control versus negative control data set and the data were log transformed to obtain equality of the variances.
The MN-RET data and the data on percent RET were analyzed by a one-way analysis of variance (Winer, 1971). The dose effect was not significant for the negative control versus treated groups analysis so Dunnett’s t-test was not performed nor were, (Winer 1971) linear dose-related trend tests performed. 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 was based on both statistical outcome and scientific judgment.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Remarks:
All dose levels tested.
Toxicity:
yes
Remarks:
Animals dosed at 0, 375 or 750 mg/kg bw/day had no indications of toxicity during the in-life portion of the MNT. One mouse dosed with 1500 mg/kg bw/day had urine perineal soiling on day two of dosing which resolved by day three.
Vehicle controls validity:
valid
Negative controls validity:
other: Vehcle control used as the negative control.
Positive controls validity:
valid
Remarks on result:
other: Results for Micronucleus Assay (MNT)
Additional information on results:
Dose Range-Finding Test (RFT):
Phase I:
Targeted dose levels of 1000 and 2000 mg/kg bw/day 2-butoxyethyl benzoate were used in the initial phase of the RFT using male and female mice. None of the mice (male and female) dosed with 1000 mg/kg bw/day displayed clinical observations, or significant changes in body weight or body temperature. One female mouse dosed with 1000 mg/kg bw/day displayed a slight decrease in body temperature of 2.2°C on day one approximately two hours after receiving the first dose.
Two of the male mice dosed with 2000 mg/kg bw/day displayed no clinical observations but had decreases in body temperature. One male mouse had a body temperature decrease of 3.6 ºC two hours after the first dose, but had mostly recovered by day two of dosing. The second male mouse had a body temperature decrease of 4.4 ºC two hours after the first dose, and lost 2.0 g of body weight over the course of the study but survived until the end of the study with no clinical observations or further substantial decreases in body temperature. The third male mouse dosed with 2000 mg/kg bw/day displayed red-colored urine on day two of dosing, but had fully recovered by day three with no further clinical signs or substantial decreases in body temperature.
The first female mouse dosed with 2000 mg/kg bw/day displayed no clinical observations or significant changes in body weight or body temperature. The next female mouse displayed no clinical observations or changes in body weight, but had a slight decrease in body temperature of 2.6°C two hours and five hours after receiving the first dose. The third female mouse dosed with 2000 mg/kg bw/day displayed red urine, dehydrated skin, slow and labored respiration, decreased feces, was absent of behavior, and had a body temperature decrease of 9.0 ºC approximately two hours after receiving the second dose at which point, the animal was declared moribund and euthanized. Gross necropsy observations by a pathologist revealed the lungs, adrenals, and urinary bladder as diffuse and red; red urine soiling was also observed. There was no evidence of gavage error.

Phase II:
Based on the mortality and clinical observations observed in the mice dosed with 2000 mg/kg bw/day, a lower dose level of 1750 mg/kg bw/day was given to additional mice (three/sex/dose). The first male mouse had decreased feces on day 3, but had no decreases in body weight or body temperature. The second male mouse had a body temperature decrease of 4.4 ºC and exhibited red urine approximately two hours after receiving the first dose. On day two, approximately five hours after the second dose, the same animal displayed red urine, dehydrated skin, slow respiration, had a body temperature decrease of 14.2 ºC, and was absent of behavior. The animal was declared moribund, was euthanized and examined by a pathologist where the bladder was observed as being diffuse and red in color, the spleen was enlarged and dark, and red urine perineal soiling was also observed. It was determined that no gavage error had occurred. The third male mouse dosed with 1750 mg/kg bw/day displayed red urine and had a body temperature decrease of 3.3 ºC on day one of dosing, but had mostly recovered by day two. On day three decreased feces were observed, but the animal had largely recovered by day four with no further clinical observations or decreases in body weight or body temperature.
The first female mouse dosed with 1750 mg/kg/bw/day had no significant changes in body weight, but had a body temperature decrease of 5.5 ºC approximately five hours after receiving the first dose, but had fully recovered by day two. This same animal was observed as having decreased feces on day three, which persisted through to day four. The next female mouse dosed with 1750 mg/kg/bw/day had a body temperature decrease of 4.3 ºC approximately two hours after receiving the first dose, and after five hours, the temperature had not recovered and was down 3.4 ºC. This same animal was observed with red urine on day one, which had resolved by day two and decreased feces on day three, which had resolved by day four. The last female animal dosed with 1750 mg/kg/bw/day had a body temperature decrease of 4.2 ºC approximately five hours after the second dose, this same animal was also observed has having decreased feces on day three, which persisted through to day four.

Phase III:
Based on the mortality and clinical observations observed in the mice dosed with 1750 mg/kg/bw/day, a lower dose of 1500 mg/kg/bw/day was given to additional mice (three/sex/dose).
All male mice dosed with 1500 mg/kg/bw/day had no significant changes in body weight or body temperature, but displayed decreased feces periodically throughout the study. All female mice dosed with 1500 mg/kg/bw/day had no significant changes in body weight or body temperature. The first female had decreased feces on day three, which had resolved by day 4. The second female mouse had red urine on day one, which was resolved by day two. On day three, the same animal had decreased feces, which had resolved by day four. The last female mouse dosed with 1500 mg/kg/bw/day, was observed with red urine on day one, which had resolved by day two with no further clinical observations.


Any other information on results incl. tables

Micronucleus Assay (MNT):

Based upon the results of the range-finding study where no appreciable differences were noted between sexes, only males were evaluated in the main study and the maximum tolerated dose was determined to be a dose of 1500 mg/kg bw/day. The middle- and low doses were 750 mg/kg bw/day and 375 mg/kg bw/day, respectively. The analytically determined concentrations of the test material in the dosing solutions used for the micronucleus test ranged from 91.5% to 102.2%% of the targeted values.

The treatments did not have a remarkable effect on the body weight or body temperature of the animals. Animals dosed at 0 (negative control), 375 mg/kg bw/day, or 750 mg/kg bw/day had no indications of toxicity based upon daily observations during the in-life portion of the MNT. One mouse dosed with 1500 mg/kg bw/day, demonstrated urine perineal soiling on day two of dosing, which was resolved by day three, with no further clinical observations. No other mice dosed at 1500 mg/kg bw/day displayed any clinical signs of toxicity throughout the study.

There were no significant differences in MN-RET frequencies between the groups treated with the test material and the negative control. 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 RET in the positive control group.

The percent RET values observed in the test material-treated animals were not significantly different from the negative control values. The percent RET values of the positive control animals were found to be significantly lower than those of the negative control animals.

Applicant's summary and conclusion

Conclusions:
2-butoxyethyl benzoate administered orally with two consecutive daily doses of ≥1500 mg/kg bw/day resulted in lethality and clear indications of systemic toxicity including an increase in the percent reticulocytes, indicating systemic availability of the test material. These data generated in the range-finding portion of the study indicate that 1500 mg/kg bw/day was the maximum tolerated dose (MTD) under the conditions of this study. Data from the RFT clearly indicated systemic availability of the test material when administered by oral gavage. Based upon the results of the study reported herein, it was concluded that the 2-butoxyethyl benzoate, did not induce a significant increase in the frequency of micronucleated reticulocytes in the peripheral blood when given as a single oral dose on two consecutive days to male Crl:CD1(ICR) mice. Hence, 2-butoxyethyl benzoate is considered negative in this test system under the experimental conditions used.
Executive summary:

The in vivo genotoxic potential of 2-butoxyethyl benzoate was evaluated by examining the incidence of micronucleated reticulocytes (MN-RET) in the peripheral blood. The test material was administered to male Crl:CD1(ICR) mice by single oral gavage on two consecutive days at dose levels of 0 (negative control), 375, 750, and 1500 mg/kg body weight (bw). The highest dose level was based upon the results of a range-finding test where at higher doses treatment-related deaths were observed in male and female mice.

The analytically determined concentrations of the test material in the dosing solutions used for the first day of dosing in the micronucleus ranged from 91.5% to 102.2% of the targeted concentrations. All animals were observed for clinical signs prior to dosing and at 2, 5, and 24 hours following each dosing. Groups of animals were euthanized 48 hours after the second treatment for the collection of peripheral blood and evaluation of RET (approximately 5,000/animal) for MN by flow cytometry. The proportion of RET was also determined based upon 5,000 RET per animal and the results expressed as a percentage. Mice treated with 40 mg/kg bw cyclophosphamide monohydrate by a single gavage dose and euthanized 48 hours later served as positive controls.

There were no treatment-related deaths or treatment-related clinical signs in the observation period of the definitive micronucleus test. There were no statistically significant increases in the frequencies of MN-RET or statistically significant effects on the percent RET in groups treated with the test material as compared to the negative controls. 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. Based upon the results of the study reported herein, 2-butoxyethyl benzoate is considered negative in this test system under the experimental conditions used.