Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Standard Guideline study conducted according to GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Supplier, City, State (Lot, Reference Number)
ANGUS Chemical Company, a wholly owned subsidiary of The Dow Chemical
Company, Buffalo Grove, Illinois (lot # CEC-200700226-56)

Purity/Characterization (Method of Analysis and Reference)
The purity of the test material was determined to be 97.9% on an anhydrous basis by gas
chromatography flame ionization detection with 0.279% water by Karl Fischer coulometric
titration. Identification was determined by gas chromatography mass spectrometry and
proton and carbon-13 nuclear magnetic resonance

Appearance:
Semi-solid, opaque white

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male
Details on test animals and environmental conditions:
Species and Sex: Male and female mice were used for the range-finding test. Only males were used for the micronucleus test due to the results of the range-finding test, which showed no apparent difference in the toxicity between the sexes.

Strain: Outbred Crl:CD1(ICR) (referred to as CD-1) 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 Laboratories (Portage, Michigan, USA)

Age at Study Start: 8 weeks

Physical Acclimation:
Each animal was evaluated by a laboratory veterinarian, or a trained animal/toxicology technician under the direct supervision of a laboratory veterinarian, to determine the general health status and acceptability for study purposes upon arrival at the laboratory (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.
Temperature: 22°C with a tolerance of ± 1°C (and a maximum permissible excursion of ± 3°C)
Humidity: 40-70%
Air Changes: 12-15 times/hour
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, USA) that were correlated to unique alphanumeric identification numbers.

Feed and Water
Animals were provided LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri, USA) in pelleted form. Feed and municipal water were provided ad libitum. Analyses of the feed were performed by PMI Nutrition International to confirm that 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 at Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan, USA.

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 and Animal ID 01.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Propylene glycol
Details on exposure:
Oral gavage was chosen as the route. Dosing solutions were prepared 1 our prior to dosing on both dsing days. Concentrations of test materials in the dosing solutions was verified using HPLC/MS and qualified using a dueterium internal standard.

Dose Range-Finding Test
This assay was conducted to aid the selection of dose levels for the micronucleus test. In phase I of the range-finding test animals were dosed with 0, 1000, and 2000 mg/kg bw/day (1/sex/dose) of the test material. The male and female mouse dosed with 2000 mg/kg bw/day and the female mouse dosed with 1000 mg/kg bw/day spontaneously died prior to the second day of dosing. Phase II of the range-finding test consisted of dose levels of 0, 250, 500, and 750 mg /kg bw/day administered (1/sex/dose) and the same doses given to an additional 3 animals/sex/dose . Animals were observed for at least 72 hours after dosing for any signs of toxicity.
Duration of treatment / exposure:
single oral gavage dose
Frequency of treatment:
once per day for 2 consecutive days
Post exposure period:
48 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 150, 300, 600 mg/kg bw/day
Basis:
actual ingested
Control animals:
yes, concurrent vehicle
Positive control(s):
Positive control:
Cyclophosphamide Monohydrate (CP). Sourec: Sigma, St. Louis, Missouri, USA.
The CP was dosed in distilled water. Dosing solution was prepared on the day of exposure, approximately 1 hour prior to dosing.

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

Examinations

Tissues and cell types examined:
Cells Examined
Approximately 5,000 reticulocytes 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 reticulocytes 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, MN-NCE, and MN-RET observed. The analyses were conducted utilizing a Coulter EPICS XL-MCL flow cytometer (Beckman Coulter).
Details of tissue and slide preparation:
Peripheral blood samples were collected from all animals approximately 48 hours after the last dosing.

Micronucleus incidence 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, USA). 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 CO2. 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 –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 emits 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 Lankes, New Jersey, USA). Wedge smears were prepared, fixed in methanol, and stored at room temperature.
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.

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.
• 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 % 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.
Statistics:
MN-RET and % RET were tested for equality of variance using Bartlett's test (alpha = 0.01; Winer, 1971). If the results from Bartlett's test were significant, then the data for the parameter may be subjected to a transformation to obtain equality of the variances. The transformations examined were the common log, the inverse, the square root, and arcsine square root in that order. The data were reviewed and an appropriate form of the data selected and subjected to the following analysis.

The MN-RET data and the data on % RET were analyzed by a one-way analysis of variance (Winer, 1971). 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 (Winer 1971). 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 were not analyzed statistically and were 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
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Range-Finding Test
Phase I
Targeted dose levels of 0 (negative control), 1000, and 2000 mg 3-amino-4-octanol/kg bw/day were used in the initial range-finding portion of the assay using one male and one female mouse at each dose level. Propylene glycol was used as the vehicle to administer the test material by oral gavage at a dosing volume of 10 ml/kg bw.

The male and female mouse dosed at the 2000 mg/kg bw/day dose level spontaneously died within the first two hours of the initial dose (Summary data, Table 3; Individual data, Tables 4 and 5). The female mouse dosed with 1000 mg/kg bw/day also spontaneously died within two hours of dosing on the initial day (Summary data Table 3; Individual data, Table 5). Examination of all three mice by a pathologist did not reveal any abnormal lesions. The male mouse at the 1000 mg/kg bw/day dose level had clinical signs including slow and deep respiration, decreased activity, partially closed eyelids, incoordinated gait, and was cold to touch after the initial dose (Summary data, Table 3; Individual data, Table 4). This mouse also had a decrease in body temperature of 16.1°C at five hours following the initial dose (Table 6). Due to this drop in body temperature and the clinical observations noted, the 1000 mg/kg bw/day dose level was declared as exceeding the maximum tolerated dose (MTD), therefore the mouse was not dosed on the second day.

Animals dosed with the vehicle (propylene glycol) exhibited no clinical signs in male or female mice (Summary data, Table 3; Individual data, Tables 4 and 5). There were no changes in body temperatures or body weights in these negative control animals (Tables 1 and 2; Tables 6 and 7).

Phase II
A second range-finding test using lower doses was performed to establish a maximum tolerated dose. Dose levels of 0 (vehicle control), 250, 500, and 750 mg/kg bw/day were selected using one animal/dose/sex. One male mouse at the 750 mg/kg bw/day dose level (animal number 08A5797) had decreased activity on both days of dosing, but no significant change in body temperature five hours post-dosing (Summary data, Table 3; Individual
data, Table 4; Table 6). The remaining male and female mice administered 0 (vehicle control), 250, 500, or 750 mg/kg bw/day (animal numbers 08A5785, 08A5801, 08A5789, 08A5805, 08A5793, 08A5809, 08A5812, respectively) had no clinical observations (Summary data, Table 3; Individual data, Tables 4 and 5), no notable changes in body temperature (Tables 6 and 7), and no significant changes in body weight (Tables 1 and 2). Based on these results, an additional three mice/sex/dose were dosed with 0 (vehicle control), 250, 500, and 750 mg/kg bw/day. No remarkable signs of toxicity were observed in any of the 0, 250 or 500 mg/kg bw/day mice, including body temperature at five hours post-dosing (Summary data, Table 3; Individual data, Tables 4 and 5; Tables 6 and 7). One male mouse at the 750 mg/kg bw/day dose level spontaneously died after dosing on the second day (Summary data, Table 3; Individual data, Table 4). The remaining two male mice at this dose level had no remarkable clinical signs or a significant change in body temperature (Summary data, Table 3; Individual data, Table 4; Table 6). In the female mice at the 750 mg/kg bw/day dose level, one mouse spontaneously died after the second day of dosing and another had perineal urine soiling on the second day (Summary data, Table 3; Individual data, Table 5). All remaining female mice at this dose level had neither clinical signs nor remarkable changes in body temperature. There were no remarkable changes in the body weights of any of the animals (Tables 1 and 2). Animals dosed with the vehicle control (propylene glycol) exhibited no clinical signs in male or female mice (Summary data, Table 3; Individual data, Tables 4 and 5).

Based upon these range-finding results it was determined that 750, 1000, and 2000 mg/kg bw/day exceeded the MTD in both male and female mice while the 500 mg/kg bw/day dose level showed no significant levels of toxicity in either sex. Based on these results, 600 mg/kg bw/day was deemed to be an appropriate high dose for the micronucleus test. No apparent sex-related differences in toxicity were noted in the range-finding test; therefore,
only male mice were evaluated in the main study.

Micronucleus Test
Based upon the results of the range-finding study, male mice were evaluated in the micronucleus test at dose levels of 0 (vehicle control), 150, 300, and 600 mg 3-amino-4- octanol/kg bw/day. The analytically determined concentrations of the test material in the dosing solutions used for the first day of dosing in the micronucleus test ranged from 103.3 to 105.5% of the targeted values (Table 8) and these values also indicated acceptable levels of stability of the test material in the vehicle. No treatment related toxicity upon daily observation was observed in any of the mice at dose levels of 0 and 150 mg/kg bw/day. At the 300 mg/kg bw/day dose level one male mouse had a decrease in body weight of 6 grams and had clinical observations of perioral soiling, noisy and labored respiration on day 2 of dosing and again on day 4 prior to necropsy (Summary data, Table 10; Individual data, Table 11; Table 9). All other animals at the 300 mg/kg bw/day dose level did not have signs of toxicity and their body weight gain was within an acceptable range (Summary data, Table 10; Individual data, Table 11; Table 9). At the 600 mg/kg bw/day dose level one mouse exhibited clinical signs of slow respiration, muscle tremors and incoordinated gait after the second day of dosing (Summary data, Table 10; Individual data, Table 11) however, these observation largely recovered by day 3 and were not observed for the remainder of the study duration.

The other five mice at the 600 mg/kg bw/day dose level showed no clinical signs (Summary data, Table 10; Individual data, Table 11) and there were no remarkable effects on the body weight of these animals (Table 9). A summary of the data on the frequencies of MN-RET and percent RET observed in
various treatment groups of male mice is presented in Table 12. The individual animal data are presented in Table 13 and the laboratory historical control data are shown in Table 14. There were no significant differences in MN-RET frequencies between the groups treated 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 RET in the positive control group (Table 12).

The percent RET values observed in the test material-treated animals were not significantly different from the negative control values (Table 12). 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:
Interpretation of results (migrated information): negative
Based upon the results of the study reported herein, it was concluded that the test material, 3-amino-4-octanol, did not induce a significant increase in the frequencies of micronucleated reticulocytes in peripheral blood when given as a single oral dose on two consecutive days to male CD-1 mice. Hence, 3-amino-4-octanol is considered nongenotoxic in this test system under the experimental conditions used.