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in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, near guideline study, available as unpublished report, fully adequate for assessment

Data source

Referenceopen allclose all

Reference Type:
study report
Report date:
Reference Type:
Genotoxicity testing of the Halon replacement candidates trifluoroiodomethane(CF3I) and 1,1,1,2,3,3,3-heptafluoropropane(HFC227ea) using the Salmonella typhimurium and L5178Y mouse lymphoma mutation assays and the mouse micronucleus test.
Dodd DE, Ledbetter AD, Mitchell AD
Bibliographic source:
Inhalation Toxicology 9(2):111-31

Materials and methods

Test guideline
equivalent or similar to guideline
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
The test material, a colorless gas, was received in a steel gas container from ManTech/Dayton on April 4, 1994, then transfeeerd to Allen Ledbetter, ManTech/RTR, who was responsible for handling, storage, and dilution of the test material. The substance was stored at ManTech/RTR at room temperature (approximately 72 F). Man?tech/Daoton documented the strength, purity, and composition of the test material and provided a Material safety and Data Sheet (MSDS) from Great Lakes Chemical Corporation for the substance. Upon acceptance of the final report, the remaining test material will be returne d for the Sponsor. No reserve sample will be retained by ManTech/RTP.

Test animals

Swiss Webster
Details on test animals or test system and environmental conditions:
Thirty male and 30 female Swiss Webster mice, approximately 42 days old at receipt, were purchased, by ManTech/RTP, from Charles River Laboratories in Raleigh, NC. The mice were held in quarantine for approximately 1 week and examined carefully to ensure their health and suitability as test subjects.

During all non-exposure periods, the mice were individually housed in suspended stainless steel wire-mesh cages with dimensions of 7" x 4" x 5" (L x W x H, 28 in2 floor space). The animal rooms were maintained at approximately 71°F (range 65-78°F) and 57% (range 46-64%) relative humidity. Fluorescent lighting was provided automatically on a 12 hours light: 12 hours dark regimen.

Certified rodent feed (Purina@ Certified Rat Chow 5002, St. Louis MO) and water, via automatic watering, were available ad libitum, except during the actual inhalation exposure periods. The mice were identified by cage number. All mice were weighed at arrival, at randomization and just prior to sacrifice. At the end of the quarantine period, the mice were given a physical examination, weighed and assigned to study groups based on a weight stratified method. Each study group for the negative control, the three concentrations of the test substance and the positive control consisted of 5 male and 5 female mice. Thus, a total of 50 mice were used for the assay. Necropsy was not done on any of the mice.

All mice were observed during the exposure period and immediately upon removal from the exposure chamber and daily during the post-exposure observation period. Necropsy was not done on any of the mice.

Administration / exposure

Route of administration:
inhalation: gas
Details on exposure:
Four groups of Swiss Webster mice were exposed for six hours per day for three consecutive days at concentrations of either 0, 2.6%, 5.3% or 10.5% HFC-227ea via nose-only inhalation. An additional group served as a positive control and was dosed on exposure day 3 via i.p. injection. The HFC-227ea mice were exposed in Cannon 52-port nose only chambers (Lab Products, Maywood, NJ) and the negative control mice were exposed in a nose-only chamber made by IN-TOX Products (Albuquerque, NM) due to a shortage of the Cannon Chambers. Forty of the 52 ports of the Cannon chambers were sealed to allow for reduced air flow in an attempt to conserve the test material.

For each exposure group, the test atmosphere was generated by metering the HFC-227ea gas from the cylinder into either a 2000 or 4000 ml Erlenmeyer flask that served as a mixing plenum. Air was metered into the flask to provide the desired exposure concentration. Each flowmeter used to deliver the gases to the generation system was calibrated with the gas for which it was to be used. The air/HFC-227ea mixture exited the flask and entered the top of the exposure chambers.

The test mixture was distributed to each animal port at a rate of approximately 52 ml/min (total chamber flow was 621 ml/min).
To maintain proper chamber airflow, the exhaust line controller was adjusted so the static pressure inside the chamber was approximately zero, indicating that the amount of flow entering the chamber matched the amount of flow exiting the chamber. This approach was required because it was not possible to accurately measure the exhaust flow using a rotameter since the flow properties change as the percentage of the test substance changes (the test substance is heavier than air).

The nominal concentration for each exposure was determined by dividing the total amount of the test material consumed (weight of the gas cylinder determined before and after each exposure) by the total exposure chamber airflow.

Actual chamber concentrations were determined by infrared analyses (IR) (Miran lA, Foxboro Analytical, South Norwalk, CT). The IR analyzer was calibrated using a closed-loop calibration method with HFC-227ea gas either just prior to the exposure or, if an existing calibration was used, the calibration was checked, via the closed loop method, just prior to exposure, and the IR calibration was rechecked after each exposure.

The IR absorbance response, expressed in recorder chart lines, was determined for each known quantity of HFC-227ea injected. A least-squares regression was determined using a Texas TI-60 calculator. Samples for injection into the IR analyzer were collected using a gas-tight syringe from an unused animal port. The IR settings were: pathlength, 15.75 meters; wavelength, 9.05 microns; absorbance, 0.25; response, x 1; slit, 1; and meter response, 4. Oxygen levels from the chamber exhausts were determined using an 02lExpiosion meter (MSA Model 421).
Duration of treatment / exposure:
3 consecutive days
Frequency of treatment:
6 hours/day
Post exposure period:
24 hours
Doses / concentrations
Doses / Concentrations:
2.6%, 5.3%, 10.5%
nominal conc.
No. of animals per sex per dose:
Control animals:
Positive control(s):
5 male and 5 female mice served as a positive control. Approximately 24 hours prior to sacrifice each positive control animal for the micronucleous test received one i.p dose of 0.4 mg/kg triethylenemelamine, which gives a reproducible increase in the frequency of micronucleated PCEs in Swiss Webster mice.


Tissues and cell types examined:
peripheral blood erythrocytes
Details of tissue and slide preparation:
Slides of peripheral blood smears were made for all animals at 24 ± 3 hours after the last exposure by the following procedure. Bovine calf serum, 2-3 μl, was placed on a slide pre-cleaned with methanol. Each mouse was sacrificed by cervical dislocation and 2-3 μI of blood per slide was obtained from the mid-ventral tail vein of a mouse and placed on top of the serum. The blood was mixed with the serum and spread on the slide to produce a thin, even film, then the slide was allowed to air-dry. Three slides were prepared per mouse, and, after the slides were dry, the erythrocytes were fixed by placing the slides in absolute methanol for two minutes; then they were allowed to air-dry vertically. ManTech/RTP was responsible for disposal of the carcasses.
The slides were stained for 20 minutes in 5% Giemsa stain in phosphate buffer containing 3% methanol and 3% 0.1 M citric acid, rinsed by dipping them in deionized water until clear, and allowed to air dry vertically. Covers lips were attached with Permount before the erythrocytes were analyzed at 100X, oil immersion, magnification.
Micronuclei were scored in slides from male and female mice from each HFC-227ea exposure group, and in slides from the positive and negative control animals. The treated and control slides were divided into three identical groups. Two groups of slides were coded by an individual not involved in the scoring or analysis, and the third group of slides was held in reserve, uncoded. Two observers were utilized, one for each set of coded slides.
Each bone marrow smear was inspected under low power to observe the distribution of cells and to select an area with good cell morphology and thin, even density (without overlapping cells) for scoring. Each slide was then scored for micronuclei using oil immersion objectives. The criteria which distinguish micronuclei from artifacts have been described by Schmid (1976). Micronuclei are identified as round or oval shaped bodies found in the cytoplasm of erythrocytes. Bodies which are refractile, improperly shaped or stained, or which are not in the focal plane of the cell are not scored as micronuclei. Cells containing more than one micronucleus are scored as a single micronucleated cell.
Evaluation criteria:
Positive: A test material is considered to have elicited a positive response in the mouse erythrocyte micronucleus test if there is a dose-related increase in micronuclei and if one or more of the doses induces a statistically significant (p < 0.05) increase in micronuclei induction.
Negative: A test material is considered to have elicited a negative response if the criteria for a positive response are not met.
When the slides from the micronucleus assay had been decoded and tabulated, the ratios (‰) of PCEs per 5000 erythrocytes were calculated for all groups of mice. Treatment groups in which the ‰ micronuclei ratios were elevated above the untreated mice were then compared with the concurrent, untreated negative control group for that sex using Student's t test, as described in Hayes (1989).

Results and discussion

Test results
no effects
Vehicle controls validity:
not applicable
Negative controls validity:
Positive controls validity:
Additional information on results:
No mice died during the study, and all mice appeared normal throughout the study. When weighed before exposure to HFC-227ea in the micronucleus assay, male mice weighed 23.5 to 32.5 g and female mice weighed 21.9 to 27.2 g. When weighed after exposure and immediately before sacrifice, male mice weighed 27.1 to 33.4 g and female mice weighed 21.2 to 29.0 g. The majority of the mice in all exposure groups, including the negative control group, lost weight from day -1 (randomization) to day 4 (sacrifice). Weight loss during exposure was not treatment related and averaged 0.4 g for the male mice and 0.9 g for the female mice. Otherwise, all mice appeared to be normal from the time of dosing until the times of sacrifice; no toxic symeffects were noted.

In the negative control mice, PCE ratios were 11.55‰ for male mice and 10.76‰ for female mice, values which were low but within historical ranges for the laboratory. Appropriately low numbers of MN/PCE were observed in the negative control mice: 1.0‰ MN/PCE in male mice and 2.0‰ MN/PCE in female mice, and, although PCE/ erythrocyte ratios were not depressed in the positive control mice, MN /PCE ratios were elevated. In response to 0.4 mg/kg TEM, the PCE ratios were 12.96‰ in male mice and 9.36‰ in female mice, and 21.0‰ MN/PCE were observed in cells from male mice and 18.0‰ MN/PCE in cells from female mice.

In comparison to the negative controls, PCE/erythrocyte ratios were not significantly depressed in male or female mice exposed to HFC-227ea, and neither statistically significant increases in MN (at p < 0.05) nor dose-related increases in MN were observed in male or female mice. Therefore, since neither criterion for a positive response was met in mice of either sex, HFC-227ea was clearly negative for the induction of micronuclei, which indicates that HFC-227ea is not capable of inducing structural chromosomal aberrations in vivo.

Applicant's summary and conclusion