Hexafluoropropene

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Remarks:

Study was conducted in accordance with requirements outlined by the U.S. Environmental Protection Agency in the Fluoralkenes Final Test Rule, promulgated under the provisions of Section 4 of the Toxic Substances Control Act (TSCA).

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: USEPA Fluoroalkenes Final Test Rule

Deviations:

not applicable

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Hexafluoropropene

EC Number:

204-127-4

EC Name:

Hexafluoropropene

Cas Number:

116-15-4

Molecular formula:

C3F6

IUPAC Name:

1,1,2,3,3,3-hexafluoroprop-1-ene

Constituent 2

Reference substance name:

1-Propene, hexafluoro-

IUPAC Name:

1-Propene, hexafluoro-

Details on test material:

- Name of test material (as cited in study report): 1-Propene, 1,1,2,3,3,3-hexafluoro-.- Physical state: Gas.- Purity: 100 %

Test animals

Species:

mouse

Strain:

other: Crl:CD-1(ICR)BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS:
- Age at study initiation: 49 days.
- Housing: Three mice per cage in stainless steel, wire mesh cages suspended above Upjohn Deotized Animal Cage Boards or R-2 Reemay-backed cage boards. Rats and mice were housed in separate Bioclean rooms dedicated to the study, males and females on separate cage racks. A system of rotation was employed to relocate cage racks within the animal rooms each week and to reposition the cages on the racks every two weeks.
- Diet (e.g., ad libitum): ad libitum ground Purina Certified Rodent Chow (GPCRC).
- Water (e.g., ad libitum): ad libitum tap water in water bottles refilled at least twice each week, clean water bottles furnished once each week. Tap water supplied by the Wilmington Suburban Water Corporation.
- Acclimation period: Pretest quarantine period lasted for 16 days.

ENVIRONMENTAL CONDITIONS:
- Temperature (°C): Target 23 +/- 2.
- Humidity (%): Target 50 +/- 10.
- Photoperiod (hrs dark/hrs light): 12/12.

Administration / exposure

Route of administration:

inhalation: gas

Type of inhalation exposure:

whole body

Vehicle:

other: Air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE/CHAMBER DESCRIPTION:
- Exposure apparatus: Stainless steel and glass inhalation chamber, 4 cubic meters in size.
- Method of conditioning air: Chambers operated in a one-pass, flow-through mode.
- System of generating atmospheres: Atmospheres were generated by mixing HFP gas from metered cylinders with room air in dilutions that would achieve the target chamber concentrations.
- Temperature, humidity in air chamber: Mean temperatures in the exposure chambers for all exposure days were 21.6 to 22.2°C; daily mean temperatures ranged from 19.9 to 24.4° C. The mean relative humidity for all exposures were 62.5 to 63.7 percent; daily mean values ranged between 51.0 and 79.5 percent.
- Air flow rate: 1000 L/min.

TEST ATMOSPHERE:
- Brief description of analytical method used: Concentrations of the test substance were determined by injecting atmospheric samples into a Hewlett Packard 5830A gas chromatograph/flame ionization detector.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Purity analyses of the test material were conducted prior to study inception and study completion. Prestudy samples were taken from the main supply tank used to fill the cylinders shipped to Haskell Laboratory for study use. Results indicated that the material was 100.0%. The sample analyzed at study completion was taken from one of the partially empty cylinders used in the study. Sample results for the second sample indicate the material consisted of 100.0%.

Two air samples were taken from the generation chambers every 20 minutes during the exposure period each day. Mean concentrations in exposure chambers targeted at 10, 50 and 150 ppm were 10.1+/- 0.3, 50.1 +/- 1.1 and 150 +/- 2.7 ppm respectively, during the study. Daily means were within +/- 10 percent of the targeted concentrations on all but two occasions: on exposure day 7, the mean concentration of the 10 ppm chamber was 8.9 ppm and on exposure day 56 the mean concentration in the 150 ppm chamber was 134 ppm.

Duration of treatment / exposure:

92 days

Frequency of treatment:

6 hours/day, 5 days/week.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
10.1 ppm +/- 0.3
Basis:
analytical conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
50.1 ppm +/- 1.1
Basis:
analytical conc.

Doses / concentrations 3

Remarks:

Doses / Concentrations:
150 ppm +/- 2.7
Basis:
analytical conc.

No. of animals per sex per dose:

25 mice per sex per exposure group.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses were selected based on the results of previously conducted 2-week inhalation studies conducted on rats and mice. Mild nephrosis in the kidneys was observed in male rats exposed to 200 ppm for 2 weeks. Kidney nephrosis was also observed in male mice exposed to 75 or 200 ppm for 2 weeks. The objective of this study was to evaluate subchronic inhalation toxicity during a longer time period of 90 days.
- Rationale for animal assignment: Following the quarantine period, 100 mice of each sex were selected for the study based on adequate body weight gain and freedom from any ophthalmological abnormalities or clinical signs of disease or injury. Animals were divided by computerized, stratified randomization into four groups of 25 male and 25 female mice, distributed such that there were no statistically significant differences among group body weight means within a sex for each species. Due to an error in sexing animals, there were 24 male and 26 female mice in the respective control groups.
- Rationale for selecting satellite groups: 10 animals at each treatment level were designated as recovery animals.
- Post-exposure recovery period in satellite groups: 4 weeks.

Examinations

Observations and examinations performed and frequency:

CAGE-SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed at least once daily.
- Cage-side observations included observations for mortality, clinical signs of toxicity and changes in appearance or behavior. Moribund animals were euthanized and necropsied.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once a week at the time of body weight determination

BODY WEIGHT: Yes
- Time schedule for examinations: Mice were weighed on test day -2 and test day 4, then weekly during the inhalation phase of the study. Mice were weighed on test day 88 during the last week of exposure. Beginning on test day 92, mice designated for recovery were weighed once each week for the recovery period. Animals scheduled for sacrifice were weighed the day of sacrifice.

FOOD CONSUMPTION:
- Food consumption for each animal determined: Yes. Records of individual food consumption were maintained weekly during exposure and recovery phases.

FOOD EFFICIENCY: No

WATER CONSUMPTION: Yes
- Water consumption for each animal determined: Yes. Records of individual water consumption were maintained weekly during exposure and recovery phases.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Two ocular examinations were conducted - during the quarantine period on test day -17 and on test day 82.
- Dose groups that were examined: All rats and mice received for the study participated in the first examination, the second examination was performed on all surviving rats and mice participating in the study.
- Anaesthetic used for examination: Yes (one drop of 1 percent atropine sulfate).

HEMATOLOGY: Yes
- Time schedule for collection of blood: During the exposure period and at the end of the recovery period. Blood collection occurred on study days 45 and 86 during the exposure period, and on days 120 (males) and 121 (females) at the end of the recovery period. Pretest evaluations were not conducted for mice due to low body weights prior to study inception.
- Anaesthetic used for blood collection: Yes (carbon dioxide)
- Animals fasted: No.
- How many animals: Five male and five female mice per group were randomly selected and sampled at 45 days, then sacrificed. Ten male and 10 female mice from each group were randomly selected for examination near the end of the study, then designated for the 90-day sacrifice. Surviving male and female mice were retained for recovery and bled at the end of the recovery period.
- Parameters evaluated include erythrocyte, leukocyte and platelet counts; analysis of hemoglobin concentration and hematocrit; and a differential leukocyte determination. Mean corpuscular hemoglobin, mean corpuscular volume and mean corpuscular hemoglobin concentration were calculated. Blood smears for reticulocyte counts were prepared at each sample event and for animals sacrificed in extremis, but evaluation was not required. Bone marrow smears were prepared from all animals at the 90-day and end of recovery sacrifices, but they were not evaluated.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: During the exposure period and at the end of the recovery period. Blood collection occurred on study days 45 and 86 during the exposure period, and on days 120 (males) and 121 (females) at the end of the recovery period. Pretest evaluations were not conducted for mice due to low body weights prior to study inception.
- Anesthetic used for blood collection: Yes (carbon dioxide).
- Animals fasted: No.
- How many animals: Five male and five female mice per group were randomly selected and sampled at 45 days, then sacrificed. Ten male and 10 female mice from each group were randomly selected for examination near the end of the study, then designated for the 90-day sacrifice. Surviving male and female mice were retained for recovery and bled at the end of the recovery period.
- Parameters evaluated include creatinine and blood urea nitrogen. Due to the limited quantity of serum available for study mice, only these two parameters were assayed.

URINALYSIS: No (rats only)

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

Gross pathological and histopathological examinations were performed.
Following the bleeding for the 45-day clinical pathology evaluations, the 5 mice/sex/group were sacrificed and necropsied. Kidneys were removed and preserved for histopathologic evaluation. No other pathology evaluations were performed on this group of mice.

At the end of the exposure period, 10 mice/sex/group were sacrificed on days 94 and 95. Following the recovery period, remaining mice were sacrificed on days 122 and 123. Animals were killed by injection of sodium pentobarbital and exsanguination, then examined for gross pathological lesions. The brain, heart, liver, lungs, spleen, kidneys, adrenals and testes/ovaries of each animal were weighed. The following tissues were collected from all animals whenever tissue integrity permitted: skin, bone marrow, lymph nodes (mandibular and mesenteric), spleen, thymus, aorta (thoracic), heart, trachea, lungs, nose (four cross sections), salivary glands, esophagus, stomach, liver, pancreas, small intestine (duodenum, jejunum and ileum), large intestine (cecum, colon and rectum), gall bladder (mice only), kidneys, bladder, pituitary, thyroid-parathyroid, adrenals, prostate, testes, epididymides, seminal vesicles, mammary gland, ovaries, uterus, vagina, brain (includes sections of medulla/pons, cerbellar cortex and cerebral cortex), spinal cord, peripheral nerve (sciatic), muscle (thigh), bone (sternum), eyes, exorbital lacrimal glands, Harderian glands and all gross lesions.

All tissues from mice in the 150 ppm exposure and control groups sacrificed at the end of the exposure or recovery periods, and from all mice that were found dead (tissue integrity permitting) or were sacrificed in extremis, were further processed to slides and examined microscopically. Lungs, liver, kidneys and all gross lesions from mice in the groups exposed to 10 or 50 ppm were also examined microscopically. Kidneys from all mice sacrificed on day 45 were also examined.

Statistics:

Body weights, body weight gains, food and water consumption, organ weights and clinical laboratory measurements were analyzed by a one-way analysis of variance (ANOVA). When the test for differences among test group means (F-test) was significant, pairwise comparisons between test and control groups were performed using the Dunnett's test. Pairwise comparisons for organ weights were also performed using the least significant differences (LSD) test.

Bartlett's test for homogeneity of variance was performed on the clinical laboratory data. When the results of this test were significant at a level of 0.05, the Kruskal-Wallis test was employed and the Mann-Whitney U test was used to compare means from the treatment and control groups.

Incidences of clinical observations and selected kidney lesions were analyzed by Fisher's Exact test with a Bonferroni correction.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY. A statistically significant number of male mice in the groups exposed to 50 or 150 ppm had a blue color to the abdominal skin, which was first observed early in the exposure period. No statistically significant observations were made for male mice during the recovery phase. The relationship of these observations to test substance exposure is unclear. None of the individual signs were significant among female mice, but the total number of female mice in the 150 ppm group with signs during the exposure period was statistically significant compared to controls. During the recovery phase, the total number of female mice in the group exposed to 50 ppm with an onset of clinical signs was significantly higher than the number of control mice with clinical signs. The relationship of these elevated number of mice with clinical signs to test substance exposure is unclear. There were several early deaths among mice. One mouse was accidentally killed during chamber loading. Seven were accidentally killed during or found dead following the orbital sinus bleeding for clinical pathology. Four unscheduled mortalities were considered not to be accidental in nature. None of the mortalities could be directly attributed to inhalation exposure of the test substance.

BODY WEIGHT AND WEIGHT GAIN. Mean body weights for male and female mice exposed to the test substance were similar to body weights of controls on all but a few intermittent occasions when mice were weighed. These differences were considered incidental and not a result of test substance exposure. Body weight gains of mice during the entire exposure period (days -2 to 88) and the recovery phase (days 92 to 120) showed no significant differences for test substance-exposed male or female mice when compared to controls.

FOOD CONSUMPTION. No statistically significant differences in food consumption were observed that could be attributed to test substance exposure.

WATER CONSUMPTION. Mean daily volumes of water consumed by male and female mice exposed to 150 ppm were greater than mean water consumption by male and female controls. Although values were only statistically significant for female mice, the differences for both sexes were considered biologically significant and related to test substance exposure. Mice exposed to 10 or 50 ppm did not show significant differences in water consumption for test days 1 to 92, and none of the groups exhibited significant differences during the recovery phase.

OPHTHALMOSCOPIC EXAMINATION. All animals used in the study were free of ocular lesions at study inception. Lesions were observed in five study mice, but these were considered a result of trauma. None of the observed lesions were considered related to test substance exposure.

HEMATOLOGY. There were no biologically significant differences in lymphocyte counts in male or female mice and the remaining hematological parameter measurements were considered unremarkable.

CLINICAL CHEMISTRY. Due to the limited quantity of serum available for study mice, only creatinine and blood urea nitrogen were assayed, and many of the creatinine levels were lower than the acceptable limit of detectability for the instrument used. There were no biologically significant differences in the results from the blood urea nitrogen assays performed for mice.

ORGAN WEIGHTS. No statistically significant differences in organ weights were observed for the male treatment groups compared to controls. Statistically significant differences in organ weights observed for females included reduced mean relative heart weight present at the end of the exposure period for the group exposed to 150 ppm, and reduced mean and absolute kidney weights following the recovery period for all female treatment groups.

GROSS PATHOLOGY. Gross pathological observations in mice were considered unremarkable.

HISTOPATHOLOGY: NON-NEOPLASTIC. Biologically and statistically significant incidences of histopathologic lesions were present in the kidneys of male and female mice. Kidney lesions included regeneration of the inner cortical tubules, cytomegaly of tubular epithelium, tubular epithelial necrosis and nephropathy. Tubular regeneration was noted in males and females exposed to 150 ppm, and in females exposed to 50 ppm sacrificed at the end of the exposure period. Regeneration was observed in these same exposure groups at 45 days. Male mice also exhibited additional lesions in the kidneys. Mice in the 50 ppm and 150 ppm exposure groups sacrificed at 45, 94 and 122 days exhibited incidences of cytomegaly of kidney epithelial cells. Necrosis of kidney tubules was observed in two male mice in the 150 ppm groups and in one mouse exposed to 50 ppm sacrificed in extremis on the 6th day of the study. Five male mice exposed to 150 ppm also exhibited kidney nephropathy at the end of the recovery period. Although this lesion is common in mice of this age and strain, it is not generally present in high incidences. The histopathologic kidney lesions observed in the male and female mice exposed to 50 or 150 ppm were considered to be compound-related. However, a potential relationship between organ weight differences and microscopic observations for male and female mice in this study was not apparent.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

10 ppm

Sex:

male/female

Basis for effect level:

other: Due to the microscopic pathology effects found at 50 and 150 ppm.

2

Dose descriptor:

LOAEL

Effect level:

50 ppm

Sex:

male/female

Basis for effect level:

other: Histopathologic kidney lesions present during and at the end of the inhalation phase included regeneration of the inner cortical tubules, cytomegaly of tubular epithelium, and tubular epithelial necrosis.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

The notable effects in mice were the microscopic lesions observed in the kidneys of mice exposed to 50 of 150 ppm. The no-observable-effect level for 90 days of repeated inhalation exposure in mice is 10 ppm. The no-effect level following 28 days of recovery is also 10 ppm.

Applicant's summary and conclusion

Conclusions:

The no-observable-effect level for 90 days of repeated inhalation exposure in mice is 10 ppm. The no-effect level following 28 days of recovery is also 10 ppm.

The study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).

Executive summary:

Four groups each of 25 male and 25 female Crl:CD-1(ICR)BR mice were exposed to target concentrations of 0, 10, 50 or 150 ppm for 6 hours/day, 5 days/week for a period of approximately 90 days. Subsequently, 10 mice/sex/group were maintained without exposure for an additional 28 days. The animals were weighed once each week and food and water consumption were monitored throughout the study. Ophthalmological examinations were conducted prior to study start and at the end of the inhalation phase. Clinical pathology evaluations were conducted at approximately 45 days, 90 days and at the end of the recovery period. At 45 days, five mice/sex/group that were evaluated for clinical pathology were also sacrificed and necropsied. At approximately 90 days, 10 mice/sex/group were sacrificed and at the end of the recovery period all surviving animals were sacrificed and necropsied. Selected tissues were evaluated microscopically.

The mean concentrations for the study exposures in the inhalation chambers targeted at 10, 50 or 150 ppm were 10.1, 50.1 and 150 ppm HFP, respectively. Temperature and humidity conditions in the chambers were consistently within acceptable ranges for the study.

Mean body weights and body weight gains for male and female mice were unremarkable and not affected by exposures to the test material. No differences in food consumption were noted for male or female mice during the study. Elevated levels of water consumption were observed in mice exposed to 150 ppm; these differences were statistically significant for female mice. Statistically significant observations of blue skin color to the abdomen were observed in male mice exposed to 50 or 150 ppm. The relationship of this observation to the test material is not clear. A number of early mortalities occurred during the study, but were related to blood sampling for the clinical pathology examinations. The incidences of early mortalities were considered inconsequential to test substance exposure. Hematology conducted in mice showed no effects that were related to test substance exposures.

The notable effects in mice were the microscopic lesions observed in the kidneys of mice exposed to 50 of 150 ppm. The kidney lesions included regeneration of the inner cortical tubules, cytomegaly of tubular epithelium, and tubular epithelial necrosis.The no-observable-effect level for 90 days of repeated inhalation exposure in mice is 10 ppm. The no-effect level following 28 days of recovery is also 10 ppm. At recovery, cytomegaly and kidney nephropathy were present in male mice.