Fluoroethylene

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

repeated dose toxicity: inhalation

Remarks:

combined repeated dose and carcinogenicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: EPA Fluoroalkenes Final Test rule 40 CFR 799.1700

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OTS 798.3300 (Carcinogenicity)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Fluoroethylene

EC Number:

200-832-6

EC Name:

Fluoroethylene

Cas Number:

75-02-5

Molecular formula:

C2H3F

IUPAC Name:

fluoroethene

Details on test material:

- Analytical purity: >99.9%

Test animals

Species:

rat

Strain:

other: Crl:CD®BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Approximately 40 days of age
- Weight at study initiation: Range of 143.8 – 215.8 grams for males; range of 67.0 – 240.5 grams for females
- Fasting period before study: Not reported
- Housing: One per cage
- Diet (e.g. ad libitum): ad libitum, except during exposures.
- Water (e.g. ad libitum): ad libitum, except during exposures.
- Acclimation period: 17 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-25°C
- Humidity (%): 30-70%
- Air changes (per hr): Set to provide a minimum of 10 fresh air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:

inhalation: gas

Type of inhalation exposure:

whole body

Vehicle:

other: Filtered air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Four 1.4 m3 chambers constructed of stainless steel or glass.
- Method of holding animals in test chamber: Rats were transferred from their individual cages to exposure cages, with 2 or 3 rats per cage.
- Source and rate of air: The chambers were operated in a one-pass flow-through mode with airflow rates adequate to provide sufficient oxygen for test animals, to prevent contamination from volatiles derived from animal excreta, and to enable adequate distribution of test substance in the chambers.
- Method of conditioning air: One-pass flow-through mode. Test substance was metered into filtered air to the desired concentrations for each of the 3 test chambers and filtered air alone was passed into the control chamber.
- Temperature, humidity, pressure in air chamber: See Table 1.
- Air flow rate: See Table 1.
- Air change rate: Not reported.
- Treatment of exhaust air: Chamber exhaust was vented to the atmosphere through stacks which extended 40 feet above the ground.

TEST ATMOSPHERE
- Brief description of analytical method used: Chamber atmospheres were analyzed by gas chromatography. The analytical method had a precision and accuracy of ± 10%. Periodically, the overall nominal chamber concentrations were calculated by dividing the flow rate of test substance by the total chamber airflow. Chamber atmospheres were analyzed at approximately 30 minute intervals during each six-hour exposure period by gas chromatography and compared against a standard curve prepared daily.
- Samples taken from breathing zone: Yes

VEHICLE
- Filtered air

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chamber concentrations of test substance over the entire study were within one percent of target concentration. The overall standard deviations of the analyses were within three percent of the mean.
See Table 1.

Duration of treatment / exposure:

up to 24 months

Frequency of treatment:

6 hours per day, 5 days per week, weekends and holidays excluded

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 25, 250, 2500 ppm
Basis:
nominal conc.

No. of animals per sex per dose:

95 animals per sex per group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Concentrations of test substance for this oncogenicity study were selected based on the results of the urinary fluoride and cell proliferation data obtained from the 90-day subchronic inhalation study where rats and mice were exposed to test substance at concentrations of 200, 2000, or 20000 ppm. A concentration-related increase in urinary fluoride was observed which reached a plateau in the 2000 ppm exposure groups, evidence of test substance metabolism. Cell proliferation evaluations following 90 days of exposure revealed exposure-related increases in the liver at all test substance concentrations. The increased hepatic cell proliferation was considered biologically significant at all exposure levels.
- Rationale for selecting satellite groups: Cell proliferation evaluation studies were conducted as an adjunct to the core oncogenicity study. Five rats/sex/group were evaluated for cell proliferation.
- Post-exposure recovery period in satellite groups: No.

Positive control:

None

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily.

CAREFUL CLINICAL OBSERVATIONS: Yes
- Time schedule: At every weighing. Once per week during the first 3 months, once every other week for the remainder of the study.

BODY WEIGHT: Yes
- Time schedule for examinations: Once per week during the first 3 months, once every other week for the remainder of the study, and prior to scheduled necropsies.

FOOD CONSUMPTION
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No.

FOOD EFFICIENCY
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pretest for all animals received for the study, prior to selection and grouping. All surviving rats prior to final sacrifices.
- Dose groups that were examined: All groups

CLINICAL PATHOLOGY EVALUATION: Yes
Ten animals/sex/group were randomly selected for first evaluation at 3 months, and these same 10 animals were used for the 6- and 12-month evaluations and were designated for the 12-month sacrifice. Ten animals/sex/group were randomly selected for the 18-month evaluation and sacrifice. Ten animals/sex from the control and low concentration groups were randomly selected for the 24-month evaluation and were then sacrificed. Intermediate and high concentration groups were not evaluated at the 24-month time period due to early sacrifices. For all sampling periods, selected animals that died prior to the clinical evaluations were replaced by the next available animal on the randomization list to maintain group total at ten.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Approximately 3, 6, 12, 18, and 24 months after study initiation.
- Anaesthetic used for blood collection: Yes, light carbon dioxide anaesthesia.
- Animals fasted: Yes.
- How many animals: 10 animals/sex/group were evaluated at 3, 6, 12, and 18 months and 10 animals/sex were evaluated from the control and low-concentration groups at 24 months due to early sacrifices. The 10 animals/sex/group selected for the first evaluation at 3 months, were used for the 6- and 12-month evaluations and were designated for the 12-month sacrifice. Ten animals/sex/group were randomly selected for the 18-month evaluation and sacrifice. Ten animals/sex from the control and low concentration groups were randomly selected for the 24-month evaluation and were then sacrificed. Intermediate and high concentration groups were not evaluated at the 24-month time period due to early sacrifices.
- Parameters checked: See Table 2.

CLINICAL CHEMISTRY: Yes.
- Time schedule for collection of blood: Approximately 3, 6, 12, 18, and 24 months after study initiation.
- Anaesthetic used for blood collection: Yes, light carbon dioxide anaesthesia.
- Animals fasted: Yes.
- How many animals: 10 animals/sex/group were evaluated at 3, 6, 12, and 18 months and 10 animals/sex were evaluated from the control and low-concentration groups at 24 months due to early sacrifices. The 10 animals/sex/group selected for the first evaluation at 3 months, were used for the 6- and 12-month evaluations and were designated for the 12-month sacrifice. Ten animals/sex/group were randomly selected for the 18-month evaluation and sacrifice. Ten animals/sex from the control and low concentration groups were randomly selected for the 24-month evaluation and were then sacrificed. Intermediate and high concentration groups were not evaluated at the 24-month time period due to early sacrifices.
- Parameters checked: See Table 3.

URINALYSIS: Yes.
- Time schedule for collection of urine: Approximately 3, 6, 12, 18, and 24 months after study initiation.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- How many animals: 10 animals/sex/group were evaluated at 3, 6, 12, and 18 months and 10 animals/sex were evaluated from the control and low-concentration groups at 24 months due to early sacrifices. The 10 animals/sex/group selected for the first evaluation at 3 months, were used for the 6- and 12-month evaluations and were designated for the 12-month sacrifice. Ten animals/sex/group were randomly selected for the 18-month evaluation and sacrifice. Ten animals/sex from the control and low concentration groups were randomly selected for the 24-month evaluation and were then sacrificed. Intermediate and high concentration groups were not evaluated at the 24-month time period due to early sacrifices.
- Parameters checked: See Table 4.

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. See Table 5.
ORGAN WEIGHTS: Yes. See Table 5.
HISTOPATHOLOGY: Yes. See Table 5.

All animals that were sacrificed by design, designated for Cell Proliferation Evaluation, found dead, accidentally killed, or were sacrificed in extremis were necropsied. Interim sacrifices of 10 animals/sex/group were conducted at 12 and 18 months. Due to early mortality, final sacrifice of the intermediate- and high-concentration groups occurred prior to 24 months and the control and low-concentration groups were sacrificed at approximately 24 months. The 10 animals/sex/group selected for Clinical Pathology evaluations at 3 months, were used for the 6- and 12-month evaluations and were designated for the 12-month sacrifice. Ten animals/sex/group were randomly selected for the 18-month evaluation and sacrifice. Ten animals/sex from the control and low concentration groups were randomly selected for the 24-month Clinical Pathology evaluation and were then sacrificed.

Bone marrow smears were prepared from all animals sacrificed by design; evaluation was not required. Blood smears were prepared from all animals sacrificed by design or in extremis; evaluation was not necessary. All tissues collected from animals in the control and high concentration groups and animals that were found dead (tissue integrity permitting) or were sacrificed in extremis, were further processed to slides and examined microscopically. Nose, lungs, liver, kidneys, gross lesions, and target organs from animals in the low and intermediate concentration groups were examined microscopically. Remaining tissues collected from animals in the low and intermediate concentration groups were evaluated microscopically if needed to establish a no-observable-effect level for potential test substance-related abnormalities.

Cell Proliferation:
Tissues listed in Table 5 were collected, weighed, and fixed. Liver, lung, nose, and kidneys from animals in control and high-concentration groups were processed for immunohistochemical analysis of 5-bromo-2’deoxyuridine (BrdU) incorporation into nuclei, and tissues were evaluated for cell proliferation. Remaining tissues from all animals were processed and evaluated for cell proliferation as needed to determine a no-observable-effect level. The liver was evaluated for cell proliferation and examined microscopically for alterations which might influence cell labelling indices at all exposure concentrations.

Other examinations:

CELL PROLIFERATION EVALUATION: Yes.
Sacrifices at approximately 2 weeks, 3 months, and 12 months.

Statistics:

See Table 6.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

effects observed, treatment-related

Details on results:

CLINICAL SIGNS AND MORTALITY
Early mortality precluded meaningful statistical evaluation of clinical observations data among the 250 and 2500 ppm groups. There were no unusual incidences of clinical observations in any of the 250 or 2500 ppm exposure groups compared to controls, nor was there the appearance of any unique clinical observation. There was a significant increase in the incidence of “weak” and “coloured discharge eye(s)” among 25 ppm female rats compared to controls. There were no clinical observations specifically associated with the early deaths noted. Mortality in all exposed animals was sudden and without clinical observation associated with morbidity. There was a statistically significant decrease in survival among male and female rats of the 250 and 2500 ppm groups compared to controls. The decreased survival of 25 ppm female rats was considered toxicologically relevant, although not statistically significant (p = 0.053). With the exception of 2 deaths related to hemangiosarcoma, no exposure-related increases in mortality occurred until after the 12-month interim sacrifice. Mortality related to test substance-induced hepatichemangiosarcoma became more significant during the second year of exposure. The total incidences of male rats of the 0, 25, 250, and 2500 ppm groups that died due to hemangiosarcoma (hepatic and extrahepatic) over the course of the study were 1/80, 2/80, 25/80, and 15/80, respectively. These incidences in female rats were 0/80, 7/80, 14/80, and 15/80, respectively.

OPHTHALMOLOGY
There were no ophthalmoscopic observations in rats that occurred with an incidence suggestive of a compound-related effect. The most frequent finding was pale ocular fundi which is common and, in many instances, a sign of anaemia.

BODY WEIGHT AND WEIGHT GAIN
There were no statistically significant changes in mean body weight of either male or female rats through test day 583. A 4% and 8% decrease in mean body weight gain of 250 ppm male and female rats, respectively, was noted when evaluated over the 0-583 day interval. These changes do not appear to be concentration-related, although early mortality could have skewed mean body weight of the 2500 ppm rats towards higher values. Decreases of 15% and 11% among male and female rats, respectively, of the 250 ppm groups were observed when mean body weight gain was evaluated over the 1-652 day interval. Mean body weight gain of the 25 ppm male and female rats was reduced 6% and 10%, respectively, when evaluated over the entire study.

HAEMATOLOGY
There were no biologically significant effects on haematological parameters measured at any of the evaluations. All significant observations noted were attributed to individual variation within a group or were within the expected ranges of normal biologic variation.

CLINICAL CHEMISTRY
There were no biologically significant effects on clinical chemical parameters measured at any of the evaluations. All significant observations noted were attributed to individual variation within a group or were within the expected ranges of normal biologic variation.

URINALYSIS
There were no biologically significant effects on urinalysis parameters measured at any of the evaluations. All significant observations noted were attributed to individual variation within a group or were within the expected ranges of normal biologic variation. Rat urinary fluoride measurements demonstrated a concentration- and time-dependent increase in fluoride ion excretion. The dose response appeared nonlinear. Although a plateau was apparent at the first measurement, it became less obvious with time. Fluoride ion excretion was greater in males than females. In both sexes, there was a time-dependent increase observed.

ORGAN WEIGHTS
Aside from sporadic, statistically significant changes, there were no compound-related changes in mean final body weight or mean final absolute or relative organ weight at either the 12-month, 18-month, or final sacrifices. Due to differences in final sacrifice dates of the 250 and 2500 ppm groups relative to control and 25 ppm groups, statistical comparisons were not performed on organ weights collected at final sacrifice.

GROSS PATHOLOGY
Refer to Section 7.7 (Carcinogenicity) DI.K1.24Mon.InhGas.R.HLR-480-91.KD for details of gross pathology effects.

HISTOPATHOLOGY: NON-NEOPLASTIC
Refer to Section 7.7 (Carcinogenicity) DI.K1.24Mon.InhGas.R.HLR-480-91.KD for details of non-neoplastic effects.

HISTOPATHOLOGY: NEOPLASTIC
Refer to Section 7.7 (Carcinogenicity) DI.K1.24Mon.InhGas.R.HLR-480-91.KD for details of neoplastic histopathology findings.

CELL PROLIFERATION EVALUATION
Refer to Section 7.7 (Carcinogenicity) DI.K1.24Mon.InhGas.R.HLR-480-91.KD for details of cell proliferation effects.

Effect levels

Dose descriptor:

LOAEL

Effect level:

25 other: ppm

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability). Under the conditions of this study, the test substance was carcinogenic in male and female rats at concentrations greater than or equal to 25 ppm. A no-observable adverse effect level was not determined. Based on this information, a LOAEL for repeated dose toxicity in rats was determined to be 25 ppm (47 mg/m3).

Executive summary:

The purpose of this study was to assess the oncogenic potential of the test substance in rats when administered by inhalation. Groups of 95 male and 95 female Crl:CD^®BR rats were exposed to either 0, 25, 250, or 2500 ppm test substance for 6 hours per day, 5 days per week, for up to 2 years, weekends and holidays excluded. Test material was analyzed for purity and stability several times during the study. Chamber atmospheres were analyzed during the exposure period for test substance concentration, temperature, humidity, and airflow rate. Body weights were obtained weekly during the first 3 months of the study and every other week for the remainder of the study. Clinical signs of toxicity were monitored throughout the study. An ophthalmological examination was performed on all animals prior to study start. All rats were examined prior to final sacrifice. At 3, 6, 12, 18, and 24 months (control and 25 ppm groups only at 24 months), 10 male and 10 female rats per group were subjected to a clinical evaluation that included measures of haematology, clinical chemistry, and urinalysis. Necropsy, and gross and microscopic evaluations of animals were as follows. Interim sacrifices of 10 rats per group were conducted at 12 and 18 months. Due to early mortality, final sacrifice of rats of the 250 and 2500 ppm groups occurred on test days 586 and 657, respectively. Control and 25 ppm groups of rats were sacrificed at approximately 24 months. Selected organs from 5 animals per group were evaluated for gross and microscopic observations and for cell proliferation at approximately 2 weeks, 3 months, and 12 months.

 

The mean purity of all test substance batches was 99.99%. The mean purity of all stability analyses was 99.98%. All chamber analyses indicated that concentrations were within 1% of target concentration. Chamber temperature ranged from 22.0 to 23.1°C. Chamber humidity ranged from 52.7% to 60.7%. Chamber airflow rate ranged from 868.4 L/min to 1060.2 L/min. Slight decreases in mean body weight gain (6-15%) were noted among rats of the 25 and 250 ppm groups, but not the 2500 ppm groups, when evaluated through final sacrifice. There were no unique or unusual incidences of clinical signs that were associate with test substance toxicity. The only possible exception was an increased incidence of "weak" and "coloured discharge eye(s)" among 25 ppm female rats compared to controls. Survival was decreased in male rats of the 250 and 2500 ppm groups and female rats of all test substance-exposed groups compared to controls. There were no ophthalmoscopic observations that occurred with an incidence suggestive of a compound-related effect. There were no biologically significant effects on haematological, clinical chemical, or urinalysis parameters measured in rats at any of the evaluations. Urinary fluoride excretion was concentration- and time-dependent. At necropsy, the following main gross observations were made in rats that were related to test substance exposure: masses, nodules, discoloration and haemorrhage of the liver; mass/nodules and discoloration of the lungs, and fluid of the peritoneal cavity; and masses of the head, face and periaural area; and abscesses of the face. Microscopically, these lesions were correlated with hepatic hemangiosarcoma, hepatocellular adenoma and carcinoma, foci of clear cell and basophilic alteration, and sinusoidal dilatation, metastatic lung tumours, and Zymbal's gland tumours. The incidences of these lesions were concentration-related in all exposed groups. Hepatic hemangiosarcoma appeared to be the sentinel lesion in rats. The first hepatic hemangiosarcoma appeared on test day 362. Early mortality was primarily related to haemorrhage from hepatic hemangiosarcoma. There were no increases in cell proliferation of the organs examined that were consistent and could be related to test substance exposure. The spectrum of test substance-induced tumours is similar to that induced by other similar test substances in rats. Under the conditions of this study, the test substance was carcinogenic in male and female rats at concentrations greater than or equal to 25 ppm. A no-observable adverse effect level was not determined. Based on this information, a LOAEL for repeated dose toxicity in rats was determined to be 25 ppm (47 mg/m³).