Chlorotrifluoroethylene

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 weeks

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Published literature fulfilled basic scientific principles.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1988

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

GLP compliance:

no

Remarks:

Research paper

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Chlorotrifluoroethylene

EC Number:

201-201-8

EC Name:

Chlorotrifluoroethylene

Cas Number:

79-38-9

Molecular formula:

C2ClF3

IUPAC Name:

1-chloro-1,2,2-trifluoroethene

Constituent 2

Reference substance name:

Chlorotrifluoroethylene (CTFE)

IUPAC Name:

Chlorotrifluoroethylene (CTFE)

Details on test material:

CTFE was supplied as a liquid (boiling point 279.9 centigrades) in pressurized stainless steel cylinders (Allied Corporation, Chemical Sector). It was analyzed by gas chromatographic mass spectroscopy before and immediately after the study, establishing the sample to be at least 99.9% pure CTFE and not containing any detectable perfluoroisobutylene (PFIB, detection limit < 1 ppm) or other identifiable contaminants.

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

All animals were housed individually in suspended stainless steel mesh cages. All animals were given food (Purina Rat Chow 5002) and water ad libitum during nonexposure periods only.

Administration / exposure

Route of administration:

inhalation: gas

Type of inhalation exposure:

whole body

Vehicle:

clean air

Remarks on MMAD:

MMAD / GSD: Not applicable for gas

Details on inhalation exposure:

Exposure chamber designs and operations
The subchronic study was conducted in stainless steel and glass exposure chambers having a volume of 1 m3 for periods of 6 hr/day. The chambers used in the subchronic study were operated on a conditioned (charcoal and HEPA filtered, dehumidified by chilling and then reheated) isolated air supply, under negative pressure, at an average airflow of 350 L/min. This provided an equilibrium time of 13.1 min. Subchronic study animals were exposed 5 days/week for 13 consecutive weeks.

Test atmosphere generation procedure
Exposure atmospheres were generated by metering the pure test material at known flow rates into the chamber air inlet, where it was then diluted to the desired concentration with chamber ventilation air. The cylinder containing CTFE was fitted with an appropriate pressure regulator that was connected to four Fischer-Porter flowmeters specifically calibrated with CTFE.

Measured mean exposure concentrations (± standard deviation) were 0, 29.4 (± 2.9), 61.5 (± 4.7), and 121.2 (± 8.8) for the control and 30, 60, and 120 ppm target exposure groups, respectively.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For the subchronic study, the chambers were routinely monitored using a Wilks Scientific Miran 1-A Ambient Air Analyzer. The CTFE exposure levels were verified periodically using a Perkin Elmer Model Sigma 2 gas chromatograph with a multiple gas sampling valve attachment and Sigma 10 data station. Chamber exposure levels were measured six times per chamber per day during all studies.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 hr/day, 5 days/week for 13 weeks

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0
Basis:
analytical conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
29.4 ± 2.9 ppm (V)
Basis:
analytical conc.

Doses / concentrations 3

Remarks:

Doses / Concentrations:
61.5 ± 4.7 ppm (V)
Basis:
analytical conc.

Doses / concentrations 4

Remarks:

Doses / Concentrations:
121.2 ± 8.8 ppm (V)
Basis:
analytical conc.

No. of animals per sex per dose:

Each group consisted of 20 males and 20 females. See the experimental design for the study reported in the field "Any other information on results including tables".

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
- Rationale for selecting satellite groups: These rats were assigned randomly to groups by a censored randomization procedure designed to ensure comparable mean body weights between groups.
- Post-exposure recovery period in satellite groups: 2 weeks

Positive control:

Not required.

Examinations

Observations and examinations performed and frequency:

All animals were observed twice daily for general condition and given detailed clinical assessments for signs of toxicity twice during the first 2 weeks of exposure and once per week thereafter. A complete neurobehavioral screen was performed on all animals five times during the study. Body weights were measured before the first exposure, twice during each of the first 2 weeks and weekly thereafter. Serum chemistries (BUN, SGPT, alkaline phosphatase, glucose, creatinine, total protein, albumin, globulin-by difference, Ca, P, Cl, Na, and K) and urinalysis (volume, osmolality, pH, and creatinine) were determined after both 1 and 2 months (5 animals/sex/group), during the final week of exposure (15 animals/sex/group), and at 2 weeks after the final exposure (in the retained group of 5 animals/sex/group). In addition, determination of urinary and serum fluoride levels were made at 1 month, during final exposure, and 2 weeks after the final exposure using the same animals selected for serum chemistry determinations.

Sacrifice and pathology:

Gross necropsy examinations were conducted on all animals. Organ weights were taken, and relative (to body weight) ratios were calculated on the following organs: brain, lungs (with trachea), liver, spleen, heart, thymus, each kidney, and combined gonads. Tissues were saved from 40 major organs (fixation in 10% neutral buffered formalin, H & E stain) and examined microscopically from all animals in the control and high level exposure groups killed at the end of the exposure period. Tissues examined included the nasal turbinates and upper respiratory tract. In addition, sections of the kidneys, lungs, liver, brain, spinal cord, optic nerves, heart, spleen, testes, and lymph nodes were examined from all other animals in the study.

Other examinations:

No data

Statistics:

The results of measurements of all quantitative continuous variables, such as body weight, hematology, and clinical chemistry data were intercompared (exposure groups to controls) by the use of the following tests: Bartlett's homogeneity of variance, analysis of variance, and Duncan's procedure. The latter was used if F for analysis of variance was significantly high to delineate which groups differ from the controls. If Bartlett's test indicated heterogeneous variance, the Fmax test was used for each group versus the control. If these individual Fmax tests were not significant of N1 = N2, Student's t-test was used; if significant, the means were compared by the Cochran t-test or the Wilcoxon rank sum test. Body weights on the subacute study were compared using Dunnett's procedure.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

Five animals died during the study (one 29 ppm, two 62 ppm, and two 121 ppm), all due to mishap during the course of anesthesia and interim blood sample collection.

Mortality:

no mortality observed

Description (incidence):

Five animals died during the study (one 29 ppm, two 62 ppm, and two 121 ppm), all due to mishap during the course of anesthesia and interim blood sample collection.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In the males, the 121 ppm group had significantly (P≤0.05) reduced body weights on days 4, 60, and 67. In the females, all three exposure groups had decreased body weights intermittently throughout the study but not in a dose-responsive manner.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no observed gross changes in the respiratory tract or nasal turbinates.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Inhalation of CTFE resulted in microscopic tubular changes in the kidneys of both male and female rats.

Histopathological findings: neoplastic:

no effects observed

Details on results:

In the males, the 121 ppm group had significantly (P ≤ 0.05) reduced body weights on days 4, 60, and 67 (3 %, 6 %, and 5 %, respectively) but not at other intervals. In the females, all three exposure groups had decreased body weights intermittently throughout the study but not in a dose-responsive manner.
In the 121 ppm exposure level, the RBC was decreased at the end of the recovery period in males (5.6%) and at 60 days in females (4.7%). All other hematology parameters appeared normal. In the males, serum albumin levels were increased in a dose-responsive manner at the end of 60 days, significant only at 121 ppm (9.4 %), and in all groups at 90 days (5.0 % at 29 ppm, 7.0 % at 62 ppm, and 7.0 % at 121 ppm). At 30 days, BUN was reduced in both males and females at 121 ppm (9 % and 17%, respectively). It was comparable to control values at other intervals. Alkaline phosphatase was significantly reduced in 121 ppm males at 30 days (17%) but was normal at other intervals. Serum creatinine levels in 62 and 121 ppm groups tended to be lower than controls. This was especially noted in females at 30 days and males at 60 days. Serum fluorides were increased in a dose-responsive manner to a significant level in males in all groups at 30 and 90 days and in females in all groups at 30 and 90 days, except the 29 ppm group at 30 days, but were comparable to controls by the end of the recovery period. Serum fluoride levels were also higher at 90 days than at 30 days.
In the males, both absolute and relative liver weights were increased in a dose-responsive manner in both the main study group and the recovery group. The increases in absolute weights (7.9 % at 29 ppm, 8.4 % at 62 ppm, and 8.9 % at 121 ppm) were significant in all main study exposure groups but not in the recovery groups. The increases in relative liver weights were also significant in all treatment groups terminated at the end of exposure, and the 121 ppm group was significantly increased at the end of the recovery period. Liver weights of CTFE-exposed females were comparable to weights of livers in controls. Both absolute and relative male kidney weights were increased in a dose-responsive manner and to a statistically significant extent in all male exposure groups at 90 days. The differences in weights were 9.3 %, 10.9 %, and 16.6 %, respectively. At the end of the recovery period, there were no effects on absolute kidney weights, but the relative weights were increased significantly for both kidneys in the 121 ppm animals and only for right kidneys in the 62 ppm males. Only recovery group females showed dose-responsive increases in both absolute and relative kidney weights. Of these, the 121 ppm right absolute kidney weights were statistically significantly increased (13%).
In the males, urine creatinines were decreased in a dose-responsive manner at 90 days, with the decrease being significant in the 62 and 121 ppm groups (9 %, 30 %, and 46 %). Urine creatinines measured at other intervals and in all CTFE-exposed females were comparable to controls. Urine fluorides were increased in a dose-responsive manner at 30 and 90 days and at the end of the recovery period, with the increase being significant in the 29 ppm males at 30 and 90 days, 29 ppm females at 30 days, and in the two higher exposure groups at all three measurement periods.
Inhalation of CTFE resulted in microscopic tubular changes in the kidneys of both male and female rats. These tubular changes were most frequent in rats exposed to the highest concentration (121 ppm) but were observed also in rats exposed to the intermediate concentration (62 ppm). No definitive kidney toxicity was observed in either males or females of the lowest exposure groups (29 ppm). These changes occurred in the middle portion of the cortex and were characterized by large, usually dilated tubules in 14 of 15 males at 121 ppm, 8 of 5 at 62 ppm, 0 of 15 at 29 ppm, and 2 of 15 controls. These were lined by enlarged epithelial cells with enlarged nuclei in 15 of 15 males at 121 ppm, 7 of 15 males at 62 ppm, and 0 of 15 males at both 29 and 0 ppm. Other kidney findings occurred in rats of CTFB-exposed groups without a definitive concentration relationship. These findings had frequencies or severities or both that were greater than those observed in the control groups. These tubular findings included atrophy, basophilia, hyperplasia, proteinaceous deposits, casts, concretions, and yellow-brown intracellular pigments. These microscopic findings are consistent with early progressive renal disease or glomerulonephrosis, which is observed commonly in old rats. The findings suggest that CTFE exposure was accelerating a concurrent or inherent disease procless in addition to the enlargement of tubular cells and their nuclei. There were no observed gross or microscopic changes in the respiratory tract or nasal turbinates. Other kidney findings occurred in rats of CTFE-exposed groups without a definitive concentration relationship. These findings had frequencies or severities or both that were greater than those observed in the control groups. These tubular findings included atrophy, basophilia, hyperplasia, proteinaceous deposits, casts, concretions, and yellow-brown intracellular pigment.

Effect levels

Key result

Dose descriptor:

NOAEC

Effect level:

29 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

62 ppm

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Any other information on results incl. tables

The observations suggest that these CTFE exposures may have accelerated a concurrent or inherent disease process (in addition to causing an enlargement of tubular cells and their nuclei). These findings imply that exposure level may be much more important than the total number of exposures. This would support the suggestion that the CTFE nephrotoxicity may be related to the concentration of the CTFE-cysteine metabolite in the kidney. Observations in recovery group animals clearly established that the effects observed were reversible on cessation of exposure. Elevations were seen in urinary fluoride and fluorine levels in the CTFE-exposed animals. With the urine samples, the elevations in fluoride, even for the high level exposure group, decreased as the study went on and were more marked in males than in females. Serum fluoride levels in the high exposure group also were generally within an order of magnitude of control values.

Data on serum creatinine levels (mg/dl), serum fluoride levels (g/ml) and urine fluoride levels (g/ml) are reported in the fiels"Attached background material".

Tab I

Experimental design for subchronic study: number of animals equally divided male and female:

Target/actual exposure level (ppm)	Exposed	Hematology and clinical chemistry intervals				Fluoride determining intervals			Necropsy intervals
		No. of animals				No. of animals			No. of animals
		Day 30	60	90	105	Day 30	90	105	Day 90	105
0/0	40	10	10	30	10	10	30	10	30	10
30/29	40	10	10	30	10	10	30	10	29a	10
60/62	40	10	10	30	10	10	30	10	29a	10
120/121	40	10	10	30	10	10	30	10	29a	10
Total	160	40	40	120	40	40	120	40	115	40

a Animals died due to mishap during the process of interuim blood collections, reducing the sizes of groups at necropsy.

Data on serum creatinine levels (mg/dl), serum fluoride levels (g/ml) and urine fluoride levels (g/ml) are reported in the fiels"Attached background material".

Applicant's summary and conclusion

Conclusions:

It can be concluded that the CTFE has adverse effects on the kidneys included increased organ weights, alterations in clinical chemistry parameters and clinical observations, and alterations in microscopic structure at concentration of above 29 ppm in air.

Executive summary:

In a subchronic study, male and female F-344 rats were exposed 6 hr/day, 5 days/ week for 13 weeks at mean levels of 0, 29, 62, and 121 ppm via inhalation. No animals died as a result of being exposed to CTFE. Indications of effects on the kidneys included increased organ weights, alterations in clinical chemistry parameters and clinical observations, and alterations in microscopic structure. Males also showed alterations in body weights and absolute and relative liver weights, which were reversible after cessation of exposure. When compared to the total body burden of fluoride that could have resulted from these exposures (almost 25 mg/day) these increases are slight and would support observations for other fluoroalkenes that these materials are only moderately absorbed or are rapidly eliminated once absorbed. Dose-response was evident in both sexes, although males were more sensitive, with 29 ppm being a limited effect level in the males. A group of animals maintained for 2 weeks after the completion of exposure showed marked remission from the observed effects. The data from this study indicate that an operating standard for worker exposure could be set from a starting point of using 29 ppm as a NOAEC.