Carbon tetrafluoride

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 2012 - March 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Perfluoroethane

EC Number:

200-939-8

EC Name:

Perfluoroethane

Cas Number:

76-16-4

Molecular formula:

C2F6

IUPAC Name:

hexafluoroethane

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Name of test material : perfluoroethane (CAS 76-16-4) named H-30188 in the study
- Lot/batch number of test material: 1011MT0161 ; 1110CW0033 and 0911MT0144
- Purity: 99.99% according to Certificate of Analysis (COA - appendix A of the study)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability under storage conditions: Stable based on analyses conducted by the sponsor. The test substance appeared to be stable under the storage conditions.
- Stability under test conditions: The test substance appeared to be stable under the conditions of the study; no evidence of instability was observed.
- Solubility and stability of the test substance in the dispersant/vehicle/test medium: Test substance is a gas miscible with the houseline air used in the study.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: No treatment prior to testing
- Preliminary purification step: No

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Remarks:

nulliparous, non-pregnant rats

Details on species / strain selection:

The Crl:CD(SD) strain was chosen because extensive background data are available from the literature, the supplier, and previous studies at DuPont Haskell. This strain is also considered suitable relative to longevity, hardiness, and incidence of spontaneous disease.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories International, Inc., Kingston, New York
- Age at Arrival: Approximately 51 days
- Age at Start of Inhalation: Approximately 70 days
- Weight on day after arrival: 198-240 grams (males); 148-183 grams (females)
- Weight at start of inhalation: 317-445 grams (males) and 201-269 grams (females)
- Assigned to test groups randomly: Yes, under following basis: Computerized, stratified randomization so that no statistically significant group mean body weight differences occur within a sex.
- Housing: Individually in solid bottom caging with bedding, and enrichment as appropriate; sexes on separate racks except during cohabitation (for reproduction group)
- Cohabitation: Mating pairs (females placed in males’ cages) on a 1:1 basis in solid bottom caging with bedding and enrichment. Fourteen days after the first day of cohabitation, females without evidence of copulation were housed singly.
- Diet: PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002 ad libitum except when fasted.
- Water: Tap water ad libitum
- Acclimation period: approximately 20 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26ºC (68-79ºF)
- Humidity (%): Relative humidity of 30-70%
- Photoperiod (hrs dark / hrs light): Artificial (fluorescent light) on an approximate 12-hour light/dark cycle (except during eye examinations and motor activity).

Administration / exposure

Route of administration:

inhalation: gas

Type of inhalation exposure:

whole body

Vehicle:

other: air and supplemental oxygen

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus:
For exposure numbers 1 to 10, all exposure chambers were constructed of stainless steel and glass (NYU style) with a nominal internal volume of 350 L. For exposure numbers 11 to 71, control animals were exposed to air in the same chamber while H-30188 (test material) test exposures were performed in stainless steel and glass (NYU style) chamber with a nominal internal volume of 150 L, due to technical issues regarding exhaust emissions. A stainless steel baffle below the chamber turret inside the chambers promoted uniform chamber distribution of the test atmosphere.

- Method of holding animals in test chamber:
exposure in a chamber. Whole body exposure.

- Exposure mode :
During exposure, animals were individually placed in stainless steel, wire-mesh modules and exposed whole-body inside the exposure chamber, when animals were housed as mating pairs and exposed in the same manner.

- Atmosphere Generation:
H-30188 (test material) vapor and supplemental oxygen were metered into a 1-liter 3-neck mixing flask by Brooks model 5850 E or 5850 I mass flow controllers. The mixture left the mixing flask and entered the glass transfer tube where chamber air supply was added to the mixture by Brooks model 5850 E or 5851 I mass flow controllers. The gas mixture then entered the top of the exposure chamber through a turret. The air-control atmosphere was similarly generated in a different room without the H-30188 vapor.
Test atmospheres were exhausted from the bottom of each chamber through MSA filters using vacuum pumps and discharged into the fume hood.

- Chamber Distribution of Test Substance:
Uniform distribution of the test substance in the 150-L and 350-L exposure chambers was determined.


TEST ATMOSPHERE
- Brief description of analytical method used: During each exposure, the vapor concentration of H-30188 was determined by gas chromatography at least once an hour from the test chambers.
- Samples taken from breathing zone: yes

VEHICLE
- Justification for use and choice of vehicle: The test material is a gas. The mode of exposition is via inhalation.
- Composition of vehicle: Chamber atmospheres were generated by dilution of the tested substance in houseline air and oxygen.

Analytical verification of doses or concentrations:

yes

Remarks:

gas chromatography

Details on analytical verification of doses or concentrations:

The control chamber was sampled at least twice a day. Samples of chamber atmosphere were continually drawn from the sampling port and were directly injected into an Agilent model 6890 gas chromatograph (GC) equipped with an automated gas sample valve and a flame ionization detector. All samples were chromatographed isothermally at 80ºC on a 30m x 0.53mm (nominal diameter) RTX-502.2 fused silica glass column. The vapor concentration of H-30188 was determined from a standard curve derived from gas standards. Standards were prepared prior to exposure by injecting known volumes of the vapor test substance into Tedlar® bags containing known volumes of air.

Duration of treatment / exposure:

approximately 20 days of exposure followed by approximately 28 days of no-exposure (recovery)

Frequency of treatment:

6 hours/day, 5 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Doses / concentrations 2

Dose / conc.:

2 500 ppm

Doses / concentrations 3

Dose / conc.:

10 000 ppm

Doses / concentrations 4

Dose / conc.:

50 000 ppm

No. of animals per sex per dose:

5 rats/sex/dose (subchronic with recovery subset)

Control animals:

yes

Details on study design:

- Dose selection rationale: In a previous acute inhalation study the inhalation LC50 in male and female rats was greater than 502,000 ppm. Based on these results, exposure concentrations of 0, 2500, 10,000, and 50,000 ppm were selected for the current study.
- Quarantine Period: 7 days
- selection criteria: Animals with adequate body weight gain and free of any clinical signs of disease or injury. Animals that did not meet these criteria were eliminated from consideration for use in the study. Weight variation of individual rats did not exceed ± 20% of the mean weight for each sex.
- Rationale for animal assignment: Randomization. Computerized, stratified randomization so that no statistically significant group mean body weight differences occur within a sex.

Examinations

Observations and examinations performed and frequency:

Body weight, clinical signs, food consumption were recorded throughout the study (during the treated period and the recovery period). Two ophtalmology evaluations were conducted by a veterinary ophtalmologist. Both eyes were examined by focal illumination and indirect ophtalmoscopy after mydriasis was produced.
On test day 29, blood and urine samples were collected from 5 male and 5 female rats per group for measurement of hematology, clinical chemistry and urinalysis parameters and again near the end of the recovery period on test day 57.
An abbreviated neurobehavioral evaluation was conducted on the 5 males and 5 females prior to test substance administration in order to obtain baseline measurements. This neurobehavioral evaluation was conducted again following approximately 4 weeks of exposure for the animals.

Sacrifice and pathology:

Following collection of terminal blood samples from the rats, the rats were euthanized, selected organs were weighed and selected tissues were evaluated microscopically.
The following organs were weighed fresh at necropsy from all adult animals: adrenals, brain, heart, kidneys, liver, lungs, spleen, thymus, ovaries, uterus, testes, epididymides, prostate, seminal vesicles, accessory sex organs.

Statistics:

Yes. For each parameter analyzed with a trend test the test was applied to the data sequentially.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related clinical signs of toxicity were observed in males or females.
Incidental clinical signs common to the age and strain of rat were occasionaly observed during the exposure and/or recovery periods.

Mortality:

no mortality observed

Description (incidence):

Unscheduled deaths did not occur in the animals.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No adverse test substance-related effects on body weight or weight gain occurred in males or females.
The body weight changes observed at 10000 ppm (females) and 2500 ppm (females) were not dose-dependent and were considered to be spurious.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No test substance-related effects or statistically differences in food consumption were observed for males or females.

Food efficiency:

no effects observed

Description (incidence and severity):

No test substance-related effects or statistically differences in food efficiency were observed for males or females.

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No adverse test substance-related ophtalmologic effects occurred in males or females.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No exposure-related changes in any group mean hematology parameter in male or female rats.
The following statistically significant difference was not considered to be related to exposure to the test substance: absolute large unstained cells were statistically significantly higher in female rats exposed to 50000 ppm. There were no other statistically significant differences in other white blood cell parameter in any subsets of male or female rats at any concentration tested. Therefore, this change was considered unrelated to exposure and non-adverse.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No exposure-related changes in group mean clinical chemistry parameters in male or female rats.
The following statistically significant difference was not considered to be related to exposure to the test substance: blood urea nitrogen (BUN) was minimally lower in female rats exposed to 50000 ppm. However, there were no statistically significant differences in other kidney-related clinical pathology parameters and no exposure-related changes in renal histopathology in this group. Therefore, the lower BUN in the 50000 ppm females was considered unrelated to exposure and non-adverse.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no statistically significant differences in group mean urinalysis parameters in male or female rats at any concentration tested.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No test substance-related or statistically significant effects occurred on :
- on forelimb or hindlimb grip strength
- on any behavioral parameter evaluated
- on motor activity
for males and females exposed to the substance.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related organ weight effects. All individual and mean organ weight differences were considered spurious and unrelated to test substance exposure.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test substance-related gross observations in either male or female rats.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Microscopic findings: no test substance-related microscopic findings in either male or female rats in the 50000 ppm inhalation exposure group.

Effect levels

Key result

Dose descriptor:

NOAEC

Effect level:

ca. 50 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

food efficiency

gross pathology

haematology

mortality

ophthalmological examination

organ weights and organ / body weight ratios

urinalysis

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study, the NOAEC of perfluoroethane for systemic toxicity in male and female rats was 50000 ppm, the highest concentration tested, based on the absence of effects in all endpoints.

Executive summary:

The purpose of this study was to determine the potential subchronic and reproductive toxicity from repeated inhalation exposure of male and female rats to 0, 2500, 10,000, or 50,000 ppm of the test substance.   

Groups of 20 young adult male or nulliparous, non-pregnant female Crl:CD(SD) rats were divided into the following subsets: Subchronic (including a micronucleus evaluation), Subchronic with Recovery (including a neurobehavioral evaluation), and Reproduction (including a neurobehavioral evaluation). 

Chamber atmospheres of test substance were generated by dilution of the test substance vapor in air and supplemental oxygen. An air control group was also evaluated using a similar generation method. Vapor concentrations of the test substance were measured by gas chromatography (GC). 

Body weight, clinical signs, food consumption were recorded throughout the study (during the treated period and the recovery period). Two ophtalmology evaluations were conducted by a veterinary ophtalmologist. Both eyes were examined by focal illumination and indirect ophtalmoscopy after mydriasis was produced.

On test day 29, blood and urine samples were collected from 5 male and 5 female rats per group for measurement of hematology, clinical chemistry and urinalysis parameters and again near the end of the recovery period on test day 57.

An abbreviated neurobehavioral evaluation was conducted on the 5 males and 5 females prior to test substance administration in order to obtain baseline measurements. This neurobehavioral evaluation was conducted again following approximately 4 weeks of exposure for the animals.

No test substance-related or adverse effects were observed.

Under the conditions of the study, the no-observed-adverse-effect level (NOAEL) for systemic toxicity in males and females was 50,000 ppm, the highest concentration tested, based on the absence of effects in all endpoints.