Hexafluoropropene, oxidized, oligomers, reduced, fluorinated

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 July 2001 to 01 October 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD Guideline-conform study conducted under GLP.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Ethene, tetrafluoro-, oxidized, polymd., reduced, decarboxylated

IUPAC Name:

Ethene, tetrafluoro-, oxidized, polymd., reduced, decarboxylated

Constituent 2

Reference substance name:

Tetrafluoroethylene, oxidized, oligomers, reduced, decarboxylated

EC Number:

500-539-6

EC Name:

Tetrafluoroethylene, oxidized, oligomers, reduced, decarboxylated

Cas Number:

161075-02-1

Molecular formula:

CF2H-O-(CF2-CF2-O)m-(CF2-O)n-CF2H

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): HFPE, H-Galden 8004X
- Molecular formula (if other than submission substance): HCF2O(C2F4O)m(CF2O)nCF2H
- Molecular weight (if other than submission substance): 230-350
- Smiles notation (if other than submission substance): not applicable
- InChl (if other than submission substance): not applicable
- Structural formula attached as image file (if other than submission substance): see Illustration
- Substance type: Pure substance
- Physical state: Liquid
- Analytical purity: >99%
- Impurities (identity and concentrations): not reported
- Composition of test material, percentage of components: (n=0 m=0): 0,3%, (n=1 m=0): 5,6%, (n=0 m=1): 43%, (n=2 m=0): 2,7 %, (n=1 m=1): 18 %, (n=0 m=2): 25%, (n=2 m=1): 5%, (n=1 m=2): 0,2%, (n=0 m=3): 0,1%, (n=2 m=2): 0,1%
- Isomers composition: not reported
- Purity test date: not reported
- Lot/batch No.: 04/01
- Expiration date of the lot/batch: April 2005
- Stability under test conditions: Stable
- Storage condition of test material: Ambient temperature in the dark

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: livestock farming
- Age at study initiation: approximately 8 weeks
- Weight at study initiation: males: 266-324 g, females: 185-245 g
- Fasting period before study: no. Animals had no access to food and water during the 6-hour exposure period and overnight prior to sampling for laboratory investigations.
- Housing: 5 animals/sex/cage
- Diet (e.g. ad libitum): pellet diet ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: approximately 2 weeks, between arrival of animals and the commencement of exposure.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-22 (measured)
- Humidity (%): 46-75 (measured)
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12 hour cicle (6 a.m.-6 p.m.)

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Whole body exposure chambers
- Method of holding animals in test chamber: Animals were placed into individual compartment of the exposure chamber. A separate exposure chamber was used for each group.
- Method of conditioning air: Air supplied to the vapour generators and secondary dilution vessels was filtered to remove any residual particulate and was dried.
- System of generating vapour: The chamber atmospheres were produced by metering the liquid test substance into glass vapour generators through which dried and heated air was passed at a group dependent flow rate ranging from 90 to 150 l/minute and subsequently 60 to 100 l/minute from exposure 40. The flow rate was decrease to conserve test material. For all exposed groups, the vapour/air mixture produced in the vapour generators was passed into the base of a secondary dilution vessel. A further supply of clean and dry air was supplied to Groups 2 and 3. The air supply to Group 4 was provided solely by the vapour generation system.
- Temperature, humidity, pressure in air chamber: The mean temperatures in chambers ranged between 22.6 °C +-1.13 (Group 1 Chamber) and 24.3 22.6 °C +- 0.99 (Group 4 Chamber). The mean relative humidity (RH) in chambers ranged between 30% +- 6.2 (Group 4 Chamber) and 42% +- 6.6 (Group 1 Chamber) .
- Air flow rate: about 150 l/minute initially and about 100 l/minute from exposure 40 onward to give the final chamber conconcentrations.
- Air change rate: not reported
- Method of particle size determination: not relevant
- Treatment of exhaust air: not reported

TEST ATMOSPHERE
- Brief description of analytical method used: Gas Chromatograph was used to measure the concentration of H GALDEN in the test atmospheres within the four inhalation chambers.
- Samples taken from breathing zone: yes. Chamber atmosphere was sampled in sequence from each of the four exposure chambers and from one point within each chamber. Air from each chamber was continually drawn through a transfer line, which was therefore equilibrated with the mean concentration from each chamber. Every seven minutes, air from the transfer lines was swiched to the injection loop pf the gas chromatograph for automated analysis and data logging. When not being sampled, these transfer lines were pumped to waste.

VEHICLE (if applicable)
- Justification for use and choice of vehicle: air
- Composition of vehicle: air
- Type and concentration of dispersant aid (if powder): not relevant
- Concentration of test material in vehicle: 1014, 3297, 10075 ppm
- Lot/batch no. of vehicle (if required): not relevant
- Purity of vehicle: not relevant

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The samples of chamber atmosphere were injected into a gas chromatograph, which was calibrated using vapour standards prepared in gas sampling bags.

Vapour samples are collected using an automated system fitted with electrically controlled valves, which are manipulated using Chamber Environment Monitoring System (CEMS-2) software.
The test atmosphere is drawn directly from the inhalation chamber through the sample line to the gas-sampling valve located on the GC. Initially, the gas-sampliong valve of the GC is set to the "load" position after 60 seconds. Simultaneously, the GC activates the start of the run sequence.

Chromatographic conditions:
Analytical column: DB.1, 5 micrometer film thickness, 30 m x 0.53 mm i.d.
Carrier gas: Helium (2.1 ml/minute)
Split ratio 1:25
Oxidant: Air ( 330 ml/minute)
Fuel: Hydrogen (33 ml/minute)
Injection volume: 500 microliter via an automated gas valve
Injection temperature: 200 °C
Detector temperature: 200 °C
Range: 1000
Gas sample valve: Off at 2.5 minute
Retention time H GALDEN: approximately 0.6-2.3 minutes (analysis for major peak at 0.9 minutes)

Duration of treatment / exposure:

6 hours/day

Frequency of treatment:

5 days/week for 13 consecutive weeks.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm (analytical)

Doses / concentrations 2

Dose / conc.:

1 014 ppm (analytical)

Remarks:

Target: 1000 ppm

Doses / concentrations 3

Dose / conc.:

3 297 ppm (analytical)

Remarks:

Target: 3300 ppm

Doses / concentrations 4

Dose / conc.:

10 075 ppm (analytical)

Remarks:

Target: 10000 ppm

No. of animals per sex per dose:

20 (10 males + 10 females)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: the dose levels (1000, 3300, 10000 ppm) were selected in consultation with the sponsor, on the results obtained in a 14-day preliminary toxicity study performed in the same laboratory.
- Rationale for animal assignment (if not random): random

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS: Yes
Dated and signed records of appearence, change and disappearence of clinical signs were maintened in clinical history sheets for individual animals.
Sign of ill health, together with any behavioural change or response to tratment, were recorded.
In addition detailed observations were made according to the following time schedule:
-Daily during exposure period as following:
1. Pre-exposure observation, 2. During exposure, 3. As each animal is returned to its home cage, 4. As late as possible in the working day.
Throughout the study, checks were made early in the working day and again in the afternoon to look for dead or moribund animals.

DETAILED PHYSICAL EXAMINATION AND ARENA OBSERVATION
Once prior to the study start, in each week of exposures, a detailed physical examination was performed on all animals. The observations took place, at the weekend, when there were no exposures, at approximately the same time of day on each occasion. After removal from the cage, the animal was assessed for physical condition and behaviour during handling and after being placed in a standard arena. Particular attention was made to possible signs of neurotoxicity, such as convulsions, tremor and abnormal gait or behaviour, as well as to any audible respiratory noise. Any deviations from normal were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: one week before the start of exposure, on the day that treatment commenced, each week thereafter and also prior to necropsy.

FOOD CONSUMPTION: Yes
The quantity of food consumed by each cage of rats was recorded on a weekly basis, commencing 1 week before the start of exposures.
Food intake per rat (g/rat/week) was calculated using the total amount of food given to and left by each cage in each group and the number of rats in each cage.

FOOD EFFICIENCY: No

WATER CONSUMPTION: Yes
The quantity of water consumed by each cage of rats was recorded on a daily basis, commencing 1 week before the start of exposures.
Water intake per rat (g/rat/day) was calculated using the total amount of water given to and left by each cage in each group and the number of rats in each cage.

OPHTHALMOSCOPIC EXAMINATION: Yes
The eyes of all rats were examined once before the start of dosing.
All control and high dose group rats were examined during Week 13 of dosing (prior to dosing on the day).
The following ocular structures were examined: adnexa, conjunctiva, cornea, sclera, anterior chamber, iris (pupil dilated), lens, vitreous and ocular fundus.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on Week 13 of exposure from all rats.
- Animals fasted: Yes, overnight
- How many animals: 80
- The following parameters were examined: Haematocrit, Haemoglobin, Erythrocyte count, Mean cell haemoglobin concentration , Mean cell volume, Mean cell haemoglobin, Total leucocyte count (Neutrophilis, Lymphocytes, Eosinophils, Basophils, Monocytes, Large unstained cells), Plateled count, Reticulocyte count, Cell morphology, Prothrombin time, Activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on Week 13 of exposure from all rats.
- Animals fasted: Yes, overnight
- How many animals: 80
- The following parameters were examined: Glucose, Total protein, Albumin/Globulin (A/G) ratio, Urea, Creatinine, Alkaline phosphatase, Alanine amino-transferase, Aspartate amino-transferase, Gamma Glutamyl transpeptidase, Creatine phosphokinase, Total bilirubin, Sodium, Potassium, Calcium, Inorganic phosphorus, Chloride, Cholesterol, Triglycerides.

URINALYSIS: Yes
- Time schedule for collection of urine: on Week 13 of exposure from all rats
- Metabolism cages used for collection of urine: Yes. Rats were placed overnight in individual urine cages.
- Animals fasted: Food and water were withheld overnight
- The following parameters were examined: Appearance, Volume, pH, Specific gravity, Protein, Sodium, Potassium, Chloride, Total reducing substances, Glucose, Ketones, Bilirubin, Blood pigments.

For microscopic examination, a portion of the urine sample was centrifuged and the resulting deposit was examined for the presence of the following: Epithelial cells, Leucocytes, Erythrocytes, Crystals, Renal tubule casts, Spermatozoa and precursors, Other abnormal constituents

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During Week 12; prior to the start of exposure; during the weekend, when no exposures were in progress.
- Dose groups that were examined: All animals
- Battery of functions tested: sensory activity, grip strength, motor activity
Sensory reactivity: Approach response, touch response, auditory startle response, tail pinch response.
Grip strength: forelimb and hindlimb grip strength was measured. Two trials were performed.
Motor activity: motor activity was recorded using an infra-red detector. The following categories were recorded: time spent in locomotory activity, non-locomotor activity, in no movement. The number of occurrences (events) of each category is also recorded. The test session for each animal was one hour, with data being collected every 2 minutes.

Sacrifice and pathology:

Following 13 weeks of exposure the rats were sacrificed. The examinations took place over 2 days, and, for animals sacrifieced on the second day, exposure were performed on Day 1 of Week 14.

GROSS PATHOLOGY: Yes
All rats were subjected to a detailed macroscopic examination.
The following organs from all animals were dissected free of fat and weighed: adrenals, kidney, ovaries, thymus, brain, liver, spleen, uterus with cervix, epididymides, lungs including bronchi, testes, heart.

HISTOPATHOLOGY: Yes
The following tissues were examined: abnormalities*, adrenals, aorta (thoracic), brain, caecum, colon, duodenum, epididymides, eyes, femur (longitudinal section through joint), heart, ileum jejunum, kidneys, larynx (2 levels), liver (section from 2 lobes)*, lungs (section from 4 major lobes including bronchi)*, lymph nodes (mandibular, mesenteric and tracheobronchial), nasal turbinates, oesophagus, ovaries, pancreas, pituitary, rectum, salivary grands, sciatic nerves, seminal vesicles, skeletal muscle (thigh), spinal cord, spleen, sternum, stomach, testes (PAS staining), thymus, thyroid with parathyroids, trachea (including bifurcation), urinary bladder, uterus with cervix.
Groups 1 and 4: all above listed tissues.
Groups 2 and 3: only star(*)marked tissues and, due to treatment-related changes, liver

Other examinations:

BONE MARROW EXAMINATION
Prior the post mortem examination, a tibial/femoral bone marrow sample was obtained from each animal. Each smear was air-dried and held pending a possible future requirements for examination (a visual assessment for cellularity, distribution and morphology).

Statistics:

All statistical analysis were performed separately for males and females.
Data relating to food and water consumption were analyzed fon a cage basis. For all other parameters the analysis were performed using the individual animal as experimental unit. Bodyweight data were analysed using weight gains.
A sequence of statistical analysis was used for bodyweight, food and water consumption, clinical pathology, organ weight and grip strength data. Details on the sequence of statistical analysis are reported in the section "Any other information on materials and method".

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

There were no treatment-related effects during the 13 weeks of the treatment period. No observations were observed during or immediately post exposure in any group.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were considered to be no effects of treatment on the variable pattern of bodyweight change between the groups.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

There were considered to be no effect of treatment on water consumption of males during the 13 weeks of treatment.
Group mean water intake of Group 4 (High dose) females was slightly greater that the concurrent controls over the 13 weeks of exposure, although the differences did not attain statistical significance. There were no dosage related effects on water consumption amongst Low and Intermediate dose females.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related findings observed amongst High dose animals during the ophthalmic examinations in Week 13.

Haematological findings:

no effects observed

Description (incidence and severity):

There were considered to be no treatment-related findings in the haematological parameters investigated in Week 13.

Occasional values were statistically different from Controls (i.e. mean cell haemoglobin concentration (MCHC) levels of High dose female and prothrombin time (PT) and activated partial thromboplastin time (APTT) of treated females). In the absence of dosage-responses and correlation amongst sexes, these findings are considered not to be of toxicological significance.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Group mean creatine phosphokinase (CPK) levels were lower in all treated groups (statistically significantly lower for all treated females) compared with the control in Week 13. However, the Control values for males and females were remarkably high (expected values are in the region of 150 U/L) and as such these changes are of no toxicological importance.
Group mean urea levels were slightly lower than Control amongst High dose animals, with statistical significance being attained for males. In the absence of a dose response this change is considered to be of no toxicological significance.
Group mean total protein levels (due to higher albumin levels) for Intermediate and High dose males and females were slightly higher than the Controls with the values for females attaining statistical significance. In the absence of a dose response this change is considered to be of no toxicological significance.
Although other slight inter-group differences in the biochemical parameters investigated in Week 13 occasionally attained statistical significance, these findings were considered incidental and of no toxicological significance.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Group mean urinary volume was lower in High dose males when compared with the Controls with a consequent lower pH and higher specific gravity. However, in the absence of similar findings amongst females, and no effects on water consumption, these changes are no of toxicological significance.
There were considered to be non effects of treatment on the remaining urinalysis parameters investigated.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

SENSORY REACTIVITY and GRIP STRENGTH: In week 12 group mean forelimb grip strength of Intermediate dose and High dose female rats was slightly but statistically significantly lower than the Controls. In the absence of effects in males, no dosage-relationship and no similar effect on hindlimb grip strength, this change is considered to be of no toxicological significance.
There were considered to be no effects of treatment on group mean hindlimb grip strength.
Sensory reactivity was considered unaffected by treatment in Week 12.

MOTOR ACTIVITY: There were considered to be no treatment-related effects in the time spent in locomotory activity in Week 12.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Following 13 weeks of treatment, group mean body weight-adjusted kidney weight were statistically significantly higher than Controls, for Intermediate dose females and High dose males and females. However, in the absence of any pathological changes, this change is considered to be of no toxicological significance.

Group mean body weight-adjusted liver weights were statistically significantly higher in High dose animals following 13 weeks of treatment.

Group mean thymus weights ( bodyweight-adjusted fro females) were higher in High dose animals, with statistically significance being attained for female. However, since no dosage related trend was apparent the change is considered to be of no toxicological significance.
There were considered to be no further effects on the organ weight investigated.

Gross pathological findings:

no effects observed

Description (incidence and severity):

The macroscopic examinations performed at termination revealed no changes attributable to treatment with HFPE.
The incidence and distribution of all findings were considered to fall within the expected background range of macroscopic changes.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Findings considered to be related with treatment were reported in the liver:
Centrilobular hepatocyte hypertrophy was reported in all Group 4 males, the majority of Group 4 female and a proportion of Group 3 males and females, but no in Group 2 or Control animals.

All other findings were considered to be incidental and of no toxicological significance.

Histopathological findings: neoplastic:

no effects observed

Effect levels

1

Key result

Dose descriptor:

NOEC

Effect level:

1 014 ppm (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

2

Key result

Dose descriptor:

NOAEC

Effect level:

10 075 ppm (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

RESULTS of MICROSCOPIC EXAMINATION

Treatment related findings

		MALES				FEMALES
Group / (Concentration)		1 Control group	2 (1014 ppm)	3 (3297 ppm)	4 (10075 ppm)	1 Control group	2 (1014 ppm)	3 (3297 ppm)	4 (10075 ppm)
Centrilobular hepatocyte hypertrophy	Total	0	0	5a	10c	0	0	3	9c
	Minimal	0	0	5	3	0	0	3	6
	Slight	0	0	0	7	0	0	0	3
Number of livers examined		10	10	10	10	10	10	10	10
Statistical significance compared to Control Group 1:a: p < 0,05 ; c : p < 0.001

Applicant's summary and conclusion

Conclusions:

The only biologically significant change seen in this study on H GALDEN was centrilobular hypertrophy of the liver in the majority of High dose animals (correlating with an increased bodyweight-adjusted liver weight) and a proportion of Intermediate animals. There was no concurrent alteration of biochemical parameters (e.g., hepatic enzymes). As this is considered to be an adaptive response to exposure of the test material rather than an indication of toxicity it is concluded that No Adverse Effect Concentration (NOAEC) was 10075 ppm (115209 mg/m3) and that the No Effect Concentration (NOEC) was 1014 ppm (11595 mg/m3).

Executive summary:

H GALDEN was administered to rats by whole body inhalation exposure for 13 weeks. Three groups (each of 10 males and 10 females) were exposed to target concentrations of 1000, 3300 and 10000 ppm, 6 hours a day, 5 days a week for 13 consecutive weeks. A fourth group acting as a control was exposed to air alone.

During the study clinical signs, physical examination, arena observations were recorded. Bodyweight and food consumption were recorded weekly. Water consumption was recorded daily. Sensory reactivity, grip strength and motor activity were recorded for all animals in Week 12. Opthalmoscopy was performed on all animals pre-dose and in Week 13. In Week 13, haematology, biochemistry and urinalysis was undertaken. Following the 13-week period, all animals were sacrificed on the day following the last exposure

and subjected to a full macroscopic examination and a range of organ weights recorded. Histopathological examinations were carried out on selected tissues from all animals (dependent upon dose group).

Mean analysed vapour concentrations of HFPE were 1014, 3297 and 10075 ppm. These levels were in good agreement with the target exposure levels of 1000, 3300 and 10000 ppm.

The only biologically significant change seen in this study was centrilobular hypertrophy of the liver in the majority of high dose animals (correlating with an increased in bodyweight-adjusted liver weight) and a proportion of intermediate dose animals. As this is considered to be an adaptive response to exposure of the test material rather than an indication of toxicity it is concluded that the No Adverse Effect Concentration (NOAEC) was 10075 ppm (115209 mg/m3) and that the No Effect Concentration (NOEC) was 1014 ppm (11595 mg/m3).