[No public or meaningful name is available]

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22/Mar/04 - 24/Jan/05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)

Deviations:

not specified

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: MITI Handbook 1996 - Guidelines for Industrial Chemicals

Deviations:

not specified

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

1,1,2,2-tetrafluoroethyl-2,2,2-trifluoroethyl ether

EC Number:

609-858-6

Cas Number:

406-78-0

Molecular formula:

C4H3F7O

IUPAC Name:

1,1,2,2-tetrafluoroethyl-2,2,2-trifluoroethyl ether

Test material form:

liquid

Specific details on test material used for the study:

Name: HFE-347pc-f-
Source: Asahi Glass Co., Ltd. Chemicals Company.
Grade of material: Technical
Colour: Clear colourless
Physical state: Liquid
Boiling point: 56.22°C
Vapour pressure: 31kPa at 25°C
Batch reference number: 50312080
CTL test substance reference number: Y12880/001
Purity (% w/w): 99.9
Storage conditions: Ambient temperature in the dark

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

60 males and 60 females, plus 2 per sex as spares (including 20 males and 20 females for recovery groups).
During the acclimatisation period prior to the study, animals were randomly allocated to holding cages and treatment groups through a method based on individual body weights. Animals were identified by ear-punching.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Before the start of the study, animals were accommodated in stainless steel cages (3.4m3). Environmental conditions were maintained as appropriate to the species and study type (temperature: 22 ± 3°C, humidity: 30-70%, air changes: at least 12/h, diet type: CT1, light cycle: artificial, 12 hours light and 12 hours dark). Any deviations from the temperature and humidity ranges were not considered to have affected the study integrity. Diet and mains water were supplied ad libitum, except during exposure and diet was withheld during urine collection. The diet and water provided were routinely analysed for contaminants. For environmental enrichment, rats were provided with nylabones and cardboard tunnels, which were removed during the exposure period.

Animals were acclimatised for at least 1 week before the start of the study.

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

clean air

Details on inhalation exposure:

Test atmospheres were generated by metering liquid test substance onto the top of a jacketed glass condenser column heated to 40°C using Techne water baths. A counter flow of air was used to pass the resultant vapour to the input point of the long-term chamber. Watson Marlowe pumps were used to deliver liquid test substance to the condenser. Pump settings were as follows: Group 1 and 5; control no pump required, Group 2; 5-9%, Group 3; 11-22%, Group 4 and 6; 37.5-60%. Generation settings were 25L/minute air generation. Conditioned air, into which the test substance was introduced, was supplied directly to the exposure chamber at a nominal flow rate of 500L//minute. The chamber containing control animals was supplied with conditioned air at 500L/minute only. Atmosphere generation and chamber air flow rates were monitored continuously using variable area flowmeters.
Nominal concentrations of the test substance during the exposure generation period were calculated using the following formulae:
Concentration (mg/l) = weight loss (mg) / (time (minutes x airflow) (1/minute))
Concentration (ppm) = (concentration (mg/l) x 24000) / molecular weight
Concentration (mg/m3) = concentration (ppm) x 8.179
The test atmospheres were sampled manually using a suitable gas tight syringe and analysed using gas chromatography. Each test atmosphere was analysed approx. hourly during each exposure. The analysis system was calibrated using an appropriate range of freshly prepared standards prior to the study and at intervals thereafter. The peak obtained was used to calculate the atmosphere concentration.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Trial generations were carried out prior to the start of exposure in order to:
- Determine the requisite air and test compound flow rates to achieve the appropriate concentrations
- Determine the appropriate method of atmosphere generation
- Determine the most appropriate method of analysis
- Determine the distribution of test substance in the exposure chambers

Duration of treatment / exposure:

Up to 13 weeks, with the rats being retained for a further 28 days post the 90 day exposure period without treatment

Frequency of treatment:

6 hours per day, 5 days per week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

151.2 ppm (nominal)

Doses / concentrations 2

Dose / conc.:

318.5 ppm (nominal)

Doses / concentrations 3

Dose / conc.:

1 036.6 ppm (nominal)

Doses / concentrations 4

Dose / conc.:

104 ppm (analytical)

Doses / concentrations 5

Dose / conc.:

310 ppm (analytical)

Doses / concentrations 6

Dose / conc.:

1 000 ppm (analytical)

Doses / concentrations 7

Dose / conc.:

1 236.66 mg/m³ air (nominal)

Doses / concentrations 8

Dose / conc.:

2 605.01 mg/m³ air (nominal)

Doses / concentrations 9

Dose / conc.:

8 478.35 mg/m³ air (nominal)

Doses / concentrations 10

Dose / conc.:

850.616 mg/m³ air (analytical)

Doses / concentrations 11

Dose / conc.:

2 535.49 mg/m³ air (analytical)

Doses / concentrations 12

Dose / conc.:

8 179 mg/m³ air (analytical)

No. of animals per sex per dose:

10

Details on study design:

The study had a replicate design with each replicate consisting of 5 animals per exposure group

Positive control:

Control animals were exposed to air only, but were otherwise treated in a similar manner to test animals

Examinations

Observations and examinations performed and frequency:

Prior to the start of the study, all rats were examined to ensure they were physically normal and exhibited normal activity. Detailed clinical observations, including the finding of no abnormalities detected, were recorded weekly prior to exposure. Rats were observed frequently during exposure period and immediately after exposure periods. All rats were checked at least twice daily for morbidity or mortality during exposure periods and at least once a day during the recovery period.

Observations performed included: Body weight, food consumption and utilisation, ophthalmoscopy, functional observation battery, motor activity, urinalysis, haematology, blood clinical chemistry

Sacrifice and pathology:

All rats were killed by over-exposure to halothane Ph Eur vapour, followed by exsanguination. The animals were then examined post-mortem. This involved an external observation and detailed examination of thoracic and abdominal viscera.

The following organs were weighed for all animals at scheduled termination: adrenal glands, kidneys, liver, ovaries, pituitary gland, brain, lungs (trachea attached but larynx removed), testes, uterus with cervix, thyroid gland, spleen, thymus, heart, epididymides. Paired organs were weighed together. Thyroid gland included parathyroid gland which was weighed 24 hours post-fixation and dissection from the larynx

Tissue submission: The following were taken from all main study and recovery animals and preserved in appropriate fixatives: Abnormal tissues, adrenal gland, aorta, bone (femur, including joint), bone marrow (femur), brain (medulla/pons, cerebellar cortex, cerebral cortex), cecum, cervix, colon, duodenum, epididymis, eyes (retina, optic nerve), harderian gland, heart, ileum, jejunum, kidney, lachrymal gland, larynx, liver, lung, lymph node (cervical, mesenteric), mammary gland (female only), nasopharynx, sciatic nerve, oesophogus, oral cavity, ovary, pancreas, parathyroid gland, peyer's patch, pharynx, pituitary gland, prostate gland, rectum, salivary gland, seminal vesicle, skin (right flank), spinal cord (cervical, mid-thoracic, lumbar), spleen, sternum with bone marrow, stomach, testis, thymus, thyroid gland, trachea, urinary bladder, uterus, vagina, voluntary muscle.

All submitted tissue from control and high exposure main study animals and respiratory tract and abnormal tissues from the remaining main study exposure groups were embedded in paraffin wax. 5um sections were cut and stained with haematoxylin and eosin. At examination postmortem, the lungs (with trachea and larynx attached) were excised, trimmed and weighed following removal of the larynx. The lungs, inflated with 10% formol saline and the trachea and larynx were fixed in 10% formol saline, routinely processed to paraffin blocks, cut into 5um sections and stained with haematoxylin and eosin for histopathological examination. Three standard sections of larynx were produced, taken at the level of the base and epiglottis, through the ventral pouch and the cricoid cartilage to include different epithelial cell types of the larynx and underlying seromucinous glands. The heads from all animals were removed along with excess skin and muscle. The brain was excised, and the nasal cavity perfused with 10% formol saline through the nasopharynx. The head was then immersed in formol saline, followed by decalcification with 20% formic acid. After processing, six standard sections were produced to include all different epithelial cell types and accessory nasal structures. The six sections were routinely stained with haematoxylin and eosin.

All submitted tissues from the control and high exposure groups were examined by light microscopy. Lungs, trachea and abdominal tissues were examined from the low and mid exposure groups.

Other examinations:

Determination of cyanide insensitive Palmitoyl CoA oxidation: Liver samples were taken from 5 males and 5 females per group (from replicates 3 and 4) for the assessment of palmitoyl CoA. An accurately weighed sample of approx. 5g was placed in 15ml ice cold 0.25M sucrose, homogenised and centrifuged. The oxidation of palmitoyl CoA is a measure of the degradation of saturated fatty acids to acetyl CoA.

Statistics:

All data were evaluated using ANOVA and/or covariance for each specified parameter using the MIXED procedure in SAS.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There were no treatment related effects on body weight adjusted for initial weight for males, either in the main study or recovery groups. Statistically significant differences from control seen at 817.9 or 2453.7 mg/m3 (100 or 300 ppm) did not form part of a dose-response relationship and was therefore considered to be incidental to treatment.
The difference from control in week 2 for females exposed to 817.9, 2453.7, or 8179 mg/m3 (100, 300 or 1000ppm) in the main study was due to a high weight for control female number 67 and was considered incidental to treatment. Bodyweight for female’s exposure to 8179 mg/m3 (1000ppm) was statistically lower than the control in the main study animals during weeks 5, 6, 12 and 15 (max difference 5%) and in weeks 4 and 6 (maximum 6%) in the recovery group. The difference from control was small (5 to 6%) and was not reflected in the same weeks (except for week 6) between main and recovery groups. Any effects were therefore concluded to not be adverse.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption for males exposed to 8179 mg/m3 (1000ppm) was lower than control in week 1 (main study) and weeks 12 and 14 (recovery). Food consumption for females exposed to 8179 mg/m3 (1000ppm) was lower than control in weeks 2, 3, 4, 6 and 15 (recovery only). The differences from control were small and/ or were not reflected in the same weeks between main and recovery group, meaning none were considered to be adverse.

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

The eyes of all examined pre-study animals were normal. The eyes of all animals in control and 8179 mg/m3 (1000ppm) exposure groups (main study) examined in week 13 were normal. Consequently, the eyes of the intermediate and recovery groups were not examined prior to termination.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At week 14, neutrophil count was higher in males exposed to 8179 mg/m3 (1000ppm). The mean value was within historical control mean data and the difference from control was considered incidental to treatment. Reticulocyte count and prothrombin time were higher than control in females exposed to 8179 mg/m3 (1000ppm).
The isolated statistically significant differences from control seen at week 14 at 817.9 mg/m3 (100ppm) in males and 2453.7 mg/m3 (300ppm) in females were not dose related and considered to be incidental to treatment.
At week 18, in males exposed to 8179 mg/m3 (1000ppm), red blood cell count was higher and mean cell volume, neutrophil count, white blood cell count and monocyte count were lower than control. The difference from control for white blood cell count was exaggerated by a high control value for male 43. In females, reticulocyte count and prothrombin time were not statistically different from control showing evidence of recovery; in addition, mean cell haemoglobin and cell haemoglobin were higher than control.
None of the differences above were considered to be of any haematological or toxicological significance.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

A small number of differences from control were seen at 2453.7 mg/m3 (300ppm) (glucose in females, phospholipid and cholesterol in males) however, these were not dose related and were considered incidental to treatment. At 8179 mg/m3 (1000ppm), after the recovery period, plasma y-glutamyl transferase activity was lower than control in males. This was attributed to a high value in control (male 50) and a low value at 8179 mg/m3 (1000ppm) (male 51). At 8179 mg/m3 (1000ppm), after the recovery period, plasma aspartate transaminase activity was higher than control in females.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

There was no difference from control in urine clinical chemistry in males in the main study or females in the main study/ recovery groups. Urine volume was lower and urine specific gravity was slightly higher than control for males at 8179 mg/m3 (1000ppm) in week 18 (recovery group). There differences from control were small and considered to be of no toxicological significance.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no effects of treatment on on landing foot splay, grip strength, time to tail flick or motor activity measurements. For landing foot splay measurements, the difference from control in males exposed to 2453.7 mg/m3 (300ppm) was not dose related and so was considered incidental to treatment. The occasional differences from control seen at 817.9 and 2453.7 mg/m3 (100ppm and 300 ppm respectively) in motor activity measurements were not dose related and so were also considered incidental to treatment. The small difference from control for females exposed to 8179 mg/m3 (1000ppm) in minutes 6-10 of motor activity measurements was isolated and again considered incidental to treatment.

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At week 14, spleen weight was lower than control for males exposed to 8179 mg/m3 (1000ppm). However, this was attributed to the high spleen weight for control male number 8. The differences from control in spleen weight at 817.9 mg/m3 (100ppm) and lung weight at 2453.7 mg/m3 (300ppm) were not dose related and considered incidental to treatment. Thymus weight was higher than control in all exposure concentrations for females. However, the control mean was low, and all treatment group means fell within the historical control date range. Absolute weights for adrenal gland, kidney and liver were lower than control for females exposed to 8179 mg/m3 (1000ppm), but not when adjusted for final bodyweight. At week 18, absolute thymus weight was lower than control in females, but there was no difference from control when adjusted for final bodyweight.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A small number of lesions were observed which were not related to treatment

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

A small number of spontaneous lesions were observed, which were not related to treatment. There was a minimal increase in the incidence of eosinophilic casts (unilateral) in the kidney of males exposed to 8179 mg/m3 (1000ppm) and a decrease in the incidence of tubular basophilia in the kidney of males in the same group. Both changes were considered unrelated to treatment. No treatment related changes were observed in the respiratory system.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

There were no effects on liver palmitoyl CoA activity measured in the main study animals.

Details on results:

There were no treatment related clinical observations or deaths during the study. There were no adverse treatment related effects on bodyweight, food consumption or utilisation. There were also no treatment related effects seen during the FOB. There were some changes in haematology parameters in animals exposed to 8179 mg/m3 (1000ppm), which had returned to control levels at the end of the recovery period or were within the historical control data base or, were different from control after the recovery period only. None of these differences were considered to be adverse. Although there were no treatment-related effects on blood or urine clinical chemistry at the end of the exposure period, females had higher plasma aspartate transaminase and males had lower urine volume and minimally higher specific gravity following the recovery period. In the absence of any adverse treatment related effects on organ weights or macroscopic or microscopic findings, these small differences from control were considered to be of no toxicological significance.

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

1 036.6 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects seen

2

Key result

Dose descriptor:

NOAEL

Effect level:

8 478.4 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects seen

Target system / organ toxicity

1

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

1 036.6 ppm (nominal)

2

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

8 478.4 mg/m³ air (nominal)

Any other information on results incl. tables

The overall mean concentrations from the weight loss of test substance were calculated to be as follows:

Target concentration (ppm)	Target concentration (mg/m3)	Nominal concentration (ppm)	Nominal concentration (mg/m3)
100	817.9	151.2	1236.7
300	2453.7	318.5	2605.0
1000	8719.0	1036.6	8478.4

Applicant's summary and conclusion

Conclusions:

Administration of up to 8179 mg/m3 (1000ppm) of HFE-347pc-f by inhalation for up to 90 consecutive days resulted in no adverse treatment related findings.

Executive summary:

Groups of 10 male and 10 female Alpk:APfSD (Wistar-derived) rats were exposed whole body for 6 hours per day to 0, 817.9, 2453.7, and 8179 mg/m3 (0, 100, 300 and 1000ppm) for 5 days per week, over a minimum of 13 weeks. An additional control group and treatment group exposed to the highest concentration was included for assessment of recovery. Recovery animals were retained without treatment at the end of the 90 day exposure period for a further 28 days.

Clinical observations, body weight and food consumption were measured throughout the study. A functional observation battery (FOB) including clinical assessments, measurements of grip strength, time to tail flick, landing foot splay and motor activity were conducted during week 12 of the study. Urine sample collected during week 13 (main study) or 17 (recovery) of the study were analysed. At the end of the study, the rats were killed and examined post-mortem. Cardiac blood samples were taken for clinical pathology, selected organs were weighed, liver samples were taken for the determination of palmitoyl co enzyme A activity and specified tissues were taken for histopathological examination.

During the study, there were no deaths or treatment related clinical observations. There were no adverse treatment related effects on bodyweight, food consumption or utilisation. There were also no treatment-related effects during the FOB. There were some changes to haematology parameters in animals exposed to 8179 mg/m3 (1000ppm), which had returned to control levels at the end of the recovery period/ were within the historical control data base/ were different from control after the recovery period, of which none were considered to be adverse. Although there were no treatment related effects on blood or urine clinical chemistry at the end of the exposure period, females had higher plasma aspartate transaminase and males had lower urine volume and minimally higher specific gravity following the recovery period. In the absence of any adverse treatment related effects on organ weights or macroscopic/ microscopic findings, these small deviances from the control were considered to be of no toxicological significance.

Overall, administration of up to 8179 mg/m3 (1000ppm) HFE-347pc-f by inhalation for up to 90 consecutive days resulted in no adverse treatment related findings. The pathological NOEL of HFE-347pc-f was therefore determined as 8179 mg/m3 (1000ppm).