1,1,2,2,3,3,4-heptafluorocyclopentane

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 1997-09-03 to 1997-10-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

1986

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

430-710-1

EC Name:

-

Cas Number:

15290-77-4

Molecular formula:

C5H3F7

IUPAC Name:

1,1,2,2,3,3,4-heptafluorocyclopentane

Specific details on test material used for the study:

Batch No.: 9706-1A
Purity: 98.65 %

Test animals

Species:

rat

Strain:

other: CD

Details on species / strain selection:

The rat was chosen because of its acceptance as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies. The CD strain was used because of the historical control data available in ths laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited (Margate, Kent, England)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Ca. 38 days
- Weight at study initiation: Male: 147-162 g; Female: 127-165 g
- Fasting period before study: no
- Housing: The animals were housed five of one sex per cage, unless this number was reduced by mortality, in TR18 cages which were made of a stainless steel body with a stainless steel mesh lid and floor. The cages were suspended above absorbent paper which was changed at appropriate intervals. Cages, cage-trays, food boppers and water bottles were changed at appropriate intervals.
- Diet: Expanded rodent diet (Rat and Mouse No. 1 Maintenance Diet from Special Diets Services Limited, Witham, Essex, England), ad libitum
- Water: ad libitum
- Acclimation period: 10 days

DETAILS OF FOOD AND WATER QUALITY:
The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent. No contaminants that might have prejudiced the study were present in the diet or water.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): 55
- Air changes (per hr): at least 10
- Photoperiod: 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

An appropriate quantity of dose for the animal of the group (the required amount for the animal plus 0.5 mL (the volume of the 8 choke catheter) was removed from the vial via a 16 gauge hypodermic needle; the needle was removed, the catheter attached and the excess dose was expelled. The animal was then dosed. After the first animal the catheter was removed and kept horizontal (to avoid loss of dose form contained within the catheter); the next aliquot of dose was removed from the vial (via syringe and 16 gauge needle), the catheter attached to the syringe and the animal dosed. After the last animal in the group had received its dose, the dose form contained within the catheter was sucked back and expelled into the vial (via the 16 gauge needle). A fresh syringe, needle and catheter was employed for each group of animals.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulation took place, slightly warmed (23-25°C) in a sealed unit with no air flow. Appropriate quantities of the test substance were measured volumetrically into a large glass vial containing a magnetic stirrer bar and sealed immediately. The vehicle, corn oil (warmed to 23-25°C), was added through the septum, the vial shaken and placed on a magnetic stirrer.
Bulk mixes of each test formulation were prepared weekly and aliquoted for daily administration; each aliquot was stored at 4 °C prior to use and allowed to come up to room temperature before administration to the animals.

VEHICLE
- Concentration in vehicle: 3, 30, 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The formulation samples, supplied as unit doses, were diluted with toluene and dissolved test item further diluted to a concentration within a nominal range. The concentration of test item in the dilution solution was determined by GC using a flame ionization detector.

Duration of treatment / exposure:

28 d

Frequency of treatment:

once each day, seven days per week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

15 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

150 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: selected based on the preliminary study results (Report No. ZCE020/972390)
- Fasting period before blood sampling for clinical biochemistry: overnight
- Recovery group: A further five male and five female animals assigned to the Control and high dosage groups completed a further two weeks without treatment to assess the recovery from any treatment-related changes.

Positive control:

None

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily
- Any deviations from normal were recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Immediately before dosing; Immediately after dosing on return of the animal to its cage; On completion of dosing of each group; Between one and two hours after completion of dosing of all groups; As late as possible in the working day.

BODY WEIGHT: Yes
- Time schedule for examinations: during the acclimatization period, on the day that treatment commenced, at twice weekly intervals throughout the treatment and recovery periods and before necropsy

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g/animal.

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: Yes

WATER CONSUMPTION: Yes, assessed by visual observations

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 29 for all main study animals, Day 43 for all recovery phase animals
- Anaesthetic used for blood collection: Yes (Halothane/nitrous oxide)
- Animals fasted: Yes, overnight
- How many animals: 5 male and 5 female per group
- Parameters examined: Packed cell volume (PCV); Hemoglobin concentration (HB); Erythrocyte count (RBC); Total and differential leucocyte count (WBC); Platelet count (PLAT); Mean cell hemoglobin concentration (MCHC); Mean cell hemoglobin (MCH); Mean cell volume (MCV); Prothrombin time (PT); Activated partial thromboplastin time (PTTK); Blood film - Romanowsky stain, examined by light microscopy for abnormal morphology and unusual cell types, including normoblasts.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 29 for all main study animals, Day 43 for all recovery phase animals
- Animals fasted: Yes
- How many animals: 5 male and 5 female per group
- Parameters examined.: Alkaline phosphatase (ALP); Alanine aminotransferase (ALT); Aspartate aminotransferase (AST); Gamma-glutamyl transpeptidase (GGT); Total bilirubin concentration (BILT); Glucose concentration (GLUC); Urea concentration (UREA); Creatinine concentration (CREA); Total cholesterol concentration (CHOL); Total triglyceride concentration (TRlG); Total protein concentration (TP); Albumin concentration (CHEM ALB); Albumin/globulin ratio (A/G); Sodium (Na); Potassium (K); Chloride (Cl); Calcium (Ca); Inorganic phosphorus (Phos)

URINALYSIS: Yes
- Time schedule for collection of urine: after 28 days treatment and after 14 days of recovery
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: Appearance (APP); Volume (VOL); pH; Specific gravity (SG); Protein (PROT); Glucose (GLUC); Ketones (KET); Bilirubin (BIL); Blood; Microscopy - the sediment from centrifugation was examined for epithelial cells (EP), polymorphonuclear leucocytes (CP), erythrocytes (RBC), crystals (CRY), spermatozoa and precursors (S) or other abnormalities (A).

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (See Terminal observations - 28d test in attached backgroud material)

HISTOPATHOLOGY: Yes (See Terminal observations - 28d test in attached backgroud material)

Statistics:

The significance of inter group differences in hematology (excluding the incidence of morphological abnormalities evident on blood smears), blood chemistry and urinalysis (volume, specific gravity and pH) were assessed by Student's t-test using a pooled error variance. Statistical significances for eosinophil, basophil, monocyte and large unstained cell counts are not reported as these data are not normally distributed.
For organ weights and bodyweight changes, homogeneity of variance was tested using Bartlett's test. Whenever this was found to be statistically significant a Behrens-Fisher test was used to perform pairwise comparisons, otherwise a Dunnett's test was used.
Inter-group differences in macroscopic pathology and histopathology were assessed using Fisher's Exact test.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Transient post-dosing salivation was evident amongst animals treated at 1000 mg/kg/day; this sign is common on studies of this tye and is not considered to be of toxicological significance

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Although when compared to the Controls, male animals treated at 1000 mg/kg/day showed a marginally low bodyweight gain, the females at this dosage showed a marginally high gain and this, therefore, was considered to be fortuitous. Bodyweight change during the recovery phase was similar to the Controls.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Overall food consumption during the treatment period was marginally lower in males receiving 1000 mg/kg/day when compared with the Controls, although this was likely to have been fortuitous. During the recovery phase the food consumption of animals previously given 1000 mg/kg/day was similar to the Controls.

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There was no change that could be ascribed to treatment with test item. Inter-group variations which attained statistical significance were either minor, lacked dosage-relationship or were confined to one sex.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

After four weeks of treatment individual male and female animals receiving 150 or 1000 mg/kg/day showed, when compared with the Controls, slightly high plasma triglyceride concentrations, although a dosage-relationship was not always evident. It is likely that these isolated high values were fortuitous.
Although there were a few differences from the Control values, with some statistical significance, they either lacked dosage-relationship or were confined to one sex and are considered to be unrelated to treatment. Lower gamma glutamyl transpeptidase or total bilirubin values, when compared with the Control animals, were evident in males treated at 15 and 1000 mg/kg/day or females treated at 150 and 1000 mg/kg/day, respectively, but are not considered to be of toxicological significance.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Bodyweight-relative liver weights were marginally high in individual animals treated a 1000 mg/kg/day, when compared with the Controls, however, this was considered to be fortuitous. Absolute and bodyweight-relative adrenal weights were slightly high for females treated at this dosage but this change was minor, lacked a histopathological correlate and was not seen in the males; it is, therefore, considered to be fortuitous.
Following the recovery phase, the liver and adrenal weights were not dissimilar to the Controls.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Intra-cytoplasmic protein droplets were seen in the kidneys of male rats at all dosages

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: not relevant to human

2

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: the intra-cytoplasmic protein droplets have no relevance to human health

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

The No-Observed-Adverse-Effect Level (NOAEL) was considered to be 1000 mg/kg/day.
Although a No-Observed-Effect Level (NOEL) in male rats has not been detailed by this investigation, the intra-cytoplasmic protein droplets have no relevance to human health, especially in the absence of any blood or urinary abnormality. With the exclusion of these findings, seen only in the kidneys of treated male rats, the NOEL for both sexes is considered to be 1000 mg/kg/day.

Executive summary:

The 28d test was conducted in compliance with EC B.7.

Three groups of rats comprising five males and five females received test by oral gavage at dosages of 15, 150 or 1000 mg/kg/day at a constant volume-dosage of 5 mL/kg bodyweight in corn oil for 28 days. A similarly constituted Control group received vehicle alone at the same volume dosage. A further five male and five female animals assigned to the Control and high dosage groups completed a further two weeks without treatment to assess the recovery from any treatment-related changes.

There was no death or sign considered to be attributable to treatment. Bodyweight gain, overall food consumption and food conversion efficiencies were considered to have been unaffected by treatment with test item. There was no change in the cellularity or chemical composition of the blood, or in the urine, which could unequivocally be attributed to treatment. There was no effect of treatment on the absolute and bodyweight- relative organ weights.

There was no macroscopic finding recorded at necropsy which could be attributed to treatment. Microscopic examination revealed intra-cytoplasmic protein droplets in the kidneys of males at all dosages; the incidence of this lesion was lower after the recovery period. This change is generally considered to be a sex- and species-specific effect and to have no human health relevance.

The No-Observed-Adverse-Effect Level (NOAEL) was considered to be 1000 mg/kg/day.

Although a No-Observed-Effect Level (NOEL) in male rats has not been detailed by this investigation, the intra-cytoplasmic protein droplets have no relevance to human health, especially in the absence of any blood or urinary abnormality. With the exclusion of these findings, seen only in the kidneys of treated male rats, the NOEL for both sexes is considered to be 1000 mg/kg/day.