[No public or meaningful name is available]

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:

14/Jan/2000 - 08/May/2000

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

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:

Test substance: 1,1,2,2-tetrafluoroethyl-2,2,2-trifluoroethyl ether (HFE-347pc-f; CAS No. 406-78-0)
Description: Clear liquid
Lot number: 991224
Purity: 99.82%
Storage: Stored in airtight containers at room temperature, protected from light

A predetermined amount of HFE-347pc-f was weighed before use and dissolved in Macorgol 400 JP to prepare a 500mg/mL solution. In addition, the solution was diluted stepwise with the vehicle to prepare a 50mg/mL solution and a 5mg/mL solution. The preparations were stored at room temperature under airtight conditions and protected from light. Administration occurred within 4 hours of preparation.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

50 each of male and female Sprague-Dawley rats [Crj:CD(SD)IGS] ages 5 weeks were purchased for the study and quarantined/ acclimatised for 11 days. During this period, the health conditions of the animals was evaluated and 40 each of male and female animals were selected from the healthy cohort. The selected animals were assigned to each group by stratified randomisation of body weight. The age at the start of administration was 6 weeks and the body weight ranged from 216-245g for males and 155-184g for females.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals were housed and kept individually in stainless steel bracket cages for rats (260W x 380D x 180H mm) in an animal room, where the temperature was set at 22 ± 3°C, the humidity was set at 50 ± 20%, the ventilation at more than 10 times/ hour (all fresh air system) and lighting for 12 hours/ day (from 6:00 to 18:00), illuminanace of 150 to 300 lux). A chow for experimental animals and domestic tap water in a polycarbonate water bottle were provided ad libitum.
Cages, feeders, saucers and water bottles were all used post autoclaving and cages/ feeders were changed more than once every 5 weeks. Saucers and water bottles were changed more than twice per week. In addition, the animal room was cleaned daily after the end of work and the floor was wiped and disinfected with a 400-fold diluted solution of benzethonium chloride.
Animals were identified with tattoos describing the abbreviation of animal number on the root of the tail and cages were identified by attaching colour labels describing the study number and root of administration.
The food and water provided were analysed for contaminants/ pollutants. The results were within range of the reference values in the acceptance criteria specified, meaning there was no abnormal value considered to affect the study.

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

For administration, the dosing volume was calculated from the most recent body weight and the dosing solution was administered once per day, using a disposable syringe equipped with an oral sound for rats. For the water injection JP and vehicle control groups, water and macrogel 400 were administered in a similar manner.

Vehicle:

macrogel ester

Details on oral exposure:

The oral route by gavage was selected for administration because oral ingestion was considered as the possible route of exposure in humans.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

In a preliminary 7-day repeated dose toxicity study (dose: 10, 100 and 1000mg/kg) conducted prior to the main study, no toxic change was observed in any group. As a result of this, 1000mg/kg was selected as the highest dose for this study, with 100mg/kg and 10mg/kg doses being set as the mid-dose and low-dose values, in a geometric ratio of 10.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once a day

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

10 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Details on study design:

AE 3000 was orally administered repeatedly to Sprague-Dawley rats at dose levels of 0 (water for injection JP control group), 0 (vehicle control group), 10, 100 and 1000mg/kg for 28 days, in order to examine its toxicity. 10 each of males and female rats were used in the control groups and 1000mg/kg group and 5 each of male and female rats in the 10 and 100mg/kg groups. The drug was withdrawn for 14 days after the end of the administration period to examine the recovery. In addition, water for injection JP and Macrogol 400 were administered in a similar manner in the water for injection JP control group and the vehicle control group, respectively.

Examinations

Observations and examinations performed and frequency:

The starting date of administration was considered as day 1 of administration and the starting date of recovery was considered as day 1 of recovery. The following observations, determinations and examinations were conducted on all animals.

Cllinical signs including death and moribund animals were observed more than once per day during the administration period and once per day during the recovery period. Body weight was determined once before administration and once per week during administration using an electronic balance.
Water/ food consumption were calculated by determining the amount supplied and the residual amount on the following day. Measurements were taken once before the start of administration and once per week during the administration and recovery periods, using an electronic balance. Additionally, food efficeincy was calculated by the following equation - (Body weight gain (g/period) / Food consumption (g/period)) x 100.

Urinalysis: 5mL of water for injection JP was administered orally by gavage to each animal for scheduled necropsy at the end of the administration period and at the end of the recovery period. Urine was collected for ~3 hours (under fasting and prohibition of drinking) and this urine was considered as the fresh urine to conduct examinations on occult blood, ketones, glucose, protein, pH, urobilinogen, bilirubin and colour tone.

Haematology: At the end of the administration and recovery periods, animals were opened under anaesthesia with ether, after fasting for about 20h from the previous day, and blood was collected from the abdominal aorta. Using the blood anticoagulated with EDTA-2K or 3.8% sodium citrated, the following parameters were determined: Red blood cell count, white blood cell count, platelet count, haematocrit, haemoglobin, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, differential white blood cell count, reticulocyte count, prothrombin time and activated partial thromboplastin time.

Blood biochemistry: Blood was collected simultaneously with that for haematology and serum was obtained by centrifuging the collected blood. The serum was used to determine the following parameters: Aspartate and alanine aminotransferase, alkaline phosphatase, y-glutamyl transpeptidase, choline esterase, albumin, glucose, total cholesterol, triglyceride, total bilirubin, urea nitrogen, creatinine, inorganic phosphorus, Ca/ Na/K/Cl and A/G ratio.

Sacrifice and pathology:

At the end of the recovery and administration periods, the animals were opened under anaesthesia and the blood was collected. The animals were exsanguinated to death by cutting the abdominal aorta. The body surface and various intracranial, intrathoracic and intraperitoneal organs and tissues were observed macroscopically.

Post necropsy, the following organs were excised and weight with an electronic balance: Brain, liver, kidneys, spleen, adrenals, testes, prostate, ovaries and uterus. The relative weights of the organs were calculated from the body weight on the day of necropsy.

The following organs and tissues were excised and fixed and preserved in 10% neutral buffered formalin solution: Brain, pituitary, thyroid gland (including parathyroid), thymus, submandibular/ sublingual salivary glands, heart, trachea, lungs, liver, stomach, duodenum, kidneys, spleen, adrenals, prostate, epididymis, seminal vesicles, ovaries, uterus, urinary bladder, bone and bone marrow (sternum and femur) and organs and tissues with macroscopic abnormalities. The eyeballs and harderian gland were prefixed in glutaraldehyde-formalin solution and the testes were prefixed in Bouin's solution and preserved in 10% neutral buffered formalin solution.
At the end of the administration period, microscopic examinations were conducted on the organs and tissues in the water for injection control group, the vehicle control group and the 1000mg/kg group after having excised, embedded in paraffin, sectioned and stained with haematoxylin and eosin. The organs and tissue examined were the heart, liver, spleen, kidneys, adrenals, testes, prostate, ovaries, uterus, stomach, bone marrow and bone (femur, sternum) and the organs and tissues with macroscopic abnormalities (epididymis in one male of the vehicle control group and uteri in one female in the 10mg/kg group and one female in the 100mg/kg group). Since there was no effect of the test substance in the histopathological examination at the end of the administration period, no histopathological examination of organs and tissues were conducted in the mid/low-dose groups or the recovery groups

Statistics:

In the 28-day treatment group, Bartlett's test for homogeneity of variance was conducted and if homogeneity of variance was observed, one-way analysis of variance was conducted. Thus, if there was some significant difference between groups, a paired comparative test of mean values was conducted with the water for injection JP control group using Dunnett's method. If variance was not uniform, Kruskal-Wallis' H-test was conducted and if there was significant difference between groups, a paired comparative test of mean rank was conducted with the water for injection JP control group, using the Dunnett's method.

In the 14-day recovery group, F-test was conducted and if uniformity of variance was observed, student's t-test was conducted and if no uniformity of variance was observed, Aspin-Welch's t-test was conducted to compare with the water for injection JP control group. For clinical signs and the result of ophthalmoscopy, urinalysis, necropsy and histopathology, no statistical analysis was conducted.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation was sporadically observed in 1 to 3 animals per day on and after 10 days of administration for males and on and after 9 days of administration for females, in the 1000mg/kg group. No other clinical signs were observed in any group throughout the administration period and recovery period.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

A significant increase was observed in males in the vehicle control group on Day 17 after administration

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In the examination at the end of the administration period, significantly shortened activated partial thromboplastin time was observed in males in the 1000mg/kg group. A significantly decreased reticulocyte count was also seen in females in the 10mg/kg group, compared with the water for injection JP control group.
In the examination at the end of the recovery period, a significant decrease in the eosinophil ration was observed in females in the 1000mg/kg group. In addition, a significant decrease in the band nucleic neutrophil ratio was observed in males in the vehicle control group.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In the examination at the end of the administration period, a significant increase in total cholesterol was observed in males in the 10mg/kg group and a significant decrease in triglyceride in females in the 1000mg/kg group, compared with the water for injection JP control group.
In the examination at the end of recovery period, significant decreases in γ-GTP and BUN and significant increases in triglyceride, glucose, albumin and A/G ration were observed in the male 1000mg/kg group, compared with the water for injection JP control groups.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

A significant decrease in the absolute weight of right adrenal was observed in males in the 10 and 100mg/kg groups, compared with the water for injection JP control group. No change was observed in in males of the 1000mg/kg group or females of any group. A significant decrease in the relative brain weight was observed in males in the vehicle control group.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Swollen uteri were observed in 1 to 4 females in all treated groups, but there was no difference in its incidence compared to the water for injection JP control group. In addition, nodes at the cauda epididymis were observed in one male in the vehicle control group and atrophied ovaries and uterus were observed in one female in the vehicle control group. In the examination at the end of the recovery period, no abnormality was observed in any group.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In the examination at the end of the administration period, infiltration of mononuclear cells and microgranulomas in the liver, infiltration of mononuclear cells, basophilic tubules, fibrosis and cysts in the kidneys, increased extramedullary hematopoiesis in the spleen, infiltration of mononuclear cells in the prostate and a diluted lumen of the uterus were observed in males and females in the 1000mg/kg group, which were similarly observed in the water for injection JP control group or the vehicle control group.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects observed

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

In the 1000mg/kg group, salivation was observed sporadically in 1 to 3 animals per day after administration on and after 10 days of administration in males and on and after 9 days of administration in females. It was a transient finding and was not observed before administration on the following day, meaning it was considered as a reflective response induced by stimulation of the test substance. Since no macroscopic or histopathological changes were observed in the oral cavity, salivary gland or stomach, this finding was of no toxicological significance.

Significantly shortened activated partial thromboplastin time and significantly decreased triglyceride observed in the haematology and blood biochemistry at the end of administration period were both mild changes. Since there was no histopathologically related change in the liver, these changes were considered to be of no toxicological significance. For the significantly decreased reticulocyte count and increased total cholesterol, there was no relationship to doses adn these changes were not considered attributable to administration of the test substance. Significantly decreased eosinophil ratio observed at the end of the recovery period was a mild change and considered as a physiological change. Since the change was not observed at the end of the administration period, it was considered to be of no toxicological significance. Decreased y-GTP and BUN and increased triglyceride, glucose, albumin and A/G ration were not significantly different from the vehicle control group and so were inferred as not related to the administration of the test substance.

Significantly decreased absolute weight of the right adrenal was not a dose-dependent change and there was no change in relative weight. Therefore, it was considered to not be related to the administration of the test substance.

There was no abnormality attributable to administration of the test substance in body weight, food consumption, food efficiency, water intake, ophthalmoscopy, urinalysis, necropsy and histopathology.

Based on the observations made, since no toxic change was observed even after the addition of 1,1,2,2-tetrafluoroethyl-2,2,2-trifluoroethyl ether at a dose of 1000mg/kg, the No Observed Adverse Effect Level (NOAEL) of 1,1,2,2-tetrafluoroethyl-2,2,2-trifluoroethyl ether under the conditions of this study, was considered to be 1000mg/kg for both males and females.

Executive summary:

AE 3000 was orally administered repeatedly to Sprague-Dawley rats at dose levels of 0 (water for injection JP control group), 0 (vehicle control group), 10, 100 and 1000mg/kg for 28 days, in order to examine its toxicity. 10 each of males and female rats were used in the control groups and 1000mg/kg group and 5 each of male and female rats in the 10 and 100mg/kg groups. The test substance was withdrawn for 14 days after the end of the administration period to examine the recovery. In addition, water for injection JP and Macrogol 400 were administered in a similar manner in the water for injection JP control group and the vehicle control group, respectively.

There was no toxicological change seen post administration of the test substance in clinical signs, body weight, food consumption/ efficiency, water intake, ophthalmolscopy, urinalysis, hematology, blood biochemistry, organ weight, necropsy or histopathology.

Since no toxicological change was observed in this study, even after administration of 1,1,2,2-tetrafluoroethyl-2,2,2-trifluoroethyl ether at a dose of 1000mg/kg, the NOEL of 1,1,2,2-tetrafluoroethyl-2,2,2-trifluoroethyl ether was considered as 1000mg/kg for both male and female rats.