Octane, 1,1,1,2,2,3,3,4,4,5,5,6,6-tridecafluoro-

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1,1,1,2,2,3,3,4,4,5,5,6,6-tridecafluorooctane

EC Number:

700-684-7

Cas Number:

80793-17-5

Molecular formula:

C8H5F13

IUPAC Name:

1,1,1,2,2,3,3,4,4,5,5,6,6-tridecafluorooctane

Details on test material:

- Name of test material (as cited in study report): 1,1,1,2,2,3,3,4,4,5,5,6,6-Tridecafluorooctane
- Physical state: colourless, transparent liquid
- Batch number: #060616
- Purity: 99.9%
- Storage condition of test material: at room temperature
- Stability under storage conditions: stable

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan, Inc. (Hino Breeding Center; 735; Shimokomatsuki, Hino-cho, Gamo-gun, Shiga 529-1633, Japan)
- Age at study initiation: 5 weeks
- Weight at study initiation: 117.5-143.4 g (males), 109.2-128.5 g (females)
- Fasting period before study: no
- Housing: hanging stainless steel cages with wire-mesh floor at 1 animal/cage (165 Wx300 Dx150 H mm, TOKIWA KAGAKU KIKAI)
- Diet: MF pelleted diet (lot no. 060601 and 060802, Oriental Yeast), ad libitum
- Water: chlorinated water from the Hita City supply via automatic watering system with sipper tubes, ad libitum
- Acclimation period: 7 days (including 6 days quarantine)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.7-23.8
- Humidity (%): 48.5-65.1
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was accurately weighed and mixed with olive oil to prepare 2.0 w/v% formulation. The lower concentrations of 0.08 and 0.4 w/v% were diluted from 2.0 w/v%. These were prepared twice a week. The formulations were stored in a dark and cold place.

VEHICLE
- Justification for use and choice of vehicle: The test substance was not dissolved in purified water and olive oil, and the condition of the olive oil suspension was good. Therefore, olive oil (Fujimi Pharmaceutical) was used as the vehicle.
- Concentration in vehicle: 0.08, 0.4 and 2.0 w/v%
- Amount of vehicle (if gavage): 10 ml/kg bw
- Lot/batch no.: 038OHS

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples were taken from the middle layer of dose formulations immediately after preparation for the subject study. The samples were pre-treated and measured with gas chromatography (GC). The concentrations of 0.08, 0.4 and 2.0 w/v% formulations were confirmed to be within 100 ± 10% of each nominal concentration at the first preparation of the dosing period.

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

200 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: 7-day oral repeated dose toxicity study at 4 doses (25, 250, 500 and 1000 mg/kg bw/day)
- Post-exposure recovery period in satellite groups: 14 days

Positive control:

none

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: three times a day during the dosing period, once daily during the recovery period

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once weekly

BODY WEIGHT: Yes
- Time schedule for examinations: before dosing, and on days 1, 3, 8, 12, 17, 21, 26 and 28 during the dosing period; on days 1, 5, 10 and 14 during the recovery period

FOOD CONSUMPTION:
- Time schedule: before dosing, on days 1, 3, 8, 15, 22 and 28 during the dosing period; on days 1, 4, 8 and 14 during the recovery period

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at end of dosing period and at end of recovery period
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: all animals of the respective groups
- Parameters examined were red blood cell count (RBC), white blood cell count (WBC), hemoglobin concentration (Hb), hematocrit value (Ht), mean corposcular volume (MCV), mean corposcular hemoglobin (MCH), mean corposcular hemoglobin concentration (MCHC), platelet count, reticulocyte count, prothrombin time (PT), activated partial thromboplastin time (APTT), differentiation of leukocytes (neutrophils, eosinophils, basophils, lymphocytes, monocytes, large unstained cells (LUC).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at end of dosing period and at end of recovery period
- Animals fasted: Yes
- How many animals: all animals of the respective groups
- Parameters examined were aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), cholinesterase (ChE), γ-glutamyl transpeptidase (γ-GTP), total cholesterol (T-Cho), triglyceride (TG), glucose, total protein, albumin, A/G ratio, blood urea nitrogen (BUN), creatinine, total bilirubin (T-Bil), calcium, inorganic phosphorous (IP), sodium, potassium, chloride.

URINALYSIS: Yes
- Time schedule for collection of urine: at end of dosing period and at end of recovery period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined were urine volume, colour, turbidity, urine specific gravity, pH, protein, glucose, occult blood, urinary sediments.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: week 4
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity, grip strength, motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The weights of the following organs were measured in all animals: liver, heart, kidneys, testes, epididymides, ovaries, brain, spleen thymus, adrenals. The relative organ weight was also calculated based on the body weight at the time of necropsy.

HISTOPATHOLOGY: Yes
Organs and tissues examined were trachea, lungs, incisors, stomach, intestine (duodenum to rectum, with Peyer's patches), liver, heart, kidneys, urinary bladder, testes, epididymides, prostate, seminal vesicle, ovaries, uterus, vagina, brain (cerebrum, cerebellum and pons), spinal cord, sciatic nerve, bone marrow (femur), axillar and mesenteric lymph nodes, spleen, thymus, pituitary gland, thyroids (with parathyroids), adrenals, eye ball.

Other examinations:

none

Statistics:

Data regarding body weights (excluding those at the time of necropsy) food intakes, hematological examinations, blood chemical examinations, urine volume and specific gravity, organ weights, grip strength and locomotor activity count were analyzed using the Bartlett's test for homogeneity of variance. If the variances were homogeneous at a significance level of 5%, one way analysis of variance was performed. If there was a significant difference in this analysis, the difference between the vehicle control group and each of the treatment groups was analyzed by the Dunnett's test.
If the variances were not homogeneous, the Kruskal-Wallis's test was used. If there was a significant difference in this analysis, the difference between the vehicle control group and each of the treatment group was analyzed by the nonparametric Dunnett's test.
FOB numerical data was analyzed using the Kruskal-Wallis's test. If there was a significant difference in this analysis, the difference between the vehicle control group and each of the treatment groups was analyzed by the nonparametric Duenna's test.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

During Dosing Period
-Males: Salivation only after administration was observed in one animal of the 8 mg/kg group two animals of the 40 mg/kg group and six animals of the 200 mg/kg group.
-Females: Salivation only after administration was observed in two animals of the 40 mg/kg group and seven animals of the 200 mg/kg group.

During Recovery Period
-Males: Mottled teeth were observed in three animals of the 200 mg/kg group.
-Females: No abnormalities noted.

Although salivation was observed in males of all treatment groups and females of the 40 and 200 mg/kg treatment group, it was only observed transiently (only after dosing), and no other related effects were observed. Therefore, salivation was not considered to be toxicologically significant.

Mottled teeth in the males of the 200 mg/kg recovery group was considered to be related to enamel dysplasia, however; histopathological signs of enamel dysplasia (decreased iron pigments in the ameloblast, impaired iron-pigment secretion to the enamel and degeneration or necrosis of the enamel) were not observed. Therefore, the mottled teeth were considered to be a slight and reversible change and not treatment-related.

Mortality:

no mortality observed

Description (incidence):

No mortality was observed in any treatment group.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No abnormalities in body weight were observed in any treatment group.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

During Dosing Period
-Males: No abnormalities were observed in any treatment group.
-Females: Food consumption was decreased in the 40 and 200 mg/kg treatment groups on day 22, however; this was transient, not dose-related, and no changes in body weight were observed. Therefore, this change was not considered to be treatment-related.

During Recovery Period
-No abnormalities were observed in any treatment group.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no ophthalmological abnormalities observed.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At Termination of Dosing Period
-Males: No abnormalities were observed in any treatment group.
-Females: WBC was increased in the 8 mg/kg treatment group.

At Termination of Recovery Period
-Males: MCV was decreased in the 200 mg/kg treatment group.
-Females: Eosinophils were decreased in the 200 mg/kg treatment group.

These changes only occurred sporadically, and did not exhibit a dose-response relationship. Therefore, these changes are not considered to be treatment-related.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

At Termination of Dosing Period
-Males: Total cholesterol and albumin were decreased in the 200 mg/kg groups.
-Females: Cholinesterase was decreased in the 40 and 200 mg/kg groups.

At Termination of Recovery Period
-Males: No abnormalities were noted.
-Females: Total cholesterol was decreased in the 200 mg/kg group.

These changes are not considered to be treatment-related, because the changes are slight and fall within the historical background range for Crl:CD(SD) rats at this laboratory.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No abnormalities were detected in the urinalysis of any treatment group.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no behavioural abnormalities observed in any treatment group.

Immunological findings:

no effects observed

Description (incidence and severity):

There were no immunological abnormalities observed in any treatment group.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At Termination of Dosing Period
-Males: Relative liver and kidney weights were increased in the 200 mg/kg group.
-Females: Relative liver weight was increased in the 200 mg/kg group.

At Termination of Recovery Period
-Males: No abnormalities were noted.
-Females: Absolute and relative kidney weights and relative ovary weights were increased in the 200 mg/kg group.

Relative liver weight increase in both males and females was considered to be treatment-related, and is supported by histopathological findings (centrilobular hypertrophy of the hepatocytes).

Increased relative kidney weights without kidney lesion in males of the 200 mg/kg group were not considered to be treatment-related, since this was a slight change within the historical background range. Increased absolute and relative kidney weight and increased relative ovary weight in females of the 200 mg/kg group were not considered to be treatment-related, because these changes occurred sporadically with no dose-response relationship.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At Termination of Dosing Period
-Males: Enlargement of the liver was observed in two animals (nos. 22 and 25) of the 200 mg/kg group.
-Females: A decrease in size of the left lobe in the thyroid was observed in one animal (no. 43) of the 8 mg/kg group. Enlargement of the liver in two animals (nos. 53 and 55) and pelvic dilatation in the kidneys in one animal (no. 53) were observed in the 200 mg/kg group.

At Termination of Recovery Period
-Males: Mottled teeth were observed in three animals (nos. 26, 29 and 30) were observed in the 200 mg/kg group.
-Females: No abnormalities were noted.

Liver enlargement in both males and females of the 200 mg/kg treatment group was considered to be treatment-related, and is supported by organ weight increases and histopathological findings (centrilobular hypertrophy of the hepatocytes).

Pelvic dilatation and the decreased size of the left lobe of the thyroid observed in females in the 200 mg/kg treatment group were not considered to be treatment related, as these were single spontaneous occurrences.

Mottled teeth in the males of the 200 mg/kg recovery group was considered to be related to enamel dysplasia, however; histopathological signs of enamel dysplasia (decreased iron pigments in the ameloblast, impaired iron-pigment secretion to the enamel and degeneration or necrosis of the enamel) were not observed. Therefore, the mottled teeth were considered to be a slight and reversible change and not treatment-related.

Neuropathological findings:

no effects observed

Description (incidence and severity):

No neuropathological abnormalities were observed.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At Termination of Dosing Period
-Males: Basophilic tubules in the kidney in one animal (no. 4) and round cell infiltration in the prostate in one animal (no. 5) were observed in the vehicle control group. Centrilobular hypertrophy of the hepatocytes in the liver in four animals (nos. 21, 22, 24, 25), solitary cyst in the medulla in one animal (no. 23) and subcapsular solitary cyst in one animal (no. 21) in the kidney were observed in the 200 mg/kg group.
-Females: Mineralization in the corticomedullary junction of the kidney in two animals (nos. 31 and 32) and ultimobranchial rest in the thyroid in one animal (no. 33) were observed in the vehicle control group. Aplasia of the left lobe in the thyroid in one animal (no. 43) was observed in the 8 mg/kg group. Pelvic dilatation in the kidney was observed in one animal (no. 53) of the 200 mg/kg group.

At Termination of Recovery Period
-Males: No abnormalities were noted.
-Females: Solitary cyst in the medulla of the kidney was observed in one animal (no. 38) of the vehicle control group.

Centrilobular hypertrophy of the hepatocytes in the liver of four male animals in the 200 mg/kg treatment group was considered to be treatment-related, and is supported by increased liver organ weight and liver enlargement of both male and female animals of the 200 mg/kg treatment group.

The solitary cyst in the medulla in one male and subcapsular solitary cyst in the kidney of another male in the 200 mg/kg treatment group, the aplasia of the left lobe in the thyroid of one female in the 9 mg/kg treatment group, and pelvic dilatation in the kidney of one female in the 200 mg/kg treatment group were considered to be single spontaneous instances and not treatment-related.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

No neoplastic histopathological abnormalities were observed.

Other effects:

not examined

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: increased relative liver weights and enlargement of the liver in males and females and centrilobular hypertrophy of the hepatocytes and increased relative kidney weights in males of the 200 mg/kg bw/day groups

Target system / organ toxicity

Critical effects observed:

not specified

System:

hepatobiliary

Organ:

liver

Any other information on results incl. tables

Discussion of the results

No death occurred in the dosing and recovery periods.

Salivation was transiently observed in males of all treatment groups and females of the 40 and 200 mg/kg bw groups; however, it was not considered to be toxicologically significant since animals salivated just after dosing and there were no related changes to this.

Food consumption was decreased in females of the 40 and 200 mg/kg groups on day 22 of the dosing period. This was transient and not dose-related, and there were not body weight changes related to the decreased food intake on the same day. Therefore this change was not considered to be treatment-related.

In blood chemistry, decreased cholinesterase in females of the 40 and 200 mg/kg groups and decreased total cholesterol and albumin in males of the 200 mg/kg group, were noted, and these were not considered related to the test article, since they were slight changes within the background value of the laboratory and lesion of the liver in males was more severe than that in females.

In organ weights, relative liver weights were increased in males and females of the 200 mg/kg bw/day groups. In necropsy, enlargement of the liver was observed in males and females of the 200 mg/kg bw/day groups. In histopathological examinations, centrilobular hypertrophy of the hepatocytes was observed in males of the 200 mg/kg bw/day group.

Increased relative kidney weights without kidney lesion in males of the 200 mg/kg group were considered to be treatment related. Pelvic dilatation in female and solitary cyst in the medullar and subcapsular solitary cyst in males were noted in the kidney in the 200 mg/kg groups, and they were single occurrences and observed spontaneously.

No abnormalities were noted in the FOB, body weights and urinalysis.

In the recovery groups, mottled teeth were observed in males of the 200 mg/kg group, This was considered related to dysplasia of the enamel. In an animal which has brown teeth like a rat, decreased iron pigments in the ameloblast, impaired iron-pigment secretion to the enamel and degeneration or necrosis of the enamel are generally observed by fluoride ingestion (Dokusci Byori Soshiki Gaku, 2000). In this study, these signs were not observed in histopathological examinations at the end of the dosing and recovery periods. Therefore, the mottled teeth were considered to be slight and reversible changes, and considered disappeared with the growing of teeth because of no histopathological changes. Decreased MCV in males, decreased eosinophils, decreased total cholesterol, increased absolute and relative kidney weights and increased relative ovary weights were considered to be not treatment-related since they were slight changes within the background values in our laboratory. No abnormalities were noted in body weights, food consumption, FOB, urinalysis and histopathological examinations.

Applicant's summary and conclusion

Conclusions:

The effects of AC-6000 were observed in the liver, however; these changes were reversible. The NOEL for AC-6000 was considered to be 40 mg/kg/day under the conditions tested due to increased relative liver weights and enlargement of the liver in males and females and centrilobular hypertrophy of the hepatocytes in males of the 200 mg/kg groups.

Executive summary:

In a 28-day repeated oral dose toxicity GLP study according to OECD guideline 407 and EU method B.7, groups of five male and five female Crl: CD(SD) rats were dosed with AC-6000. The high dose was set at 200 mg/kg bw/day, and altogether 3 doses including 40 and 8 mg/kg bw/day were employed. Recovery groups were also set for the 200 mg/kg bw/day and vehicle control groups.

 

No death occurred in the dosing and recovery periods. In organ weights, relative liver weights were increased in males and females of the 200 mg/kg bw/day groups. In necropsy, enlargement of the liver was observed in males and females of the 200 mg/kg bw/day groups. In males of the 200 mg/kg bw/day group increased relative kidney weights were observed. In histopathological examinations, centrilobular hypertrophy of the hepatocytes was observed in males of the 200 mg/kg bw/day group. No treatment-related/ toxicologically significant abnormalities were noted in clinical signs, FOB, body weights, food consumption, hematology, blood chemistry and urinalysis.

 

In the recovery groups, mottled teeth were observed in males of the 200 mg/kg bw/day group. No abnormalities were noted in FOB, body weights, food consumption, hematology, blood chemistry, urinalysis and histopathology.

 

In conclusion, the effects of AC-6000 were observed in the liver. Even though these effects are reversible, the NOAEL was considered to be 40 mg/kg bw/day due to increased relative liver weights and enlargement of the liver in males and females and centrilobular hypertrophy of the hepatocytes in males of the 200 mg/kg bw/day groups.