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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 April - 20 August 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl 2-chloroprop-2-enoate
EC Number:
801-260-5
Cas Number:
96383-55-0
Molecular formula:
C11H6ClF13O2
IUPAC Name:
3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl 2-chloroprop-2-enoate
Details on test material:
- Name of test material (as cited in study report): C6SFCLA
- Physical state: colourless liquid
- Lot/batch No.: 110907
- Stability under test conditions: stable during the experimental period (measured by infrared absorption spectrometry)
- Storage condition of test material: stored in a cold place (2-6.9°C), dark and in a tight container

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc. (Hino Breeding Center)
- Age at study initiation: 6 weeks
- Weight at study initiation (day 1): 202.4 - 228.0 g (males), 131.8 - 167.2 g (females)
- Fasting period before study: 18-23 h before scheduled necropsy
- Housing: single housing in stainless-steel cages
- Diet: Autoclave-sterilized pellet diet (CRF-1, Oriental Yeast Co, Ltd.)
- Water: Well water admixed with NaClO (free residual chlorine level: about 2 ppm), ad libitum (except during motor activity measurements)
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.5 - 24.8
- Humidity (%): 39.5 - 65.0
- Air changes (per hr): 10-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 5 April 2012 To: 11 May 2012 (dosing group), 25 May 2012 (recovery group)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: olive oil containing cremophor (10%)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations of 2 and 8 mg/mL were prepared every 7 days. Test substance was weighed and transferred to a beaker. About 50% of the total volume of the vehicle was added to the beaker and the dosing formulation was stirred with a magnetic stirrer. After stirring, the dosing formulation was transferred to the mesuring cylinder. The beaker and stirring bar were washed with the vehicle, and the washing was transferred tot he measuring cylinder. The final volume was adjusted by adding a proper quantity of the vehicle to the required concentrations of 2 and 8 mg/mL.

VEHICLE
- Concentration in vehicle: 2 and 8 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity and stability of the test substance preparations were confirmed with an analytical GC method according to an internal protocol. The relative standard deviation (RSD) of analytical values (concentration) was within the criteria (actual RSD: 0.2% to 0.3%, criteria: not more than 10%), and measured concentration (mean) was within the criteria (actual concentration 98.7% to 100.5% of nominal concentrations, criteria: nominal concentration ± 10%)
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
2, 10, 40 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
Main study: 10 (control, mid and high dose), 5 (low dose)
Recovery period: 5 (control, mid and high dose), 0 (low dose) animals from the main study each
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In two 7-day screening studies animals were dosed with 50, 250, and 100 mg/kg bw/day (5 animals per sex and dose) or 1, 5, and 20 mg/kg bw/day (3 male animals per dose). Death and moribund animals (both sexes) were found in the 250 and 1000 mg/kg bw dose group. High AST and liver and kidney weights were observed in both sexes of the 50 mg/kg bw/day group. 1 male of this group showed discoloration of the heart, dark reddish macule of the lung, low values of body weight, food consumption and reticulocytes, and high values of platelets and monocytes. Effects on body weight and food consumption was also observed in 1 female of the 50 mg/kg bw dose group. Discoloration of the heart was also observed in dead animals of the 250 and 1000 mg/kg bw/day groups. No treatment-related changes were observed in the 1, 5, and 20 mg/kg bw/day groups. Thus 40 mg/kg bw/day was set as the high dose level in the main study expecting that there would be no death or some toxicity change.

- Post-exposure recovery period in satellite groups: 14 days

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (dosing period), once daily (recovery period)
- Cage side observations checked: mortality and general signs of toxicity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before start of dosing and once a week during the dosing and recovery periods.

BODY WEIGHT: Yes
- Time schedule for examinations: Days 1, 8, 15, 22 and 28 (during dosing period), Days 29, 36, and 42 during recovery period

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

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: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: Days 29 and 43 (dosing and recovery period, respectively)
- Anaesthetic used for blood collection: Yes (sodium pentobarbital, 30 mg/kg bw)
- Animals fasted: Yes (18.5-23 h)
- How many animals: all animals
- Parameters examined: leukocytes, erythrocytes, hemoglobin, hematocrit, MCV, MCH, MCHC, reticulocyte ratio and count, platelets, Differential leukocyte ratio and count, prothrombin time (PT), Activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Days 29 and 43 (dosing and recovery period, respectively)
- Animals fasted: Yes (18.5-23 h)
- How many animals: all animals
- Parameters examined: total protein, albumin and albumin/globulin ratio, AST, ALT, gamma-Glutamyltranspeptidase, alkaline phosphatase, total bilirubin, total bile acids, total cholesterol, triglycerides, glucose, blood urea nitrogen, creatinine, calcium, inorganic phosphorus, sodium, potassium, chloride

URINALYSIS: Yes
- Time schedule for collection of urine: Day 27
- Metabolism cages used for collection of urine: No
- Animals fasted: Yes (only access to water)
- Parameters examined: pH, Protein, Glucose, ketone body, occult blood

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule: all animals, once in week 4 of the dosing period
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity / grip strength/ motor activity
Sacrifice and pathology:
SACRIFICE
- All animals were euthanized after blood sampling by exsanguination and were subjected to necropsy

GROSS NECROPSY
Necropsy of all males and females of the control and 40 mg/kg bw/day group was performed.
The following organs/tissues were collected and examined:
Brain (cerebrum, cerebellum and pons), heart, ovaries (females), liver, uterus (females), kidneys, adrenal glands, prostate, seminal vesicles with coagulating glands as a whole (males), thymus, urinary bladder, thyroid (incl. parathyroid), spleen, mammary glands, all gross lesions, eye, lymphnodes (submaxillary), submaxillary gland, parathyroid, stomach, duodenum, jejunum, ileum (including Peyer´s patches), cecum, colon, rectum. mesenteric lymph node, pancreas, testis (males), epididymides, vagina, femur, sternum, bone marrow (femur and sternum), M. biceps femoris, peripheral nerve (e.g. sciatic nerve), pituitary gland, spinal cord, trachea, lungs (including bronchus)


HISTOPATHOLOGY / ORGAN WEIGHTS
All full histopathology was carried out on the organs and tissues of all males and females of the control and 40 mg/kg bw/day group. Examination of heart, lung, and liver was performed in all animals of all dose groups (end of dosing period) and in all animals of the control and high dose group (end of recovery period)

The following tissues were prepared for microscopic examination:
With weight determination:
brain (cerebrum, cerebellum and pons), heart, ovaries (females), liver, uterus (females), kidneys, adrenal glands, prostate, seminal vesicles with coagulating glands as a whole (males), thymus, urinary bladder, thyroid (incl. parathyroid), spleen, mammary glands, all gross lesions

Without weight determination:
Eye, lymphnodes (submaxillary), submaxillary gland, parathyroid, stomach, duodenum, jejunum, ileum (including Peyer´s patches), cecum, colon, rectum. mesenteric lymph node, pancreas, testis (males), epididymides, vagina, femur, sternum, bone marrow (femur and sternum), M. biceps femoris, peripheral nerve (e.g. sciatic nerve), pituitary gland, spinal cord, trachea, lungs (including bronchus)
Statistics:
Statistical analysis was performed with a computer system (MiTOX-PPL, Mitsui Zosen Systems Research Inc.). For the numeral data, Bartlett´s test was performed to compare variances among groups (significance level: 5%). When variance of data was homogenous, Dunnett´s multiple comparison test was performed to compare with the control group. When variance of data was heterogenous, Steel´s multiple comparison test was performed to compare with the control group. For results of urinalysis with reagent strip, Steel´s multiple comparison test was performed after the grades were converted into numeric values. Mann-Whitney´s U test was used for the histopathological findings after the grades were converted into numeric values. In all cases, levels of p<0.01 (1%) and p<0.05 (5%) were considered to be significant and two-tailed test was used.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
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:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
40 mg/kg bw: high values of monocytes in both sexes at the end of the dosing period (non-adverse). Several non-adverse and not treatment-related effects were observed in all dose groups.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
40 mg/kg bw: high values of AST (both sexes), ALT (males), and inorganic phosphorus (males) and low levels of glucose (males) (non-adverse). Several non-adverse and not treatment-related effects were observed in the 10 and 40 mg/kg bw/day dose groups.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
40 mg/kg bw: high values or tendencies of absolute and relative heart (both sexes) and kidney weights (females) after dosing and recovery period (adverse)
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
40 mg/kg bw: degeneration/necrosis (myocardial cell) and inflammatory cellular infiltration in the myocardium(both sexes, adverse) after dosing period, fibrosis in the myocardium after the recovery period (both sexes, adverse), effects on lung and liver
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No treatment-related changes were observed.

BODY WEIGHT AND WEIGHT GAIN
No treatment-related changes were observed.

FOOD CONSUMPTION
No treatment-related changes were observed.

HAEMATOLOGY
40 mg/kg bw/day: high values of monocytes in both sexes at the end of the dosing period. High value of platelets and low values of basophils and the ratio were noted (males, end of recovery period)
10 mg/kg bw/day: high value of platelets (males, end of recovery period), prolongations of PT and PTT (females) (within mean±2SD of historical control data)
2 mg/kg bw/day: high values of eosinophil ratio males at the end of the dosing period (no dose-response correlation).

CLINICAL CHEMISTRY
40 mg/kg bw: High values of AST (both sexes), ALT (males), and inorganic phosphorus (males) and low levels of glucose (males) (end of dosing period, non-adverse). High values of ALP and sodium were noted in the males (within mean±2SD of historical control data). High sodium (males), potassium, and chlorine (males) values at the end of the recovery period (within mean±2SD of historical control data).
10 mg/kg bw/day: high potassium values (within mean±2SD of historical control data), high A/G ratio (males, no dose-response correlation)

URINALYSIS
No treatment-related changes were observed.

NEUROBEHAVIOUR
No treatment-related changes were observed.

ORGAN WEIGHTS
40 mg/kg bw: high values or tendencies of absolute and relative heart (both sexes) and kidney weights (females) after dosing and recovery period (adverse)

GROSS PATHOLOGY
No treatment-related changes were observed.

HISTOPATHOLOGY: NON-NEOPLASTIC
40 mg/kg bw:
Heart:degeneration/necrosis (myocardial cell) and inflammatory cellular infiltration in the myocardium(5/5 males, 5/5 females) after dosing period, fibrosis in the myocardium after the recovery period (1/5 males, 1/5 females)
Lung: degeneration/necrosis (myocardial cell around the vein) and inflammatory cellular infiltration in the myocardium around the vein (2/5 males, 2/5 females)
Liver: mild hypertrophy of the centrilobular hepatocytes (2/5 males, 3/5 females)
10 mg/kg bw/day: mild and focal inflammatory cellular infiltration in the myocardium (2/5 males, 1/5 females) at the end of the dosing period and in 1/5 males after the recovery period
Control group: mild and focal inflammatory cellular infiltration in the myocardium (3/5 males) at the end of the recovery group

HISTOPATHOLOGY: NEOPLASTIC
No findings were reported.

HISTORICAL CONTROL DATA
Historical control data were provided and used for discussion for endpoints where data have been observed that were outside the range of the actual control group.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Degeneration/necrosis in the myocardial cell and in myocardial cells around the vein at 40 mg/kg bw/day.
Dose descriptor:
LOAEL
Effect level:
40 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Degeneration/necrosis in the myocardial cell and in myocardial cells around the vein at 40 mg/kg bw/day.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1: Histopathological findings in males after the dosing period.

Organ and findings

 

Dose groups

 

 

 

 

 

Control

2 mg/kg bw/day

10 mg/kg bw/day

40 mg/kg bw/day

Liver

Hypertrophy, hepatocyte, centrilobular

0/5

0/5

0/5

2/5 (mild)

 

Cellular infiltration, inflammatory, focal

2/5 (mild)

0/5

2/5 (mild)

3/5 (mild)

Lung

Degeneration/necrosis, myocardial cell, vein, focal

0/5

0/5

0/5

2/5 (mild)

 

Cellular infiltration, vein, myocardium, inflammatory, focal

0/5

0/5

0/5

2/5 (mild)

Heart

Degeneration/necrosis, myocardial cell, diffuse

0/5

0/5

0/5

5/5 (mild)**

 

Cellular infiltration, myocardium, inflammatory, focal

0/5

0/5

2/5 (mild)

0/5

 

Cellular infiltration, myocardium, inflammatory, diffuse

0/5

0/5

0/5

5/5 (mild)**

 

Fibrosis, myocardium, diffuse

0/5

0/5

0/5

1/5 (mild)

**: p<0.01 (significantly different from control)


Table 2: Histopathological findings in females after the dosing period. 

Organ and findings

 

Dose groups

 

 

 

 

 

Control

2 mg/kg bw/day

10 mg/kg bw/day

40 mg/kg bw/day

Liver

Vacuolation, hepatocyte, periportal

0/5

1/5 (mild)

0/5

0/5

 

Hypertrophy, hepatocyte, centrilobular

0/5

0/5

0/5

3/5 (mild)

 

Cellular infiltration, inflammatory, focal

1/5 (mild)

0/5

0/5

0/5

Lung

Degeneration/necrosis, myocardial cell, vein, focal

0/5

0/5

0/5

2/5 (mild)

 

Cellular infiltration, vein, myocardium, inflammatory, focal

0/5

0/5

0/5

2/5 (mild)

 

Cellular infiltration, inflammatory, focal

0/5

1/5 (mild)

0/5

0/5

Heart

Degeneration/necrosis, myocardial cell, diffuse

0/5

0/5

0/5

5/5 (mild)**

 

Cellular infiltration, myocardium, inflammatory, focal

0/5

0/5

1/5 (mild)

0/5

 

Cellular infiltration, myocardium, inflammatory, diffuse

0/5

0/5

0/5

5/5 (mild)**

 

Fibrosis, myocardium, diffuse

0/5

0/5

0/5

1/5 (mild)

**: p<0.01 (significantly different from control)


 

Table 3: Histopathological findings in males and females after the recovery period.

Organ and findings

 

Dose groups

 

 

 

 

Control

10 mg/kg bw/day

40 mg/kg bw/day

Liver

Necrosis, hepatocyte, focal

Males: 0/5

Males: 0/5

Males: 1/5 (mild)

 

Cellular infiltration, inflammatory, focal

Males: 1/5 (mild) Females: 0/5

Males: 1/5 (mild) Females: 1/5 (mild)

Males: 2/5 (mild) Females: 1/5 (mild)

Lung

 

0/5

0/5

0/5

Heart

Cellular infiltration, myocardium, inflammatory, focal

Males: 3/5 (mild)

Males: 1/5 (mild)

Males: 1/5 (mild)

 

Fibrosis, myocardium, diffuse

Males: 0/5

Females: 0/5

Males: 0/5

Females: 0/5

Males: 4/5 (mild)*

Females: 3/5

*: p<0.05 (significantly different from control)


 

Table 4: Selected organ weights of males and females after the dosing and recovery period.

Absolute organ weight (relative organ weight)

 

Dose groups

 

 

 

 

 

Control

2 mg/kg bw/day

10 mg/kg bw/day

40 mg/kg bw/day

after dosing period

males

 

 

 

 

 

Heart

1.43±0.15 (0.38±0.03)

1.36±0.21 (0.37±0.03)

1.39±0.09 (0.37±0.03)

1.61±0.06 (0.42±0.01)

 

Liver

12.01±0.8 (3.21±0.09)

11.83±1.39 (3.23±0.17)

12.26±0.87 (3.25±0.20)

13.38±1.49 (3.49±0.21)

 

Females

 

 

 

 

 

Heart

0.81±0.05 (0.38±0.03)

0.84±0.09 (0.41±0.03)

0.87±0.10 (0.40±0.04)

0.99±0.08** (0.51±0.04)**

 

Liver

6.54±0.31 (3.11±0.14)

6.48±0.82 (3.16±0.21)

7.25±0.64 (3.33±0.22)

7.31±1.07 (3.72±0.29)**

after recovery period

males

 

 

 

 

 

Heart

1.52±0.15 (0.36±0.02)

-

1.68±0.15 (0.38±0.05)

1.57±0.10 (0.37±0.02)

 

Liver

13.25±1.34 (3.14±0.20)

-

12.75±1.13 (2.87±0.17)

12.88±1.02 (2.98±0.20)

 

Females

 

-

 

 

 

Heart

0.88±0.04 (0.38±0.03)

-

0.85±0.11 (0.37±0.05)

1.03±0.10 (0.44±0.05)

 

Liver

6.71±0.57 (2.91±0.06)

-

6.62±1.21 (2.88±0.12)

7.35±0.20* (3.14±0.16)*

*: p<0.05 (significantly different from control)

**: p<0.01 (significantly different from control)

 

Applicant's summary and conclusion

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