1,1,2,2,3,3,4,4-octafluoro-1,4-diiodobutane

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12-JAN-2006 to 06-FEB-2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

This GLP-compliant study was conducted according to a protocol equivalent to OECD guideline 412.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1,1,2,2,3,3,4,4-octafluoro-1,4-diiodobutane

EC Number:

206-788-4

EC Name:

1,1,2,2,3,3,4,4-octafluoro-1,4-diiodobutane

Cas Number:

375-50-8

Molecular formula:

C4F8I2

IUPAC Name:

1,1,2,2,3,3,4,4-octafluoro-1,4-diiodobutane

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): 1,4-Diiodoperfluorobutane

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: HanRcc:WIST(SPF)
- Source: RCC Ltd, Laboratory Animal Services / CH-4414 Füllinsdorf / SWITZERLAND
- Age at study initiation: 7 weeks for males; 10 weeks for females
- Weight at study initiation: the weight variation in animals entering the study did not exceed ± 7% of the mean weight of the corresponding sex.
- Fasting period before study: no data available
- Housing: animals of the same sex were housed in groups of up to 5 in Makrolon type-IV cages with wire mesh tops and standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz / SWITZERLAND).
- Diet: ad libitum (pelleted standard Kliba-Nafag 3433 rat maintenance diet, batch no. 63/05 and 76/05), except during the daily exposure periods and during 15 h prior to clinical laboratory investigations
- Water: ad libitum (community tap-water from Füllinsdorf, chlorinated to ca. 0.5 ppm), except during the daily exposure periods
- Acclimation period: 14 days, under laboratory conditions, after clinical health examination

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 to 25°C
- Humidity: 38 to 58%
- Air changes: 10 to 15 changes per hr
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From 18-JAN-2006 to 28-MAR-2006

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

other: compressed filtered air

Remarks on MMAD:

MMAD / GSD: Not applicable

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: inhalation by whole-body vapour exposure was performed in sealed chambers used for group isolation. These chambers were constructed of stainless steel. Glass doors were equipped with silicone rubber seals. The data of the exposure chambers are summarised in Table 1 below.
- Method of holding animals in test chamber: animals were accustomed to the whole-body inhalation chambers and the exposure conditions for 3 daily periods of ca. 1, 3 and 6 hours, respectively.
- Source and rate of air: the oxygen content was equal to 20.6%.
- Method of conditioning air: no data available
- System of generating particulates/aerosols: for groups 2 (0.2 ppm) and 3 (0.632 ppm), gas bags were prepared at concentrations of 215.42 ppm and 673.20 ppm, respectively. From the gas bags, the vapour was transferred with a gas pump to a pipe and was then diluted with 420 L/min filtered air to the inlet duct of the exposure chamber.
For group 4 (2 ppm), the test substance was pumped at a specific rate into a round bottomed glass flask to be vaporised. Compressed air (40 L/min) was supplied into the glass flasks and allowed the liquid to equilibrate to the temperature of the walls of the container. The vapour produced then was passed through a pipe and was then diluted with 380 L/min of filtered air to the inlet duct of the exposure chamber.
Gas bags and flasks containing the test substance were kept at room temperature.
- Temperature, humidity, pressure in air chamber: ca. 23.3°C; ca. 41%; negative pressure of ca. 2 mm H2O
- Air flow rate: no data available
- Air change rate: 10 to 15 air changes per hour
- Method of particle size determination: not applicable
- Treatment of exhaust air: no data available

TEST ATMOSPHERE
- Brief description of analytical method used: The test item vapour was measured by on-line gas chromatography (GC).
- Samples taken from breathing zone: yes

VEHICLE: not applicable

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The chamber concentration and stability of the test substance over the duration of ca. 6 hours was determined on at least two occasions prior to the start of the animal exposures. The test item was generated in the chambers at nominal concentrations of 0.2, 0.632 and 2 ppm and measured by on-line gas chromatography (GC).
During the treatment period, the concentration of the test substance in the chamber was determined daily, at least 5 times each hour of exposure. Start of recording was after the chamber equilibration time T90, and continued until pump off.
The method of analysis corresponded to the following:
- Column: DB-5 (No. 163755)
- Oven: 100°C, isothermal
- Runtime: 3 min
- Injector: 250°C
- Detector: 250°C
- Carrier gas: helium
- Sample loop: ca. 1700 µL
- Retention time: 0.90 ± 0.01 min

Duration of treatment / exposure:

4 consecutive weeks

Frequency of treatment:

6 hours/day and 5 days/week

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0.2, 0.632 and 2 ppm (equivalent to 0.0037, 0.0117, and 0.037 mg/L)
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0.199, 0.633, and 2.03 ppm (equivalent to 0.0037, 0.0117, and 0.0376 mg/L)
Basis:
analytical conc.

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: vapour concentrations were chosen on the basis of the results of the 5-day dose range finding inhalation toxicity study in the rat (see ESR entitled "I-(CF2)4-I - Repeat. dose tox.: 5-day inhal. - K1 2008RCC").
- Rationale for animal assignment (if not random): not applicable
- Rationale for selecting satellite groups, post-exposure recovery period in satellite groups: in order to assess the reversibility of any treatment-related findings satellite groups of animals were maintained for a subsequent period of 4 weeks without treatment.
- Section schedule rationale (if not random): not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: all animals were observed for mortality/morbidity twice daily, once prior to and once following exposure, during the treatment period and at least once daily on non-exposure days of treatment period and during the acclimatisation period and recovery periods.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: clinical signs were recorded on the first day of the acclimatisation period. During the exposure period, clinical signs were observed twice daily, prior to and following exposure. On non-exposure days of the treatment period and during the recovery period, clinical signs were observed once daily. During exposure, only grossly abnormal signs were observed, as the animals were housed in the whole-body inhalation chambers. Observations included, but were not limited to: changes in the skin and fur, eyes and mucous membranes and also respiratory, circulatory, autonomic and central nervous system, somatomotor activity and behaviour pattern.

BODY WEIGHT: Yes
- Time schedule for examinations: each animal was weighed on the first day of the acclimatisation period (used for randomised), and thereafter at least once weekly during the acclimatisation, treatment and recovery periods.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes; food consumption was measured per cage of 5 animals once weekly during the acclimatisation, treatment and recovery periods.

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: Yes
- Time schedule for examinations: water consumption was measured per cage of 5 animals at least twice weekly during the acclimatisation, treatment and recovery periods.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations, dose groups that were examined: ophthalmoscopic examinations was performed in all animals before treatment (during acclimatisation [24-JAN-2006]) and in all surviving animals in week 4 of the treatment period [24-FEB-2006] and in week 4 of the recovery period [24-MAR-2006]. Observations were performed after instillation of a mydriaticum, using a Heine Bifocal Type Miroflex ophthalmoscope (Eisenhut Vet. AG, CH-4123 Allschwil / SWITZERLAND).

HAEMATOLOGY: Yes
- Time schedule for collection of blood, how many animals: blood samples from the retro-orbital plexus were collected for haematology from all animals of all groups under isofluorane (Forene) anaesthesia before necropsy. The samples were collected early in the working day to reduce biological variation caused by circadian rhythm.
- Anaesthetic used for blood collection: Yes (isofluorane)
- Animals fasted: Yes; the animals were fasted in metabolism cages for ca. 15 hours before blood sampling, but water was provided ad libitum.
- Parameters checked: erythrocyte count (RBC), haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration, reticulocyte count, red blood cell morphology (red cell volume distribution width, haemoglobin concentration distribution width), reticulocyte maturity index, platelet count, total leukocyte count, differential leukocyte count, coagulation (prothrombin time, activated partial thromboplastin time (PTT))

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood, how many animals: blood samples from the retro-orbital plexus were collected for clinical biochemistry from all animals of all groups under isofluorane (Forene) anaesthesia before necropsy. The samples were collected early in the working day to reduce biological variation caused by circadian rhythm.
- Animals fasted: Yes; the animals were fasted in metabolism cages for ca. 15 hours before blood sampling, but water was provided ad libitum.
- Parameters checked: glucose, total cholesterol, creatinine (CREAT), phospholipids, urea, total bilirubin, triglycerides (TRIGLY), alanine aminotransferase, aspartate aminotransferase (ASAT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), glutamate dehydrogenase, creatinine kinase (CK), gamma-glutamyl-transferase, chloride, calcium, sodium, potassium, total protein, phosphorus, albumin, globulin, albumin/globulin ratio, alpha-glutathione S-transferase (alpha-GST)

URINALYSIS: Yes
- Time schedule for collection of urine: sampling during the 15-h period before blood sampling
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: volume, relative density, osmolality, colour, appearance, pH, protein, glucose, ketone, nitrite, bilirubin, erythrocytes, urobilinogen, leukocytes

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes; all animals of all groups were necropsied. The surviving animals were fasted overnight before necropsy, but water was provided. All animals were anaesthetised by an intraperitoneal injection of sodium pentobarbitone and sacrificed by exsanguination.
HISTOPATHOLOGY: Yes; samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4% formaldehyde solution unless otherwise stated. Lungs were instilled with the fixative at a hydrostatic pressure of 30 cm.
Adrenal glands
Aorta
Brain (cerebrum, cerebellum, brain stem)
Cecum
Colon
Duodenum
Epididymides (fixed in Bouin's solution)
Oesophagus
Extraorbital lacrimal glands
Eyes with optic nerves (fixed in Davidson's solution)
Femur, including joint
Harderian glands (fixed in Davidson's solution)
Heart
Ileum
Jejunum
Kidneys
Larynx (three transversal sections, at least levels II, III and IV)
Liver
Lungs
Lymph nodes - bronchial, mandibular, mesenteric
Mammary gland area
Nasal cavities (infused with formalin) with "paranasal sinuses" (Levels I to IV)
Nasopharyngeal duct and pharynx (1 longitudinal section)
Ovaries
Pancreas
Pituitary gland
Prostate
Rectum
Salivary glands - mandibular, sublingual
Sciatic nerve
Seminal vesicles (fixed in Bouin's solution)
Skeletal muscle (thigh region)
Skin
Spinal cord - cervical, midthoracic, lumbar
Spleen
Sternum with bone marrow
Stomach
Testes (fixed in Bouin's solution)
Thymus
Thyroid gland with parathyroid gland
Tongue
Trachea (one transversal section below larynx)
Tracheal bifurcation, carina and mainstem bronchi
Urinary bladder (infused with formalin)
Uterus with uterine cervix
Vagina
All gross lesions

Other examinations:

ORGAN WEIGHTS: the following organ weights were recorded at the scheduled dates of necropsy. Organ to body weight and organ to brain weight ratios were calculated. Paired organs were weighed together.
Adrenal glands
Brain
Epididymides
Heart
Kidneys
Liver
Lungs
Ovaries
Spleen
Testes
Thymus
Uterus

Statistics:

The Dunnett test (many-to-one t-test) based on a pooled variance estimate was used to analyse body weight, organ weights and food and water consumption and clinical laboratory parameters.
The Steel test (many-one rank test) was applied instead of the Dunnett test for those clinical laboratory parameters that could not be assumed to follow a normal distribution.
The Fisher's exact test was applied to macroscopic findings and ophthalmoscopy data where appropriate.
Group means and standard deviations were calculated for continuous data.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

There were no treatment-related clinical signs during the treatment period. Localized hair loss was observed in single females of groups 3 and 4. There were no clinical signs during the recovery period.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths during the course of this study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

see below in "Any other information on results incl. tables"

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

see below in "Any other information on results incl. tables"

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

There were no treatment related effects on water consumption and relative water consumption.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Animals treated with the test substance did not distinguish from controls.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

see below in "Details on results"

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

see below in "Details on results"

Urinalysis findings:

not specified

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

see below in "Details on results"

Gross pathological findings:

no effects observed

Description (incidence and severity):

see below in "Details on results"

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

BODY WEIGHT (Table 4 in attachment)
During the treatment period, body weight and body weight gain were marginally to slightly reduced in both genders of treated groups for the majority of determinations. The reduced body weight gain attained statistical significance in males of group 4 for all determinations.
During the recovery period, body weight was still slightly decreased in both genders of group 4 with a tendency towards marginally increased body weight gain at the end of the recovery period.

FOOD AND WATER CONSUMPTION (Table 4 in attachment)
During the treatment period, food consumption and relative food consumption were marginally to slightly reduced in both genders of treated groups for the majority of determinations. Statistical significance was not attained.
During the recovery period, food consumption and relative food consumption were still slightly reduced in both genders of group 4 except for relative food consumption in males.

HAEMATOLOGY (Table 5 in attachment)
After 4 weeks of treatment, activated partial thromboplastin time (PTT) significantly decreased in males of group 3.
After 4 weeks of recovery, males of group 4 showed a significant increase in absolute and relative count of large unstained cells (Luc); females of group 4 displayed a significant increase in red blood cell (RBC) count and a significant decrease in mean corpuscular haemoglobin (MCH).

CLINICAL CHEMISTRY (Table 5 in attachment)
After 4 weeks of treatment, creatinine significantly increased in males of group 3. Triglycerides increased in a significant manner in females of group 4. Aspartate aminotransferase activity significantly increased in males of groups 2 and 4. Inorganic phosphorus decreased significantly in males of groups 2 and 3. There was no significant change in plasma alpha-GST levels in all treated males and females when compared to control (group 1).
After 4 weeks of recovery, only females of group 4 displayed significant biochemistry changes when compared to control: increase in lactate dehydrogenase (LDH), creatinine kinase and phosphorus, and decrease in alkaline phosphatase and chloride. With the exception of LDH levels, all other clinical laboratory parameters in both genders of group 4 were considered to be similar to control. Plasma alpha-GST levels were not significantly changed in treated rats of both sexes when compared to control after 4 weeks of recovery.

ORGAN WEIGHTS (Table 2 in “Any other information on results incl. tables”)
After 4 weeks of treatment, lungs/body weight ratio significantly increased in males of group 3 when compared to control (group 1). Liver/body weight ratio increased in a significant manner in females of group 4. Kidneys/body weight ratio significantly increased in males of group 4.
After 4 weeks of recovery, relative kidney weight was still slightly but significantly increased in females of group 4 compared to control. All other organ weights in both genders of group 4 were considered to be similar to control.

GROSS PATHOLOGY (Table 3 in “Any other information on results incl. tables”)
After 4 weeks of treatment, thickened liver and foci in the thymus were observed in one male of group 1. In group 3, hardened liver, pelvic dilation and skin alopecia were observed in single females. In group 4, one female showed a watery cyst in the ovaries and four females showed skin alopecia.
After 4 weeks of recovery, pelvic dilation was observed in one female of group 4. Dilation of uterus was observed in one female of group 1 and in three females of group 4. There were no macroscopic findings in recovery males.
The macroscopic observations described above were not attributed to treatment.

HISTOPATHOLOGY: NON-NEOPLASTIC
The test item caused no morphological alterations in the organs and tissues examined. There were no histological alterations that could be attributed to treatment.

Effect levels

1

Key result

Dose descriptor:

NOAEC

Effect level:

>= 2 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: body weight; food consumption; haematology; clinical chemistry; organ weights

2

Key result

Dose descriptor:

NOAEC

Effect level:

>= 0.037 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: converted from 2 ppm

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Table 2: Summary of significant results on relative organ weight after 4 weeks of treatment and after 4 weeks of recovery (organ/body weight ratio (%))

	4-week treatment								4-week recovery
Gender	Males				Females				Males		Females
Group	1	2	3	4	1	2	3	4	1	4	1	4
Body weight (g)	332	314	320	304	215	205	209	204	356	342	233	224
Lungs	0.384	0.398	0.429*	0.413	0.491	0.507	0.506	0.503	0.349	0.369	0.468	0.471
Liver	2.63	2.66	2.66	2.90	2.72	2.72	2.74	3.07*	2.49	2.63	2.68	2.83
Kidneys	0.619	0.632	0.627	0.711*	0.681	0.688	0.691	0.724	0.551	0.635	0.624	0.728*

* = statistically significant

 

Table 3: Summary of significant macroscopical findings after 4 weeks of treatment and after 4 weeks of recovery

	4-week treatment								4-week recovery
Gender	Males				Females				Males		Females
Group	1	2	3	4	1	2	3	4	1	4	1	4
LIVER Thickened Hardened	1/5 -	0/5 -	0/5 -	0/5 -	- 0/5	- 0/5	- 1/5	- 0/5	- -	- -	- -	- -
THYMUS - Focus/foci	1/5	0/5	0/5	0/5	-	-	-	-	-	-	-	-
KIDNEYS - Pelvic dilation	-	-	-	-	0/5	0/5	1/5	0/5	-	-	0/5	1/5
OVARIES - Watery cyst	-	-	-	-	0/5	0/5	0/5	1/5	-	-	-	-
SKIN - Alopecia	-	-	-	-	0/5	0/5	2/5	4/5	-	-	-	-
UTERUS - Dilation	-	-	-	-	-	-	-	-	-	-	1/5	3/5

* = statistically significant

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, the no-observed-adverse-effect-concentration (NOAEC) was considered to be 2 ppm (0.037 mg/L).

Executive summary:

The purpose of this 28-day inhalation toxicity study was to evaluate the range of toxicity and to indicate potential target organs (if any) associated with the exposure of the test substance when administered to rats by whole-body inhalation for 6 hours/day for 5 days/week for 4 consecutive weeks. In order to assess the reversibility of any treatment-related findings, satellite groups of animals were maintained for a subsequent period of 4 weeks without treatment.

 

Groups of 5 male and 5 female Wistar rats were exposed to the test substance at target concentrations of 0.2, 0.632, 2 ppm for groups 2 to 4, respectively. The rats of group 1 served as air controls. Additional satellite groups of 5 males and 5 females were kept in groups 1 and 4 to investigate the potential for recovery. Mortality, clinical signs, body weight, food consumption, water consumption, ophthalmoscopic examinations, clinical laboratory investigations, organ weights, macroscopic and microscopic findings were recorded.

 

The mean analytical vapour concentrations of the test substance were 0.199, 0.633 and 2.03 ppm for groups 2 to 4. Temperature, relative humidity and oxygen concentration during exposure were considered to be suitable for this type of study.

 

There were neither unscheduled deaths nor treatment-related clinical signs during the course of the study. There were no treatment related effects on water consumption and relative water consumption. Ophthalmoscopic examinations did not distinguish treated groups from control.

 

There were neither gross lesions nor histological alterations that could be attributed to treatment of the test item at the tested concentrations. Under the conditions of this study, the test substance did not reveal signs of toxicity in any of the organs and tissues examined.

 

Nevertheless, in animals treated with 2 ppm potential treatment-related findings manifested after 4 weeks of treatment in liver and kidney by increased absolute and relative organ weights. The slight to moderate increase in liver weight may be considered to be reflected in females by a pronounced increase in triglycerides after 4 weeks of treatment and by a moderate to pronounced increase in lactate dehydrogenase after 4 weeks of recovery. These changes attained statistical significance.

In view of the adverse effects on the liver, mid-zonal steatosis combined with hepatocellular hypertrophy, observed in an oral study with an analogous substance, the effects of the test item on the liver in animals treated at 2 ppm may be considered as indicative of an initial adverse effect.

 

Treatment-related findings were slightly decreased body weight/body weight gain together with slightly reduced food consumption/relative food consumption in treated animals compared to control.   

 

The treatment-related and potentially treatment-related findings did only comprise slight changes/effects and, thus, were not considered to be of adverse nature. These changes included slight increase in the following organ weights: lung, liver, kidney in the high dose group, still present after the 4-week recovery period.

 

Based on the results of this study, the no-observed-adverse-effect-concentration (NOAEC) was considered to be 2 ppm (equivalent to 0.037 mg/L).