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Description of key information

Key value for chemical safety assessment

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Forty young adult Wistar rats (Crl: (WI) WU®BR) 33-37 days at delivery were supplied. At the start of the study, the animals were about 8 weeks old. After delivery, the animals were carefully inspected by the veterinarian or his designate. The study consisted of 5 males and 5 females in each treatment and control group.

Prior to the treatment period, the rats were acclimatized for about 3 weeks to the housing conditions in the animal room. During this time they were trained to become accustomed to the nose-only inhalation tubes. During acclimatization, animals were observed once daily. Body weight was measured at the end of the acclimatization.

Each animal in the study was assigned a unique six digit individual number by a numbered plate on the cage. The number assigned was GG00NN, where GG denotes the two digit (01-99) group number, 00 is a common separator and NN are the (01-99) consecutive animal number. In addition, the animals were tattooed on the ear with their identification number.

Animals were housed in Makrolon® (polycarbonate) type III (37.5 x 21.5 x 19cm) cages in the animal house. Absorbent softwood was used as bedding material in the cages (Softwood, altromin ¾, Altromin International, Lage, Germany). The cages were changed twice weekly or more often when necessary. Food was offered fresh daily. The diet used was (1324N specially prepared), supplied by Altromin International, Lage, Germany. Drinking water from the Hanover city water supplier was offered fresh daily in Makrolon® bottles, ad libitum. Food and water were not offered during the exposure period.
Temperature and relative humidity were recorded continuously. The values in the animal room were set at 22 ± 2°C for temperature and 30-70% for relative humidity. The animal room lighting was on a 12 hour light/dark cycle controlled by an automatic timing device. Air flow rate was adjusted to 12 – 15 changes per hour.
Route of administration:
inhalation: vapour
Type of inhalation exposure:
nose only
Vehicle:
air
Details on inhalation exposure:
Generation of the test atmosphere
The exposure atmosphere was generated by injecting a defined mass flow of PFBE into a constant air flow. The test substance was injected into a slightly heated flask using a pump. The resulting vapour was then withdrawn by air (dilution airflow approximately 15l/min via the animal house air conditioning system). The air flow rates were controlled by mass flow meters. A long tube to the inhalation unit ensured homogenous mixing of the test atmosphere and temperature equalization with that of the inhalation unit.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The actual PFBE concentration was measured using a flame ionization detector (FID). The FID was calibrated against n-butane so that changes of response could be easily detected using routine control of the FID with n-butane. Calculation of the PFBE concentration from the n-butane base signals were performed using PFBE samples with subsequent (twice weekly) chemical analysis by gas chromatography (GC). This calibration was done for three PFBE concentrations in the range 400, 2000 and 10000 ppmv. The routine measurements of the test atmosphere was achieved by connecting the inhalation units to the FID at 20 minute intervals.
Duration of treatment / exposure:
28 consecutive days
Frequency of treatment:
6 hours / day
Remarks:
Doses / Concentrations:
401, 2069, 9879 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
Daily observations

All animals were observed especially for the following symptoms daily:
General condition
Visible mucous membranes
Behaviour and locomotor activity
Central nervous symptoms

Body weight, Food and Water consumption

Individual body weights, food and water consumption were recorded weekly.

Haematology and Clinical Chemistry

Haematological, clinico-chemical and urine analyses were performed before final sacrifice. Blood samples were collected from each animal prior to necropsy following an overnight fasting period. Blood was obtained by puncture of the retrobulbar venous plexus under light halothane anaesthesia and collected in tubes coated with K2-EDTA (haematology) and heparin lithium salt (clinical chemistry). For determination of prothrombin time and partial thromboplastin time, blood was drawn at necropsy from the vena cava using a syringe with 10% (v/v) of 0.11 mmol/l citric acid sodium salt as the anticoagulant. The samples were analysed in a randomised sequence.
Sacrifice and pathology:
Each animal was subjected to a full necropsy. During the study period, any animal judged to be moribund by the veterinarian or his designate and all animals at terminal sacrifice were killed with an overdose of CO2, exsanguinated and necropsied immediately. The physical condition of the animal
prior to euthanasia and the examination of the internal organs were described in detail on individual autopsy protocol sheets. Qualified prosectors
under the supervision of a veterinarian / pathologist were available seven days a week, so that the necropsies were performed on a timely basis. In all cases, dead animals were identified by reference to each animal’s identification number.

The following organs and tissues were collected from each rat and fixed in 10% neutral buffered formalin (lungs were inflated at approximately 20 cm water pressure with formalin):
Brain, pituitary, tongue, eyes (optic nerve), extra orbital lacrimal glands, Harderian glands, nasal and paranasal cavities, larynx, pharynx, trachea,
thyroid, parathyroids, lungs, thymus, heart, aorta, lung associated lymph nodes, salivary glands, mandibular lymph nodes, liver, pancreas, spleen,
kidneys, adrenals, oesophagus, forestomach, glandular stomach, duodenum, jejunum, ileum, caecum, colon, rectum, mesenterium and lymph nodes, urinary bladder, testes, epididymides, prostate, seminal vesicles, ovaries, uterus, vagina/cervix skeletal muscle, femur including joint, spinal cord,
skin, peripheral nerve (N. ischiadicus), sternum and bone marrow.
In addition to the terminal body weight, the lung, liver, kidneys, adrenals, testes were weighed (paired organs) separately). Relative organ weight data were also computed.

Histopathology

Tissues for histological examination were embedded in paraffin, sectioned at 4 μm and stained with haematoxylin and eosin.

Complete histopathological examination (according to Bahnemann et al., 1995) were performed in all animals of the control and high exposure
groups.

The slides were examined by light microscopy and the observations were recorded with an on-line computer programme.
Statistics:
Statistical tests on the comparison of treatment groups were performed at the level of α = 0.05. Bodyweight, food and water consumption, and haematology data were analysed using analysis of variance as a global test. Pair-wise comparison of the means of treatment groups with the means of the clean air control group were performed using Dunnett’s modification of the t-test. Thus the experiment-wise error rate was controlled in this multiple testing procedure. For comparisons between two treatment groups, the two-sided t-test at a level of α = 0.05 was used. If applicable other statistical tests were used.
Statistical evaluation for histopathological findings: significance of differences of the frequencies were evaluated as pair-wise comparison between clean air control and treatment groups using Fisher’s exact test. These tests were performed at the local significance level of α = 0.05.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
In males, decreased mean partial thromboplastin time was found in the high dose group (10,000 ppm PFBE p<0.01)
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In males, mildly increased mean cholinesterase and urea were found in the medium dose group (2000 ppmv PFBE, p ,0.05). No clear cut dose dependence was present. In females mean inorganic phosphate was observed in the high dose group (10000 ppmv, p<0.05).
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see below
Dose descriptor:
NOAEC
Effect level:
2 000 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
ophthalmological examination
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Remarks on result:
other: histopathology only conducted on the liver in the 400 and 2000 ppm groups
Critical effects observed:
not specified

Histopathology

Histopathological changes were observed in the larynx, the lungs and the liver that were probably treatment-related. With the exception of the liver, which was histopathologically examined in all groups, all other organs were examined only in the control and high dose groups (10000 ppmv PFBE).

Larynx

Very slight (minimal) focal to multifocal submucosal inflammatory cell infiltration occurred in 1/5 males and 2/5 females of the high dose groups (10000 ppmv PFBE) and was absent in the controls. The effects were probaly treatment-related.

Lungs

Very slight (minimal) focal alveolar histiocytosis was seen in 1/5 rats each of the high dose (10000ppmv PFBE) and very slight focal interstitial inflammatory cell infiltration was seen in another high dose female. These effects were possibly treatment-related reactive changes since they were not observed in controls. However, like the other observed lung lesions (pleural fibrosis, osseous metaplasia), these findings could have occurred incidentally.

Liver

Very slight (minimal) to slight (multifocal) fatty hepatocellular cytoplasmic vacuolation was seen in 4/5 male rats of the high dose group (10000ppmv PFBE). In addition, one male of this group showed a slight focal (lobular) necrosis accompanied by a slight inflammatory cell infiltration. These degenerative lesions are considered to be treatment-related. For hepatocellular cytoplasmic vacuolation, the difference to the control males was statistically significant.

Hepatic microgranulomas were seen in the majority of both the control and PFBE-exposed rats, but were not treatment-related.

Other organs

A large variety of other findings in other organs were diagnosed which did not show significant differences between treatment and control groups or which are considered not unusual for rats of this strain and age.

Conclusions:
Based on the results of this study, the No Observed Adverse Effect Concentration (NOAEC) of PFBE (3,3,4,4,5,5,6,6,6-nonafluorohexene) under the experimental conditions of the study in the Wistar (WU) rat was judged to be 2000 ppmv.
Executive summary:

Five male and five female Wistar (WU) rats (Crl : (WI) WU®BR) per group, about 8 weeks old at study start, were exposed in nose-only exposure units to clean air (control) and to target concentrations of 400 ppmv (low dose), 2000 ppmv (medium dose) and 10000 ppmv (high dose) PFBE (3,3,4,4,5,5,6,6,6-nonafluorohexene). The duration of the exposure was 6 hours per day for 28 consecutive days. The mean actual concentrations measured during the exposure periods 401±19 ppmv, 2069±179 ppmv and 9879±391 ppmv PFBE for the low, medium and high dose groups respectively.

The exposure was well tolerated by all rats with no signs of intoxication. No treartment-related mortality occurred. No influence on bodyweight, food or water consumption was observed.

No findings that could be related to treatment were observed at final sacrifice. No influence on organ weights was observed.

A decrease in mean partial thromboplastin time was seen in the high dose group (10000 ppmv PFBE). Small, although not clearly dose-dependent, increases in mean cholinesterase and urea were also reported. These effects were attributed to an effect on the liver.

Probable test substance-related histopathological changes were observed in the larynx, the lungs and the liver. With the exception of the liver, which was histopathologically examined in all groups, all other organs were examined only in the control and high dose groups (10000 ppmv PFBE).

Larynx

Very slight (minimal) focal to multifocal submucosal inflammatory cell infiltration occurred in 1/5 males and 2/5 females of the high dose groups (10000 ppmv PFBE).

Lungs

Very slight (minimal) focal alveolar histiocytosis was seen in 1/5 rats each of the high dose (10000ppmv PFBE) and very slight focal interstitial inflammatory cell infiltration was seen in another high dose female. These effects were possibly treatment-related reactive changes since they were not observed in controls. However, like the other observed lung lesions (pleural fibrosis, osseous metaplasia) these findings could have occurred incidentally.

Liver

Very slight (minimal) to slight (multifocal) fatty hepatocellular cytoplasmic vacuolation was diagnosed exclusively in 4/5 male rats of the high dose group (10000ppmv PFBE). In addition, one male of this group showed a slight focal (lobular) necrosis accompanied by a slight inflammatory cell infiltration. These degenerative lesions are considered to be treatment-related. For hepatocellular cytoplasmic vacuolation, the difference to the control males was statistically significant.

Based on the results of this study, the No Observed Adverse Effect Concentration (NOAEC) of PFBE

(3,3,4,4,5,5,6,6,6-nonafluorohexene) under the experimental conditions of the study in the Wistar (WU) rat is judged to be 2000 ppmv.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
20 123 mg/m³
Study duration:
subacute
Species:
rat

Additional information

Two studies are available for repeated exposure by inhalation to PFBE (3,3,4,4,5,5,6,6,6-nonafluorohexene).

1) In a 28-day study conducted to OECD Test Guideline 412, five male and five female Wistar (WU) rats (Crl : (WI) WU®BR) per group, about 8 weeks old at the start of the study, were exposed in nose-only exposure units to clean air (control) or to target concentrations of 400 ppmv (low dose), 2000 ppmv (medium dose) and 10000 ppmv (high dose) PFBE. The duration of the exposure was 6 hours per day for 28 consecutive days. The mean actual concentrations measured during the exposure periods 401±19 ppmv, 2069±179 ppmv and 9879±391 ppmv PFBE for the low, medium and high dose groups respectively.

The exposure was well tolerated by all rats with no signs of intoxication. No test substance-related mortality occurred. No influence on bodyweight, food or water consumption was observed.

No gross findings that could be related to treatment were observed at final sacrifice. No influence on organ weights was observed.

A decrease in mean partial thromboplastin time was seen in the high dose group (10000 ppmv PFBE). Small, although not clearly dose-dependent, increases in mean cholinesterase and urea were also reported. These effects were attributed to an effect on the liver.

Probable test substance-related histopathological changes were observed in the larynx, the lungs and the liver. With the exception of the liver, which was histopathologically examined in all groups, all other organs were examined only in the control and high dose groups (10000 ppmv PFBE).

Larynx

Very slight (minimal) focal to multifocal submucosal inflammatory cell infiltration occurred in 1/5 males and 2/5 females of the high dose groups (10000 ppmv PFBE).

Lungs

Very slight (minimal) focal alveolar histiocytosis in 1/5 rats each of the high dose (10000ppmv PFBE) and very slight focal interstitial inflammatory cell infiltration in another high dose female were further possible treatment-related reactive changes since they were not observed in controls. However, like the other observed lung lesions (pleural fibrosis, osseous metaplasia) these findings could have occurred incidentally.

Liver

Very slight (minimal) to slight (multifocal) fatty hepatocellular cytoplasmic vacuolation was diagnosed exclusively in 4/5 male rats of the high dose group (10000ppmv PFBE). In addition, one male of this group showed a slight focal (lobular) necrosis accompanied by a slight inflammatory cell infiltration. These degenerative lesions are considered to be treatment-related. For hepatocellular cytoplasmic vacuolation, the difference to the control males was statistically significant.

Based on the results of this study, the No Observed Adverse Effect Concentration (NOAEC) of PFBE

(3,3,4,4,5,5,6,6,6-nonafluorohexene) under the experimental conditions of the study in the Wistar (WU) rat is judged to be 2000 ppmv (20123 mg/m3).

2) A 2 -week repeated inhalation toxicity study was conducted in the rat to a protocol equivalent to OECD Test Guideline 412.

In 2 separate experiments, male and female Cr1 : CD® rats were exposed to PFBE in air. In each study, groups of 10 rats were exposed to target concentrations of either 0, 500, 5000 or 50000 ppm for periods of 6 hours/day, 5 days/week for 2 weeks. The actual test concentrations for the males were 541, 4750 and 46,300 ppm and for the females were 473, 5140 and 46800 ppm.

Clinical observations of rats during exposure were indistinguishable from those of controls. Male and female rats in all test groups showed a normal rate of weight gain throughout the study.

Clinical chemistry measurements on rats exposed to 500 ppm PFBE showed no treatment-related changes. Following 10 exposures, male rats exposed to 50000 ppm PFBE tended to have significantly lower platelet counts and to excrete more fluoride in the urine than controls. Female rats exposed to 50000 ppm PFBE tended to excrete more fluoride and urobilinogen in the urine than the controls. Females exposed to 5000 or 50000 ppm PFBE had significantly more monocytes in the blood than the controls. No treatment-related changes were observed 14 days post exposure.

Pathologic examination showed no treatment-related macroscopic or microscopic changes in any rats exposed to PFBE.

A comparison of organ to bodyweight ratios between exposed rats and controls showed that following the 10thexposure, kidney weights were significantly higher than controls in both male and female rats exposed to 50000ppm PFBE. Fourteen days later, these liver and kidney effects were still seen in male rats but had subsided in females. The significance of these effects is difficult to interpret since no specific microscopic lesions were observed in any of the rats exposed to PFBE.

Under the Annex IX Requirements a Sub-chronic 90-day study is required, and a testing proposal has been made for a study in the rat according to OECD Test Guideline 413.


Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
GLP Guideline study

Justification for classification or non-classification

No adverse effects were seen in rats exposed to 2000 ppm (20123 mg/m3) PFBE (3,3,4,4,5,5,6,6,6-nonafluorohexene) in a 28-day repeated inhalation study conducted to OECD Test Guideline 412.

Therefore classification for STOT-RE is not warranted according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation EC No. 1272/2008 because toxic effects were only seen in rats exposed to concentrations well in excess of 3 mg/l (3000 mg/m3), 6 hr/day for 28 days.

Based also on this study, classification is not warranted according to EU Directive 67/548/EEC (R48) because significant toxicity was only seen in the rat following exposure to concentrations well in excess of 0.075 mg/l (75 mg/m3), 6 hr/day for 28 days.