Registration Dossier

Administrative data

Description of key information

The gaseous substance could only be tested by the inhalation route. The effects obtained in 2 reliable studies were slightly different in intensity, possibly because of the different rat strains tested (a 28-day inhalation study in Sprague-Dawley rats and an OECD421 by inhalation in Wistar rats).

Based on effects observed mainly on relative organ weights, the kidneys seem to be the target organ.

After 28 days of exposure, the main effects were reduced body weight gain (especially in females at 50 ppm), significant increase in relative / adjusted kidney weights at 15 and 50 ppm (males, females) and 5 ppm (females), significant increase in adjusted liver weights at 15 and 50 ppm (but not observed in Wistar exposed at the same concentrations and for the same duration). These changes were not associated with significant lesions in the kidneys or liver.

In Sprague-Dawley, the 28-day exposure was associated with microscopic changes in the nasal turbinates (pseudogland formation) and decreased body weight gain. The local effects on nasal turbinates were not observed in Wistar rats in the OECD421 study indicating differences in susceptibility depending on the rat strain. They were considered to be a minor response.

Key value for chemical safety assessment

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01-OCT-2002 to 29-MAY-2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Crl:CD (SD) IGS BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (Uk) Ltd / Margate, Kent / ENGLAND
- Age at study initiation: 55 to 62 days of age
- Weight at study initiation: 271 to 320 g for males; 207 to 260 g for females
- Fasting period before study: no data available
- Housing: inside a rodent facility with a positive pressure, five of one sex per stainless steel cage (North Kent Plastic; 35 cm x 53 cm x 25 cm)
- Diet: ad libitum, except during the 6-h exposure or when urine was being collected and overnight before routine blood sampling; Rat and Mouse No. 1 Maintenance Diet (Special Diet Services / Witham / ENGLAND)
- Water: ad libitum, except during the 6-h exposure or when urine was being collected; community tap-water
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 22.5°C
- Humidity: 31 to 52%
- Air changes: no data available
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From 09-OCT-2002 to 19-NOV-2002 (main study) and 03-DEC-2002 (recovery study)
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: clean air
Remarks on MMAD:
MMAD / GSD: Not applicable
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: whole-body exposure chambers constructed from stainless steel and glass, with an internal volume of 0.75 cm3
- Source and rate of air, method of conditioning air: no data available
- System of generating atmosphere: the test atmosphere was produced by metering the test liquid from a stainless steel pressure resistant cylinder, followed by dilution with clean air prior to the resultant vapour atmosphere passing into the exposure chamber.
- Temperature, humidity, pressure in air chamber, air flow rate, air change rate, treatment of exhaust air: no data available

TEST ATMOSPHERE
No data available

VEHICLE
No data available
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
No data available
Duration of treatment / exposure:
4 consecutive weeks
Frequency of treatment:
6 hours/day
Dose / conc.:
0 ppm (nominal)
Dose / conc.:
5 ppm (nominal)
Remarks:
corresponding to 5 ppm (analytical) or 0.033 mg/L (converted) or 33 mg/m3 (converted)
Dose / conc.:
15 ppm (nominal)
Remarks:
corresponding to 15 ppm (analytical) or 0.099 mg/L (converted) or 99 mg/m3 (converted)
Dose / conc.:
50 ppm (nominal)
Remarks:
corresponding to 51 ppm (analytical) or 0.338 mg/L (converted) or 338 mg/m3 (converted)
No. of animals per sex per dose:
5 per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the target exposure levels used in this study were selected based on a previous work with this compound performed in the laboratory (see ESR entitled "SIFREN - Repeat. dose tox.: 14-day inhal. - K1 2003HLS"). In this study, rats were exposed to target concentrations of 10, 30 and 100 ppm, where clinical effects were seen at the higher exposure levels.
- 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 2 weeks without treatment. Male and female rats (5/sex/dose) were exposed either to clean air (control) or 50 ppm of the test item daily for 4 weeks followed by the 2-week recovery period.
Observations and examinations performed and frequency:
Debilitated animals were observed carefully and, where necessary, isolated to prevent cannibalism. Animals judged in extremis were killed. Animals were also killed to prevent unnecessary or prolonged suffering. Where possible, blood samples were taken ante mortem and analysed for haematology and blood chemistry parameters. A complete necropsy was performed in all cases.

CAGE SIDE OBSERVATIONS: Yes
- Time schedule, cage side observations checked: animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages and cage-trays were inspected daily for evidence of ill-health amongst the occupants, such as loose faeces.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: detailed observations were recorded daily, on the days of exposure as follows: pre-exposure observation, during exposure, as each animal was returned to its home cage, and as late as possible in the working day. In addition, a more detailed weekly physical examination was performed on each animal to monitor general health. During the acclimatisation and recovery periods, observations of animals and their cages were recorded at least once per day.
Before treatment and during each week of treatment and week 2 of recovery period, a detailed physical examination and arena observations were performed on each animal. Examinations were performed at approximately the same time of day.
After removal from home cage, animals were assessed for physical condition and behaviour during handling and after being placed in standard arena. Particular attention was paid to possible signs of neurotoxicity such as convulsions, tremors and abnormalities of gait or behaviour. Attention was also given to the detection of audible respiratory noise.

BODY WEIGHT: Yes
- Time schedule for examinations: the weight of each rat was recorded 1 week prior to the start of exposure (week -1), on the day that treatment started (day 0), weekly throughout the exposure and recovery periods, and before necropsy.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes; the weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded 1 week prior to the start of exposure (week -1), and weekly throughout the exposure and recovery periods. From these records the mean weekly consumption per animal (g/animal) was calculated for each cage.

FOOD EFFICIENCY: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: the quantity of water consumed by each cage of rats was recorded daily, commencing 1 week prior to the start of exposures and throughout the exposure and recovery periods.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to the start of exposures and during week 4 of treatment
- Dose groups that were examined: all animals before the beginning of exposures and animals from groups 1 (control) and 4 (high dose) during week 4 of treatment
As no treatment-related changes were observed, the examination was not performed during the recovery phase or for animals of groups 2 and 3.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to sacrifice following the last day of exposure and the last day of the recovery period
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes; overnight starvation
- How many animals: all animals of control and treated groups
- Parameters checked: haematocrit (Hct); haemoglobin (Hb); red blood cell count (RBC); mean cell haemoglobin (MCH); mean cell haemoglobin concentration (MCHC); mean cell volume (MCV); total white cell count (WBC); differential WBC count (neutrophils (N), lymphocytes (L), eosinophils (E), basophils (B), monocytes (M), large unstained cells (LUC)); platelet count (Plt); reticulocyte count (Retic); blood cell morphology/type; prothrombin time (PT); activated partial thromboplastin time (APPT); cellularity (cellular), distribution (Distrib) of cell types and cell morphology (Morp) of tibial bone marrow samples

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: immediately prior to sacrifice following the last day of exposure and the last day of the recovery period
- Animals fasted: Yes; overnight starvation
- How many animals: all animals of control and treated groups
- Parameters checked: alkaline phosphatase (ALP); alanine aminotransferase (ALT); aspartate aminotransferase (AST); gamma-glutamyl transpeptidase (gGT); creatine phosphokinase (CPK total); total bilirubin (Bili); urea; creatinine (Creat); glucose (Gluc); total cholesterol (Chol); triglylcerides (Trig); sodium (Na); potassium (K); chloride (Cl); calcium (Ca); inorganic phosphorus (Phos); Total protein (total Prot); albumin (Alb); α1 globulin (a1); α2 globulin (a2); β globulin (Beta); γ globulin (Gamma); albumin/globulin ratio (A/G ratio)

URINALYSIS: Yes
- Time schedule for collection of urine: during week 4 of treatment and week 2 of recovery
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes; from approximately 17:40 hours (week 4 of treatment) and 16:30 hours (week 2 of recovery) until approximately 07:30 hours the following day
- Parameters checked: appearance (App); volume (Vol); pH; specific gravity (SG); protein (Prot); sodium (U-Na); potassium (U-K); chloride (U-Cl); glucose (Gluc); ketones (Keto); bile pigments (Bili); heam pigments (Blood); epithelial cells (Epi); leucocytes (Leuc); erythrocytes (RBC); crystals (Cryst); spermatozoa and precursors (Sperm); casts (Casts); other abnormal components (Abn.)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during week 4 of treatment, as well as during week 2 of recovery.
- Dose groups that were examined: all animals of control and treated groups were investigated. However, animals were not necessarily all tested on the same day, but the number of animals was balanced across the groups on each day of testing.
- Battery of functions tested: sensory activity (i.e., approach response, touch response, auditory startle reflex, tail pinch response), grip strength (i.e., forelimb and hindlimb), motor activity

OTHER:
* URINARY FLUORIDE: following assessment of the above urinary parameters, the residual urine samples were kept at approximately -20°C for further analysis of urinary fluoride by ion specific electrode.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes; animals killed during the study and those surviving until the end of the scheduled treatment or recovery period were killed by an intraperitoneal injection of pentobarbitone sodium followed by exsanguination from the brachial arteries. All animals were then subjected to a detailed necropsy. A full macroscopic examination of tissues was performed. The cranial roof was removed to allow observation of brain, pituitary gland and cranial nerves. After ventral mid-line incision, the neck and associated tissues and the thoracic, abdominal and pelvic cavities and their viscera were exposed and examined in situ. The requisite organs were weighed and external and cut surfaces of organs and tissues were examined. Any abnormality in the appearance or size of any organ and tissue was recorded and the required tissue samples preserved in appropriate fixative.

HISTOPATHOLOGY: Yes; testes and epididymides were fixed in Bouin's solution and eyes were fixed in Davidson's fluid, prior to transfer of these tissues to 70% industrial methylated spirit. Samples (or the whole) of the other tissues listed below from main study and recovery animals were preserved in 10% neutral buffered formalin, dehydrated, embedded in paraffin wax, sectioned at 4- to 5-µm thickness and stained with hematoxylin and eosin, except the testes which were stained using a standard periodic acid/Schiff (PAS) method:
Adrenals - cortex and medulla
Aorta - thoracic
Brain - cerebellum, cerebrum and midbrain
Caecum
Colon
Duodenum
Epididymides
Eyes
Femur with joint - longitudinal section through the joint
Harderian glands
Head (not examined histologically but retained against any requirement for microscopic examination)
Heart - including cortex, medulla and papilla regions
Ileum
Jejunum
Kidneys
Lachrymal glands
Larynx - 2 levels
Liver - section from all major lobes
Lungs - section from all major lobes, including bronchi
Lymph nodes - mandibular, mesenteric, tracheo-bronchial
Mammary area - caudal (not examined histologically but retained against any requirement for microscopic examination)
Nasal turbinates - 3 levels
Nasopharynx
Oesophagus
Optic nerves (not examined histologically but retained against any requirement for microscopic examination)
Ovaries
Pancreas
Pituitary
Prostate (not examined histologically but retained against any requirement for microscopic examination)
Rectum
Salivary glands - submandibular/sublingual
Sciatic nerves
Seminal vesicles
Skeletal muscle - thigh
Skin (not examined histologically but retained against any requirement for microscopic examination)
Spinal cord - transverse and longitudinal section at the cervical, lumbar and thoracic levels
Spleen
Sternum - including bone marrow
Stomach - including keratinised, glandular and antrum in sections
Testes
Thymus
Thyroid - including parathyroids in section where possible
Tongue (not examined histologically but retained against any requirement for microscopic examination)
Trachea - including bifurcation
Urinary bladder
Ureter (not examined histologically but retained against any requirement for microscopic examination)
Uterus and cervix
Vagina (not examined histologically but retained against any requirement for microscopic examination)
For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required for microscopic pathology.
Samples of any abnormal tissues were also retained and processed for examination. In those cases where a lesion was not clearly delineated, contiguous tissue was fixed with the grossly affected region and sectioned as appropriate.

Microscopic examination was performed as follows: all tissues preserved for examination were investigated for all main study animals of groups 1 (control) and 4 (high dose) sacrificed on completion of the scheduled treatment period, and for all animals killed or dying during the study. Microscopic examinations for all main animals of groups 2 and 3 and all recovery animals was limited to adrenals, epididymides, femur, heart, kidneys, liver, lungs, spleen and testes.
Tissues reported at macroscopic examination as being grossly abnormal were examined for all animals of main and recovery studies.
In addition, the following tissues, considered to exhibit a reaction to treatment at the high exposure level, were examined for all animals of groups 2 and 3 in the main study and in all animals of recovery study: pancreas (males only) and nasal turbinates.
Other examinations:
ORGAN WEIGHTS: the following organs, taken from each animal killed after 4 weeks of treatment or 2 weeks of recovery, were dissected free of adjacent fat and other contiguous tissue and the weighs were recorded:
Adrenals
Brain
Epididymides
Heart
Kidneys
Liver
Lungs including bronchi
Ovaries
Spleen
Testes
Thymus
Uterus with cervix
Bilateral organs were weighed together.
Statistics:
All statistical analyses were carried out separately for males and females.
The following data types were analysed at each time point separately:
- Grip strength and motor activity
- Bodyweight (using gains)
- Blood chemistry, haematology and urinalysis (including urinary fluoride)
- Organ weights (absolute and adjusted for terminal bodyweight)
- Pathological findings (for the number of animals with and without each finding)
For categorical data, including pathological findings, the proportion of animals was analysed using Fisher's exact test for each treated group vs. the control.
For continuous data, Bartlett's test was first applied to test the homogeneity of variance between groups. Using tests dependent on the outcome of Bartlett's test, treated groups were then compared with the control group, incorporating adjustment for multiple comparisons where necessary.
Clinical signs:
no effects observed
Description (incidence and severity):
there were no clinical signs observed at any time prior to exposure, during exposure, post-exposure or during the weekly physical examination and arena observations.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
there was one unscheduled death.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
see in "Details on results"
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
see in "Details on results"
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
see in "Details on results"
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
see in "Details on results"
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
see in "Details on results"
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
see in "Details on results"
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
see in "Details on results"
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
see in "Details on results"
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see in "Details on results"
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
One female of group 4 was sacrificed following exposure on day 14 of the main study due to suspected broken bone. There were no clinical signs observed at any time prior to exposure, during exposure, post- exposure or during the weekly physical examination and arena observations. Brown staining on head and red/brown staining around eyes were noted for a small proportion of rats on a number of occasions following exposure.

BODY WEIGHT AND WEIGHT GAIN (see Table 3 in "Any other information on results incl. tables")
The loss in bodyweight seen for some groups at week 4 of exposure period was considered to be caused by overnight deprivation of food, on day 28, prior to bleeding for haematology and blood chemistry investigations.
Examination of weight gain up to week 3 of exposure period indicated a significant decrease in weigh t gain for females of group 4 when compared to controls. During the recovery period, bodyweights at recovery week 2 were again affected by overnight starvation. At week 1 of recovery, bodyweight gains of groups 1 and 4 were similar.


FOOD AND WATER CONSUMPTION (see Table 3 in "Any other information on results incl. tables")
The reduction in food and water consumption observed during week 4 of exposure period and week 2 of recovery period was considered attributable to the overnight deprivation of food, on day 28 of exposure period and day 14 of recovery period, respectively, prior to bleeding for haematology and blood chemistry investigations and was considered not to be treatment-related.
Examination of food and water consumption up to week 3 of exposure period and at week 1 of recovery indicated no statistically significant change in all treated groups.

OPHTHALMOSCOPIC EXAMINATION
There were no significant abnormalities observed during the pre-treatment ophthalmic examination, as well as during the examination at week 4 of exposure period.

HAEMATOLOGY
In peripheral blood, differences between groups were generally small, independent of dosage and largely inconsistent between sexes. They were considered not to be of toxicological importance. Visual assessment of bone marrow smears for cellularity, distribution and morphology indicated that there were no treatment-related effects on bone marrow.

CLINICAL CHEMISTRY (see Table 4 in "Any other information on results incl. tables")
Blood urea levels were higher than control in all treated groups following 4 weeks of exposure, however, this was not dose-related. Statistical significance was achieved for male rats of groups 3 and 4 and female rats of group 4. At the end of recovery period, urea levels were similar for groups 1 and 4.

Other differences were generally small, independent of dosage and largely inconsistent between the sexes. They were considered unlikely to be of toxicological importance.

URINALYSIS (see Table 5 in "Any other information on results incl. tables")
Group mean urine volume was lower than control for all treated groups and although the effect was greatest in group 4 there was no clear dose-relationship. None of the differences were statistically significant. The reduced urine volume in group 4 was associated with increased specific gravity, urinary K and Cl concentrations, which were statistically significant for females. The effect on urine volume and electrolyte concentration was not apparent after the 2-week recovery period and was therefore considered likely to be a treatment-related effect.
Other differences were generally small, independent of dosage and largely inconsistent between the sexes. They were considered unlikely to be of toxicological importance.

NEUROBEHAVIOUR (see Table 2 in "Any other information on results incl. tables")
There were no treatment-related effects on sensory reactivity and motor activity.
In contrast, a significant increase in hindlimb grip strength was evident for females of group 4 following 4 weeks of exposure. Moreover, a non-significant increase in forelimb grip strength was also evident for females of groups 3 and 4 following 4 weeks of exposure. A trend towards increased hindlimb grip strength was seen for males and females of group 4 following the 2-week recovery period; an upward trend in fore limb grip strength was also observed for females of group 4 at the end of recovery period. However, these increases were very slight.
The apparent increases in grip strength noted at the end of exposure and recovery periods were considered not to be treatment-related but rather to be due to natural variation. In addition, due to the isolated nature of the finding (i.e., in the absence of other sensory reactivity and motor activity treatment-related findings) and as the direction of the change (increase) for grip strength at the end of exposure period occurred while bodyweight was experiencing reduced gains, it was considered unlikely that the increase in grip strength was treatment-related.

ORGAN WEIGHTS (see Table 6 in "Any other information on results incl. tables")
Kidney weights (absolute and bodyweight adjusted values) for test groups were greater than control at week 4 of exposure period. Statistical analysis of the bodyweight adjusted values indicated that statistical significance was achieved for all female groups and males of group 4.
Liver weights (absolute and bodyweight adjusted values) were slightly higher than control values for all test groups but the effect was not dose-related, since weights of group 2 were higher than those of groups 3 and 4. In the absence of microscopic findings, it was considered that this increase in liver weight was of no toxicological importance. Statistical significance was achieved for bodyweight adjusted values for all female groups and males of group 4.
At the end of recovery period, kidney weights (absolute and bodyweight adjusted values) for females of group 4 remained higher than control.
All other statistically significant differences were considered not to be of toxicological significance.

GROSS PATHOLOGY
The macroscopic examination performed at termination revealed no lesions attributable to treatment. The incidence and distribution of all findings were considered to fall within the expected background range of macroscopic changes.

HISTOPATHOLOGY: NON-NEOPLASTIC (see Table 7 in "Any other information on results incl. tables") The following microscopic changes were considered to be treatment-related:
- Nasal turbinates: in the respiratory epithelium, an increased incidence of pseudogland formation was seen in males of group 4 compared to controls. Pseudogland formation was considered a minor response and was probably related to local goblet cell hyperplasia. After 2 weeks of recovery, the respiratory epithelium of nasal turbinates showed no more impairment, demonstrating a complete recovery.
- Kidneys: cortical tubules with hyaline droplets were noted in all male treated groups but not in any controls. After 2 weeks of recovery, only a single male in group 4 showed cortical tubules with hyaline droplets in the kidney.

In addition, an increased incidence and severity of acinar cell apoptosis were noted in males of group 4, compared with controls, but not in females of this group. As this finding was seen in controls of both sexes and the severity was not high compared to controls, its relationship to treatment was uncertain. After 2 weeks of recovery, no acinar cell apoptosis was seen in the pancreas of any animal, demonstrating a complete recovery.

No histological findings were observed in the liver to explain the increased organ weights recorded in high dose groups.

All other microscopic findings in animals from main and recovery studies were considered to be incidental and of no toxicological importance.

OTHER FINDINGS (see Table 5 in "Any other information on results incl. tables")
* URINARY FLUORIDE: a dose-related increase in urinary fluoride was evident in both sexes of all test groups following 4 weeks of exposure and attained statistical significance for groups 3 and 4. An increase in urinary fluoride was still evident for rats of group 4 following the 2-week recovery period. This was statistically significant but markedly less than that measured at week 4 of exposure period.
Key result
Dose descriptor:
NOAEC
Effect level:
5 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Critical effects observed:
yes
Lowest effective dose / conc.:
5 ppm (nominal)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Critical effects observed:
yes
Lowest effective dose / conc.:
51 ppm (nominal)
System:
respiratory system: upper respiratory tract
Organ:
nasal cavity
Treatment related:
yes
Dose response relationship:
yes

The pseudogland formation observed in the nasal turbinates of the nasal cavity of animals treated for 28 -day by inhalation was no longer present following the 2 week-recovery period.

The hyaline droplet formation in the cortical tubules of kidneys of male rats was reversed after the 2 week-recovery period (except 1 animal) and may be related to accumulation of alpha-2u globulin, an effect specific to male rats, and which is usually not considered relevant to humans.

The increased body weight-adjusted liver weight observed at the end of the 28-day treatment period was no longer significant after the 2-week recovery period and was considered adaptative in nature.

Conclusions:
The only effects seen in rats exposed to 5 ppm of test item were a slight increase in hyaline droplet formation in the cortical tubules of kidneys (males only) and an increase in urinary fluoride (males and females). The former was considered to be of no significance to man and the latter was a consequence of the metabolic breakdown of the test substance.
At 15 and 51 ppm the adverse effects included microscopic changes in the nasal turbinates (statistically significant in males at 51 ppm) and a slightly reduced weight gain (statistically significant in females at 51 ppm). Based on these observations, the no-observed-adverse-effect-level, for male and female rats, was considered to be 5 ppm (eq. to 33 mg/m3).
Executive summary:

The objective of this study was to assess the systemic toxicity potential of the test substance to Crl:CD (SD)IGS BR rats by repeated inhalation administration over a period of 4 weeks, followed by a 2-week recovery period. The study was conducted according to OECD guideline 412 and in compliance with GLP.

Three groups of male and female rats (5/sex/dose) were exposed to a vapour of the test substance by inhalation at nominal concentrations of 5, 15, and 50 ppm, 6 hours a day for 4 consecutive weeks using a whole-body exposure system. A similarly constituted control group was exposed to clean air only. A further 5 male and 5 female rats were assigned to each of the control and high dose groups for a 2-week recovery period following the 4-week treatment period. During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity, grip strength, motor activity, bodyweight, food and water consumption, ophthalmic examination, urinary fluoride, organ weight, macroscopic and microscopic pathology investigations were undertaken.

The study mean analysed concentrations of the test item were 5, 15 and 51 ppm for the low, intermediate and high dose groups, respectively. These concentrations were equivalent to 0.033, 0.099 and 0.338 mg/L (at 25°C and 760 mmHg) for groups 2 to 4, respectively.

There was one unscheduled death. A female of group 4 was sacrificed on humane grounds following exposure on day 14 due to a suspected broken bone. There were no treatment-related clinical signs observed pre-exposure, during exposure, post-exposure or during the weekly physical examination and arena observations.

There were no treatment-related effects on sensory reactivity, grip strength or motor activity.

Reduced mean bodyweight gains were evident for rats of groups 3 and 4 during the exposure period and attained statistical significance for female rats of group 4.

A slight reduction in food and water consumption was evident for females of group 3, and males (food only) and females (food, water) of group 4 during the exposure period. A slight reduction in food and water consumption was still evident for females of group 4 during the recovery period.

There were no significant abnormalities observed during the pre-treatment ophthalmic examination. There were no treatment-related effects noted during the examination in week 4.

There were no treatment-related effects on haematology parameters including those examined in the bone marrow.

Blood urea levels were higher than control in all treated groups following 4 weeks of exposure and attained statistical significance for male rats of groups 3 and 4, and females of group 4. At the end of recovery period, urea levels were similar for groups 1 and 4. The elevation of blood urea levels was considered likely to be a consequence of changes in renal function and was considered to be of no toxicological importance due to the lack of a dose-relationship.

Group mean urine volume was lower than control for all treated groups. The reduced urine volume in rats of group 4 was associated with increased specific gravity, urinary potassium concentration and urinary chloride concentration, which were statistically significant for females. It is possible that this was an anomalous finding as the usual effect of fluoride containing compounds is polyuria. It is possible that the reduced urine output during the overnight collection period was consequence of increased urine output during the exposure period.

A dose-related increase in urinary fluoride was evident in both sexes of all treated groups following 4 weeks of exposure and attained statistical significance for groups 3 and 4. Urinary fluoride levels for rats of group 4 remained higher than controls following 2 weeks of recovery and remained statistically significant. The presence of fluoride ion in urine is commonly associated with the metabolic breakdown of fluoride-containing compounds such as the test substance.

Kidney and liver weights for test groups were greater than controls following 4 weeks of exposure and attained statistical significance for groups 3 and 4. Kidney weights of females from group 4 remained higher than controls following 2 weeks of recovery. The changes in kidney weight were considered to be treatment-related and potentially associated with changes in renal function. In the absence of microscopic findings, it was considered that the increase in liver weight was of no toxicological importance.

The macroscopic examinations performed at termination revealed no treatment-related abnormalities. At the end of the exposure period, the microscopic examinations revealed treatment-related increased incidence of pseudogland formation in the respiratory epithelium in the nasal turbinates of males from group 4 and increased incidences of cortical tubules with hyaline droplets in the kidneys of males from all treated groups. At the end of the recovery period, the microscopic examinations revealed recovery to control level for pseudogland formation in the nasal turbinates of the respiratory epithelium. Almost complete recovery had occurred for cortical tubules with hyaline droplets of the kidney. The presence of hyaline droplets in cortical tubules in the kidneys of male rats is a common non-specific response to a range of chemicals and is considered to be due to exacerbated accumulation of alpha-2µ-globulin in the kidneys and probably of no significance to man.

At 15 and 51 ppm the adverse effects included microscopic changes in the nasal turbinates of minor significance and a reduced weight gain. Partial or complete recovery from all the effects seen in this study was considered to have been demonstrated. In this study the no-observed-adverse-effect-level, for male and female rats, was considered to be 5 ppm (i.e. 33 mg/m3).

Endpoint:
repeated dose toxicity: inhalation, other
Remarks:
Repro./Develop. screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
7-SEP-2016 to 21-NOV 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
provides information relevant to the repeated dose toxicity although it is a reproductive and developmental toxicity study screening
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD Guideline 421
Version / remarks:
Reproduction/developmental toxicity study with additional parameters
Deviations:
no
Principles of method if other than guideline:
- Principle of test: OECD421 including haematology and clinical biochemistry
- Short description of test conditions: 28 day treatment in males, 50-53 days in females
- Parameters analysed / observed: haematology and clinical biochemistry, gross pathology and histopathology
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- batch No.of test material: SMF271SG08 to SMF271SG17 (same production batch supplied in 10 cylinders)
- Expiration date of the lot/batch: 11-08-2021

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Wistar Han IGS (Crl:WI(Han))
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulsfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: at arrival females were about 10 weeks old and males were about 9 weeks old; Females were 12 weeks old and males were 11 weeks old at the start of pre-treatment period, 14 weeks (F) / 13 weeks (M) at the start of exposure, and 16 weeks (F) / 15 weeks (M) at the start of mating.
The age difference between males and females is deliberate, to avoid mating of siblings.

- Weight at study initiation:
On day 0 of treatment (pre-mating), mean BW were:
For males: controls: 342.17 +/- 14.54 g, low dose: 339.52 +/- 14.28 g, mid-dose: 335.67 +/- 14.67 g, high dose: 337.07 +/- 13.98 g
For females: controls: 224.65 +/- 7.97 g, low dose: 223.86 +/- 5.20 g, mid-dose: 225.43 +/- 6.84 g, high dose: 226.08 +/- 8.39 g

- Fasting period before study: not applicable
- Housing: Makrolon cages, after allocation, during pre-mating period, the animals were housed 4 or 5/cage (separated by sex). For mating, one male and one female were housed together. Mated females were house individually. During exposure periods, the rats were individually housed in the exposure unit without access to food or water. Immediately after exposure, the animals were returned to their home cage.
- Diet : ad libitum. Cereal-based (closed formula) powder rodent diet (VRF1(FG)). Each batch of this diet is analyzed by the supplier for nutrients and contaminants.
- Water : ad libitum. domestic mains tap water suitable for human consumption. The water was given in polypropylene bottles, which were cleaned weekly and filled as needed. Results of the routine physical, chem ical and microbiological examination of drinking water as conducted by the supplier are made available to the test facility
- Acclimation period: at least 14 days prior to the start of exposure

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 45-65%
- Air changes (per hr): 10 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hrs dark/ 12 hrs light

IN-LIFE DATES: From: 7-SEP-2016 (arrival of the animals) To: 22-NOV-2016 (necropsy of dams and pups)
Route of administration:
inhalation: gas
Type of inhalation exposure:
whole body
Vehicle:
clean air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
The exposure conditions are reported under Toxicity to reproduction section/ OECD421 screening study.

TEST ATMOSPHERE
- Brief description of analytical method used: the actual concentrations were analysed by
photoacoustic infrared for groups 2 and 3, or total carbon analysis for group 4.
- Samples taken from breathing zone: yes

VEHICLE
- Composition of vehicle: clean air
- Oxygen concentration during exposure: 20.6 - 20.8 % (v/v)
- Carbon dioxide concentration: 0.079 - 0.116 % (v/v)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The details on analytical verification are reported under Toxicity to reproduction section/ OECD421 screening study.
Duration of treatment / exposure:
Whole-body inhalation during 6 hours per exposure day.
Total exposure duration (see details under "Frequency of treatment"):
- Male animals were sacrificed after 29 days of exposure.
- Female animals were sacrificed on day 13 of lactation or shortly thereafter. The total number of exposure days was 40-47 in females.
- Female animals that failed to mate or appeared not pregnant after mating were sacrificed at least 21 days after the last mating date or at least 25 days after the presumed mating date, respectively
Frequency of treatment:
Exposure to the test substance during the different study phases:
- Pre exposure period: male and females were not exposed.
- Premating period: male and female animals were exposed during 2 weeks prior to mating to the test substance for 5 days/week (i.e. 10 exposure days in total)
- Mating period: male and female rats were exposed daily until confirmation of mating.
- Post-mating period: males and non-mated females were exposed daily until sacrifice.
- Gestation period: mated female animals were exposed daily from successful mating (i.e. the finding of a sperm positive vaginal smear considered gestation day 0) until gestation day 19 (included).
- Lactation period: Females were exposed daily starting lactation day 5 until lactation day 12 (included).
There was no exposure of females between GD19 and LD4 to allow the females to litter. Daily exposure was resumed on lactation day 5 up to day 13 of lactation.
Dose / conc.:
0 ppm (nominal)
Dose / conc.:
5 ppm (nominal)
Remarks:
corresponding to 4.95 ppm (analytical) or 0.033 mg/L (converted) or 33 mg/m3 (converted)
Dose / conc.:
4.95 ppm (analytical)
Remarks:
SD: +/- 0.24 ; average exposure
corresponds to 0.0328 mg/L (converted) or 32.8 mg/m3 (converted)
Dose / conc.:
15 ppm (nominal)
Remarks:
corresponding to 15.04 ppm (analytical) or 0.0994 mg/L (converted) or 99.4 mg/m3 (converted)
Dose / conc.:
15.04 ppm (analytical)
Remarks:
SD: +/- 1.05 ; average exposure
corresponds to 0.0997 mg/L (converted) or 99.7 mg/m3 (converted)
Dose / conc.:
50 ppm (nominal)
Remarks:
corresponding to 50.69 ppm (analytical) or 0.331 mg/L (converted) or 331 mg/m3 (converted)
Dose / conc.:
50.69 ppm (analytical)
Remarks:
SD: +/- 2.16; average exposure
corresponds to 0.336 mg/L (converted) or 335.9 mg/m3 (converted)
No. of animals per sex per dose:
12/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The exposure levels were selected on the basis of the results of a 28-day inhalation study in rats.
- Rationale for animal assignment: computer randomization proportionally to body weight.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily observation in the morning hours, before the exposure.
- Cage side observations listed in Attachment 2 were included.
- Animals were also observed halfway through the 6-hour exposure period to monitor any breathing abnormalities and any restlessness. Animals were again observed post-exposure.

BODY WEIGHT: Yes
- Time schedule for examinations: once during the acclimatization period, at initiation of treatment,
then weekly (males, and females during the pre-mating and mating period). Mated females were weighed on days 0, 7, 14 and 20 during presumed gestation and on day 0, 4, 7 and 13 of lactation. Non-mated females were weighed once per week after the mating period. All the animals were weighed on their scheduled necropsy date.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as gfood/kg body weight/day: Yes
Food was refreshed once a week. The food consumption was measured per cage when body weight was measured, except during the mating period.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on day of necropsy, blood will be sampled from aorta. For males: on day 30, for females: on day 51-53.
- Anaesthetic used for blood collection: Yes (sodium pentobarbital anaesthesia)
- Animals fasted: Yes, fasted overnight (water available)
- How many animals: 5/sex/group
- Parameters: listed below "under Any other information on materials and method" were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on day of necropsy. For males: on day 30, for females: on day 51-53.
- Animals fasted: Yes, fasted overnight (water available)
- How many animals: 5/sex/group
- Parameters: listed below "under Any other information on materials and method" were examined

OTHER: (see IUCLID 7.8.1)
- Estrous cyclicity (parental animals)
Vaginal smears were made daily in all females from the start of the pre-treatment period until confirmation of mating. An additional smear was made at the day of sacrifice.
- Litter observations
- Hormone analysis
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table)
HISTOPATHOLOGY: Yes (see table)
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A lower mean body weight (5%), but non-statistically significant, was observed in males of the mid and high concentration groups at the end of the exposure period.
A transient decrease in mean body weight gain in the males in the mid and high concentration groups, concurrent to a transient decrease in mean food consumption in the males in the high concentration group, which was limited to the first two weeks of exposure (premating phase).
No treatment related effects were observed in body weight in females (despite generally lower body weight gain in treated females during pre-mating phase compared to the control group, with no difference in food consumtion). Body weight change was statistically lower in the first week of gestation in females of the mid and high concentration groups, compared to the control group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A transient decrease in mean food consumption was observed in the males in the high concentration group, which was limited to the first two weeks of exposure (premating period).
No treatment related effects were observed in food consumption in females.
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):
No statistically significant changes were observed in red blood cell parameters and in prothrombin time (males, females).
In males, a statistically significant lower absolute number of neutrophils in the low concentration group was considered a chance finding in absence of a concentration response relationship.
In females, a statistically significant increase in the percentage lymphocytes was observed at the mid and high concentration groups, and a decrease in the percentage neutrophils. In absence of statistically significant differences in absolute lymphocytes and neutrophils this was considered not toxicologically relevant.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Glucose plasma levels were increased in males in the low and high concentration groups. However, in the absence of dose-dependence and no similar findings in females, the effect was not considered related to treatment.
A statistically significant decreased mean creatinine levels in females in the high concentration group were considered to be related to treatment.
Urinalysis findings:
not examined
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):
An increased relative mean relative kidney weight was observed in males of the mid and high concentration group. The mean absolute kidney weight did not reach statistical significance.
In females absolute and relative mean kidney weight were increased in the high concentration group.
In addition mean relative lung weight was increased in the females in the high concentration group.
No effects were observed on absolute and relative reproductive organ weights.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Microscopic evaluation did not reveal treatment related histopathological changes. The increase in absolute and relative kidney weights in the high concentration females and the increase in relative kidney weights in the mid and high concentration males were statistically significantly higher than in controls. However, this could not be explained by microscopical findings in the kidneys.
In general, the histopathological changes observed were about equally distributed amongst the different treatment groups or occurred in one or a few animals only. They are common findings in rats of this strain and age or occurred as individual chance findings. Therefore, they were not considered to be related to treatment.
All animals in the high concentration group showed stripy pattern on the teeth, which is considered to be related to the fluor in the test substance. Animals in the intermediate groups were not examined.
Histopathological findings: neoplastic:
no effects observed
Details on results:
OTHER EFFECTS
* Estrus cycle: No effects observed
* Hormone analysis in male F0, in male and female pups (F1) sacrificed on lactation day 13: no effects observed
* Fertility and reproductive performance (see details in IUCLID section 8.7.1) : No effects on fertility parameters, length of gestation.
* litter data (see details in IUCLID section 8.7.1)
Key result
Dose descriptor:
NOAEC
Effect level:
15 ppm (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
organ weights and organ / body weight ratios
Key result
Dose descriptor:
NOEC
Effect level:
5 ppm (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
Critical effects observed:
yes
Lowest effective dose / conc.:
50 ppm (analytical)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes

The body weight was found slightly lower (not statistically significant) in the mid and high concentrations groups. The relative kidney weight was significantly increased in males and females at the high concentration (50 ppm), and in males of the mid-concentration (15 ppm) group. However, these changes were not associated with major histopathological findings.

Among blood chemistry parameters, the only noticeable finding was a decrease in serum creatinine in females treated at the high concentration (50 ppm).

Conclusions:
Information on the general toxicity in this reproductive toxicity screening study are relevant to the repeated dose toxicity endpoint.
Based on the effects on absolute and relative kidney weight, relative lung weight and mean creatinine level in females in the high concentration group, the No Observed Adverse Effect concentration (NOAEC) for maternal toxicity was placed at the mid concentration (15 ppm, i.e., 99 mg/m3).
Based on the effects on relative kidney weight in males in the mid and high concentration group and the transient effects on body weight and food consumption in the first two weeks of exposure in males in the mid and high concentration groups, the No Observed Effect Concentration (NOEC) was placed at the low concentration (5 ppm, i.e. 33 mg/m3).
In the absence of effects on fertility and reproductive parameters, the NOEC for reproduction was 50 ppm (i.e., 338 mg/m3), the highest concentration tested.
Based on a slightly lower mean pup weight in the high concentration group, the NOAEC for developmental toxicity was placed at the mid concentration (15 ppm, Ie., 99 mg/m3).
Executive summary:

The possible effects of hexafluorobutadiene on reproductive performance and pup development were investigated in a reproductive and developmental toxicity screening study (Test guideline OECD421) conducted in Wistar rats. The test substance was administered by inhalation (6 hours/day) at concentrations of 0 (control), 5, 15 and 50 ppm during a premating period of 2 weeks (5 days/week, 10 exposure days in total), then daily during mating and post-mating up to sacrifice for the males.

Females were exposed to the test atmospheres during a premating period of 2 weeks, during mating, gestation and lactation. Mated females were not exposed to the test substance between gestation day 19 and lactation day 4 in order to allow the females to litter. Daily exposure was resumed on lactation day 5 up to day 13 of lactation. The total number of exposure days was 28 days for males and 40-47 days for females.

The test atmospheres were stable and the actual concentrations were close to intended.

Exposure to 0, 5, 15 or 50 ppm hexafluorobutadiene resulted in:

• No treatment-related mortality and morbidity.

• A transient decrease in mean body weight gain in the males in the mid and high concentration groups and a transient decrease in mean food consumption in the males in the high concentration group, which was limited to the first two weeks of exposure.

No treatment related effects were observed in food consumption or body weight in females.

• An increased relative mean relative kidney weight in males in the mid and high concentration group. In females absolute and relative mean kidney weight were increased in the high concentration group. In addition mean relative lung weight was increased in the females in the high concentration group.

• All animals in the high concentration group showed stripy pattern on the teeth, which is considered to be related to the fluor in the test substance. Animals in the intermediate groups were not examined.

• No other treatment-related findings were observed in histopathological examination.

• No effects on red blood cell parameters, white blood cell parameters and prothrombin time.

• A statistically significant decrease in mean creatinine level in females in the high concentration group.

• No effects on fertility or reproduction parameters.

• No effects on T4 and TSH hormone levels in adult males, and in male and female pups (lactation day 13).

• No effects on pup sex and survival, pup observations, anogenital distance or nipple retention. Macroscopic and microscopic examination of the pup thyroid did not show any effects.

• A slight decrease in mean pup weight in the high concentration group was considered related to treatment. No effects were observed on pup weight gain.

Based on the effects on absolute and relative kidney weight, relative lung weight and mean creatinine level in females in the high concentration group, the No Observed Adverse Effect concentration (NOAEC) for maternal toxicity was placed at the mid concentration (15 ppm hexafluorobutadiene).

Based on the effects on relative kidney weight in males in the mid and high concentration group and the transient effects on body weight and food consumption in the first two weeks of exposure in males in the mid and high concentration groups, the No Observed Effect Concentration (NOEC) was placed at the low concentration (5 ppm hexafluorobutadiene).

In the absence of effects on reproductive parameters, the No Observed Effect Concentration (NOEC) for reproductive toxicity/fertility was placed at the high concentration (50 ppm hexafluorobutadiene).

In absence of effects on pup sex and survival, pup observations, anogenital distance and nipple retention, but based on a slightly lower mean pup weight in the high concentration group, the No Observed Adverse Effect Concentration (NOAEC) for developmental toxicity was placed at the mid concentration (15 ppm hexafluorobutadiene).

Results of the bone marrow micronucleus test combined to the repeated exposure are reported under the in vivo genotoxicity section. Hexafluorobutadiene, at concentrations up to 50 ppm by inhalation, did not show any indication of chromosomal damage and/or damage to the mitotic spindle apparatus of the bone marrow target cells of male rats. Systemic availability of the test substance was demonstrated by slight effects on body weight change and food consumption in males, indicating that the negative response observed in this bone marrow micronucleus test is not due to lack of systemic availability of the test substance or its metabolites.

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
16-JUL-2002 to 14-AUG-2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
yes
Remarks:
adapted for Dose-range finding study
Principles of method if other than guideline:
Deviations: the exposure duration was shortened to 14 days as the test was a preliminary study (Dose-range finding study).
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Crl:CD (SD)IGS BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (Uk) Ltd / Margate, Kent / ENGLAND
- Age at study initiation: 49 to 56 days of age
- Weight at study initiation: 247 to 275 g for males; 189 to 222 g for females
- Fasting period before study: no data available
- Housing: inside a rodent facility with a positive pressure, five of one sex per stainless steel cage (North Kent Plastic; 35 cm x 55 cm x 25 cm)
- Diet: ad libitum, except during the 6-h exposure or when urine was being collected; Rat and Mouse No. 1 Maintenance Diet (Special Diet Services / Witham / ENGLAND)
- Water: ad libitum, except during the 6-h exposure or when urine was being collected; community tap-water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21 to 23.5°C
- Humidity: 36 to 59%
- Air changes: no data available
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From 17-JUL-2002 to 07-AUG-2002
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
clean air
Remarks on MMAD:
MMAD / GSD: Not applicable
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: exposure chambers constructed from stainless steel and glass, with an internal volume of 0.75 m3
- Source and rate of air, method of conditioning air: no data available
- System of generating atmosphere: the test atmosphere was produced by metering the test liquid from a stainless steel pressure resistant cylinder, followed by dilution with clean air prior to the resultant vapour atmosphere passing into the exposure chamber.
- Temperature, humidity, pressure in air chamber, air flow rate, air change rate, treatment of exhaust air: no data available

TEST ATMOSPHERE
No data available

VEHICLE
No data available
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
No data available
Duration of treatment / exposure:
14 consecutive days
Frequency of treatment:
6 hours/day
Dose / conc.:
10 ppm (nominal)
Remarks:
corresponding to 11 ppm (analytical)
Dose / conc.:
30 ppm (nominal)
Remarks:
corresponding to 30 ppm (analytical)
Dose / conc.:
100 ppm (nominal)
Remarks:
corresponding to 99 ppm (analytical)
No. of animals per sex per dose:
5 per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the exposure levels used in this study were selected based on a previous work with this compound performed in the laboratory (see ESR entitled "SIFREN - Repeat. dose tox.: 7-day inhal. - K1 2003HLS"). In this study, rats were exposed to target concentrations of 20, 70 and 200 ppm, where clinical effects were seen at the higher exposure levels.
- Rationale for selecting satellite groups, post-exposure recovery period in satellite groups: no data available
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages and cage-trays were inspected daily for evidence of ill-health amongst the occupants, such as loose faeces.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: detailed observations were recorded daily, on days of exposure as follows: pre-exposure observation, during exposure, as each animal was returned to its home cage, and as late as possible in the working day. In addition, a more detailed weekly physical examination was performed on each animal to monitor general health. During acclimatisation period, observations of animals and their cages were recorded at least once per day.

BODY WEIGHT: Yes
- Time schedule for examinations: the weight of each rat was recorded daily, commencing 1 week prior to the start of exposure, on the day that exposures commenced (day 0), daily throughout the exposure period, and before necropsy.


FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes; the weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded daily, commencing 1 week prior to the start of exposure, and daily throughout the exposure. From these records the daily consumption per animal (g/animal) was calculated for each cage.

FOOD EFFICIENCY: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: the quantity of water consumed by each cage of rats was recorded daily, commencing 1 week prior to the start of exposures until the end of the study.

OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: Yes
- Time schedule for collection of urine: during week 2 of treatment, overnight urine samples were collected from all animals from ca. 16.00 hours until ca. 07.30 hours the following day.
- Metabolism cages used for collection of urine, animals fasted: animals were deprived of food and water from ca. 16.00 hours and placed in an individual metabolism cage.
- Parameters checked: urinary fluoride by ion specific electrode

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes; animals surviving until the end of the scheduled treatment were killed by an intraperitoneal injection of pentobarbitone sodium followed by exsanguination from the brachial arteries. All animals were then subjected to a detailed necropsy. All external features and orifices were examined. The cranial roof was removed to allow observation of brain, pituitary gland and cranial nerves. After ventral mid-line incision, the neck and associated tissues and the thoracic, abdominal and pelvic cavities and their viscera were exposed and examined in situ. The requisite organs were weighed and external and cut surfaces of organs and tissues were examined. Any abnormality in the appearance or size of any organ and tissue was recorded and the required tissue samples preserved in appropriate fixative.

HISTOPATHOLOGY: Yes; samples (or the whole) of tissues listed below from all animals were preserved in 10% neutral buffered formalin, dehydrated, embedded in paraffin wax, sectioned at ca. 4 to 5-µm thickness and stained with hematoxylin and eosin:
Head (for nasal turbinates)
Kidneys - including cortex, medulla and papilla regions
Larynx
Liver - section from all main lobes
Lungs and bronchi - section from all major lobes
Nasal turbinates - 3 levels
Trachea - including bifurcation
For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required for microscopic pathology.
Samples of any abnormal tissues were also retained and processed for examination. In those cases where a lesion was not clearly delineated, contiguous tissue was fixed with the grossly affected region and sectioned as appropriate.
Other examinations:
ORGAN WEIGHTS: the following organs, taken from each animal killed after 14 days of treatment, were dissected free of adjacent fat and other contiguous tissue and the weighs were recorded:
Adrenals
Brain
Kidneys
Liver
Lungs with mainstem bronchi
Bilateral organs were weighed together.
Statistics:
All statistical analyses were carried out separately for males and females.
The following data types were analysed at each time point separately:
- Bodyweight (using gains)
- Urinary fluoride
- Organ weights (absolute and adjusted for terminal bodyweight)
- Pathological findings (for the number of animals with and without each finding)
For categorical data, including pathological findings, the proportion of animals was analysed using Fisher's exact test for each treated group vs. the control.
For continuous data, Bartlett's test was first applied to test the homogeneity of variance between groups. Using tests dependent on the outcome of Bartlett's test, treated groups were then compared with the control group, incorporating adjustment for multiple comparisons where necessary.
Clinical signs:
no effects observed
Description (incidence and severity):
there were no clinical signs observed at any time during the study.
Mortality:
no mortality observed
Description (incidence):
there were no unscheduled deaths.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
see in "Details on results"
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
see in "Details on results"
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, non-treatment-related
Description (incidence and severity):
see in "Details on results"
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
see in "Details on results"
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):
see in "Details on results"
Gross pathological findings:
no effects observed
Description (incidence and severity):
the macroscopic examinations performed at termination revealed no lesions attributable to treatment with the test item. The incidence and distribution of all findings were considered to fall within the expected background range of macroscopic changes.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see in "Details on results"
Histopathological findings: neoplastic:
no effects observed
Details on results:
BODY WEIGHT (Table 2 in "Any other information on results incl. tables")
After the first treatment day, males of group 4 lost weight; females of group 4 did not gain weight in that same period. Actual weight loss, attributable to treatment, was not seen after day 2.
At the end of treatment period, males of group 4 gained significantly less weight, compared to the control group.

FOOD CONSUMPTION (Table 2 in "Any other information on results incl. tables")
A reduction in food consumption was evident for rats of groups 3 and 4 during the exposure period.
The apparent marked reduction in food consumption observed on day 13 was considered attributed to the overnight deprivation of food for the collection of urine and was therefore unlikely to be treatment-related.

WATER CONSUMPTION (Table 2 in "Any other information on results incl. tables")
An increase in water consumption was evident for rats of group 4 during the exposure period and was marked for females of group 4. A slight reduction in water consumption was evident for males of group 3 during the same period.
The apparent marked reduction in water consumption observed on day 13 was considered attributed to the overnight deprivation of water for the collection of urine and was therefore unlikely to be treatment-related.

URINALYSIS (Table 3 in "Any other information on results incl. tables")
A treatment-related increase in urinary fluoride was evident across groups for both sexes, and attained statistical significance for females of group 2 and rats of both sexes from groups 3 and 4.

ORGAN WEIGHTS (Table 4 in "Any other information on results incl. tables")
Kidney weights of males from group 3 and male and female rats from group 4 were greater than control values. Liver and lung weights of females from group 4 were also higher than controls. These findings were considered to be treatment-related.

HISTOPATHOLOGY: NON-NEOPLASTIC (Table 5 in "Any other information on results incl. tables")
The following microscopic changes were considered to be treatment-related:
- Kidneys: slight or moderate tubular basophilia in the inner cortex and outer medulla (proximal straight tubules) was present in all male and female rats exposed to 100 ppm (group 4). Minimal tubular basophilia in the inner cortex and outer medulla (proximal straight tubules) was seen in a single female to 30 ppm (group 3).
- Lungs: minimal to slight prominent alveolar macrophages were present in the air control (group 1) and all exposure groups, with increased incidence prevalence in the groups 3 and 4, and an increased severity in group 4.
- Nasal turbinates: respiratory epithelial pseudogland formation was seen in males and females of groups 3 or 4.

In kidneys, minimal corticomedullary, medullary or papillary mineralisation was identified in males and females exposed to the test item; and medullary mineralisation was found in a single female air control. Intratubular mineralisation develops spontaneously in the kidneys of laboratory animals and is often an incidental finding in both sexes. Consequently, this minimal finding was not considered treatment-related.
All other microscopic findings were considered to be incidental and of no toxicological importance.
Dose descriptor:
NOAEC
Effect level:
10 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
NOAEC
Effect level:
11 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Critical effects observed:
yes
Lowest effective dose / conc.:
30 ppm (analytical)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Critical effects observed:
yes
Lowest effective dose / conc.:
30 ppm (analytical)
System:
respiratory system: upper respiratory tract
Organ:
nasal cavity
Treatment related:
yes
Dose response relationship:
yes
Conclusions:
Under the conditions of this study, the substance induced significant effects at the two highest doses (30 and 99 ppm) in both males and females, while the only noticeable effect at the lowest dose (11 ppm) was the presence of alveolar macrophages in the lungs of females, although without statistical significance. Based on the results of this study, the concentrations of 5, 15, and 50 ppm were selected for the 28-day inhalation study.
Executive summary:

The objective of this study was to assess the systemic toxicity potential of the test substance to Crl:CD (SD)IGS BR rats by repeated inhalation administration over a period of 14 days. The study was conducted according to a protocol based on OECD guideline 412, and in compliance with GLP.

Three groups of male and female rats (5/sex/dose) were exposed to a vapour of the test substance by inhalation at nominal concentrations of 10, 30 and 100 ppm, 6 hours a day for 14 consecutive days using a whole-body exposure system. A similarly constituted control group was exposed to clean air only. During the study, clinical condition, detailed physical observations, bodyweight, food and water consumption, urinary fluoride, organ weight, macroscopic and microscopic pathology investigations were undertaken.

The study mean analysed concentrations of test item were 11, 30 and 99 ppm for the low, intermediate and high dose groups, respectively.

There were no unscheduled deaths and there were no clinical signs observed at any time during the study.

After the first treatment day, males of group 4 (high dose) lost weight; females of group 4 did not gain weight in that same period. Actual weight loss attributable to treatment was not seen after day 2. At the end of the treatment period, males of group 4 gained significantly less weight, compared to the control group.

A reduction in food consumption was evident for rats of groups 3 and 4 and an increase in water consumption was evident for rats of group 4. A slight reduction in water consumption was evident for males of group 3 during the same period.

A treatment-related increase in urinary fluoride was evident across treated groups of both sexes, and attained statistical significance for females of group 2 and rats of both sexes from groups 3 and 4.

There were treatment-related differences in kidney weight for males of group 3 and male and female rats from group 4, and in liver and lung for females of group 4.

The macroscopic examinations performed at termination revealed no treatment-related abnormalities. The microscopic examinations revealed in the kidneys, slight or moderate tubular basophilia in the inner cortex and outer medulla (proximal straight tubules), which was seen in all rats of group 4, and was noted at a minimal level in a single female rat of group 3. This finding correlated with the increase in the mean kidney weight of rats from group 4. The effect in group 3 was only marginal, and there was no effect observed in group 2 (low dose). In the lungs, prominent alveolar macrophages were identified in the majority of animals exposed to the test substance and in a single male and two females of group 1 (control). Animals of groups 2 and 3 demonstrated a minor increase in incidence, but not severity, with increasing exposure level. Rats from group 4 had increased prevalence and severity. In the nasal turbinates, minimal or slight respiratory epithelial pseudogland formation was found in groups 3 and 4, with evidence of dose-relationship in the group prevalences. This finding was not seen in group 2. Additional findings in the kidney were not considered treatment-related and all other microscopic findings were considered to be incidental and of no toxicological importance.

In view of the findings summarised above, target concentrations of 5, 15 and 50 ppm were considered suitable for a subsequent 28-day study. In addition to bodyweight and food consumption effects, these target concentrations for the 28-day study were selected specifically with relation to water consumption and urinary fluoride, together with the microscopic findings mentioned above.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
99.4 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
Reliable studies in two different rat strains showed relatively consistent results although with variable intensity. Therefore the dose descriptor for the risk assessment was selected using a weight of evidence approach considering data from all three repeated dose toxicity studies.
System:
urinary
Organ:
kidney

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01-OCT-2002 to 29-MAY-2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Crl:CD (SD) IGS BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (Uk) Ltd / Margate, Kent / ENGLAND
- Age at study initiation: 55 to 62 days of age
- Weight at study initiation: 271 to 320 g for males; 207 to 260 g for females
- Fasting period before study: no data available
- Housing: inside a rodent facility with a positive pressure, five of one sex per stainless steel cage (North Kent Plastic; 35 cm x 53 cm x 25 cm)
- Diet: ad libitum, except during the 6-h exposure or when urine was being collected and overnight before routine blood sampling; Rat and Mouse No. 1 Maintenance Diet (Special Diet Services / Witham / ENGLAND)
- Water: ad libitum, except during the 6-h exposure or when urine was being collected; community tap-water
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 22.5°C
- Humidity: 31 to 52%
- Air changes: no data available
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From 09-OCT-2002 to 19-NOV-2002 (main study) and 03-DEC-2002 (recovery study)
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: clean air
Remarks on MMAD:
MMAD / GSD: Not applicable
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: whole-body exposure chambers constructed from stainless steel and glass, with an internal volume of 0.75 cm3
- Source and rate of air, method of conditioning air: no data available
- System of generating atmosphere: the test atmosphere was produced by metering the test liquid from a stainless steel pressure resistant cylinder, followed by dilution with clean air prior to the resultant vapour atmosphere passing into the exposure chamber.
- Temperature, humidity, pressure in air chamber, air flow rate, air change rate, treatment of exhaust air: no data available

TEST ATMOSPHERE
No data available

VEHICLE
No data available
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
No data available
Duration of treatment / exposure:
4 consecutive weeks
Frequency of treatment:
6 hours/day
Dose / conc.:
0 ppm (nominal)
Dose / conc.:
5 ppm (nominal)
Remarks:
corresponding to 5 ppm (analytical) or 0.033 mg/L (converted) or 33 mg/m3 (converted)
Dose / conc.:
15 ppm (nominal)
Remarks:
corresponding to 15 ppm (analytical) or 0.099 mg/L (converted) or 99 mg/m3 (converted)
Dose / conc.:
50 ppm (nominal)
Remarks:
corresponding to 51 ppm (analytical) or 0.338 mg/L (converted) or 338 mg/m3 (converted)
No. of animals per sex per dose:
5 per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the target exposure levels used in this study were selected based on a previous work with this compound performed in the laboratory (see ESR entitled "SIFREN - Repeat. dose tox.: 14-day inhal. - K1 2003HLS"). In this study, rats were exposed to target concentrations of 10, 30 and 100 ppm, where clinical effects were seen at the higher exposure levels.
- 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 2 weeks without treatment. Male and female rats (5/sex/dose) were exposed either to clean air (control) or 50 ppm of the test item daily for 4 weeks followed by the 2-week recovery period.
Observations and examinations performed and frequency:
Debilitated animals were observed carefully and, where necessary, isolated to prevent cannibalism. Animals judged in extremis were killed. Animals were also killed to prevent unnecessary or prolonged suffering. Where possible, blood samples were taken ante mortem and analysed for haematology and blood chemistry parameters. A complete necropsy was performed in all cases.

CAGE SIDE OBSERVATIONS: Yes
- Time schedule, cage side observations checked: animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages and cage-trays were inspected daily for evidence of ill-health amongst the occupants, such as loose faeces.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: detailed observations were recorded daily, on the days of exposure as follows: pre-exposure observation, during exposure, as each animal was returned to its home cage, and as late as possible in the working day. In addition, a more detailed weekly physical examination was performed on each animal to monitor general health. During the acclimatisation and recovery periods, observations of animals and their cages were recorded at least once per day.
Before treatment and during each week of treatment and week 2 of recovery period, a detailed physical examination and arena observations were performed on each animal. Examinations were performed at approximately the same time of day.
After removal from home cage, animals were assessed for physical condition and behaviour during handling and after being placed in standard arena. Particular attention was paid to possible signs of neurotoxicity such as convulsions, tremors and abnormalities of gait or behaviour. Attention was also given to the detection of audible respiratory noise.

BODY WEIGHT: Yes
- Time schedule for examinations: the weight of each rat was recorded 1 week prior to the start of exposure (week -1), on the day that treatment started (day 0), weekly throughout the exposure and recovery periods, and before necropsy.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes; the weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded 1 week prior to the start of exposure (week -1), and weekly throughout the exposure and recovery periods. From these records the mean weekly consumption per animal (g/animal) was calculated for each cage.

FOOD EFFICIENCY: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: the quantity of water consumed by each cage of rats was recorded daily, commencing 1 week prior to the start of exposures and throughout the exposure and recovery periods.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to the start of exposures and during week 4 of treatment
- Dose groups that were examined: all animals before the beginning of exposures and animals from groups 1 (control) and 4 (high dose) during week 4 of treatment
As no treatment-related changes were observed, the examination was not performed during the recovery phase or for animals of groups 2 and 3.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to sacrifice following the last day of exposure and the last day of the recovery period
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes; overnight starvation
- How many animals: all animals of control and treated groups
- Parameters checked: haematocrit (Hct); haemoglobin (Hb); red blood cell count (RBC); mean cell haemoglobin (MCH); mean cell haemoglobin concentration (MCHC); mean cell volume (MCV); total white cell count (WBC); differential WBC count (neutrophils (N), lymphocytes (L), eosinophils (E), basophils (B), monocytes (M), large unstained cells (LUC)); platelet count (Plt); reticulocyte count (Retic); blood cell morphology/type; prothrombin time (PT); activated partial thromboplastin time (APPT); cellularity (cellular), distribution (Distrib) of cell types and cell morphology (Morp) of tibial bone marrow samples

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: immediately prior to sacrifice following the last day of exposure and the last day of the recovery period
- Animals fasted: Yes; overnight starvation
- How many animals: all animals of control and treated groups
- Parameters checked: alkaline phosphatase (ALP); alanine aminotransferase (ALT); aspartate aminotransferase (AST); gamma-glutamyl transpeptidase (gGT); creatine phosphokinase (CPK total); total bilirubin (Bili); urea; creatinine (Creat); glucose (Gluc); total cholesterol (Chol); triglylcerides (Trig); sodium (Na); potassium (K); chloride (Cl); calcium (Ca); inorganic phosphorus (Phos); Total protein (total Prot); albumin (Alb); α1 globulin (a1); α2 globulin (a2); β globulin (Beta); γ globulin (Gamma); albumin/globulin ratio (A/G ratio)

URINALYSIS: Yes
- Time schedule for collection of urine: during week 4 of treatment and week 2 of recovery
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes; from approximately 17:40 hours (week 4 of treatment) and 16:30 hours (week 2 of recovery) until approximately 07:30 hours the following day
- Parameters checked: appearance (App); volume (Vol); pH; specific gravity (SG); protein (Prot); sodium (U-Na); potassium (U-K); chloride (U-Cl); glucose (Gluc); ketones (Keto); bile pigments (Bili); heam pigments (Blood); epithelial cells (Epi); leucocytes (Leuc); erythrocytes (RBC); crystals (Cryst); spermatozoa and precursors (Sperm); casts (Casts); other abnormal components (Abn.)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during week 4 of treatment, as well as during week 2 of recovery.
- Dose groups that were examined: all animals of control and treated groups were investigated. However, animals were not necessarily all tested on the same day, but the number of animals was balanced across the groups on each day of testing.
- Battery of functions tested: sensory activity (i.e., approach response, touch response, auditory startle reflex, tail pinch response), grip strength (i.e., forelimb and hindlimb), motor activity

OTHER:
* URINARY FLUORIDE: following assessment of the above urinary parameters, the residual urine samples were kept at approximately -20°C for further analysis of urinary fluoride by ion specific electrode.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes; animals killed during the study and those surviving until the end of the scheduled treatment or recovery period were killed by an intraperitoneal injection of pentobarbitone sodium followed by exsanguination from the brachial arteries. All animals were then subjected to a detailed necropsy. A full macroscopic examination of tissues was performed. The cranial roof was removed to allow observation of brain, pituitary gland and cranial nerves. After ventral mid-line incision, the neck and associated tissues and the thoracic, abdominal and pelvic cavities and their viscera were exposed and examined in situ. The requisite organs were weighed and external and cut surfaces of organs and tissues were examined. Any abnormality in the appearance or size of any organ and tissue was recorded and the required tissue samples preserved in appropriate fixative.

HISTOPATHOLOGY: Yes; testes and epididymides were fixed in Bouin's solution and eyes were fixed in Davidson's fluid, prior to transfer of these tissues to 70% industrial methylated spirit. Samples (or the whole) of the other tissues listed below from main study and recovery animals were preserved in 10% neutral buffered formalin, dehydrated, embedded in paraffin wax, sectioned at 4- to 5-µm thickness and stained with hematoxylin and eosin, except the testes which were stained using a standard periodic acid/Schiff (PAS) method:
Adrenals - cortex and medulla
Aorta - thoracic
Brain - cerebellum, cerebrum and midbrain
Caecum
Colon
Duodenum
Epididymides
Eyes
Femur with joint - longitudinal section through the joint
Harderian glands
Head (not examined histologically but retained against any requirement for microscopic examination)
Heart - including cortex, medulla and papilla regions
Ileum
Jejunum
Kidneys
Lachrymal glands
Larynx - 2 levels
Liver - section from all major lobes
Lungs - section from all major lobes, including bronchi
Lymph nodes - mandibular, mesenteric, tracheo-bronchial
Mammary area - caudal (not examined histologically but retained against any requirement for microscopic examination)
Nasal turbinates - 3 levels
Nasopharynx
Oesophagus
Optic nerves (not examined histologically but retained against any requirement for microscopic examination)
Ovaries
Pancreas
Pituitary
Prostate (not examined histologically but retained against any requirement for microscopic examination)
Rectum
Salivary glands - submandibular/sublingual
Sciatic nerves
Seminal vesicles
Skeletal muscle - thigh
Skin (not examined histologically but retained against any requirement for microscopic examination)
Spinal cord - transverse and longitudinal section at the cervical, lumbar and thoracic levels
Spleen
Sternum - including bone marrow
Stomach - including keratinised, glandular and antrum in sections
Testes
Thymus
Thyroid - including parathyroids in section where possible
Tongue (not examined histologically but retained against any requirement for microscopic examination)
Trachea - including bifurcation
Urinary bladder
Ureter (not examined histologically but retained against any requirement for microscopic examination)
Uterus and cervix
Vagina (not examined histologically but retained against any requirement for microscopic examination)
For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required for microscopic pathology.
Samples of any abnormal tissues were also retained and processed for examination. In those cases where a lesion was not clearly delineated, contiguous tissue was fixed with the grossly affected region and sectioned as appropriate.

Microscopic examination was performed as follows: all tissues preserved for examination were investigated for all main study animals of groups 1 (control) and 4 (high dose) sacrificed on completion of the scheduled treatment period, and for all animals killed or dying during the study. Microscopic examinations for all main animals of groups 2 and 3 and all recovery animals was limited to adrenals, epididymides, femur, heart, kidneys, liver, lungs, spleen and testes.
Tissues reported at macroscopic examination as being grossly abnormal were examined for all animals of main and recovery studies.
In addition, the following tissues, considered to exhibit a reaction to treatment at the high exposure level, were examined for all animals of groups 2 and 3 in the main study and in all animals of recovery study: pancreas (males only) and nasal turbinates.
Other examinations:
ORGAN WEIGHTS: the following organs, taken from each animal killed after 4 weeks of treatment or 2 weeks of recovery, were dissected free of adjacent fat and other contiguous tissue and the weighs were recorded:
Adrenals
Brain
Epididymides
Heart
Kidneys
Liver
Lungs including bronchi
Ovaries
Spleen
Testes
Thymus
Uterus with cervix
Bilateral organs were weighed together.
Statistics:
All statistical analyses were carried out separately for males and females.
The following data types were analysed at each time point separately:
- Grip strength and motor activity
- Bodyweight (using gains)
- Blood chemistry, haematology and urinalysis (including urinary fluoride)
- Organ weights (absolute and adjusted for terminal bodyweight)
- Pathological findings (for the number of animals with and without each finding)
For categorical data, including pathological findings, the proportion of animals was analysed using Fisher's exact test for each treated group vs. the control.
For continuous data, Bartlett's test was first applied to test the homogeneity of variance between groups. Using tests dependent on the outcome of Bartlett's test, treated groups were then compared with the control group, incorporating adjustment for multiple comparisons where necessary.
Clinical signs:
no effects observed
Description (incidence and severity):
there were no clinical signs observed at any time prior to exposure, during exposure, post-exposure or during the weekly physical examination and arena observations.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
there was one unscheduled death.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
see in "Details on results"
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
see in "Details on results"
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
see in "Details on results"
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
see in "Details on results"
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
see in "Details on results"
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
see in "Details on results"
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
see in "Details on results"
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
see in "Details on results"
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see in "Details on results"
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
One female of group 4 was sacrificed following exposure on day 14 of the main study due to suspected broken bone. There were no clinical signs observed at any time prior to exposure, during exposure, post- exposure or during the weekly physical examination and arena observations. Brown staining on head and red/brown staining around eyes were noted for a small proportion of rats on a number of occasions following exposure.

BODY WEIGHT AND WEIGHT GAIN (see Table 3 in "Any other information on results incl. tables")
The loss in bodyweight seen for some groups at week 4 of exposure period was considered to be caused by overnight deprivation of food, on day 28, prior to bleeding for haematology and blood chemistry investigations.
Examination of weight gain up to week 3 of exposure period indicated a significant decrease in weigh t gain for females of group 4 when compared to controls. During the recovery period, bodyweights at recovery week 2 were again affected by overnight starvation. At week 1 of recovery, bodyweight gains of groups 1 and 4 were similar.


FOOD AND WATER CONSUMPTION (see Table 3 in "Any other information on results incl. tables")
The reduction in food and water consumption observed during week 4 of exposure period and week 2 of recovery period was considered attributable to the overnight deprivation of food, on day 28 of exposure period and day 14 of recovery period, respectively, prior to bleeding for haematology and blood chemistry investigations and was considered not to be treatment-related.
Examination of food and water consumption up to week 3 of exposure period and at week 1 of recovery indicated no statistically significant change in all treated groups.

OPHTHALMOSCOPIC EXAMINATION
There were no significant abnormalities observed during the pre-treatment ophthalmic examination, as well as during the examination at week 4 of exposure period.

HAEMATOLOGY
In peripheral blood, differences between groups were generally small, independent of dosage and largely inconsistent between sexes. They were considered not to be of toxicological importance. Visual assessment of bone marrow smears for cellularity, distribution and morphology indicated that there were no treatment-related effects on bone marrow.

CLINICAL CHEMISTRY (see Table 4 in "Any other information on results incl. tables")
Blood urea levels were higher than control in all treated groups following 4 weeks of exposure, however, this was not dose-related. Statistical significance was achieved for male rats of groups 3 and 4 and female rats of group 4. At the end of recovery period, urea levels were similar for groups 1 and 4.

Other differences were generally small, independent of dosage and largely inconsistent between the sexes. They were considered unlikely to be of toxicological importance.

URINALYSIS (see Table 5 in "Any other information on results incl. tables")
Group mean urine volume was lower than control for all treated groups and although the effect was greatest in group 4 there was no clear dose-relationship. None of the differences were statistically significant. The reduced urine volume in group 4 was associated with increased specific gravity, urinary K and Cl concentrations, which were statistically significant for females. The effect on urine volume and electrolyte concentration was not apparent after the 2-week recovery period and was therefore considered likely to be a treatment-related effect.
Other differences were generally small, independent of dosage and largely inconsistent between the sexes. They were considered unlikely to be of toxicological importance.

NEUROBEHAVIOUR (see Table 2 in "Any other information on results incl. tables")
There were no treatment-related effects on sensory reactivity and motor activity.
In contrast, a significant increase in hindlimb grip strength was evident for females of group 4 following 4 weeks of exposure. Moreover, a non-significant increase in forelimb grip strength was also evident for females of groups 3 and 4 following 4 weeks of exposure. A trend towards increased hindlimb grip strength was seen for males and females of group 4 following the 2-week recovery period; an upward trend in fore limb grip strength was also observed for females of group 4 at the end of recovery period. However, these increases were very slight.
The apparent increases in grip strength noted at the end of exposure and recovery periods were considered not to be treatment-related but rather to be due to natural variation. In addition, due to the isolated nature of the finding (i.e., in the absence of other sensory reactivity and motor activity treatment-related findings) and as the direction of the change (increase) for grip strength at the end of exposure period occurred while bodyweight was experiencing reduced gains, it was considered unlikely that the increase in grip strength was treatment-related.

ORGAN WEIGHTS (see Table 6 in "Any other information on results incl. tables")
Kidney weights (absolute and bodyweight adjusted values) for test groups were greater than control at week 4 of exposure period. Statistical analysis of the bodyweight adjusted values indicated that statistical significance was achieved for all female groups and males of group 4.
Liver weights (absolute and bodyweight adjusted values) were slightly higher than control values for all test groups but the effect was not dose-related, since weights of group 2 were higher than those of groups 3 and 4. In the absence of microscopic findings, it was considered that this increase in liver weight was of no toxicological importance. Statistical significance was achieved for bodyweight adjusted values for all female groups and males of group 4.
At the end of recovery period, kidney weights (absolute and bodyweight adjusted values) for females of group 4 remained higher than control.
All other statistically significant differences were considered not to be of toxicological significance.

GROSS PATHOLOGY
The macroscopic examination performed at termination revealed no lesions attributable to treatment. The incidence and distribution of all findings were considered to fall within the expected background range of macroscopic changes.

HISTOPATHOLOGY: NON-NEOPLASTIC (see Table 7 in "Any other information on results incl. tables") The following microscopic changes were considered to be treatment-related:
- Nasal turbinates: in the respiratory epithelium, an increased incidence of pseudogland formation was seen in males of group 4 compared to controls. Pseudogland formation was considered a minor response and was probably related to local goblet cell hyperplasia. After 2 weeks of recovery, the respiratory epithelium of nasal turbinates showed no more impairment, demonstrating a complete recovery.
- Kidneys: cortical tubules with hyaline droplets were noted in all male treated groups but not in any controls. After 2 weeks of recovery, only a single male in group 4 showed cortical tubules with hyaline droplets in the kidney.

In addition, an increased incidence and severity of acinar cell apoptosis were noted in males of group 4, compared with controls, but not in females of this group. As this finding was seen in controls of both sexes and the severity was not high compared to controls, its relationship to treatment was uncertain. After 2 weeks of recovery, no acinar cell apoptosis was seen in the pancreas of any animal, demonstrating a complete recovery.

No histological findings were observed in the liver to explain the increased organ weights recorded in high dose groups.

All other microscopic findings in animals from main and recovery studies were considered to be incidental and of no toxicological importance.

OTHER FINDINGS (see Table 5 in "Any other information on results incl. tables")
* URINARY FLUORIDE: a dose-related increase in urinary fluoride was evident in both sexes of all test groups following 4 weeks of exposure and attained statistical significance for groups 3 and 4. An increase in urinary fluoride was still evident for rats of group 4 following the 2-week recovery period. This was statistically significant but markedly less than that measured at week 4 of exposure period.
Key result
Dose descriptor:
NOAEC
Effect level:
5 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Critical effects observed:
yes
Lowest effective dose / conc.:
5 ppm (nominal)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Critical effects observed:
yes
Lowest effective dose / conc.:
51 ppm (nominal)
System:
respiratory system: upper respiratory tract
Organ:
nasal cavity
Treatment related:
yes
Dose response relationship:
yes

The pseudogland formation observed in the nasal turbinates of the nasal cavity of animals treated for 28 -day by inhalation was no longer present following the 2 week-recovery period.

The hyaline droplet formation in the cortical tubules of kidneys of male rats was reversed after the 2 week-recovery period (except 1 animal) and may be related to accumulation of alpha-2u globulin, an effect specific to male rats, and which is usually not considered relevant to humans.

The increased body weight-adjusted liver weight observed at the end of the 28-day treatment period was no longer significant after the 2-week recovery period and was considered adaptative in nature.

Conclusions:
The only effects seen in rats exposed to 5 ppm of test item were a slight increase in hyaline droplet formation in the cortical tubules of kidneys (males only) and an increase in urinary fluoride (males and females). The former was considered to be of no significance to man and the latter was a consequence of the metabolic breakdown of the test substance.
At 15 and 51 ppm the adverse effects included microscopic changes in the nasal turbinates (statistically significant in males at 51 ppm) and a slightly reduced weight gain (statistically significant in females at 51 ppm). Based on these observations, the no-observed-adverse-effect-level, for male and female rats, was considered to be 5 ppm (eq. to 33 mg/m3).
Executive summary:

The objective of this study was to assess the systemic toxicity potential of the test substance to Crl:CD (SD)IGS BR rats by repeated inhalation administration over a period of 4 weeks, followed by a 2-week recovery period. The study was conducted according to OECD guideline 412 and in compliance with GLP.

Three groups of male and female rats (5/sex/dose) were exposed to a vapour of the test substance by inhalation at nominal concentrations of 5, 15, and 50 ppm, 6 hours a day for 4 consecutive weeks using a whole-body exposure system. A similarly constituted control group was exposed to clean air only. A further 5 male and 5 female rats were assigned to each of the control and high dose groups for a 2-week recovery period following the 4-week treatment period. During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity, grip strength, motor activity, bodyweight, food and water consumption, ophthalmic examination, urinary fluoride, organ weight, macroscopic and microscopic pathology investigations were undertaken.

The study mean analysed concentrations of the test item were 5, 15 and 51 ppm for the low, intermediate and high dose groups, respectively. These concentrations were equivalent to 0.033, 0.099 and 0.338 mg/L (at 25°C and 760 mmHg) for groups 2 to 4, respectively.

There was one unscheduled death. A female of group 4 was sacrificed on humane grounds following exposure on day 14 due to a suspected broken bone. There were no treatment-related clinical signs observed pre-exposure, during exposure, post-exposure or during the weekly physical examination and arena observations.

There were no treatment-related effects on sensory reactivity, grip strength or motor activity.

Reduced mean bodyweight gains were evident for rats of groups 3 and 4 during the exposure period and attained statistical significance for female rats of group 4.

A slight reduction in food and water consumption was evident for females of group 3, and males (food only) and females (food, water) of group 4 during the exposure period. A slight reduction in food and water consumption was still evident for females of group 4 during the recovery period.

There were no significant abnormalities observed during the pre-treatment ophthalmic examination. There were no treatment-related effects noted during the examination in week 4.

There were no treatment-related effects on haematology parameters including those examined in the bone marrow.

Blood urea levels were higher than control in all treated groups following 4 weeks of exposure and attained statistical significance for male rats of groups 3 and 4, and females of group 4. At the end of recovery period, urea levels were similar for groups 1 and 4. The elevation of blood urea levels was considered likely to be a consequence of changes in renal function and was considered to be of no toxicological importance due to the lack of a dose-relationship.

Group mean urine volume was lower than control for all treated groups. The reduced urine volume in rats of group 4 was associated with increased specific gravity, urinary potassium concentration and urinary chloride concentration, which were statistically significant for females. It is possible that this was an anomalous finding as the usual effect of fluoride containing compounds is polyuria. It is possible that the reduced urine output during the overnight collection period was consequence of increased urine output during the exposure period.

A dose-related increase in urinary fluoride was evident in both sexes of all treated groups following 4 weeks of exposure and attained statistical significance for groups 3 and 4. Urinary fluoride levels for rats of group 4 remained higher than controls following 2 weeks of recovery and remained statistically significant. The presence of fluoride ion in urine is commonly associated with the metabolic breakdown of fluoride-containing compounds such as the test substance.

Kidney and liver weights for test groups were greater than controls following 4 weeks of exposure and attained statistical significance for groups 3 and 4. Kidney weights of females from group 4 remained higher than controls following 2 weeks of recovery. The changes in kidney weight were considered to be treatment-related and potentially associated with changes in renal function. In the absence of microscopic findings, it was considered that the increase in liver weight was of no toxicological importance.

The macroscopic examinations performed at termination revealed no treatment-related abnormalities. At the end of the exposure period, the microscopic examinations revealed treatment-related increased incidence of pseudogland formation in the respiratory epithelium in the nasal turbinates of males from group 4 and increased incidences of cortical tubules with hyaline droplets in the kidneys of males from all treated groups. At the end of the recovery period, the microscopic examinations revealed recovery to control level for pseudogland formation in the nasal turbinates of the respiratory epithelium. Almost complete recovery had occurred for cortical tubules with hyaline droplets of the kidney. The presence of hyaline droplets in cortical tubules in the kidneys of male rats is a common non-specific response to a range of chemicals and is considered to be due to exacerbated accumulation of alpha-2µ-globulin in the kidneys and probably of no significance to man.

At 15 and 51 ppm the adverse effects included microscopic changes in the nasal turbinates of minor significance and a reduced weight gain. Partial or complete recovery from all the effects seen in this study was considered to have been demonstrated. In this study the no-observed-adverse-effect-level, for male and female rats, was considered to be 5 ppm (i.e. 33 mg/m3).

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
16-JUL-2002 to 14-AUG-2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
yes
Remarks:
adapted for Dose-range finding study
Principles of method if other than guideline:
Deviations: the exposure duration was shortened to 14 days as the test was a preliminary study (Dose-range finding study).
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Crl:CD (SD)IGS BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (Uk) Ltd / Margate, Kent / ENGLAND
- Age at study initiation: 49 to 56 days of age
- Weight at study initiation: 247 to 275 g for males; 189 to 222 g for females
- Fasting period before study: no data available
- Housing: inside a rodent facility with a positive pressure, five of one sex per stainless steel cage (North Kent Plastic; 35 cm x 55 cm x 25 cm)
- Diet: ad libitum, except during the 6-h exposure or when urine was being collected; Rat and Mouse No. 1 Maintenance Diet (Special Diet Services / Witham / ENGLAND)
- Water: ad libitum, except during the 6-h exposure or when urine was being collected; community tap-water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21 to 23.5°C
- Humidity: 36 to 59%
- Air changes: no data available
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From 17-JUL-2002 to 07-AUG-2002
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
clean air
Remarks on MMAD:
MMAD / GSD: Not applicable
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: exposure chambers constructed from stainless steel and glass, with an internal volume of 0.75 m3
- Source and rate of air, method of conditioning air: no data available
- System of generating atmosphere: the test atmosphere was produced by metering the test liquid from a stainless steel pressure resistant cylinder, followed by dilution with clean air prior to the resultant vapour atmosphere passing into the exposure chamber.
- Temperature, humidity, pressure in air chamber, air flow rate, air change rate, treatment of exhaust air: no data available

TEST ATMOSPHERE
No data available

VEHICLE
No data available
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
No data available
Duration of treatment / exposure:
14 consecutive days
Frequency of treatment:
6 hours/day
Dose / conc.:
10 ppm (nominal)
Remarks:
corresponding to 11 ppm (analytical)
Dose / conc.:
30 ppm (nominal)
Remarks:
corresponding to 30 ppm (analytical)
Dose / conc.:
100 ppm (nominal)
Remarks:
corresponding to 99 ppm (analytical)
No. of animals per sex per dose:
5 per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the exposure levels used in this study were selected based on a previous work with this compound performed in the laboratory (see ESR entitled "SIFREN - Repeat. dose tox.: 7-day inhal. - K1 2003HLS"). In this study, rats were exposed to target concentrations of 20, 70 and 200 ppm, where clinical effects were seen at the higher exposure levels.
- Rationale for selecting satellite groups, post-exposure recovery period in satellite groups: no data available
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages and cage-trays were inspected daily for evidence of ill-health amongst the occupants, such as loose faeces.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: detailed observations were recorded daily, on days of exposure as follows: pre-exposure observation, during exposure, as each animal was returned to its home cage, and as late as possible in the working day. In addition, a more detailed weekly physical examination was performed on each animal to monitor general health. During acclimatisation period, observations of animals and their cages were recorded at least once per day.

BODY WEIGHT: Yes
- Time schedule for examinations: the weight of each rat was recorded daily, commencing 1 week prior to the start of exposure, on the day that exposures commenced (day 0), daily throughout the exposure period, and before necropsy.


FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes; the weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded daily, commencing 1 week prior to the start of exposure, and daily throughout the exposure. From these records the daily consumption per animal (g/animal) was calculated for each cage.

FOOD EFFICIENCY: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: the quantity of water consumed by each cage of rats was recorded daily, commencing 1 week prior to the start of exposures until the end of the study.

OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: Yes
- Time schedule for collection of urine: during week 2 of treatment, overnight urine samples were collected from all animals from ca. 16.00 hours until ca. 07.30 hours the following day.
- Metabolism cages used for collection of urine, animals fasted: animals were deprived of food and water from ca. 16.00 hours and placed in an individual metabolism cage.
- Parameters checked: urinary fluoride by ion specific electrode

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes; animals surviving until the end of the scheduled treatment were killed by an intraperitoneal injection of pentobarbitone sodium followed by exsanguination from the brachial arteries. All animals were then subjected to a detailed necropsy. All external features and orifices were examined. The cranial roof was removed to allow observation of brain, pituitary gland and cranial nerves. After ventral mid-line incision, the neck and associated tissues and the thoracic, abdominal and pelvic cavities and their viscera were exposed and examined in situ. The requisite organs were weighed and external and cut surfaces of organs and tissues were examined. Any abnormality in the appearance or size of any organ and tissue was recorded and the required tissue samples preserved in appropriate fixative.

HISTOPATHOLOGY: Yes; samples (or the whole) of tissues listed below from all animals were preserved in 10% neutral buffered formalin, dehydrated, embedded in paraffin wax, sectioned at ca. 4 to 5-µm thickness and stained with hematoxylin and eosin:
Head (for nasal turbinates)
Kidneys - including cortex, medulla and papilla regions
Larynx
Liver - section from all main lobes
Lungs and bronchi - section from all major lobes
Nasal turbinates - 3 levels
Trachea - including bifurcation
For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required for microscopic pathology.
Samples of any abnormal tissues were also retained and processed for examination. In those cases where a lesion was not clearly delineated, contiguous tissue was fixed with the grossly affected region and sectioned as appropriate.
Other examinations:
ORGAN WEIGHTS: the following organs, taken from each animal killed after 14 days of treatment, were dissected free of adjacent fat and other contiguous tissue and the weighs were recorded:
Adrenals
Brain
Kidneys
Liver
Lungs with mainstem bronchi
Bilateral organs were weighed together.
Statistics:
All statistical analyses were carried out separately for males and females.
The following data types were analysed at each time point separately:
- Bodyweight (using gains)
- Urinary fluoride
- Organ weights (absolute and adjusted for terminal bodyweight)
- Pathological findings (for the number of animals with and without each finding)
For categorical data, including pathological findings, the proportion of animals was analysed using Fisher's exact test for each treated group vs. the control.
For continuous data, Bartlett's test was first applied to test the homogeneity of variance between groups. Using tests dependent on the outcome of Bartlett's test, treated groups were then compared with the control group, incorporating adjustment for multiple comparisons where necessary.
Clinical signs:
no effects observed
Description (incidence and severity):
there were no clinical signs observed at any time during the study.
Mortality:
no mortality observed
Description (incidence):
there were no unscheduled deaths.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
see in "Details on results"
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
see in "Details on results"
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, non-treatment-related
Description (incidence and severity):
see in "Details on results"
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
see in "Details on results"
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):
see in "Details on results"
Gross pathological findings:
no effects observed
Description (incidence and severity):
the macroscopic examinations performed at termination revealed no lesions attributable to treatment with the test item. The incidence and distribution of all findings were considered to fall within the expected background range of macroscopic changes.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see in "Details on results"
Histopathological findings: neoplastic:
no effects observed
Details on results:
BODY WEIGHT (Table 2 in "Any other information on results incl. tables")
After the first treatment day, males of group 4 lost weight; females of group 4 did not gain weight in that same period. Actual weight loss, attributable to treatment, was not seen after day 2.
At the end of treatment period, males of group 4 gained significantly less weight, compared to the control group.

FOOD CONSUMPTION (Table 2 in "Any other information on results incl. tables")
A reduction in food consumption was evident for rats of groups 3 and 4 during the exposure period.
The apparent marked reduction in food consumption observed on day 13 was considered attributed to the overnight deprivation of food for the collection of urine and was therefore unlikely to be treatment-related.

WATER CONSUMPTION (Table 2 in "Any other information on results incl. tables")
An increase in water consumption was evident for rats of group 4 during the exposure period and was marked for females of group 4. A slight reduction in water consumption was evident for males of group 3 during the same period.
The apparent marked reduction in water consumption observed on day 13 was considered attributed to the overnight deprivation of water for the collection of urine and was therefore unlikely to be treatment-related.

URINALYSIS (Table 3 in "Any other information on results incl. tables")
A treatment-related increase in urinary fluoride was evident across groups for both sexes, and attained statistical significance for females of group 2 and rats of both sexes from groups 3 and 4.

ORGAN WEIGHTS (Table 4 in "Any other information on results incl. tables")
Kidney weights of males from group 3 and male and female rats from group 4 were greater than control values. Liver and lung weights of females from group 4 were also higher than controls. These findings were considered to be treatment-related.

HISTOPATHOLOGY: NON-NEOPLASTIC (Table 5 in "Any other information on results incl. tables")
The following microscopic changes were considered to be treatment-related:
- Kidneys: slight or moderate tubular basophilia in the inner cortex and outer medulla (proximal straight tubules) was present in all male and female rats exposed to 100 ppm (group 4). Minimal tubular basophilia in the inner cortex and outer medulla (proximal straight tubules) was seen in a single female to 30 ppm (group 3).
- Lungs: minimal to slight prominent alveolar macrophages were present in the air control (group 1) and all exposure groups, with increased incidence prevalence in the groups 3 and 4, and an increased severity in group 4.
- Nasal turbinates: respiratory epithelial pseudogland formation was seen in males and females of groups 3 or 4.

In kidneys, minimal corticomedullary, medullary or papillary mineralisation was identified in males and females exposed to the test item; and medullary mineralisation was found in a single female air control. Intratubular mineralisation develops spontaneously in the kidneys of laboratory animals and is often an incidental finding in both sexes. Consequently, this minimal finding was not considered treatment-related.
All other microscopic findings were considered to be incidental and of no toxicological importance.
Dose descriptor:
NOAEC
Effect level:
10 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
NOAEC
Effect level:
11 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Critical effects observed:
yes
Lowest effective dose / conc.:
30 ppm (analytical)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Critical effects observed:
yes
Lowest effective dose / conc.:
30 ppm (analytical)
System:
respiratory system: upper respiratory tract
Organ:
nasal cavity
Treatment related:
yes
Dose response relationship:
yes
Conclusions:
Under the conditions of this study, the substance induced significant effects at the two highest doses (30 and 99 ppm) in both males and females, while the only noticeable effect at the lowest dose (11 ppm) was the presence of alveolar macrophages in the lungs of females, although without statistical significance. Based on the results of this study, the concentrations of 5, 15, and 50 ppm were selected for the 28-day inhalation study.
Executive summary:

The objective of this study was to assess the systemic toxicity potential of the test substance to Crl:CD (SD)IGS BR rats by repeated inhalation administration over a period of 14 days. The study was conducted according to a protocol based on OECD guideline 412, and in compliance with GLP.

Three groups of male and female rats (5/sex/dose) were exposed to a vapour of the test substance by inhalation at nominal concentrations of 10, 30 and 100 ppm, 6 hours a day for 14 consecutive days using a whole-body exposure system. A similarly constituted control group was exposed to clean air only. During the study, clinical condition, detailed physical observations, bodyweight, food and water consumption, urinary fluoride, organ weight, macroscopic and microscopic pathology investigations were undertaken.

The study mean analysed concentrations of test item were 11, 30 and 99 ppm for the low, intermediate and high dose groups, respectively.

There were no unscheduled deaths and there were no clinical signs observed at any time during the study.

After the first treatment day, males of group 4 (high dose) lost weight; females of group 4 did not gain weight in that same period. Actual weight loss attributable to treatment was not seen after day 2. At the end of the treatment period, males of group 4 gained significantly less weight, compared to the control group.

A reduction in food consumption was evident for rats of groups 3 and 4 and an increase in water consumption was evident for rats of group 4. A slight reduction in water consumption was evident for males of group 3 during the same period.

A treatment-related increase in urinary fluoride was evident across treated groups of both sexes, and attained statistical significance for females of group 2 and rats of both sexes from groups 3 and 4.

There were treatment-related differences in kidney weight for males of group 3 and male and female rats from group 4, and in liver and lung for females of group 4.

The macroscopic examinations performed at termination revealed no treatment-related abnormalities. The microscopic examinations revealed in the kidneys, slight or moderate tubular basophilia in the inner cortex and outer medulla (proximal straight tubules), which was seen in all rats of group 4, and was noted at a minimal level in a single female rat of group 3. This finding correlated with the increase in the mean kidney weight of rats from group 4. The effect in group 3 was only marginal, and there was no effect observed in group 2 (low dose). In the lungs, prominent alveolar macrophages were identified in the majority of animals exposed to the test substance and in a single male and two females of group 1 (control). Animals of groups 2 and 3 demonstrated a minor increase in incidence, but not severity, with increasing exposure level. Rats from group 4 had increased prevalence and severity. In the nasal turbinates, minimal or slight respiratory epithelial pseudogland formation was found in groups 3 and 4, with evidence of dose-relationship in the group prevalences. This finding was not seen in group 2. Additional findings in the kidney were not considered treatment-related and all other microscopic findings were considered to be incidental and of no toxicological importance.

In view of the findings summarised above, target concentrations of 5, 15 and 50 ppm were considered suitable for a subsequent 28-day study. In addition to bodyweight and food consumption effects, these target concentrations for the 28-day study were selected specifically with relation to water consumption and urinary fluoride, together with the microscopic findings mentioned above.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
99.4 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
One key study in Sprague-Dawley rats (28-day inhalation) showed pseudogland formation (minimal grade) in the respiratory epithelium. This was considered a minor response. The local effects on nasal turbinates were also observed in a 14-day study at highest concentration (99 ppm). However, in the OECD421, the effects were however not observed in Wistar rats exposed at the same concentrations and for the same duration (or longer in females) as the 28-day study. The susceptibility may vary between rat strains and species. Therefore the dose descriptor for the risk assessment was selected using a weight of evidence approach considering data from all three repeated dose toxicity studies. As statistical significance was reached at 51 ppm (28-day males) and 99 ppm (14-day males and females) with minor effects, and no effects were observed in the OECD421 Wistar rats (males treated for 28 days, and females for 40 to 47 days) at up to 50 ppm, the exposure concentration of 15 ppm was considered as starting point for the DNEL derivation (local effects, long-term).

Mode of Action Analysis / Human Relevance Framework

There are insufficient data to conclude on the mode of action and relevance to human.

Effects on the kidney were mainly organ weight changes. The effects observed in kidney were either specific of male rats (hyaline droplets in cortical tubules in male rats of the 28-day study were considered related to alpha 2u globulin and not relevant to human) or minimal effects on the cortical tubules, not consistently observed in the various studies.

Information on the potential metabolic pathways associated with halo- and fluoroalkenes seem to indicate kidney is the target organ. The relevance of these findings to human is currently unknown due to the complexity of the multiple metabolic pathways and steps leading to a potential renal toxicity.

Additional information

Justification for classification or non-classification

The systemic adverse effects observed in the available repeated dose toxicity studies were relatively minor at the concentrations tested and consisted mainly of a slightly lower body weight, and greater kidney or liver weight (relative and/or absolute) indicative of metabolic activity. No major histopathological findings were observed. A similar concentration range was then selected for the reproductive/developmental screening due to the uncertainty of effects in pregnant female rats.

However, hexafluorobutadiene is a fluoroalkene and glutathione-dependent metabolic activation taking place in the liver and/or in the kidney have been proposed for haloalkenes to explain their specific nephrotoxicity. The effects seem dependent on the nature and ratio of conjugates (e.g. cysteine S-conjugates) and reactions taking place in the renal tubules. In the absence of further metabolic characterisation and nature of effects at higher concentrations, uncertainty remains on the actual nephrotoxic potential of hexafluorobutadiene metabolites because a number of variables may influence the severity of effects in the ultimate target organ. Despite the limited effects reported in rats at the exposure levels used in the present dataset, it is proposed to consider Hexafluorobutadiene as STOT RE category 2 as a protective approach, on the suspicion of a common mechanism shared with other fluoroalkenes.