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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.This study is used for read-across and therefore has been assigned a reliability of 2 (reliable with restrictions). Otherwise the study has a reliability of 1 (reliable without restriction).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
yes
Remarks:
Serum gamma glutamyl transpeptidase and ornithine decarboxylase were not evaluated.
Qualifier:
according to
Guideline:
other: EEC Methods for the Determination of Toxicity Method B.8 Directive 92/69/EEC
Deviations:
no
Remarks:
Serum gamma glutamyl transpeptidase was not evaluated.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Purity: 99.94%

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: approximately 8 weeks
- Weight at study initiation: between 235 and 300 grams (males); between 174 and 217 grams (females)
- Fasting period before study: no
- Housing: solid bottom caging with Sheperd’s(TM) Cob + PLUS(TM) as bedding and enrichment.
- Diet (e.g. ad libitum): ad libitum except during exposure and fasting periods prior to necropsy
- Water (e.g. ad libitum): ad libitum except during exposure
- Acclimation period: at least 6 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 23° C
- Humidity (%): 43 to 70%
- Air changes (per hr): 6 to 10
- Photoperiod (hrs dark / hrs light): 12-hours dark / 12-hours light

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Stainless steel and glass (NYU style) with a nominal internal volume of 750 L (test chambers) or 1000 L (control chamber). A tangential feed inside the chamber promoted uniform chamber distribution of the test atmosphere. The chamber volume was chosen so that the total body volume of the test animals did not exceed 5% of the chamber volume
- Method of holding animals in test chamber: Animals were individually placed in stainless steel, wire-mesh cages and exposed, whole-body, inside the exposure chamber.
- Source and rate of air: Houseline
- Method of conditioning air: Not reported
- System of generating test atmosphere: Chamber atmospheres for the low level chamber were generated by vaporisation of the test substance in air by passing air over the test substance contained in an impinger. Chamber atmospheres for the intermediate- and high-level chambers were generated by flash evaporation of the test substance in air with a heated, glass mixing flask. The test substance was metered into the mixing flask with a syringe infusion pump. The mixing flask was heated to approximately 175-180°C to vaporise the test substance. Houseline generation air, metered to the mixing flask by a mass flow controller, carried the vaporised test substance into a filtered and conditioned dilution air supply leading to the exposure chamber. The filtered and conditioned dilution air supply was monitored with a thermoanemometer. Heat tape was used to heat the glass connecting tube from the heated flask to the top of the chamber to ensure the test substance remained in the vapour state. Chamber atmospheres for the control chamber were generated similarly to the intermediate- and high-levels chambers except without the infusion pump or heat tape on the glass connecting tube from the heated flask to the chamber. The heating mixing flask, heat tapes, and dilution air were controlled and monitored by the Inhalation Toxicology Automated Data System. Chamber concentrations of test substance were controlled by varying the test substance feed rate to the heated flask, varying the amount of generation air, and/or varying the amount of dilution air.
- Temperature, humidity, pressure in air chamber: Temperature ranged from 17 to 23°C (targeted at 20-24°C). Relative humidity ranged from 43 to 70% (targeted at 30-70%). Oxygen concentration was 21%. Air pressure was not reported.
- Air flow rate: Not reported
- Air change rate: 6 to 10 air changes per hour
- Treatment of exhaust air: Exhausted through an exhaust manifold prior to discharge into the fume hood

TEST ATMOSPHERE
- Brief description of analytical method used: Atmospheric concentrations of the test substance were determined by gas chromatography at approximately 60-minute intervals during the daily exposures.
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Known volumes of chamber atmosphere were drawn from the sampling port through a sampling train consisting of a midget, fritted, glass impinger containing acetone as the collection medium. Aliquots of impinger solution were injected into a gas chromatograph equipped with a flame ionisation detector. All samples were chromatographed isothermally at 95°C on a fused silica glass column with a 30-meter length, 530 μm inside diameter, and 3.0 μm film thickness. The atmospheric vapour concentration of the test substance was determined from a standard curve derived from liquid standards. Standards were prepared weekly by quantitative dilution of the liquid test substance in acetone. Upon completion of the exposures, sample results were transferred to the Inhalation Reporting and Analysis System, which collated sample calculations.
Duration of treatment / exposure:
6 hours/day
Frequency of treatment:
23 exposures over a 4-week period (weekends excluded)
Doses / concentrations
Remarks:
Doses / Concentrations:
0 ± 0, 1.0 ± 0.0084, 10 ± 0.057 or 100 ± 0.33 ppm (mean ± S.E.M.)
Basis:
analytical conc.
No. of animals per sex per dose:
20 males and 20 females in the control and 100 ppm dose groups; 10 males and 10 females in the 1 and 10 ppm dose groups.
The first 10 male and female rats per dose level were designated for sacrifice at the end of the exposure period. The remaining 10 male and female rats per dose level were designated for sacrifice at the end of the 1-month recovery period.
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: A previously performed acute inhalation study indicated low toxicity where the LC50 for the test substance was greater than 5.2 mg/L but less than 9.9 mg/L, where the 5.2 and 9.9 mg/L exposure atmospheres contained both vapour and aerosol phases of the test substance. Results from the acute and a one-week subchronic inhalation exposure with the test substance indicated that the maximum practically attainable vapour-only exposure atmosphere was approximately 100 ppm. Exposure of male and female rats to 100 ppm for 5 days resulted in minimal body weight losses that were approximately 6-8% less than the control animals and microscopic pathological findings that were consistent with exposure to a fluorine-containing test material. Therefore, the high concentration of the test substance for the 4-week study was selected to be 100 ppm with the intermediate and low concentrations being log orders of magnitude less than the high concentration.
- Rationale for animal assignment (if not random): Animals of each sex were selected for use on study from those released from quarantine based on adequate body weight gain and freedom from any ophthalmology abnormalities or clinical signs of disease or injury. They were distributed by computerised, stratified randomisation into study groups so that there were no statistically significant differences among group body weight means within a sex. The weight variation of selected rats did not exceed ± 20% of the mean weight for each sex. The first 10 rats in each group were designated for subchronic toxicity.
- Rationale for selecting satellite groups: The remaining rats in the control and high groups were designated for recovery.
- Post-exposure recovery period in satellite groups: At the conclusion of the 4-week exposure period, all animals designated for recovery were allowed to recover for approximately 4 weeks.
Positive control:
None.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Examinations to detect moribund or dead animals and abnormal behaviour and appearance among animals were conducted at the time of loading the animals into the chamber on exposure days, and at least once daily on non-exposure days.

GENERAL CLINICAL OBSERVATIONS: Yes
During the daily exposures, the response to an alerting stimulus was determined for the animals as a group within each exposure chamber. The alerting response was determined prior to the initiation of each exposure, 3 times during exposure, and after the conclusion of each exposure just prior to animal removal from the exposure chamber. Study technicians judged whether the group of animals within a given exposure chamber displayed a normal, diminished, enhanced, or absent alerting behaviour in response to a standardised auditory stimulus. In addition, study technicians observed the animals for clinical signs 3 times. Clinical signs observed were recorded for the animals collectively within a chamber, since individual animal identification was not visible to the observer. Immediately following each exposure, each rat was individually handled and examined for abnormal behaviour and appearance. Clinical signs of hair loss, and fur/skin stains associated with urination. During the recovery period clinical observations were continued.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At weighing, detailed clinical observations were conducted including (but not limited to) evaluation of fur, skin, eyes, mucous membranes, occurrence of secretions and excretions, autonomic nervous system activity (lacrimation, piloerection, and unusual respiratory pattern), changes in gait, posture, response to handling, presence of clonic, tonic, stereotypical, or bizarre behaviour. Any abnormal clinical signs noted were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: At least once a week before exposure, and at least once a week during the recovery period.

FOOD CONSUMPTION: Yes
- The amount of food consumed by each animal over each weighing interval was determined by weighing each feeder at the beginning and end of the interval and subtracting the final weight and the amount of spillage from the feeder during the interval from the initial weight. From these measurements, mean daily food consumption over the interval was determined.

FOOD EFFICIENCY: Yes
- From the food consumption and body weight data, the mean daily food efficiency was calculated.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
All surviving rats were examined prior to the 4-week sacrifice. Both eyes of each rat were examined by focal illumination and indirect ophthalmoscopy. The eyes were examined in subdued light after mydriasis had been produced.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On test day 31 for males and females at the end of the exposure period and at the end of the recovery period.
- Anaesthetic used for blood collection: Yes (isofluorane)
- Animals fasted: Yes
- How many animals: All animals.
- Parameters checked in Table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On test day 31 for males and females at the end of the exposure period and at the end of the recovery period.
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked in Table 2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: On test day 31 for males and females at the end of the exposure period.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in Table 3 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations and dose groups examined: All animals prior to test substance exposure and during the fourth week of exposure. Control and 100 ppm recovery group animals during the 8th week of the study.
- Battery of functions tested:
Abbreviated Functional Observation Battery: Sensory and motor function assessments were conducted by evaluating grip strength, responses to approach/touch, sharp auditory stimulus, and tail pinch, and pupillary constriction.
Motor Activity Evaluation: Rats were individually tested for 90 minutes in automated activity monitors.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see Table 4)
After approximately 28 days on study, 10 rats/sex/exposure concentration were sacrificed and necropsied for evaluation of subchronic toxicity. The rats were euthanized by isofluorane and exsanguination. Rats sacrificed by design were fasted on the afternoon before their scheduled sacrifice. A final sacrifice was performed on surviving rats following the final clinical pathology evaluation. The order of sacrifice was stratified across the groups with in a sex. Gross examination was performed on all rats and final body weights and organ weights were recorded. Of the 80 rats in the main study groups (10/sex/exposure level), 2 died before the 90-day terminal sacrifice. One group 1 male rat (108) and a group 3 female rat (358) were accidentally killed on Day 31 during blood collection and received a gross and microscopic examination. All other rats survived until scheduled termination.
After a post-exposure period of approximately 1 month, surviving rats from control and the high exposure level (100 ppm) were sacrificed and necropsied. Rats were euthanized, tissues collected, and organ weights recorded as described for the main study.

ORGAN WEIGHTS: Yes (See Table 4)
Mean absolute and mean relative organ weights (relative to final body weight; relative to brain weight) for weighed organs were calculated. Final body weights determined just prior to necropsy were used in the assessment of organ weight changes. Organs from rats accidentally killed were not weighed.

HISTOPATHOLOGY: Yes (see Table 4)
All tissues collected from rats designated for subchronic toxicity in the high-concentration and control groups, and tissues collected from early decedents were further processed to slides, stained with haematoxylin and eosin, and examined microscopically. Target organs from rats in the low- and intermediate-concentration groups were evaluated microscopically. The tibiofemoral joint (femur and tibia) and nose (teeth: sections from nose levels I and II contain the upper incisors) collected from rats in the low- and intermediate-concentration groups designated for subchronic toxicity were evaluated microscopically.
All 40 rats (20 males, 20 females) designated for the 1-month recovery period survived until the scheduled sacrifice. Potential target organs (incisor teeth and femur/knee joint) were examined microscopically from all recovery rats. As in the main study, examination of teeth in the recovery rats was limited to the 2 upper incisors present within the first 2 sections of the nose (levels I and II).
Statistics:
See Table 5.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
HAEMATOLOGY
There were no adverse changes in group mean haematology parameters in male or female rats at the end of the exposure period (test day 31). Minimal decreases in red blood cell mass parameters (red blood cell, haemoglobin, and haematocrit) in female rats exposed to 100 ppm were considered to be test substance-related, however, not adverse as decreases in red cell mass parameters of this magnitude (decreases in group means for red cell mass parameters were ≤ 5%) are not expected to affect red cell function-delivery of oxygen to tissues. After 1 month of recovery, group mean values for these measurements were similar to the respective control group and not statistically significant.

CLINICAL CHEMISTRY
Following 4 weeks of exposure to the test substance, concentration-related and adverse changes in clinical chemistry parameters were observed in male and female rats exposed to 100 ppm. In these animals, increased mean serum bilirubin levels were observed when compared to the control group and several females exposed to 100 ppm test substance exhibited increased alanine aminotransferase (ALT). Increased albumin (ALB) in male and female rats exposed to 100 ppm was considered to be test substance-related, however, not adverse. Following 4 weeks of recovery, group mean values for these clinical pathology parameters in both male and females were similar to the respective control group and not statistically different. The following statistically significant changes in group mean clinical chemistry parameters at test day 31 or after 1 month of recovery (test days 57/58) were considered to be unrelated to test substance exposure (and non-adverse) because the change did not occur in a concentration related pattern or occurred only after 1 month of recovery: cholesterol (CHOL) was minimally decreased in male rats exposed to 100 ppm after 1 month of recovery (25% below the control); triglycerides (TRIG) were minimally increased in male rats exposed to 1 or 10 ppm (30% and 37% above the control, respectively) at test day 31; total protein (TP) and globulin (GLOB) were minimally decreased in male rats exposed 10 ppm (3% and 5% below the control, respectively) at test day 31; and calcium (CALC) was minimally decreased in female rats exposed to 1 ppm (3% below the control) at test day 31.

URINALYSIS
There were no adverse changes in group mean urinalyses parameters in male or female rats following the exposure period (test day 31). The following statistically significant changes in mean urinalyses parameters were related to test substance, however, not adverse: Urine volume (UVOL) mildly increased in male rats exposed to 100 ppm (130% above the control). This increase in UVOL was accompanied by an appropriate decrease in urinary specific gravity (SG) and in urine total protein (UMTP) in male rats exposed to 100 ppm (2% and 46% below the control, respectively). These changes in UVOL, SG, and UMTP were considered to be related to treatment and represent the expected finding following exposure to a fluorine-containing compound. The diuretic response to a fluorine-containing compound is characterised by increase in the UVOL, and associated decrease in urinary specific gravity. Although changes in these urinary parameters were related to test substance exposure, they were considered to be non-adverse because there were no changes in renal histopathology or clinical chemistry values (Serum creatinine, BUN) suggestive of exposure related adverse renal functional or morphological effect. After 1 month of recovery, group means for UVOL, SG, and UMTP in males exposed to 100 ppm were similar to the respective control group and not statistically significant.

PLASMA AND URINE FLUORIDE
As expected following exposure to a fluoride-containing compound, plasma and urine fluoride were increased in some exposure groups. Statistically significant increases in plasma fluoride concentrations were observed at the end of exposure period (test day 31) in male and female rats exposed to 10 (females only) or 100 ppm. Following the one-month recovery (test day 57/58) group mean plasma fluoride values in male and female rats exposed to 100 ppm were lower, however, they remained statistically significantly increased compared to the control group.
Statistically significant increases in mean urine fluoride were present in male and female rats exposed to 1 (males only), 10 or 100 ppm at the end of the exposure period (test day 31). Following the one-month recovery (test day 57/58), mean urine fluoride was reduced; however, it remained statistically significantly increased in male and female rats exposed to 100 ppm.

NEUROBEHAVIOUR
Male rats exposed to 100 ppm test substance demonstrated decreased motor activity during the fourth week of exposure. The decreased motor activity in male rats exposed to 100 ppm had resolved following the 4-week recovery period. In the absence of other neurobehavioral changes in 100 ppm male rats or in female rats, the lower motor activity in 100 ppm males appears to be secondary to other systemic effects of the test substance, instead of a primary effect on the nervous system. No adverse effects on motor activity were observed in either males exposed to 1 or 10 ppm or female rats exposed to 1, 10, or 100 ppm.

ORGAN WEIGHTS
Male and female rats exposed to 100 ppm test substance demonstrated increased mean absolute liver weights as well as increased mean liver weights relative to body and brain weight one day following the last exposure. By the end of the recovery period, the liver weights were similar to control.
The following changes in group mean organ weight parameters were considered to be unrelated to test substance exposure and non-adverse:
At the end of the exposure period, mean relative (to body and brain weight) adrenal weight was higher in 100 ppm males; mean adrenal weight relative to body weight was also higher in the 10 ppm male group. These differences were statistically significant. However, these changes in adrenal weight parameters were not associated with changes in mean absolute adrenal weights, and no correlative microscopic findings were present in any animal in these groups. In addition, similar changes in adrenal weight were not present in any treated female group or in any sex following 1 month recovery. Therefore, increases in adrenal gland weight parameters were considered unrelated to test substance exposure.
Mean relative (to body weight) kidney weights and mean absolute and relative (to body weight and brain weight) uterine weights were higher compared to controls in the 100 ppm female recovery group. Both the kidney and uterine weight differences in the recovery animals were interpreted to be spurious since test substance-related kidney and uterine weight effects were not observed following the 28-day exposure period.
All other organ weight changes were considered incidental and unrelated to test substance administration.

HISTOPATHOLOGY
There were no test substance-related adverse microscopic findings at any of the exposure concentrations tested. Test substance-related, but non-adverse, microscopic findings were present in the teeth (incisors) and sections of long bones (femur and tibia) at the termination of exposure and following the 1-month recovery period. These changes consisted of increased lamination of dentin of the incisor teeth and incomplete decalcification of enamel of the incisors and the bone trabeculae in the tibia and femur. These findings were consistent with exposure to a fluorine-containing test substance and were not associated with any histopathological changes suggestive of tissue injury or any adverse functional consequences in these tissues. Therefore, these microscopic findings were not considered to be adverse.

Effect levels

open allclose all
Dose descriptor:
NOAEC
Effect level:
10 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based upon increased serum ALT and bilirubin levels (females) and increased liver weighs in both male and females exposed to 100 ppm.
Dose descriptor:
LOAEC
Effect level:
100 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based upon increased serum ALT and bilirubin levels (females) and increased liver weighs in both male and females exposed to 100 ppm.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).
The no-observed-adverse-effect conentration (NOAEC) was 10 ppm and the LOAEC was 100 ppm for male and female rats based upon increased serum ALT and bilirubin levels (females) and increased liver weighs in both male and females exposed to 100 ppm.
Executive summary:

Four groups male and female Crl:CD(SD) rats were exposed whole body 6 hours/day, 5 days/week to 0 ± 0, 1.0 ± 0.0084, 10 ± 0.057 or 100 ± 0.33 ppm test substance over a 4-week period for a total of 23 exposures. Ten male and 10 female rats exposed to 0 (control) or 100 ppm were then subject to an approximate 4-week recovery period.

There were no adverse effects on body weight, body weight gains, food consumption, or food efficiency, and there were no test substance-related or adverse clinical signs of toxicity in males or females exposed to any concentration over the course of this study.

No adverse effects on motor activity were observed in either males exposed to 1 or 10 ppm or female rats exposed to 1, 10, or 100 ppm. Male rats exposed to 100 ppm demonstrated decreased motor activity during the fourth week of exposure. The decreased motor activity in male rats exposed to 100 ppm had resolved following the 4-week recovery period. In the absence of other neurobehavioral changes in 100 ppm male rats or in female rats, the lower motor activity in 100 ppm males appears to be secondary to other systemic effects of the test substance, instead of a primary effect on the nervous system. There were no adverse effects observed in the ophthalmological evaluations at any exposure level in any of the animals in this study.

Following 4 weeks of exposure, concentration-related and adverse changes in clinical chemistry parameters were observed in male and female rats exposed to 100 ppm. In these animals, increased mean serum bilirubin levels were observed when compared to the control group and several females exposed to 100 ppm exhibited increased alanine aminotransferase (ALT). Following 4 weeks of recovery, group mean values for these clinical pathology parameters in both male and females were similar to the respective

control group and not statistically different. No effects on clinical chemistry parameters were observed in males or females exposed to 1 or 10 ppm.

There were no test substance-related adverse effects on gross pathological evaluation in male or females at any exposure level. Male and female rats exposed to 100 ppm demonstrated increased mean absolute liver weights as well as increased mean liver weights relative to body and brain weight one day following the last exposure. By the end of the recovery period, the liver weights were similar to control.

There were no test substance related adverse microscopic findings at any of the exposure concentrations tested. Test substance-related, but non-adverse, microscopic findings were present in the teeth (incisors) and sections of long bones (femur and tibia) at the termination of exposure and following the 1-month recovery period. These changes consisted of increased lamination of dentin of the incisor teeth and incomplete decalcification of enamel of the incisors and the bone trabeculae in the tibia and femur. These findings were consistent with exposure to a fluorine-containing test substance and were not associated with any histopathological changes suggestive of tissue injury or any adverse functional consequences in these tissues. Therefore, these microscopic findings were not considered to be adverse.

There were no portal of entry effects noted in this study; no adverse findings were observed in the respiratory tract tissues at any exposure level. Therefore, the adverse effects observed in this study are considered to be systemic effects and are consistent with those observed in a previous oral repeated dose study with the test substance.

Under the conditions of this study, the no-observed-adverse-effect concentration (NOAEC) for the test substance was 10 ppm for male and female rats based upon increased serum ALT and bilirubin levels (females) and increased liver weights in both male and females exposed to 100 ppm.