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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: 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.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report Date:
1996

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
Chamber air flow was not reported; see Table 2 in Materials and Methods for clinical chemistry parameters not examined
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Purity: 2 lots of the test substance were used with purity of 99.78% and 99.66%

Test animals

Species:
rat
Strain:
other: Crl:CD®BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: Approximately 56 days
- Weight at study initiation: Not reported
- Fasting period before study: No
- Housing: Individually in stainless steel, wire-mesh cages suspended above cageboards, males and females on separate cage racks
- Diet (e.g. ad libitum): ad libitum, except during exposure
- Water (e.g. ad libitum): ad libitum, except during exposure
- Acclimation period: Approximately 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23±2°C
- Humidity (%): 55±15%
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Three 750 L chambers (test substance exposure) and a 1000 L chamber (control exposure). The chambers and all surfaces that the test substance contacted prior to exhaust from the chamber were constructed of stainless steel and glass.
- Method of holding animals in test chamber: Two per cage in wire basket exposure modules, except for approximately 2 weeks when rats were housed individually in wire basket exposure modules.
- Source and rate of air: Not reported
- Method of conditioning air: Not reported
- System of generating particulates/aerosols: The chambers were operated in a one-pass, flow-through mode. The test substance was generated by metering the test substance into a glass J-tube filled with glass beads. Heated air was blown through the glass beads to evaporate the liquid test substance and the resulting vapour was diluted to the desired concentration with filtered air for each of the 3 test chambers. Chamber concentrations were controlled by varying the test substance flow rates into the exposure chamber. Heated air alone was metered in an identical manner into the control chamber.
- Temperature, humidity, pressure in air chamber: 19-24°C; 30-70%; pressure not reported
- Air flow rate: 110-211 L/min (control chambers); 100-180 L/min (test chambers; except test days 1 and 2, for males at 500 ppm where it was 72-73 L/min)
- Air change rate: Not reported
- Method of particle size determination: Not applicable
- Treatment of exhaust air: Diluted with room air and exhausted through a roof stack

TEST ATMOSPHERE
- Brief description of analytical method used: GC with a flame ionization detector; analyzed at 30-minute intervals during each 6-hour exposure period. Diastereomer ratio (area percent) from a test chamber was determined once daily (except test days 3, 29, 46, and 67). During prestudy method development, distribution of test substance in the chamber was determined.
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Due to a spill of the test substance in the chamber room on test day 57, a small quantity of test substance (8.5 ppm) was detected in the control chamber since the chambers were operated under a negative pressure relative to the chamber room. However, the test substance was only detected in 1 of the 10 samples collected, and the mean concentration for the day was 0.8 ppm. For the 500 ppm chamber, mean daily concentrations ranged between 475-530 ppm. Mean chamber concentration was lower (92% of nominal) than this range on test day 58 and was higher (112% of nominal) than this range on test day 4. For the 2000 ppm chamber, mean daily concentrations ranged between 1800 and 2200 ppm. Mean chamber concentration was lower (81-88% of nominal) on test days 3, 17, and 33. For the 3500 ppm chamber, mean daily concentration ranged between 3150-3600 ppm. Mean chamber concentration was lower (87% of nominal) on test day 3.
Duration of treatment / exposure:
90 days (weekends and 1 holiday excluded)
Frequency of treatment:
6 hours/day, 5 days/week
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 500, 2000, 3500 ppm (5154; 20618; 36081 mg/m3, respectively)
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0, 503, 1982, 3473 ppm (0; 5189; 20433; 35802 mg/m3, respectively)
Basis:
analytical conc.
No. of animals per sex per dose:
20 animals/sex, for which 10 animals/sex were used for neurotoxicity substudy
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on an ALC in rats of 10000 ppm (103088 mg/m3) and 2-week inhalation range finding studies where clinical signs of toxicity indicative of seizure-like behaviour occurred during exposure to the test material at concentrations as low as 3700 ppm (38143 mg/m3).
- Rationale for selecting satellite groups: To evaluate potential neurotoxicity based on clinical signs observed in a 2-week range-finding study.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily on exposure days and at least once daily on weekend and holidays

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

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

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Once during the pretest period and prior to the 90-day sacrifice

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Approximately 45 and 90 days after exposure initiation
- Anaesthetic used for blood collection: Yes (light carbon dioxide anaesthesia)
- Animals fasted: Yes
- How many animals: All
- Parameters checked in table [No. 1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Approximately 45 and 90 days after exposure initiation
- Animals fasted: Yes
- How many animals: All
- Parameters checked in table [No. 2] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Approximately 45 and 90 days after exposure initiation
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [No. 2] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes (see study DI.K1.90-day.InhVap.Neuro.R.HLR-24-94 in section 7.9.1 for details)
- Time schedule for examinations: Prior to initial exposure and during weeks 4, 8, and 13
- Dose groups that were examined: 10 rats/sex/concentration
- Battery of functions tested: Functional observational battery (FOB), grip strength, motor activity, neuropathology

OTHER: Liver samples were collected from the first 5 surviving rats per group for determination of peroxisome proliferation. The radioactivity was measured by liquid scintillation spectrometry.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table No. 3)

HISTOPATHOLOGY: Yes (see table No. 3)

Tissues collected from rats in the high-concentration and control groups were further processed to slides and examined microscopically. Eyes collected from rats in the low- and intermediate-concentration groups as well as additional sections of eyes collected from control and high-concentration groups were evaluated microscopically to establish whether the eye was a target organ. Nose, lungs, liver, kidneys, and gross lesions, from rats in the low- and intermediate-concentration groups were also processed to slides and examined microscopically.
Statistics:
See Table 4

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
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:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY: Two male rats at 3500 ppm were sacrificed in extremis due to an injury or spontaneous disease. One female at 500 ppm was accidently killed; one female at 2000 ppm was sacrificed in extremis due to stem cell leukaemia; one female at 3500 ppm was found dead due to a fractured skull. During non-exposure periods there was a significantly higher incidence of stained fur in females exposed to ≥500 ppm; however, there was no dose-response relationship, and the median day on which this sign was first observed was similar to controls for all exposures. In addition, there were no gross or microscopic morphological changes, or changes in any other parameter that could be correlated with the increased incidence of stained fur. Since the higher incidence of stained fur could not be correlated with any other findings and did not affect the health of the animals, it was not considered to be adverse or toxicologically significant. During the exposure periods, rats at 2000 and 3500 ppm, which were visible through the chamber window were observed to exhibit abnormal behaviour including jerking/jumping, pawing the air, flinching, abnormal gait, convulsions, tremors, excessive grooming, and high activity. In general, these clinical signs occurred within the first hour of exposure, and most frequently during the 20-30 minute period after vapour generation was initiated and the concentration in the chamber was increasing rapidly toward the target concentrations. Within 1-2 hours after the chamber concentration reached the target concentrations, the animals appeared to adapt, and these clinical signs were rarely observed during the remainder of the daily exposure period. These signs were not observed at all during non-exposure periods. In the 2000 ppm chamber, these clinical signs were observed on 4 exposure days over the course of the study. At 3500 ppm, these signs were observed on the majority of exposure days (beginning with test day 1); although by test week 7, it appeared that toward the end of each exposure week, the clinical signs did not occur during exposure, which may reflect adaptation to the test substance. These signs were also observed during the range-finding study, and their occurrence in rats exposed to 2000 or 3500 ppm was considered compound related.

BODY WEIGHT AND WEIGHT GAIN: The significantly higher body weight gain over test days 1-8 for 500 ppm males and the significantly lower body weight gain for males at ≥500 ppm over test days 8-15 were considered to be spurious since the changes were not dose-related and were not consistent over time. Females exposed to 2000 or 3500 ppm had a higher, although not statistically significant weight gain over the interval of test days 1-92 compared to control. In addition, body weight for test days 85 and 92, and weight gain over test days 78-85 for 3500 ppm females were significantly higher compared to controls. However, there were no compound-related morphological changes that correlated wit the higher weight gain in 2000 and 3500 ppm females, and therefore, it was not considered to be adverse or toxicologically significant.

FOOD CONSUMPTION: Food consumption for control males over test days 36-43 was higher compared to values for ≥500 ppm males; however, this value was also higher than both previous and succeeding control values and was considered to be a spurious event. Similarly, food consumption values for 2000 and 3500 ppm females over the interval of test days 43-49 were higher than control values, and were higher than either the previous or succeeding values for 2000 and3500 ppm females and were considered to be spurious.

OPHTHALMOSCOPIC EXAMINATION: Near the end of the study, the incidences of male rats with eye lesions were 0, 0, 5, and 6% for 0, 500, 2000, and 3500 ppm, respectively. The incidences of eye lesions in female rats were 0, 5, 0, and 0% for the 0, 500, 2000, and 3500 ppm groups, respectively. Since the observed lesions did not exhibit a dose-related trend relative to the structures affected, and since the lesions are commonly seen in rats of this strain and age, they were considered to be spontaneous or due to trauma.

CLINICAL CHEMISTRY: Significantly decreased serum concentration at the 90-day evaluation in male and female rats exposed to 3500 ppm (slightly decreased at the 45-day evaluation) indicated a possible progressive effect, and was considered to be a compound-related, toxicologically significant effect. Statistically significant decreased in serum sorbital dehydrogenase, bilirubin, and blood urea nitrogen concentration were not considered to be adverse compound-related effects because decreases in these parameters are not biologically important.

URINALYSIS: Females at 3500 ppm had a compound-related decrease in urine osmolality and increase in urine volume at the 45-day and 90-day evaluations. These changes were not correlated with changes in other parameters that assess kidney function. Urinary fluoride ion concentration was significantly increased in 2000 and 3500 ppm males and females, however, this was not considered to be an adverse effect since the higher fluoride ion concentration reflects absorption and metabolism of the test substance with release and urinary excretion of fluoride ion. In addition, urinary fluoride ion was increased in the 2000 ppm females; however, the value was not statistically significant. Other statistically significant differences (decreased urobilinogen concentration) were not considered to be adverse because a decrease in this analyte is not biologically significant.

NEUROBEHAVIOUR: See report DI.K1.90-dayInhVap.Neuro.R.HLR-24-94.KD in section 7.9.1 for details.

ORGAN WEIGHTS: Absolute and relative adrenal weights were increased in males at 3500 ppm, however, there were no gross or microscopic morphological changes that correlated with the increased weight, and therefore they were considered to be spurious. Females exposed to 3500 ppm had significantly increased absolute and relative liver weights and increased absolute lung weight; however, there were no gross or microscopic morphological changes that correlated with the increased weight, and therefore, they were considered to be spurious.

GROSS PATHOLOGY: The incidence of retinal rosettes/folds was slightly increased (27%) in 3500 ppm males and females; however, in all affected animals the lesions were unilateral and have been observed previously in recent historical controls (the incidence ranged between 0-20% for males and 0-10% for females from 90-day studies conducted in 1992 and 1993). However, since only 10 animals per group are routinely evaluated in 90-day subchronic studies, one additional animal with the lesion can vary the incidence by 10%. Based on the unilateral nature of these lesions and the spontaneous occurrence of these lesions in historical controls, the retinal lesions observed were considered to be spontaneous.

OTHER FINDINGS: Hepatic Peroxisome Proliferation: A concentration-dependent significant decrease in peroxisomal beta-oxidation activity was observed in males at ≥ 500 ppm. However, since there were no apparent changes in fat metabolism as indicated by serum cholesterol concentrations, serum triglyceride concentrations, and body weights, the reduced hepatic peroxisomal beta-oxidation activity was not considered to be a biologically significant event.

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 500 other: ppm (15463 mg/m3)* - 500 ppm (5154 mg/m3)
Sex:
male/female
Basis for effect level:
other: see 'Remark'

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).

NOAEL = 500 ppm
Executive summary:

Four groups of 20 male Crl:CD®BR rats per group and four groups of 20 female rats per group were exposed to atmospheres of the test substance vapour for 6 hours per day, 5 days per week, over a 90-day period at concentrations of 0, 500, 2000, or 3500 ppm. During the exposures, the concentration of the test substance was determined by gas chromatographic analysis of the chamber atmosphere. Ophthalmic examinations were conducted prior to initiation of exposures and after approximately 13 weeks of exposure. Body weights and food consumption were determined weekly. Evaluations of haematology, clinical chemistry, and urinalysis parameters were conducted at approximately 7 and 13 weeks. A functional observational battery (FOB) and assessment of motor activity (MA) were conducted on 10 rats per sex per concentration prior to initiation of the study. Similar evaluations were conducted at approximately 4, 8, and 13 weeks after initiation of exposure. Approximately 13 weeks after study initiation, all surviving rats designated for the subchronic toxicity subset (approximately 10 rats per sex per concentration) were necropsied, selected organs were weighed, and tissues were examined for gross and microscopic lesions. In addition, all surviving rats designated for the neurotoxicity subset (approximately 10 rats per sex per concentration) were necropsied and evaluated for morphological changes in nervous tissues.

 

The overall mean concentrations of the test substance in the exposure chambers for the 13-week test period were 0.0, 503.3, 1981.9, and 3472 ppm corresponding to the nominal concentrations of 0, 500, 2000, and 3500 ppm, respectively. A small amount (8.5 ppm) of the test substance was detected in the control chamber on test day 57 during one of the sampling intervals due to a small spill of the test substance in the chamber room.

 

Rats exposed to 2000 or 3500 ppm exhibited clinical signs of central nervous system toxicity during the exposure period, which included: jerking/jumping, pawing the air, flinching, abnormal gait, convulsions, tremors, excessive grooming, and elevated activity level. In addition, a compound-related decrease in motor activity was observed in males and females exposed to 3500 ppm. FOB and MA were determined on weekends approximately 18-34 hours following exposure. There were no adverse compound-related effects on body weight, body weight gain, food consumption, food efficiency, mortality, the incidence of ophthalmoscopically visible alterations to the structures of the eye, or the incidence of clinical signs during non-exposure periods. In addition, no compound-related effects were detected on forelimb grip strength, hindlimb grip strength, foot splay, or other FOB parameters evaluated in this study. Compound-related haematological alterations were not observed at any exposure concentration in either males or females. A compound-related decrease in serum albumin was observed in 3500 ppm males and females. In addition, 3500 ppm females had a compound-related decrease in urine osmolality and increased urine volume. Urine fluoride concentration was also elevated in male and females exposed to 2000 and 3500 ppm; however, this is indicative of metabolism and is not considered to be adverse. There were no compound-related effects on organ weight parameters or on the incidence of gross or microscopic morphological changes in any tissue, including nerve and muscle tissue. Adverse effects on hepatic peroxisomal beta-oxidation activity also were not observed at any exposure concentration. If this study is taken in isolation, the NOAEL was 500 ppm for males and females based on the clinical signs of central nervous system toxicity observed during exposure at concentrations of 2000 ppm and above. However, in the 90-day study, all adverse central nervous system effects occurred very early in the exposure and presented in the same manner as the effects in the acute inhalation and neurotoxicity studies. The adverse effects did not increase in severity or occurrence with increasing exposure duration. Therefore, the nature of the adverse effects associated with test substance provided a unique opportunity to use a 2-hr neurotoxicity study (DI.K2.2-hr.NEURO.HLR-395-95) to evaluate the toxicity between the 500 and 2000 ppm exposure levels used in the 90-day study. When all of the relevant data were considered, the NOAEL for central nervous system effects (in both extended repeated exposures or acute exposures) was 15463 mg/m3.