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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:
guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
Remarks:
Conducted according to the guideline in effect at the time of study conduct.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Clear gas
Details on test material:
- Purity: >99.964%

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at arrival: Males: 7 weeks; Females: 8 to 10 weeks
- Weight at study initiation: Males ranged from 263 - 269 g; Females ranged from 181 - 186 g
- Fasting period before study: no data
- Housing: In groups of maximally five in Makrolon type-4 cages with wire mesh tops and sterilized standard softwood bedding including paper enrichment
- Diet: ad libitum except during the periods when the animals were restrained in the exposure tubes and prior to blood sampling for clinical laboratory investigations
- Water: ad libitum in water bottles except during the periods when the animals were restrained in the exposure tubes
- Acclimation period: 12 or 13 days under test conditions after health examination. Animals were accustomed to the restraining tubes for 3 daily periods of approximately 1, 2, and 4 hours, respectively.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70%
- Air changes (per hr): Air-conditioned with 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour fluorescent light / 12-hour dark cycle with at least eight hours music during the light period

Administration / exposure

Route of administration:
inhalation: gas
Type of inhalation exposure:
nose only
Vehicle:
air
Details on inhalation exposure:
Inhalation Exposure System
Inhalation exposure was performed using a flow-past system. Ports for animal exposure were positioned radially around the nose-only, flow-past exposure chamber on several different levels. The animals were confined separately in restraint tubes. The atmosphere was discharged constantly through the exposure system and exhausted using a tubing/filter system. The exposure system ensured a uniform distribution and provided a constant flow of test material to each exposure tube. The flow of air at each tube was 1 L/min, which was sufficient to minimize re-breathing of the test atmosphere as it was more than twice the respiratory minute volume of a rat. Before commencement of the exposure of the groups, technical trials were conducted (without animals) using the inhalation system foreseen for the study. Technical trial data are documented in the raw data after review but not reported.

Test Atmosphere Generation
The concentration of the test item in the inhalation chamber was controlled by regulating the flow of the test item to the inhalation tower and by the addition of dilution air.

Exposure System Monitoring
Atmosphere concentration, relative humidity, temperature and oxygen concentration were measured on test atmosphere samples taken at a representative exposure port. In the chamber, temperature was 22.2 - 22.9°C, relative humidity was 0.0 - 4.8%, and oxygen concentration was 19.5 - 20.4%.
All airflow rates (including those for concentration and particle size measurements) were determined using calibrated gas meters and pressure gauges or flow meters.

Determination of Nominal Atmosphere Concentration
Nominal concentrations were determined in groups 2 to 4 by weighing the cylinders before and after each exposure to determine the quantity of test item used. This was done weekly for group 2 and once per exposure for groups 3 and 4. The weight used for gas generation was then divided by the total airflow volume to give the nominal concentration.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of the test substance was measured twice or three times per hour of exposure per online GC for groups 1 to 4. Analytical concentrations were determined by GC. The exposure airflow rate was adjusted as appropriate before the start of the exposure using calibrated flow-meters and / or pressure gauges. The actual airflow rate was monitored hourly during each exposure. Additional measurements were performed if considered necessary.
Duration of treatment / exposure:
28 days
Frequency of treatment:
6 hours/day, 5 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
1 000 ppm (nominal)
Dose / conc.:
10 000 ppm (nominal)
Dose / conc.:
20 000 ppm (nominal)
Remarks:
20,000/15,000
Dose / conc.:
1 000 ppm (analytical)
Remarks:
± 3
Dose / conc.:
10 010 ppm (analytical)
Remarks:
± 40
Dose / conc.:
20 239 ppm (analytical)
Remarks:
20239 ± 557/15076 ± 145
No. of animals per sex per dose:
Main Study: 10 animals/sex.
Recovery / Micronuclei Study: 5 animals/sex.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The initial target atmosphere concentrations were selected by the Sponsor based on a previously performed acute inhalation study that demonstrated a threshold for seizure activity in rats of 33000 ppm. The target atmosphere concentration in group 4 was reduced due to the premature death of two male animals during the 3rd exposure and moderate body weight loss in several males in this group from day 1 to day 4 of exposure.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily during exposure, once daily during acclimatization and recovery

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Recorded twice daily before and after exposure, once daily on week-ends and once weekly during acclimatization and recovery

BODY WEIGHT: Yes
- Time schedule for examinations: Recorded twice weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes; Recorded weekly

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were drawn from the retro-orbital plexus from all allocated animals at the end of exposure and end of recovery. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms.
- Anaesthetic used for blood collection: Yes, light isoflurane anesthesia
- Animals fasted: Yes; in metabolism cages for 15 - 18 hours before blood sampling but allowed access to water ad libitum
- How many animals: all animals from all dose groups
- Parameters checked in Table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Same as Haematology above
- Parameters checked in Table 2 were examined.

URINALYSIS: Yes
- Time schedule for collection of blood: Same as Haematology above
- Parameters checked in Table 3 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Animals were observed for behavior, reflexes, activity, responsiveness, urine or feces, posture and general abnormalities once during acclimatization and once at the end of the exposure period. Any abnormal findings were recorded and, where appropriate, graded in severity. Additionally, locomotor activity was measured quantitatively for the same animals. Due to an error the measurement for locomotor activity during acclimatization could not be reported. Activity was measured with an Activity Monitor AMS-0151 (FMI, Germany). Activity of the animals (based on beam count) was recorded for 6-minute intervals over a period of 30 minutes. These data and the total activity over 30 minutes were reported.
- Dose groups that were examined: all animals
- Battery of functions tested: See Table 4.

OTHER: Micronucleus Evaluation
See Section 7.6.2, report summary, DI.K1.MicroNuc.R.DuPont-19179-782.KD for detailed summary of micronucleus evaluation.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see Table No. 5)
All animals were weighed and necropsied. Descriptions of all macroscopic abnormalities were recorded. All animals surviving to the end of the observation period were anesthetized by intraperitoneal injection of pentobarbitone and killed by exsanguination.

From representative test item-related macroscopic findings photographs were taken during the necropsy, if appropriate. Samples of the following tissues and organs were collected from all animals at necropsy and, unless otherwise indicated, fixed in neutral phosphate buffered 4% formaldehyde solution. Additional tissues (such as animal identification) were retained in accordance with standard operating procedures but were not processed or examined further.

Organ Weights
The weights of the organs from allocation of main study and recovery/micronuclei animals were recorded on the scheduled dates of necropsy listed and their organ to terminal body weight ratios as well as organ to brain weight ratios determined.

Histotechnique
All organ and tissue samples to be examined by the study pathologist were processed, embedded and cut at an approximate thickness of 2 - 4 micrometers and stained with hematoxylin and eosin.

HISTOPATHOLOGY: Yes (see Table 5)
Slides of all organs and tissues listed were collected at terminal sacrifice from the main study animals of the control and high-concentration groups (allocation from main study animals) were examined by the study pathologist. The same applied to all occurring gross lesions (allocations from main study and recovery/micronuclei), the lungs of the low and mid concentration group (allocation main study animals, the lungs of all allocation recovery / micronuclei animals) and to all animals, which died spontaneously or had to be terminated in extremis.
Other examinations:
Micronucleus Evaluation
See Section 7.6.2, report summary, DI.K1.MicroNuc.R.DuPont-19179-782.KD for detailed summary of micronucleus evaluation.
Statistics:
The following statistical methods were used to analyze the grip strength, locomotor activity, food consumption, body weight, macroscopic findings, organ weights and ratios, as well as clinical laboratory data:

• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.

• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.

• Fisher's exact-test was applied to the macroscopic findings.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
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
No exposure related clinical signs were noted during the course of the study.
Two male animals exposed to 20000 ppm test substance died during the 3rd exposure. Thereafter, the target concentration was reduced from 20000 ppm to 15000 ppm. However, two further male animals died during the exposure in the last week of the exposure phase. Neither clinical signs nor relevant effects on the body weight development were noted in these animals. In addition, the cause of these deaths could not be established histopathologically. All other animals survived the scheduled exposure and recovery periods.

BODY WEIGHT AND WEIGHT GAIN
There was no effect noted on animal body weights during the course of this study. Male rats exposed to 10000 ppm test substance demonstrated statistically significant reductions in body weight gains from test days 4 through 22, while males exposed to 20000/15000 ppm demonstrated statistically significant reductions in body weight gains from test days 4 through 28 and increases from days 4 through 11 during the recovery period. Females exposed to 20000/15000 ppm demonstrated a statistical reduction in body weight gain on test day 4 of the exposure. While there were statistically significant reductions in body weight gains, they were not considered adverse since the animals body weights were not affected except for the males exposed to 20000/15000 ppm, where the reduction in body weight gain correlated with lethality and, therefore, was considered test substance-related and adverse.

FOOD CONSUMPTION
Reduced food consumption was noted during the first week of exposure in males of group 4 (attaining statistical significance on that single occasion when compared to controls). This reduction in food consumption was considered spurious and not related to test substance-exposure. No effects on food intake were noted in females.

HAEMATOLOGY
The absolute and relative reticulocyte count were statistically significantly increased in males exposed to 10000 and 20000/15000 ppm test substance, when compared to control group. However, due to the lack of correlative changes in total red blood cell counts, haemoglobin, haematocrit and red cell mass parameters, the changes in reticulocytes were considered non-adverse.

The following statistically significant changes in mean haematology parameters were not exposure-related and non-adverse:
• Red cell distribution width (RDW) was lower in males exposed to 1000 ppm test substance when compared to the control value. There were no associated changes in red cell mass parameters or red blood cell morphology and the values were within the historical control range and did not demonstrate a concentration-response relationship. Therefore, this change was not considered test substance-related or adverse.
• Absolute neutrophils were decreased in male rats exposed to 10000 ppm test substance when compared to the control value. Since the value of absolute neutrophils was in the historical control range and the decrease did not demonstrate a concentration-response relationship, this change was not considered test substance-related or adverse.
• The absolute number of eosinophils was reduced in male rats exposed to 10000 and 20000/15000 ppm test substance as well as the relative number of eosinophils in males exposed to 20000/15000 ppm. While these reductions demonstrated statistical significance, they were within the range of historical control data and small reductions in eosinophils are of questionable physiological relevance. Therefore, these changes were not considered test substance-related or adverse.
• The relative number of lymphocytes was increased in male rats exposed to 1000 ppm test substance. This increase was minor in magnitude (5%), did not affect the absolute number of circulating lymphocytes, was within the historical control range and did not demonstrate a concentration-response relationship. Therefore, this change was not considered test substance-related or adverse.
• Females exposed to 10000 ppm test substance demonstrated a reduction in absolute basophils. The decrease was minor in magnitude (0.01 G/L), was within the historical control range and did not demonstrate a concentration-response relationship. Therefore, this change was not considered test substance-related or adverse.
• Females exposed to 10000 ppm test substance demonstrated a small (8%) reduction in PTT time. Since reductions in PTT time are of questionable physiological significance, the value was within the historical control range and the reduction did not demonstrate a concentration-response relationship, this change was not considered test substance-related or adverse.

CLINICAL CHEMISTRY
Potassium and phosphorus levels were statistically significantly increased in males and females of group 4 at the end of the exposure period when compared to controls. These increases were considered test substance-related; however, they were not considered adverse since the values were within the historical control range. Complete reversal was observed for these parameters at the end of recovery period.

The following statistically significant changes in mean clinical chemistry parameters were not exposure-related and non-adverse:
• Total bilirubin was reduced in males exposed to 10000 ppm test substance. Since this value was within the historical control range and did not demonstrate a concentration-response relationship, this reduction was not considered test substance-related or adverse.
• Triglycerides were reduced in males exposed to 1000 and 20000/15000 ppm test substance. Since these values were within the historical control range and did not demonstrate a concentration-response relationship, this reduction was not considered test substance-related or adverse.
• Total protein was minimally (3%) reduced in male rats exposed to 20000/15000 ppm test substance. Since this value was within the historical control range and minimal in nature it was not considered test substance-related or adverse.
• Serum calcium was minimally reduced (5%) in male rats exposed to 10000 ppm test substance following the recovery period. Since this change was not concentration-dependent and was only observed following the recovery period, it was not considered test substance-related or adverse.
• Female rats exposed to 20000/15000 ppm test substance demonstrated a reduction in serum glucose. Since this value was within the historical control range it was not considered test substance-related or adverse.
• Serum LDH was decreased in female rats exposed to 1000 ppm test substance. Since this value was within the historical control range, did not demonstrate a concentration response relationship and the physiological relevance of reduced LDH is questionable, this change was not considered test substance-related or adverse.
• Serum alanine amino transferase (ALAT) was increased in females exposed to 20000/15000 ppm test substance following the recovery period. Since no changes in ALAT were observed following the exposure period and this value was within the historical control range, this increase was not considered test substance-related or adverse.
• Serum phosphorus was decreased in female rats exposed to 10000 ppm test substance. Since this reduction was only observed after the recovery period and within the historical control range, it was not considered test substance-related or adverse.

URINALYSIS
The pH value in males exposed to 20000/15000 ppm test substance was statistically significantly decreased. While statistically significant, this reduction in pH was within the range of all values observed for urine pH on the study and therefore, was not considered test substance-related or adverse.

NEUROBEHAVIOUR
• Functional Observational Battery - Observations
No test substance-related effects were noted in the functional observational battery at the end of the exposure period. Observations in exposed animals were also observed in the control animals at a similar incidence and demonstrated no concentration-response relationship and were therefore, considered to be non-adverse and not related to test substance exposure.
• Functional Observational Battery - Measurements
There were no effects on the grip strength (fore and hind leg) or body temperature at the end of the exposure period that were considered to be test substance-related. A statistically significant decrease in forelimb and hindlimb strength in male and female rats, respectively was observed at 1000 ppm test substance when compared to the air control group. However, these decreases demonstrated no concentration-response relationship and the magnitude of differences observed were within that observed during the pre-exposure acclimation period. Therefore, the statistical findings were considered non-adverse and not related to test substance exposure. Statistically significant differences that were noted on two occasions when compared to controls were considered to be incidental in the absence of a dose-relationship.
• Locomotor Activity
Measurement of the locomotor activity at the end of the exposure period by low beams count after 6, 12, 18, 24 and 30 min gave no indication of a test substance-related effect. Statistically significant reductions were observed at the 12 and 18 minute time points in males exposed to 10000 ppm test substance. Statistically significant increases in locomotor activity were observed in all treated females at the 6 minute interval; females exposed to 10000 also demonstrated increased activity during the 12 minute interval as well as the total activity. These differences were considered to be sporadic and incidental, did not demonstrate a concentration response relationship and therefore, were considered non-adverse and not related to test substance exposure.

ORGAN WEIGHTS
There were no adverse changes in organ weight parameters at any of the exposure concentrations tested. All statistically significant differences were considered unrelated to exposure and/or nonadverse, as discussed below. In male rats, statistically significant organ weight changes were limited to a statistically higher adrenal weight relative to body weight at 20000/15000 ppm test substance at the end of exposure and following the recovery period. These changes could be secondary to stress associated with the systemic toxicity (mortality) observed in males at this exposure concentration. However, there were no changes in other adrenal weight parameters (absolute adrenal weight or adrenal weight relative to brain weight) and no exposure-related microscopic changes in the adrenals at the end of the exposure period. Therefore, the higher adrenal weight relative to body weight observed in the 20000/15000 ppm male group was likely unrelated to exposure and was considered non adverse. In females, all adrenal weight parameters were statistically higher in the 10000 and 20000/15000 ppm recovery groups. These changes were likely due to a spuriously low control group mean in the female control recovery group, as the mean absolute adrenal weight in the female control recovery group (0.066 grams) was approximately 12% lower than that of the female control group sacrificed at the end of exposure (0.075 grams). In addition, there were no statistically significant changes in any adrenal weight parameter and no exposure-related microscopic changes in the adrenal glands in the 10000 and 20000/15000 ppm female groups at the end of the exposure period. Therefore, the statistically significant differences in adrenal weights in these recovery group females were considered to be unrelated to exposure to test substance. The only other statistically significant differences in organ weight parameters was a decrease in lung weight relative to both brain and body weight in the 10000 ppm female recovery group. These differences were considered spurious and unrelated to exposure, as they did not occur in a concentration-related manner.

GROSS PATHOLOGY
No gross findings at necropsy, considered to distinguish exposed rats from controls, were recorded at necropsy. A dark red discoloration of various organs and incompletely collapsed lungs were recorded in animals that died prematurely. These lesions are common alterations in decedents and, therefore, were considered to be related to a certain degree of tissue autolysis before necropsy of these animals and not to represent a primary exposure-related effect.

HISTOPATHOLOGY: NON-NEOPLASTIC
All microscopic findings recorded were within the range of normal background lesions which are commonly noted in animals of this strain and age.

OTHER FINDINGS
Micronucleus Evaluation
The test item did not induce micronuclei as determined by the micronucleus test in peripheral blood cells on day 4 or at the end of exposure. See also Section 7.6.2, report DI.K1.MicroNuc.R.DuPont-19179-782.KD.

Effect levels

Dose descriptor:
NOAEC
Effect level:
10 010 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reduction in body weight gain and mortality at 20239/15076 ppm

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
The study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).
Male/Female NOAEC = 10010 ppm
Executive summary:

The purpose of this inhalation study was to assess the cumulative toxicity of the test substance when administered 6 hours daily and 5 days per week to rats by nose-only, flow-past inhalation exposure for a period of 28 days. The reversibility or progression of any test substance related effects or any delayed toxicity was assessed during a 2-week exposure free recovery period. In addition, potential genotoxicity was investigated by the evaluation of micronuclei level in the blood. Groups of 10 male and 10 female Wistar rats each were exposed by nose-only flow-past inhalation to target concentrations of 1000, 10000 and 20000 / 15000 ppm test substance in each of groups 2 to 4, respectively. The rats of the control group were exposed to filtered air only (group 1). An additional 5 male and 5 female rats were kept for a 2-week recovery period. Mortality, clinical signs, functional observation battery, grip strength, body temperature, locomotor activity, body weights, food consumption, clinical laboratory investigations, micronucleus evaluation, organ weights, macroscopic and microscopic findings were recorded.

 

Exposure to chemically determined atmosphere concentrations of 1000, 10010 and 20239 / 15076 ppm test substance were achieved in groups 2 to 4, respectively, and were close to the respective targets. Temperature, relative humidity and oxygen concentration during exposure were considered to be suitable for this type of study.

 

Four male animals died during the 20000 / 15000 ppm exposure period. The cause of these deaths could not be established. No exposure related clinical signs were noted during the course of the study. In addition, no test substance related effects were noted in the functional observational battery, on the grip strength, the body temperature and on the locomotor activity at the end of the exposure period. Reduced food consumption was noted in males exposed to 20000/15000 ppm. There was no effect on mean animal body weights during the course of this study. Body weight gain was concentration-dependently reduced in males exposed to 10000 ppm and 20000/15000 ppm during the exposure period; however, this was not considered adverse since the animal’s body weight was not affected. Potassium and phosphorus levels were increased in both sexes of group 4 at the end of the exposure period. These increases were considered test substance-related; however, they were not considered adverse since the values were within the historical control range. Complete reversal was observed for these parameters at the end of recovery period.

 

Based on the reduction in body weight gain and mortality observed in group 4 (20000/15000 ppm), the No-Observed-Adverse-Effect-Concentration (NOAEC) was considered to be 10010 ppm.