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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:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified
Details on test material:
- Name of test material: Aminoethylpiperazine (AEP)
- Physical state: Liquid
- Analytical purity: 98% gas chromatography
- Lot/batch No.: 3C27081150
- Stability under test conditions:The test substance is considered to be stable under the storage conditions.
- Storage condition of test material:Ambient

Test animals

Species:
rat
Strain:
other: F344/DuCrl
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (Kingston, New York)
- Age at study initiation: Animals were ~5 weeks at arrival and ~9 weeks of age at initiation of treatment
- Housing: After assignment to the study, animals were housed two or three per cage in stainless steel cages. Cages had solid floors with corncob bedding and paper nesting material for enrichment. Cages contained a feed crock and a pressure activated lixit valve-type watering system.
- Diet: ad libitumexcept during acclimation to the nose-only exposure tubes and during nose-only exposure
- Water: ad libitumexcept during acclimation to the nose-only exposure tubes and during nose-only exposure
- Acclimation period: Rats were acclimated to nose-only exposure tubes prior to starting exposures to test material. Rats were loaded into exposure tubes and remained in the tubes for increasing amounts of time. They remained in the exposure tubes for one hour on the first day of acclimation. The amount of time in the tubes was increased by one hour/day until the rats remained in the exposure tubes for six consecutive hours.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C with a range of 20°C-26°C
- Humidity (%): 50% with a range of 30-70%
- Air changes (per hr): 10-15 times/hour (average)
- Photoperiod (hrs dark / hrs light): 12-hour light/dark (on at 6:00 a.m. and off at 6:00 p.m.)

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure:
nose only
Vehicle:
clean air
Remarks on MMAD:
MMAD / GSD: The average mass median aerodynamic diameter (MMAD) of aerosol present in the exposure chamber test atmospheres was 0.85 ± 1.52, 1.96 ± 1.58, or 1.04 ± 2.39 microns (MMAD ± geometric standard deviation; GSD) for the 0.2, 5.1, or 53.5 mg AEP/m3 exposure chambers, respectively.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Forty-two liter, Dow-modified ADG flow-past, nose-only chambers [30 centimeters (cm) in diameter by 60 cm high]
- Source and rate of air: Compressed air supplied to the chamber was at ambient temperature. Airflow through the chamber was determined with a manometer which measured the pressure drop across a calibrated orifice plate and was maintained at approximately 30-35 liters per minute, which was sufficient to provide the normal concentration of oxygen to the animals and approximately 43-50 air changes per hour.
- Method of conditioning air: The 53.5 mg/m3 AEP exposure atmosphere was generated by metering the liquid test material with a syringe pump (KD Scientific, Inc., Holliston, Massachusetts) into an aerosol spray nozzle (TSI Inc., Shoreview, Minnesota). The test material was mixed with air in the spray nozzle, and passed through an aerosol mixing/conditioning chamber (TSE Systems, Inc., Chesterfield, Missouri) wrapped with heat tape before being passed into the exposure chamber. The carrier air and heat tape were heated to the minimum extent necessary to vaporize the test material aerosol. The air and test material vapors were mixed with the appropriate amount of humidified dilution air. The 0.2 and 5.1 mg/m3 AEP exposure atmospheres were generated using a glass J-tube method (Miller et al., 1980). Liquid test material was metered into a glass J-tube assembly with a syringe pump (KD Scientific, Inc., Holliston, Massachusetts) and vaporized by passing air (approximately 30 liters per minute) through the bead bed in the glass J-tube. The air was heated with a flameless heat torch to the minimum extent necessary to vaporize the test material. The mid (5.1 mg/m3) and low (0.2 mg/m3) exposure chamber atmospheres were generated using separate J-tube/syringe pump generation systems. The generation systems were electrically grounded and the J-tubes were changed as needed. The air and test material vapors were mixed with the appropriate amount of humidified dilution air to achieve the target chamber concentration. The test material was not recycled.
On the first day of exposure (Test Day 1), all chambers were monitored to ensure sufficient O2 levels via oxygen sensors with high and low alarms set at 24% and 19% O2, respectively. CO2 levels were also monitored.

- System of generating particulates/aerosols:
- Temperature, humidity, pressure in air chamber: Exposure room temperature and chamber temperature, humidity, and airflow were recorded approximately every hour during the exposure periods from each exposure chamber.
- Air flow rate: Based on the approximately 30-35 liter per minute flow rate for the exposure chambers, the theoretical equilibrium time to 99% (T99) of the target concentration was ~5.5-6.5 minutes. The animals were placed on the chamber after the T99 had elapsed and were removed after ~360 minutes of exposure.
- Method of particle size determination: Due to the formation of AEP-carbamate, some aerosol was present in each of the exposure chambers. The mass median aerodynamic diameter (MMAD) was determined at least once per week for each exposure chamber either by drawing samples from within the animal breathing zone, at a set rate using a constant flow air sampling pump through a multi-stage cascade impactor (Sierra Instruments, Inc., Monterey, California), or by using a time-of-flight aerodynamic particle size spectrometer (APS 3321; TSI Incorporated, Shoreview, Minnesota). The MMAD and geometric standard deviation (σg) was determined for each sample as well as the average of the samples.
- Treatment of exhaust air:

TEST ATMOSPHERE
- Brief description of analytical method used: The concentration of AEP present in each chamber was determined analytically, for each exposure group, 1-3 times during each six-hour exposure period. Each sample was taken by drawing chamber atmosphere from within the animal breathing zone at a set rate using a constant flow air sampling pump and collecting the test material using NITC treated XAD-2 sorbent tubes. Analyses of the chamber atmosphere samples were conducted using liquid chromatography with ultraviolet detection (LC/UV). This method measured the total concentration of AEP, both vapor and AEP-carbamate, present in the exposure atmosphere. See Appendix B for details of the analytical method used.
The time-weighted average (TWA) exposure concentration was calculated from the analytical measurements for each chamber.
Prior to exposure of animals to the test material, the distribution of the test material in the breathing zone of each chamber was determined.
Nominal concentrations for the exposure system which supplied test material to the exposure chambers were calculated from the amount of test material used in the generation apparatus each day and the total airflow through the exposure system for each exposure period.



VEHICLE (if applicable)
- Justification for use and choice of vehicle:
- Composition of vehicle:
- Type and concentration of dispersant aid (if powder):
- Concentration of test material in vehicle:
- Lot/batch no. of vehicle (if required):
- Purity of vehicle:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of AEP present in each chamber was determined analytically, for each exposure group, 1-3 times during each six-hour exposure period. Each sample was taken by drawing chamber atmosphere from within the animal breathing zone at a set rate using a constant flow air sampling pump and collecting the test material using NITC treated XAD-2 sorbent tubes. Analyses of the chamber atmosphere samples were conducted using liquid chromatography with ultraviolet detection (LC/UV). This method measured the total concentration of AEP, both vapor and AEP-carbamate, present in the exposure atmosphere. The time-weighted average (TWA) exposure concentration was calculated from the analytical measurements for each chamber.
Duration of treatment / exposure:
6 hours/day
Frequency of treatment:
5 days/week for 13 weeks (65 days of exposure)
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/m3
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0.2 mg/m3
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
5 mg/m3
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
50 mg/m3
Basis:
nominal conc.
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The target exposure levels selected, 0, 0.2, 5, or 50 mg/m3, were based on the results of a two-week nose-only inhalation study with AEP (Hotchkiss et al., 2015). The high-exposure concentration (53.5 mg/m3) was expected to result in exposure-related point-of-contact effects in the respiratory tract with no fatalities. The mid- and low-exposure concentrations were fractions of the high-exposure concentration and were expected to provide exposure-response data for any treatment-related effects observed in high-exposure group. A filtered air control group was included. A recovery group of males (most sensitive sex in the two-week study) was included to evaluate the reversibility of any treatment-induced effects.
Positive control:
Not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once a day, at approximately the same time each day (usually in the morning).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Pre-exposure and at least weekly throughout the exposure period

BODY WEIGHT: Yes
- Time schedule for examinations: All rats were weighed during the pre-exposure period, on test day 1 prior to the first exposure, at least twice per week during the first 4 weeks of exposure, and at least weekly thereafter.

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

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: No data

WATER CONSUMPTION: No data
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pre-exposure and prior to the scheduled necropsy (test day 86) using indirect ophthalmoscopy
- Dose groups that were examined: All animals

HAEMATOLOGY: Yes / No / No data
- Time schedule for collection of blood:
- Anaesthetic used for blood collection: Yes (identity) / No / No data
- Animals fasted: Yes / No / No data
- How many animals:
- Parameters checked in table [No.?] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were obtained from the orbital sinus following anesthesia with isoflurane/O2 at the scheduled necropsy. Blood was not obtained from animals that were euthanized prior to their scheduled necropsy.

URINALYSIS: Yes
- Time schedule for collection of urine: Urine samples were obtained from all animals the week prior to the scheduled necropsy. Animals were housed in metabolism cages and the urine collected overnight (approximately 16 hours). Feed and water were available during this procedure.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
Means and standard deviations were calculated for all continuous data. Feed consumption, body weights, terminal body weight, organ weight (absolute and relative), urine volume, urine specific gravity, hematologic parameters (excluding RBC indices and differential WBC), coagulation and clinical chemistry parameters (excluding globulin and albumin/globulin ratio) were evaluated by Bartlett's test for homogeneity of variances. Based on the outcome of Bartlett's test, exploratory data analysis was performed by a parametric ANOVA (Steel and Torrie, 1960) or nonparametric ANOVA (Hollander and Wolfe, 1973). If significant at alpha = 0.05, the ANOVA was followed respectively by Dunnett's test or the Wilcoxon Rank-Sum test (Hollander and Wolfe, 1973) with a Bonferroni correction for multiple comparisons to the control. The experiment-wise alpha level of 0.05 was reported for these two tests. Statistical outliers were identified by a sequential test (Grubbs, 1969).
DCO incidence data (scored observations only) were statistically analyzed by a z-test of proportions comparing each treated group to the control group at alpha = 0.05 (Bruning and Kintz, 1987). Data collected at different time points were analyzed separately.
Descriptive statistics only (means and standard deviations) were reported for body weight gains, globulin and albumin/globulin ratio, RBC indices, differential WBC counts, chamber concentration, temperature, relative humidity and airflow and exposure room temperature.
Data collected in the recovery phase was analyzed by the same methods as indicated during the exposure phase.
Because numerous measurements were statistically compared in the same group of animals, the overall false positive rate (Type I errors) was greater than the nominal alpha levels. Therefore, the final toxicologic interpretation of the data considers other factors, such as dose-response relationships, biological plausibility and consistency, and historical control values.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
Clinical ObservationsExaminations performed on all animals pre-exposure and at least weekly throughout the study noted thin appearance in two (of 20) male rats exposed to 0.2 mg/m3 on test day 47 (caused by an abnormally working lixit valve with restricted water flow in the animal housing cage), a broken incisor in one (of 20) male 53.5 mg/m3 group rat on test day 81, and a kinked tail tip due to a mechanical injury in 1 (of 10) female 53.5 mg/m3 group rat on test day 88. Mortality: Animal 15A0444, a female in the 5.1 mg/m3 group, was found in a cage with two male rats on test day 27; this animal was subsequently removed from the study and taken to necropsy on test day 29 due to a sperm positive slide. There were no instances of mortality related to test material exposure throughout this study.
Mortality:
no mortality observed
Description (incidence):
Clinical ObservationsExaminations performed on all animals pre-exposure and at least weekly throughout the study noted thin appearance in two (of 20) male rats exposed to 0.2 mg/m3 on test day 47 (caused by an abnormally working lixit valve with restricted water flow in the animal housing cage), a broken incisor in one (of 20) male 53.5 mg/m3 group rat on test day 81, and a kinked tail tip due to a mechanical injury in 1 (of 10) female 53.5 mg/m3 group rat on test day 88. Mortality: Animal 15A0444, a female in the 5.1 mg/m3 group, was found in a cage with two male rats on test day 27; this animal was subsequently removed from the study and taken to necropsy on test day 29 due to a sperm positive slide. There were no instances of mortality related to test material exposure throughout this study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no statistically identified differences in the body weights of any treated groups when compared to their respective controls. Body weight gains were also unaffected by treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Statistically identified lower mean feed consumption values were observed from test days 1-5 in male recovery group rats exposed to 0.2, 5.1, or 53.5 mg/m3 and female rats exposed to 5.1 or 53.5 mg/m3 when compared to their respective controls.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Examinations performed on all animals pre-exposure and at termination revealed no treatment-related findings.
Haematological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related alterations in any of the hematology parameters of males or females at any exposure level.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no treatment-related alterations in clinical chemistry parameters of males or females at any exposure level.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no treatment-related or statistically significant changes in urinalysis parameters of males or females at any exposure level.
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
. Final body weights and all other organ weights in both sexes were similar to controls.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related gross pathologic observations in males or females at any exposure level. All gross pathologic observations were interpreted to be spontaneous alterations, unassociated with exposure to AEP.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Changes in the nasal tissues in mid and high dose groups and nasal tissue larynx, trachea, and lungs of high dose group
Histopathological findings: neoplastic:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY:
Animal 15A0444, a female in the 5.1 mg/m3 group, was found in a cage with two male rats on test day 27; this animal was subsequently removed from the study and taken to necropsy on test day 29 due to a sperm positive slide. There were no instances of mortality related to test material exposure throughout this study.
No treatment-related clinical observations were observed during the study. Examinations performed on all animals pre-exposure and at least weekly throughout the study noted thin appearance in two (of 20) male rats exposed to 0.2 mg/m3 on test day 47 (caused by an abnormally working lixit valve with restricted water flow in the animal housing cage), a broken incisor in one (of 20) male 53.5 mg/m3 group rat on test day 81, and a kinked tail tip due to a mechanical injury in 1 (of 10) female 53.5 mg/m3 group rat on test day 88.

BODY WEIGHT AND WEIGHT GAIN: There were no statistically identified differences in the body weights of any treated groups when compared to their respective controls. Body weight gains were also unaffected by treatment.

FOOD CONSUMPTION: There were no treatment-related effects in the amount of feed consumed by any treated group when compared to their respective controls. Statistically identified lower mean feed consumption values were observed from test days 1-5 in male recovery group rats exposed to 0.2, 5.1, or 53.5 mg/m3 and female rats exposed to 5.1 or 53.5 mg/m3 when compared to their respective controls. In addition, male 90-day exposure groups rats exposed to 5.1 mg/m3 were noted in one instance (test days 75-81) to have statistically identified higher mean feed consumption values when compared to their respective controls. The increases and/or decreases in feed consumption noted throughout the study were considered unrelated to treatment related due to their random occurrence.

OPHTHALMOSCOPIC EXAMINATION: Examinations performed on all animals pre-exposure and at termination revealed no treatment-related findings.

HAEMATOLOGY: There were no treatment-related alterations in any of the hematology parameters of males or females at any exposure level. Females exposed to 0.2 mg/m3 had statistically significant decreases in mean red blood cell count and mean hemoglobin concentration, relative to controls, which were interpreted to be unrelated to treatment because of the lack of a dose response for these parameters, and the values were within historical control ranges of studies recently conducted at this laboratory.

CLINICAL CHEMISTRY: There were no treatment-related alterations in clinical chemistry parameters of males or females at any exposure level. Males exposed to 53.5 mg/m3 had a statistically significant lower mean cholesterol concentration which was interpreted to be unrelated to treatment because of the lack of a clear dose-related response in the low- and mid-exposure levels, the minimal difference of the high-exposure level cholesterol concentration as compared to the historical control group range, and as compared to the concurrent control group.

URINALYSIS: There were no treatment-related or statistically significant changes in urinalysis parameters of males or females at any exposure level.

ORGAN WEIGHTS: Males and females exposed to 53.5 mg/m3 had higher absolute and relative mean lung weights as compared to controls. The higher relative lung weights of males and females exposed to 53.5 mg/m3 were statistically significant, relative to controls. The higher absolute and relative lung weight of females exposed to 53.5 mg/m3 were interpreted to be treatment-related because the values were clearly higher than the historical control ranges from studies recently conducted at the laboratory. The higher absolute and relative lung weights of males exposed to 53.5 mg/m3 were within or near the historical control range, were minimally different from the concurrent control values, and were therefore interpreted to be unrelated to treatment. Final body weights and all other organ weights in both sexes were similar to controls.

GROSS PATHOLOGY: There were no treatment-related gross pathologic observations in males or females at any exposure level. All gross pathologic observations were interpreted to be spontaneous alterations, unassociated with exposure to AEP.

HISTOPATHOLOGY: NON-NEOPLASTIC
There was no histopathological evidence of any primary systemic toxicity in any AEP exposure group (systemic NOEC = 53.5 mg/m3). The nasal tissue was the most sensitive target tissue in rats exposed to AEP. Compared to air-exposed (0 mg/m3) control rats of the same sex, male and female rats exposed to 5.1 or 53.5 mg/m3 had exposure-related histopathological changes in the nasal tissues. The larynx, trachea, and lungs of male and female rats exposed to 53.5 mg/m3 also had exposure-related histopathological effects. All of the exposure-related changes were consistent with localized irritant effects of the test material at the point of contact with the airway epithelium.
The majority of the nasal tissue effects were present in the anterior and dorsal aspects of the nasal passages. The most frequently affected site in the nasal tissue was the transitional epithelium lining the nasoturbinates, maxilloturbinates, and lateral walls of the nasal passages. All males exposed to 5.1 or 53.5 mg/m3, all females exposed to 53.5 mg/m3, and the majority of females exposed to 5.1 mg/m3 had multifocal atrophy (very slight, slight, or moderate), hyperplasia (very slight or slight), chronic-active inflammation (very slight or slight), and squamous metaplasia (very slight or slight) of the transitional epithelium. Focal or multifocal, very slight ulcers of the transitional epithelium were also present in a few males and females exposed to 5.1 or 53.5 mg/m3. Focal or multifocal atrophy (very slight, slight, or moderate) of the anterior respiratory epithelium occurred in the majority of males exposed to 53.5 mg/m3, and in lesser numbers of females exposed to 53.5 mg/m3 and males and females exposed to 5.1 mg/m3. Multifocal hyperplasia (very slight or slight) of the anterior respiratory epithelium occurred in the majority of males exposed to 53.5 mg/m3 and in lesser numbers of females exposed to 53.5 mg/m3 and males exposed to 5.1 mg/m3. Additional treatment-related effects of the anterior respiratory epithelium consisted of: increased incidence of multifocal, very slight or slight hyperplasia and hypertrophy of mucous cells lining the septum and lateral walls of the ventral meatus in males and females exposed to 5.1 or 53.5 mg/m3, as compared to the incidence in same sex control group animals; multifocal, very slight or slight chronic active inflammation in some males exposed to 5.1 or 53.5 mg/m3; multifocal, slight squamous metaplasia in 3/10 males exposed to 53.5 mg/m3; focal, slight mineralization in 2/10 males exposed to 5.1 mg/m3; and focal slight necrosis in 1/10 males exposed to 5.1 mg/m3. Focal or multifocal atrophy (very slight, slight or moderate) of the anterior olfactory epithelium occurred in the majority of females exposed to 5.1 or 53.5 mg/m3, and in lesser numbers of males exposed to 5.1 or 53.5 mg/m3. Additional treatment-related effects of the anterior olfactory epithelium consisted of: multifocal, very slight or slight chronic active inflammation in 1/10 males exposed to 5.1 mg/m3 and 1/10 females exposed to 53.5 mg/m3; focal or multifocal, very slight or slight respiratory epithelial metaplasia in a few males and females exposed to 5.1 or 53.5 mg/m3, and a focal, very slight ulcer with accompanying slight olfactory epithelial mineralization in 1/10 males exposed to 5.1 mg/m3. A very slight or slight suppurative exudate was present in the lumen of the nasal passages in some males exposed to 5.1 or 53.5 mg/m3, and in 2/10 females exposed to 53.5 mg/m3.
Some treatment-related histopathological effects were present in the posterior and ventral aspects of the nasal passages. The majority of males and females exposed to 53.5 mg/m3 had very slight or slight hyaline droplet formation in the olfactory epithelium. Very slight or slight hyaline droplet formation was also present in the respiratory epithelium in 5/10 males and 5/10 females exposed to 53.5 mg/m3. The hyaline droplet formation in the olfactory and respiratory epithelium was most commonly present on the ventral ethmoid turbinates and the ventral septum in the posterior aspect of the nasal passages. An increase in the incidence of multifocal, very slight or slight eosinophilic inflammation was present in males exposed to 53.5 mg/m3 and females exposed to 5.1 or 53.5 mg/m3, as compared to the incidence of same sex control group animals. The inflammation, which consisted of mainly eosinophils, was present in the lamina propria of the ventral aspect of the ethmoid turbinates, subjacent to the dorsal aspect of the pharyngeal duct, and/or in the lamina propria of the ventral and posterior aspects of the nasal septum. Very slight mucous cell hypertrophy of cells lining the pharyngeal duct was present in 6/10 males and 5/10 females exposed to 53.5 mg/m3. One male exposed to 53.5 mg/m3 had slight hyperplasia and hypertrophy of mucous cells lining the pharyngeal duct. One male exposed to 5.1 mg/m3 had a focal, very slight ulcer of the epithelium of the pharyngeal duct, which was accompanied by slight granulomatous inflammation in the underlying lamina propria.
The most common treatment-related effects of the larynx, present in all males and females exposed to 53.5 mg/m3, consisted of: multifocal, slight squamous metaplasia of the respiratory epithelium; multifocal, slight or moderate subacute to chronic inflammation of the lamina propria; and multifocal, slight or moderate fibrosis in the lamina propria. The chronic inflammation was composed of a mixture of neutrophils, lymphocytes and plasma cells, with nodular clusters of lymphocytes present in the lamina propria at the base of the epiglottis. Treatment-related multifocal, slight or moderate hyperplasia of the respiratory epithelium was present in the larynx of all males and 9/10 females exposed to 53.5 mg/m3. All of these laryngeal effects were most prominent in the anterior larynx, near the base of the epiglottis at the level of the seromucinous gland, and some of these effects were present with lesser frequency on the medial aspect of the arytenoid cartilage. Additional treatment-related effects of the larynx in rats exposed to 53.5 mg/m3 consisted of: multifocal, very slight erosions of the respiratory epithelium at the base of the epiglottis in 1/10 males; slight suppurative exudate in lumen of the larynx in 1/10 males and 4/10 females; multifocal, very slight necrosis of individual cells in the respiratory epithelium of ventral aspect of the larynx at the level of the ventral pouch in 1/10 males; and focal or multifocal, very slight, slight, or moderate ulceration of the respiratory epithelium in 4/10 males and 5/10 females. The locations of the laryngeal ulcers varied from animal to animal, and were noted in the respiratory epithelium at the base of the epiglottis, dorsal to the ventral pouch, and/or on the medial aspect of the arytenoid cartilage.
The most common treatment-related effect of the lungs was multifocal, very slight epithelial alteration of the bronchi, observed in all males and females exposed to 53.5 mg/m3. The epithelial alteration was characterized by loss of cilia, flattening, and minimal stratification of the bronchial epithelium. The epithelial alteration was noted in the epithelium that lines the portion of the bronchial walls which project into the airways at some of the branching points of the bronchi. One male exposed to 53.5 mg/m3 had focal, slight bronchiolo-alveolar hyperplasia of the lungs, which was interpreted to be treatment related. Alveolar macrophages and perivascular eosinophils were present in the focal area of bronchiolo-alveolar hyperplasia.
Treatment-related focal or multifocal, very slight epithelial alteration of the respiratory epithelium of the trachea was present in all males and 7/10 females exposed to 53.5 mg/m3. The epithelial alteration was characterized by loss of cilia, flattening, and minimal stratification of respiratory epithelial cells at the level of bifurcation of the trachea.
There were no treatment-related histopathological effects in the upper or lower respiratory tract of males or females exposed to 0.2 mg/m3.
All males from the control and high-dose (53.5 mg/m3) groups had degeneration of testicular seminiferous tubules and associated degenerative spermatic elements of the epididymides of variable severities and distributions. These alterations were caused by physical compression of the testes when the rats were present in the nose-only exposure chambers. All other histopathologic observations were interpreted to be spontaneous alterations, unassociated with exposure to AEP.

Effect levels

open allclose all
Dose descriptor:
NOEC
Effect level:
0.2 mg/m³ air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: point of contact irritant effects
Dose descriptor:
NOEC
Effect level:
53.5 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: systemic toxicity

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Female Red Blood Cell Counts and Hemoglobin Concentrations

 Sex  Females            
 Exposure Level (mg/m3)  Historical Control@  0  0.2  5.1  53.5
 Red Blood Cell Count (106/µ L)  8.35 -9.41  8.92  8.73*  8.85  9.03
 Hemoglobin Concentration (g/dL)  14.5 -16.8  15.4  15.0*  15.4  15.6

@Historical control from data obtained from two nose-only 90-day inhalation toxicity studies and eleven data sets with dietary 90-day toxicity endpoints in F344 rats reported from 2011-2015 (seven 90-day studies and four 2-year studies using 90-day time points).

*Statistically different from control mean by Dunnett’s test, alpha=0.05. Male Cholesterol Concentrations
 Sex  Males            
 Dose (mg/m3)  Historical Control@  0  0.2  5.1  53.5
 Cholesterol Concentration (mg/dL)  52 -70  54  49  50  48*

@Historical control from data obtained from two nose-only 90-day inhalation toxicity studies and eleven data sets with dietary 90-day toxicity endpoints in F344 rats reported from 2011-2015 (seven 90-day studies and four 2-year studies using 90-day time points).

*Statistically different from control mean by Dunnett’s test, alpha=0.05.

Applicant's summary and conclusion

Conclusions:
There were no treatment-related changes in in-life observations, ophthalmological exams, body weight or body weight gains, feed consumption, clinical chemistry, coagulation, hematology, urinalysis, or gross pathologic observations in male or female rats at any exposure level.
Females exposed to 53.5 mg/m3 had higher absolute and relative mean lung weights (statistically significant for relative weight) as compared to controls, which were interpreted to be treatment-related.
In the 13-week exposure group, there was no histopathological evidence of any primary systemic toxicity in any AEP exposure group (systemic NOEC = 53.5 mg/m3). The nasal tissue was the most sensitive target tissue in rats exposed to AEP. Compared to air-exposed (0 mg/m3) control rats of the same sex, male and female rats exposed to 5.1 or 53.5 mg/m3 had exposure-related histopathological changes in the nasal tissues. The larynx, trachea, and lungs of male and female rats exposed to 53.5 mg/m3 also had exposure-related histopathological effects. All of the exposure-related changes were consistent with localized irritant effects of the test material at the point of contact with the airway epithelium.
Treatment-related histopathologic effects persisted following 13-weeks of recovery in the nasal tissues of males exposed to 5.1 or 53.5 mg/m3, and in the larynx and lungs of males exposed to 53.5 mg/m3.
Based on treatment-related effects in the nasal tissues of males and females exposed to 5.1 or 53.5 mg/m3, the no-observed-effect concentration (NOEC) for point of contact irritant effects for male and female F344/DuCrl rats repeatedly exposed to AEP for 13 weeks (65 exposures) was 0.2 mg/m3. The no-observed-effect concentration (NOEC) for systemic toxicity was 53.5 mg/m3.
Executive summary:

This study was designed to evaluate the potential for local (portal-of-entry) and systemic toxicity from inhalation of aminoethylpiperazine (AEP). Groups of ten male and ten female F344/DuCrl rats were exposedvianose-only inhalation to AEP six hours/day, five consecutive days/weekfor 13 weeks (a total of 65 exposures). Additional groups of 10 male rats per exposure concentration were contemporaneously exposed and held (unexposed) for an additional 13 weeks following the last exposure day to serve as post-exposure recovery groups to determine the persistence or reversibility of any AEP-dependent effects identified at the end of exposure. The exposure atmosphere contained both AEP vapor and an AEP-carbamate aerosol that formed spontaneously in the humidified chamber atmosphere. The rats were exposed to analytically-determined concentrations of0, 0.2 ± 0.1, 5.1 ± 1.1, or 53.5 ± 6.0 mg AEP/m3(study mean±standard deviation). The analytical method measured total AEP present in the exposure chambers (AEP vapor + AEP-carbamate aerosol). The average mass median aerodynamic diameter (MMAD) of the aerosol fraction in each of the exposure chambers was0.85±1.52,1.96 ± 1.58, or 1.04 ± 2.39microns (MMAD ± geometric standard deviation; GSD) for the 0.2, 5.1, or 53.5 mg AEP/m3exposure chambers, respectively. In-life observations(including ophthalmology), body weights/body weight gains, feed consumption,urinalysis,hematology, coagulation, clinical chemistry, and organ weights were evaluated. A gross necropsy was conducted and a detailed histopathological examination of the entire respiratory tract was performed to assess treatment-dependent portal-of-entry effects. In addition, a detailed histopathologic examination of other specified tissues/organs was performed on the control- and high-exposure group rats to identify treatment-related systemic toxicity.

There were no treatment-related changes in in-life observations, ophthalmologic examinations, body weight/body weight gains, feed consumption, clinical chemistry, coagulation, hematology, urinalysis, or gross pathologic observations in male or female rats at any exposure level.

Females exposed to 53.5 mg/m3had higher absolute and relative mean lung weights (statistically significant for relative weight) as compared to controls, which were interpreted to be treatment-related.

In the 13-week exposure group, there was no histopathological evidence of any primary systemic toxicity in any AEP exposure group (systemic NOEC ≥ 53.5 mg/m3). The nasal mucosa was the most sensitive target tissue in rats exposed to AEP. Compared to air-exposed (0 mg/m3) control rats of the same sex, male and female rats exposed to 5.1 or 53.5 mg/m3had exposure-related histopathological changes in the nasal tissues. The larynx, trachea, and lungs of male and female rats exposed to 53.5 mg/m3also had exposure-related histopathological effects. All of the exposure-related changes were consistent with localized irritant effects of the test material at the point of contact with the airway epithelium.

There were no treatment-related histopathologic effects in males or females exposed to 0.2 mg/m3. In the 13-week recovery group, treatment-related histopathological effects persisted in the nasal tissues of males exposed to 5.1 or 53.5 mg/m3, and in the larynx and lungs of males exposed to 53.5 mg/m3.

Based on treatment-related effects in the nasal tissues of males and females exposed to 5.1 or 53.5 mg/m3, the no-observed-effect concentration (NOEC) for point of contact irritant effects for male and female F344/DuCrl rats repeatedly exposed toAEPfor 13 weeks (65 exposures) was 0.2 mg/m3. The no-observed-effect concentration (NOEC) for systemic toxicity was 53.5 mg/m3.