2-piperazin-1-ylethylamine

Endpoint summary

Description of key information

An OECD 422 guideline study, "Combined Repeated Dose Toxicity Study with the Reproductive/Developmental Toxicity Screen Test, was conductedon Sprague Dawley rats at doses of 0, 500, 2000 ppm and 8000 ppm of aminoethyl piperazine (AEP) administered via the drinking water
An OECD 90 day rat inhalation study was conducted with 90 day recovery via nose only exposures. Male and female F34 rats were exposed 6 h/day, 5 days/wk for 13 wks (a total of 65 exposure days) to 0, 0.2, 5.1, or 53.5 mg AEP/m3.  All exposure-related effects were consistent with point-of-contact irritation of the upper and lower airway epithelium.  No treatment-related histopathologic lesions observed in the upper or lower respiratory tract of male or female rats exposed to 0.2 mg AEP/m3 (NOEC).  No histopathologic evidence of treatment-related systemic toxicity was observed in any AEP-exposed rats including rats exposed to the highest concentration of 53.5 mg AEP/m3. 
Female rats exposed to 53.5 mg AEP/m3 had higher absolute and relative mean lung weights, compared to control rats
Exposure-related lesions in the larynx, trachea and lungs were limited to rats exposed to 53.5 mg AEP/m3 
Rats exposed to 5.1 and 53.5 mg AEP/m3 had concentration-dependent nasal airway lesions
Exposure-related lesions in the 13-week recovery groups persisted in the nasal tissues of male rats exposed to 5.1 and 53.5 mg AEP/m3 and the larynx and lungs of males exposed to 53.5 mg AEP/m3 
The repeated dose dermal administration of AEP resulted in dose-related iritative effects at the test site in male rats at 100, 500, or 1000 mg/kg/day and in female rats at 500 and 1000 mg/kg/day.   There was no systemic toxicity at any of the dose levels.  The NOEL was considered to be 1000 mg/kg/day.  

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5 March 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP/Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:Charles River Laboratories, Inc.
- Age at study initiation: 91-101 days
- Weight at study initiation: Males 300-500 gm; Females 200-300 gm
- Housing:All animals were individually housed in clean suspended wire mesh cages in an environmentally controlled room during the acclimation period and continuing until mating. Following successful mating, the females were housed individually in a plastic cage containing ground
corncob nesting material (Bed O'Cobs) and remained in these cages until euthanasia on lactation day 4.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Actual mean daily temperature ranged from 70.5°F to 71.4°F (21.4°C to 21.9°C).
- Humidity (%): Mean daily relative humidity ranged from 38.2% to 52.0% during the study.
- Air changes (per hr):10 room air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hour light/dark photoperiod

IN-LIFE DATES: From: 5 March 2010 To: 27 April 2010

Route of administration:

oral: drinking water

Vehicle:

water

Details on oral exposure:

VEHICLE: Water
The test substance was administered as a constant concentration (mg/ml) in reverse osmosis-treated drinking water.

The test item formulations were prepared approximately weekly as single formulations for each dosage level; the pH of each formulation was adjusted to 9.0 ± 0.1 with 1 N HCl (prepared using 37% hydrochloric acid, NF; lot nos. YT0470 and YW0968, exp. date: 5 June 2012 and 30 November 2012, respectively, received from Spectrum Chemical Manufacturing Corporation, New Brunswick, NJ). The test item formulations were transferred into 10-L plastic carboys for administration and stored at room temperature. The test item formulations were stirred continuously throughout the preparation and sampling.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical concentrations were within 98.6 to 110% of target doses.

The analyzed drinking water formulations were stable for 14 days of room temperature storage.

Duration of treatment / exposure:

Males/Females: At least 28-Days

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
500, 2000 or 8000 ppm
Basis:
nominal in water

No. of animals per sex per dose:

12

Control animals:

yes

Details on study design:

- Dose selection rationale:Due the steep dose response curve (in the range-finding/palatability study) between 10000 ppm, which exceeded the
maximum tolerated dose, and 7500 ppm which resulted in a slight, transient reduction in body weights, food consumption, and water consumption, the high-dose level of 8000 ppm for the definitive study was chosen.

Males: Individual body weights were recorded twice weekly, beginning one week prior to test substance administration, on the first day of dosing and twice weekly thereafter until euthanasia. Individual food consumption and water consumption were recorded twice weekly, beginning one week prior to test substance administration, on the first day of dosing and twice weekly thereafter until animals were paired for breeding. Following evidence of mating, males continued to have individual food consumption and water consumption recorded twice weekly thereafter until euthanasia.

Females: Individual body weights were recorded twice weekly, beginning one week prior to test substance administration, on the first day of dosing and twice weekly thereafter until evidence of copulation was observed. Individual food consumption and water consumption were recorded twice weekly, beginning one week prior to test substance administration, on the first day of dosing and twice weekly thereafter until animals were paired for breeding. Females with no evidence of mating had body weights, food consumption, and water consumption recorded twice weekly upon completion of the breeding period through euthanasia.

Positive control:

Not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
All rats were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
A detailed physical examination was conducted weekly on each animal and on the day of necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: Twice weekly

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

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Individual water consumption were recorded twice weekly, beginning one week prior to test substance administration, on the first day of dosing and twice weekly thereafter until animals were paired for breeding.

OPHTHALMOSCOPIC EXAMINATION: As part of the Functional Observation Battery which included a pupil response

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the scheduled necropsies (study day 28, males and lactation day 4, females).
- Anaesthetic used for blood collection: Yes - isoflurane
- Animals fasted: All males were fasted overnight prior to blood collection.
- How many animals:All animals (with the exception of the female that failed to deliver)
- Blood for hematology was collected from the retro-orbital sinus following isoflurane anesthesia.
- Blood for coagulation parameters was collected from the vena cava at the time of necropsy. Blood was collected into tubes containing EDTA (hematology) or sodium citrate (clotting determinations).
- Parameters examined
Total leukocyte count (WBC)
Erythrocyte count (RBC)
Hemoglobin (HGB)
Hematocrit (HCT)
Mean corpuscular volume (MCV)
Mean corpuscular hemoglobin (MCH)
Mean corpuscular hemoglobin concentration (MCHC)
Platelet count (PLATELET)
Prothrombin time (PT)
Activated partial thromboplastin time (APTT)
Reticulocyte count Percent (RETIC)
Absolute (RETIC ABSOLUTE)
Mean Platelet Volume (MPV)
Red cell distribution width (RDW)
Hemoglobin Distribution Width (HDW)
Differential leukocyte count –
Percent and absolute
-Neutrophil (NEU)
-Lymphocyte (LYMPH)
-Monocyte (MONO)
-Eosinophil (EOS)
-Basophil (BASO)
-Large unstained cell (LUC)
Platelet estimate
Red cell morphology (RBC Morphology)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the scheduled necropsies (study day 28, males and lactation day 4, females).
- Animals fasted: All males were fasted overnight prior to blood collection.
- How many animals:All animals (with the exception of the female that failed to deliver)
- Blood for serum chemistry was collected from the retro-orbital sinus following isoflurane anesthesia.
- Blood was collected into tubes containing no anticoagulant (serum chemistry).
- Parameters examined
Albumin
Total protein
Globulin [by calculation]
Albumin/globulin ratio (A/G Ratio) [by calculation]
Total bilirubin (Total Bili)
Urea nitrogen
Creatinine
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Gamma glutamyltransferase (GGT)
Glucose
Total cholesterol (Cholesterol)
Calcium
Chloride
Phosphorus
Potassium
Sodium
Triglycerides (Triglyceride)
Bile acids

URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:Females:Prior to dosing and on lactation day 4. Males:Prior to dosing and following approximately 28 days of dose administration
- Dose groups that were examined: All
- Battery of functions tested: sensory activity / grip strength / motor activity / body temperature

OTHER:

Sacrifice and pathology:

UNSCHEDULED DEATHS
Gross necropsies were performed on the males and females that were euthanized (by carbon dioxide inhalation) in extremis or found dead during the course of the study. A gross necropsy was performed. The animals were then discarded.

SCHEDULED EUTHANASIA
All surviving F0 adults were euthanized by carbon dioxide inhalation. Males were euthanized following completion of the mating period. Females that delivered were euthanized on lactation day 4; the numbers of former implantation sites and corpora lutea were recorded. Females that failed to deliver were euthanized on post-cohabitation day 25 (females with no evidence of mating). Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss (Salewski, 1964). Necropsy included examination of the external surface, all orifices and the cranial cavity, the external surface of the brain, and the thoracic, abdominal, and pelvic cavities, including viscera.

At the time of necropsy, the following tissues and organs were placed in 10% neutral-buffered formalin (except as noted):
Adrenal glands (2)
Aorta Axillary
Bone with marrow (sternebrae)
Bone marrow smear
Brain
Cerebrum level 1
Cerebrum level 2
Cerebellum with medulla/pons
Coagulating glands
Eyes with optic nerve (2)b
Gastrointestinal tract
- Esophagus
- Stomach
- Duodenum
- Jejunum
- Ileum
- Cecum
- Colon
- Rectum
Heart
Kidneys (2)
Liver (sections of 2 lobes)
Lungs (including bronchi, fixed by inflation with fixative)
Lymph node
- Axillary
- Mesenteric
- Mandibular
Ovaries and oviducts (2)
Pancreas
Peripheral nerve (sciatic)
Pituitary gland
Prostate gland
Salivary gland [mandibular (2)]
Seminal vesicles (2)
Skeletal muscle (rectus femoris)
Skin with mammary glandc
Spinal cord (cervical)
Spleen
Testes with epididymidesd (2)
Thymus gland
Thyroids [with parathyroids, if present (2)]
Trachea
Urinary bladder
Uterus with vagina
All gross lesions (all groups)

The following organs were weighed from all F0 animals at the scheduled necropsies:
Adrenal glands
Brain
Epididymidesa
Heart
Kidneys
Liver
Ovaries with oviducts
Spleen
Testes
Thymus gland
Thyroids with parathyroids

MICROSCOPIC EXAMINATIONS
After fixation, protocol-specified tissues were trimmed. Trimmed tissues were processed into paraffin blocks, sectioned at 4 to 8 microns, mounted on glass microscope slides, and stained with hematoxylin and eosin, with the exception of the testes and epididymides which were stained with PAS and hematoxylin to allow for a detailed histopathological examination.

Microscopic examination was performed on all tissues listed from all animals in the control and 8000 ppm groups at the scheduled necropsies and from the males and females that died or were euthanized in extremis. Missing tissues were identified as not found at necropsy, lost at necropsy, lost during processing, not in plane of section, or other reasons as appropriate.

Other examinations:

LITTER VIABILITY AND DEATHS
Each litter was examined daily for survival, and all deaths were recorded. All pups were individually identified by application of tattoo markings on the digits following completion of parturition. A daily record of litter size was maintained. Intact offspring dying were necropsied using a fresh dissection technique, which included examination of the heart and major vessels (Stuckhardt and Poppe, 1984). Tissues were preserved in 10% neutral-buffered formalin for possible future histopathologic examination only as deemed necessary by the gross findings. The carcass of each pup was then discarded.

CLINICAL OBSERVATIONS
Litters were examined daily for survival and any adverse changes in appearance or behavior. Each pup received a detailed physical examination on PND 1 and 4. Any abnormalities in nursing behavior were recorded.

BODY WEIGHTS
Pups were individually weighed on PND 1 and 4. Mean pup weights were presented by sex for each litter and by dose group. When body weights could not be determined for a pup during a given interval (due to an unscheduled death, weighing error, etc.), group mean values were calculated for that interval using the available data. The time periods a given pup was not weighed were left blank or designated as “NA” on the individual
report tables.

SEX DETERMINATION
Pups were individually sexed on PND 0 and 4.

CALCULATION OF LITTER PARAMETERS
Litter parameters were defined as follows:
Mean Live Litter Size = Total No. of Viable Pups on PND 0/No. of Litters with Viable Pups PND 0

Postnatal Survival Between Birth and PND 0 or PND 4 (% Per Litter) =
Sum of (Viable Pups Per Litter on PND 0 or PND 4/No. of Pups Born Per Litter)/No. of Litters Per Group x 100

Postnatal Survival for All Other Intervals (% Per Litter) =
Sum of (Viable Pups Per Litter at End of Interval N/Viable Pups Per Litter at Start of Interval N)/No. of Litters Per Group x 100

Where N = PND 0-1 and 1-4

SCHEDULED EUTHANASIA
On PND 4, surviving F1 rats were euthanized via an intraperitoneal injection of sodium pentobarbital and discarded.

Statistics:

Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean. Data obtained from nongravid females were excluded from statistical analyses following the mating period. Where applicable, the litter was used as the experimental unit.

Analyses
were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test item-treated group to the control group by sex.

Parental mating, fertility, conception, and copulation indices were analyzed using the Chi-square test with Yates’ correction factor (Hollander and Wolfe, 1999). Mean parental body weights (weekly, gestation, and lactation), body weight changes, and food consumption, offspring body weights and body weight changes, gestation length, numbers of former implantation sites and corpora lutea, number of pups born, live litter size on PND 0, unaccounted-for sites, absolute and relative organ weights, clinical pathology values (except gamma glutamyltransferase), and pre-coital intervals were
subjected to a parametric one-way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences between the control and test item-treated groups. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett’s test (Dunnett, 1964) was used to compare the test item-treated groups to the control group. FOB parameters (sensory observations) that yield scalar or descriptive data and histopathological findings in the test item-treated groups were compared to the control group using Fisher’s Exact test (Steel and Torrie, 1980).

Continued below

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Increased mortaility in 8000 ppm group

Mortality:

mortality observed, treatment-related

Description (incidence):

Increased mortaility in 8000 ppm group

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean male body weight in 8000 ppm group was up to 11.0% lower than the control group during the treatment period. Lower mean body weight gain was noted during the first week of treatment (study days 0-6) in female rats in 8000 ppm group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In the 8000 ppm male and female rats reduced feed consumption was observed.

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

Lower mean water consumption observed in the high dose group.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Only examined as part of FOB

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

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:

not specified

Details on results:

OTHER FINDINGS: In a previous range-finding/palatability study (WIL-768001, Kuhl, Draft), groups of three male and female Crl:CD(SD) rats were administered 0, 750, 1875, 3750, 7500,10000, or 15000 ppm AEP in pH 9.0 drinking water for 14 days. The 15000 and 10000 ppm groups exceeded the maximum tolerated dose after seven days of treatment, as evidenced by significant body weight losses (~20-30%) that was driven by feed and water consumption of less than 5g/animal/day. Based on these findings, animals in these dose groups were euthanized for humane reasons on study day (SD) 7. Animals in the 7500 ppm group had slight, but not statistically identified, effects on body weightlbody weight gain that were likely driven by lower feed and water consumption upon initiation of treatment (SD 1-4). This effect was transient and recovered by SD 7. There were no effects at concentrations of 3750 ppm AEP or lower.

F0 GENERATION
CLINICAL OBSERVATIONS AND SURVIVAL
In the 8000 ppm group, 5 males (nos. 67248, 67254, 67256, 67258, and 67273) and 1 female (no. 67217) were euthanized in extremis between study days 7-13. The moribundity of these animals occurred following body weight losses (46 g to 122 g) with reduced food (≤19 g/day) and water consumption (≤22 g/day) from study day 0 through the day of death/euthanasia. Clinical findings noted for these animals on the days prior to
or on the day of death/euthanasia were limited to decreased defecation, red material around the eye or nose, and/or hair loss on the forelimb. In addition, 1 female (no. 67216) was found dead on study day 20. Upon microscopic examination, the cause of death for this female was determined to be hydrocephalus, which was presumed to be an incidental finding that was unrelated to administration of the test item. The cause of moribundity could not be determined microscopically for any the animals euthanized in extremis because no significant internal findings were observed. However, the moribundity of these animals occurred in the presence of test item-related reductions in body weight and food and water consumption noted at this same dosage level, and therefore was considered to be test item-related. All other animals survived to the scheduled necropsies.

For animals that survived to the scheduled necropsy, clinical findings were limited to hair loss on the limbs. These findings occurred infrequently and/or at similar frequencies in the control group, and were not attributed to test item administration.

BODY WEIGHTS
MALES
Test item-related mean body weight losses were noted in the 8000 ppm group males during study days 0-10 followed by a lower mean body weight gain during study days 10-13; differences from the control group were significant (p<0.01) during study days 0-3 and 3-6. Mean body weights in the 8000 ppm group were up to 11.0% lower compared to the control group during study days 0-13; differences from the control group were significant (p<0.05) on study day 6. These body weight effects were primarily due to the 5 males in this group euthanized in extremis during study days 7-13. Mean body weights and body weight gains for the surviving males were comparable to the control group during the remainder of the treatment period (study days 13-31). Due to the initial mean body weight losses and lower mean body weight gain in the 8000 ppm group, a significantly (p<0.05) lower mean body weight gain was noted for the overall pre-mating period (study days 0-13) and a lower (not statistically significant) mean body weight gain was noted when the entire generation (study days 0-31) was evaluated compared to the control group.

Mean male body weights and body weight gains in the 500 and 2000 ppm groups were similar to the control group throughout the treatment period. Differences from the control group were slight and not statistically significant.

FEMALES
PRE-MATING
A test item-related, significant (p<0.01) mean body weight loss followed by an absence of body weight gain were noted in the 8000 ppm group during the first week of the treatment period (study days 0-6). As a result, mean body weight in this group was 5.0% lower (not statistically significant) compared to the control group on study day 6. During the remainder of the pre-mating period (study days 6-13), mean body weight gain in the 8000 ppm group was similar to the control group. The mean body weight loss and absence of body weight gain noted in the 8000 ppm group during the first week of treatment were of sufficient magnitude to result in a significantly (p<0.05) lower mean body weight gain when the entire pre-mating period (study days 0-13) was evaluated, and thus were considered to be adverse.

Mean female body weights and body weight gains in the 500 and 2000 ppm groups were similar to the control group during the pre-mating period. Differences from the control group were slight and not statistically significant.

GESTATION
Slightly lower (not statistically significant) mean body weight gains were noted in the 2000 and 8000 ppm groups generally throughout gestation compared to the control group. As a result, significantly (p<0.05 or p<0.01) lower mean body weight gains were noted in these groups when the overall gestation treatment period (gestation day 0-20) was evaluated, and mean body weight in the 8000 ppm group was 5.8% lower (not statistically significant) than the control group on gestation day 20. Conversely, the lower mean body weight gains noted in the 2000 ppm group were not of sufficient magnitude to affect mean body weight, and therefore were not considered to be test item-related.

Mean body weights and body weight gains in the 500 ppm group were generally similar to those in the control group throughout gestation. Differences from the control group were slight and not statistically significant.

LACTATION
Mean maternal body weight gains were unaffected by test item administration during lactation days 1-4. However, mean body weights in the 8000 ppm group were up to 6.6% lower (not statistically significant) than the control group during lactation days 1-4 as a result of the lower mean body weights noted in this group during the pre-mating period and gestation.

Mean body weights in the 500 and 2000 ppm groups were unaffected by test item administration during lactation days 1-4. Differences from the control group were slight and not statistically significant.

FOOD CONSUMPTION
MALES
Test item-related lower mean male food consumption, evaluated as g/animal/day and g/kg/day, was noted in the 8000 ppm group during the pre-mating period (study days 0-13); differences from the control group were significant (p<0.05 or p<0.01). The lower mean food consumption corresponded to the overall lower mean body weight gain noted in this group during the pre-mating period and was primarily due to the 5 males in this group euthanized in extremis during study days 7-13. Mean food consumption in this group was similar to the control group during study days 27-30.

Mean male food consumption in the 500 and 2000 ppm groups was similar to the control group during the pre-mating period (study days 0-13). Differences from the control group were slight and not statistically significant.

FEMALES
PRE-MATING
Test item-related lower mean female food consumption, evaluated as g/animal/day and g/kg/day, was noted in the 8000 ppm group during the first week of treatment (study days 0-6); differences from the control group were significant (p<0.01) and corresponded to a period of mean body weight loss. Mean food consumption in this group was similar to the control group during study days 6-13.

Mean food consumption in the 500 and 2000 ppm groups was unaffected by test item administration during the pre-mating period (study days 0-13). Prior to the initiation of treatment (study days -7 to -3), significantly (p<0.01) higher mean food consumption was noted in the 2000 ppm group. No other statistically significant differences were noted when the test item-treated groups were compared to the control group.

GESTATION
Mean food consumption in the 500, 2000, and 8000 ppm groups was unaffected by test item administration during gestation. Significantly (p<0.05) higher mean food consumption (g/animal/day value only) was noted in the 500 ppm group during gestation days 0-4. However, in the absence of an exposure-related response, the increased mean food consumption was not considered to be treatment-related. No other statistically significant differences were noted when the test item-treated groups were compared to the control group.

LACTATION
Mean food consumption in the 500, 2000, and 8000 ppm groups was unaffected by test item administration during lactation days 1-4. Differences from the control group were slight and not statistically significant.

WATER CONSUMPTION
MALES
Mean water consumption, evaluated as g/animal/day and g/kg/day, in the 8000 ppm group males was lower than the control group during study days 0-10. Differences from the control group were generally significant (p<0.01) and corresponded to a test item-related lower mean body weight gain noted during the same interval. In addition, a significant (p<0.01) decrease in mean water consumption was noted in the 8000 ppm group during study days 27-31.

Mean water consumption in the 500 and 2000 ppm group was similar to that in the control group throughout the study. Lower (p<0.05) mean water consumption was also noted in the 2000 ppm group compared to the control group during study days 27-31. Due to the lack of a concurrent effect on mean body weight gain during this interval, the decreased mean water consumption in this group was not considered to be test item-related. No other statistically significant differences were noted when the test item-treated groups were compared to the control group.

FEMALES
PRE-MATING
Lower mean water consumption, evaluated as g/animal/day and g/kg/day, was noted in the 8000 ppm group during study days 0-10; differences from the control group were significant (p<0.01) during study days 0-3 and corresponded to a test-item-related mean body weight loss during the first week of dose administration. Mean water consumption in this group was similar to the control group during study days 10-13.

Mean water consumption in the 500 and 2000 ppm groups was unaffected by test item administration during the pre-mating period (study days 0-13). Increased mean water consumption was noted in both groups during study days 10-13; differences from the control group were generally significant (p<0.05 or p<0.01). However, in the absence of a similar effect in the high-dose group, the increased mean water consumption noted in
the 500 and 2000 ppm group was not considered to be test item-related. No other statistically significant differences were noted when the test item-treated groups were compared to the control group.

GESTATION
Mean maternal water consumption, evaluated as g/animal/day and g/kg/day, was unaffected by test item administration during gestation. Differences between the control, 500, 2000, and 8000 ppm groups were slight and not statistically significant.

LACTATION
Mean maternal water consumption, evaluated as g/animal/day and g/kg/day, was unaffected by test item administration during lactation days 1-4. Differences between the control, 500, 2000, and 8000 ppm groups were slight and not statistically significant.

TEST ITEM CONSUMPTION
The average quantities of aminoethylpiperazine consumed during the F0 generation are presented below (Table 1). Values for the entire lactation period in all groups were elevated, as is commonly seen in nursing animals.

FUNCTIONAL OBSERVATIONAL BATTERY (FOB)
SENSORY OBSERVATIONS
Sensory parameters were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the control group during study day 27 (males) or on lactation day 4 (females).

NEUROMUSCULAR OBSERVATIONS
Neuromuscular parameters were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the control group during study day 27 (males) or on lactation day 4 (females).

PHYSIOLOGICAL OBSERVATIONS
A lower (6.7%) mean body weight was noted for the 8000 ppm group females compared to the control group at the physiological observations on lactation day 4; the difference from the control group was significant (p=0.002). The lower mean body weight corresponded to the lower mean body weight recorded on lactation day 4. Mean body temperature was unaffected by test item administration at all dosage levels. There were no other statistically significant differences for the test item-treated groups when compared to the control group on study day 27 (males) or lactation day 4 (females).

LOCOMOTOR ACTIVITY
Locomotor activity patterns (total activity) in F0 animals were unaffected by test item administration at all concentrations when evaluated on study day 27 (males) and lactation day 4 (females). Values obtained from the 6 subintervals evaluated (0-10, 11-20, 21-30, 31-40, 41-50 and 51-60 minutes) and the overall 60-minute test session values were comparable to the concurrent control values with the following exceptions. Mean total activity counts for females in the 500 and 8000 ppm groups at the lactation day 4 evaluation were higher for the 6 subintervals as well as the overall 60-minute test session; differences from the control group achieved significance (p≤0.003) for these groups when the overall 60-minute test session was evaluated for total motor activity counts by a repeated measures analysis. However, no dose-related trend was apparent and the increase in motor activity was primarily attributed to individual females in the 500 and 8000 ppm groups with atypically high total motor activity values. In addition, mean total motor activity counts in the 500 and 8000 ppm groups on lactation day 4 were generally similar to pretest values during the first subinterval (0-10 minutes) and differences between the pretest and lactation evaluations were limited to greater habituation on lactation day 4. Therefore, the increased mean total activity counts for females in the 500 and 8000 ppm groups were not considered to be test item-related.

No remarkable shifts in the pattern of habituation occurred in any of the test item-treated groups when the F0 animals were evaluated at study day 27 (males) and lactation day 4 (females).

CLINICAL PATHOLOGY
HEMATOLOGY
There were no test item-related alterations in hematology and coagulation parameters. A significantly (p<0.05) lower mean corpuscular hemoglobin (MCH) value was noted in the 2000 ppm group males. This group mean difference was not considered to be test item-related because the value did not show a dose- or time-related response. No other statistically significant differences were noted when the test item-treated groups were compared to the control group.

SERUM CHEMISTRY
There were no test item-related effects on serum chemistry parameters. Significantly (p<0.05) higher mean glucose and triglyceride values were noted in the 500 ppm group males and females, respectively. These group mean differences were not considered to be test item-related because the values did not show a dose- or time-related response. No other statistically significant differences were noted when the test item-treated groups were compared to the control group.

REPRODUCTIVE PERFORMANCE
F0 male and female reproductive parameters are presented in Table 2.

No test item-related effects on reproductive performance were observed at any exposure level. No statistically significant differences were noted between the control and test item-treated groups. One mating pair in the 8000 ppm group did not produce a litter. The mean numbers of days between pairing and coitus in the test item-treated groups were similar to the control group value. None of these differences were statistically significant.

GESTATION LENGTH AND PARTURITION
Mean gestation lengths in the 500, 2000, and 8000 ppm groups were similar to those in the control group. No statistically significant differences were noted. No signs of dystocia were noted in these groups.

ANATOMIC PATHOLOGY
MACROSCOPIC EXAMINATIONS
In the 8000 ppm group, 5 males and 1 female were euthanized in extremis between study days 7-13, and 1 female was found dead study day 20. All other animals survived to the scheduled necropsies. No test item-related internal findings were observed at any dosage level in males and females that died, were euthanized in extremis, failed to deliver, or at the scheduled necropsies. Macroscopic findings observed in the test item-treated groups, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related. The mean numbers of unaccounted-for sites, implantation sites, and corpora lutea in the 500, 2000, and 8000 ppm groups were similar to the control group values.

ORGAN WEIGHTS
The mean final body weight for the 8000 ppm group females was 6.4% lower (not statistically significant) than the control group females. This lower body weight was considered an adverse test item-related effect. A significantly (p<0.05) higher mean relative (to body weight) thyroid/parathyroid weight was noted in the 8000 ppm group females when compared to the control group; however, this change was attributed to the test item-related decrease in mean body weight and was not considered to be a direct effect of the test item. In addition, lower mean absolute and relative (to body and brain weight) spleen weights were noted in the 8000 ppm group females compared to the control group. The differences were significant (p<0.05 or p<0.01), but the splenic weight differences were not considered test item-related given the lack of microscopic changes consistent with cell loss. No other statistically significant differences were noted when the test item-treated groups were compared to the control group.

MICROSCOPIC EXAMINATIONS
There were no test item-related histologic changes. Five males and 1 female in the 8000 ppm group were euthanized in extremis between study days 7-13; a specific cause of death was not determined microscopically for these animals. In addition, female no. 67216 was found dead on study day 20. Microscopically, all ventricles of the brain were markedly dilated (hydrocephalus) and were lined by hyperplastic and hypertrophic ependymal cells. The surrounding neuropil contained increased numbers of cells consistent with gliosis and chronic inflammation. The ependymal and surrounding neuropil changes were most evident in the lateral and third ventricles. A specific etiology for the ventricular changes in the brain was not determined. The cause of death for this rat was hydrocephalus, which was presumed to be an incidental finding that was unrelated to administration of the test item.

Remaining histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than administration of the test item. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Dose descriptor:

NOAEL

Effect level:

2 000 mg/L drinking water

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

Table 1 Mean Calculated F0 Test Item Consumption mg/kg/day

	Males			Females
Target Exposure Level	Prior to Mating	After Mating	TWA^a	Prior to Mating	Gestation	Lactation
500 ppm	41	37	40	61	57	83
2000 ppm	162	126	152	224	216	285
8000 ppm	416	404	409	598	899	1376

^aTime Weighted Average

Conclusions:

Based on test item-related moribundity at 8000 ppm, reduced mean body weights, body weight gains, and food and water consumption in the 8000 ppm group males and females, the NOAEL for systemic toxicity was considered to be 2000 ppm.

Executive summary:

This study was designed to investigate the potential toxic effects of the test item when administered to rats for 28 days and to evaluate the potential of the test item to effect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition, and early postnatal development.

The test item, aminoethylpiperazine (AEP), was administered continuously in reverse osmosis-purified drinking water to 3 groups of Crl:CD(SD) rats, each group consisting of 12 males and 12 females. Exposure levels were 500, 2000, and 8000 ppm. A concurrent

control group of 12 rats/sex received the vehicle (reverse osmosis-purified drinking water) on a comparable regimen. Males and females were approximately 14 weeks of age at the beginning of test item administration. The test item was offered to males for a

minimum of 14 days prior to mating. Males continued to be exposed to the test item throughout mating and through the day of euthanasia. Females were exposed to the test item for a minimum of 14 days prior to mating through lactation day 4; the female that

failed to deliver was exposed to the test item through the day of euthanasia (post-cohabitation day 25). Males were exposed for 32 consecutive days and females were exposed for 39-53 consecutive days.

All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food and water consumption were recorded at appropriate intervals. FOB assessments and locomotor activity data were recorded for 12 animals/sex/group prior to the initiation of exposure and for 7-12 males/group following approximately 28 days of exposure and for 9-12 females/group on lactation day 4. All F0 females were allowed to deliver and rear their pups until lactation day 4.

F1 clinical observations and body weights were recorded on PND 1 and 4. Pups were necropsied on PND 4. Clinical pathology evaluations (hematology and serum chemistry) were performed on all available F0 animals (7-12/sex/group) at necropsy.

FOB assessments and locomotor activity and clinical pathology evaluations were not conducted for the female that failed to deliver. F0 males were euthanized following completion of the mating period. F0 females were euthanized on lactation day 4 for females that delivered and post-cohabitation day 25 for the female that failed to deliver. Complete necropsies were conducted on all F0 animals, and selected organs were weighed. Selected tissues were examined microscopically from all F0 animals in the control and high-dose groups; gross lesions from all animals in all dosage groups were also examined microscopically.

Five males and 1 female in the 8000 ppm group were euthanized in extremis during the treatment period following body weight losses and reduced food and water consumption. The cause of moribundity of these animals could not be determined microscopically. In

addition, 1 female in the 8000 ppm group was found dead on study day 20; the cause of death for this female was determined to be an incidental finding (hydrocephalus) that was unrelated to administration of the test item. The moribundity observed in the 8000 ppm

group males was considered to be test item-related as it occurred in the presence of effects on body weight and food and water consumption noted at this same dosage level. All other animals survived to the scheduled necropsies.

In the 8000 ppm group males, test item-related lower mean body weight gains were noted when the overall pre-mating (study days 0-13) and treatment (study days 0-31) periods were evaluated; correspondingly lower mean food and water consumption were noted

during the pre-mating period. As a result, mean male body weight in this group was up to 11.0% lower than the control group during the treatment period. In addition, a test item-related lower mean body weight gain with corresponding reduced food and water

consumption was noted during the first week of treatment (study days 0-6) in the 8000 ppm group females, resulting in a lower (5.0%) mean body weight on study day 6. As a result of these body weight effects, lower mean body weights and/or body weight gains in the absence of effects on food and water consumption continued to be observed in the 8000 ppm group females throughout gestation and lactation.

Mean body weights, body weight changes, and food and water consumption were unaffected by test item administration in the 500 and 2000 ppm group males throughout the study and in the 500 and 2000 ppm group females during the pre-mating, gestation,

and lactation periods.

No test item-related effects were noted during the FOB assessments or locomotor activity evaluations at any dosage level.

At the scheduled necropsy, a test item-related lower mean final body weight was noted in the 8000 ppm group females. No other changes in clinical pathology parameters, gross necropsy observations, or organ weight changes associated with test item administration were observed.

Endpoint conclusion

Dose descriptor:

NOAEL

152 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

no

Qualifier:

according to guideline

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

Species:

rat

Strain:

other: F344/DuCrl

Sex:

male/female

Details on test animals or test system 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:

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)

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

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.

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.

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

Critical effects observed:

not specified

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 (10^6/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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

53.5 mg/m³

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

no

Qualifier:

according to guideline

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

Species:

rat

Strain:

other: F344/DuCrl

Sex:

male/female

Details on test animals or test system 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:

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)

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

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.

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.

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

Critical effects observed:

not specified

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 (10^6/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.

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

0.2 mg/m³

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Not applicable

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: 1a: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

GLP compliance:

yes

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Type of coverage:

occlusive

Vehicle:

other: distilled water

Details on exposure:

TEST SITE
- Area of exposure: no data
- % coverage: The test material was applied to an area which was approximately 10% of the total body surface area.
- Type of wrap if used: The exposure site was occluded with an absorbent gauze pad and non-absorbent cotten and the animal was wrapped in an elastic wrap and tape to hold the test material, gause pad and cotton in place.
- Time intervals for shavings or clipplings: no data


REMOVAL OF TEST SUBSTANCE
- Washing (if done): The application site was wiped with a water-dampened towel to remove any residual test material.
- Time after start of exposure: Wraps were removed approximately 6 hours after application and the application site was wiped with a water-dampened towel to remove any residual test material.


TEST MATERIAL
- Amount(s) applied (volume or weight with unit): Dose volume of 4 ml/kg yielded the appropriate dose.
- Concentration (if solution): AEP was administered as a solution in distilled water such that a dose volume of 4 ml/kg yielded the appropriate dose.
- Constant volume or concentration used: yes



VEHICLE
- Amount(s) applied (volume or weight with unit): no data
- Concentration (if solution): no data
- Purity: no data


USE OF RESTRAINERS FOR PREVENTING INGESTION: no data

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

6 hours/application

Frequency of treatment:

5 days/week for 29 days

Remarks:

Doses / Concentrations:
100, 500 or 1000 mg/kg/application
Basis:
nominal per unit body weight

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: no data

DETAILED CLINICAL OBSERVATIONS: Yes

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: no data

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

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: No data
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: No data
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: all

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: No data
- Animals fasted: Yes
- How many animals: all

URINALYSIS: Yes
- Time schedule for collection of urine: Urinalysis was conducted during the week prior to termination of the study.
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes / No / No data ******

NEUROBEHAVIOURAL EXAMINATION: Yes / No / No data *****
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

All parameters examined statistically were first tested for equality of variance using Bartlett’s test. If the results from Bartlett’s test were significant, then the data for the parameter were subjected to a transformation to obtain equality of the variances. Transformations examined included common log, inverse and square root, in that order with a Bartlett’s test following each transformation. When Bartlett’s test was satisfied no further transformations were applied. In-life body weights were evaluated using a three wayrepeated measures analysis of variance for time, sex and dose. Terminal body weight, organ weight (absolute and relative, excluding ovaries and testes), hematologic parameters (excluding differential WBC and RBC indices), clinical chemistry parameters, and urine specific gravity were evaluated using a two-way ANOVA with the factors of sex and dose. Results for ovaries and testes weight (absolute and relative) were analyzed using a one-way ANOVA. Final interpretation of numerical data considers statistical analyses along with other factors, such as dose-response relationships and whether the results were plausible in light of other biological and pathological findings. Descriptive statistics only were reported for feed consumption, feed efficiency, WBC differential counts and RBC indices.

Clinical signs:

effects observed, treatment-related

Dermal irritation:

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:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

There was no evidence of dermal irritation in male and female rats administered 100 mg/kg/application AEP during the course of the study.

Three male rats administered 500 mg/kg/application AEP were observed to have very slight erythema (barely perceptible) at the test site on the last day of study. Two of five male rats administered the highest dose of 1000 mg/kg/application AEP had very slight erythema (barely perceptible) following two applications of test material. All high dose male rats were observed to have very slight erythema at the test site on test day 10 that continued throughout the duration of the study. Scabbing, scaling and fissuring were observed at the test site of 4/5 on test day 24. All high dose male rats had scabbing, scaling and fissuring observed at the test site at study end.

Female rats administered 500 and 1000 mg/kg/application AEP had very slight erythema (barely perceptible) observed on test day 24 that remained evident throughout the duration of the study. Additionally, scabbing was observed on 1/5 and 3/4 female rats administered 500 and 1000 mg/kg/application AEP, respectively, on test day 24. Scaling and fissuring were observed on all female rats given 500 or 1000 mg/kg/application AEP on test day 24. At the end of the study (test day 29), 4/5 and 3/4 female rats given 500 and 1000 mg/kg/application AEP, respectively, had scabbing at the test site. On test day 29, scaling and fissuring were observed at the test site on 2/5 and 2/4 female rats given 500 and 1000 mg/kg/application AEP, respectively.

In life body weights, feed consumption, clinical pathology including hematology, clinical chemistry and urinalysis, and organ weights exhibited no treatment-related effects.

Gross and histopathological findings considered to be treatment-related were confined to the application site. Treatment-related gross necropsy observations at the dermal test site were present in rats from the 500 and 1000 mg/kg/application groups. All males and females from the 500 and 1000 mg/kg/application groups had erythema at the dermal test site. Multifocal scabs at the dermal test site were present in 2/5 and 4/5 females from the 500 mg/kg/application group, and in 5/5 males and 5/6 females in the 1000 mg/kg/application group. Male and female rats from the 100 mg/kg/application group had no gross observations at the dermal test site.

Treatment-related histopathologic skin lesions at the dermal test site were present in males exposed to 100, 500 or 1000 mg/kg/application, and females exposed to 500 or 1000 mg/kg/application. Females exposed to 100 mg/kg/application had no significant histopathologic lesions at the dermal test site. In general, there was a dose-related increase in the incidence and/or severity of skin lesions from all dose groups. More extensive lesions at the dermal test site, consisting of sebaceous gland hyperplasia, dermal chronic-active inflammation, parakeratosis, ulcers and epidermal pustules, were present in some male and female rats from the 500 and/or 1000 mg/kg/application groups.

Given that testicular and ovarian weights of high dose animals were comparable to control values and there were no grossly visible effects considered to be treatment related, histopathologic examination of testes and ovaries were not performed on all animals.

Testes of a limited number of animals were examined from the 100 (1 rat) and 500 (3 rats) mg/kg groups. One rat from the 500 mg/kg/application group was observed upon gross examination to have flaccid testis unilateral. This animal along with 3 others were examined histopathologically. Moderate tubule atrophy along with tubule mineralization, multifocal, was observed in one testis. The other testes examined were within normal limits. Testes from the highest dose, 1000 mg/kg/application, were not examined.

Under conditions of this study, the repeated dermal administration of AEP resulted in dose-related irritative effects at the dermal test site in male rats administered 100, 500 or 1000 mg/kg/application, and in female rats administered 500 or 1000 mg/kg/application. There was no evidence of systemic toxicity in rats administered AEP at dosages of 100, 500 or 1000 mg/kg/application. Therefore, the no-observed-effect-level (NOEL) for systemic toxicity was considered 1000 mg/kg/application AEP for male and female Fischer 344 rats following dermal administration

Dose descriptor:

NOEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: No systemic effects were seen at the highest dose level of 1000 mg/kg/application

Critical effects observed:

not specified

Conclusions:

Under conditions of this study, the repeated dermal administration of AEP resulted in dose-related irritative effects at the dermal test site in male rats administered 100, 500 or 1000 mg/kg/application, and in female rats administered 500 or 1000 mg/kg/application. There was no evidence of systemic toxicity in rats administered AEP at dosages of 100, 500 or 1000 mg/kg/application. Therefore, the no-observed-effect-level (NOEL) for systemic toxicity was considered 1000 mg/kg/application AEP for male and female Fischer 344 rats following dermal administration.

Endpoint conclusion

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Additional information

Oral:

In the OECD 422 repeat dose study, male and female rats were administered AEP in drinking water at 500, 2000, and 8000 ppm. Male rats were exposed 14 days before mating, during breeding and until necropsy for a period of 32 consecutive days. Female rates were exposed 14 days before mating, during breeding and gestation, and through lactation day 4 for a period of 39 -53 consecutive days.

In the 8000 ppm male and female group, lower mean body weights with corresponding reduced food and water intake was noted during the premating period. Lower mean body weights in the males persisted throughout the study period. In females, lower mean body weights and body weight gains weight gains persisted in the absence of effects on food and water consumption throughout gestation and lactation. Five males and 1 female in the 8000 ppm group were euthanized in extremis during the treatment period following body weight losses and reduced food and water consumption.

Based on the treatment related moribundity at 8000 ppm, reduced mean body weights and body weight gains, and food and water consumption in both males and females at 8000 ppm, the NOAEL for systemic toxicity was considered to be 2000 ppm (approximately 152 mg/kg/day).

Inhalation:

In an inhalation 90 -day repeated dose study (OECD 413) rats were administrated 0, 0.2 ± 0.1, 5.1 ± 1.1, or 53.5 ± 6.0 mg AEP/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.

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.

Dermal:

The repeated dose dermal administration of AEP resulted in dose-related irritative effects at the test site in male rats at 100, 500, or 1000 mg/kg/day and in female rats at 500 and 1000 mg/kg/day. There was no systemic toxicity at any of the dose levels. Observations included gross pathology, histopathology, clinical chemistry and urinalysis. The NOEL was considered to be 1000 mg/kg/day the highest dose tested.

Justification for classification or non-classification

According to the GHS criteria the results of the inhalation repeat dose toxicity data warrant a classification for target organ toxicity-repeat exposure 1 for local effects on the respiratory system.