1,4-diazabicyclooctane

Endpoint summary

Description of key information

Repeated exposure to 1,4-Diazabicyclo[2.2.2]octaneby inhalation at dose levels as high as 0.41 mg/L/6h/day caused localized toxicity at the site of contact, namely, the upper respiratory tract and the eyes of rats. No systemic toxicity was seen at 0.41 mg/L/6h/day. Repeated exposure to 1,4-Diazabicyclo[2.2.2]octaneby oral gavage at dose levels as high as 1000 mg/kg/day caused reversible inflammatory and proliferative effects in the kidneys and urinary bladder of rats. The NOAEL for systemic oral toxicity was 300 mg/kg/day for females and 100 mg/kg/day for males.   

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

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:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study was conducted accorduing to OECD 422 guidelines and GLP principles

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

All deviations were minor and did not affect the outcome of the study

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Sixty male and sixty female Crl:CD®(SD)IGS BR rats, were received in good health from Charles River Laboratories, Raleigh, North Carolina, on August 10, 1999. The rats were approximately 58 days old. Upon receipt, each animal was examined by a qualified technician. All rats were weighed on the day following receipt. Animals were uniquely identified by a Monel® metal eartag displaying the animal number and housed for 14 days for acclimation purposes. During the acclimation period, the animals were observed twice daily for mortality and moribundity.

Upon arrival and until pairing, all animals were housed in clean, wire-mesh cages suspended above cage-board which was changed at least three times per week. The Reproductive/Developmental Toxicity Phase animals were paired for mating in the home cage of a male from the same treatment group. Following positive identification of mating or upon completion of the mating period, these females were housed individually in plastic maternity cages containing ground corncob nesting material (Bed-O'Cobs®; The Andersons, Industrial Products Division, Maumee, Ohio) and remained in these cages until euthanasia on lactation day 4.

Animals were housed in accordance with the "Guide for the Care and Use of Laboratory Animals." The animal facilities at WIL Research Laboratories, Inc., are accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International).

The basal diet used in this study, PMI Nutrition International, Inc., Certified Rodent LabDiet® 5002, is a certified feed with appropriate analyses performed by the manufacturer and provided to WIL Research Laboratories, Inc. Feeders were changed and sanitized once per week, with the following exception. Documentation of feeder sanitization was not found for the week of August 9, 1999. This deviation did not affect the outcome of the study. Municipal water supplying the facility is sampled for contaminants according to standard operating procedures. The results of these analyses are maintained at WIL Research Laboratories, Inc. Contaminants were not present in animal feed or water at concentrations expected to interfere with the objectives of this study. Reverse-osmosis-purified (on-site) drinking water delivered by an automatic watering system and the basal diet were provided ad libitum throughout the acclimation period and during the study.

All animals were housed throughout the acclimation period and during the study in an environmentally-controlled room. Controls were set to maintain a temperature of 72±4°F and a relative humidity between 30% and 70%. Room temperature and relative humidity were recorded once daily with the following exception. Temperature and humidity were inadvertently not recorded on study day 12 (September 5, 1999). This deviation would not be expected to have an adverse impact on the quality or outcome of the study due to its singular occurrence. Temperature ranged from 70.1° to 73.5° F and relative humidity ranged from 34.0% to 61.1% during the study period. Light timers were calibrated to provide a 12-hour light/ 12-hour dark photoperiod. Air handling units were set to provide approximately 10 fresh air changes per hour.

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

deionized

Details on oral exposure:

The test article in the vehicle, deionized water, was administered orally to three groups of Sprague-Dawley Crl:CD®(SD)IGS BR rats once daily at dosage levels of 100, 300, and 1000 mg/kg/day at a dose volume of 5 ml/kg. A concurrent control group received the vehicle, deionized water, on a comparable regimen at 5 ml/kg. 
The test mixtures were administered by gavage, via a 16-gauge stainless-steel gavage cannula (Popper and Sons, Inc., New Hyde Park, New York), as a single daily dose.
Individual dosages were calculated based on the most recently recorded body weights to provide the correct mg/kg/day dose. All animals were dosed at approximately the same time each day with the following exception. The males were dosed three hours later on study day 27 (September 20, 1999) than on study day 26 due to the performance of Functional Observational Battery evaluations. This deviation would not be expected to have an adverse impact on the quality or integrity of the data or the outcome of the study due to its singular occurrence.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The dosing formulations were solutions; therefore, homogeneity analyses were not performed. Prior to the initiation of dosing, a representative batch of each dosing formulation was prepared. Two 10-ml samples were collected from the middle of the control group formulation and two 10-ml samples were collected from the middle of each treated group formulation. One set of samples was analyzed to determine the concentration of the test article in the formulations at time 0. The remaining set of samples was stored under normal laboratory conditions for eight days and then analyzed to ascertain the stability of the test article in the vehicle. The protocol states that stability of the test article formulations will be established prior to the initiation of dosing. However, since the initial 200 mg/ml formulation prepared prior to the initiation of dosing was not within the WIL SOP AC-086 requirement for acceptability (+ 10% of target), a second 200 mg/ml was prepared. The results of the stability analysis for the 200 mg/ml formulation were not obtained until after the initiation of dosing. This deviation had no impact on the quality or integrity of the study because the dosing formulations were shown to be stable for eight days. One 10-ml sample was also collected from the middle of each test article formulation prepared during study weeks -1, 0, 2, and 5 (August 20, August 27, September 10 and October 1, 1999, respectively) rather than those prepared during study weeks 0, 3 and 6 as specified in the protocol. These formulations were used for dosing during study weeks 0, 1, 3, and 6, respectively. This deviation would not be expected to have an adverse impact on the integrity or outcome of the study since these dosing formulations contained the amount of test article prescribed in the protocol. These samples were analyzed for concentration by the Analytical Chemistry Department at WIL Research Laboratories, Inc. The dosing preparations were stable for eight days and contained the amount of test article specified in the protocol.

Duration of treatment / exposure:

All animals were dosed for 14 days prior to mating and through the day prior to necropsy (29 days for males and through lactation day 4 or post-mating/post-cohabitation day 25 for females). The females assigned to the Recovery Phase were dosed through the first lactation day 4 for the Reproduction Phase (40 days of dosing).

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

Ten animals/sex per dose were assigned to each dose group (Groups 1-4) for evaluation of repeated-dose toxicity and reproductive toxicity. An additional five rats/sex were assigned to the control and high dose groups for evaluation of repeated-dose toxicity only and evaluated following a 14-day recovery period. Recovery animals were not evaluated for reproductive parameters.

Control animals:

yes, concurrent vehicle

Details on study design:

Ten males and 10 females in each treatment group were dosed for 14 days prior to mating and through the day prior to necropsy. An additional five males/group in the control and 1000 mg/kg/day groups were assigned to the recovery phase and were dosed for a minimum of 28 days (duration of pre-mating and mating periods for the reproductive phase). An additional five females/group in the control and 1000 mg/kg/day groups were assigned to the recovery phase and were dosed for 40 days (until the first lactation Day 4 of the reproductive phase). Recovery phase animals were maintained for a minimum 14-day (nondosing) recovery period.
The following deviation occurred. In order to allow sufficient time to review the data from the recovery period, the Recovery Phase females were maintained for a 15-day recovery period instead of 14 days as specified in the protocol. This deviation would not be expected to affect the quality or integrity of the data or the outcome of the study since all Recovery Phase animals were treated similarly

Positive control:

None

Observations and examinations performed and frequency:

Clinical observations were recorded daily and detailed physical examinations were conducted weekly for all F0 animals. Throughout the study period, all rats were also observed at the time of dosing and approximately one hour following dosing. Parental body weights and food consumption were recorded at appropriate intervals. Functional observational battery evaluations and locomotor activity evaluations were performed on F0 males following the completion of dosing (approximately 28 days), on lactation Day 4 for females that delivered and at study week 5 for the recovery phase females. 

Sacrifice and pathology:

A complete necropsy was performed on all F0 animals. Clinical pathology evaluations (hematology and serum chemistry) were conducted on five rats/sex/group at the scheduled necropsies and on the recovery females (serum chemistry only) at the scheduled necropsy. Selected organs were weighed from all F0 animals at the scheduled necropsies (primary and recovery).
The following tissues were examined microscopically in the control and high-dose rats: adrenal glands, aorta, bone marrow - sternal, brain, cecum, colon, duodenum, epididymides, esophagus, lacrimal glands, eyes with optic nerve, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes (submandibular and mesenteric), pancreas, pituitary gland, prostate, rectum, salivary glands, sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid/parathyroids, trachea, urinary bladder, and any gross lesions.

Statistics:

All analyses were conducted using two-tailed tests for a minimum significance level of 5%, comparing each treated group to the vehicle control group. Means were presented with the standard deviation (S.D.) and the number of animals (N) used to calculate the mean. Data obtained from nongravid females were excluded from statistical analysis following the mating period. Statistical tests were performed using appropriate computing devices or programs.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no treatment-related findings at the detailed physical examinations. No test article-related differences in Functional Observational Battery parameters (home cage, handling, open field, sensory, neuromuscular and physiological observations) were apparent at any dose level for either sex.

Mortality:

no mortality observed

Description (incidence):

There were no treatment-related findings at the detailed physical examinations. No test article-related differences in Functional Observational Battery parameters (home cage, handling, open field, sensory, neuromuscular and physiological observations) were apparent at any dose level for either sex.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weight gain in the 1000 mg/kg/day group males was statistically reduced during the last week of dosing (p<0.01) and mean body weight gain in the 1000 mg/kg/day group females was reduced during gestation Days 17-20 and 0-20 (p<0.05).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was decreased in the 1000 mg/kg/day group males during week 0-1 and in the 1000 mg/kg/day group females during gestation Days 4-7, 7-11, 11-14, 14-17, 17-20, and 0-20 with sporadic statistical significance.

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Hematology parameters were not adversely affected by treatment.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Test article-related changes in serum chemistry parameters were observed in the 1000 mg/kg/day group females at the lactation Day 4 evaluation and consisted of statistically increased mean alkaline phosphatase (ALP) concentration (p<0.01)

Urinalysis findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The 1000 mg/kg/day group females had statistically increased mean liver weights (absolute (p<0.05) and relative (p<0.01) to final body weight) at the lactation Day 4 necropsy, which were consistent with the increased mean ALP concentrations.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test article-related gross findings in the F0 animals.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test article-related microscopic changes consisted of inflammatory and/or proliferative changes in the kidneys and urinary bladder.

Details on results:

Motor activity (total and ambulatory) in the 1000 mg/kg/day group females was reduced at the lactation Day 4 evaluation. However, no reductions in motor activity in the 1000 mg/kg/day group females in the recovery phase were observed at the end of the dosing period.

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

gross pathology

haematology

mortality

organ weights and organ / body weight ratios

water consumption and compound intake

Dose descriptor:

LOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

gross pathology

mortality

organ weights and organ / body weight ratios

water consumption and compound intake

Critical effects observed:

not specified

Test article-related microscopic changes consisted of inflammatory and/or proliferative changes in the kidneys of the 300 and 1000 mg/kg/day group males and the kidneys and urinary bladder of the 1000 mg/kg/day group females. At the Reproductive/Developmental Phase necropsy (study week 4), chronic inflammation of the kidneys was noted in one and two males in the 300 and 1000 mg/kg/day groups, respectively, and acute pyelitis was noted in a single 1000 mg/kg/day group male. In the females, inflammatory and/or proliferative lesions of the kidneys were noted in all groups, including the control group, but were noted at increased incidence in the 1000 mg/kg/day group. Kidney lesions (suppurative pyelonephritis, subacute pyelitis, uroepithelial hyperplasia of the renal papilla and/or pelvis and/or chronic inflammation) were seen in one female in each of the control, 100 and 300 mg/kg/day groups; however, three females in the 1000 mg/kg/day group had similar kidney lesions. It is likely that the kidney lesions seen in the 1000 mg/kg/day group males and females, although diagnosed separately, represent a continuum of renal inflammatory changes of similar etiology and were considered test article-related effects. Subacute inflammation and uroepithelial hyperplasia of the urinary bladder were also seen in two females in the 1000 mg/kg/day group. Following a 14-day recovery period, test article-related findings were limited to subacute pyelitis and uroepithelial hyperplasia of the kidney and subacute inflammation and uroepithelial hyperplasia of the urinary bladder in a single 1000 mg/kg/day group female.

Conclusions:

Oral administration of Triethylenediamine resulted in F0 toxicity in both males and females at a dose level of 1000 mg/kg/day as evidenced by changes in clinical conditions of the animals, reduced body weight and food consumption, reduced motor activity (females only), increased serum alkaline phosphatase concentrations (females only), increased liver weights (females only) and microscopic changes (inflammatory and/or proliferative lesions) in the kidneys (both sexes) and urinary bladder (of a single 1000 mg/kg/day group female), none of the above findings persisted to the end of the 14-day recovery period. F0 toxicity in the 300 mg/kg/day groups was limited to chronic inflammation of the kidneys in the males. There were no indications of F0 toxicity in the 100 mg/kg/day group males and females. Based on the data obtained, the NOAEL for F0 parental systemic toxicity was considered to be 100 mg/kg/day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study, GLP study

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

no

GLP compliance:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Kleintierfarm Madoerin AG 4414 Fuellinsdorf / Switzerland
- Age at study initiation: 6 weeks
- Weight at study initiation: males: 168-201 grams, females: 144-165 grams
- Housing: individually in Makrolon type-3 cages
- Diet: pelleted standard Kliba no. 343 rat maintenance diet (Klingentalmuehle AG, Kaiseraugst/Switzerland), ad libitum.
- Water: Treated community tap water from Geneva, ad libitum.
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 40 -70
- Air changes (per hr): 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12h/12h

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

other: water

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The exposure system used was of the flow-past nose-only design. This system has been developed based upon fluid dynamic modeling of the aerosol flow from the entry to the animal's nose. The internal active volume of the chamber for exposing 40 animals by nose-only was one liter. The resulting time for the concentration at an animal port to reach 99 % of its ultimate value (T99) was 10 seconds for the 40 animal chamber. These designs have been integrated into a general purpose flow-past nose-only exposure system by RCC. This system was constructed of anodised aluminium and readily accepts a variety of the different size Makrolon animal restraint tubes (which have been designed with consideration of the anatomy and physiology of the rodent). Each level has 8 ports and can be rotated, allowing close observation of all the animals without interruption of exposure. The entire unit is modular, permitting easy cleaning and the choice of how many levels of animals will be used .
The system is unique by comparison with conventional nose-only exposure systems by eliminating the problem of depletion of aerosol at lower chamber levels by animals in the levels above by supplying "fresh" aerosol to each animal. The design ensures uniform exposure to all animals in the system and provides that the air exhaled by one animal does not reach any other animal in the chamber.
The exposure aerosol enters the inlet at the top under a slight positive pressure and is distributed to the entrance of each of feed tubes. The aerosol is then delivered through these tubes to the animal's nose and is then extracted away through a second concentric tube which is maintained under a negative pressure through aspiration at the OUTLET. Thus, a constant stream of exposure aerosol is fed past the animals' nose, eliminating the problems of rebreathing and depletion of aerosol that are common in standard exposure systems.

- Method of holding animals in test chamber: The animals were confined separately in Makrolon tubes which were positioned radially around the exposure chamber.
- System of generating particulates/aerosols: The test article atmosphere was generated using a constant volume reservoir feeding a Hospitak No. 950 nebuliser. The liquid aerosol was then diluted with clean air to achieve the high concentration required for this study and discharged into the first exposure chamber. The medium and low concentrations were achieved by further dilution of the high concentration through airvac systems. This generation procedure was chosen to achieve the intended aerosol concentrations with a mass median aerodynamic diameter of 3 µm or less.
The untreated control group was exposed to nebulised distilled water (solvent) using a similar system.

- EXPOSURE SYSTEM MONITORING: Determinations of concentration, particle size distribution, oxygen concentration, relative humidity and temperature were performed at the position of the animal's snout in the exposure system. All measurements of the test atmosphere as described below were taken directly from the test atmosphere feed tube on the flow-past exposure system which delivers fresh test article to the animal's nose. Thus all sampling was isoaxial, and represents exactly what is delivered to the animal's nose.

- Samples taken from breathing zone: yes
Samples for the analytical determination of concentrations were collected once weekly for each group during exposure.
Samples for the gravimetric determination of concentrations were collected daily in the high dose group.
No samples were taken for the medium and low dose group due to the volatility of the test article.
The aerosol concentration was also continuously monitored during exposure using a RAM-1 light scattering device.

RESULTS: 0, 0.06 (+/- 0.01), 0.41 (+/- 0.08) mg/L air [mean (+/- S.D.)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Sampling for Gravimetric Determination of Concentration: The test article, sampled from the exposure unit as described above, was collected on Gelman Type A/E 47 mm diameter glass fiber filters using a stainless steel filter sampling device (Gelman). The exact airflow rates during sampling were measured with calibrated measuring systems and recorded in the raw data. Only the particulate fraction remaining on the filter was measured with this method. Any vapour or volatile fractions would not be indicated by this method. The relative aerosol concentration was monitored using a RAM-1 light scattering type aerosol monitor (GCA Corp.).

Analytical Determination of Concentration:
The test article was sampled as described above and passed through three bottles, each filled with 80 mL of acetone and cooled to -70 °C with dry ice. These solutions were transferred into 100 mL volumetric flasks, the bottles were rinsed with acetone and the volume made up to 100 mL. These solutions were analyzed by photometric analysis.

Particle Size Determination:
The particle size of the test atmosphere was determined using a Mercer 7 stage cascade impactor (In-Tox Products, Model 02-1300). The sampling airflow rate was 1.0 L/min. The test atmosphere was impacted at each stage onto stainless steel slips which were weighed before and after sampling using a Model M3 balance from Mettler AG, Switzerland. Note that only the solid or non evaporated fraction of the test article is collected with the impactor. Any vapour component or any fraction which might evaporate or sublimate is not accounted for.
Seven determinations of the particle size distribution were performed in the high dose group during the study period .

Duration of treatment / exposure:

28 days

Frequency of treatment:

6 hours/day, 5 days/week for a total of 20 exposures

Remarks:

Doses / Concentrations:
0.0058, 0.063 or 0.62 mg/L as aerosol
Basis:
nominal conc.

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:
Viability / Mortality: Each animal was observed twice daily during the week and once daily during the weekends.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Clinical Signs: Each animal was observed at least once daily for any sign of toxicity or clinical symptoms.

BODY WEIGHT: Yes
The body weight of each animal was recorded once during the acclimation period and weekly thereafter, using an electronic balance (Mettler PE 2000).
FOOD CONSUMPTION:
- The food consumption (per cage) was recorded once during the acclimation period and weekly thereafter using an electronic balance (Mettler PE 2000).

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.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at week 4 of treatment
- Dose groups that were examined: All animals.
Ten minutes after the application of a mydriatic solution (Dispersa AG, Hettlingen/Switzerland) the cornea, lens, anterior chamber, vitreous body and ocular fundus of both eyes were examined under dimmed light using a Heine Miroflex 2 Ophthalmoscope (Eisenhut Vet AG, Allschwil/Switzerland). A description of any abnormality was recorded.

HAEMATOLOGY:
Yes, blood samples were drawn from the retro-orbital plexus. The following anticoagulant was used during blood collection : EDTA-K2.
- Time schedule for collection of blood: Blood samples were collected from each animal between the hours of 6.45 and 7.45 a.m. to reduce biological variation caused by circadian rhythm.
- Anaesthetic used for blood collection: Yes, light ether anesthesia.
- Animals fasted: Yes, for 18 hours before blood sampling, but water was provided.
- How many animals: all animals.
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY:
Yes
- Time schedule for collection of blood: see Haematology.
- Animals fasted: Yes, see Haematology.
- How many animals: see Haematology.
- Parameters checked in table [No.2] were examined.

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data.

Recording was done on data sheets.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
ORGAN WEIGHTS: The following organ weights of all animals necropsied at study termination were recorded: adrenal glands, kidneys, liver, lungs, and testes.
NECROPSY AND HISTOPATHOLOGY: Yes
All animals were necropsied. All surviving animals were anesthetized by intraperitoneal injection of sodium pentobarbital (approximately 300 mg/kg bw) and exsanguinated. All organs were examined and all findings recorded. All collected tissues were fixed in 4 % buffered formaldehyde solution and embedded in paraffin. Sections were cut at an approximate thickness of 4 µm and stained with hematoxylin and eosin.
All organs and tissue samples described in the Pathology Report under "Necropsy and Histopathology" from rats of the control and high dose groups, as well as the larynx and tissues with macosocopical abnormalities from rats of the low and medium dose groups were examined microscopically.
Recording was done on-line.

Statistics:

Statistical methods were used to analyze the body weights, food consumption, organ weights and clinical laboratory data:
Univariate one-way analysis of variance was used to assess the significance of intergroup differences.
If the variables could be assumed to follow a normal distribution, the Dunnett-test (many to one t-test) based on a pooled variance estimate was applied for the comparison between the treated groups and the control groups.
The Steel-test (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution. For the overall spontaneous mortality data, the Fisher' s exact test for 2 x 2 tables was applied.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables.
Individual values, means, standard deviations and statistics were rounded off before printing. For example, test statistics were calculated on the basis of exact values for means and pooled variances and then rounded off to two decimal places. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values .

Details on results:

CLINICAL SIGNS AND MORTALITY
In the high dose, one female, died on day 5 of the study. The animals of the high dose group showed inflammation (necrotic dermatitis) of the ears, nose and eyes. No clinical signs were noted in the lower dose groups.

BODY WEIGHT AND WEIGHT GAIN
no treatment-related changes

FOOD CONSUMPTION
The food consumption was significantly reduced in the high dose group in males between days 8 and 29 of the treatment period .

OPHTHALMOSCOPIC EXAMINATION
The ophthalmoscopic examination indicated corneal opacity, ulceration, and granulosis in the male and female animals of the high dose group.

HAEMATOLOGY
The assessment of haematological and biochemical data indicated no changes of toxicological significance at the end of the 4 weeks exposure period.
CLINICAL CHEMISTRY
For biochemical data no adverse changes of toxicological significance were noted at the end of the 4 weeks exposure period. However, the following effects were noted:
- slightly increased urea level for females of the mid and high dose group and for both sexes of the high dose group.
- slightly increased aspartate aminotransferase activity for males of the high dose group.
- slightly decreased sodium level for both sexes of the high dose group.
- slightly decreased chloride level for males of the high dose group.
• The relation of these findings to the treatment, if any, remain, of doubtful significance. The effects noted are therefore, considered to be of an adaptive nature.
All other differences in the results of the clinical biochemistry parameters were considered to be incidental and of normal biological variation (treatment-unrelated).

ORGAN WEIGHTS
The following statistically significant changes in absolute and relative organ weights were noted in the intermediate and high dose groups as compared to controls:
In males: - Testes displayed increased weights in the high dose group (P < 0 .05) and increased ratios (organ/bodyweight) in the intermediate and high dose groups (P < 0 .01).
- Adrenals displayed increased ratios in the high dose group (P < 0 .01).

GROSS PATHOLOGY
MACROSCOPIC FINDINGS:
• Macroscopic findings which were considered related to treatment or experimental procedures included:
- skin auricles, eschars or nodules in the high dose group.
- eye region, red-brown foci or eschars in the high dose group.
The remaining macroscopic findings were considered spontaneous and amongst those commonly recorded in this age and strain of rat.
MICROSCOPIC FINDINGS:
• Microscopic correlates of the macroscopic findings listed above were:
- larynx chronic laryngitis, severity grade minimal to moderate in three animals of the intermediate dose group and seven animals in the high dose group (dose related).
- acute necrotizing laryngitis (severity grade: severe) recorded in one animal of the high dose group was considered to have contributed to the animal´s death.
- skin either an exudative or a necrotic dermatitis of a slight to moderate degree of severity could be determined on the pinna of the ear or on the eyelids in six high dose group rats.
These findings were considered evidence of irritation produced by the test article to the mucous membrane of the larynx at the high and intermediate dose levels and to the skin at the highest dose level. The low dose level was free of adverse pathology findings.

The following microscopic findings, whilst infrequent in this age and strain of rat, were nevertheless considered spontaneous:
- lung multifocal granulomatous pneumonitis, severity grade moderate in one animal of the control.
- pancreas focal exocrine atrophy, severity grade minimal to slight in one animal of the control and one of the high dose group.
- testes bilateral atrophy of the seminiferous epithelium accompanied by reduced sperm content of the epididymides, severity grade moderate in one animal of the control.
- epididymides unilateral sperm granuloma, severity grade moderate in one animal of the high dose group.

Some rats from all dose groups had a minimal to slight degree of goblet cell hypertrophy, chiefly in the anterior nasal cavity, which is considered to be a non-specific adaptive alteration.

Other microscopic findings recorded are spontaneous in nature and are within the normal range of background pathology observed in this age and strain of rat. They may be attributed to sub-clinical illness, spontaneous congenital abnormalities or physiological status.

Dose descriptor:

NOAEL

Remarks:

local

Effect level:

0.006 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

food efficiency

haematology

mortality

Dose descriptor:

LOAEL

Remarks:

local

Effect level:

0.063 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

food efficiency

haematology

mortality

ophthalmological examination

other: organ weights: testes (increased ratios); histopathology: larynx (chronic laryngitis).

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

0.62 mg/L air (nominal)

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

food efficiency

haematology

mortality

ophthalmological examination

Critical effects observed:

not specified

EXPOSURE SYSTEM MONITORING:

The table presents the test article concentrations applied (nominal) and the concentrations measured by gravimetry and chemical analysis

Group	Target concentration (mg/L air)	Input to the system	Output from the system (= inhaled concentrations)
		Nominal concentration (mg/L air)	Gravimetric (mg/L air) Mean, (S.D.)	Analytical (mg/L air) Mean, (S.D.)
1 (Control)	0	0	--	--
2	0.005	0.0058	*	**
3	0.05	0.063	*	0.06 (0.01)
4	0.5	0.62	0.517 (0.098)	0.41 (0.08)
Validation of the System (Test without Animals)
2	0.005	0.011		0.010-0.012
4	0.5	0.76		0.65-0.69

* Due to the volatility of the aerosol particles of the test article, it was not possible to obtain gravimetric concentrations and particle size measurements for groups 2 and 3.

The experience of this laboratory has repeatedly indicated that with water based solutions, the Hospitak 950 nebulizer used for aerosol generation produces aerosols of less than approximately 3 micrometers mass medium aerodynamic diameter.

** Below the limit of analytical detection.

SUMMARY OF PARTICLE SIZE DISTRIBUTION (% )

It should be noted that only the particle fraction remaining on the impactor stages is indicated by these measurements.

Evaporation of the test article from the impactor stages would tend to be greater for the smaller particles thus producing a bias towards the larger sizes.

The experience of this laboratory with the Hospitak 950 nebulizer is that it consistently produces aerosols of approximately 3 µm less with water based solutions.

Listed below are the data sets for which weights were obtainable. Due to the above, considerations, caution should be used in their interpretation.

 

Dose (0.5 mg/L)

 

ECD µm	Day 1 Cumulative (%)	Day 2 Cumulative (%)	Day 7 Cumulative (%)	Day 23 Cumulative (%)	Day 28 Cumulative (%)
4.6	100	100	100	100	100
3.0	46.4	74.9	54.2	44.3	54.6
2.13	15.1	30.7	24.8	18.4	28.8
1.60	13.5	20.1	6.3	14.4	13.0
1.06	13.5	13.5	2.8	13.7	11.8
0.715	12.0	10.3	2.8	11.1	10.2
0.325	8.3	7.1	2.6	8.9	10.2
<0.325	3.1	4.9	1.3	4.1	3.4

 

 

Dose (0.05 mg/L)

 

ECD µm	Day 9 Cumulative (%)	Day 11 Cumulative (%)
4.6	100	100
3.0		100
2.13		99.5
1.60	73.1	97.5
1.06	62.9	97.5
0.715	58.9	94.0
0.325	53.8	83.1
<0.325	23.4	42.3

 

Conclusions:

The No Observed Adverse Effect Concentration (NOAEC) for systemic toxicity was 0.41 mg/L/6h/day, which was the highest dose tested.

Executive summary:

In a 4-week inhalation study, 5 rats/dose/sex were exposed whole body to an aerosol of 1,4-Diazabicyclo[2.2.2]octane 6 hours/day, 5 days/week for four weeks (20 exposures) at nominal concentrations of 0, 0.0058, 0.063 and 0.62 mg/L (analytical concentrations were 0, <0.011, 0.06 and 0.41 mg/L). The low dose was below the analytical limit of detection (0.011 mg/L). The control animals were exposed to the vehicle (distilled water) only. One female in the high dose group died on Day 5. The high-dose animals exhibited necrotic dermatitis of the ears, nose and eyes. Ophthalmoscopic exams revealed corneal opacity, ulceration and granulosis in the high-dose group. Food consumption and body weight gain were decreased in the high-dose males. Hematology and clinical chemistry values were normal. No organ weight changes were observed in the female groups. Absolute testes weights were increased in the high-dose group (p<0.05). Relative adrenal weights were increased in the high-dose group (p<0.001). Relative testes weights were increased in the mid- and high-dose groups (p<0.01). Histopathology indicated minimal to moderate chronic laryngitis in the mid- and high-dose groups (both sexes). The frequency of this finding was dose-related (7 out of 10 high-dose and 3 our of 10 mid-dose). The female that died had severe acute necrotizing laryngitis. Increased organ weights could not be correlated histopathologically. No compound-related effects were seen at the lowest dose level. This study indicated that for 1,4-Diazabicyclo[2.2.2]octane, the point of contact was the site of action, namely, the upper respiratory tract and the eyes. Since no systemic toxicological effects were observed, the No Observed Adverse Effect Concentration (NOAEC) for systemic toxicity was 0.41 mg/L/6h/day, which was the highest dose tested. The NOAEC for local toxicity, based on the lack of an effect observed in the larynx could not be ascertained with confidence, since the lowest dose level could not be measured analytically. The Lowest Observed Adverse Effect Concentration (LOAEC) was 0.06 mg/L/6h/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

410 mg/m³

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study, GLP study

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

no

GLP compliance:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Kleintierfarm Madoerin AG 4414 Fuellinsdorf / Switzerland
- Age at study initiation: 6 weeks
- Weight at study initiation: males: 168-201 grams, females: 144-165 grams
- Housing: individually in Makrolon type-3 cages
- Diet: pelleted standard Kliba no. 343 rat maintenance diet (Klingentalmuehle AG, Kaiseraugst/Switzerland), ad libitum.
- Water: Treated community tap water from Geneva, ad libitum.
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 40 -70
- Air changes (per hr): 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12h/12h

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

other: water

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The exposure system used was of the flow-past nose-only design. This system has been developed based upon fluid dynamic modeling of the aerosol flow from the entry to the animal's nose. The internal active volume of the chamber for exposing 40 animals by nose-only was one liter. The resulting time for the concentration at an animal port to reach 99 % of its ultimate value (T99) was 10 seconds for the 40 animal chamber. These designs have been integrated into a general purpose flow-past nose-only exposure system by RCC. This system was constructed of anodised aluminium and readily accepts a variety of the different size Makrolon animal restraint tubes (which have been designed with consideration of the anatomy and physiology of the rodent). Each level has 8 ports and can be rotated, allowing close observation of all the animals without interruption of exposure. The entire unit is modular, permitting easy cleaning and the choice of how many levels of animals will be used .
The system is unique by comparison with conventional nose-only exposure systems by eliminating the problem of depletion of aerosol at lower chamber levels by animals in the levels above by supplying "fresh" aerosol to each animal. The design ensures uniform exposure to all animals in the system and provides that the air exhaled by one animal does not reach any other animal in the chamber.
The exposure aerosol enters the inlet at the top under a slight positive pressure and is distributed to the entrance of each of feed tubes. The aerosol is then delivered through these tubes to the animal's nose and is then extracted away through a second concentric tube which is maintained under a negative pressure through aspiration at the OUTLET. Thus, a constant stream of exposure aerosol is fed past the animals' nose, eliminating the problems of rebreathing and depletion of aerosol that are common in standard exposure systems.

- Method of holding animals in test chamber: The animals were confined separately in Makrolon tubes which were positioned radially around the exposure chamber.
- System of generating particulates/aerosols: The test article atmosphere was generated using a constant volume reservoir feeding a Hospitak No. 950 nebuliser. The liquid aerosol was then diluted with clean air to achieve the high concentration required for this study and discharged into the first exposure chamber. The medium and low concentrations were achieved by further dilution of the high concentration through airvac systems. This generation procedure was chosen to achieve the intended aerosol concentrations with a mass median aerodynamic diameter of 3 µm or less.
The untreated control group was exposed to nebulised distilled water (solvent) using a similar system.

- EXPOSURE SYSTEM MONITORING: Determinations of concentration, particle size distribution, oxygen concentration, relative humidity and temperature were performed at the position of the animal's snout in the exposure system. All measurements of the test atmosphere as described below were taken directly from the test atmosphere feed tube on the flow-past exposure system which delivers fresh test article to the animal's nose. Thus all sampling was isoaxial, and represents exactly what is delivered to the animal's nose.

- Samples taken from breathing zone: yes
Samples for the analytical determination of concentrations were collected once weekly for each group during exposure.
Samples for the gravimetric determination of concentrations were collected daily in the high dose group.
No samples were taken for the medium and low dose group due to the volatility of the test article.
The aerosol concentration was also continuously monitored during exposure using a RAM-1 light scattering device.

RESULTS: 0, 0.06 (+/- 0.01), 0.41 (+/- 0.08) mg/L air [mean (+/- S.D.)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Sampling for Gravimetric Determination of Concentration: The test article, sampled from the exposure unit as described above, was collected on Gelman Type A/E 47 mm diameter glass fiber filters using a stainless steel filter sampling device (Gelman). The exact airflow rates during sampling were measured with calibrated measuring systems and recorded in the raw data. Only the particulate fraction remaining on the filter was measured with this method. Any vapour or volatile fractions would not be indicated by this method. The relative aerosol concentration was monitored using a RAM-1 light scattering type aerosol monitor (GCA Corp.).

Analytical Determination of Concentration:
The test article was sampled as described above and passed through three bottles, each filled with 80 mL of acetone and cooled to -70 °C with dry ice. These solutions were transferred into 100 mL volumetric flasks, the bottles were rinsed with acetone and the volume made up to 100 mL. These solutions were analyzed by photometric analysis.

Particle Size Determination:
The particle size of the test atmosphere was determined using a Mercer 7 stage cascade impactor (In-Tox Products, Model 02-1300). The sampling airflow rate was 1.0 L/min. The test atmosphere was impacted at each stage onto stainless steel slips which were weighed before and after sampling using a Model M3 balance from Mettler AG, Switzerland. Note that only the solid or non evaporated fraction of the test article is collected with the impactor. Any vapour component or any fraction which might evaporate or sublimate is not accounted for.
Seven determinations of the particle size distribution were performed in the high dose group during the study period .

Duration of treatment / exposure:

28 days

Frequency of treatment:

6 hours/day, 5 days/week for a total of 20 exposures

Remarks:

Doses / Concentrations:
0.0058, 0.063 or 0.62 mg/L as aerosol
Basis:
nominal conc.

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:
Viability / Mortality: Each animal was observed twice daily during the week and once daily during the weekends.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Clinical Signs: Each animal was observed at least once daily for any sign of toxicity or clinical symptoms.

BODY WEIGHT: Yes
The body weight of each animal was recorded once during the acclimation period and weekly thereafter, using an electronic balance (Mettler PE 2000).
FOOD CONSUMPTION:
- The food consumption (per cage) was recorded once during the acclimation period and weekly thereafter using an electronic balance (Mettler PE 2000).

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.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at week 4 of treatment
- Dose groups that were examined: All animals.
Ten minutes after the application of a mydriatic solution (Dispersa AG, Hettlingen/Switzerland) the cornea, lens, anterior chamber, vitreous body and ocular fundus of both eyes were examined under dimmed light using a Heine Miroflex 2 Ophthalmoscope (Eisenhut Vet AG, Allschwil/Switzerland). A description of any abnormality was recorded.

HAEMATOLOGY:
Yes, blood samples were drawn from the retro-orbital plexus. The following anticoagulant was used during blood collection : EDTA-K2.
- Time schedule for collection of blood: Blood samples were collected from each animal between the hours of 6.45 and 7.45 a.m. to reduce biological variation caused by circadian rhythm.
- Anaesthetic used for blood collection: Yes, light ether anesthesia.
- Animals fasted: Yes, for 18 hours before blood sampling, but water was provided.
- How many animals: all animals.
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY:
Yes
- Time schedule for collection of blood: see Haematology.
- Animals fasted: Yes, see Haematology.
- How many animals: see Haematology.
- Parameters checked in table [No.2] were examined.

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data.

Recording was done on data sheets.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
ORGAN WEIGHTS: The following organ weights of all animals necropsied at study termination were recorded: adrenal glands, kidneys, liver, lungs, and testes.
NECROPSY AND HISTOPATHOLOGY: Yes
All animals were necropsied. All surviving animals were anesthetized by intraperitoneal injection of sodium pentobarbital (approximately 300 mg/kg bw) and exsanguinated. All organs were examined and all findings recorded. All collected tissues were fixed in 4 % buffered formaldehyde solution and embedded in paraffin. Sections were cut at an approximate thickness of 4 µm and stained with hematoxylin and eosin.
All organs and tissue samples described in the Pathology Report under "Necropsy and Histopathology" from rats of the control and high dose groups, as well as the larynx and tissues with macosocopical abnormalities from rats of the low and medium dose groups were examined microscopically.
Recording was done on-line.

Statistics:

Statistical methods were used to analyze the body weights, food consumption, organ weights and clinical laboratory data:
Univariate one-way analysis of variance was used to assess the significance of intergroup differences.
If the variables could be assumed to follow a normal distribution, the Dunnett-test (many to one t-test) based on a pooled variance estimate was applied for the comparison between the treated groups and the control groups.
The Steel-test (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution. For the overall spontaneous mortality data, the Fisher' s exact test for 2 x 2 tables was applied.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables.
Individual values, means, standard deviations and statistics were rounded off before printing. For example, test statistics were calculated on the basis of exact values for means and pooled variances and then rounded off to two decimal places. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values .

Details on results:

CLINICAL SIGNS AND MORTALITY
In the high dose, one female, died on day 5 of the study. The animals of the high dose group showed inflammation (necrotic dermatitis) of the ears, nose and eyes. No clinical signs were noted in the lower dose groups.

BODY WEIGHT AND WEIGHT GAIN
no treatment-related changes

FOOD CONSUMPTION
The food consumption was significantly reduced in the high dose group in males between days 8 and 29 of the treatment period .

OPHTHALMOSCOPIC EXAMINATION
The ophthalmoscopic examination indicated corneal opacity, ulceration, and granulosis in the male and female animals of the high dose group.

HAEMATOLOGY
The assessment of haematological and biochemical data indicated no changes of toxicological significance at the end of the 4 weeks exposure period.
CLINICAL CHEMISTRY
For biochemical data no adverse changes of toxicological significance were noted at the end of the 4 weeks exposure period. However, the following effects were noted:
- slightly increased urea level for females of the mid and high dose group and for both sexes of the high dose group.
- slightly increased aspartate aminotransferase activity for males of the high dose group.
- slightly decreased sodium level for both sexes of the high dose group.
- slightly decreased chloride level for males of the high dose group.
• The relation of these findings to the treatment, if any, remain, of doubtful significance. The effects noted are therefore, considered to be of an adaptive nature.
All other differences in the results of the clinical biochemistry parameters were considered to be incidental and of normal biological variation (treatment-unrelated).

ORGAN WEIGHTS
The following statistically significant changes in absolute and relative organ weights were noted in the intermediate and high dose groups as compared to controls:
In males: - Testes displayed increased weights in the high dose group (P < 0 .05) and increased ratios (organ/bodyweight) in the intermediate and high dose groups (P < 0 .01).
- Adrenals displayed increased ratios in the high dose group (P < 0 .01).

GROSS PATHOLOGY
MACROSCOPIC FINDINGS:
• Macroscopic findings which were considered related to treatment or experimental procedures included:
- skin auricles, eschars or nodules in the high dose group.
- eye region, red-brown foci or eschars in the high dose group.
The remaining macroscopic findings were considered spontaneous and amongst those commonly recorded in this age and strain of rat.
MICROSCOPIC FINDINGS:
• Microscopic correlates of the macroscopic findings listed above were:
- larynx chronic laryngitis, severity grade minimal to moderate in three animals of the intermediate dose group and seven animals in the high dose group (dose related).
- acute necrotizing laryngitis (severity grade: severe) recorded in one animal of the high dose group was considered to have contributed to the animal´s death.
- skin either an exudative or a necrotic dermatitis of a slight to moderate degree of severity could be determined on the pinna of the ear or on the eyelids in six high dose group rats.
These findings were considered evidence of irritation produced by the test article to the mucous membrane of the larynx at the high and intermediate dose levels and to the skin at the highest dose level. The low dose level was free of adverse pathology findings.

The following microscopic findings, whilst infrequent in this age and strain of rat, were nevertheless considered spontaneous:
- lung multifocal granulomatous pneumonitis, severity grade moderate in one animal of the control.
- pancreas focal exocrine atrophy, severity grade minimal to slight in one animal of the control and one of the high dose group.
- testes bilateral atrophy of the seminiferous epithelium accompanied by reduced sperm content of the epididymides, severity grade moderate in one animal of the control.
- epididymides unilateral sperm granuloma, severity grade moderate in one animal of the high dose group.

Some rats from all dose groups had a minimal to slight degree of goblet cell hypertrophy, chiefly in the anterior nasal cavity, which is considered to be a non-specific adaptive alteration.

Other microscopic findings recorded are spontaneous in nature and are within the normal range of background pathology observed in this age and strain of rat. They may be attributed to sub-clinical illness, spontaneous congenital abnormalities or physiological status.

Dose descriptor:

NOAEL

Remarks:

local

Effect level:

0.006 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

food efficiency

haematology

mortality

Dose descriptor:

LOAEL

Remarks:

local

Effect level:

0.063 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

food efficiency

haematology

mortality

ophthalmological examination

other: organ weights: testes (increased ratios); histopathology: larynx (chronic laryngitis).

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

0.62 mg/L air (nominal)

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

food efficiency

haematology

mortality

ophthalmological examination

Critical effects observed:

not specified

EXPOSURE SYSTEM MONITORING:

The table presents the test article concentrations applied (nominal) and the concentrations measured by gravimetry and chemical analysis

Group	Target concentration (mg/L air)	Input to the system	Output from the system (= inhaled concentrations)
		Nominal concentration (mg/L air)	Gravimetric (mg/L air) Mean, (S.D.)	Analytical (mg/L air) Mean, (S.D.)
1 (Control)	0	0	--	--
2	0.005	0.0058	*	**
3	0.05	0.063	*	0.06 (0.01)
4	0.5	0.62	0.517 (0.098)	0.41 (0.08)
Validation of the System (Test without Animals)
2	0.005	0.011		0.010-0.012
4	0.5	0.76		0.65-0.69

* Due to the volatility of the aerosol particles of the test article, it was not possible to obtain gravimetric concentrations and particle size measurements for groups 2 and 3.

The experience of this laboratory has repeatedly indicated that with water based solutions, the Hospitak 950 nebulizer used for aerosol generation produces aerosols of less than approximately 3 micrometers mass medium aerodynamic diameter.

** Below the limit of analytical detection.

SUMMARY OF PARTICLE SIZE DISTRIBUTION (% )

It should be noted that only the particle fraction remaining on the impactor stages is indicated by these measurements.

Evaporation of the test article from the impactor stages would tend to be greater for the smaller particles thus producing a bias towards the larger sizes.

The experience of this laboratory with the Hospitak 950 nebulizer is that it consistently produces aerosols of approximately 3 µm less with water based solutions.

Listed below are the data sets for which weights were obtainable. Due to the above, considerations, caution should be used in their interpretation.

 

Dose (0.5 mg/L)

 

ECD µm	Day 1 Cumulative (%)	Day 2 Cumulative (%)	Day 7 Cumulative (%)	Day 23 Cumulative (%)	Day 28 Cumulative (%)
4.6	100	100	100	100	100
3.0	46.4	74.9	54.2	44.3	54.6
2.13	15.1	30.7	24.8	18.4	28.8
1.60	13.5	20.1	6.3	14.4	13.0
1.06	13.5	13.5	2.8	13.7	11.8
0.715	12.0	10.3	2.8	11.1	10.2
0.325	8.3	7.1	2.6	8.9	10.2
<0.325	3.1	4.9	1.3	4.1	3.4

 

 

Dose (0.05 mg/L)

 

ECD µm	Day 9 Cumulative (%)	Day 11 Cumulative (%)
4.6	100	100
3.0		100
2.13		99.5
1.60	73.1	97.5
1.06	62.9	97.5
0.715	58.9	94.0
0.325	53.8	83.1
<0.325	23.4	42.3

 

Conclusions:

The No Observed Adverse Effect Concentration (NOAEC) for systemic toxicity was 0.41 mg/L/6h/day, which was the highest dose tested.

Executive summary:

In a 4-week inhalation study, 5 rats/dose/sex were exposed whole body to an aerosol of 1,4-Diazabicyclo[2.2.2]octane 6 hours/day, 5 days/week for four weeks (20 exposures) at nominal concentrations of 0, 0.0058, 0.063 and 0.62 mg/L (analytical concentrations were 0, <0.011, 0.06 and 0.41 mg/L). The low dose was below the analytical limit of detection (0.011 mg/L). The control animals were exposed to the vehicle (distilled water) only. One female in the high dose group died on Day 5. The high-dose animals exhibited necrotic dermatitis of the ears, nose and eyes. Ophthalmoscopic exams revealed corneal opacity, ulceration and granulosis in the high-dose group. Food consumption and body weight gain were decreased in the high-dose males. Hematology and clinical chemistry values were normal. No organ weight changes were observed in the female groups. Absolute testes weights were increased in the high-dose group (p<0.05). Relative adrenal weights were increased in the high-dose group (p<0.001). Relative testes weights were increased in the mid- and high-dose groups (p<0.01). Histopathology indicated minimal to moderate chronic laryngitis in the mid- and high-dose groups (both sexes). The frequency of this finding was dose-related (7 out of 10 high-dose and 3 our of 10 mid-dose). The female that died had severe acute necrotizing laryngitis. Increased organ weights could not be correlated histopathologically. No compound-related effects were seen at the lowest dose level. This study indicated that for 1,4-Diazabicyclo[2.2.2]octane, the point of contact was the site of action, namely, the upper respiratory tract and the eyes. Since no systemic toxicological effects were observed, the No Observed Adverse Effect Concentration (NOAEC) for systemic toxicity was 0.41 mg/L/6h/day, which was the highest dose tested. The NOAEC for local toxicity, based on the lack of an effect observed in the larynx could not be ascertained with confidence, since the lowest dose level could not be measured analytically. The Lowest Observed Adverse Effect Concentration (LOAEC) was 0.06 mg/L/6h/day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEC

60 mg/m³

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In a 4-week inhalation study, 5 rats/dose/sex were exposed whole body to an aerosol of 1,4-Diazabicyclo[2.2.2]octane 6 hours/day, 5 days/week for four weeks (20 exposures) at nominal concentrations of 0, 0.0058, 0.063 and 0.62 mg/L (analytical concentrations were 0, <0.011, 0.06 and 0.41 mg/L). The low dose was below the analytical limit of detection (0.011 mg/L). The control animals were exposed to the vehicle (distilled water) only. One female in the high dose group died on Day 5. The high-dose animals exhibited necrotic dermatitis of the ears, nose and eyes. Ophthalmoscopic exams revealed corneal opacity, ulceration and granulosis in the high-dose group. Food consumption and body weight gain were decreased in the high-dose males. Hematology and clinical chemistry values were normal. No organ weight changes were observed in the female groups. Absolute testes weights were increased in the high-dose group (p<0.05). Relative adrenal weights were increased in the high-dose group (p<0.001). Relative testes weights were increased in the mid- and high-dose groups (p<0.01). Histopathology indicated minimal to moderate chronic laryngitis in the mid- and high-dose groups (both sexes). The frequency of this finding was dose-related (7 out of 10 high-dose and 3 our of 10 mid-dose). The female that died had severe acute necrotizing laryngitis. Increased organ weights could not be correlated histopathologically. No compound-related effects were seen at the lowest dose level. This study indicated that for 1,4-Diazabicyclo[2.2.2]octane, the point of contact was the site of action, namely, the upper respiratory tract and the eyes.  Since no systemic toxicological effects were observed, the No Observed Adverse Effect Concentration (NOAEC) for systemic toxicity was 0.41 mg/L/6h/day, which was the highest dose tested. The NOAEC for local toxicity, based on the lack of an effect observed in the larynx could not be ascertained with confidence, since the lowest dose level could not be measured analytically. The Lowest Observed Adverse Effect Concentration (LOAEC) was 0.06 mg/L/6h/day.

In a Combined Repeated Dose with Reproduction/Developmental Toxicity Screening study (OECD 422), rats were exposed orally to 1,4-Diazabicyclo[2.2.2]octane at dose levels of 0, 100, 300 and 1000 mg/kg/day for 28 days. Dosing solutions were administered by gavage at a dose volume of 5 ml/kg. The control group received the vehicle (deionized water) only. Oral administration of 1,4-Diazabicyclo[2.2.2]octaneresulted in parental (F₀) systemic toxicity in both males and females at a dose level of 1000 mg/kg/day. This was evidenced by changes in clinical condition of the animals, reduced body weight and food consumption, reduced motor activity (females only), increased serum alkaline phosphatase concentrations (females only), increased liver weights (females only) and microscopic changes (inflammatory and/or proliferative lesions) in the kidneys and/or urinary bladder. With the exception of lesions in the kidneys and urinary bladder of a single 1000 mg/kg/day group female, none of the above findings persisted to the end of the 14-day recovery period. F₀ systemic toxicity in the 300-mg/kg/day group was limited to chronic inflammation of the kidneys in the males. There were no indications of F₀ systemic toxicity in the 100 mg/kg/day group males and females.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Guideline study conducted under GLP

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
Guideline study conducted under GLP. 4-week study with 6h per day exposures and analytically measured concentrations. High-dose produced moderate-severe local effects.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
Guideline study conducted under GLP. 4-week study with 6h per day exposures and analytically measured concentrations. High-dose produced moderate-severe local effects.

Justification for classification or non-classification

No STOT is warranted as only local irritative effects or reversible effects were observed.