1,4-diazabicyclooctane

Repeated dose toxicity: oral

Administrative data

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

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

1,4-diazabicyclooctane

EC Number:

205-999-9

EC Name:

1,4-diazabicyclooctane

Cas Number:

280-57-9

Molecular formula:

C6H12N2

IUPAC Name:

1,4-diazabicyclooctane

Test material form:

other: white crystalline powder

Details on test material:

A sufficient volume of the vehicle, deionized water, was dispensed into a labeled storage container for administration to the control group. The solution was stirred continuously throughout use, using a magnetic stir bar.Each test article formulation was prepared by weighing the appropriate amount of the test article, Triethylenediamine, into a calibrated, labeled storage container. A sufficient volume of vehicle, deionized water, was added to each container. The pH was adjusted to 7.58-8.30 using concentrated hydrochloric acid (received from Sigma-Aldrich, St. Louis, Missouri, lot no. 17H3495 and from Spectrum Quality Products, Inc., New Brunswick, New Jersey, lot nos. LP0l89 and OR0022). An additional volume of the vehicle was added to the calibration mark and the formulations were stirred until uniform and throughout use, using a magnetic stir bar.The formulations for each group were prepared weekly (August 20, 27, September 3, 10, 17, 24, October 1 and 8, 1999) and were stored at room temperature. The formulations were dispensed into aliquots for daily dose administration. The dosing preparations were visually inspected by the study director on August 20, 1999, and were found to be visually homogeneous and acceptable for dose administration.

Test animals

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.

Administration / exposure

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

Doses / concentrations

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

Examinations

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.

Results and discussion

Results of examinations

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.

Effect levels

1

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

2

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

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.