1,4-diazabicyclooctane

Repeated dose toxicity: inhalation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1987

Report date:

1987

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

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

Details on test material:

- Name of test material (as cited in study report): Triethylendiamine- Physical state: Crystalline solid- Analytical purity: ca. 97 %- Lot/batch No.: 257 125 1086, Fluka AG - Storage condition of test material: At room temperature in the dark.

Test animals

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

Administration / exposure

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

Doses / concentrations

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

Examinations

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 .

Results and discussion

Results of examinations

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.

Effect levels

1

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

2

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).

3

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

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

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

 

Applicant's summary and conclusion

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.