1,4-diazabicyclooctane

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1987

Report date:

1987

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

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): Triethylenediamine - Physical state: solid- Analytical purity: > 97 %- Lot/batch No.: 2544 39 886, Fluka AG

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Zentralinstitut fuer Versuchstierzucht, Hannover, F.R.G .
- Number of animals 108: 54 males (m) and 54 females (f)
- Number of animals treated per group (controls; test groups): 12 (6 m; 6 f)
- Number of animals analysed per group (controls; treated animals): 10 (5 m; 5 f)
- Age at study initiation: 2.5 - 4 months
- Assigned to test groups randomly: yes, under following basis: random selection within the sexes.
- Fasting period before study: the period of 18 h before administration of the test substance, positive and negative controls.
- Housing: Macrolon, type I, 1 animal per cage.
- Diet: ad libitum, ALTROMIN standard diet (ALTROMIN Tier-Labor Service, Lage, Germany)
- Water: ad libitum, water from the water supply of the Southern Hessian Gas and Water Board, Darmstadt, Germany)
- Acclimation period: two weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 3
- Humidity (%): 40 - 60, not regulated
- Air changes (per hr): 10 changes of air per hour
- Photoperiod (hrs dark / hrs light): 12h/12h

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: water
- Amount of vehicle (if gavage or dermal): 10 mL/kg b.w.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Solution prepared on day of administration

Duration of treatment / exposure:

24 h, 48 h, 72 h after the treatment with the high dose. 24 h after the treatment with the medium and low dose.

Frequency of treatment:

Single dose

Post exposure period:

Rats were killed after 24, 48 and 72 hours

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

90 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

900 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

CPA; Cyclophosphamide; 2-(Bis(2-chloroethyl)amino)tetrahydro (2H)-1,3,2-oxazophosphorine-2-oxid.
- Route of administration: oral (gavage), single
- Doses / concentrations: 30 mg/kg b.w. dissolved in 0.9 % NaCl-solution, Solution prepared on day of administration
- Volume 10 mL/kg b.w.

Examinations

Tissues and cell types examined:

bone marrow of the femora

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Dosing was chosen with regard to the results of pre-experiments.
In a first pre-experiment each of 2 males and 2 females received 1800; 1200; or 900 mg/kg bw of the test substance.
900 mg/kg b.w. was the highest non-lethal dose. With this dose the animals expressed toxic reactions: incomplete eyelid closure, abdominal position, reduction of spantaneous activity.
With higher doses of the sustance animals died: 1800 mg/kg b.w. dosing: 2 out of 4 treated animals died within 1 hour.
1200 mg/kg b.w. dosing: 2 out of 4 treated animals died within 1 hour.
In a second pre-experiment each 5 males and 5 females received 900 mg/kg b.w. of the test substance orally. None of the treated animals died within the observation period (72 hours).

DETAILS OF SLIDE PREPARATION:
The animals were killed by cervical dislocation. The femora were removed and freed from muscles and tissue. The epiphyses were cut off with scissors and the marrow was flushed out with 0.3 mL fetal calf serum, using a 1 mL syringe, into 5 mL fetal calf serum. The cell suspension was centrifuged at 1000 rpm for 5 minutes. The cell pellet was spread on a slide. The smear was air-dryed and then stained with May-Gruenwald/Giemsa. Cover slips were mounted with EUKITT (KINDLER, Freiburg, Germany), 3 slides were made from each animal.

METHOD OF ANALYSIS:
Analysing of the micronucleus rates was performed using Nikon microscopes with 100 x oil immersion objectives.
1000 polychromatic erythrocytes (PCE) were analysed per animal. The relationship of polychromatic and normochromatic erythrocytes was determined in cell samples containing 1000 polychromatic erythrocytes to obtain information on cytotoxic effects of the treatment. The analysis was performed with coded slides.

Statistics:

Not necessary to perform as all mean micronucleus rates in the groups treated with the test substance are in the range of the negative controls.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

(dose dependent decrease of PCE/NCE ratio)

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): There was no enhancement of the number of cells with micronuclei in the groups treated with the test substance as compared with the negative controls treated with the solvent.

Any other information on results incl. tables

Group	Substance	Dose (mg/kg)	Sex	post exposure period (h)	PCE with micronuclei	Ratio PCE/NCE
1	solvent	0	males/females	24	0.07	2.08
2	solvent	0	males/females	48	0.13	1.04
3	solvent	0	males/females	72	0.04	1.61
4	CPA	30	males/females	24	0.8	2.70
5	test substance	90	males/females	24	0.13	1.51
6	test substance	300	males/females	24	0.07	1.36
7	test substance	900	males/females	24	0.09	1.31
8	test substance	900	males/females	48	0.02	1.04
9	test substance	900	males/females	72	0.08	1.59

PCE = polychromatic erythrocyts (1000 were scored for micronuclei)

NCE = normochromatic erythrocyts

The NCE/PCE ratio is based on the scoring of 1000 erythrocyts