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Diss Factsheets

Toxicological information

Endpoint summary

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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

1,4-Diazabicyclo[2.2.2]octane gave no indication of in vitro mutagenic activity or in vivo clastogenic potential.

Link to relevant study records
Reference
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
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay
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
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
Dose / conc.:
90 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
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.
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.
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.

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

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

In a guideline (OECD 471) study, 1,4-Diazabicyclo[2.2.2]octane was tested at doses of up to 5000 µg/plate in Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538, both with and without metabolic activation and yielded negative results.

In a guideline (OECD 474) study, 1,4-Diazabicyclo[2.2.2]octane was tested for its ability to induce micronuclei in bone marrow erythrocytes in mice using oral doses of up to 900 mg/kg bw and was found to be negative. The test material was dissolved in water for dosing. Two out of 4 mice tested at both 1200 and 1800 mg/kg bw, died in a range-finding experiment. The highest non-lethal dose tested was 900 mg/kg. At this dose, incomplete eyelid closure and a reduction in spontaneous activity were observed. In the main study, 6 male and 6 female mice were treated in each group (0, 90, 300 and 900 mg/kg). Three high-dose and three negative control groups were treated so that the bone marrow cell preparation could be done at 24, 48, and 72 hours post-dose. The positive control and the low- and mid-dose groups were only analyzed at 24 hours post-dose. 1000 cells from each of 5 male and 5 female mice per group were analyzed. There was no enhancement of the number of cells with micronuclei in the test substance treated groups as compared to the distilled water control. The positive control (cyclophosphamide) significantly enhanced micronuclei rates by 8-fold or more.

Justification for selection of genetic toxicity endpoint
Guideline in vivo study conducted under GLP

Justification for classification or non-classification

Data is sufficient and no classification is required.