Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted according to GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report Date:
1991

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
other: US EPA Guidelines from 1985
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): diethylaminoethanol
- Analytical purity: > 99%
- Lot/batch No.: G-9-A
- Physical state: clear colourless liquid
- Storage condition of test material: ambient temperature in the dark

Test animals

Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Olac Limited, England
- Age at study initiation: 4-5 week old mice
- Weight at study initiation: 17.7 - 27.8 g
- Diet: Laboratory animal diet LAD 1 (Biosure, Manea, Cambridgeshire, England), ad libitum
- Water: Drinking water was supplied to each cage via a polythene bottle and sipper-tube
- Acclimation period: at least four days prior to treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2 °C
- Humidity (%): 55 ± %
- Air changes (per hr): 15
- Photoperiod 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
0.9 % saline
Details on exposure:
Dosing volume: 10 mL/kg
Sampling times: 24 hr (all groups), 48 hr (control + high dose) and 72 hr (control + high dose).
Duration of treatment / exposure:
single oral dose
Frequency of treatment:
only one application
Post exposure period:
24 hr (20 and 100 mg/kg bw dose)
72 hr (control + high dose)
Doses / concentrations
Remarks:
Doses / Concentrations:
20, 100 and 500 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
five/sex/dose/sampling time
Control animals:
yes, concurrent vehicle
Positive control(s):
chlorambucil, 30 mg/kg bw in 10% aqueous ethanol

Examinations

Tissues and cell types examined:
Animals were inspected daily throughout the acclimatisation period and the dosing period for signs of ill-health or reaction to treatment. Any deviation from normal was recorded. All animals were weighed on the day of treatment and again immediately before termination, and bodyweights were recorded. In addition, the animals in the preliminary toxicity test were weighed immediately prior to dosing and daily thereafter until termination.
The frequency of micronucleated cells per at least 1000 polychromatic erythrocytes, as well as the frequency of micronucleated cells per at least 1000 mature erythrocytes was determined. The ratio of polychromatic to mature cells was also determined.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
The doses were chosen based on a preliminary toxicity test using 2 animals/sex/group treated with 312.5, 625, 1250 and 2500 mg/kg bw.
DETAILS OF SLIDE PREPARATION:
Animals were killed by cervical dislocation following carbon dioxide inhalation. Femurs from each animal were rapidly dissected out and cleaned of adherent tissu . The epiphyses were cut off to obtain access to the marrow canal. Marrow cells were flushed out with 2.5 ml foetal calf serum using a syringe and needle. The recovered cells were centrifuged at 1000 rpm for five minutes . The bulk of the supernatant fluid was discarded and the cell pellet resuspended in the remaining fluid. Single drops of the cell suspension were transferred to clean, dry slides, two or three smears (for the preliminary toxicity test or main micronucleus test respectively) prepared, and the slides left to air-dry. Following fixation in methanol for ten minutes, they were stained manually, using the Schmid (May-Grunwald and Giemsa) staining technique.





Statistics:
Mann-Whitney U-test.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
at 100 mg/kg bw 1 male had hunched posture and piloerection. At 500 mg/kg bw, signs of toxicity were more pronounced and 1 male needed to be killed in extremis 42 hrs post-dosing.
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
TOXICITY
No adverse reactions to treatment were recorded for any animal exposed to diethylaminoethanol at 20 mg/kg bw. However, one male mouse dosed at 100 mg/kg bw showed signs including hunched posture and piloerection. One male mouse treated at 500 mg/kg bw was killed in extremis approximately 42 hours after dosing; signs included rales, piloerection, hunched posture and swollen abdomen. Sixteen of the remaining animals dosed at this level showed adverse reactions to treatment intermittently after dosing including rales (13 mice), piloerection (11), hunched posture (10) and irregular respiration (1).

EFFECT ON PCE/NCE RATIO PCE/NCE ratio (males and females combined):
at 24 hours: 0.9 (all doses); 0.8 (saline control);
at 48 hours: 0.9 (500 mg/kg bw ); 0.9 (saline control);
at 72 hours: 0.7 (500 mg/kg bw ); 0.7 (saline control);

GENOTOXIC EFFECTS
Mean number of micronucleated PCE/1000 PCE (males and females combined):
- 0.9 % saline control: 1.2, 0.6 and 0.6 at 24, 48 and 72 hours, respectively;
- 20 mg/kg bw: 0.8 at 24 hours;
- 100 mg/kg bw: 0.7 at 24 hours;
- 500 mg/kg bw: 0.6, 0.8 and 0.3 at 24, 48 and 72 hours, respectively.

STATISTICAL RESULTS
Frequencies of micronucleated polychromatic erythrocytes were not significantly different from controls at any dose or sampling time.

PRELIMINARY STUDY
The PCE/NCE ratio in the preliminary study was 0.9, 0.8 and 0.5 in the 312.5 mg/kg bw , 625 mg/kg bw and 1250 mg/kg bw dose groups, respectively. This indicated that the test substance reached the bone marrow.

POSITIVE CONTROL
Positive controls gave the expected response.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative