Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 March 1988 - 14 June 1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
GLP study performed according to a method similar to OECD guideline 474 without significant deviations.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report Date:
1988

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
- Name of test material (as cited in study report): 5601-98-1, Order #88-004
- Substance type: clear colorless liquid
- Physical state: liquid
- Analytical purity: responsibility of the Sponsor
- Lot/batch No.: Order #88-004, recieved on March 25, 1988
- Stability under test conditions: no apparent change in its physical state
- Storage condition of test material: room temperature

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Wilmington, Massachusetts
- Age at study initiation: 7 weeks
- Weight at study initiation: males: 28-32g, females: 22-27g
- Assigned to test groups randomly: yes, randomized by bodyweight, assigned to groups by use of a random number table
- Fasting period before study: no
- Housing: 5 per cage (according to sex and dose group)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30 - 70%
- Air changes (per hr): no info
- Photoperiod (hrs dark / hrs light): 12 h dark/ 12 h light

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: distilled water
Details on exposure:
one single intraperitoneal dose per mouse at a volume of 10 mL/kg body weight
Duration of treatment / exposure:
1 single dose
Frequency of treatment:
once
Post exposure period:
test substance: 30, 48 and 72h
positive control: 30h
negative control: 48h
Doses / concentrations
Dose / conc.:
75 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
- triethylenemelamine
- Route of administration: intraperitoneal
- Doses / concentrations: 0.5 mg/kg

Examinations

Tissues and cell types examined:
bone marrow of femur
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Based on dose range finding study with following doses: 10, 50, 100, 200, 500, 1000 and 2000 mg/kg bw .
All mice died from 200 mg/kg bw up to 2000 mg/kg bw dose groups. Due to the mortality observed and the appearance of mild to severe pharmacotoxic signs in the 100 mg/kg bw dose group, 75 mg/kg bw was selected as an estimate of the maximum tolerated dose.

DETAILS OF SLIDE PREPARATION:
All mice were sacrified by cervical dislocation. Femora were opened carefully at the proximal end with a scissors until a small opening to the marrow canal became visible. A 1 ml tuberculin syringe filled with approx. 0.2 ml fetal bovine serum was inserted into the marrow cavity and the marrow was gently flushed (to assure maximum dispersion) into 1.0 ml of fetal bovine serum in a 3 ml conical centrifuge tube. The femora were flushed with fetal bovine serum until all the marrow was out and the bone appeared almost translucent. The suspension was centrifuged at 1000 rpm for 5 minutes. The supernatant was removed leaving a small amount of fetal bovine serum with the remaining cell button. The button was mixed with a pasteur pipette to assure a homogenous mixture. The marrow smears were made by placing immediately a small drop of the cell suspension near the frosted end of a glass slide pre-cleaned in absolute ethanol and smeared by pulling the cell suspension behind with another pre-cleaned slide at a 45° angle. The slides were quickly dried on a slide warmer set at 56°C, dipped in absolute methanol and air dried.
Stained with Giemsa.

METHOD OF ANALYSIS:
Slides were screened for good preparation, i.e. well spread, undamaged, perfectly stained.
1000 polychromatic erythrocytes (PCE)(immature) were counted for the presence of micronuclei per animal. Also the number of normochromatic erythrocytes (NCE) (mature) present in these 1000 PCE was recorded.
Data are expressed as the number of micronucleated PCE versus total normal PCE in 1000 PCE per animal.
A total of 1000 PCE and NCE was also counted per animal. These data were expressed as the ratio PCE/NCE.
OTHER: Slides were coded randomly by study number and number designation. The code was kept on a separate sheet until the slides were evaluated. Following evaluation, slides were decoded. Coding of the slides was carried out by an individual not involved in the actual scoring.
Evaluation criteria:
If the spontaneous rate of micronuclei in the PCE is less than 0.2% and the positive control is statistically greater (p<0.05) than the spontaneous rate and at least seven animals per group survived the treatment, the results are deemed acceptable.
Statistics:
- one-tailed t-tests were used to make pairwise comparisons between each treatment group and its concurrent vehicle control for statistically significant increases in the number of micronucleated PCE.
- the proportion of PCE per 1000 erythrocytes per animal were evaluated by pairwise two-tailed t-tests after an arcsin transformation was performed.
All comparisons were made for each sacrifice time separately comparing treated groups versus the vehicle control group.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
abnormal gait, decreased activity, piloerection, decreased body tone, no mortality observed
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The substance did not produce any statistically significant increases in the number of micronucleated PCEs. There was a significant increase in PCE/NCE ratios for the 72h treated group as compared to the vehicle control. However, biological significance of this positive result, if any, is unknown.

RESULTS OF RANGE-FINDING STUDY
- Dose range: 10 - 2000 mg/kg
- Clinical signs of toxicity in test animals:
all mice died from 200 mg/kg up to 2000 mg/kg dose groups.
no mice died at the three lower dose groups.
100 g/kg: writhing, abnormal gait, decreased activity (immediately after treatment), decreased body tone, piloerection, abnormal gait and decreased activity (all, at 24h, 48h, 72h), abnormal stance, tremors, ptosis, cyanosis and hypothermia (1 female, 72h after treatment)


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay):
total micronucleated PCE: 17/10000 (30h), 9/10000 (48h), 15/10000 (72h), 14/10000 (neg control), 462/10000 (pos control)
- Ratio of PCE/NCE (for Micronucleus assay):
1.031 (30h), 1.384 (48h), 1.711 (72h), 1.191 (neg control), 0.758 (pos control)
- Appropriateness of dose levels and route: no data
- Statistical evaluation:
results of positive control are statistically different from results of negative control (induction of micronuclei and PCE/NCE ratio)
number of induced micronuclei is not statistically different from negative control.
PCE/NCE ratio is statistically different from negative control, only at 72h sacrifice time.

Applicant's summary and conclusion

Conclusions:
The substance, at 75 mg/kg bw, was considered negative in the micronucleus test at the time intervals evaluated under the experimental conditions of this assay.