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:
1988-04-25 to 1988-05-25
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The OECD Guideline 474 states that samples of bone marrow should be taken at least twice, starting not earlier than 24h after treatment, but not extending beyond 48h after treatment with appropriate interval(s) between samples. In this assay, bone marrow samples are taken at 30, 48 and 72h after treatment. 6 Animals died minutes after administrating the dose in the definitive study. However, these animals were replaced.

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:
yes
Principles of method if other than guideline:
The OECD Guideline 474 states: that samples of bone marrow should be taken at least twice, starting not earlier than 24h after treatment, but not extending beyond 48h after treatment with appropriate interval(s) between samples. In this assay, bone marrow samples are taken at 30, 48 and 72h after treatment.
Also, the likelihood that the test substance reach the general circulation or the target tissue has not been discussed.
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Name of test material: Trimethylolpropanepoly(oxypropylene)triamine
EC no.: 500-105-6
CAS no.: 39423-51-3
Physical state: clear colourless liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Order #88-006, recieved on March 25, 1988

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: no apparent change in its physical state

OTHER SPECIFICS:
- Name of test material (as cited in study report): 5601-96-1

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: 8 weeks
- Weight at study initiation: males: 29-37g, females: 26-32g
- 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: max 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: 12 days


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


IN-LIFE DATES: From: April 1988 To: May 1988

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: deionized water
Details on exposure:
one single intraperitoneal dose per mouse
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.:
2.5 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: 50, 100, 250, 500 and 1000 mg/kg bw .
Pharmacotoxic signs were observed in the 100 mg/kg group and at higher dose levels evaluated. Mortality occurred in the 250 mg/kg bw group (1/4). All animals but one, died within 2 hours post-dose in the 500 and 1000 mg/kg bw dose groups. Therefore, 150 mg/kg bw was selected as dose.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
All mice were sacrified by cervical dislocation.

DETAILS OF SLIDE PREPARATION:
One end of each femur (one proximal, one distal to the iliac end) was opened carefully with 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 a 5ml round bottom culture tube containing 1.0 ml of fetal bovine serum. Each femur was 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 pellet. The pellet was resuspended with a pasteur pipette to assure a homogenous mixture.
A small drop of the cell suspension was spread immediately onto an ethanol pre-cleaned glass slide. The smears were quickly dried on a slide warmer set at approx. 56°C, dipped in absolute methanol (2sec) 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) per animal were scored for the presence of micronuclei, as well as for the number of micronucleated normochromatic erythrocytes (NCE)(mature) present in the optical field containing these 1000 PCE.
Data are expressed as the total number of mean percentage of micronucleated PCE for 10000 PCE per group (1000 PCE per mouse, whenever possible). An additional 1000 erythrocytes (PCE and NCE) were also scored per mouse and the proportion was expressed as the PCE/NCE ratio.
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 0.5% and the positive control is statistically significantly 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 erytrocytes per animal were evaluated by pairwise two-tailed t-test 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:
decreased activity, abnormal gait, body drop and decreased body tone, six animals were dead within minutes of dose administration
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 0.75 - 50 mg/kg
- Clinical signs of toxicity in test animals:
2.5 mg/kg: decreased activity, body drop and abnormal gait (immediately after treatment), no signs (24h), decreased body tone (most animals at 48h), body drop, abnormal gait (1 female, 48h), piloerection, decreased body tone (all, 72h)
1 mg/kg: no signs (immediately to 24h after treatment), decreased body tone (most animals, at 48 and 72h after administration)
0.75 mg/kg: no signs (immediately to 24h after treatment), piloerection, decreased body tone, abnormal gait (all males, 48h), decreased body tone (all, 72h).
2.5 mg/kg was selected for the definitive study

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay):
8/10000 (30h), 13/10000 (48h), 10/10000 (72h), 11/10000 (neg control), 342/10000 (pos control)
- Ratio of PCE/NCE (for Micronucleus assay):
1.248 (30h), 1.639 (48h), 1.358 (72h), 1.246 (neg control), 0.821 (pos control)
- Appropriateness of dose levels and route: dose level seems appropriate and was based on the range finding study. However, six animals (4 males, 2 females) were dead within minutes of dose administration, indicating that the dose (2.5 mg/kg) was near or at the maximum tolerated dose. Death occurred in 2 males of the 30h group, 2 males of the 48h group, 2 females of the 72h group. Necroscopy for all six mice did not reveal visible lesions. Mice were replaced so that there were 10 mice per dose group.
- Statistical evaluation: OK.
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 between the test substance and the negative control (at none of the sacrifice times).
PCE/NCE ratio was significantly increased for the 48h treated group as compared to the vehicle control. The significance of this positive result is undetermined.

Applicant's summary and conclusion

Conclusions:
The test article, administered in single intraperitoneal dose at 2.5 mg/kg bw, did not induce a statistically positive increase in micronuclei in the hemopoietic cells of the mouse bone marrow at the time intervals evaluated under the experimental condition of this assay.