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:
16 July 1985 - 18 Dec 1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study followed OECD guideline 474. In compliance with GLP Regulation. 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. Statistically significant increase observed in micronuclei at 72h sacrification time. In retest, this positive result could not be confirmed.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1986
Report Date:
1986

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
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 reaches 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
Specific details on test material used for the study:
- Name of test material (as cited in study report): 5601-57-2 Order #J-257
- Physical state: liquid
- Analytical purity: responsability of the Sponsor
- Lot/batch No.: Order #J-257, recieved on July 1, 1985
- 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 Breeding Laboratories, Wilmington, Massachusetts
- Age at study initiation: 7,5 weeks
- Weight at study initiation: males: 28-34g, females: 26-29g
- 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): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data


IN-LIFE DATES: From: July 1985 To: December 1985

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: distilled 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.:
1 666 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: 500, 1000, 1666, 3000 and 5000 mg/kg bw .
Mortality was observed at the 3000 and 5000 mg/kg dose levels. The dose selected for the micronucleus test is 1666 mg/kg bw as an estimate of the maximum tolerated dose.

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

DETAILS OF SLIDE PREPARATION:
Femurs 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 transparant. If necessary, the distal ends were opened and flushed.
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 approx. 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 were recorded.
Data are expressed as the number of micronucleated PCE versus total normal PCE in 1000 PCE per animal.
A total of 100 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 0.5% 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 resultsopen allclose all
Sex:
male/female
Genotoxicity:
negative
Remarks:
RETEST
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Sex:
male/female
Genotoxicity:
positive
Remarks:
TEST: increase in incidence of micronuclei, after 72h
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 500-5000mg/kg
- Clinical signs of toxicity in test animals:
Pharmacotoxic signs (decreased body tone, writhing, decreased activity) were observed at all levels evaluated and death occured at the 2 highest doses

RESULTS OF DEFINITIVE STUDY (TEST - see also RETEST below)
- Induction of micronuclei (for Micronucleus assay):
2/10000 (neg control), 542/10000 (pos control), 5/10000 (30h), 3/10000 (48h), 12/10000 (72h)
- Ratio of PCE/NCE (for Micronucleus assay):
1.61 (neg control), 0.77 (pos control), 1.76 (30h), 1.43 (48h), 1.59 (72h)
- Statistical evaluation: OK
Statistically significant increase in the incidence of micronuclei in animals sacrified at 72h. The effect was generally observed in males.
No increases in other animal groups. No effect in the PCE/NCE ratio in test groups.
Positive control: Statistically significant increase in the incidence of micronucleated PCE and a depression of the PCE/NCE ratio.

RETEST: Additional testing was performed to confirm the results.
10 male + 10 female CD-1 mice (7 weeks) were administered the test substance for sacrifice at 72h.
Concurrent negative (distilled water) (5 males, 5 females) + positive control (0.5 mg TEM/kg) (5 males, 5 females) were included with sacrifice at 72h and 30h respectively. All other technical aspects of the study were done according to the original protocol.
RESULTS OF DEFINITIVE STUDY (RETEST)
- Induction of micronuclei (for Micronucleus assay):
5/10000 (neg control), 485/10000 (pos control), 4/10000 (female, 72h), 2/10000 (male, 72h)
- Ratio of PCE/NCE (for Micronucleus assay):
1.92 (neg control), 0.83 (pos control), 1.77 (combined, male and female, 72h)
- Statistical evaluation:
No significant increases were observed in the incidence of micronuclei in the treated animals.
No statistically significant difference in PCE/NCE ratio observed between results of the tested dose and the negative control.
The original positive finding was judged to be of no biological significance.

Any other information on results incl. tables

Other observations:

- Test experiment: abnormal gait, decreased body tone, no mortality observed.

- Retest experiment: All animals (both male and female) exhibited writhing immediately after dosing. Some animals exhibited decreased body tone at 4, 24, 48 and 72 hours; additional signs at 24 hours included diarrhea in one male and vocalization in one female.

Applicant's summary and conclusion

Conclusions:
Result of the study: negative
Results in the initial micronucleus test indicated a statistically significant increase in the incidence of micronuclei in animals administered 1666 mg/kg bw and sacrificed at 72h. The results of an additional re-test with 10 males and 10 females (with concurrent controls) were negative in the micronucleus test at a dose level of 1666 mg/kg at all of the time intervals evaluated. The findings are based on the inability of the test article to give a reproducible statistically significant increase in the incidence of micronuclei per 1000 PCE per animal in the treated group versus the negative control group under the conditions of this assay.
Based on the overall results, the test article was negative in the micronucleus test at a dose level of 1666 mg/kg administered in single intraperitoneal doses with sacrifice times of 30, 48 and 72h.