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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1980
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
Significant deficiencies from guideline e.g. samples were taken 6 hrs after the last dose as opposed to 18-24 hrs

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1980

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
Significant deficiencies from guideline e.g. samples were taken 6 hrs after the last dose as opposed to 18-24 hrs
GLP compliance:
no
Type of assay:
mammalian germ cell cytogenetic assay

Test material

Constituent 1
Chemical structure
Reference substance name:
1,3,5-tris(2,3-dibromopropyl)-1,3,5-triazine-2,4,6(1H,3H,5H)-trione
EC Number:
257-913-4
EC Name:
1,3,5-tris(2,3-dibromopropyl)-1,3,5-triazine-2,4,6(1H,3H,5H)-trione
Cas Number:
52434-90-9
Molecular formula:
C12H15Br6N3O3
IUPAC Name:
tris(2,3-dibromopropyl)-1,3,5-triazinane-2,4,6-trione
impurity 1
Reference substance name:
unknown impurities
IUPAC Name:
unknown impurities
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Test Material generically named as : FR 930

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals or test system and environmental conditions:
Adult male and female mice, strain CD-1, from a randomly bred closed colony were purchased from Charles River Breeding Laboratories, Inc., Wilmington, MA. This healthy, random-bred strain has been selected to maximize genetic heterogeneity and at the same time assure access to a common source.

Animals were group-housed up to 15 mice per cage. A commercial diet (Purina Laboratory Chow) and water were offered ad libitum unless contraindicated by the particular experimental design.
Thirty-two outbred mice, 4 males and 4 females per dose level, were used. Animals were assigned to study groups at random according to Litton Bionetics, Inc. (LBI) Standard Operating Procedures (SOP). Prior to study initiation, animals were weighed to calculate dose levels according to SOP “Animal Weight Determinazion.” The volume of test article administrered per animal was established using this method unless there was significant variation among individuals, in which case individual calculations were made. Animals were uniquely identified by ear tag or ear punch. Dose or treatment groups were identified by cage card.

Sanitary cages and bedding was used. Personnel handling animals or working within the animal facilities were required to wear suitable protective garments. When appropriate, individuals with respiratory or other overt infections were excluded from the animal facilities.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
DMSO
Duration of treatment / exposure:
Two days split dose
Frequency of treatment:
24h/dose
Post exposure period:
14 days
Doses / concentrationsopen allclose all
Dose / conc.:
10 000 mg/kg bw/day (actual dose received)
Remarks:
highest dose, FR 930
Dose / conc.:
1 250 mg/kg bw/day (actual dose received)
Remarks:
lowest dose, FR930
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Triethylenemelamine
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
DMSO
No. of animals per sex per dose:
4 male and 4 female for each four different dose
Control animals:
yes, concurrent vehicle
Positive control(s):
Triethylenemelamine (TEM) at 1.0 mg/kg was dissolved in saline and used as the positive control article and was administered via a split dose intraperitoneal (IP) injection. The negative control article was the vehicle used for the test article (DMSO) and was administered by the same route as, and concurrently with, the test article and in volumes equal to the maximum amount administered to the experimental animals (0.25 ml per male mouse, and 0.20 ml per female mouse).

Examinations

Tissues and cell types examined:
Mouse Bone Marrow
Details of tissue and slide preparation:
EXTRACTION OF BONE MARROW
Six hours after the last dose, animals were killed with 〖CO〗_2; and the adhering soft tissue and epiphyses of both tibia were removed. The marrow was aspirated from the bone and transferred to centrifuge tubes containing 5 ml fetal calf serum (one tube for each animal).

PREPARATION OF SLIDES
Following centrifugation to pellet the tissue, the supernatant was drawn off, cells resuspended in a drop of serum, and suspension spread on slides and air dried. The slides were then fixed in methanol, stained in May-Gruenwald solution followed by Giemsa, and rinsed in deionized water (Scmid, 1975).

SCREENING THE SLIDES
A thousand PCE’s per animal were scored. The frequency of micronucleated cells was expressed as percent micronucleated cells based on the total PCE’s present in the scored optic field. The normal frequency of micronuclei in this mouse strain in between 0.3 and 0.4%.
Evaluation criteria:
In tests performed for this evaluation only PCE’s were scored for micronuclei. Mature erythrocytes and other cells in the field were noted but not scored.

Two dose levels were established to ensure that a nontoxic level of the test article was scored. Dose response data are not necessary to define a test article as active. Responses considered positive are assumed to reflect clastogenic and related activities of test articles. Agents which break chromosomes and induce nondisjunction and other events which produce structural or numerical changes in chromosomes can produce micronuclei.

The data were analyzed by a student t-test (Brancroft 2957). Individual animal results were used as data points in the analysis. The set of micronuclei frequencies among the controls was compared to the set for each treatment level. Male and female animal data were analyzed separately and combined. Increases above the negative control frequency that are significant at p < 0.01 are considered indicative of an active agent.

For control of bias, all slides were coded prior to scoring and scored blind.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: not induce micronuclei
Additional information on results:
The test material, FR930 is evaluated here for its ability to induce micronuclei in polychromatic erythrocytes (PCE, i.e. immature red blood cells) in mouse bone marrow.

Stock solutions of the test compound were prepared in dimethylsulfoxide (DMSO) at initial concentrations of 800 mg/ml and 100 mg/ml for administering the high (5 g/kg/day) and low (0.63 g/kg/day) doses respectively. (total doses were 10 g/Kg and 1.25 g/kg).
The frequency of micronuclei in marrow from the negative control animals is within the normal range for this laboratory. The positive control compound, triethylenemelamine (TEM) induced a large increase in micronuclei, to a total of about 7% . There is no evidence for induction of micronuclei by the test compound.

Applicant's summary and conclusion

Conclusions:
FR930 did not induce micronuclei at the dose tested.

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