1,3,5-tris(2,3-dibromopropyl)-1,3,5-triazine-2,4,6(1H,3H,5H)-trione

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1980

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1980

Report date:

1980

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

Significant deficiencies from current Guideline, e.g. samples were taken 6 hours after the last dose.

GLP compliance:

no

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

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: Interstab 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:

A subchronic dosing regimen was used. It consisted of two administrations approximately 24 hours apart. Treatment covered a 30-hour period or two cell cycles.

Frequency of treatment:

24h/dose

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

10 000 mg/kg bw/day (actual dose received)

Remarks:

highest dose, FR 930

Doses / concentrations 2

Dose / conc.:

1 250 mg/kg bw/day (actual dose received)

Remarks:

lowest dose, FR930

No. of animals per sex per dose:

4 males and 4 females

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 maximum amount administered to the experimental animals was 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 CO2 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 (Schmid, 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 1957). 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: did not induce micronuclei at the doses tested

Additional information on results:

The test material, Interstab FR930 is evaluated here for its ability to induce micronuclei in polychromatic erythrocytes (PCE, i.e. immature red blood cells) in mouse bone marrow.
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.
Further details on results are present in the attache full study report.

Applicant's summary and conclusion

Conclusions:

The test item Interstab FR930 did not induce micronuclei at the doses tested.

Executive summary:

The objective of this study was to evaluate the test article for clastogenic activity in polychromatic erythrocyte (PCE) stem cells in treated mice.

Adult male and female mice, strain CD-1, healthy and random-bred strain have been selected to maximize genetic heterogeneity and at the same time assure access to a common source.

Thirty-two outbred mice, 4 males and 4 females per dose level, were used. 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. 

The route of administration was oral gavage, the most common route of administration for this test procedure. A subchronic dosing regimen was used: it consisted of two administrations approximately 24 hours apart. Treatment covered a 30-hour period or two cell cycles. 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. The total doses were 10 g/Kg (highest dose) and 1.25 g/kg (lowest dose).

The negative control article 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).

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 frequency of micronuclei in mouse bone 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. 

In conclusion, the test item did not induce micronuclei at the doses tested.