Registration Dossier

Administrative data

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: No internationally accepted guideline followed but study under GLP, well documented and robust protocol.

Data source

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

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Test substance was administered by intraperitoneal injection to induce chromosomal damage in bone marrow cells of Fischer-344 rats. In the definitive stuy, male rats were given DBPP at doses of 0, 40, 200, and 400 mg/kg bw; female rats were given DBPP at doses of 0, 60, 300, and 600 mg/kg bw. Groups of animals were sacrificed 6, 12, and 24 hr after treatment. A concurrent positive control group received triethylenemelamine.
Cells from males exposed to 0 and 400 mg/kg bw and from females exposed to 0 and 600 mg/kg bw DPP and from animals in the positive control groups were evaluated microscopically for mitotic indec and chromosomal abnormalities.
GLP compliance:
yes
Type of assay:
mammalian germ cell cytogenetic assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Liquid
Details on test material:
- Name of test material (as cited in study report): DBPP
- Physical state: clear, colorless liquid
- Lot/batch No.: SA-84048
- Storage condition of test material: stored in original bottle placed in a secondary lightproof and unbreakable container at room temperature

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Simonsen Laboratories Inc., 1180C Day road, Gilroy, CA 95020
- Age at study initiation: not specified
- Weight at study initiation: males: 203 g, females: 191 g.
- Assigned to test groups randomly: yes
- Housing: 2 or 3 per polycarbonate cage with wood shavings as bedding
- Diet (e.g. ad libitum): Certified Purina Lab Chow, ad libidum
- Water (e.g. ad libitum): fresh purified water, ad libidum
- Acclimation period: 5 day quarantaine

ENVIRONMENTAL CONDITIONS
- Temperature (°C): not specified
- Humidity (%): not specified
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): not specified

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
Details on exposure:
ADMINISTRATION OF TEST ARTICLE
DBPP was diluted with corn oil. Fresh dilutions were made for each pilot and definitive study. All animals were treated with a single intraperitoneal injection. Dosing injection volumes were 1 to 5 mg/kg bw depending on the study.
Duration of treatment / exposure:
Single injection
Frequency of treatment:
1x
Post exposure period:
Dose range-finding study: 14 days.
Pilot studies: 24 hours or 14 days.
Definitive study: 6, 12 and 24 hours
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
8, 40, 200, 1000 and 5000 mg/kg
Basis:
other: injected (dose range-finding)
Remarks:
Doses / Concentrations:
312, 625, 1250, 2500 and 5000 mg/kg
Basis:
other: injected (pilot)
Remarks:
Doses / Concentrations:
40, 60, 200, 300, 400 and 600 mg/kg
Basis:
other: injected (definitive)
No. of animals per sex per dose:
dose range-finding: 2 rats per sex per dose
pilot: 7 rats per sex per dose (2 sacrificed after 24 hrs, 5 after 14 days)
definitive: 6 rats of one sex per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
- yes: triethylenemelamine (TEM)
- Route of administration: ip injection, solution in Hanks' balanced salt solution (HBSS)
- Doses / concentrations: 0,2 mg/kg

Examinations

Tissues and cell types examined:
bone marrow cells
Details of tissue and slide preparation:
DETAILS OF SLIDE PREPARATION:
Femoral bone marrow cells were prepared from each rat according to a modification of the approach specified by Nichols et al. (1972). Approximately 2 to 3 hrs prior to sacrifice, each rat was injected ip with colchicine dissolved in HBSS. The animals were sacrificed by CO2 asphyxiation followed by cervical dislocation. Skin and muscle tissue were carefully removed from the femur immediately after sacrifice. The femur, exposed knee to hip, was cut just above the knee, and cells were aspirated from the distal end of the femur through an 18-gauge needle into a 5-mL syringe containing 2 mL of warm HBSS. The procedure was then repeated for the other leg, and HBSS and bone marrow cells were dispensed along the inner side of a 15-mL centrifuge tube. The suspension was mixed by aspirating it up and down at least 5 times through the 18-gauge needle to break the fibrin and ensure cell dissociation. Care was taken to avoid pushing HBSS into the femur.
The tubes were centrifuged at 700 rpm for 10 minutes and the supernatant was carefully discharged so as not to disturb the cell pellet. Then 0,55% KCl (warmed to 37°C) was added to each tube, the pellet was gently disrupted and gross debris was removed. Next the tubes were incubated in a 37°C water bath for 20 minutes. Then 1 mL of fixative (methanol : glacial acetic acid 3:1) was added and mixed with the hypotonic solution. The tubes were cetrifuged, the supernatant discarded and fixative was added and mixed with the pellet. Tubes were centrifuged, supernatant discarded and fresh fixative was added. Subsequently, tubes were allowed to stand for 10 to 30 min at room temperature and the fixation was repeated.

Slides were prepared by dropping cell suspensions onto water-dipped slides, passing the slides through the flame of an alcohol lamp several times, and checking the cell density using a phase-contrast microscope. If cell density was too thick, more fixative was added to dilute the suspension. Based on the amount of cell suspension, six or fewer slides were prepared from each tube.
The slides were placed in 7% Giemsa for 5 to 20 minutes, rinsed in distilled water, soaked in xylene and mounted with Permount to make permanent slides for scoring under bright-field or phase-contrast optics.

METHOD OF ANALYSIS:
Prepared slides from males and females exposed to 0 mg/kg (negative controls), from males exposed to 400 mg/kg, from females exposed to 60 mg/kg and from all animals exposed to TEM (positive controls) were divided into identical groups and coded by an individual not involved in the microscopic evaluation. Slides from 5 animals (if available) in each of the above treatment groups per sex per sacrifice period were coded after choosing the animals randomly or from the results of initial evaluation of the quality of the slides. The slides were decoded only after all slides in each group had been analyzed completely.
For this stuy, two individuals separately analyzed coded slides from each animal. Slides were evaluated for mitotic index (based on at least 1000 cells/animal), and 60 cells per animal, when possible, were evaluated for chromosomal aberrations. Because a sufficient number of cells (60 or nearly 60) was analyzed for the majority of animals in each treatment group, the chromosomal preparations from the additional animals were not evaluated.
For analysis of the slides, score sheets were used, in which was noted, i.a., quality of the slide, vernier settings, MI, chromosome number and numbers of various categories of chromatid and chromosomal aberrations for each cell scored. Chromatid and isochromatid gaps were recorded for each cell but were not considered chromosomal aberrations in the analysis of the data. After analysis was completed, the slides were decoded and the score sheets were summarized.
Evaluation criteria:
Positive:
A test article was considered to have elicited a positive response in the in vivo bone marrow cytogenetic assay if either the mean aberrant cell frequency or the mean chromosomal aberration frequency per cell, or both, was significantly greater (p<0,05) in the test article-treated animals than in the negative control animals.

Negative:
A test article was considered to have alicited a negative response if the results obtained by each cytogeneticist were in general agreement and the criteria for a positive response were not met.

Inconclusive:
The results of this assay were considered inconclusive if there was reason to believe that the concentrations of the test article selected for evaluation were inappropriate (i.e. excessive toxicity or lack of any toxicity)
Statistics:
The mean number of chromosomal aberrations per cell, the mean frequency of aberrant cells, the mean MI, the mean frequency of cells with structurally aberrant chromosomes and the mean frequency of aneuploid (hyperploid) cells were calculated for each treatment group x sex x sacrifice-time subclass from the individual animal means. For the first 3 of these values, Bartlett's test was employed to investigate homogeneity among treatment variances. A one-way analysis of variance was used to analyze the data from each sex x sacrifice time subclass. If a significant (i.e. p<0,05) F-value was obtained, Dunnett's test was used to compare the test article treatment means with the appropriate negative control mean. Student's t-test was used to evaluate the significance of differences between the negative control and positive control groups.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
at > 625 mg/kg
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
24h PILOT STUDY
- No significant deleterious effect of treatment with DBPP on mitotic indices.
- All rats treated with DBPP lost more weight than did the negative control rats.
- Toxicity in male rats:
* 5000 mg/kg (2/2): mortality
* 2500 mg/kg (2/2) and 1250 mg/kg (1/2): exudate anround the penis and a lower abdominal region:
* 625 mg/kg or less: no toxic symptoms
- Toxicity in female rats:
* 5000 mg/kg: animal 1: moderately humped back; animal 2: no toxicity
* 2500 mg/kg (2/2): no toxic symptoms
* 1250 mg/kg (1/2): marked hyperpnea and lack of activity + marked diarrhea at time of sacrifice
* 625 mg/kg (1/2): lack of activity, slight hyperpnea, exudate from both eyes + moderate diarrhea, slight red-brown exudate around the anus and left eye and a moderate humped back at time of sacrifice.
* 312 mg/kg or less: no toxic symptoms

14d PILOT STUDY
- Toxicity in male rats:
* 5000 mg/kg: (5/5) mortality, no surviving animals by day 7.
* 2500 mg/kg: (4/5) mortality, 1 animal surviving by day 7. Remaining animal showed the following symptoms: an exudate arount he penis, lower abdomen, a midly humped back, mildly to moderately rough fur and an abdominal lump around the site of injection.
* 1250 mg/kg: (1/5) mortality, 4 animals surviving by day 7. Remaining animals showed one or more of the following symptoms: exudate from the eye(s), exudate from the mouth, exudate around the penis, exudate from the anus, lower abdominal region, mild diarrhea, mild hyperpnea, mildly humped back, mildly rough fur. One animal in this dose group exhibited no toxic symptoms.
* 625 mg/kg: (1/5) mortality, 4 animals surviving by day 7. Remaining animals showed one or more of the following symptoms: slight ataxia, decreased activity, sligh rough fur, slight exudate from the eye(s), slight exudate from the mouth, slight exudate from the nose, mild diarrhea. Two animals in this dose group exhibited no toxic symptoms.
* 312 mg/kg: no clinical signs of toxicity
- Toxicity in female rats:
* 5000 mg/kg: (3/5) mortality, 2 animals surviving by day 7. Remaining animals showed one or more of the following symptoms: exudate around the vagina, lower abdomen, mildly humped back, mildly rough fur, abdominal lumps around the site of injection.
* 2500 mg/kg: (3/5) mortality, 2 animals survivyng by day 7. Remaining animals showed one or more of the following symptoms: exudate around the vagina, lower abdomen, mildly humped back, abdominal lumps at site of injection.
* 1250 mg/kg: (3/5) mortality, 2 animals surviving by day 7. Remaining animals showed one or more of the following symptoms: mildlly rough fur, mild exudate around the vagina and lower abdomen, lack of activity, mild to marked hyperpnea, marked serous descharge from one eye, moderate wheezing.
* 625 mg/kg: no mortality. The animals showed one or more of the following symptoms: mild to moderate hyperpnea, mildly humped back, decreased activity, exudate around the eyes, exudate around the vagina and abdomen, mildly rough fur, marked diarrhea, mild exudate from the mouth and nose, paler eyes. One animals showed no toxic symptoms.
* 312 mg/kg or less: one animal had a mild exudate around the eye, another one favored its left hind leg. The other animals showed no symptoms of toxicity.

DEFINITIVE STUDY - CLINICAL OBSERVATIONS
Males:
* 400 mg/kg - 6h dose group: decreased activity (1/5), marked ataxia (1/5)
* 400 mg/kg - 12h dose group: mild to moderate diarrhea (5/5), decreased activity (3/5)
* 200 mg/kg - 6h dose group: slight discharge from the nose (1/5)
Females:
* 600 mg/kg - 6h dose group: decreased activity (5/5), marked ataxia (4/5), slight exudate from the eye (1/5)
* 600 mg/kg - 12h dose group: decreased activity (5/5), moderately humped back (3/5),
* 600 mg/kg - 24h dose group: slight to moderate ataxia (4/5), decreased activity (3/5), slightly humped back (4/5)
The rats treated with the test substance lost weight when compared to their concurrent negative controls.

DEFINITIVE STUY - CYTOLOGICAL OBSERVATIONS
The 3 measures of cytological damage that were statistically evaluated were the MI, the percentage of chromosomally aberrant cells and the frequency of chromosomal aberrations per cell. Prior to analysis of variance, Bartlett's test was conducted on these variables to investigate differences in treatment-group variances. The variances among treatment groups were found to be homogeneous.
At 6, 12 and 24h post treatment, no significant differences in the % of chromosomally aberrant cells or the frequency of chromosomal aberrations per cell were observed between the negative control and DBPP-treatment groups in male or female rats? The only significant difference in MI was between the female negative control and DBPP-treated groups 24h after treatment: a higher MI was noted for females treated with DBPP.
Animals in the concurrent 24h-post treatment positive control groups had significantly higher percentages of aberrant cells and significantly higher frequencies of aberrations per cell than did animals in the negative control groups.

From the results of these studies, it is concluded that DBPP does not induce chromosomal damage in male or female Fischer-344 rats under the conditions used in this study.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
From the results of these studies, it is concluded that DBPP does not induce chromosomal damage in male or female Fischer-344 rats under the conditions used in this study.