Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Initiation Date: January 24, 1990 Experimental Start Date: Febru3ry 12, 1990 Experimental Termination Date: March 19, 1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report Date:
1990

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): NBPT
- Substance type: Organic
- Physical state: Fluffy white powder
- Date received: January 22, 1990
- Genetics Assay No.: 12025
- Storage condition of test material : Not reported

Test animals

Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague-Dawley. Inc., Frederick, MD.
- Age at study initiation: 8 weeks
- Weight at study initiation:
26.8 - 37.0 grams (males) and 20.4 - 30.2 grams (females).
- Assigned to test groups randomly: yes
- Fasting period before study: Not reported.
- Housing: Animals were group-housed by sex up to five per cage
- Diet: A commercial diet (Purina Certified Laboratory Cho* #15002)
- Water: water were available ad libitum for the duration of the study
- Acclimation period: seven days.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72± 6°F
- Humidity (%): Set to acheive limits of 50 ± 20%
- Air changes (per hr): Not stated
- Photoperiod (hrs dark / hrs light): Lighting was controlled by a time switch to give twelve hours light and twelve hours darkness.


Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used:
Based upon the solubility data from the dose range finding assay, the vehicle used to suspend the test article for the bone marrow micronucleus assay was corn oil.
Details on exposure:
The test article was suspended in corn oil and dosed by intraperitoneal injection at 100, 333, and 1000 mg/kg based upon the results of a previously conducted dose range finding assay (HLA Study No.: 12025-0459).
Duration of treatment / exposure:
Dosed once.
Frequency of treatment:
Dosed once.
Post exposure period:
72 hours.
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 333 and 1000 mg/kg body weight
Basis:
nominal conc.
No. of animals per sex per dose:
15 mice per sex per dose .
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): Known to produce micronuclei under the conditions of the test.
- Route of administration: Dosed orally.
- Doses / concentrations: 80 mg/kg

Examinations

Tissues and cell types examined:
Bone marrow polychromatic Erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
The dose levels used in this assay were based upon the results of the previously conducted dose range finding assay (HLA Study No.: 12025-0459). The doses selected for this assay were 100, 333 and 1000 mg/kg body weight and were administered by intraperitoneal injection.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
At the appropriate harvest time (24, 48 and 72 hours following dosing) the animals were euthanatized with CO2 and the adhering soft tissue and epiphyses of both tibia were removed. The marrow was flushed into a centrifuge tube (one tube for each animal) with 3 ml fetal calf serum. Following centrifugation to pellet the tissue most of the supernatant was drawn off, the cells were resuspended and the suspension spread on slides and air-dried.

DETAILS OF SLIDE PREPARATION:
The slides were then fixed in methanol, stained in May-Gruenwald solution followed by Giemsa. and rinsed in deionized water (Schmid. 1975). After being air-dried. the slides were coverslipped using Depex· mounting medium.

METHOD OF ANALYSIS:
The coded slides were then scored for micronuclei and the polychromatic (PCE) to normochromatic (NCE) cell ratio. Standard forms were used to record these data. Where possible one thousand PCEs per animal were scored. The frequency of micronucleated cells as expressed as percent micronucleated cells based n the total PCEs present in the scored optic field. The normal frequency of micronuclei in this mouse strain is about 0.0-0.4%.

The frequency of PCEs versus NCEs was determined by scoring the number of NCEs observed in the optic fields while scoring the 1000 PCEs for micronuclei. Inseveral instar.ces if 5000 NCEs had been analyzed for the PCE:NCE ratio prior to completing the analysis of the 1000 PCEs for micronuclei. colJe~Lion of data for the PCE:NCE ratio was stopped at 5000 NCEs.

Evaluation criteria:
The criteria for determining a positive response involved a statistically significant dose-related increase in micronucleated PCEs, or the detection of a reproducible and statistically significant positive response for at least one dose level. A test article that induced neither a statistically significant dose response nor a statistically significant and reproducible increase at one dose level was considered negative. In either case, the final decision was based on scientific judgment.
Statistics:
The analysis of the data was performed using an Analysis of Variance (p≤0.05) on the square root arcsine transformation (Sokal and Rohlf, 1981) which was performed on the proportion of cells with micronuclei per animal (square root arcsine proportion). Once the Analysis of Variance had been performed, Tukey's Studentized range test (HSD) with adjustment for multiple comparisons (p≤0.05) was used at each harvest time to determine which dose groups, if any, were significantly different from the vehicle control. Analyses were performed separately for each harvest time and sex combination, and also at each harvest time for the sexes combined.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF DEFINITIVE STUDY
All animals in the 100 mg/kg, vehicle, and positive control groups appeared normal after dosing and remained healthy until the appropriate harvest times. All animals in the 333 and 1000 mg/kg dose groups became prostrate within five minutes of dosing. Within 30 minutes of dosing the animals in the 333 mg/kg dose group had recovered slightly and appeared languid with squinted eyes. Approximately two hours after dosing the high dose (1000 mg/kg) animals remained prostrate with dyspnea and squinted eyes; however, all animals in the 333 mg/kg group appeared normal and they remained healthy until the appropriate harvest times. The following morning, approximately 18 hours after dosing, five high dose animals (male "s 5893, 5945, 6011 and 6035; female 16019) were found dead. Four additional high dose animals (male 16045; female I's 5923, 5978 and 6017) expired within 22 hours of dosing. The remaining high dose animals were either prostrate with dyspnea or languid. All high dose animals had rough haircoats and squinted eyes. These conditions remained unchanged until the appropriate harvest times with two additional high dose deaths (male #5951; female 15939) observed approximately 44 hours after dosing.


Evaluation of Bone Marrow Slides:

The test article, NBPT, induced no significant increases in micronucleated polychromatic erythrocytes over the levels observed in the vehicle controls in either sex or at any of the harvest times. Significant reductions in the PCE:NCE ratio were detected in the male high dose groups at both the 48 and 72 hour harvest times. The positive control, CP, induced significant increases in micronucleated PCEs in both sexes, with means and standard errors of 0.92% ± 0.36% and 1.32% ± 0.48% for the males and females, respectively. The test material was found not to produce a significant increase in the frequency of micronuclei in polychromatic erythrocytes of mice under the conditions of the test.

The test material, NBPT, did not induce a significant increase in micronuclei in bone marrow polychromatic erythrocytes under the conditions of this assay and is considered negative in the mouse bone marrow micronucleus test.








Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The test material, NBPT, did not induce a significant increase in micronuclei in bone marrow polychromatic erythrocytes under the conditions of this assay and is considered negative in the mouse bone marrow micronucleus test.
Executive summary:

Introduction: The objective of this in vivo assay was to evaluate the ability of the test article, NBPT, to induce micronuclei in bone marrow polychromatic erythrocytes of ICR mice. This study was conducted using modifications of the procedures suggested by Heddle et al. (1983).

Method: The test article was suspended in corn oil and dosed by intraperitoneal injection at 100, 333, and 1000 mg/kg based upon the results of a previously conducted dose range finding assay (HLA Study No.: 12025-0459). Ten animals (five males and five females) were randomly assigned to each dose/harvest time group. Vehicle and positive control groups euthanatized 24 hours after dosing were included in the assay. The animals were dosed with the test article and were euthanatized 24, 48 and 72 hours after dosing for extraction of the bone marrow.

Conclusion: The test material, NBPT, did not induce a significant increase in micronuclei in bone marrow polychromatic erythrocytes under the conditions of this assay and is considered negative in the mouse bone marrow micronucleus test.