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Toxicological information

Genetic toxicity: in vivo

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in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Results of this study are scentifically acceptable, the testing procedures and resulting data are sufficient and well documented.

Data source

Reference Type:

Materials and methods

Test guideline
equivalent or similar to guideline
EPA OPPTS 870.5385 (In Vivo Mammalian Cytogenetics Tests: Bone Marrow Chromosomal Analysis)
Version / remarks:
and OPPTS 870.5395 (In Vivo Mammalian Cytogenetic Tests: Erythrocyte Micronucleus Assay)
GLP compliance:
not specified
Type of assay:
chromosome aberration assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium fluoride
EC Number:
EC Name:
Sodium fluoride
Cas Number:
Molecular formula:
sodium fluoride
Details on test material:
- Name of test material (as cited in study report): sodium fluoride from Aldrich Chemical Co., Milwaukee, WI

Test animals

Details on test animals or test system and environmental conditions:
- Source: No data
- Age at study initiation: 10 Weeks
- Diet (e.g. ad libitum): Low F ion diet (NIH-07) ad libitum
- Water (e.g. ad libitum): Deionized drinking water ad libitum

Administration / exposure

Route of administration:
oral: drinking water
- Vehicle(s)/solvent(s) used: Drinking water
- Justification for choice of solvent/vehicle: Most likely route of exposure to sodium fluoride
- Concentration of test material in vehicle: 0, 5, 10, 50, 100, 200 or 400 ppm fluoride (as sodium fluoride)
- Amount of vehicle (if gavage or dermal):
Duration of treatment / exposure:
6 weeks
Frequency of treatment:
continuous for 7 days/week for 6 weeks
Doses / concentrations
Doses / Concentrations:
0, 5, 10, 50, 100, 200 or 400 ppm fluoride (as sodium fluoride)
nominal in water
No. of animals per sex per dose:
Ten to 16 male mice were used in each dose group to ensure that at least 8 animals would be available at the end of the exposure period.
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (CP)
- Justification for choice of positive control(s): Substance known to cause chromosome aberrations in bone marrow and micronucleated peripheral blood erythrocytes.
- Route of administration: In drinking water
- Doses / concentrations: 114 ug/mL, prepared fresh daily


Tissues and cell types examined:
Bone marrow and peripheral blood
Details of tissue and slide preparation:
- Slides for scoring chromosome aberrations (ABS) were prepared from 8 mice/dose group, selected at random from among the surviving treated animals. Coded slides were prepared from bone marrow using 2 femurs/mouse. As many slides as possible were prepared from each mouse for chromasome analyses. This assured that a sufficient number of slides would be available for distribution to the scoring laboratories. It alsso permitted a greater number of anaphase preparations to be scored. The sodium fluoride-treated, water control and positive control slides were coded. All slides were prepared and stained using buffered Giemsa. Mitotic indices were determined in the bone marrow cells prior to scoring for ABS by examinations of 500 cells/animal. The animals treated, and slides prepared, at Environmental Health Research and Testing Inc.
- Blood smears were prepared from peripheral blood (by tail-snip) for micronuclei (MN) scoring after 1 week of sodium fluoride treatment, and when the mice were killed after 6 weeks; 4 slides were made/mouse. Five mice were randomly selected from the 0, 100, 200 and 400 ppm and positive conrol CP group for this procedure. The same mice were used for the week 1 and week 6 samples. The smears were stained with acridine orange and scored using a fluorescent microscope at 1000X magnification. In this procedure, MN appear as bright yellow stained, round or ring-shaped figures having diameters ranging from 1/20 to 1/5 of that of an erythrocyte.

- At 6-weeks, bone marrow preparations from mice treated with 0, 100, 200 and 400 ppm fluoride and the positvie control CP group were scored for the presence of ABS. An attempt was made to score 50 metaphase cells/mouse. Because colchicine was not administered, and because each laboratory had its own criteria for identifying acceptable metaphase and anaphase preparations to be scored, it was not always possible to score 50 cells/animal. If positve responses would have been seen in the 100-400 ppm sodium fluoride range, the laboratories would have been given the coded lower-dose slides to score. Not attempts were made to standardize cell acceptability or scoring criteria among laboratories.
- For metaphase analyses, at Environmental Health Research and Testing Inc., 50 metaphase spreads (25 cells by each of two independent scorers) were evaluated/animal for ABS. At U. Missouri, Kansas City, an attempt was made to score up to 25 metaphase spreads/slide, from 4 slides/mouse, for ABS. All scoring was performed by a single individual.
- For anaphase analyses, at Environmental Health Research and Tesitng Inc., attempts were made to evaluate up to a maximum of 50 anaphase cells/mouse (25 cells/independent scorer). The incidences of early anaphase cells with chromosome stickiness, fragments, bridges, anaphase cells in multipolar phase and last anaphase cells were recorded. At the U. Missouri, Kansas City, a single scorer evaluated as many early anaphase cells as could be found in 4 slides/mouse. Chromosome fragments and bridges were recorded, as well as cells containing micronuclei. A National Institute of Environmental Health Sciences (NIEHS), early and late anaphase cells, and cells with decomposed chromosomes, were scored for the presence of bridges and fragments. For most mice, 100 cells were scored. Additional cells were scored on different sections of the slides from some randomly-selected animals.
- For MN analyses, the increases of micronucleated polycrhomatic (PCE) and normochromatic erythrocytes (NCE) at each time-point were determined by two independent scorers who each evaluated 1000 PCEs and 1000 NCEs.

OTHER: Fluoride Analyses
One humerus was removed from each mouse at the time of killing, labeled and frozen in individual tubes for fluoride analyses. After thawing, all soft tissue was removed from the bones by boiling in deionized water for 30 minutes, followed by scraping to remove any remaining tissue. The bones weere ether-extracted (2Xdaily for 3 days) to remove lipids. They were then dried overnight at 100 degrees C and ashed overnight at 600 degrees C. Before analysis, a portion of midshaft bone (4-5 mm in length) was crushed and 3 samples, weighing between 0.1 and 0.3 mg each, were taken from each bone and placed in diffusion cells for fluoride analysis. Fluoride analyses were done by the established acid diffusion, ion-selective electrode method.
Peripheral blood micronucleus (MN) data were analyzed using the Micronucleus Assay Data Management and Statistical software package (version 1.4), which was designed specifically for in vivo MN data (Integrated Laboratory Systems, Research Triangle Park, NC, USA). To determine whether a specific treatment resulted in a significant increase in MN, the numbers of MN were pooled within each dose group and analyzed by a one-tailed trend test. In the software package used, the trend test incorporates a variance inflation factor to account for excess animal variability. Pairwise comparisons consisted of a Student's t-test for each dose against the solvent control. Because no ABS were observed in the untreated control slides of the metaphase study performed at the U. of Missouri, Kansas City, the 95% binomial confidence limits were calculated for all the mean metaphase aberration values. The same procedure was used for the analysis of aberrant anaphase cells.

Results and discussion

Test results
No increases in peripheral blood micronuclei were seen following 1 or 6 weeks fluoride treatment up to 400 ppm. No increases in ABS were seen in bone marrow cells when metaphase and anaphase cells were examined after 6 weeks of fluoride treatment.
Three of 16 mice receiving 400 ppm fluoride died during week 6 of the treatment period. A decrease in body weight was noted in mice treated with 200 (during weeks 5 and 6 of treatment) and 400 ppm fluoride (from week 2 to week 6).
Vehicle controls validity:
Negative controls validity:
not examined
Positive controls validity:

Applicant's summary and conclusion

Interpretation of results (migrated information): negative
Sodium fluoride, at concentrations up to 400 ppm fluoride in the drinking water of male B6C3F1 mice, did not cause either increases in micronuclei in peripheral erythrocytes at either 1 or 6 weeks following treatment or increases in chromosome aberrations in bone marrow cells when metaphase and anaphase cells were examined following 6 weeks of treatment.