Registration Dossier

Administrative data

Description of key information

In rodents, repeated exposure to fluoride salts, such as sodium fluoride, causes alteration of teeth at daily doses of 4 mg NaF/kg bw/day or greater.  Higher repeated doses of 10 and 25 mg NaF/kg bw/day increase dental effects as well as cause toxic effects in bone and the stomach.  The severity of the toxic effects is related to increasing dose and duration of exposure.  In a life-time chronic exposure study in the rats, an extremely high concentration of 175 ppm sodium fluoride in drinking water was associated with an increased incidence of osteosclerosis in female rats and equivocal evidence of osteosarcoma in male rats. 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
repeated dose toxicity: oral
Remarks:
combined repeated dose and carcinogenicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to generally valid procedures and according to GLP guidelines. All parameters described are closely related or comparable to guideline methods.
Qualifier:
equivalent or similar to
Guideline:
other: EPA OPP 83-5 (Combined Chronic Toxicity / Carcinogenicity)
GLP compliance:
yes
Limit test:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Inc., Wilmington, MA
- Age at study initiation: 6 weeks
- Weight at study initiation: males, ~175 g; females ~ 150 g
- Housing: individually housed in stainless steel cages with screen bottoms
- Diet (e.g. ad libitum): either Purina Certified Rodent Chow (laboratory chow) or a specially formulated low-fluoride control diet
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 19 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.2 +/- 1.7
- Humidity (%): 50 +/- 20
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
Treatment groups were fed a low-fluoride diet to which NaF was added at a dose of 4, 10 or 25 mg/kg/day. The amount of NaF added to the low-fluoride diet was adjusted weekly for the first 26 weeks and every 4 weeks thereafter to keep the daily fluoride intake constant. Control rats received either the low-fluoride control diet or laboratory chow.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Each preparation of the low-fluoride diet containing added fluoride was analyzed for fluoride content. Diet consumption records confirmed that target doses were met throughout the study.
Duration of treatment / exposure:
The study was designed to continue treatments for 24 months or until survival for a single group of males or females was 20% or less. When this survival rate was reached, all groups of that sex were killed - at 95 weeks for males and 99 weeks for females.
Frequency of treatment:
continuous
Remarks:
Doses / Concentrations:
0 mg NaF/kg/day in normal laboratory chow; 0 mg NaF/kg/day in low-fluoride diet; 4 mg NaF/kg/day in low-fluoride diet; 10 mg NaF/kg/day in low-fluoride diet; and 25 mg NaF/kg/day in low-fluoride diet
Basis:
actual ingested
No. of animals per sex per dose:
70 animals per sex per dose
Control animals:
other: One control group of male and females was given the normal laboratory chow and a second control group of males and females was given a low-fluoride diet.
Details on study design:
The study was designed to continue for 24 months or until survival for a single group of males or a single group of females was 20% or less. When this survival rate was reached, all groups of that sex were killed (in this study, 95% for males and 99 weeks for females). The sutdy design included killing of rats at 26 and 53 weeks for assessment of fluoride toxicity. At 26 weeks, sufficient rats were killed to retain 60 rats for each sex in each group in the study; at 53 weeks, 10 rats of each sex in each group were killed.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data

DERMAL IRRITATION (if dermal study): No data

BODY WEIGHT: Yes
- Time schedule for examinations: weekly for the first 26 weeks, and every 4 weeks thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Hematology measurements were conducted on rats killed according to the study schedule. Blood was collected from all rats killed at 26 and 53 weeks and from 10 rats of each sex in each treatment group killed at 95 (males) and 99 (females) weeks.

CLINICAL CHEMISTRY: Yes
- Clinical chemistry measurements were conducted on rats killed according to the study schedule. Blood was collected from all rats killed at 26 and 53 weeks and from 10 rats of each sex in each treatment group killed at 95 (males) and 99 (females) weeks.

URINALYSIS: Yes
- Urinalysis was conducted on rats prior to scheduled sacrifce. Unine was collected from all rats killed at 26 and 53 weeks and from 10 rats of each sex in each treatment group killed at 95 (males) and 99 (females) weeks. Uriine was collected overnight after the rats were placed in metabolism cages with access to water, but not diet.

NEUROBEHAVIOURAL EXAMINATION: No data
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- After the rats were killed, terminal body weight was obtained, and adrenals, right femur, brain, heart, kidneys, liver, stomach, ovaries, pituitary, spleen, testes with epididymides, and thyroids with parathyroids were weighed. All gross lesions and a broad sampling of tissues from rats that were killed according to schedule, died, or were killed when they became moribund were preserved in 10% buffered neutral formalin.
- The following bone specimens were collected: femurs, cervical vertebrae, skulls, and mandibles.

HISTOPATHOLOGY: Yes
- The following bones and teeth were examined microscopically: cranium, femur, premaxillae with incisors, maxillae with first molars, and mandible with incisor and first molar.
- Microscopic examination was performed on all tissues from rats on the low-fluoride control diet and from all rats treated with 25 mg NaF/kg/day that were killed at 26 and 53 weeks.
- In addition, microscopic examination was performed on the stomach, bones and teeth from rats treated with 10 mg NaF/kg/day and killed at 26 weeks and from rats treated with 4 and 10 mg NaF/kg/day and killed at 53 weeks.
- For groups of males killed at 95 weeks, and groups of females killed at 99 weeks, all tissues from 10 randomly selected rats of each sex in each treatment group given the low-fluoride diet, control laboratory chow, or a NaF dose of 25 mg/kg/day were examined microscopically. Tissues examined included the stomach, liver, kidneys, cranium femur, incisors, adrenals, brain, heart, lungs, ovaries, uterus, pancreas, pituitary, prostate, seminal vescicles, spleen, urinary bladder, testes, epididymides, thyroids, parathyroids, and spinal cord.
- Because treatment-related bone thickening in the cranium had been seen at necropsy, the cranium from each of 5 rats of each sex from the laboratory chow control group was also examined microscopically.
Other examinations:
Samples of diets and samples of bone (radius and ulna) and urin from animals killed at 95 (males) and 99 (females) weeks were analyzed for fluoride levels. An ion-specific fluoride electrode on a digital voltmeter reading to 0.1 mV was used for all fluoride analyses of diet, bone and urine. Bone fluoride was measured after bones were ashed in a muffle furnace at 550 degrees C overnight.
Statistics:
Analysis of variance was used for statistical evaluation. Provided that the value from Bartlett's test of homogeneity of variance was not significant, treated groups were compared with the low-fluoride control group by the least significant difference criterion. The laboratory chow controls were compared with low-fluoride diet controls by Student't t-test. For survival analysis, the generalized Kuskal-Wallis test was used.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
effects observed, treatment-related
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
-The incidence of fluoride-related dental changes was increased in rats given NaF at 4 mg/kg/day or more. Incisors were thickened, white, and often pitted.
- The last surviving males in each group were killed at 95 weeks, and the last surviving females in each group were killed at 99 weeks, because survival was 20% or less in the males receiving NaF at a dose of 4 mg/kg/day and in females in the low-fluoride control group at these times.

BODY WEIGHT AND WEIGHT GAIN
-The low-fluoride control rats and the rats given 4 or 10 mg NaF/kg/day gained more weight than the control rats fed the laboratory chow and those fed 25 mg NaF/kg/day.
- Reduced weight gain associated with the dose of 25 mg/kg/day was manifested early in the study and a comparison with the low-fluoride controls showed a decrement in weight gain of approximately 30% at this dose of NaF. This decrement was largely attributable to reduced diet consumption, but for the males, this fluoride dose was also associated with reduced efficiency of conversion of ingested diet to body weight (feed efficiency) during the first year.
- Controls fed laboratory chow gained significantly less weight than controls fed the low-fluoride diet while consuming significantly more diet.
- Although rats ingesting 25 mg NaF/kg/day had an approximately 30% decrement in weight gain and an early decrement in feed efficiency, tis dose did not adversely affect survival in either sex.
-At 4 and 10 mg NaF/kg/day, females but not males had longer survival than female or male low-fluoride controls, respectively.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
- At 25 mg NaF/kg/day, reduced diet consumption was observed.

FOOD EFFICIENCY
- In males dosed at 25 mg NaF/kg/day, reduced efficiency of conversion of ingested diet to body weight (feed efficiency) was measured during the first year.

CLINICAL CHEMISTRY
-There were several statistically significant differences in clinical pathology parameters between control rats fed the low-fluoride diet and rats given 4,10 or 25 mg NaF/kg/day. At 25 mg/kg/day, the blood glucose and specific gravity of urine were moderately lower in both sexes, and total protein and globulin were mildly to moderately lower in males.
- Other differences between control rats fed low-fluoride diet and those given NaF were small and were not consistent over time.

URINALYSIS
- Levels of fluoride in the urine were increased as the level of NaF in the diet increased. urinary fluoride concentration was a linear function of the ingested dose, regardless of whether the fluoride was endogenous to the diet or added as NaF.

ORGAN WEIGHTS
- Except for stomach and femur, statistically significant diferences in absolute and relative organ weights were inconsistent across the various times when rats were killed and/or were secondary to changes in body weight.

GROSS PATHOLOGY
- Macroscopically, clear evidence of fluoride toxicity was seen in the teeth, bones and stomach. The incidence of these changes were related to dose and duration of the test.

HISTOPATHOLOGY: NON-NEOPLASTIC
- Microscopically, clear evidence of fluoride toxicity was seen in the teeth, bones and stomach.
- Nonneoplastic ameloblastic displasia, fractured or malformed incisors, and enamel hypoplasia were observed. Ameloblastic dysplasia was characterized by flattening of the ameloblastic layer, herniation of ameloblasts into the enamel, and inclusions of enamel in the ameloblasic layer.
- Enamel hypoplasia was observed in rats killed at 26, 53, 95 or 99 weeks. Upper incisors were affected more than lower incisors, and the incidence and severity of these changes increased as the dose of NaF and the time in the study increased.
- The only treatment-related effect associated with the molars was the occurrence of basophilic granules, which were also associated with incisors.
- The incidence of fluoride-related bone changes were obvious in the skull of rats receiving NaF at doses of 10 mg/kg/day or more. The incidence and severity of these changes increased as the dose of NaF and the time in the study increased.
- Mild hyperkeratosis and acanthosis were observed in the nonglandular portion of the stomach of rats given NaF at doses of 10 mg/kg/day or more and that were killed at 26, 95 or 99 weeks.
- Microscopically, chronic inflammation of the gastric mucosa was increased in indicence and severity in rats dosed at 10 mg NaF/kg/day or more when compared to controls.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
- No neoplasms associated with NaF treatment were observed.

Dose descriptor:
LOAEL
Remarks:
toxicity
Effect level:
ca. 4 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOAEL
Remarks:
carcinogenicity
Effect level:
ca. 25 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: No neoplastic changes associated with NaF were observed.
Critical effects observed:
not specified
Conclusions:
Fluoride toxicity included a 30% decrement in weight gain occurring at 25 mg NaF/kg/day,and macroscopic and microscopic changes in teeth, bone and stomach of the rats of both sexes. The incidence and severity of these changes were related to increased dose of NaF and increased duration of exposure. Despite clear evidence of toxicity in male and female rats receiving sodium fluoride in the diet for 95 and 99 days, respectively, sodium fluoride did not alter the incidence of preneoplastic and neoplastic lesions at any site in rats of either sex.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
9.5 mg/kg bw/day
Study duration:
chronic
Species:
rat

Additional information

Subacute Toxicity:

A 28-day repeated oral toxicity study (Proctor & Gamble, Co., 1991) was conducted to characterize the systemic toxicity of sodium fluoride. Weanling male and female Sprague-Dawley rats were dosed by gavage at concentrations of 2.5, 25 or 250 ppm sodium fluoride. The NOEL was 25 ppm. The following effects were observed at 250 ppm; hematology - significant depression in mean cell volume of males and females and mean cell hemoglobin of males; clinical chemistry - significant decrease in total protein of males and females and significant increase in alanine aminotransferase, potassium and chloride of females only; several changes in the mineral analysis of teeth and bone; and increased absolute and relative stomach weights.

Subchronic Toxicity:

The U.S. National Toxicology Program (NTP, 1990) evaluated the toxicological effects of continuous exposure to 0, 30, 100 or 300 ppm sodium fluoride in drinking water on F344 male and female rats for a 6-month period. Sodium fluoride caused weight loss at 300 ppm, fluorosis of the teeth at 100 and 300 ppm, minimal hyperplasia of the gastric mucosa of the stomach at 100 and 300 ppm (however, one high dose rat of each sex had an ulcer), a dose-related increase in fluoride content of bone and urine with increasing fluoride concentration in the drinking water, and a significant increase in fluoride content in the plasma at 300 ppm. No significant signs of toxicity were observed at concentrations of 10 or 30 ppm.

The U.S. National Toxicology Program (NTP, 1990) evaluated the toxicological effects of continuous exposure to 0, 10, 50, 100, 200, 300 or 600 ppm sodium fluoride in drinking water of male and female B6C3F1 mice for a 6-month period. Sodium fluoride caused death in some animals at 600 ppm and in a single male animal at 300 ppm, weight loss at 200 to 600 ppm, fluorosis of the teeth at 100, 200, 300 and 600 ppm, acute nephrosis and/or lesions in the liver and myocardium in mice that died early, minimal alterations in bone growth/remodeling in the long bones at 50 to 600 ppm, a dose-related increase in fluoride content of bone and urine with increasing fluoride concentration in the drinking water, and a possible dose-related increase in fluoride content in the plasma. No signs of toxicity were observed at the low dose of 10 ppm sodium fluoride.

Chronic Toxicity

The U.S. National Toxicology Program (NTP, 1990) evaluated the toxicological and carcinogenic effects of continuous exposure to 0, 25, 100 or 175 ppm sodium fluoride in drinking water on male and female F344/N rats for a 2 -year period. Survival and weight gains of the male and female rats were not affected by fluoride treatment. Rats receiving sodium fluoride developed effects typical of dental fluorosis at 25, 100 and 175 ppm, and female rats had increased osteosclerosis at the high-dose of 175 ppm.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Key study. In the registration dossier for KF and NaF, a LOAEL of 10 mg/kg/by, based on NaF was selected from a chronic study as the relevant effect level. This is re-calculated to 9.5 mg/kg/day based on K2TiF6, based on the fluoride content of both substances.

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: stomach; other: bone

Justification for classification or non-classification

Based on the results described above, classification for repeated dose toxicity is not warrented.