Disodium fluorophosphate

Repeated dose toxicity: oral

Administrative data

Endpoint:

repeated dose toxicity: oral

Remarks:

combined repeated dose and carcinogenicity

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Study was performed in accordance with acceptable procedures and under the conditions of GLP. This study is considered to be comparable to a guideline study. The reliability has been restricted to reliability 2 on the basis that the study was conducted on an analogous substance (NaF). NaF is considered to be analogous to disodium fluoride in terms of toxicological profile for the following reasons; - A combined 28-day reprotoxicity screening toxicity study (OECD 422) performed on disodium fluorophosphate confirmed the leading health effects to be consistent with the effects reported for exposure to inorganic fluorides (fluorosis). In particular severe effects were noted in the mucosal lining of the stomach and are consistent with exposure to fluoride. - Results of studies on NaF are considered to present a worst case for exposure as NaF contains approximate 47% F compared to 13% F in disodium fluorophosphate. - The phosphate content of sodium fluorophosphate is not considered to present a hazard in terms of systemic toxicity. Phosphate is an essential element; a maximum tolerable daily intake value (MTDI) for phosphates is reported to be 70 mg/kg bw/day. This value greatly exceeds the limit value placed on an inorganic fluoride (OEL = 2.5 mg/kg bw/day F) and as such is not considered to be relevant for risk assessment.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1990

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

other: EPP OPP 83-5

Deviations:

not applicable

GLP compliance:

yes

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Sodium fluoride

EC Number:

231-667-8

EC Name:

Sodium fluoride

Cas Number:

7681-49-4

Molecular formula:

FNa

IUPAC Name:

sodium fluoride

Details on test material:

- Name of test material (as cited in study report): sodium fluoride obtained from J.T. Baker Chemical Co., Phillipsburg, NJ
- Physical state: solid
- Analytical purity: >99%

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system 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

Administration / exposure

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 analysed 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.

Doses / concentrations

Remarks:

Doses / Concentrations:
0 (control), 4, 10, 25 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

70 animals per sex per dose.

Control animals:

other: yes, two control groups were observed: one group containing male and female rats was given the normal laboratory chow and the second group of male and female rats were 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 study 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.

Positive control:

Not applicable.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data

DERMAL IRRITATION (if dermal study): Not applicable.

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): Not applicable.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Haematology 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 sacrifice. Urine 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. Urine 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 urine from animals killed at 95 (males) and 99 (females) weeks were analysed for fluoride levels. An ion-specific fluoride electrode on a digital voltmeter reading to 0.1 mV was used for all fluoride analysis of diet, bone and urine. Bone fluoride was measured after bones were ashed in a muffle furnace at 550°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.

Results and discussion

Results of examinations

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 examined

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, this 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 differences 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.
- The absolute and relative stomach weights of rats given NaF doses of 10 mg/kg bw/day or more were increased. These increases were associated with inflammatory reactions in stomach.

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.
- Non-neoplastic ameloblastic displasia, fractured or malformed incisors, and enamel hypoplasia were observed. Ameloblastic dysplasia was characterised 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 incidence 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.

Effect levels

1

Dose descriptor:

LOAEL

Remarks:

toxicity

Effect level:

4 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

2

Dose descriptor:

NOAEL

Remarks:

carcinogenicity

Effect level:

25 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: No neoplastic changes associated with NaF were observed.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The doses of NaF ingested induced the expected gastric toxic effects and bone and dental fluorosis. Despute this evidence of chronic fluoride toxicity, there was no evidence of of preneoplastic and neoplastic lesions at any site in rats of either sex.