Calcium fluoride

Endpoint summary

Description of key information

The results of studies of repeated dose oral toxicity indicate that calcium fluoride exhibits typical fluoride toxicity, however the low water solubility of the substance indicates that the oral bioavailability of fluoride from the substance is less than other salts such as sodium fluoride, thereby limiting its toxicity.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: NTP study

Qualifier:

according to guideline

Guideline:

other: NTP protocol

Principles of method if other than guideline:

6-month repeated dose study.

GLP compliance:

not specified

Limit test:

no

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

Progeny of C57BL/N6 female and C3H/HeN male mice, obtained from Charles River Laboratories, Wilmington, DE, were used in this study. Mice were 4 to 6 weeks old when placed on study. Their diet consisted of a semisynthetic low fluoride diet. The mice were kept in groups of ten in cages set at a temperture of 22-24 degC and of relative humidity 40-60%. The fluorescent light was 12 hours per day.

Route of administration:

oral: drinking water

Vehicle:

water

Details on oral exposure:

Groups of 8-12 mice of each sex received 0, 10, 30, 100, 200, 300 or 600 ppm sodium fluoride in deionised water ad libitum for 6 months.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

The concentrations are nominal concentrations.

Duration of treatment / exposure:

0, 10, 50, 100, 200, 300 or 600 ppm sodium fluoride in deionised water

Frequency of treatment:

Continuous (ad libitum, in drinking water)

Dose / conc.:

0 ppm

Remarks:

Concentration of NaF in drinking water.

Dose / conc.:

10 ppm

Remarks:

Concentration of NaF in drinking water.

Dose / conc.:

50 ppm

Remarks:

Concentration of NaF in drinking water.

Dose / conc.:

100 ppm

Remarks:

Concentration of NaF in drinking water.

Dose / conc.:

200 ppm

Remarks:

Concentration of NaF in drinking water.

Dose / conc.:

300 ppm

Remarks:

Concentration of NaF in drinking water.

Dose / conc.:

600 ppm

Remarks:

Concentration of NaF in drinking water.

No. of animals per sex per dose:

The method stated ten mice of each sex per dose group. However, some of the test animals were incorrectly sexed, resullting in 8-12 mice per sex per dose group.

Control animals:

other: One control group received de-ionised water and a low fluoride diet. One control group received sodium chloride and a low fluoride diet. An additional control group received standard diet.

Details on study design:

Groups of 8-12 mice of each sex received 0, 10, 30, 100, 200, 300 or 600 ppm sodium fluoride in deionised water ad libitum for 6 months. All test animals receiving water supplemented with sodium fluroide were provided with a low fluoride (<2.1 ppm) semisynthetic diet throughout the study. Three control groups were included in the studies of male and female mice; one received deionised drinking water and a low fluoride, semisynthetic diet, the second received sodium chloride supplemented deionized drinking water and a low fluoride, semisynthetic diet and the third received deionized water and a standard NIH-07 diet. At termination of the studies, the fluoride concentrations in urine, blood and bone were determined from samples collected from all surviving mice. Necropsy was performed on all animals, with histopathology at 300 and 600 ppm.

Positive control:

Not relevant

Observations and examinations performed and frequency:

Animals were observed twice daily for mortality and morbidity, weighed initially, once weekly and at termination. Clinical observations recorded daily. Food consumption recorded every other week for the first 13 weeks and for 1 week during each of the last 3 months. Water consumption was recorded daily.

Sacrifice and pathology:

Necropsy was performed on all animals, with histopathological investigation of animals at 300 and 600 ppm.

Other examinations:

Fluoride concentrations in bone, blood and urine measured prior to necropsy

Statistics:

Not reported.

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:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

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

Deaths occurred at 600 ppm (4 males, 9 females) and at 300 ppm (1 male). Signs of toxiicity (weakness, thin appearance, hunched posture) were seen at 600 ppm. Mice at 100, 200m 300 and 600 ppm had chalky white teeth; the lower incisors were more affected and were also chipped at higher dose levels.

BODY WEIGHT AND WEIGHT GAIN

Reduced weight gain was seen at 200, 300 and 600 ppm; food consumption was reduced in males at 600 ppm. Water consumption was unaffected by treatment.

GROSS PATHOLOGY

None

HISTOPATHOLOGY

Treatment-related findings were noted in the kidney, liver, testes and myocardium of decedents. Acute nephrosis was characterised by extensive multifocal degeneration and tubular necrosis and was diagnosed as the cause of death in these animals. Multifocal myocardial degeneration was also seen in two 600 ppm females. Liver changes consisted of scattered heptocellular hypertrophy and megalocytosis. The effects on the testes (degeneration/necrosis of the seminiferous tunules) were not considered to be directly related to treatment, but occur frequently in moribund mice. Effects were also noted on teh femur and (to a lesser extent) the tibia of mice at 50 ppm and greater. Changes are considered to be indicative of altered rates of bone deposition and remodelling. Effects on the teeth were seen at 300 and 600 ppm.


OTHER FINDINGS

The fluoride content of plasma, bone and urine increased with dose level.

Dose descriptor:

NOEL

Effect level:

< 1.93 mg/kg bw/day (actual dose received)

Based on:

element

Remarks:

fluoride

Sex:

male

Basis for effect level:

other: Effects on bone

Remarks on result:

other: Corresponding NaF concentration in the water: 50 ppm

Dose descriptor:

NOEL

Effect level:

< 4.3 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks:

NaF

Sex:

male

Basis for effect level:

other: Effects on bone

Dose descriptor:

LOEL

Effect level:

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

Based on:

element

Remarks:

fluoride

Sex:

male

Basis for effect level:

other: Effects on bone

Remarks on result:

other: Corresponding NaF concentration in the water: 50 ppm

Dose descriptor:

LOEL

Effect level:

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

Based on:

test mat.

Remarks:

NaF

Sex:

male

Basis for effect level:

other: Effects on bone

Dose descriptor:

NOEL

Effect level:

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

Based on:

element

Sex:

female

Basis for effect level:

other: Effects on bone

Remarks on result:

other: Corresponding NaF concentration in the water: 50 ppm

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Remarks:

NaF

Sex:

female

Basis for effect level:

other: Effects on bone

Critical effects observed:

not specified

Dose (ppm)	Survival	Mean Body Weight			Final Weight relative to control (%)
		Initial	Final	Change
Male
Control^a	9/9	16.9±0.4	40.2±1.0	23.3±1.1	100
Control^b	10/10	18.6±0.4*	41.6±0.6	23.0±0.7	103
Control^c	11/11	17.8±0.4	39.2±1.0	21.4±1.0	97
10	9/9	17.3±0.5	43.1±1.5	25.8±1.8	107
50	10/10	18.0±0.6	41.1±1.1	23.1±1.3	102
100	10/10	19.2±0.8	41.5±1.1	22.3±1.3	103
200	10/10	17.9±0.7	36.5±1.2	18.6±1.4*	91
300	7/8	18.8±0.7	38.1±1.1	19.0±1.4*	95
600	5/9	17.4±0.4	32.0±1.6**	14.8±1.9**	80
Female
Control^a	11/11	16.9±0.6	30.2±1.4	13.3±1.6	100
Control^b	10/10	18.6±0.4	31.5±1.0	12.9±1.1	104
Control^c	9/9	16.6±0.2	28.7±0.9	12.1±0.8	95
10	11/11	17.1±0.4	29.6±1.1	12.5±1.1	98
50	10/10	16.4±0.3	32.2±1.1	15.8±1.2	107
100	10/10	17.2±0.4	30.6±1.5	13.4±1.4	101
200	10/10	17.2±0.4	25.3±0.6**	8.1±0.7*	84
300	12/12	16.9±0.3	26.2±0.8*	9.3±0.7*	87
600	2/11	16.6±0.4	24.5±1.5	9.0±1.0	81

*Significantly different (P≤0.05) from the control group by Dunn’s or Shirley’s test

**P<0.01

^a         Control group receiving semisynthetic, low fluoride diet and deionised water.

^b      Control group receiving semisynthetic, low fluoride diet and sodium chloride supplemented deionised water

^c      Control group receiving standard NIH-07 diet and deionised water.

Organs and Diagnoses	300 ppm	600 ppm
Male Animals initially in study Early deaths   Kidney Nephrosis, multifocal   Liver Megalocytosis, multifocal Syncytial alteration, multifocal   Myocardium Mineralization, multifocal   Testis Necrosis Tubule, degeneration, multifocal Tubule, multinucleated giant cells, multifocal   Female   Animals initially in study Early deaths   Kidney Nephrosis, multifocal   Liver Megalocytosis, multifocal Syncytial alteration, multifocal   Myocardium Degeneration, multifocal Mineralization, multifocal	8 1     1     1 1     1     1   1       12 0     0     0 0     0 0	9 4     2     4 4     4     3 2 1       11 9     2     7 7     2 4

Conclusions:

Skeletal effects of fluoride were seen at all dose levels in this study.

Executive summary:

In the 6 month studies in mice, 4/9 males and 9/11 females receiving 600 ppm sodium fluoride and 1/8 male given water containing 600 ppm died.

The fluoride content of urine and bone increased with the concentration of sodium fluoride in the drinking water in both sexes of mice. Bone fluoride concentration were as high as 14.8 µg/mg of ashed bone in male mice receiving 600 ppm sodium fluoride in water. The bone fluoride content found in mice was somewhat greater than that found in rats given comparable sodium fluoride content. This maybe due to a greater water intake on a body weight basis by mice than by rats resulting in higher exposures. Plasma fluoride concentrations in mice showed a good dose relationship and appeared increased in groups receiving water concentrations of 50 ppm of sodium fluoride or higher.

Histopathologic findings for mice are consistent with previously recognised toxic effects. The acute nephrosis observed in the kidneys was probably the most likely cause of death. Lesions were also observed on the incisor teeth, femur and tibia of mice.

Based on these observations, the LOEL in male mice is 50 ppm NaF and the NOEL in female mice is 50 ppm NaF. Taking into account the typical water consumption, this corresponds to a LOEL of 1.93 mg F- /kg bw/d or 4.3 mg NaF/kg bw/d for male mice and a NOEL of 2.89 mg F-/kg bw/d or 6.4 mg NaF/kg bw/d for female mice.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

1.2 mg/kg bw/day

Study duration:

subchronic

Species:

mouse

Quality of whole database:

Reliable read across data based on sodium fluoride investigations.

System:

musculoskeletal system

Organ:

bone

tooth

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Additional information

Oral studies

In a study using 12 paired groups of albino rats, Lawrenz et al (1939) administered fluoride in the form of calcium fluoride or cryolite in the drinking water for 14 weeks. Total fluoride intake (from the basal diet and drinking water) amounted to a dose level of approximately 0.75 mg/kg bw/d, which corresponds to 1.54 mg CaF₂/kg bw/d. Bodyweight gain and food consumption were retarded in both dose groups. Clinical signs were limited to transient haematuria in both groups, and the appearance of dental striations in all animals from Week 8 -10. Analytical determinations at necropsy revealed fluoride retention of approximately 59%, and that the large majority of the fluoride in the body was present in the skeleton (~96%). No differences were apparent between the treated groups. The authors conclude that the effects of fluoride from these two insoluble salts are comparable.

Smith & Leverton (1934) investigated the comparative toxicity of various forms of inorganic fluoride in a 6-week feeding study in male rats. They noted that overt effects of calcium difluoride toxicity (bodyweight reduction, mortality) were seen only at very high concentrations compared to other, water-soluble fluorides. Mortality was observed at fluoride concentrations well above 1000 mg/kg bw/d. In contrast, mild dental effects were seen at comparable fluoride levels for all of the compounds investigated, however the more severe effects were only seen at much comparatively much higher concentrations of calcium difluoride. The results of the study therefore indicate that the toxicity of calcium difluoride is markedly influenced by its low water solubility.

Sodium fluoride studies

The much greater water solubility of sodium fluoride (41300 mg/L) compared to calcium fluoride (15 mg/L) means that the bioavailability of fluoride from NaF is likely to be much greater than that of fluoride from CaF₂ and therefore represents a worst case.

The toxicity of NaF has been extensively examined under the American National Toxicology Program (NTP). The dataset contains a 14 day dose range-finder test in rat and in mice, a 6 month study in rat and in mice and a 2 year chronic study in rat and in mice. The chronic data are entered in IUCLID under the carcinogenicity endpoint. The animals were administered with NaF via the drinking water.

In the 14-day range finder study in rat, mortality was seen at the highest dose level of 800 ppm, and at lower NaF concentrations, effects of reduced body weight gain were observed. Based on these findings, the registrants have deduced a NOAEL of 50 ppm NaF for male rats and 100 ppm NaF for female rats. Based on estimated water uptake information, this corresponds to ca. 4.8 mg NaF/kg bw/d for male rats and 11.3 mg NaF/kg bw/d for female rats.

In the 14-day range finder study in mice, similar observations were made, with mortality only at the highest dose level of 800 ppm and body weight changes and reduced water consumption at lower dose levels. Based on these findings, the registrants have deduced a NOAEL of 400 ppm NaF for male and female mice. Based on estimated water uptake information, this corresponds to ca. 50.2 mg NaF/kg bw/d for male mice and 76.0 mg NaF/kg bw/d for female mice.

In the 6-month rat study, reduced body weights and reduced food and water consumption were observed at the highest dose level of 300 ppm. At lower concentrations, signs of dental fluorosis were noted. Based on these findings, the registrants have deduced a NOAEL of 100 ppm NaF for male and female rats. Based on estimated water uptake information, this corresponds to ca. 5.6 mg NaF/kg bw/d for male rats and 6.7 mg NaF/kg bw/d for female rats.

In the 6-month mice study, mortality was observed at 600 ppm NaF concentration in the drinking water. At lower dose levels, skeletal effects were seen. Based on these findings, the registrants have deduced a LOEL of 50 ppm NaF for male mice and a NOEL of 50 ppm NaF for female mice. Based on estimated water uptake information, this corresponds to ca. 4.3 mg NaF/kg bw/d for male mice and 6.4 mg NaF/kg bw/d for female mice.

In the 2-year rat study, dental effects were observed at all dose levels, including the lowest applied level of 25 ppm. The LOAEL was thus determined to be 25 ppm, which corresponds to 1.3 mg NaF/kg bw/d for male and female rats.

In the 2-year mice study, the NOAEL for male and female mice was determined to be 175 ppm NaF, which corredsponds to 16.7 mg NaF/kg bw/d for male mice and 18.8 mg NaF/kg bw/d for female mice.

The available studies provide a consistent picture in which fluoride toxicity results in mortality at high dose levels, and dental and skeletal effects at lower dose levels. The lowest dose level at which dental effects are observed are found in the 2 year study in rats. Here, a LOAEL of 1.3 mg NaF/kg bw/d is determined. Taking into account that CaF₂ contains 2 equivalents of fluoride per mole, this corresponds to a dose level of 1.2 mg CaF₂/kg bw/d.

As mentioned before, this is to be considered a conservative estimate, as the difference in bioavailability of fluoride from CaF₂ versus NaF is not accounted for.

Dermal studies

A waiver is proposed: fluoride toxicity has been adequately characterised by the oral route of exposure and dermal absorption is likely to be negligible.

Inhalation study

A waiver is proposed: fluoride toxicity has been adequatly characterised by the oral route.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
While it was necessary to use data from studies investigating sodium fluoride toxicity, the NTP investigations into the subchronic exposure of rats and mice are considered to be reliable and represent a worst case for the assessment of calcium fluoride toxicity as a result of the differing water solubility of the two salts.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
Repeated exposure guideline compliant investigation of fluoride toxicity was considered appropriate for assessing calcium fluoride effects

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
Repeated exposure guideline compliant investigation of fluoride toxicity was considered appropriate for assessing calcium fluoride effects

Repeated dose toxicity: inhalation - systemic effects (target organ) respiratory: lung

Justification for classification or non-classification

The available data do not indicate that the substance meets the criteria for classification for repeated dose toxicity according to the CLP Regulation (1272/2008/EC).