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Toxicological information

Carcinogenicity

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Description of key information

High quality NTP studies in the rat and mouse are available for sodium fluoride  The EU RAR for HF concludes that the data are sufficient to suggest that fluoride is not carcinogenic in animals.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records
Reference
Endpoint:
carcinogenicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
7th October 1985 to 27th September 1987
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: NTP study
Qualifier:
according to
Guideline:
other: NTP protocol
Principles of method if other than guideline:
NTP protocol for the assessment of carcinogenicity
GLP compliance:
yes
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
Male and female F344/N rats were used, obtained from the National Cancer Institute's Frederick Cancer Research Facility. Rats were 4 weeks old on arrival, and were quarantined for 12 days. Rats were placed on study when they were 6 weeks old. Rats were housed 5 per cage in polycarbonate cages rotated every other week,and identified by toe marks. Specially formulated low fluoride feed (NIH-07 Rat and Mouse Pellets Low Fluoride) and deionised water were available ad libitum. Beta-Chip hardwood chips were provided as bedding as changed twice weekly. Temperature was maintained at 19.4-26.1oC, humidity: 22-76%, fluorescent light was provided 12 hours/day, and there were 10 room air changes per hour.

The study began on 7th October 1985, the 105 week scheduled termination took place on 27th September 1987.
Route of administration:
oral: drinking water
Vehicle:
water
Details on exposure:
Rats were given sodium fluoride in deionised drinking water at 0, 25, 100 or 175 ppm ad libitum for up to 103 weeks.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation analyses were conducted utilising a potentiometric method with a fluoride ion electrode. Analyses were conducted weekly on all dose formulations during the first 6 months, then every 8 weeks for the remainder of the study. Analyses indicated that all dose formulations were within ±10% of target concentrations throughout the study. Deionised water had a fluoride concentration of ≤0.1ppm.
Duration of treatment / exposure:
Fluoridated drinking water was available ad libitum, daily for up to 103 weeks.
Frequency of treatment:
Daily.
Post exposure period:
Rats were sacrificed approximately 1 week after the final dose.
Dose / conc.:
0 ppm
Remarks:
Concentration of NaF in drinking water.
Dose / conc.:
25 ppm
Remarks:
Concentration of NaF in drinking water.
Dose / conc.:
100 ppm
Remarks:
Concentration of NaF in drinking water.
Dose / conc.:
175 ppm
Remarks:
Concentration of NaF in drinking water.
No. of animals per sex per dose:
Groups of 100 rats of each sex received 0 or 175ppm sodium fluoride, and groups of 70 rats of each sex received 25 or 100 ppm. An additional group of 50 animals of each sex was included to provide paired age-matched controls, these animals received deionised water.
Control animals:
yes, concurrent vehicle
Details on study design:
Groups of 100 rats of each sex received 0 or 175ppm sodium fluoride, and groups of 70 rats of each sex received 25 or 100 ppm. The doses were based on the lower weight gain of male and female rats given 300ppm in a 6 month study, and the occurrence of potentially life-threatening lesions in the stomach of these rats. Also, an initial 2 year study had been conducted using 100ppm as the top dose, but it was felt that the rats could tolerate higher concentrations, therefore the top dose chosen for the present study was 175ppm. An additional group of 50 animals of each sex was included to provide paired age-matched controls, these animals received deionised water. During every study week that one or more rats from any group receiving sodium fluoride in the water was found dead or killed in a moribund condition, one animal of the same sex was chosen at random from the paired control group and killed. For group assignment, animals were assigned to weight classes, then randomised to test and control groups by partitioning algorithm using Xybion Pathology/Toxicology Data System.
Positive control:
Not required.
Observations and examinations performed and frequency:
Rats were observed twice daily for mortality and morbidity. Initially weights and clinical signs were recorded weekly through to week 13, then monthly thereafter. Every 4 weeks feed and water consumption was recorded for a 7 day consecutive period.
Sacrifice and pathology:
Necropsy was performed on all animals including those found dead. Interim sacrifices were performed at 27 weeks and 66 weeks. Terminal sacrifice was performed at 105 weeks.
Other examinations:
In addition, studies to assess the bioavailability of fluoride in the diet were conducted in male rats at approx. 6, 12 and 18 months on study. Urinalysis, haematology and clinical chemistry were carried out at 27 and 66 weeks. The left humerus of all rats killed at interim sacrifices and of 10 randomly selected rats from each group at terminal sacrifice was evaluated for fluoride concentration. The incisors of designated rats at all scheduled sacrifices were evaluated for attrition and mottling.
Statistics:
Survival analyses: the probability of survival was estimated by the product-limit procedure of Kaplan and Meier. Analyses for possible dose-related effects on survival used Cox's method for testing two groups for equality and Tarone's life table test for dose-related trends.
The incidence of neoplastic or nonneoplastic lesions was given as the ratio of the number of animals bearing such lesions at a specific anatomic site to the number of animals in which that site was examined.
Tumour incidence was analysed by logistic regression, tumour prevalence was modeled as a logisitic function of chemical exposure and time. Also used were the life table test, the Fisher exact test, and the Cochran-Armitage trend test. Tests of significance included pairwise comparisons.
Continuous variables were analysed by comparing dosed groups to the control group using the nonparametric Dunn or Shirley multiple comparison test. Jonckheere's test was also used.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
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:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
CLINICAL SIGNS AND MORTALITY

There was no treatment-related mortality; survival was unaffected by treatment. Clinical signs were limited to the teeth of rats at 100 and 175 ppm (attrition, deformity, discoloration, mottling, malocclusion). Mottling of the teeth was apparent at 25 ppm.

BODY WEIGHT AND WEIGHT GAIN

Bodyweights and weight gain were unaffected by treatment with NaF.

FOOD CONSUMPTION

Food consumption was unaffected by treatment


WATER CONSUMPTION AND COMPOUND INTAKE

Water consumption was unaffected by treatment. The intake of sodium fluoride over the course of the study was estimated to be 1.3, 5.2 and 8.6 mg/kg bw/d in males; 1.3, 5.5 abd 9.5 mg/kg bw/d for females.


HAEMATOLOGY

Parameters were unaffected by treatment.

CLINICAL CHEMISTRY

Parameters were unaffected by treatment.

URINALYSIS

Parameters were unaffected by treatment, with the possible exception of a small increase in calcium concentration in 175 ppm females.


ORGAN WEIGHTS

Organ weights were unaffected by treatment.


HISTOPATHOLOGY: NON-NEOPLASTIC

The incidence of osteosclerosis was increased in females at 175 ppm. Aterations in the teeth (dentine dysplasia, odontoblast and ameloblast degeneration) were seen in the incisors and were more frequent in males than females.


HISTOPATHOLOGY: NEOPLASTIC

Osteocarcoma was seen in one male at 100 ppm(2%) and three males at 175 ppm (4%). Squamous cell tumours of the oral mucosa were seen in control and treated animals and were not considered to be related to treatment. A marginal increase in thyroid follicular cell neoplasms was not considered to be related to treatment. A decreased incidence of uterine stromal polyps was seen in females at 175 ppm.


HISTORICAL CONTROL DATA

Osteosarcoma was reported to occur with a mean incidence of 0.5% in untreated male rats in NTP studies and a maximum incidence of 6%. The incidences in treated males in this study are within the historical range.

OTHER FINDINGS

Plasma fluoride concentration, urine fluoride concentration and bone fluoride concentration were increased in all treated groups.
Relevance of carcinogenic effects / potential:
Based on the results of this study the NTP state that there was 'equivocal evidence of carcinogenic activity' of sodium fluoride in male F344/N rats based on the occurrence of a small number of osteosarcomas in dosed animals (equivocal evidence is a category for uncertain findings defined as studies that are interpreted as showing a marginal increase of neoplasms that may be related to chemical administration). There was no evidence of carcinogenic activity in female F344/N rats receiving sodium fluoride in their drinking water at 25, 100 or 175ppm.
Dose descriptor:
NOAEL
Effect level:
0.59 mg/kg bw/day (actual dose received)
Based on:
element
Remarks:
fluoride
Sex:
male
Basis for effect level:
other: Equivocal evidence of carcinogenicity was seen in higher dose groups.
Remarks on result:
other: Corresponding NaF concentration in the water: 25 ppm
Dose descriptor:
NOAEL
Effect level:
1.3 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
NaF
Sex:
male
Basis for effect level:
other: Equivocal evidence of carcinogenicity was seen at 100 and 175 ppm
Remarks on result:
other: Corresponding NaF concentration in the water: 25 ppm
Dose descriptor:
NOAEL
Effect level:
9.5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
NaF
Sex:
female
Basis for effect level:
other: No evidence of carcinogenicity was seen in any of the dose groups
Remarks on result:
other: Corresponding NaF concentration in the water: 175 ppm
Dose descriptor:
NOAEL
Effect level:
4.3 mg/kg bw/day (actual dose received)
Based on:
element
Remarks:
fluoride
Sex:
female
Basis for effect level:
other: No evidence of carcinogenicity was seen in any of the dose groups.
Remarks on result:
other: Corresponding NaF concentration in the water: 175 ppm
Dose descriptor:
NOAEL
Effect level:
< 0.59 mg/kg bw/day (actual dose received)
Based on:
element
Remarks:
fluoride
Sex:
male/female
Basis for effect level:
other: Dental effects were apparent in all treated groups
Remarks on result:
other: Corresponding NaF concentration in the water: 25 ppm
Dose descriptor:
NOAEL
Effect level:
< 1.3 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
NaF
Sex:
male/female
Basis for effect level:
other: Dental effects were apparent in all treated groups
Remarks on result:
other: Corresponding NaF concentration in the water: 25 ppm

Survival rates after 2 years were:

males - control 42/80; 25ppm 25/51; 100ppm 23/50; 175ppm 42/80

females - control 59/80; 25ppm 31/50; 100ppm 34/50; 175ppm 42/80.

The pairwise comparison of the incidence of osteosarcomas in the 175 ppm group versus that in the controls was not statistically significant, however osteosarcomas occurred with a statistically significant dose-response trend.

Bone osteosarcomas in male rats

Bone: Osteosarcoma

Control

25ppm

100ppm

175ppm

Overall ratesa

0/80 (0%)

0/51 (0%)

1/50 (2%)

3/80 (4%)b

Adjusted ratesc

0.0%

0.0%

4.3%

5.3%

Terminal ratesd

0/42 (0%)

0/25 (0%)

1/23 (4%)

1/42 (2%)

First incidence (days)

-

-

729 (T)

388

Logisitic regression testse

P=0.027

-f

P=0.380

P=0.099

(T) Terminal sacrifice

aNumber of tumour bearing animals/number of animals necropsied.

bOne extraskeletal osteosarcoma occurred in a high dose male rat.

cKaplan-Meier estimated tumour incidence at the end of the study after adjustment for intercurrent mortality.

dObserved incidence at terminal kill.

eBeneath the control incidence are the P values associated with the trend test. Beneath the dosed group incidence are the P values corresponding to pairwise comparisons between the controls and that dosed group. The logistic regression tests regard these lesions as non fatal.

fNo tumours in dosed group or control group; statistical test not performed.

Tooth abnormalities (gross observations) in rats

Observationa

Control

25ppm

100ppm

175ppm

Male

 

 

 

 

Attritionb

0 (0%)

0 (0%)

7 (30%)

22 (50%)

Deformityc

1 (1%)

0 (0%)

12 (17%)

27 (27%)

Discolourationc

1 (1%)

2 (3%)

15 (21%)

31 (31%)

Malocclusionc

1 (1%)

1 (1%)

2 (3%)

13 (13%)

Mottlingb

2 (5%)

22 (85%)

22 (96%)

44 (100%)

Female

 

 

 

 

Attrition

0 (0%)

0 (0%)

1 (3%)

2 (4%)

Deformity

0 (0%)

0 (0%)

1 (1%)

8 (8%)

Discolouration

0 (0%)

2 (3%)

2 (3%)

8 (8%)

Malocclusion

1 (1%)

0 (0%)

0 (0%)

1 (1%)

Mottling

0 (0%)

8 (26%)

32 (94%)

53 (98%)

aDiscolouration designates an overall effect, while mottling indicates variegated discolouration. The terms are not mutually exclusive.

bThe incidences for this observation are for the lower incisors of animals observed at week 104 only (males n=43, 26, 23, 44; females n=59, 31, 34, 54).

cThe incidences for this observation include interim and terminal sacrifice animals (males and females n = 100, 70, 70, 100).

Conclusions:
Based on the results of this study the NTP state that there was 'equivocal evidence of carcinogenic activity' of sodium fluoride in male F344/N rats based on the occurrence of a small number of osteosarcomas in dosed animals (equivocal evidence is a category for uncertain findings defined as studies that are interpreted as showing a marginal increase of neoplasms that may be related to chemical administration). There was no evidence of carcinogenic activity in female F344/N rats receiving sodium fluoride in their drinking water at 25, 100 or 175ppm.
Executive summary:

The potential carcinogenic activity of sodium fluoride was determined in a 2 year drinking water study using male and female F344/N rats. Rats were exposed to concentrations of 0, 25, 100 or 175 ppm daily in their drinking water.

Survival rates were not affected by sodium fluoride administration.

The teeth of the rats showed a dose-dependent whitish discolouration, and males had an increased incidence of tooth deformities and attrition leading on occasion to malocclusion. Osteosclerosis of long bones was increased in female rats given 175 ppm, no other significant nonneoplastic lesions appeared related to sodium fluoride administration. A small number of osteosarcomas were seen in male rats given 100 or 175ppm, none were seen in the control or 25 ppm groups. Osteosarcomas occurred with a statistically significant dose-response trend, leading to the conclusion that a weak association may exist between the occurrence of these neoplasms and the administration of sodium fluoride. Therefore, according to the NTP Explanations of Levels of Incidence, the NTP concluded that under the conditions of this study, there was 'equivocal evidence of carcinogenic activity' of sodium fluoride in male F344/N rats based on the occurrence of a small number of osteosarcomas in dosed animals. Equivocal evidence is a category for uncertain findings defined as studies that are interpreted as showing a marginal increase of neoplasms that may be related to chemical administration. There was no evidence of carcinogenic activity in female F344/N rats receiving sodium fluoride in their drinking water at 25, 100 or 175 ppm.

Based on these results, the registrants have deducted an NOAEL of 25 ppm NaF for male rats and a NOAEL of 175 ppm for female rats based on the observations of a small amount of osteosarcomas. This corresponds to a NOAEL of 0.59 mg F-/kg bw/d or 1.3 mg NaF/kg bw/d for males and 4.3 mg F-/kg bw/d or 9.5 mg NaF/kg bw/d for females.

When taking into account the dental effects, a NOAEL < 25 ppm NaF applies to both male and female rats. This corresponds to a NOAEL < 0.59 mg F-/kg bw/d or < 1.3 mg NaF/kg bw/d for males and females.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
25
Study duration:
subchronic
Species:
rat
Quality of whole database:
The database consists of two good quality NTP assessments in rats and mice , supported by read across from published data for a similar substance.
NOAELS were established in the NTP studies but no carcinogenic effects were identifed in the supporting studies.

Carcinogenicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Carcinogenicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

There are no carcinogenicity studies available for calcium difluoride, however studies are available for the water soluble salt sodium fluoride. 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.

Studies in the rat

The NTP rat study showed evidence of an effect of sodium fluoride administration on the bones and teeth, consistent with the findings of other studies. There was no effect on survival in this study; bodyweights, food and water consumption, haematological and clinical chemistry parameters and organ weights were unaffected by treatment. Serum, urine and bone fluoride concentrations were increased in all treated groups; the urine calcium concentration was also marginally higher in females at the highest dose level. Osteosclerosis was seen in females at the highest dose level. The incidence of osteosarcoma was increased in males at the intermediate dose level (2%) and the high dose level (4%) but was within the historical range (0 -6%; mean 0.5%). The NTP concluded that the study provides 'equivocal evidence' for carcinogenicity in male rats.

An additional carcinogenicity study with NaF in the rat is available (Maurer et al, 1990). No evidence of carcinogenicity was seen in this study, at dose levels sufficient to cause toxicity.

Studies in the mouse

The NTP mouse study showed evidence of an effect of sodium fluoride administration on the teeth, consistent with the findings of other studies. There was no effect on survival in this study; bodyweights, food and water consumption, haematological parameters and organ weights were unaffected by treatment. Clinical chemistry revealed elevated ALP activity in females at the highest dose level. Microscopic findings were limited to dentine dysplasia in male mice at 175 ppm. There was no evidence of carcinogenicity in either sex.

An additional carcinogenicity study with NaF is available (Maurer et al, 1993). A high level of osteosarcomas was seen in all (control and treated) groups in this study, a finding which was attributed to infection with a retrovirus. No conclusion on the carcinogenicity of sodium fluoride can be drawn from this study.


Justification for selection of carcinogenicity via oral route endpoint:
High quality NTP studies in the rat and mouse are available for sodium fluoride The EU RAR for HF concludes that the data are sufficient to suggest that fluoride is not carcinogenic in animals.

Justification for classification or non-classification

No classification is proposed according to the CLP Regulation (1272/2008/EC). The EU RAR for HF concludes that the data are sufficient to suggest that fluoride is not carcinogenic in animals.