Registration Dossier

Administrative data

Endpoint:
neurotoxicity
Remarks:
subchronic
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Published peer-reviewed study

Data source

Reference
Reference Type:
publication
Title:
Neurotoxicity of sodium fluoride in rats
Author:
Mullenix PJ, Denbesten PK, Schunior A & Kernan WJ
Year:
1995
Bibliographic source:
Neurotoxicology and Teratology, 17(2): 169-177

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Behavioural neurotoxicity study in rats exposed either prenatally, as weanlings, or as adults.
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Sodium fluoride (NaF) was the test substance, and the authors were looking at the effects of fluoride.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Pathogen free Sprague-Dawley rats obtained from Charles River Laboratories. Rats were randomly assigned to treatment groups and housed 2/cage/treatment and sex. Light was provided on a 12 h light:dark cycle, and food and deionised water were offered ad libitum except during behavioural testing.
Pregnant dams were obtained on gestation day 8, and housed individually throughout gestation and lactation. Dams were fed Purina Rat Chow (Formulab), and pups were fed Certified Purina Rat Chow (5002) after weaning.
Weanlings were obtained (with dams) at 19 days of age. The pups were weaned on gestation day 21 and maintained on a low fluoride diet (<10ppm fluoride, Purina 5010 or Teklad L356).
Adult rats were obtained at 10 weeks of age, and exposures began at 12 weeks of age. They were maintained on the same low fluoride diet as the weanlings.

Administration / exposure

Route of administration:
other: one group received subcutaneous injections, the other groups received NaF in their drinking water
Vehicle:
other: saline (s.c.) or distilled water (oral)
Details on exposure:
Prenatal exposures: On gestational days 14-18, or 17-19, dams were given subcutaneous injections of 0.13 mg/kg sodium fluoride in saline, two or three times daily, at least 4 hours apart. Controls received an equal volume/body weight s.c. injection of saline alone on days to match the treated group. Beyond the prenatal period the pups received no further NaF treatment.
Weanling exposures: From gestation day 21 (weaning) the rats were exposed to NaF in their drinking water at concentrations of 0, 75, 100, 125, or 175ppm. Each fluoride treatment group had matching controls that received deionised water only. The 175ppm was studied only in females, and resulted in mortalities within 10 days of exposure. The survivors in this group received water only for the remainder of the study. Rats were exposed for 6 or 20 weeks.
Adult exposures: male and female rats aged 12 weeks old were exposed to 0 or 100ppm NaF in their drinking water for 5 to 6 weeks.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No information available.
Duration of treatment / exposure:
Prenatal exposure: 5 or 3 days.
Weanling exposure: 6 or 20 weeks.
Adult exposure: 5 or 6 weeks.
Frequency of treatment:
Prenatal exposure: 2 or 3 times daily.
Weanling and adult exposure: daily.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 75, 100, 125 or 175 ppm
Basis:
nominal in water
175ppm was discontinued due to excessive mortality
Remarks:
Doses / Concentrations:
0.13 mg/kg
Basis:
nominal conc.
nominal in saline
No. of animals per sex per dose:
Prenatal exposures: 7 dams were treated on gestational days 14-18, 9 dams were treated on gestational days 17-19, 13 dams served as controls. Litters were culled to 10 pups per litter.
Weanling exposures: 19-27/sex/dose.
Adult exposures: 21-24/sex/dose.
Control animals:
yes, concurrent vehicle
Details on study design:
Doses were selected based on published data that showed 100 ppm fluoride in drinking water produces dental fluorosis without overt toxicity.

Examinations

Observations and clinical examinations performed and frequency:
Body weights were recorded at various points during the study.
Specific biochemical examinations:
Blood samples were collected by cardiac puncture under CO2 anaesthesia at study termination (satellite groups were used where blood samples were required prior to behavioural testing). Plasma fluoride concentrations were determined using an ion-specific electrode following the hexamethyldisiloxane diffusion method.
Brain fluoride concentrations were determined for two treatment groups in the weanling or adult exposure studies. Brains were removed and chilled, 7 sections were dissected, individually lyophilised and weighed, and the fluoride content measured following diffusion of ionic fluoride.
Neurobehavioural examinations performed and frequency:
Except for adult exposures, behaviour was tested at 9 weeks of age, and repeated at 14 and 19 weeks depending on exposure period. Tests were conducted in an isolated observation room at the same time each day. Video cameras were used to record rat behaviour at a rate of 1 frame/s. Spontaneous behaviour in a novel environment was recorded. The novel environment was a clear Plexiglass box, where the control and treated rats were separated by a clear partition with small holes allowing them to see and smell each other during exploration. The video signals were transferred to a computer for pattern analysis and behavioural classification. The behaviours identified by the computer were 5 major body positions, and the specific activity i.e. grooming, sniffing etc.
Measures of behaviour were: calculation of behavioural initiations , calculation of behavioural total time, and calculation of behavioural time structure.
Sacrifice and (histo)pathology:
Brain weights were recorded in the animals used for brain fluoride content analysis.
Other examinations:
No other examinations were made.
Positive control:
Not examined.
Statistics:
t-tests were used to compare the number of behavioural initiations and behavioural total time per groups between groups. The behavioural time structure was analysed using K functions and RS statistics.

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):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Clinical biochemistry findings:
effects observed, treatment-related
Behaviour (functional findings):
effects observed, treatment-related
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Other effects:
not examined
Description (incidence and severity):
Migrated information from 'Further observations for developmental neurotoxicity study'



Details on results (for developmental neurotoxicity):Prenatal exposures: Prenatal NaF exposure altered behavioural outcomes in male offspring when exposure occurred on gestation days 17-19. The effect was in time structure changes. (migrated information)
Details on results:
Weanling exposures: At 125ppm body weight was reduced throughout 20 weeks exposure in both sexes. The survivors of the 175ppm had stunted growth compared to matched controls, however the effects were ameliorated by 18 weeks of age. Plasma fluoride levels were significantly increased in all exposed animals; at doses of 75 and 100 ppm for 6 weeks plasma fluoride increased with dose. At 125ppm for 6 weeks plasma fluoride was increased compared to controls but not in a dose dependent fashion. Behaviour was altered in both sexes, dependent on duration and concentration of exposure. In females, 6 weeks exposure to 100 or 125ppm affected behaviour, in males 11 weeks exposure to 125ppm affected behaviour. A relationship between behavioural effects and plasma fluoride was observed in females expoed for 6 weeks to 75, 100 or 125 ppm. A common pattern amongst behavioural disturbances, irrespective of sex, exposure duration or fluoride concentration, developed.
Adult exposures: male and female rats given 100ppm for 6 weeks had significantly increased plasma fluoride levels with no effect on body weight. Behaviour was only affected in females. Compared to females exposed at weaning, females exposed as adults had a lower plasma fluoride level associated with significant behavioural changes.
Fluoride exposure elevated fluoride levels in the brain. In male and female rats exposed to 125ppm for 20 weeks starting at weaning fluoride levels were increased in all 7 brain regions examined. In rats exposed to 100 ppm for 6 weeks starting at 3 months of age fluoride levels increased in the medulla oblongata in both sexes, and in the hippocampus of females.

Any other information on results incl. tables

Fluoride exposure caused sex and dose specific behavioural disruption (as measured by computer pattern recognition in a novel environment), with a common pattern. Males were most sensitive to prenatal day 17 -19 exposure, whereas females were more sensitive to weanling and adult exposures. After fluoride ingestion, the severity of the effect on behaviour increased directly with plasma F levels and F concentrations in specific brain regions.

Applicant's summary and conclusion

Conclusions:
Fluoride exposure caused sex and dose specific behavioural disruption (as measured by computer pattern recognition in a novel environment), with a common pattern.
Executive summary:

The study was designed to assess the neurotoxicity of sodium fluoride (NaF) in Sprague-Dawley rats. Rats were exposed prenatally, as weanling, or adults. Prenatal exposure - dams were injected subcutaneously with 0.13 mg/kg NaF or saline on gestational days 14 -18 or 17 -19. Weanlings were exposed to NaF in their drinking water at 0, 75, 100 or 125 ppm for 6 to 20 weeks. 3 month old adults received water containing 100 ppm for 6 weeks. Fluoride exposure caused sex and dose specific behavioural disruption (as measured by computer pattern recognition in a novel environment), with a common pattern. Males were most sensitive to prenatal day 17 -19 exposure, whereas females were more sensitive to weanling and adult exposures. After fluoride ingestion, the severity of the effect on behaviour increased directly with plasma F levels and F concentrations in specific brain regions.