Table 3.

Animal studies published between 2005 and 2019 which investigated the effects of developmental fluoride exposure (without studies investigating neurobehavioral endpoints)

Species, strain, number of animals	Exposure duration, chemical form, route	NaF concentration in DW (mg/L)	Doses (mg/kg/d)	NOAEL (mg/L) (mg/kg/d)	LOAEL (mg/L) (mg/kg/d)	Outcome (as stated by the authors)	Limitations	References
Rat, Wistar n = 45 (F0) 7 dams/group	Multigenerational study: continuous exposure during three generations (F0, F1, F2) Sodium fluoride (DW)	1, 10, 50, 100	Dams: NaFa: 0.05, 0.5, 2.5, 5 F–a: 0.02, 0.23, 1.1, 2.3	10 mg/L NaF 0.5 mg NaF/kg/d 0.23 mg F–/kg/d	50 mg/L NaF 2.5 mg NaF/kg/d 1.13 mg F– /kg/d	50 and 100 mg/L NaF (n = 7/group): significant histopathological changes were found in the myocardial tissue: myocardial cell necrosis, extensive cytoplasmic vacuole formation, nucleus dissolution in myosits, swollen and clumped myocardial fibers, fibrillolysis, interstitial edema, small hemorrhagic areas and hyperemic vessels	Blinding: NR Control for litter effects: NR Inadequate reporting of concentration in DW: unclear whether referring to NaF or F– Group size < 10 F– concentration in DW (control) and feed: NR	Cicek et al. (2005)
Mouse (species unclear), Wistar 10 dams/group	GD 15 until PND 14	500	NaFb: 75 mg/kg/d F–b: 34 mg/kg/d		500 mg/L NaF 75 mg NaF/kg/d 34 mg F–/kg/d	Fluoride given to dams led to oxidative stress in mothers as well as in offspring, able to induce enhanced lipid peroxidation levels and protein conformational changes, as suggested by stress protein (HSP, GRP) expression changes	Species/strain unclear Characterization of the test compound: NR Single high dose tested Blinding: NR Control for litter effects: NR Treatment duration < pre- and postnatal period F– concentration in DW (control) and feed: NR	Bouaziz et al. (2007)
Rat, Wistar n = 6/group	Female rats throughout gestation and lactation, neonates received tap water until PND 90 Sodium fluoride (DW)	4.5 and 9.0	Dams: NaFa: 0.23, 0.45 F–a: 0.1, 0.2	–	4.5 mg/L NaF 0.23 mg NaF/kg/day 0.1 mg F– /kg/day	Treatment groups: (n = 6). PND 90: male pups showed changes in sperm parameters (sperm count, viability) at 4.5 and 9 mg/L NaF	Blinding: NR 2 dose levels tested, no NOAEL established Group size < 10	Reddy et al. (2007)
Rat, Sprague-Dawley n = 40 (20/sex) n = 5/group	From pre-pregnancy to PND 56 Sodium fluoride (DW)	0, 25, 50, 100	Dams: NaFa: 0, 1.25, 2.5, 5 F–a: 0, 0.57, 1.13, 2.3	25 mg/L NaF 1.25 mg NaF/kg/day 0.57 mg F–/kg/day	50 mg/L NaF 2.5 mg NaF/kg/day 1.13 mg F–/kg/day	Testes of offspring at PND 56 (n = 5/group): effects of NaF on testicular histopathology, testicular ultrastructure and germ cell apoptosis at 50 and 100 mg/L NaF	Group size < 10 Control for litter effects: NR F– concentration in DW (control) and feed: NR	Zhang et al. (2013)
Mouse, ICR, 6–8 weeks of age Number of animals unclear	3 treatment groups: Group I: male mice given 100 mg/L NaF in water for 35 days Group II: female mice mated with male mice not given NaF; 100 mg/L NaF in DW for 48 h after becoming pregnant Group III: control Sodium fluoride (DW)	100	NaFa: 9 F–a: 4.1	–	100 mg/L NaF 9 mg NaF/kg/day 4.1 mg F– /kg/day	NaF treatment disrupted DNA methylation of H19 and Peg3 in early embryonic stages of the mouse. However, there were no significant changes in DNA methylation in sperm and liver of male mice treated with NaF	Single dose tested Blinding: NR Control for litter effects: NR Number of animals: NR Experimental setting not clearly described: e.g., discrepancy between description in methods and in the abstract/figures F– concentration in DW (control) and feed: NR	Zhu et al. (2014)
Mouse, ICR, 6–8 weeks of age Number of animals during treatment unclear	4 treatment groups Group I: female mice mated with male mice not given NaF; 120 mg/L NaF in DW for 48 h after becoming pregnant Group II: male mice given 120 mg/L NaF in water for 35 days Group III: female mice mated with NaF-treated males, 120 mg/L NaF for 48 h after becoming pregnant Group IV: control Sodium fluoride (DW)	120	NaFa: 10.8 F–a: 4.9	–	120 mg/L NaF 10.8 mg NaF/kg/day 4.9 mg F– /kg/day	120 mg/L NaF in drinking water of pregnant mice for 48 h: NaF affected DNA methylation of early embryos (n = 20). H19 gene was significantly downmethylated in embryos from pregnant mice. Methylation of both H19 and Peg3 genes was disrupted when the parent male mice were treated with NaF for 35 days	Single dose tested Blinding: NR Control for litter effects: NR Number of animals during treatment: NR F– concentration in DW (control) and feed: NR	Zhao et al. (2015)
Rat, Sprague-Dawley	From pre-pregnancy to PND 56 Sodium fluoride (DW)	0, 25, 50, 100 Control: tap water (fluoride ion concentration < 1.0 mg/L)	Dams NaFa: 1.25, 2.5, 5 F–a: 0.57, 1.13, 2.3	–	25 mg/L NaF 1.25 mg NaF/kg/day 0.57 mg F–/kg/day	Testes of offspring were excised on PND 56 (n = 3/group): NaF exposure induced histological lesions in rat testes and caused extensive abnormalities in testicular tissue (all dose groups)	Group size unclear (start, pregnancy, treatment) Blinding: NR Control for litter effects: NR F– concentration in feed: NR	Zhang et al. (2016)

Search criteria were: PubMed database for animal studies published between January 2005 and February 2019 with the key words in the title/abstract including ‘fluoride’, ‘sodium fluoride’ and ‘developmental toxicity’. In addition to the PubMed search, the reference lists of included studies and records that do not contain original data (i.e., reviews, editorials, or commentaries) were checked for further studies. Publications where the full text was not available in English or not published in a journal (master thesis, dissertations etc.) were excluded

DW drinking water, GD gestation day, NaF sodium fluoride, NR not reported, PND postnatal day

^aConversion of F^–concentration in drinking water into daily doses was performed by application of default conversion factors derived by EFSA (EFSA 2012)

For chronic studies, a default factor of 0.05 for rats and 0.09 for mice should be used, e.g., 1 mg/L in water is equivalent to a dose of 0.05 and 0.09 mg/kg b.w. per day in rats and mice, respectively. For subchronic studies, a default factor of 0.09 for rats and 0.15 for mice should be used. For subacute studies, a default factor of 0.12 for rats and 0.18 for mice should be used

In case the authors only reported F^– concentration in drinking water, in the present estimation conversion factors of 0.05/0.09 were used for adult animals/the parental generation and conversion factors of 0.12/0.18 for the pups or short-term exposure of young animals

^bas reported by the authors