Caffeine and reduction of fetal ossification in the rat: fact or artifact?

This study was done to determine the gestational period during which the rat fetus is susceptible to reduction of skeletal ossification by caffeine. Caffeine, 100 mg/kg/day by gavage, caused the greatest reduction in ossification, as assessed by staining with alizarin red S, in fetuses exposed between day 16 to 19 of gestation, less in those treated between day 7 to 19, and markedly less in those receiving it between day 7 to 16; a single dose on day 19 had very little effect. This indicates that the fetus is most susceptible late in pregnancy. Bones in early stages of mineralization on day 20 showed a greater reduction in staining than those in later stages. Thus, caffeine appears to lower the rate of ossification rather than reduce its final extent. In the day 7 to 19 caffeine treatment group, but not in the day 16 to 19 group, maternal and fetal body weights were reduced, and 1.6% of the fetuses had aphalangia. The day 7 to 16 caffeine treatment reduced fetal body weight. This argues against an association between reduction in fetal weight and ossification. None of the treatments affected rates of resorption or litter size. A novel and important observation made is that the different caffeine treatments affected the staining by alizarin of both claws and bones in a qualitatively and quantitatively similar manner. Since claws are devoid of osteoid, this observation questions the specificity of alizarin for the assessment of the state of fetal ossification and raises doubt as to the significance of the observed action of caffeine on ossification.