Statistical and Experimental Approaches to Discriminate Between Substance-specific Reproductive Effects and Those Mediated by Maternal Undernutrition - a Case Study with Rosin and Derivatives

It is well-accepted that undernutrition lowers fertility of rodents and retards growth of their offspring. However, accounting for undernutrition's confounding effects in reproductive toxicology studies remains challenging. Rosin (EC 232-475-7), a substance derived from pine trees, and fourteen of its derivatives were tested in OECD test guideline 422 studies. Dietary exposure to several of the substances lowered ovulation rate and retarded offspring growth. However, the substances proved unpalatable and rats consumed lower amounts of diet. The resulting undernutrition was suspected to be the cause of the reproductive effects. Consistent with this suspicion, the extent of undernutrition correlated with size of impact on reproduction. Furthermore, after statistically controlling for undernutrition, in general there was no evidence for a direct effect of the substances on offspring growth. A pair-fed feed restriction study confirmed the hypothesis that undernutrition was the sole cause of the developmental effect of dietary Rosin. Unexpectedly, dietary Rosin did not lower ovulation rate during this study and, so, the hypothesis that undernourishment is the sole cause of this reproductive effect remains unproven. However, results from a second investigative study suggest that reduced ovulation rate is a transient response to undernutrition. It is concluded that the reproductive effects of Rosin and derivatives are generally caused by undernutrition. The approaches reported here might be of use to other workers in the field and we suggest some possible improvements to the pair-fed restriction study design.