SETAC Presentation: Factors to Consider When Interpreting Slight Effects on Eggshell Thickness

SETAC Presentation: Factors to Consider When Interpreting Slight Effects on Eggshell Thickness

Eggshell thickness is one of 15 endpoints evaluated in avian reproduction studies (OCSPP 850.2300, OECD 206). Typically, eggs are reserved for eggshell thickness on predetermined days during the 10 weeks of egg collections. Eggshells are cut open around the equator, contents are discarded, shells are washed and left to dry for ≥48 h prior to measurement with a micrometer to ±0.001 mm. Compared to the quantal endpoints in the test, eggshell thickness has a low coefficient of variation; resulting in effects as low as a 3% thinning from the control being reported as statistically significant. Subsequently, eggshell thickness has been one of the endpoints that most commonly drove avian reproduction study conclusions at Smithers.

Even though the organochlorine literature on eggshell thinning indicates population-relevant effects are unlikely below 18% thinning, the 2009 Guidance of EFSA for bird and mammal risk assessments implies that slight eggshell thinning effects, even if there is no subsequent effect on hatchability, cannot be dismissed as not biologically relevant due to potential interspecies differences, the high inter-pair variability and the need to be protective of free-ranging birds on calcium-deficient diets. In order to help determine whether slight eggshell thinning effects can be clearly linked to exposure to a given test material or if this is precluded by other factors, we evaluated the precision and accuracy with which eggshells can be measured.

Preliminary results indicate inter-analyst variation is greater than many of the slight effects that have driven study conclusions. While intra-analyst variability appears to be lower, the mean thickness of 45 eggshells still ranged by 5% when shells were measured on 10 occasions during an 8-week period; this was apparently independent of storage time, temperature and humidity. Regulatory agencies are increasingly focused on endocrine- mediated effects, yet there remains uncertainty around the exact mechanisms involved in eggshell thinning. Additionally, an investigation of mechanistic toxicity, molecular, cellular, tissue or organ-level biomarkers is not included in the current avian reproduction study.

This engenders a necessity for industry and regulators to understand the precision and accuracy with which eggshell thickness can be measured in the laboratories performing these vertebrate intensive one-shot studies so that slight effects and noise can be differentiated.

Kelley Stanfield will present the research at the SETAC North America 42nd Annual Meeting, November 14-18, 2021.

Download the poster:

Kelley Stanfield SETAC 2021 Eggshell Thickness
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