Manganese and lead co-exposure and neurodevelopment pregnancy (n = 462) or at delivery (n = 462) from 1997 through 2000 in a larger study examining plasma lead biomarkers during pregnancy and neurodevelopment in Mexico City (Surkan et al. 2008; Tellez-Rojo et al. 2006). For the present study, we included 493 children who provided a blood sample at 12 and/or 24 months of age (12 months, n = 301; 24 months, n = 482) and underwent neurodevelopmental testing. Children were followed through 36 months of age. The par- ent study for this project did not include mea- sures of blood manganese, but whole blood was archived during follow-up visits whenever possible. After exclusions [e.g., very low birth weight (< 1,500 g), severely premature birth (< 32 weeks gestation), or missing data on gesta- tional age], 486 children (12 months, n = 296; 24 months, n = 475) met eligibility criteria. Eligible mothers were informed about the study, and written consent was obtained before participation. The human subjects committees of the National Institute of Public Health of Mexico, Harvard School of Public Health, and participating hospitals approved all study materials and procedures. Measurement of blood manganese and lead. Venous whole blood samples were col- lected in trace metal-free tubes from children at 12 and/or 24 months of age and immedi- ately frozen. We prepared and analyzed sam- ples for manganese and lead concentrations at the Trace Metals Laboratory at Harvard School of Public Health in Boston. We meas- ured metals concentrations with a dynamic reaction cell/inductively coupled plasma mass spectrometer (Elan 6100; PerkinElmer, Norwalk, CT, USA), using previously described methods and quality control mea- sures (Zota et al. 2009). Recovery rates for manganese and lead in quality control and spiked samples were 78–113%, and preci- sion (percent relative standard deviation) was < 5%. The average limits of detection were 0.09 and 0.04 gμ/dL for manganese and lead, respectively, and all samples were above the limits of detection. Measurement of child development and potential confounders. We assessed child neuro- development at 6-month intervals (i.e., 12, 18, 24, 30, 36 months of age) using the Bayley Scales of Infant Development–II, Spanish ver- sion (BSID-IIS) (Bayley 1993). Trained study personnel, who were blinded to children’s manganese and lead levels, administered the test using a standardized protocol previously described by our research group (Gomaa et al. 2002). In this study, we considered scores from the Mental Development Index (MDI) and the Psychomotor Development Index (PDI) of the BSID-IIS as the primary outcomes. We collected information on demo- graphic, socioeconomic, and other factors that could confound the relationship between manganese–lead and neurodevelopment: sex, hemoglobin at 12 and 24 months, ferritin at 12 and 24 months, birth weight, birth length, head circumference at birth, gestational age, maternal age at delivery, maternal and pater- nal education, maternal marital status at enrollment, duration of breast-feeding in the infant’s first year, and child nutrition at 12 and 24 months collected via questionnaires. Umbilical cord blood lead and maternal blood lead at 1-month postpartum were also con- sidered as confounders, in order to examine effects of metals at 12 and 24 months inde- pendently from effects of exposures at birth. We assessed maternal IQ using the Wechsler Adult Intelligence Scale, Spanish version (Wechsler 1968). Statistical analyses. Our previous analysis of the main effect of manganese suggested an inverted U-shaped association between man- ganese and neurodevelopment at 12 months, whereby lower MDI scores were observed at the lowest and highest quintiles of the man- ganese distribution compared with midrange levels of manganese (Claus Henn et al. 2010). We therefore examined the manganese–lead interaction at both the lowest and highest manganese quintiles (i.e., highest quintile manganese × lead and lowest quintile manga- nese × lead, compared with middle manganese quintiles). We modeled lead as a continuous variable, centered at the arithmetic mean value of the distribution. Although manganese and lead data were moderately skewed, the Gauss– Markov model requires that only the model residuals be normally distributed. Because this assumption was satisfied, we did not log transform the exposure variables. Skewness may also arise from the presence of outliers; the influence of identified outliers on effect estimates was examined in sensitivity analyses, as described below. Neurodevelopment scores were approximately normally distributed and were modeled as continuous outcomes. We fit two (i.e., one for each exposure time point) linear mixed-effects models with repeated measures of Bayley scores to examine the manganese–lead interaction over time. Additionally, we fit separate adjusted regres- sion models for each exposure time point with each Bayley assessment (i.e., 12-month blood metals with 12-, 18-, 24-, 30-, and 36-month Bayley scores; 24-month blood metals with 24-, 30-, and 36-month Bayley scores). We adjusted all models for the same set of covari- ates described previously (Claus Henn et al. 2010): sex, hemoglobin (grams per deciliter; modeled as continuous variable), gestational age (weeks; continuous variable), maternal IQ (continuous variable), and maternal educa- tion (years; continuous variable). We included known predictors of neurodevelopment or strong potential confounders (sex, maternal IQ, and education) a priori in multivariable Environmental Health Perspectives • volume 120 | number 1 | January 2012 regression models, based on biological plau- sibility. To the model with a priori covariates, we added, one at a time, covariates that were associated in bivariate models (p < 0.10) with exposures and outcomes at any time point. Additional covariates were included in the final model if the manganese or lead effect estimates changed > 10%. Although only ≤ 8% of eligible children (12 months, 21 of 296; 24 months, 38 of 475) were excluded because of missing covari- ate data, we conducted sensitivity analyses to evaluate the appropriateness of conducting a complete case analysis by comparing crude estimates for manganese–lead interaction among all eligible children (including those missing data) with those for participants with complete data. We also examined the influ- ence of extreme observations, identified with the generalized extreme studentized deviate many-outlier procedure (Rosner 1983), by fit- ting models with and without outliers. We conducted statistical analyses using SAS (version 9.2; SAS Institute, Inc., Cary, NC, USA) and R (version 2.7.1; R Foundation for Statistical Computing, www.r-project.org). We considered results statistically significant at p < 0.05. Results Participant characteristics. From among 486 children who met eligibility criteria (12-month blood metals, n = 296; 24-month blood met- als, n = 475), 455 had complete covariate data (12 months, n = 275; 24 months, n = 437) and were included in all analyses. More blood samples were collected at 24 months than at 12 months because these were convenience samples that depended on availability of a phlebotomist. Children with blood metals at 12 months were largely a subset of the chil- dren with blood metals data at 24 months and appeared similar on sociodemographic charac- teristics [see Supplemental Material, Table 1 (http://dx.doi.org/10.1289/ehp.1003300)]. In Table 1, we present major characteristics of children included in the analyses. Children included were similar to those who were excluded on most variables. Children who provided a blood sample at a single time point appeared similar on major characteristics to those who provided blood samples at both time points (data not shown). Blood manganese and lead concentrations. The arithmetic mean (± SD) concentrations at 12 and 24 months were, respectively, for blood manganese, 24.7 ± 5.9 gμ/L (range , 15.3–73.9) and 21.5 ± 7.4 gμ/L (range , 5.1–93.5), and for blood lead, 5.1 ± 2.6 gμ/dL (range , 1.5–21.4) and 5.0 ± 2.9 μg/dL (range , 1.3–37.2). Concentrations were significantly positively correlated at 12 and 24 months for both blood manganese (Spearman correlation = 0.56, p < 0.0001) and blood lead (Spearman 127