Supporting Information

Mattison et al. 10.1073/pnas.1102187108

SI Materials and Methods trospray tandem mass spectrometry (LC-ES-MS/MS) in the mul-
Animals and Animal Care. The test subjects weighing 2.5 to 4.2 kg tiple-reaction monitoring mode by monitoring specific transitions
were received into quarantine at an Association for Assessment for labeled and unlabeled MPH and unlabeled ritalinic acid (RA,
and Accreditation of Laboratory Animal Care-accredited facility the major metabolite of MPH in NHP) using the procedures
maintained on a 12-h:12-h light:dark cycle, at a temperature of 23 to previously published (3). Quantitative accuracy ranged from 95%
29 °C, humidity of 30% to 70%, and 10 to 15 air exchanges per to 111% and the inter- and intraday method precision ranged from
hour. The animals were housed individually, provided with water 2% to 6% relative SD. The limits of quantification were 0.1 ng/mL
ad libitum, and were fed High Protein Monkey Diet Jumbo for MPH and 0.03 ng/mL for RA and the limits of detection for
(#5047, PMI Nutrition International) daily, supplemented with MPH and RA were 0.03 and 0.009 ng/mL, respectively.
children’s vitamins and fresh fruit three times a week. The mon-
keys were antibody-negative for simian type D retrovirus, simian Crown-Rump Measurements. Crown-rump measurements (cm)
immunodeficiency virus, and simian T-cell leukemia virus, and were performed monthly from month 17 through month 41. This
were released from a 1-mo quarantine period following three measurement was made using a stadiometer as the test subject
negative tuberculosis tests and veterinary health assessment in- was chaired. The base of the instrument was placed under the
dicating good health. Once released from quarantine, the test ischial callosities and the top plate was placed on top of the test
subjects underwent a 4-mo acclimation and training period in the subject’s head.
Primate Facility before the initiation of methylphenidate hydro-
chloride (MPH) dosing at ∼25 to 28 mo of age. Endocrine Analyses. Monthly serum samples were assayed for total
All test subjects on the study underwent genetic safety assess- testosterone using a solid-phase RIA (RIA; Coat-A-Count, Sie-
ments and behavioral testing during the dosing period, and the data mens), for inhibin B using an ELISA (DSL-10–8410; Diagnostic
have been reported elsewhere (1, 2). Banana-flavored reward System Labs), for primate leptin using a RIA (PL-84K; Linco
pellets (Bioserv) were used as reinforcers in the behavioral studies. Research), and for FSH using an RIA as described in Ramaswamy
Both the primate biscuits and the reward pellets were lot-tested et al. (5). Intra- and interassay coefficient of variations, respectively,
before use to confirm that the dietary constituents (e.g., fat, pro- were 17.6% and 7.3% for testosterone, 5.6% and 6.2% for inhibin
tein, and vitamins) were within the acceptable range and that B, <7% and 4% for FSH, and 8.4% and 10.0% for leptin. Sensi-
contaminant levels—including heavy metals, fumonisin, aflatoxin, tivity was 0.04 ng/mL for testosterone, 7 pg/mL for inhibin B, 0.05
and pesticides—were below maximum acceptable levels. Test ng/mL for FSH, and 0.2 ng/mL for leptin. The testosterone, inhibin
subject diets were formulated on an individual basis such that the B, and leptin assays were performed by Toxicologic Pathology
rate of body weight gain in an individual nonhuman primate Associates in Jefferson, AR. The FSH assays were conducted by the
(NHP) was at 0.1 kg per month in the initial phases of the ex- Assay Core of the Specialized Cooperative Centers Program in
periment. The rate of gain was increased to 0.2 kg per month as the Reproduction and Infertility Research at the University of Pitts-
animals approached puberty. burgh School of Medicine. The diurnal serum samples were as-
sayed for luteinizing hormone (LH) using a homologous (macaque)
Dose Selection. Animals were assigned to dose groups by a ran- RIA (6) and for total testosterone using the solid-phase RIA
domized weight ranking. Weights were obtained 2 wk before the (Siemens) as described in Ramaswamy et al. (7). Intra- and inter-
scheduled start of dosing. Thirty consecutive numbers were assay coefficients of variation were < 7% for both LH and testos-
assigned to the monkeys according to their body weights. The terone. Sensitivity was 0.12 ng/mL for LH and 0.04 ng/mL for
monkeys were allocated to three treatment groups consecutively by testosterone. These analyses were conducted by the Assay Core of
randomly generating the group numbers. The animals assigned to the Pittsburgh Specialized Cooperative Centers Program in In-
the control group weighed between 2.8 and 4.2 kg, the low-dose fertility and Reproduction Research.
group animals weighed between 2.9 and 4.2 kg, and those assigned
to the high-dose group weighed between 3.0 and 4.5 kg. Semen Collection. At each time, semen collection was attempted
It should be noted that the initial dose selection in the juvenile from each test subject on four occasions within a 2-wk period, with a
monkeys was based on two previous studies in adult monkeys. The minimum of 1 d of rest between collection attempts. Semen col-
first study was a pharmacokinetics (PK) study (3) and the second lections were attempted in the normal restraint chairs used for
was a preliminary study that we conducted to determine the most blood collection, with additional restraint provided by the animal-
appropriate vehicle for MPH. In the latter study, doses of 0.15 mg/ care staff, if necessary, to minimize stress on the test subject.
kg of MPH dispersed in Prang resulted in peak plasma levels of 2.0 Semen was collected using penile electroejaculation. Initially, the
to 5.0 ng /mL in aged, female NHP. This level closely approxi- penis was massaged from the sheath, followed by the use of a small
mated the target pediatric blood level of 2 to 10 ng/mL described vibrator for penile stimulation. Two strips (4 × 0.5 cm) of non-
in Swanson and Volkow (4) and was selected as the low dose for metallic cardiac defibrillation pads were prepared and kept moist.
the study. A 10-fold increase in the dose (1.5 mg/kg) was selected One strip was wrapped around the base of the penis and the other
as the high dose to provide a broad dose range for the genetic and just below the glans. The two electrodes from the Grass S88
behavioral toxicity studies (1). However, it was subsequently ob- stimulator were attached to the pad strips. The stimulator settings
served that this dosing schedule produced inadequate serum were 20 pulses per second, 18-ms duration, and 20-ms delay. The
concentrations, requiring an increase in dose to produce clinically voltage was varied from 0 to 50 V (50 mAmp). Stimulation was
relevant concentrations. applied at the lowest voltage and gradually increased over ∼90 s.
A warm (37 °C) glass scintillation vial with 2 mL of PBS was
Exposure Assessment. Labeled internal standard (100 μL of 1 ng/mL used to collect the semen. The head of the penis was held inside
in 0.1% formic acid) was added to each thawed plasma sample the vial opening to catch the entire ejaculate, which was im-
(10 μL) and 800 μL 0.1% formic acid. Samples were purified using mediately transported to the laboratory. The sample was viewed
solid phase extraction in 96-well plates and analyzed using elec- for the presence of a copulatory plug, which was removed from

Mattison et al. www.pnas.org/cgi/content/short/1102187108 1 of 7

the specimen, if present. If no ejaculation occurred the animal plasma concentrations similar to those observed in humans (2–20
was allowed to rest for approximately 1 min and the stimulation ng/mL). The dose for the high-dose group was 1.5 mg/kg, which
was repeated. If no ejaculation occurred within 10 min, the was expected to result in plasma concentrations 5- to 10-times
animal was returned to his cage. higher (20–200 ng/mL) than observed clinically. However, plasma
concentrations of MPH were substantially lower than expected for
Semen Evaluation. Sperm viability was determined by hypo-osmotic the first 4 mo of the study; consequently, doses for both the low-
swelling (HOS assay). Sperm motility and concentrations were and high-dose groups were slowly increased to 2.5 mg/kg twice a
measured using the computer-assisted sperm analysis system day and 12.5 mg/kg twice a day, respectively. Treatment with these
(HTM-IVOS, Hamilton-Thorn). higher doses resulted in plasma levels that were ∼10 and 100 ng/
mL for the low- (Fig. S1, Upper) and high-dose (Fig. S1, Lower)
Sperm Morphometry. Sperm morphometry was conducted on one
groups, respectively. The concentrations in the low-dose group
slide per animal providing a semen sample for each collection
period. One hundred sperm per slide were analyzed using the were similar to the clinical range of 2 to 20 ng/mL (4). Plasma
Metric program of the HTM-IVOS System (Hamilton-Thorn). levels in the high-dose group were 5- to 10-fold higher than the
therapeutic range. The data for month 0 through month 30 has
Sperm Morphology. Sperm morphology was conducted on one previously been published in Morris et al. (1).
slide per animal providing a semen sample for each collection As noted above, plasma levels of MPH were lower than expected
period. Sperm morphology was evaluated using the strict criteria and were raised to achieve the appropriate levels. PK experiments
(8) for human sperm morphology and the traditional human were conducted at quarterly intervals over a 2-y dosing period and
morphology scheme (9), modified to include the forms noted in are presently undergoing analysis. This analysis may help to de-
initial evaluation of the NHP slides. The abnormal sperm clas- termine if the lower levels observed in this cohort are because of a
sifications were: lower rate of absorption in the intestine or a difference in first-pass
metabolism in the aged animals used in the preliminary study.
Head abnormalities: bicephalic, tapering, pyriform, abnormal
head size, amorphous head, ridged head, elongated head, Crown-Rump Measurements. ANOVA revealed a significant in-
pointed head; crease (P < 0.0001) in the crown-rump measurement over the
Neck and midpiece abnormalities: neck/midpiece defect, thick course of the experiment. No significant effect of dose was de-
midpiece, bulged midpiece, bent neck; and tected either overall (P = 0.61) or for any individual time point. No
Tail defects: missing tail, tail defect, hairpin tail, coiled tail, significant differences between the control and low-dose groups or
double tail, cytoplasmic droplet. between the control and the high-dose groups were detected either
In addition to the defects listed above, the number of white overall or at any time point.
blood cells per 100 sperm and the number of immature germ cells
Endocrine Measurements. Luteinizing hormone. The diurnal pattern of
per 100 sperm were calculated. Two-hundred sperm were clas-
serum LH concentrations was also assessed at month 35 (Fig. S3,
sified, except for samples with a low sperm concentration (n = 5),
where only 100 sperm were analyzed per sample. Upper) and Month 38 (Fig. S3, Lower). No effect of dose on serum
LH levels was found at any time point (P > 0.05). A significant
SI Results diurnal effect was found at month 35 (P = 0.0002), but not at
Body Weight. Each test subject was weighed five times per week month 38 (P = 0.1688).
throughout the 40-mo dosing period. Individual weight gain was FSH levels. Significant differences (P = 0.0084) in serum FSH levels
evaluated monthly and adjustments to the diet were made as were detected as the animals matured, but no consistent overall
necessary to maintain a similar rate of gain in all animals. Dose- effect of dose was observed (Fig. S4).
related effects on performance in behavior testing were noted and
Semen Analysis. No significant differences were found in the sperm
attributed to the anorexic effect of MPH (2). However, test subjects
were given 24-h availability to their daily ration, which precluded concentration from month 27 through month 38 of treatment.
an effect on weight gain. To evaluate the effect of dose and length Significant differences between the control and high-dose groups in
of treatment on body weight, the weight for each animal on the the percentage of sperm with elongated heads (P = 0.0019) were
same date each month was analyzed by ANOVA. A significant found only at month 27 (Fig. S5). Morphometric analysis of
increase (P < 0.0001) in body weight was detected during the 40- elongation factor (width/length × 100) indicated that the average
mo exposure period. However, neither the ANOVA nor the elongation factor was significantly reduced (P = 0.0123) in the
Dunnett’s test revealed a significant effect of dose on body weight. high-dose group at month 27 (Fig. S6). A significant interaction
between dose and time was found in the percentage of sperm with
Exposure Assessment. In designing this study, the initial dose for the elongated heads (P = 0.0459) and in the percentage of sperm with
low-dose group was 0.15 mg/kg, which was expected to produce reduced elongation factor (P = 0.0103).

1. Morris SM, et al. (2009) The genetic toxicology of methylphenidate hydrochloride in 6. El Majdoubi M, Ramaswamy S, Sahu A, Plant TM; S6 (2000) Effects of orchidectomy
non-human primates. Mutat Res 673(1):59e66. on levels of the mRNAs encoding gonadotropin-releasing hormone and other
2. Rodriguez JS, et al. (2010) The effects of chronic methylphenidate administration on hypothalamic peptides in the adult male rhesus monkey (Macaca mulatta). J
operant test battery performance in juvenile rhesus monkeys. Neurotoxicol Teratol Neuroendocrinol 12(2):167e176.
32(2):142e151. 7. Ramaswamy S (2005) Pubertal augmentation in juvenile rhesus monkey testosterone
3. Doerge DR, Fogle CM, Paule MG, McCullagh M, Bajic S (2000) Analysis of production induced by invariant gonadotropin stimulation is inhibited by estrogen.
methylphenidate and its metabolite ritalinic acid in monkey plasma by liquid J Clin Endocrinol Metab 90:5866e5875.
chromatography/electrospray ionization mass spectrometry. Rapid Commun Mass 8. World Health Organization (1992) WHO Laboratory Manual for the Examination of
Spectrom 14:619e623. Human Semen and Sperm-Cervical Mucus Interaction, 3rd ed. (Cambridge University
4. Swanson JM, Volkow ND (2003) Serum and brain concentrations of methylphenidate: Press, Cambridge).
Implications for use and abuse. Neurosci Biobehav Rev 27:615e621. 9. World Health Organization (1999) WHO Laboratory Manual for the Examination of
5. Ramaswamy S, Pohl CR, McNeilly AS, Winters SJ, Plant TM (1998) The time course of Human Semen and Sperm-Cervical Mucus Interaction, 4th ed. (Cambridge University
follicle-stimulating hormone suppression by recombinant human inhibin A in the adult Press, Cambridge).
male rhesus monkey (Macaca mulatta). Endocrinology 139:3409e3415.

Mattison et al. www.pnas.org/cgi/content/short/1102187108 2 of 7

Fig. S1. Mean monthly plasma MPH levels (± SEM) in peripubertal male rhesus monkeys chronically exposed to the low (Upper) or high (Lower) dose of MPH.
Plasma levels of MPH were determined by LC-ES-MS-MS.

Mattison et al. www.pnas.org/cgi/content/short/1102187108 3 of 7

Fig. S2. Effect of dose and length of treatment on mean serum leptin concentrations (± SEM) in peripubertal male rhesus monkeys chronically exposed to
MPH. A 3- to 4-mo peak in serum leptin levels occurred initially, followed by a trough, and then a progressive increase (P < 0.0001) as the monkeys matured. A
significant (P = 0.0188) overall effect of dose on serum leptin levels was detected by ANOVA. Serum leptin levels were higher in the low- and high-dose groups
than in the control group (P = 0.0212, P = 0.0316, respectively).

Mattison et al. www.pnas.org/cgi/content/short/1102187108 4 of 7

Fig. S3. Effect of dose and time of day on mean serum LH levels (± SEM) in male rhesus monkeys chronically exposed to MPH. Samples were collected at 1500,
2100, 0300, and 0900 hours. Significant time effects were detected (P = 0.0002) at month 35 (Upper), but not at month 38 (Lower). No effect of dose (P > 0.05)
on serum LH levels was found at either time point.

Mattison et al. www.pnas.org/cgi/content/short/1102187108 5 of 7

Fig. S4. Effect of dose and length of treatment on mean serum FSH concentrations (± SEM) in peripubertal male rhesus monkeys chronically exposed to MPH.
Although serum FSH levels increased (P = 0.0084) as the animals matured, no consistent effect of dose was detected by either ANOVA or the Dunnett’s test.

Fig. S5. Effect of dose on the mean percentage of sperm with elongated heads (± SEM) in peripubertal male rhesus monkeys chronically exposed to MPH. At
month 27, a significant increase (P = 0.0019) in the percentage of sperm with elongated heads. No effect of the low dose on the percentage of abnormal sperm
was detected.

Mattison et al. www.pnas.org/cgi/content/short/1102187108 6 of 7

Fig. S6. Effect of dose on the mean elongation factor of sperm (± SEM) obtained from peripubertal male rhesus monkeys chronically exposed to MPH. At
month 27, a significant decrease (P = 0.0123) in the mean elongation factor (width/length × 100) was found in the sperm obtained from the high-dose group
animals. No effect of the low dose on this measurement was detected.

Mattison et al. www.pnas.org/cgi/content/short/1102187108 7 of 7