Alan M. Hoberman

A 90-day oral gavage toxicity study of D-methylphenidate and D, L-methylphenidate in Sprague-Dawley rats

D-methylphenidate is an enantiomer of D,L-methylphenidate and was developed as an improved treatment for attention deficit hyperactivity disorder in children. The current study was performed to determine and compare the toxicity of 2-50 mg/kg per day D-MPH and 100 mg/kg per day D,L-MPH for 90 days in rats with the top D-MPH dose being equimolar to 100 mg/kg D,L-MPH. The top D-MPH and D,L-MPH doses were at least 67 times that of the human dose and produced systemic exposures that were over 10 times higher than those typically achieved in children. During the course of the study, one male each from the 50 mg/kg per day and D,L-MPH groups and one female from the 50 mg/kg group died. Incidences of material around nose/eyes, scabbing, foot swelling, alopecia and abrasions were evident at 50 mg/kg per day D-MPH and 100 mg/kg per day D,L-MPH doses. Body weight and its changes decreased in a dose-dependent manner for D-MPH males. There were significant changes in some clinical chemistry measurements at the terminal bleed in the high dose groups of both sexes although most of these changes were resolved by the recovery bleed. Differences in absolute and relative body and certain organ weights for high dose D-MPH and D,L-MPH groups were seen at terminal necropsy with the differences no longer present after the recovery period. No abnormal or gross histopathological changes were associated with any of these organ weight changes reported for the terminal and recovery periods. Based on body weight changes, the no observed adverse effect level for D-MPH in rats was 20 mg/kg. Overall, the toxicity profile observed in rats with 50 mg/kg per day D-MPH was comparable to that of an equimolar dose of D,L-MPH (100 mg/kg per day) when given repeatedly for 90 days using a twice a day dosing regimen.

SEXUAL MATURATION DATA FOR CRL SPRAGUE-DAWLEY RATS: CRITERIA AND CONFOUNDING FACTORS

ABSTRACT Background: Considerable concern exists in the scientific community regarding potential effects of endocrine disruptive or modulating environmental agents on male and female reproductive development and capacity. Existing data show that in utero and postnatal exposure of rodents to endocrine modulating chemicals can influence the timing and progression of sexual differentiation and/or maturation (e.g., balano-preputial separation and vaginal opening). Methods: Sexual maturation data from various types of littering studies using International Gold Standard (IGS) Crl Sprague-Dawley rats were evaluated for consistency with both historical observations and published values from other laboratories. In addition, litters from two developmental neurotoxicology studies were statistically analyzed to identify whether increasing the numbers of pups per litter evaluated affected the interpretation of sexual maturation data sets. Results: Control values for preputial separation and vaginal opening ages ranged from PD 45.0 to 48.0 and from PD 32.0 to 34.0, respectively, regardless of the number of pups evaluated per litter. However, statistically significant delays in sexual maturation were present when three rats/sex/litter were evaluated that were not present when only one randomly selected rat/sex/litter was evaluated. Conclusions: Standardized procedures and criteria are required to provide consistent intra-laboratory values and reduce inter-laboratory differences in sexual maturation observations. When such criteria are used, these endpoints provide sensitive measures for detecting alterations in sexual maturation. However, our analyses demonstrate that the ability to detect statistically significant and biologically important differences in these endpoints is sometimes impaired by the currently common practice of evaluating only one randomly selected rat/sex/litter. Evaluation of three rats/sex/litter improved the sensitivity of the statistical analysis in detection of treatment-related effects and reduced the probability of identifying a false negative result.

Lack of effect of butylparaben and methylparaben on the reproductive system in male rats

BACKGROUND Parabens are widely used preservatives in cosmetics and pharmaceutical products, and approved as food additives. Parabens have been considered safe for these uses for many years. Recently, adverse effects on male reproductive parameters in rats have been reported when parabens were given orally for 8 weeks starting at three weeks of age. Our studies used two representative parabens, methyl- and butylparaben, to try to replicate these studies and thereby evaluate potential reproductive effects in male Wistar rats. METHODS Diets containing 0, 100, 1000 or 10,000 ppm of either butyl- or methylparaben were fed to male rats for eight weeks. Rats were 22 days of age at the start of exposure. Parameters evaluated included organ weights, histopathology of reproductive tissues, sperm production, motility, morphology and reproductive hormone levels (butylparaben only). RESULTS None of the parameters evaluated for either paraben showed compound- or dosage-dependent adverse effects. Metabolism experiments of butylparaben indicate that it is rapidly metabolized by non-specific esterases to p-hydroxybenzoic acid and butanol, neither of which is estrogenic. CONCLUSIONS Exposure to methyl- or butylparaben in the diet for eight weeks did not affect any male reproductive organs or parameters at exposures as high as 10,000 ppm, corresponding to a mean daily dose of 1,141.1+/-58.9 or 1,087.6+/-67.8 mg/kg/day for methyl- and butylparaben, respectively. The rapid metabolism of parabens by esterases probably explains why these weakly estrogenic substances elicit no in vivo effects when administered by relevant exposure routes (i.e., topical and oral).

Pharmaceutical toxicology: Designing studies to reduce animal use, while maximizing human translation

Evaluation of the safety of new chemicals and pharmaceuticals requires the combination of information from various sources (e.g. in vitro, in silico and in vivo) to provide an assessment of risk to human health and the environment. The authors have identified opportunities to maximize the predictivity of this information to humans while reducing animal use in four key areas; (i) accelerating the uptake of in vitro methods; (ii) incorporating the latest science into safety pharmacology assessments; (iii) optimizing rodent study design in biological development and (iv) consolidating approaches in developmental and reproductive toxicology. Through providing a forum for open discussion of novel proposals, reviewing current research and obtaining expert opinion in each of the four areas, the authors have developed recommendations on good practice and future strategy.

Juvenile animal toxicity study designs to support pediatric drug development

The objective of juvenile animal toxicity studies of pharmaceuticals is to obtain safety data, including information on the potential for adverse effects on postnatal growth and development. Studies in juvenile animals may assist in identifying postnatal developmental toxicities or other adverse effects that are not adequately assessed in the routine toxicity evaluations and that cannot be safely or adequately measured in pediatric clinical trials. Unlike the traditional reproductive and developmental toxicology studies that have been discussed in the accompanying reports, the design requirements for toxicity studies in juvenile animals are not explicitly defined in regulatory guidance. However, studies in juvenile animals can be useful in providing safety information necessary to enable pediatric clinical trials in pediatric patients or when there are special concerns for toxicities that cannot be safely or adequately measured in clinical trials. These juvenile animal toxicity studies are designed on a case-by-case basis. General design considerations and examples of study designs for assessment of juvenile animal toxicity are discussed.

The Toxicity Profile of Hydrolyzed Aqueous Olive Pulp Extract

The toxicity profile of HIDROX™ (Hydrolyzed Aqueous Olive Pulp Extract; OPE) was characterized in a series of toxicology studies. A limit dosage of 2000 mg/kg produced no toxicity in mice (acute oral NOAEL: 2000 mg/kg). In rats, an acute oral NOAEL of 2000 mg/kg was established, based on reductions in weight gains in both sexes at 5000 mg/kg. Reduced gains in female rats at 1500 and 2000 mg/kg were not significantly different from control values. Daily oral dosages of 1000, 1500 and 2000 mg/kg/day for 90 days produced small decreases in body weight gains at 2000 mg/kg/day in the male rats and in all groups of female rats. Feed consumption was comparable to controls. There were no adverse clinical, hematologic, biochemical, organ weight or gross necropsy effects. Focal, minimal or mild hyperplasia of the mucosal squamous epithelium of the limiting ridge of the forestomach occurred in some rats at 2000 mg/kg/day; this change was attributed to local irritation by repeated intubation of large volumes of viscous, granular dosing suspension. A NOAEL of 2000 mg/kg/day was established for the 90‐day study, based on the lack of significant adverse effects. Toxicokinetic data indicated that hydroxytyrosol (HT, the major component of OPE) was rapidly absorbed. Mean concentrations were measurable through 1 to 4 hours (tlast) at 1000 and 1500 mg/kg/day and through 8 hours at 2000 mg/kg/day. Dosages of OPE ranging from 500 to 2000 mg/kg/day did not adversely affect any of the mating, fertility, delivery or litter parameters investigated in an oral rat dosage‐range reproduction study. Adverse effects were also absent in a rat developmental toxicity study in which pregnant dams were treated with 1000, 1500 or 2000 mg/kg/day on days 6 through 20 of gestation. Plasma levels for pregnant and lactating rats were comparable to non‐pregnant rats; minimal levels crossed the placenta. Quantifiable levels were not identified in maternal milk or plasma from nursing pups. A bacterial reverse mutation and a CHO chromosome aberration assay revealed evidence of mutagenic activity at high dosages with S9 metabolic activation. However, three rat micronucleus evaluations performed after single and repeated (28‐day) dosages of up to 2000 mg/kg/day and dosages of 5000 mg/kg/day for 29 days resulted in negative findings; therefore, OPE was not considered to be mutagenic in this in vivo assay.

Oral (Drinking Water) Two-Generation Reproductive Toxicity Study of Dibromoacetic Acid (DBA) in Rats

In a two-generation study of dibromoacetic acid (DBA), Crl SD rats (30 rats/sex/group/generation) were provided DBA in drinking water at 0 (reverse osmosis-deionized water), 50,250, and 650 ppm (0,4.4 to 11.6,22.4 to 55.6, and 52.4 to 132.0 mg/kg/day, respectively; human intake approximates 0.1 μg/kg/day [0.0001 mg/kg/day]). Observations included viability, clinical signs, water and feed consumption, body and organ weights, histopathology, and reproductive parameters (mating, fertility, abortions, premature deliveries, durations of gestation, litter sizes, sex ratios and viabilities, maternal behaviors, reproductive organ weights, sperm parameters and implantation sites, sexual maturation). Histopathological evaluations were performed on at least 10 P and F1 rats/sex at 0 and 650 ppm (gross lesions, testes, intact epididymis; 10 F1 dams at 0, 250, and 650 ppm for primordial follicles). Developmental observations included implantations, pup numbers, sexes, viabilities, body weights, morphology, and reproductive performance. At 50 ppm and higher, both sexes and generations had increased absolute and relative liver and kidneys weights, and female rats in both generations had reduced absolute and relative adrenal weights; adrenal changes were probably associated with physiological changes in water balance. The livers and kidneys (10/sex/group/generation) had no histopathological changes. Other minimal effects at 50 ppm were reduced water consumption and a transient reduction in body weight. At 250 and 650 ppm, DBA reduced parental water consumption, body weight gains, body weights, feed consumption, and pup body weights. P and F1 generation male rats at 250 and 650 ppm had altered sperm production (retained step 19 spermatids in stages IX and X tubules sometimes associated with residual bodies) and some epididymal tubule changes (increased amounts of exfoliated spermatogenic cells/residual bodies in epididymal tubules, atrophy, and hypospermia), although inconsistently and at much lower incidences. Unilateral abnormalities of the epididymis (small or absent epididymis) at 650 ppm in four F1 generation male rats were considered reproductive tract malformations. The no-observable-adverse-effect level (NOAEL) and reproductive and developmental NOAELs for DBA were at least 50 ppm (4.5 to 11.6 mg/kg/day), 45,000 to 116,000 times the human adult exposure level. Reproductive and developmental effects did not occur in female rats exposed to DBA concentrations as high as 650 ppm. Based on the high multiples of human exposure required to produce effects in male rats, DBA should not be identified as a human reproductive or developmental risk.

Evaluation of the Developmental Toxicity of Linalool in Rats

The developmental toxicity of linalool, a widely used fragrance ingredient, was evaluated in presumed pregnant Sprague-Dawley rats (25/group). Oral dosages of 0, 250, 500, or 1000 mg/kg/day linalool were administered by gavage on gestational days 7 to 17. The presence of spermatozoa and/or a copulatory plug in situ was designated as gestational day 0. Rats were observed for viability, clinical signs, body weights, and feed consumption. Caesarean sectioning and necropsy occurred on gestational day 21. Uteri were examined for number and distribution of implantations, live and dead fetuses, and early and late resorptions. Numbers of corpora lutea were also recorded. Fetuses were weighed and examined for gender, gross external changes, and soft tissue or skeletal alterations. There were no maternal deaths, clinical signs, or gross lesions that were considered related to linalool. During the dosage period, mean relative feed consumption was significantly reduced by 7% and mean body weight gains were reduced by 11% at 1000 mg/kg/day. During the postdosage period, feed consumption values at 1000 mg/kg/day were significantly higher than vehicle control values, which corresponded to the increase in body weight gains during this period. Caesarean section and litter parameters, as well as fetal alterations, were not affected by linalool at any of the three dosages tested. On the basis of these data, the maternal no observed adverse effect level (NOAEL) of linalool is 500 mg/kg/day, whereas the developmental NOAEL is ≥ 1000 mg/kg/day. It is concluded that linalool is not a developmental toxicant in rats at maternal doses of up to 1000 mg/kg/day.

Effects of Administration of a Monoclonal Antibody against Mouse Tumor Necrosis Factor Alpha during Pregnancy and Lactation on the Pre- and Postnatal Development of the Mouse Immune System:

Monoclonal antibodies directed against tumor necrosis factor alpha (TNFα) are currently employed in the treatment of various immune-mediated diseases. These studies were designed to evaluate potential effects of anti-TNFαtreatment in mice during pregnancy and lactation on the development of the immune system in the F1 generation. Pregnant CD-1 mice were treated with vehicle or with 10 or 40 mg/kg of an anti-mouse TNFαmonoclonal antibody (mAb) (cV1q) on days 6, 12, and 18 of gestation and on days 3, 9, and 15 of lactation. Evaluation of immune system functionality was conducted in F1 generation mice at 11 weeks of age. Immune function was evaluated by splenocyte phenotyping, immunoglobulin M (IgM) antibody response to sheep red blood cells (SRBCs), spleen cell proliferative response to anti-CD3, and natural killer cell activity. Treatment of pregnant mice with cV1q produced no adverse effects in the dams and no adverse effects in the F1 generation. In general, the functioning of the immune system of the F1 generation did not appear to be adversely affected following exposure to cV1q in utero and during lactation. The only statistically significant change was a slight (~20%) reduction in the spleen cell expansion in response to SRBC immunization in the female F1 mice from the 40 mg/kg cV1q treatment group. In conclusion, administration of a monoclonal antibody against mouse TNFαduring pregnancy and lactation had little or no effect on selected immune parameters in mice, with only a possible minor attenuation of spleen cell response to immunization noted in the female F1 generation at 11 weeks of age.

The perinatal and postnatal toxicity of D-methylphenidate and D,L-methylphenidate in rats.

D-methylphenidate is an enantiomer of D,L-methylphenidate and was developed as an improved treatment for attention deficit hyperactivity disorder (ADHD) in children. The current study was performed to assess the potential perinatal and postnatal toxicity of both compounds in rats. About 125 presumed pregnant rats were assigned to five dose groups of 25 each. They were dosed with 2, 6, and 20 mg/kg/day D-methylphenidate and 40 mg/kg/day D,L-methylphenidate from gestation Day 7 to lactation Day 20. F1 generation rats were rebred to produce F2 fetuses. Various perinatal and postnatal measurements were made for the F0 and F1 rats. Among the significant findings were a reduction in maternal body weight gain for 20 mg/kg/day D-methylphenidate and D,L-methylphenidate and increased incidences of dilated pupil and vocalization for D,L-methylphenidate during the gestation period. Neither compound produced any other significant adverse findings in F0 and F1 generation rats at doses that were at least 25 times the maximum daily human therapeutic dose.