Two-generation Study Research Articles

Effects of the wood extractives dehydroabietic acid and betulinol on reproductive physiology of zebrafish ( Danio rerio)—A two-generation study

Two wood extractives, dehydroabietic acid (DHAA) and betulinol (BET), present in wood industry effluents were evaluated for their potential effects on the reproductive physiology of zebrafish. Adult zebrafish (F0) were exposed in a continuous flow-through system to 50 μg/l DHAA, 5 μg/l BET and 0.27 μg/l (1 nM) 17β-estradiol (E2) for 3 months. Eggs were collected from F0 fish and the following F1 generation was exposed for 6 months. Biomarkers analyzed in both F0 and F1 fish were plasma vitellogenin (Vtg), testosterone (T), E2 (only females) and gonadal histology. DHAA and BET affected growth in terms of increased condition factor, and spawning was stimulated in BET-exposed fish of the F0 generation. F0 males exposed to DHAA and F0 females exposed to BET showed lower plasma Vtg concentration, but F1 males exposed to BET showed an increase in Vtg. In fish exposed to E2, the positive control for estrogenic effects, a pronounced increase in Vtg concentration was observed. Plasma sex steroids were not significantly affected by the wood extractives. However, although not statistically significant, the T concentration tended to be lower in fish of all BET treatments. The histological study revealed alterations in spermatogenic stages of F0 males exposed to DHAA and BET, which were different from those caused by E2. In F1 females, the percentage of vitellogenic oocytes was decreased in DHAA, BET and E2 exposures. This study shows that DHAA and BET may contribute to growth alterations and reproductive disturbances reported in fish exposed to pulp and paper mill effluents. Further, these wood extractives may have different effects in F0 and F1 generation fish, which highlights the value of two-generation studies in investigations regarding endocrine disrupting compounds.

Edentulous Motherhood is Associated with Child Caries

Dental Caries Experience: A Two-Generation Study Bedos C, Brodeur JM, Arpin S, Nicolau B. J Dent Res 2005;84(10):931-6 3b The objective of this study was to associate the mother's dentate status with the children's prevalence of caries. The study was supported by the Ministère de la Santé et des Services Sociaux du Québec, the Canadian Institutes of Health Research (CIHR), and the Fonds de Recherche en Santé du Québec (FRSQ). Cross-Sectional Design

A retrospective analysis of the two-generation study: What is the added value of the second generation?

A retrospective analysis of the two-generation study: What is the added value of the second generation?

Evaluation of ibuprofen developmental toxicity—Two-generation study

Evaluation of ibuprofen developmental toxicity—Two-generation study

A retrospective analysis of the added value of the rat two-generation reproductive toxicity study versus the rat subchronic toxicity study

This study aims to evaluate the added value of the two-generation reproductive toxicity study when a subchronic study (90-day repeated dose toxicity study) is available. The analysis includes a total of 47 reproductive toxic and 75 non-reproductive toxic substances, for which a two-generation study was available. For each of these compounds the outcomes of both study types were compared, in view of the question what the impact would have been both for the derived NOAEL and for classification regarding toxicity to fertility. On average, only a small difference (less than twofold) in overall NOAELs was found between the rat two-generation study and the rat subchronic study. For individual compounds the differences could be larger (up to around a factor of 10), but differences of this magnitude equally occur between NOAELs of subchronic studies (testing the same substance). The two generation study did have an impact on classification for toxicity to fertility: about one-third of the substances shown to be toxic to fertility in the two-generation study did not show any sign of that in the 90-day study. If the subchronic study did show toxicity to reproductive organs this often occurred at (much) higher doses than other toxic effects in the same study. Therefore, apart from including more fertility endpoints, a larger dose spacing (or more dose groups) in the subchronic study might increase its detection rate of fertility toxic substances. The consequences that these findings may have for risk assessment and risk management are discussed, especially in the context of REACH.

A retrospective analysis of the two-generation study: What is the added value of the second generation?

Increasing pressure is exerted by some stakeholders to reduce the two-generation study to a one-generation study, a measure that would considerably reduce the number of animals and other costs involved in these lengthy studies. The present study retrospectively evaluates 176 multi-generation studies to assess potential differences between the first and the second generation, both in terms of the types of effects observed and in terms of the effective doses. All substances classified as reproductive toxicants by the Directive 92/32/EEC or considered as toxic to fertility by the California EPA for which we found a multi-generation study were included ( n = 58 studies). The second generation in the two-generation studies considered affected neither the overall NOAEL nor the critical effect. Therefore, it had no impact on the ensuing risk assessment, nor on classification and labeling. However, several substances did show an increased sensitivity of the F 1 adults in comparison to the P 0. These results support the proposal of replacing the current two-generation study by a one-generation study with a more extensive assessment of parameters at F 1 adulthood.

A two-generational reproductive toxicity study of zinc in rats

A two-generation reproductive toxicity study of zinc chloride (ZnCl2) was conducted in rats. Fo male and female rats were administered 0.00 (control), 7.50 (low), 15.00 (mid) and 30.00 (high) mg/kg/day of ZnCl2. Selected F1 male and female rats were exposed to the same doses received by their parents (Fo). Exposure of F0 parental rats to ZnCl2 showed significant reduction in fertility, viability (days 0 and 4), and the body weight of F1 pups from the high-dose group but caused no effects on litter size, weaning index, and sex ratio. Similarly, the continued exposure of F1 parental rats to ZnCl2 also reduced fertility, liter size, viability (day 0), and the body weight of F2 pups within the high-dose group but caused no effects on weaning index and sex ratio. Exposure of ZnCl2 to F0 and F1 parental males resulted in a significant reduction in their body weights, and the F0 and F1 parental females did not show any significant difference in their body weights compared to their control groups. However, the postpartum dam weights of both F0 and F1 female rats were significantly reduced compared to their controls. Exposure of ZnCl2 to Fo and F1 generation parental rats did not produce any significant change of their clinical signs as well as their clinical pathology parameters, except the alkaline phosphotase (ALK) level, which showed an upward trend in both sexes of both generations. Exposure of ZnCl2 to F0 rats resulted in a reduction of brain, liver, kidney, spleen and seminal vesicles weights of males and in the spleen and uterus of females. Similarly, exposure of F1 rats to ZnCl2 also resulted in reduction of brain, liver, kidney, adrenal, spleen, prostate and seminal vesicles weights of males and in spleen and uterus of females. ZnCl2 exposure resulted in grossly observed gastro-intestianla (GI) tract, lymphoreticular/hematopoietic, and reproductive tract lesions in parental rats in both generations. Reduced body fat was also recorded in F1 parental rats.

Edentulous Motherhood Is Associated with Child Caries

Dental Caries Experience: A Two-Generation Study Bedos C, Brodeur JM, Arpin S, Nicolau B. J Dent Res 2005;84(10):931-6 3b The objective of this study was to associate the mother’s dentate status with the children’s prevalence of caries. The study was supported by the Ministère de la Santé et des Services Sociaux du Québec, the Canadian Institutes of Health Research (CIHR), and the Fonds de Recherche en Santé du Québec (FRSQ). Cross-Sectional Design

Tools to evaluate estrogenic potency of dietary phytoestrogens:A consensus paper from the EU Thematic Network “Phytohealth” (QLKI-2002-2453)

Phytoestrogens are naturally occurring plantderived polyphenols with estrogenic potency. They are ubiquitous in diet and therefore, generally consumed. Among Europeans, the diet is rich in multiple putative phytoestrogens including flavonoids, tannins, stilbenoids, and lignans. These compounds have been suggested to provide beneficial effects on multiple menopause-related conditions as well as on development of hormone-dependent cancers, which has increased the interest in products and foods with high phytoestrogen content. However, phytoestrogens may as well have adverse estrogenicity related effects similar to any estrogen. Therefore, the assessment of estrogenic potency of dietary compounds is of critical importance. Due to the complex nature of estrogenicity, no single comprehensive test approach is available. Instead, several in vitro and in vivo assays are applied to evaluate estrogenic potency. In vitro estrogen receptor (ER) binding assays provide information on the ability of the compound to I) interact with ERs, II) bind to estrogen responsive element on promoter of the target gene as ligand-ER complex, and III) interact between the co-activator and ERs in ligand-dependent manner. In addition, transactivation assays in cells screen for ligand-induced ERmediated gene activation. Biochemical in vitro analysis can be used to test for possible effects on protein activities and E-screen assays to measure (anti)proliferative response in estrogen responsive cells. However, for assessment of estrogenicity in organs and tissues, in vivo approaches are essential. In females, the uterotrophic assay is applicable for testing ERa agonistic and antagonistic dietary compounds in immature or adult ovariectomized animals. In addition, mammary gland targeted estrogenicity can be detected as stimulated ductal elongation and altered formation of terminal end buds in immature or peripubertal animals. In males, Hershberger assay in peri-pubertal castrated rats can be used to detect (anti)androgenic/ (anti)estrogenic responses in accessory sex glands and other hormone regulated tissues. In addition to these short-term assays, sub-acute and chronic reproductive toxicity assays as well as two-generation studies can be applied for phytoestrogens to confirm their safety in long-term use. For reliable assessment of estrogenicity of dietary phytoestrogens in vivo, special emphasis should be focused on selection of the basal diet, route and doses of administration, and possible metabolic differences between the species used and humans. In conclusion, further development and standardization of the estrogenicity test methods are needed for better interpretation of both the potential benefits and risks of increasing consumption of phytoestrogens from diets and supplements.

The costs of REACH

Early next year, the European Union (EU) plans to implement a major piece of legislation—about 1,200 pages in its current form—to replace 40 different laws regulating the use of chemicals across the member states. When the European Commission (EC) presented its first draft in May 2003 (EC, 2003), it immediately drew huge attention because of its far‐reaching consequences, not only economic but also on human health and the environment. If passed, the REACH legislation will require the chemical industry to provide information on the potential negative effects—most of which are still unknown—of a vast number of chemicals used throughout the EU. Not surprisingly, this has met with criticism from all quarters. The costs of obtaining this information, and the subsequent effects on the European chemical industry, are hotly disputed. > [REACH] immediately drew huge attention because of its far‐reaching consequences, not only economic but also on human health and the environment REACH dictates new rules on the Registration, Evaluation and Authorisation of CHemicals. It requires manufacturers and downstream users of chemicals to register their substances—if they are sold in the EU as isolated compounds or in preparations—either with a national regulatory authority or a central European Chemical Agency, which the EC plans to establish next year in Helsinki, Finland. Companies are required to submit a registration dossier for each product, listing possible adverse effects on human health or the environment. REACH also requires additional authorization for substances that the EC considers of “very high concern”: chemicals that are carcinogenic, mutagenic or toxic to the reproductive system; endocrine disruptors; or persistent, bioaccumulative and toxic substances. Producers or downstream users must perform a risk assessment to ascertain whether their product creates risks to human health or the environment, which social benefits it provides, and whether safer alternatives are available. If, on the basis …

Safety studies on epigallocatechin gallate (EGCG) preparations. Part 3: Teratogenicity and reproductive toxicity studies in rats

Green tea and its principal active ingredient, epigallocatechin gallate (EGCG), have been demonstrated to have anticancer properties through interactions with multiple biochemical processes. Since these processes are often crucial in normal fetal development it is important to evaluate the potential effects of EGCG on the fetus. EGCG preparations of >91% purity were administered to pregnant rats during organogenesis and development in order to define the safety of Teavigo™, a high-concentration EGCG extract produced by the same novel method. In an initial preliminary study using subcutaneous and gavage routes, there was no evidence of any direct embryo–fetal toxicity, although some maternal toxicity was seen. In the main teratogenicity study, feeding pregnant rats diets supplemented at 1400, 4200 or 14,000 ppm during organogenesis was non-toxic to dams or fetuses. A two-generation study in rats fed 1200, 3600 or 12,000 ppm EGCG preparation showed no adverse effects on reproduction or fertility. The highest dose reduced the growth rate of offspring, and there was a slight increase in pup loss. A growth effect among pups was also seen at 3600 ppm, but in the second generation only. The lowest dose was considered the overall no-observed adverse effect level (NOAEL). As dams consumed twice the amount of feed during the crucial lactation period, the NOAEL was equivalent to 200 mg/kg/day EGCG preparation.

Dental Caries Experience: A Two-generation Study

The life-course framework stresses the importance of social, psychosocial, and biological factors in early life on the development of later disease. From this perspective, the association between edentulousness of mothers and their children's caries risk has not been studied. Therefore, a sample of 6303 mother-child pairs was randomly selected in Quebec (Canada). Mothers (6039 dentate and 264 edentulous) completed a self-administered questionnaire, and their children, aged 5 to 9 years, were clinically examined. Bivariate analyses and multiple logistic regressions showed that edentulous mothers' children are more likely to experience caries on both primary [OR=1.7 (1.3-2.3)] and permanent [OR=1.4 (1.0-2.0)] dentitions when compared with dentate mothers' children. These results are independent of socio-economic status, age, gender, and children's oral-health-related behaviors. Our study is the first to show that edentulous mothers' children constitute a group at risk of caries. It also highlights the need for a better understanding of the mother-child transmission of risk.

Two-generation reproduction and cross-foster studies of perfluorooctanesulfonate (PFOS) in rats

Perfluorooctanesulfonate (PFOS) is a persistent acid found widely distributed in wildlife and humans. To understand the potential reproductive and developmental effects of PFOS, a two-generation reproduction study was conducted in rats. Male and female rats were dosed via oral gavage at dose levels of 0, 0.1, 0.4, 1.6, and 3.2 mg/(kg day) for 6 weeks prior to mating, during mating, and, for females, through gestation and lactation, across two generations. Due to substantial F 1 neonatal toxicity observed in the 1.6 and 3.2 mg/(kg day) groups, continuation into the second generation was limited to F 1 pups from the 0, 0.1, and 0.4 mg/(kg day) groups. No adverse effects were observed in F 0 females or their fetuses upon caesarean sectioning at gestation day 10. Statistically significant reductions in body-weight gain and feed consumption were observed in F 0 generation males and females at dose levels of 0.4 mg/(kg day) and higher, but not in F 1 adults. PFOS did not affect reproductive performance (mating, estrous cycling, and fertility); however, reproductive outcome, as demonstrated by decreased length of gestation, number of implantation sites, and increased numbers of dams with stillborn pups or with all pups dying on lactation days 1–4, was affected at 3.2 mg/(kg day) in F 0 dams. These effects were not observed in F 1 dams at the highest dose tested, 0.4 mg/(kg day). Neonatal toxicity in F 1 pups, as demonstrated by reduced survival and body-weight gain through the end of lactation, occurred at a maternal dose of 1.6 mg/(kg day) and higher while not at dose levels of 0.1 or 0.4 mg/(kg day) or in F 2 pups at the 0.1 or 0.4 mg/(kg day) dose levels tested. In addition to these adverse effects, slight yet statistically significant developmental delays occurred at 0.4 (eye opening) and 1.6 mg/(kg day) (eye opening, air righting, surface righting, and pinna unfolding) in F 1 pups. Based on these data, the NOAELs were as follows: reproductive function: F 0 ≥ 3.2 and F 1 ≥ 0.4 mg/(kg day); reproductive outcome: F 0 = 1.6 and F 1 ≥ 0.4 mg/(kg day); overall parental effects: F 0 = 0.1 and F 1 ≥ 0.4 mg/(kg day); offspring effects: F 0 = 0.4 and F 1 ≥ 0.4 mg/(kg day). To distinguish between maternal and pup influences contributing to the perinatal mortality observed in the two-generation study, a follow-up cross-foster study was performed. Results of this study indicated that in utero exposure to PFOS causally contributed to post-natal pup mortality, and that pre-natal and post-natal exposure to PFOS was additive with respect to the toxic effects observed in pups.

Neonatal mortality from in utero exposure to perfluorooctanesulfonate (PFOS) in Sprague–Dawley rats: Dose–response, and biochemical and pharamacokinetic parameters

Perfluorooctanesulfonate (PFOS) is a widely distributed, environmentally persistent acid found at low levels in human, wildlife, and environmental media samples. Neonatal mortality has been observed following PFOS exposure in a two-generation reproduction study in rats and after dosing pregnant rats and mice during gestation. Objectives of the current study were to better define the dose–response curve for neonatal mortality in rat pups born to PFOS-exposed dams and to investigate biochemical and pharmacokinetic parameters potentially related to the etiology of effects observed in neonatal rat pups. In the current study, additional doses of 0.8, 1.0, 1.2, and 2.0 mg/kg/day were included with original doses used in the two-generation study of 0.4 and 1.6 mg/kg/day in order to obtain data in the critical range of the dose–response curve. Biochemical parameters investigated in dams and litters included: (1) serum lipids, glucose, mevalonic acid, and thyroid hormones; (2) milk cholesterol; and (3) liver lipids. Pharmacokinetic parameters investigated included the interrelationship of administered oral dose of PFOS to maternal body burden of PFOS and the transfer of maternal body burden to the fetus in utero and pup during lactation, as these factors may affect neonatal toxicity. Dosing of dams occurred for 6 weeks prior to mating with untreated breeder males, through confirmed mating, gestation, and day four of lactation. Dose levels for the dose–response and etiological investigation were 0.0, 0.4, 0.8, 1.0, 1.2, 1.6, and 2.0 mg/kg/day PFOS. Statistically significant decreases in gestation length were observed in the 0.8 mg/kg and higher dose groups. Decreases in viability through lactation day 5 were observed in the 0.8 mg/kg and higher dose groups, becoming statistically significant in the 1.6 and 2.0 mg/kg dose groups. Reduced neonatal survival did not appear to be the result of reductions in lipids, glucose utilization, or thyroid hormones. The endpoints of gestation length and decreased viability were positively correlated, suggesting that late-stage fetal development may be affected in pups exposed to PFOS in utero and may contribute to the observed mortality. Benchmark dose (BMD) estimates for decreased gestation length, birth weight, pup weight on lactation day 5, pup weight gain through lactation day 5, and viability resulted in values ranging from 0.27 to 0.89 mg/kg/day for the lower 95% confidence limit of the BMD 5 (BMDL 5). Results of analyses for PFOS in biological matrices indicate a linear proportionality of mean serum PFOS concentration to maternal administered dose prior to mating and through the first two trimesters of gestation. However, at 21 days of gestation, mean serum PFOS concentrations were notably reduced from values measured earlier in gestation. Urinary and fecal elimination was low as expected from prior observations in adult rats. Significant transfer of PFOS from dam to fetus in utero was confirmed, and results suggest that dam and corresponding fetal body burdens, as indicated by serum and liver PFOS levels, correlate with neonatal survival.

A safety study of oral xanthohumol administration and its influence on fertility in Sprague Dawley rats

Xanthohumol (XN) is a prenylated chalcone, which has been shown to possess a broad range of potential cancer preventive and additional biological activities. In the present study, we have determined the subchronic 4-wk toxicity of XN and monitored its influence on fertility and development of offspring in two fertility studies. Four-week-old female Sprague Dawley (SD) rats were treated with 0.5% XN in the diet or with 1,000 mg XN/kg body weight (b.w.) per day by gavage for 28 days. No remarkable treatment-related changes in general appearance and b.w. occurred during the study. After autopsy, liver, kidney, lung, heart, stomach, and spleen were examined macroscopically and histopathologically. Relative liver weights of animals in both treatment groups were significantly reduced by 30--40% in comparison with the control group, indicating weak hepatotoxicity. Also, mammary glands of treated rats appeared less developed compared to the controls. Consequently, we investigated the influence of XN on rat reproduction. In two fertility studies, XN (100 mg/kg b.w. per day), given either for 4 wk prior to or during mating, gestation, and nursing, did not cause any adverse effects on female reproduction and the development of offspring. Noteworthy, treatment of male rats prior to mating significantly (p=0.027) increased the sex ratio of male to female offspring. Overall, lifelong treatment at a daily dose of 100 mg/kg b.w. in a two-generation study did not affect the development of SD rats.

Consequences of endocrine disrupting chemicals on reproductive endocrine function in birds: Establishing reliable end points of exposure

It has been difficult to establish reliable indices of exposure to endocrine disrupting chemicals (EDCs) appropriate for a variety of avian species because of a vast array of reproductive strategies. Data from mammals, reptiles and fish provide insight on likely mechanisms of action for EDCs. However, many of the effects of EDCs are weaker than the actions of the native hormones, making it difficult to assess adverse effects in domestic and wild birds. It is clear that differential sensitivity to EDCs exists across species, due to the timing and mode of exposure, compound toxicity and age of the individual. Our studies on EDCs are conducted in the quail model system, with focus on reproductive endocrine, neuroendocrine and behavioral responses. Studies have included EDC exposure, either by egg injection or via diet. Results from egg injection studies showed the following: (1) estradiol administered by embryonic day 12 demasculinized male sexual behavior, altered hypothalamic neurotransmitters and reduced hen day production and fertility in a dose dependent fashion, (2) methoxychlor (MXC) or vinclozolin impaired male sexual behavior in adult quail and (3) DDE exposure impaired reproductive and immune related end points. Two-generation studies were conducted on Japanese and northern bobwhite quail with dietary methoxychlor (MXC) exposure (0, 5 and 10 ppm) beginning in adults (P1), continuing in their offspring (F1), with F2 offspring raised on control diet. MXC exposure impaired male sexual behavior, hypothalamic catecholamines and plasma steroid hormones. Moreover, MXC exposure had reproductive consequences observable at both the lower and higher doses of MXC in F1 and F2 generations. These data demonstrate that embryonic EDC exposure interferes with sexual differentiation of neural systems that direct reproduction.

Acute Exposure to Molinate Alters Neuroendocrine Control of Ovulation in the Rat

Molinate, a thiocarbamate herbicide, has been reported to impair reproductive capability in the male rat and alter pregnancy outcome in a two-generation study. Published data are lacking on the effects of acute exposure to molinate in the female. Based on this work and our previous observations with related dithiocarbamate compounds, we hypothesized that a single exposure to molinate during the critical window for the neural trigger of ovulation on the day of proestrus (PRO) would block the luteinizing hormone (LH) surge and delay ovulation. To examine the effect of molinate on the LH surge, ovariectomized (OVX) rats were implanted with Silastic capsules containing estradiol benzoate to mimic physiological levels on proestrus. Doses of 25 and 50 mg/kg molinate significantly suppressed LH and prolactin secretion. Intact regularly cycling females gavaged with 0, 25, or 50 mg/kg molinate at 1300 h on PRO were examined on estrus or estrus +1 day for the presence of oocytes in the oviduct. All control females had oocytes in the oviduct on estrus. Molinate doses of 6.25 to 50 mg/kg delayed ovulation for 24 h. Estrous cyclicity was examined after daily exposure to 50 mg/kg (21 days). Estrous cyclicity was irregular in the molinate group, showing extended days in estrus. Two experiments were conducted to determine whether molinate blocked the LH surge via a central nervous system (CNS) mode of action or via an alteration in pituitary response. In the first experiment, we evaluated the release of LH in control and molinate-treated rats after a bolus dose of exogenous GnRH. Luteinizing hormone release was comparable in the two groups, suggesting that the effect of molinate is centrally mediated. To further examine the potential role of the CNS, we examined the pulsatile release of LH present in the long-term OVX females. In this model, the pulsatile pattern of LH secretion is directly correlated with GnRH release from the hypothalamus. A significant decrease in the LH pulse frequency was observed in molinate-treated females. These results indicate that molinate is able to delay ovulation by suppressing the LH surge on the day of proestrus and that the brain is the primary target site for the effects on pituitary hormone secretion.

Reproduction of the tundra vole ( Microtus oeconomus) with dietary phytosterol supplement

Phytosterols (PS) are plant-derived compounds with estrogenic activity in vitro and beneficial effects on the serum lipid profile in vivo. In nature, PS exposure can derive from pulp mill effluents. The effects of a pulp-mill derived PS mixture on the reproduction, endocrine variables and enzyme activities of the tundra vole ( Microtus oeconomus) were investigated in a two-generation study. The cumulative food intake of PS-treated females was higher than in the control group supporting previous results on the effects of PS on food consumption in rodents. 85% of the PS treated pairs reproduced, but the figure was only 60% for the control pairs. The plasma and testicular testosterone concentrations were lower in the adult PS males, but the PS-treated male offspring had higher testicular testosterone concentrations than their controls. In the female offspring, the liver lipase activity was higher in the PS-treated group, which could be a result of decreased cholesterol absorption in the gut. Chronic PS treatment increased the reproduction probability of the species and had a potential effect on the sex steroid hormones of maturing offspring, which could have applications in environmental monitoring.

An intergenerational study of birthweight: investigating the birth order effect.

In a two-generation study of the nationwide 1958 British cohort and their offspring, we investigated the intergenerational influence of birth order on birthweight. Despite increases in own birthweight by birth order and a positive parent-offspring correlation in birthweights, there was a suggestion that parental birth order was inversely associated with offspring birthweight. This paradoxical finding was due to differential intergenerational birthweight associations, with a weaker association in later-born compared with first-born parents. Our findings suggest that underlying causes of a given birthweight should be taken into account in studies that investigate long term health outcomes.

Incontinentia pigmenti with painful subungual tumors: a two-generation study

We report 2 cases of painful subungual dyskeratotic tumors occurring in a mother and daughter with incontinentia pigmenti (IP) as a late manifestation of the disease. Both patients had a history of similar lesions appearing over a period of years on the digits of both the hands and feet. Biopsy specimens of the current lesions were examined and compared with the previous material available on both patients. The current tumors and the earlier lesions (the latter of which had originally been given diagnoses that included squamous cell carcinoma, keratoacanthoma, and verruca vulgaris) showed similar histopathologic features that were consistent with the late (verrucous) stage of IP. To our knowledge, this is the first reported case of numerous subungual tumors in IP involving more than 1 generation in the same family; the first report of subungual tumors in IP to include a 16-year follow-up; and the first report of a probable recurrence of subungual tumors in IP at the same site of a previously surgically removed tumor.