Evaluation of dietary dose administration as an alternative to oral gavage in the rodent uterotrophic and Hershberger assays.

The rodent uterotrophic and Hershberger assays evaluate potential estrogenic and (anti)-androgenic effects, respectively. Both US EPA and OECD guidelines specify that test substance is administered daily either by subcutaneous injection or oral gavage. However, dietary administration is a relevant exposure route for agrochemical regulatory toxicology studies due to potential human intake via crop residues. In this study, equivalent doses of positive control chemicals administered via dietary and gavage routes of administration were compared in the uterotrophic (17α-ethinyl estradiol) and Hershberger (flutamide, linuron, dichloro-2,2-bis(4-chlorophenyl) ethane; 4,4'-DDE) assays in ovariectomized and castrated rats, respectively. For all positive control chemicals tested, statistically significant changes in organ weights and decreases in food consumption were observed by both routes of test substance administration. Decreased body weight gain observed for dietary linuron and 4,4'-DDE indicated that the maximum tolerated dose was exceeded. Hershberger dietary administration resulted in a similar blood exposure (AUC24) for each positive control chemical when compared to gavage. Overall, the correlation in organ weight changes for both the uterotrophic and Hershberger assays suggest that dietary administration is an acceptable route of exposure with similar sensitivity to oral gavage dosing for evaluation of the endocrine potential of a test substance and represents a more appropriate route of test substance administration for most environmental exposure scenarios.

Reproductive toxicity of linuron following gestational exposure in rats and underlying mechanisms.

Linuron is a widely used herbicide in agriculture; its endocrine disruptive toxicity has recently received public attention. This study was designed to examine the developmental toxicity of linuron on the reproductive system of male offspring following maternal exposure. Mother rats received oral gavages of linuron, once daily, at the dose of 0, 50, 100, 150 or 200mg/kg, from gestational day (GD)13 to GD18; gonadal organs from GD20 fetuses were examined. Data indicated that exposed male offspring had a significantly shortened anogenital distance. Pathological examination further revealed a lack of fusion in the urogenital fold in treated fetuses, the damaged seminiferous tubules, and the injured Leydig cell ultrastructure. Analysis of serum testosterone concentrations at postnatal day (PND)2 showed a significant dose-related reduction (about 33.7-58.75%, r=-0.838, p<0.05) as compared to controls. Immunohistochemical results demonstrated a significantly reduced expression of enzymes pertinent to the testosterone production including P450scc, 3ß-HSD, and PCNA in Leydig cells (p<0.05). qPCR studies confirmed decreased levels of mRNAs encoding P450scc, 3ß-HSD and PCNA (p<0.05). Taken together, these data suggest that maternal exposure to linuron hampers the male gonadal organ development; this appears to be due to linuron's direct action on the production of testosterone in fetal and postnatal offspring.

Cumulative effects of in utero administration of mixtures of reproductive toxicants that disrupt common target tissues via diverse mechanisms of toxicity.

Although risk assessments are typically conducted on a chemical-by-chemical basis, the 1996 Food Quality Protection Act required the US Environmental Protection Agency to consider cumulative risk of chemicals that act via a common mechanism of toxicity. To this end, we are conducting studies with mixtures of chemicals to elucidate mechanisms of joint action at the systemic level with the goal of providing a framework for assessing the cumulative effects of reproductive toxicants. Previous mixture studies conducted with antiandrogenic chemicals are reviewed briefly and two new studies are described. In all binary mixture studies, rats were dosed during pregnancy with chemicals, singly or in pairs, at dosage levels equivalent to approximately one-half of the ED50 for hypospadias or epididymal agenesis. The binary mixtures included androgen receptor (AR) antagonists (vinclozolin plus procymidone), phthalate esters [di(n-butyl) phthalate (DBP) plus benzyl n-butyl phthalate (BBP) and diethyl hexyl phthalate (DEHP) plus DBP], a phthalate ester plus an AR antagonist (DBP plus procymidone), a mixed mechanism androgen signalling disruptor (linuron) plus BBP, and two chemicals which disrupt epididymal differentiation through entirely different toxicity pathways: DBP (AR pathway) plus 2,3,7,8 TCDD (AhR pathway). We also conducted multi-component mixture studies combining several 'antiandrogens'. In the first study, seven chemicals (four pesticides and three phthalates) that elicit antiandrogenic effects at two different sites in the androgen signalling pathway (i.e. AR antagonist or inhibition of androgen synthesis) were combined. In the second study, three additional phthalates were added to make a 10 chemical mixture. In both the binary mixture studies and the multi-component mixture studies, chemicals that targeted male reproductive tract development displayed cumulative effects that exceeded predictions based on a response-addition model and most often were in accordance with predictions based on dose-addition models. In summary, our results indicate that compounds that act by disparate mechanisms of toxicity to disrupt the dynamic interactions among the interconnected signalling pathways in differentiating tissues produce cumulative dose-additive effects, regardless of the mechanism or mode of action of the individual mixture component.

Sorption-desorption of alachlor and linuron in a semiarid soil as influenced by organic matter properties after 16 years of periodic inputs.

The effect of management practices on soil potential for regulating the residual concentration of pesticides was examined in samples from a Calcic Haploxeralf in Toledo (central Spain). Sorption-desorption of alachlor and linuron was found to depend on inputs of lignocelullosic wastes or cattle manure for the past 16 years. For a given herbicide, the soil sorption capacity (K(f)) follows the order control < crop residues < manure, which is consistent with the organic C content in the soil samples. Some structural characteristics of the soil humic acid as revealed by visible and infrared spectroscopies and analytical pyrolysis were useful to forecast the sorption-desorption intensity. Simple and multiple linear correlation analyses illustrate enhanced sorption of alachlor and linuron in soil plots where slightly altered soil organic matter accumulated (positive correlations with the intensity of infrared lignin signature band and with the methoxyphenol yields after pyrolysis of the humic acids and negative correlation with the aromaticity as pointed out by the optical density at 465 nm). Linuron showed a preference for soils with humic acids of low molecular weight and low degree of internal cross-linking, as inferred from the positive correlation with the ratio between optical densities at 465 and 665 nm. Under the conditions of the present experiment, agricultural practices including organic amendments seem to have a beneficial effect in the control of leaching and sorption of pesticides.

A mixture of the "antiandrogens" linuron and butyl benzyl phthalate alters sexual differentiation of the male rat in a cumulative fashion.

Prenatal exposure to environmental chemicals that interfere with the androgen signaling pathway can cause permanent adverse effects on reproductive development in male rats. The objectives of this study were to 1) determine whether a documented antiandrogen butyl benzyl phthalate (BBP) and/or linuron (an androgen receptor antagonist) would decrease fetal testosterone (T) production, 2) describe reproductive developmental effects of linuron and BBP in the male, 3) examine the potential cumulative effects of linuron and BBP, and 4) investigate whether treatment-induced changes to neonatal anogenital distance (AGD) and juvenile areola number were predictive of adult reproductive alterations. Pregnant rats were treated with either corn oil, 75 mg/kg/day of linuron, 500 mg/kg/day of BBP, or a combination of 75 mg/kg/day linuron and 500 mg/kg/day BBP from gestational Day 14 to 18. A cohort of fetuses was removed to assess male testicular T and progesterone production, testicular T concentrations, and whole-body T concentrations. Male offspring from the remaining litters were assessed for AGD and number of areolae and then examined for alterations as young adults. Prenatal exposure to either linuron or BBP or BBP + linuron decreased T production and caused alterations to androgen-organized tissues in a dose-additive manner. Furthermore, treatment-related changes to neonatal AGD and infant areolae significantly correlated with adult AGD, nipple retention, reproductive malformations, and reproductive organ and tissue weights. In general, consideration of the dose-response curves for the antiandrogenic effects suggests that these responses were dose additive rather than synergistic responses. Taken together, these data provide additional evidence of cumulative effects of antiandrogen mixtures on male reproductive development.

Sensitivity of macrophyte-dominated freshwater microcosms to chronic levels of the herbicide linuron. I. Primary producers.

Effects of chronic concentrations of linuron (0, 0.5, 5, 15, 50, and 150 micrograms/L) were studied in indoor, macrophyte dominated, freshwater microcosms. The concentrations were kept at a constant level for 4 weeks. This paper is the first in a series of two and summarizes the course of the linuron concentrations in time and its effects on macrophytes, periphyton, and phytoplankton. These endpoints were studied from 3 weeks before the start of the treatment until 11 weeks after the start. The degradation of linuron in the water was lower at higher treatment levels, probably due to a decrease in pH. Linuron treatment resulted in a decrease in biomass of the macrophyte Elodea nuttallii and a clear decrease in abundance of the algae Cocconeis, Chroomonas, and Phormidium foveolarum. It was found that Cocconeis first decreased in biovolume and after 2 weeks also in abundance. The alga Chlamydomonas increased in abundance at the two highest doses, resulting in higher chlorophyll-a levels. The NOECs of 0.5 micrograms/L for the inhibition of the growth and photosynthesis of Elodea nuttallii, the abundance of Cocconeis and Chroomonas, and the oxygen and pH levels were the lowest recorded in the microcosms. The safety factors adopted by the EU in the Uniform Principles appeared to ensure adequate protection for the ecosystem in the case of chronic exposure to linuron.

Sensitivity of macrophyte-dominated freshwater microcosms to chronic levels of the herbicide linuron. II. Community metabolism and invertebrates.

Effects of a chronic application of the herbicide Afalon (active ingredient linuron) on physicochemical conditions, decomposition of plant litter, and densities of zooplankton and macroinvertebrates were studied in indoor microcosms intended to model drainage ditches. For 28 days, concentrations of 0, 0.5, 5, 15, 50, and 150 micrograms/L linuron were maintained, each in two replicates. The microcosms were dominated by the macrophyte Elodea nuttallii. The functional response of the ecosystem is discussed in relation to shifts in community structure. Treatment effects of linuron on community metabolism, as a direct effect of the inhibition of the photosynthesis of macrophytes and algae, resulted in a decrease in dissolved oxygen and pH, and an increase in alkalinity and conductivity (NOEC 0.5 microgram/L). During the posttreatment period, differences between controls and highest dose fell gradually, but were still significant 7 weeks after the start of linuron application. Decomposition of particulate organic material in litter bags was not affected, despite decreases in DO. The negative effect of linuron on several algae (cryptophytes, diatoms) and the positive effect on the green alga Chlamydomonas resulted in a decrease of several Rotatoria and an increase in Copepoda, and, to a lesser extent, Cladocera. The complete disappearance of the macrophyte E. nuttallii in the 150 micrograms/L microcosms and a 50% reduction of its biomass in the 50 micrograms/L microcosms reduced the numbers of the snail Physella acuta, which normally inhabits macrophytes. Artificial substrates indicated a significant increase in the isopod Asellus aquaticus in the 50 and 150 micrograms/L microcosms during the post-treatment period. This, however, was counteracted by a significant decrease in A. aquaticus at the final harvest. Changes in the ecosystem structure (decline in macrophyte biomass) made the artificial substrates more attractive.

In vivo studies on genotoxicity of pure and commercial linuron.

The ureic herbicide linuron [3-(3, 4-dichlorophenyl)-1-methoxy-1-methylurea] (CAS 330-55-2) was investigated for genotoxicity in a series of in vivo experiments. Since human exposure to herbicides is not only to the active principles, but also to all the chemicals present in the commercial formulation, we tested both pure and commercial linuron. Groups of rats were treated with gavage containing different doses of the herbicide (pure compound or commercial formulation) for 14 days. The doses were 150, 300 and 450 mg/kg b.wt. for the pure compound and 315.8, 631.6 and 947.4 mg/kg b.wt. for the commercial formulation (47.5% of linuron). Faeces and urine were collected at regular intervals. Urine specimens were analysed for their mutagenic metabolites, thioethers and D-glucaric acid content. Faeces extracts were tested for mutagenicity. Linuron's ability to cause DNA damage and cytogenetic effects was also investigated after treating groups of rats once with different doses of pure or commercial linuron. DNA single-strand breaks were assessed in rat liver using the alkaline elution technique and the single-cell microgel electrophoresis assay (SCGE: 'comet' assay), and in rat testes cells with the SCGE assay. Micronuclei induction was analysed in rat bone marrow erythrocytes. Results obtained were mainly negative when the excretion of mutagenic metabolites in urine and faeces of animals treated with the pure compound or with the linuron-based commercial formulation were monitored, whereas an increase in the urinary excretion of thioethers and D-glucaric acid was observed in rats treated with the commercial formulation. No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the treated animals. However, linuron affected the viability of hepatocytes isolated from animals treated with higher doses. This cytotoxicity was accompanied by the induction of DNA single-strand breaks in the liver, as seen by the alkaline elution assay. The potential of pure linuron to induce in vivo DNA damage was confirmed with the microgel-electrophoresis technique ('comet' assay). Cytotoxicity was also seen in rat testes cells. However, no indication of DNA damage was visible.

Toxicokinetics and metabolism of linuron in rabbit: in vivo and in vitro studies.

1. Linuron (N'-(3,4-dichlorophenyl)-N-methoxy-N-methylurea) metabolism and kinetic behaviour were investigated after oral and i.v. administration to six New Zealand White female rabbits. 2. After i.v. dosage, linuron distributes quickly and widely to peripheral tissues and its is rapidly eliminated; rapid absorption was also observed after oral administration of the herbicide which undergoes extensive first pass metabolism in the liver. 3. The major metabolites obtained from both in vivo (serum samples) and in vitro (microsomal fractions incubated with linuron) experiments were identified by h.p.l.c.-mass spectrometry as N'-(3,4-dichlorophenyl)-N-methoxyurea, N'-(3,4-dichlorophenyl) urea, and N'-(6-hydroxy-3,4-dichlorophenyl) urea. 4. Given the common metabolites reported in rat and rabbit, and the fact that linuron is a liver enzyme inducer in rat, it may be possible that linuron also induces the P450 system in rabbit. Hence, despite the low acute toxicity of linuron in rabbit, the intake of hay and feed contaminated by the herbicide could be a health risk for these breeding animals since it could modify the effectiveness of many drugs commonly used in veterinary practice and metabolized by the same liver enzymes.

The effects of lindane and linuron on calcium metabolism, bone morphometry and the kidney in rats.

Experiments were performed to investigate the effects of lindane and linuron on calcium metabolism, femur morphometry and nephrotoxicity. Long-Evans hooded rats were dosed daily for 10 weeks with 0, 10 or 20 mg lindane or 10, 20 or 40 mg linuron/kg body weight beginning at weaning. Lindane significantly decreased urinary calcium concentration, serum alkaline phosphatase concentration and the cross-sectional medullary area of the bone. Lindane was nephrotoxic at both dose levels as demonstrated by elevated kidney weights, kidney-to-body-weight ratios, urinary LDH, tubule regeneration and hyaline droplet degeneration. Linuron significantly reduced medullary cross-sectional area at the 2 higher dose levels and decreased the total femur cross-sectional area at the highest dose level in the absence of effects on calcium excretion. Femur density and strength were also significantly reduced at the highest dose level of linuron. Neither compound affected the serum concentrations of parathyroid hormone or 1,25-dihydroxy Vitamin D-3. Both linuron and lindane exposure significantly increased serum cholesterol concentrations and reduced serum triglyceride concentrations. Both compounds affected calcium metabolism and/or bone morphometry but possibly by different mechanisms since the effects were not the same.