Inter- and intra-individual variation in urinary concentrations of parabens in male and female Japanese subjects.

We evaluated the representativeness of concentrations of parabens in a spot urine sample for the assessment of long-term exposure levels. Urine sample was taken monthly from 10 male Japanese subjects (35.9 ± 6.8 years) and 12 female Japanese university students (21.1 ± 0.4 years) for 5 months and measured for methyl (MP), ethyl (EP), propyl (PP) and butyl (BP) parabens by liquid chromatography-tandem mass spectrometry. Median (min-max) specific-gravity-adjusted urinary concentrations of the male group (n = 10) were 39.7 (2.99-268), 1.69 (< 0.045-75.2), 0.569 (< 0.11-123) and 0.0264 (< 0.020-24.4) ng mL-1 for MP, EP, PP and BP, respectively. Those of the female group (n = 12) were 283 (5.49-1687), 9.30 (0.290-487), 22.9 (< 0.11-307) and 3.76 (< 0.020-135) ng mL-1, respectively, which were significantly higher than those of the male group. Intra-class correlation coefficients (ICCs) were calculated for the four parabens to find 0.56, 0.58, 0.39 and 0.28 for MP, EP, PP and BP, respectively, in the male group, and 0.40, 0.43, 0.41 and 0.37 for MP, EP, PP and BP, respectively, in the female group. The results suggested that four paraben concentrations in a spot urine sample moderately reflected long-term paraben exposure of Japanese subjects. Source of exposure to parabens is also discussed.

Respiratory Uptake and Depuration Kinetics of Perfluorooctanesulfonate (PFOS) in a Marine Sandworm Species.

We determined the respiratory uptake and depuration kinetics of perfluorooctanesulfonate (PFOS) in Perinereis wilsoni, a polychaete sandworm used as a model species to investigate the fate of chemical pollutants in coastal environments. The sandworms were kept in gravel-packed containers, and the water levels were varied cyclically to mimic the tides. We used seawater kept at 17.1°C. A 7-day exposure period was followed by a 9-day depuration period. The dissolved PFOS concentration averaged 28 ng/L during the exposure period. Sandworm samples were collected regularly for analysis of PFOS concentrations, and a first-order-kinetics model was applied to the concentrations. The respiratory absorption efficiency of PFOS was estimated to be 11% that of oxygen, which is higher than the corresponding estimates reported for several fish species. The estimated depuration half-life of 15 days was comparable to previously reported estimates for fish and oligochaete species. The bioconcentration factor was 470.

Paraben exposure and semen quality of Japanese male partners of subfertile couples.

OBJECTIVES: Possible relationship between semen quality (semen volume, sperm concentration and sperm motility) and parabens exposure was investigated in male partners of couples who visited a gynecology clinic in Tokyo for infertility consultation (n = 42, 36.8 ± 5.4 years). METHODS: Semen parameters were measured according to WHO guideline at the clinic, and urinary methyl- (MP), ethyl- (EP), propyl- (PP) and butyl (BP) paraben concentrations were measured with liquid chromatography-tandem mass spectrometry. RESULTS: Geometric mean urinary concentrations (geometric standard deviation) of the subjects were 48.2 (4.52), 1.88 (4.72), 1.13 (6.75) and 0.184 (11.1) ng/mL for MP, EP, PP and BP, respectively. No significant association was found between semen parameters and urinary paraben concentrations in multiple regression analyses and logistic regression analyses. CONCLUSIONS: Two reasons of the absence of adverse effects on semen quality might be suggested: lower paraben exposure level of the subjects and small sample size. Further investigation of effect of paraben exposure among general male population at environmental levels is warranted.

[Cross-reactivity of Instant-View M-1 for detection of benzodiazepine-related drugs and their metabolites in urine].

Immunoassays are useful methods for the determination of regulated drugs in clinical and forensic laboratories. Although the Instant-View M-1 (IV M-1) immunoassay kit is frequently used to screen drugs in laboratories in Japan, basic information about the IV M-1 such as its specificity and reactivity is not available. In this study, we determined the specificity and cross-reactivity of IV M-1 for the detection of benzodiazepine-related drugs and their metabolites in urine. The IV M-1 could detect triazolobenzodiazepines such as triazolam in urine at concentrations > or = 300 ng/mL. However, thienodiazepines such as etizolam could not be detected because of lack of cross reactivity. A correlation was observed between the structure of the metabolites and the reactivity of the kit; 4-hydroxy metabolites of alprazolam and triazolam were detectable, whereas a-hydroxy metabolites were not. Furthermore, 7-amino metabolites such as nitrazepam could not be detected at any concentration, including high concentrations. The specificity and reactivity of various kits used for detection of drugs in urine are different. Therefore, it is necessary to consider the basic features of the kit used while assessing the results obtained.

Acute effects of a sarin-like organophosphorus agent, bis(isopropyl methyl)phosphonate, on cardiovascular parameters in anaesthetized, artificially ventilated rats.

The organophosphorus compound sarin irreversibly inhibits acetylcholinesterase. We examined the acute cardiovascular effects of a sarin-like organophosphorus agent, bis(isopropyl methyl)phosphonate (BIMP), in anaesthetized, artificially ventilated rats. Intravenous administration of BIMP (0.8mg/kg; the LD50 value) induced a long-lasting increase in blood pressure and tended to increase heart rate. In rats pretreated with the non-selective muscarinic-receptor antagonist atropine, BIMP significantly increased both heart rate and blood pressure. In atropine-treated rats, hexamethonium (antagonist of ganglionic nicotinic receptors) greatly attenuated the BIMP-induced increase in blood pressure without changing the BIMP-induced increase in heart rate. In rats treated with atropine plus hexamethonium, intravenous phentolamine (non-selective α-adrenergic receptor antagonist) plus propranolol (non-selective ß-adrenergic receptor antagonist) completely blocked the BIMP-induced increases in blood pressure and heart rate. In atropine-treated rats, the reversible acetylcholinesterase inhibitor neostigmine (1mg/kg) induced a transient increase in blood pressure, but had no effect on heart rate. These results suggest that in anaesthetized rats, BIMP induces powerful stimulation of sympathetic as well as parasympathetic nerves and thereby modulates heart rate and blood pressure. They may also indicate that an action independent of acetylcholinesterase inhibition contributes to the acute cardiovascular responses induced by BIMP.

Exposure to pyrethroids insecticides and serum levels of thyroid-related measures in pregnant women.

Possible association between environmental exposure to pyrethroid insecticides and serum thyroid-related measures was explored in 231 pregnant women of 10-12 gestational weeks recruited at a university hospital in Tokyo during 2009-2011. Serum levels of free thyroxine (fT4), thyroid stimulating hormone (TSH) and thyroid biding globulin (TBG) and urinary pyrethroid insecticide metabolite (3-phenoxybenzoic acid, 3-PBA) were measured. Obstetrical information was obtained from medical records and dietary and lifestyle information was collected by self-administered questionnaire. Geometric mean concentration of creatinine-adjusted urinary 3-PBA was 0.363 (geometric standard deviation: 3.06) µg/g cre, which was consistent with the previously reported levels for non-exposed Japanese adult females. The range of serum fT4, TSH and TBG level was 0.83-3.41 ng/dL, 0.01-27.4 µIU/mL and 16.4-54.4 µg/mL, respectively. Multiple regression analysis was carried out by using either one of serum levels of thyroid-related measures as a dependent variable and urinary 3-PBA as well as other potential covariates (age, pre-pregnancy BMI, parity, urinary iodine, smoking and drinking status) as independent variables: 3-PBA was not found as a significant predictor of serum level of thyroid-related measures. Lack of association may be due to lower pyrethroid insecticide exposure level of the present subjects. Taking the ability of pyrethroid insecticides and their metabolite to bind to nuclear thyroid hormone (TH) receptor, as well as their ability of placental transfer, into consideration, it is warranted to investigate if pyrethroid pesticides do not have any effect on TH actions in fetus brain even though maternal circulating TH level is not affected.

Prenatal exposure to permethrin influences vascular development of fetal brain and adult behavior in mice offspring.

Pyrethroids are one of the most widely used classes of insecticides and show neurotoxic effects that induce oxidative stress in the neonatal rat brain. However, little is still known about effects of prenatal exposure to permethrin on vascular development in fetal brain, central nervous system development, and adult offspring behaviors. In this study, the effects of prenatal exposure to permethrin on the development of cerebral arteries in fetal brains, neurotransmitter in neonatal brains, and locomotor activities in offspring mice were investigated. Permethrin (0, 2, 10, 50, and 75 mg/kg) was orally administered to pregnant females once on gestation day 10.5. The brains of permethrin-treated fetuses showed altered vascular formation involving shortened lengths of vessels, an increased number of small branches, and, in some cases, insufficient fusion of the anterior communicating arteries in the area of circle of Willis. The prenatal exposure to permethrin altered neocortical and hippocampus thickness in the mid brain and significantly increased norepinephrine and dopamine levels at postnatal day 7 mice. For spontaneous behavior, the standing ability test using a viewing jar and open-field tests showed significant decrease of the standing ability and locomotor activity in male mice at 8 or 12 weeks of age, respectively. The results suggest that prenatal exposure to permethrin may affect insufficient development of the brain through alterations of vascular development.

Acute intoxication caused by overdose of flunitrazepam and triazolam: high concentration of metabolites detected at autopsy examination.

A 52-year-old woman was found dead on the floor of the living room on the first floor of a house, which belonged to the man with whom she shared the house. On visiting the site, her clothes were found to be undisturbed. Packages of flunitrazepam (Silece, 2 mg/tablet) and triazolam (Halcion, 0.25 mg/tablet) were found strewn around the victim. Toxicological analysis was performed, and the concentrations of flunitrazepam, triazolam, and their metabolites in the victim's blood and urine were measured by high-performance liquid chromatography coupled with photodiode array and mass spectrometry. A high blood concentration of 7-aminoflunitrazepam was detected (1,270 ng/g), and further metabolites such as 7-acetamidoflunitrazepam, 7-acetamidodesmethylflunitrazepam, and 7-aminodesmethylflunitrazepam were detected in the blood and urine samples. In addition, 4-hydroxytriazolam and α-hydroxytriazolam were detected in her urine at a concentration of 950 and 12,100 ng/mL, respectively.On the basis of the autopsy findings and toxicology results of high concentrations of both flunitrazepam and triazolam derivatives, the cause of death was determined to be acute intoxication from flunitrazepam and triazolam.

Gender differences in D-aspartic acid content in skull bone.

In forensic medicine, the personal identification of cadavers is one of the most important tasks. One method of estimating age at death relies on the high correlation between racemization rates in teeth and actual age, and this method has been applied successfully in forensic odontology for several years. In this study, we attempt to facilitate the analysis of racemized amino acids and examine the determination of age at death on the basis of the extent of aspartic acid (Asp) racemization in skull bones. The specimens were obtained from 61 human skull bones (19 females and 42 males) that underwent judicial autopsy from October 2010 to May 2012. The amount of D-Asp and L-Asp, total protein, osteocalcin, and collagen I in the skull bones was measured. Logistic regression analysis was performed for age, sex, and each measured protein. The amount of D-Asp in the female skull bones was significantly different from that in the male skull bones (p = 0.021), whereas the amount of L-Asp was similar. Thus, our study indicates that the amount of D-Asp in skull bones is different between the sexes.

Time course of expression of the retinoid X receptor gene and induction of imposex in the rock shell, Thais clavigera, exposed to triphenyltin chloride.

To examine the role of the retinoid X receptor (RXR) in the development of imposex in gastropods, we investigated the time course of expression of the RXR gene in various tissues (ctenidium, ovary or testis, digestive gland, penis-forming area or penis, and head ganglia) of female and male rock shells (Thais clavigera) exposed to triphenyltin (TPT) in a flow-through exposure system for 3 months. Accumulations of TPT in tissues were clearly observed in exposed individuals, whereas no accumulation of TPT was observed in the control groups. In females, 3-month exposure to TPT resulted in the development of imposex, and penis lengths in imposex-exhibiting females were significantly longer in small females (shell height <20 mm) than in large females (shell height > or = 20 mm). RXR gene expression in the ovary, penis-forming area or penis, and head ganglia of females exposed for 3 months was significantly higher than expression in control females, and the highest RXR gene expression was found in the penis-forming area or penis. Moreover, RXR gene expression in the penis-forming area or penis of each female exposed to TPT seemed to be associated with an increase in penis length. In males, the ratio of penis length to shell height was significantly larger in the exposed groups than in the controls. Although RXR gene expression in males exposed for 3 months was not significantly higher than expression in control males in any tissues, the highest gene expression was observed in the penis of exposed males. These results suggest that RXR plays an important role in the development of male genitalia (i.e., penis and vas deferens) in gastropods, although RXR might also have other physiological functions.

Establishment of a polyclonal antibody against the retinoid X receptor of the rock shell Thais clavigera and its application to rock shell tissues for imposex research.

In the chain of study to further elucidate the role of retinoid X receptor (RXR) in the development of imposex caused by organotin compounds in gastropod mollusks, we established a polyclonal antibody against RXR of the rock shell Thais clavigera. Immunoblotting demonstrated that this antibody could recognize T. clavigera RXR. In males and imposex-exhibiting females, immunohistochemical staining with the antibody revealed nuclear localization of RXR protein in the epithelial and smooth muscle cells of the vas deferens and in the interstitial and epidermal cells of the penis. These results suggest that the polyclonal antibody against T. clavigera RXR can specifically recognize RXR protein in tissues of T. clavigera and therefore is useful for evaluating RXR protein localization. Furthermore, RXR may be involved in the induction of male-type genitalia (penis and vas deferens) in normal male and organotin-exposed female rock shells.

Impaired megabenthic community structure caused by summer hypoxia in a eutrophic coastal bay.

Eutrophication and hypoxia are major problems affecting the health of coastal ecosystems throughout the world. Tokyo Bay, Japan, is a eutrophic coastal area where the abundance of the megabenthic community has been decreasing. To assess factors associated with the impaired biota, seasonal surveys of the megabenthic community and water and sediment quality were conducted in the bay. Cluster analysis showed a difference in the community structure between the northern and southern parts of the bay. The density of species and species diversity were high throughout the year in the southern part of the bay, whereas in the northern part of the bay species diversity was low and defaunation occurred in August. At this time, bottom hypoxia due to temperature and salinity stratification, and high concentrations of nutrients, chlorophyll a, and organic matter in the water column and/or sediment, dominated the northern part of the bay. In October, bottom hypoxia was less severe but was still present in the northern part of the bay, and recolonization by mobile fishes and sessile mussels occurred. Multivariate analyses of the megabenthic community and environmental parameters in August showed the spatial pattern of the community could be explained by concentrations of dissolved oxygen and particulate organic carbon in the bottom water, and total sulfide and total organic carbon in the sediments. In particular, impairment of the biota in the northern area could be explained by the threshold concentrations of dissolved oxygen < 1.7 mL L(-1) and total organic carbon > 20.3 mg g(-1).

Combined impact of pesticides and other environmental stressors on animal diversity in irrigation ponds.

Rice paddy irrigation ponds can sustain surprisingly high taxonomic richness and make significant contributions to regional biodiversity. We evaluated the impacts of pesticides and other environmental stressors (including eutrophication, decreased macrophyte coverage, physical habitat destruction, and invasive alien species) on the taxonomic richness of freshwater animals in 21 irrigation ponds in Japan. We sampled a wide range of freshwater animals (reptiles, amphibians, fishes, mollusks, crustaceans, insects, annelids, bryozoans, and sponges) and surveyed environmental variables related to pesticide contamination and other stressors listed above. Statistical analyses comprised contraction of highly correlated environmental variables, best-subset model selection, stepwise model selection, and permutation tests. Results showed that: (i) probenazole (fungicide) was a significant stressor on fish (i.e., contamination with this compound had a significantly negative correlation with fish taxonomic richness), (ii) the interaction of BPMC (insecticide; also known as fenobucarb) and bluegill (invasive alien fish) was a significant stressor on a "large insect" category (Coleoptera, Ephemeroptera, Hemiptera, Lepidoptera, Odonata, and Trichoptera), (iii) the interaction of BPMC and concrete bank protection was a significant stressor on an "invertebrate" category, (iv) the combined impacts of BPMC and the other stressors on the invertebrate and large insect categories resulted in an estimated mean loss of taxonomic richness by 15% and 77%, respectively, in comparison with a hypothetical pond with preferable conditions.

Changes in feeding habits of the starspotted smooth-hound, Mustelus manazo, in Tokyo Bay between periods with different stock size levels.

We investigated differences in the feeding habits of the starspotted smooth-hound, Mustelus manazo, in Tokyo Bay between the mid-1990s (low stock size) and the late 2000s (high stock size). The frequency of M. manazo with empty stomachs increased from 5.9% in the mid-1990s to 16.1% in the late 2000s. A decrease in the relative weight of the stomach contents was evident from the mid-1990s to the late 2000s, especially in the small size classes, along with changes in the species composition in the stomach contents. Although crustaceans were the main constituents of the stomach contents, the proportion of crabs increased while those of shrimps and hermit crabs decreased. Changes in the feeding habits of M. manazo may be associated with shifts in the benthic community structure in Tokyo Bay.

Telomere shortening in Barrett's mucosa and esophageal adenocarcinoma and its association with loss of heterozygosity.

OBJECTIVE: Telomere shortening is thought to be associated with genetic instability. The purpose of this study was to measure telomere length in a series of Barrett's adenocarcinomas (BAs), focusing on the telomere/centromere fluorescent intensity ratio (TCR) with tissue quantitative fluorescent in situ hybridization (Q-FISH). MATERIAL AND METHODS: A total of 11 cases of BA were evaluated for upper esophagus (UE), lower esophagus (LE), Barrett's mucosa (BM), BA, and gastric cardiac mucosa (GC). Q-FISH was performed using two kinds of peptide nucleic acid probe, specific for telomeres and centromeres. The sections were analyzed with a CCD camera and original software (Tissue Telo) for measuring TCR. In addition, Laser Capture Microdissection and GeneScan were implemented for evaluation of genetic instability. RESULTS: The TCR values in BM and, to a lesser extent, BA were significantly lower than those in the other tissues, particularly in heterozygosity (LOH)-positive cases. However, no significant difference was evident between microsatellite instability (MSI)-positive and -negative groups. CONCLUSIONS: In our study of BA series, telomere length appeared to change with the degree of histological atypia, with decreases linked to LOH.

Mono-hydroxylated polychlorinated biphenyls are potent aryl hydrocarbon receptor ligands in recombinant yeast cells.

The toxicities of polychlorinated biphenyls (PCBs) are thought to be mediated mainly by the aryl hydrocarbon receptor (AhR). However, little is known about changes to AhR-mediated effects caused by metabolic conversion of PCBs. To investigate whether hydroxylation affects the affinity of PCBs for the AhR, we measured the AhR agonistic activity of mono-hydroxylated PCBs (mono-OH-PCBs) and their non-hydroxylated analogs (PCBs) using yeast cells transduced with the human AhR and its response pathway. Fifty-two of 84 tested OH-PCBs and 12 of 24 PCBs exhibited AhR agonistic effects. Of 49 OH-PCBs that had the same chlorination patterns as the tested PCBs, 26 had activities that were more than twice those of their analogous PCBs, or became activated if their non-hydroxylated analogs were inactive. In particular, 3',4,5'-trichlorobiphenyl-2-ol and 3',4,4'-trichlorobiphenyl-3-ol were 37- and 22-fold more potent than their non-hydroxylated analogs and were 1.42 times and 1.08 times, respectively, as active as a standard, beta-naphthoflavone. The activities of only 5 OH-PCBs were reduced to less than half those of their non-hydroxylated counterparts. No tested PCBs were inactivated by the presence of a hydroxyl group. These findings underscore the need to rethink the toxicological evaluation of hydroxylated metabolites of PCBs and their abundance in the environment.

Mechanisms of estrogen-induced effects in avian reproduction caused by transovarian application of a xenoestrogen, diethylstilbestrol.

To clarify breeding failure in avian species caused by the estrogenicity of chemicals, alterations in the reproductive systems of Japanese quail exposed in ovo to a xenoestrogen were investigated. An injection of diethylstilbestrol (DES) into the yolk before incubation decreased, after sexual maturation, egg-laying performance of female quails, which accompanied inducing abnormal development of the oviducts. All females treated with 50 ng DES/g of egg did not lay eggs, while 0.5-5 ng DES/g reduced egg weight and eggshell strength and thickness. In the uterus (shell gland), the mRNAs for calcium regulating factors, osteopontin and calbindin D28 K, were reduced dose-dependently by DES. Scanning electron microscopy showed that shell thinning was pronounced in the mammillary and cuticular layers of the eggshell, regions where osteopontin proteins are reportedly located. These indicate that transovarian exposure to xenoestrogens causes malformation and dysfunction of the oviducts, where calcium regulating molecules could play key roles in eggshell thinning.

Reproductive and developmental effects of transovarian exposure to o,p'-DDT in Japanese quails.

Avian species have the possible risk of embryonic exposure to persistent, lipophilic environmental contaminants, such as dichlorodiphenyltrichloroethane (DDT), by transfer of chemicals accumulated in mother birds to eggs. To model developmental and reproductive disorders of wild birds living in contaminated areas, we exposed Japanese quails in ovo to o,p'-DDT prior to incubation. A positive estrogenic substance diethylstilbestrol (DES; 1 and 10 ng/g of egg) and o,p'-DDT (1-100 microg/g of egg) were injected into the yolk before incubation. Treatment with o,p'-DDT (10 or 100 microg/g) but not with DES significantly reduced the hatchability of eggs. After sexual maturation, o,p'-DDT affected eggshell formation in female quails but had little influence on laying; high doses of o,p'-DDT significantly reduced eggshell strength, shell weight, and shell thickness, and several females treated with 100 microg o,p'-DDT/g laid eggs lacking shells. Diethylstilbestrol decreased egg production itself but had little effect on the eggshell. Both o,p'-DDT and DES caused dose-dependent shortening of the left oviduct and abnormal development of the right oviduct in females, while testis asymmetry was observed in males treated with a high dose of DES. In the uterus of the oviduct, the mRNAs for calcium-regulating factors osteopontin and calbindin D28K were reduced by both treatments, particularly that with o,p'-DDT. The results indicated that transovarian exposure to o,p'-DDT could bring about population declines in avian species through loss of fecundity caused by depression of hatchability and dysfunction of the reproductive tract.

Exposure to 9-cis retinoic acid induces penis and vas deferens development in the female rock shell, Thais clavigera.

To clarify how tributyltin (TBT) and triphenyltin (TPT) interact with the retinoid X receptor (RXR) to induce growth of male sex organs in female gastropods, we treated female rock shells (Thais clavigera) with three different concentrations (0.1, 1, or 5 microg/g wet wt) of 9-cis-retinoic acid (9CRA) or with a single concentration (1 microg/g wet wt) of TBT, TPT, or fetal bovine serum (as a control). The effects of each treatment were measured as the incidence of imposex, the length of the penis-like structure, and the vas deferens sequence (VDS) index. 9CRA induced imposex in a dose-dependent manner; imposex incidence was significantly higher in the rock shells that received 1 (P < 0.05) or 5 microg (P < 0.001) 9CRA than in the controls. After 1 month, the rock shells treated with 5 microg 9CRA exhibited substantial growth of the penis-like structure that was not as evident in the other treated shells. The length of the structure differed between the 0.1- and 5-microg 9CRA treatment groups (P < 0.05) but not between the 1- and 5-microg 9CRA treatment groups (P > 0.05). Compared with the control, the VDS index increased significantly in the 1- (P < 0.05) and 5-microg (P < 0.001) 9CRA groups. The penis-like structures behind the right tentacle in female rock shells treated with 5 microg 9CRA were essentially the same as the penises and vasa deferentia of normal males and of TBT-treated or TPT-treated imposexed females. These results further support the hypothesis that imposex in gastropods could be mediated by RXR.

Screening and detection of the in vitro agonistic activity of xenobiotics on the retinoic acid receptor.

The retinoic acid receptors (RARs) play key roles in various biological processes in response to endogenous retinoic acids. However, excessive embryonic exposure to specific ligands for each subtype of the RAR was reported to induce specific developmental abnormalities. We measured the RAR agonistic activity of 543 chemicals using an assay system adopting yeast cells transfected with the human RAR gamma and a coactivator. Eighty-five of the 543 chemicals, including 16 organochlorine pesticides, 14 styrene dimers, 9 monoalkylphenols and 6 parabens, exhibited RAR gamma agonistic effects in this assay. In particular, monoalkylphenols having a 6-9 carbon alkyl group para to the phenolic hydroxyl group possessed high affinity for the RAR gamma, and their activities were 1.363-0.446% of that of all-trans RA. para-Alkylphenols chlorinated at the ortho position also were about as active or more active than their unchlorinated analogs. In addition, all tested styrene dimers showed positive effects, and the activity of 1-phenyltetralin, the strongest in this category, was 1.169% that of all-trans RA. A number of chemicals having binding affinity for the RAR gamma were revealed in this study (both newly identified and confirmed), further comprehensive studies of in vitro and in vivo effects via the RARs are required for the reliable risk assessment of chemicals. In vitro receptor binding studies represent an important step in hazard identification and suggest a potential mechanism of action, which can be an important step in risk assessment and in particular for screening studies to identify potential toxicity and inform mechanistic studies.