The OECD program to validate the rat uterotrophic bioassay. phase 2: coded single-dose studies.The Organisation for Economic Co-operation and Development The Organisation for Economic Co-operation and Development (OECD), (in French: Organisation de coopération et de développement économiques; OCDE) is an international organisation of thirty countries that accept the principles of representative democracy and a free market has completed phase 2 of an international validation See validate. validation - The stage in the software life-cycle at the end of the development process where software is evaluated to ensure that it complies with the requirements. program for the rodent rodent, member of the mammalian order Rodentia, characterized by front teeth adapted for gnawing and cheek teeth adapted for chewing. The Rodentia is by far the largest mammalian order; nearly half of all mammal species are rodents. uterotrophic bioassay Bioassay A method for the quantitation of the effects on a biological system by its exposure to a substance, as well as the quantitation of the concentration of a substance by some observable effect on a biological system. . This portion of phase 2 assessed the reproducibility reproducibility Lab medicine The degree of agreement among repeated measurements of a particular parameter, presented in terms of a standard deviation or coefficient of variation of the results in a set of measurements of the assay with a battery of positive and negative test substances. Positive agonists of the estrogen receptor estrogen receptor A protein of a superfamily of nuclear receptors for small hydrophilic ligands–eg, steroid hormones, thyroid hormone, vitamin D, retinoids; the presence of ERs in breast CA generally is associated with a better prognosis, as they respond to included the potent reference estrogen 17[alpha]-ethinyl estradiol estradiol /es·tra·di·ol/ (es?trah-di´ol) (es-tra´de-ol) the most potent estrogen in humans; pharmacologically, it is often used in the form of its esters (e.g., e. cypionate, e. (EE), and the weak estrogen agonists bisphenol A Bisphenol A is a chemical compound containing two phenol functional groups. It belongs to the phenol class of aromatic organic compounds. It is widely prepared and sold and various important polymers/plastics are made from it. , genistein ge·nis·te·in n. A phytoestrogen found in soy, having antioxidant and anticancer properties. genistein (geˑ·n , methoxychlor methoxychlor one of the group of chlorinated hydrocarbon insecticides which cause typical signs of that poisoning. , nonylphenol Nonylphenol is an organic compound of the wider family of alkylphenols. It is a product of industrial synthesis formed during the alkylation process of phenols, particularly in the synthesis of polyethoxylate detergents. , and o,p'-DDT. The negative test substance or nonagonist was n-dibutylphthalate. The test substances were coded, and prescribed pre·scribe v. pre·scribed, pre·scrib·ing, pre·scribes v.tr. 1. To set down as a rule or guide; enjoin. See Synonyms at dictate. 2. To order the use of (a medicine or other treatment). doses of each test substance were administered in 16 laboratories. Two versions of the uterotrophic assay, the intact immature immature /im·ma·ture/ (im?ah-chldbomacr´) unripe or not fully developed. im·ma·ture adj. Not fully grown or developed. immature unripe or not fully developed. and the adult ovariectomized female rat, were tested and compared using four standardized standardized pertaining to data that have been submitted to standardization procedures. standardized morbidity rate see morbidity rate. standardized mortality rate see mortality rate. protocols covering both sc and po administration. Assay reproducibility was compared using a) EE doses identical to those used in phase I and in parallel dose-response studies, b) single doses of the weak agonists identical to one of five doses from the dose-response studies, and c) a single dose of the negative test substance. The results were reproducible re·pro·duce v. re·pro·duced, re·pro·duc·ing, re·pro·duc·es v.tr. 1. To produce a counterpart, image, or copy of. 2. Biology To generate (offspring) by sexual or asexual means. and in agreement both within individual laboratories and across the participating laboratories for the same test substance and protocol. The few exceptions are examined in detail. The reproducibility was achieved despite a variety of different experimental conditions (e.g., variations in animal strain, diet, housing protocol, bedding, vehicle, animal age). In conclusion, both versions of the uterotrophic bioassay and all protocols appear robust, reproducible, and transferable across laboratories and able to detect weak estrogen agonists. These results will be submitted along with other data for independent peer review to provide support for the validation of the uterotrophic bioassay. Key words: endocrine endocrine /en·do·crine/ (en´do-krin, en´do-krin) 1. secreting internally. 2. pertaining to internal secretions; hormonal. See also under system. en·do·crine adj. disruption disruption /dis·rup·tion/ (dis-rup´shun) a morphologic defect resulting from the extrinsic breakdown of, or interference with, a developmental process. , estrogen, rat uterus, uterotrophic. Environ en·vi·ron tr.v. en·vi·roned, en·vi·ron·ing, en·vi·rons To encircle; surround. See Synonyms at surround. [Middle English envirounen, from Old French environner Health Perspect 111:1550-1558 (2005). doi: 10.1289/ehp.5870 available via http://dx, doi. org/[Online 23 January January: see month. 2003] ********** The Organisation for Economic Co-operation and Development (OECD OECD: see Organization for Economic Cooperation and Development. ) has undertaken the validation of the uterotrophic bioassay. The management of the validation program and the results of other portions of the validation program have been described in other reports (Kanno et al. 2001; 2003). A central objective of the OECD validation program is to establish the reliability of standardized protocols for the uterotrophic bioassay. A demonstration of reliability is based on the transferability of a protocol among laboratories, where the protocol results are reproducible among laboratories (ICCVAM ICCVAM Interagency Coordination Committee on the Validation of Alternative Methods 1997; OECD 1998). Two aspects of reliability require demonstration in a validation program: a) the assay's sensitivity, or ability to respond to and detect positive substances, and b) the assay's specificity, or absence of response to negative substances (ICCVAM 1997; OECD 1998). Additionally, sensitivity and specificity should be assessed over time and should include data gathered using coded or blinded test substances (ICCVAM 1997; OECD 1998). The studies in this paper are intended to demonstrate the reliability of the uterotrophic bioassay, including its sensitivity and specificity with coded samples. The test substances were a potent reference agonist agonist /ag·o·nist/ (ag´ah-nist) 1. one involved in a struggle or competition. 2. agonistic muscle. 3. , five weak estrogen agonists, and a negative test substance. Four protocols are included in the validation studies to address the two primary versions of the uterotrophic assay, the intact, immature, and the adult ovariectomized (OVX OVX Ovariectomy ) female rat as well as the primary routes of administration, oral gavage gavage /ga·vage/ (gah-vahzh´) [Fr.] 1. forced feeding, especially through a tube passed into the stomach. 2. superalimentation. ga·vage n. 1. and sc injection. A previous article demonstrated the reproducibility of the dose response of the reference agonist, 17[alpha]-ethinyl estradiol (EE), with both versions and all protocols (Kanno et al. 2001). An accompanying article demonstrates the reproducibility of both versions and all protocols using dose responses of the five weak agonist test substances (Kanno et al. 2003). Because all laboratories performed the EE dose response separate from these data, and almost all laboratories performed the weak agonist dose-response and coded single-dose studies at separate times, a comparison of the data provides for an assessment of bioassay reproducibility over time. Materials and Methods Test substances. Test substances were obtained and distributed through a centralized cen·tral·ize v. cen·tral·ized, cen·tral·iz·ing, cen·tral·iz·es v.tr. 1. To draw into or toward a center; consolidate. 2. chemical repository (1) A database of information about applications software that includes author, data elements, inputs, processes, outputs and interrelationships. A repository is used in a CASE or application development system in order to identify objects and business rules for reuse. at TNO TNO Tamarindo, Costa Rica (Airport code) TNO Nederlandse Organisatie voor Toegepast Natuurwetenschappelijk Onderzoek TNO Trans-Neptunian Object TNO The New Order (paramilitary street gang) TNO Trust No One , Zeist Zeist (zīst), city (1994 pop. 59,258), Utrecht prov., central Netherlands, near Utrecht. It is largely residential; manufactures include pharmaceuticals and furniture. A settlement of the Moravian Brethren was established there in 1746. , the Netherlands Netherlands (nĕth`ərləndz), Du. Nederland or Koninkrijk der Nederlanden, officially Kingdom of the Netherlands, constitutional monarchy (2005 est. pop. 16,407,000), 15,963 sq mi (41,344 sq km), NW Europe. . This repository is described in the accompanying paper, including a full description of the chemical identities, purities, and sources (Kanno et al. 2001), with the exception of the negative test substance, n-dibutylphthalate (DBP DBP Diastolic Blood Pressure DBP Development Bank of the Philippines DBP Database Project (Visual Studio File Extension) DBP DNA Binding Protein DBP Disinfection Byproduct DBP Deutsche Bundespost ) (CAS no. 84-74-2, purity Purity: see Pearl, The. Purity See also Modesty. almond symbol of the Virgin Mary’s innocence. [O.T.: Numbers 17: 1–11; Art: Hall, 14] crystal its transparency symbolizes pureness. 99.9%) which was obtained from Sigma SIGMA - A scientific visual programming environment from NASA. http://fi-www.arc.nasa.gov/fia/projects/sigma/. Aldrich Aldrich may refer to: Places:
Louis, 1682–1712, titular duke of Burgundy; grandson of King Louis XIV of France. He became heir to the throne on the death (1711) of his father, Louis the Great Dauphin. , MO, USA). Because of the coded nature of this study, the amounts of test substance needed by each laboratory were calculated for each protocol. These amounts were preweighed into individually coded, opaque vials at the central repository prior to their shipment. Animal supply, husbandry husbandry careful management of e.g. animals. Implies thrifty, humane, caring. See also animal husbandry. , and preparation. The details of how participating laboratories obtained animals, the housing and husbandry conditions, the age of the animals, compliance with the OECD guidelines guidelines, n.pl a set of standards, criteria, or specifications to be used or followed in the performance of certain tasks. on animal care (OECD 2000) and appropriate national regulations, and the animal preparation and observation prior to test substance administration have been described previously (Kanno et al. 2001, 2003). Protocols. The details of the individual protocols have also been described previously (Kanno et al. 2001, 2003). Briefly, protocol A uses intact, immature female rats with dosing by oral garage for 3 consecutive days. Protocol B uses intact, immature female rats with dosing by sc injection for 3 consecutive days. Protocol C uses young adult OVX rats as described above with dosing by sc injection for 3 consecutive days. Protocol D [previously called protocol C' (Kanno et al. 2001)] also uses young adult OVX rats and extends the sc injection dosing to a total of 7 days. Coded samples, vehicle, test substance preparation, and dosing. For each test substance, individualized instructions Individualized instruction is a method of instruction in which content, instructional materials, instructional media, and pace of learning are based upon the abilities and interests of each individual learner. , depending on the amount to be shipped, were given to each laboratory. The instructions specifically stated the volume of test vehicle to be added to the coded vials to provide a reference dose solution for each test substance. Further instructions "Further Instructions" is the third episode of the third season of Lost. It aired on October 18, 2006, making it the 50th episode of the series. The episode was written by Carlton Cuse and Elizabeth Sarnoff and directed by Stephen Williams. were provided to adjust the administered test volume based on the recorded body weight (bw) of the animals in provide the prescribed experimental doses. Participating laboratories were asked to have one set of personnel prepare the test substance dose solutions and administer the preparations and a second set perform the necropsy necropsy /nec·rop·sy/ (nek´rop-se) examination of a body after death; autopsy. nec·rop·sy n. See autopsy. necropsy examination of a body after death. See also autopsy. and record the uterine uterine /uter·ine/ (u´ter-in) pertaining to the uterus. u·ter·ine adj. Of, relating to, or in the region of the uterus. weights. This was intended to minimize the chances of working out the code for each test substance. Material safety data sheets were provided in a sealed envelope to a nominated nom·i·nate tr.v. nom·i·nat·ed, nom·i·nat·ing, nom·i·nates 1. To propose by name as a candidate, especially for election. 2. To designate or appoint to an office, responsibility, or honor. person at each laboratory, who agreed to keep this envelope sealed except in cases of emergency. A generic material safety data sheet was prepared and supplied to cover all test substances so that the health and safety of personnel at the laboratory would not be compromised. The other details of the vehicle, test substance preparation, and animal-dosing procedures have been previously described (Kanno et al. 2001, 2003). Necropsy, dissection dissection /dis·sec·tion/ (di-sek´shun) 1. the act of dissecting. 2. a part or whole of an organism prepared by dissecting. , and uterine weight. As described previously, the animals were killed humanely hu·mane adj. 1. Characterized by kindness, mercy, or compassion: a humane judge. 2. Marked by an emphasis on humanistic values and concerns: a humane education. 24 hr after the last test substance administration in the same sequence as the test substance was administered. The dissection of the uterus and the measurement of wet and blotted uterine weights to the nearest 0.1 mg were performed as described previously (Kanno et al. 2001, 2003). Study management and quality control. The study management and quality control have been previously decribed. The VMG VMG Vineyard Music Group VMG Voice Message VMG Video Map Generator (AN/GPA-134) VMG Vertical Magnetic Gradient (geophysical surveying) VMG Visión Mundial de Guatemala requested that the studies be performed under OECD Good Laboratory Practice (GLP See gateway location protocol. ) guidelines (OECD 2002). However, full GLP compliance was not a requirement for a laboratory's participation in the validation program, and several of the laboratories did not perform their studies under GLP. Data were received, and after an initial statistical analysis was performed, all laboratories were requested to audit these raw data and respond to specific queries on outliers and questionable data. A small number of data corrections were made, and reporting errors on dilutions, samples, and identity of control groups were either corrected or clarified. Statistics. The recording and statistical procedures, data evaluation by an analysis of covariance Covariance A measure of the degree to which returns on two risky assets move in tandem. A positive covariance means that asset returns move together. A negative covariance means returns vary inversely. , logarithmic logarithmic pertaining to logarithm. logarithmic relationship when the logs of two variables plotted against each other create a straight line. transformation of uterine data, use of the Dunnett Dunnett is a surname, and may refer to
If each number in a list of numbers was replaced with their geometric mean, then multiplying them all together would still give the same result. of the uterine weights (relative to the vehicle control) after adjusting for the body weight of the animal at necropsy with upper and lower 95% confidence levels have all been previously described (Kanno et al. 2003). To draw inferences across laboratories about the reproducibility of results at a given dose for each protocol, mixed-effects linear models were used, where the laboratories were treated as the random effects Random effects can refer to:
Design of Phase 2 Coded Single-Dose Studies The objective of the coded single-dose studies was to produce the data to assess the reproducibility of the uterotrophic bioassay both within the same laboratory and across the multiple, participating laboratories. Further, the reproducibility was to be assessed over time and using blinded or coded samples. Overall design rationale In the survey on design rationale (DR) for software engineering[1] the authors give a very clear definition to design rationale, it is “the explicit listing of decisions made during a design process and the reasons why those decisions were made. . Three types of test substances were included: a potent reference test substance, EE; five weak estrogen receptor agonists: genistein (GN), methoxychlor (MX), nonylphenol (NP), bisphenol A (BPA BPA British Paediatric Association. ), and 1,1,1-trichloro-2,2-bis(o,p'chlorophenyl)ethane ethane (ĕth`ān), CH3CH3, gaseous hydrocarbon. It is a continuous-chain alkane. As a constituent of natural gas, it is used for fuel. It can be prepared by cracking and fractional distillation of petroleum. or o,p'-DDT (DDT DDT or 2,2-bis(p-chlorophenyl)-1,1,1,-trichloroethane, chlorinated hydrocarbon compound used as an insecticide. First introduced during the 1940s, it killed insects that spread disease and feed on crops. ); and a negative test substance, DBP. A robust statistical comparison required that identical doses be selected so that the same prescribed doses for each test substance were used in every laboratory. Two EE doses were selected from phase 1 (Kanno et al. 2001) to generate two additional sets of data to assess the reproducibility of the bioassay. The first EE dose for a given route of administration was the first minimally effective dose in the lower portion of the dose-response curve dose-response curve A graphic representation of the effects that varous doses of an agent–eg, ionizing radiation or a chemotherapeutic agent, have on a given parameter–eg, cell viability, mutation frequency, DNA damage, tumor growth or metastasis or that was a statistically significant response in all laboratories in phase 1. The second EE dose was then 0.5-log higher than the first dose, and this second dose had given responses near or at the maximum uterine response in phase 1. These selected doses were used as control reference doses as part of the accompanying dose-response studies (Kanno et al. 2003) and in these studies as coded samples. This design produces three data sets of replicate rep·li·cate v. 1. To duplicate, copy, reproduce, or repeat. 2. To reproduce or make an exact copy or copies of genetic material, a cell, or an organism. n. A repetition of an experiment or a procedure. doses to assess the reproducibility of the uterine response over time. The selection of the positive weak agonists and a series of five prescribed doses for each are described in the accompanying paper (Kanno et al. 2003). The participating laboratories were required in the dose-response studies to use the three intermediate doses, whereas the lowest and highest of the five doses were optional. Therefore, the third or fourth dose in the series was selected for this coded single-dose study. As a result, two sets of replicate doses would be available, one from the dose-response studies and one from the coded single-dose studies, and would include all five weak agonists in all four standardized protocols. The negative test substance, DBP, was chosen based on two lines of evidence. First, DBP does not display binding affinity for the rat uterine estrogen receptor, i.e., there is no displacement displacement, in psychology: see defense mechanism. Same as offset. See base/displacement. of bound [sup.3H] 17[beta]-estradiol at concentrations up to 1 mM concentrations in vitro in vitro /in vi·tro/ (in ve´tro) [L.] within a glass; observable in a test tube; in an artificial environment. in vi·tro adj. In an artificial environment outside a living organism. (Blair Blair , Anthony Charles Lynton Known as "Tony" Born 1953. British lawyer, politician, and Labour Party leader who was elected prime minister in 1997. et al. 2000). Second, in vivo in vivo /in vi·vo/ (ve´vo) [L.] within the living body. in vi·vo adj. Within a living organism. in vivo adv. toxicological studies, with some including gene activation activation /ac·ti·va·tion/ (ak?ti-va´shun) 1. the act or process of rendering active. 2. the transformation of a proenzyme into an active enzyme by the action of a kinase or another enzyme. 3. profiles, indicate that DBP does not elicit e·lic·it tr.v. e·lic·it·ed, e·lic·it·ing, e·lic·its 1. a. To bring or draw out (something latent); educe. b. To arrive at (a truth, for example) by logic. 2. responses indicative of an estrogen mode of action (Ema et al. 2000; Ema and Miyawaki 2001; Mylchreest et al. 1998, 1999; Schulz Schulz , Charles Monroe 1922-2000. American cartoonist who created the Peanuts comic strip. Noun 1. Schulz - United States cartoonist whose comic strip included the beagle Snoopy (1922-2000) Charles M. et al. 2001; Zacharewski et al 1998). A single data set that included data for all four standardized protocols was judged adequate for the negative chemical to conserve resources and animals. Selected doses. Two reference EE doses selected were 1 and 3 pg/kg bw/day for oral garage and 0.3 and 1 lag/kg bw/day for sc injection. For the weak estrogen receptor agonists, the selected doses for the oral gavage studies were 600 mg BPA/kg bw/day, 300 mg GN/kg bw/day; 300 mg MX/kg bw/day; 250 mg NP/kg bw/day; and 300 mg DDT/kg bw/day. Doses for the sc injection studies were 300 mg BPA/kg bw/day; 35 mg GN/kg bw/day; 500 mg MX/kg bw/day; 80 mg NP/kg bw/day; and 100 mg DDT/kg bw/day. For the negative test substance, DBP, a limit dose was selected for each route of administration: 1,000 mg/kg mg/kg Milligrams Per Kilogram bw/day for oral garage and 500 mg/kg bw/day for sc injection. Results The coded single-dose studies were performed by 16 laboratories. Laboratories 6, 7, 9, 10, and 15, which either participated in phase 1 (Kanno et al. 2001) or the dose-response studies in phase 2 (Kanno et al. 2003), did not participate in the coded single-dose studies. However, their EE results from these studies were included in the comparison of the EE results generated in the coded single-dose studies. Despite the size of this international study, the actual difficulties encountered were few. For example, laboratories 17 and 19 may lack results for MX, BPA, GN, or DDT, because some of these substances were not administered after these two laboratories experienced difficulty in solubilization during dosage dosage /dos·age/ (do´saj) the determination and regulation of the size, frequency, and number of doses. dos·age n. 1. Administration of a therapeutic agent in prescribed amounts. preparation. A few laboratories misinterpreted the EE dilution Dilution A reduction in earnings per share of common stock that occurs through the issuance of additional shares or the conversion of convertible securities. Notes: Adding to the number of shares outstanding reduces the value of holdings of existing shareholders. instructions, so that a few dose concentrations were either reversed or were incorrect (e.g., the high EE dose in laboratory 12). Except for laboratory 1, audits of the records were able to correct the data for the reversals. Finally, uterine wall punctures were reported in three animals in separate laboratories and groups during dissection. The possible losses of imbibed fluid did not affect any results. Mortalities, decreases in body weight or body weight gain, and clinical signs. Of 1,842 animals administered test substances in the coded single-dose studies, 42 mortalities were observed in eight laboratories. All mortalities in the coded single-dose studies were in protocol A (2 in GN studies, 3 in MC studies, 3 in DBP studies, 6 in BPA studies, 8 in DDT studies, and 19 in NP studies). As with the dose-response experiments, a dose-related dose-related see dose response. pattern of modest reductions in body weights and diminished di·min·ish v. di·min·ished, di·min·ish·ing, di·min·ish·es v.tr. 1. a. To make smaller or less or to cause to appear so. b. body weight gains was often observed in the immature animal studies and in the OVX studies where the dosing was extended to 7 days. Decreases in body weights at terminal sacrifice approaching or greater than 10% were observed with NP in most protocol A studies, DDT in protocol A, BPA in protocol D, MX in protocol D, and the high EE dose in some protocol D studies (data not shown), indicating that a maximum tolerated dose had been exceeded. Clinical signs were reported in conjunction with the mortalities and body weight losses, including piloerection piloerection /pi·lo·erec·tion/ (-e-rek´shun) erection of the hair. pi·lo·e·rec·tion n. Erection of hair. piloerection erection of hair. , crouched crouch v. crouched, crouch·ing, crouch·es v.intr. 1. a. To stoop, especially with the knees bent: crouched over the grate, searching for his keys. positions, and labored breathing. Ethinyl estradiol eth·i·nyl estradiol n. A synthetic estrogen derivative commonly used in oral contraceptives. Ethinyl estradiol studies. Within each protocol, the mean increases in the body weight-adjusted blotted uterine weights of both the low and high EE doses were reproducible. The low and high EE dose results for the dose-response and coded single-dose protocols are shown in Table 1 and Table 2, respectively, and the phase 1 results have been previously reported (Kanno et al. 2001). In protocol A, the results of the three sets of EE data were reproducible. The blotted uterine weight increases were statistically significant at both EE doses, and the weights increased in a dose-related manner. There were two exceptions. Laboratory 1 did not achieve statistical significance at the lower 1 [micro]g EE/kg/day dose in the dose-response studies, but had achieved statistical significance at this dose in phase 1. In laboratory 13, the ratio of mean uterine weight of the test substance group relative to the vehicle control group was nearly five at the lower EE dose in the coded single-dose studies. The ratio was a more modest value of 1.5 to 2 in phase 1 and the dose-response studies. In protocol B, the results of the three sets of EE data were reproducible with two exceptions. First, the ratio of the uterine weight increases in laboratories 9, 15, 18 at the lower EE dose was 3.5 to 5 in phase 2, compared with approximately 2 in phase 1. Second, laboratory 19 failed to achieve statistical significance at either EE dose. In protocol C, the results of the three sets of EE data were reproducible with one exception. Laboratory 19 achieved statistical significance with the low EE dose, but not the high EE dose, in the coded single-dose studies. This same laboratory had shown a low responsiveness to EE in protocol C in phase 1 (Table 5 and Figure 1C in Kanno et al. 2001). In protocol D, the results of the three sets of EE data were reproducible. As noted in phase 1 (Kanno et al. 2001), the extended dosing in protocol D again typically led to a further increase in the blotted uterine weights over protocol C at both the low and high EE doses, but the increased number of EE doses also led to decreases in body weight gains. Weak agonist studies. The results for the same BPA dose in the dose-response and coded single-dose studies are shown in Table 3. In protocol A, even at a dose of 600 mg BPA/kg/day, the relative uterine response was very weak and did not exceed a value of 2 in any laboratory. In the response distribution from this modest response, five laboratories failed to achieve statistical significance. Although all five had increased absolute uterine weights, the 95% lower confidence level did not exceed 1 as necessary for statistical significance. In three of these laboratories, animal mortalities occurred, decreasing the power. In protocol B, at a dose of 300 mg BPA/kg/day, the mean ratio values of the relative increase in uterine weight were between 1.5 and 2.8. In this response distribution, 3 of 23 experiments did not achieve statistical significance. In laboratory 12, the mean uterine weight was increased over 30%, but did not achieve significance. In laboratory 20, little or no evidence of a response was seen in either the dose-response or the coded single-dose studies. In protocol C, the ratio of the mean treated uterine weight relative co the vehicle controls was 2.3 to 3.4, and all laboratories in this response distribution were able to achieve statistical significance. This ratio value for the adult OVX animals was consistently greater than for the immature animals in protocol B. In protocol D, the mean blotted uterine weights appeared to be increased by the extended dosing period, and all six laboratories achieved statistical significance. The results for the same GN dose in the dose-response and coded single-dose studies are shown in Table 4. The mean uterine responses at the selected GN doses relative to controls were 2 or greater for most laboratories. All laboratories in their respective response distributions achieved statistical significance in each protocol. In the case of GN, the immature animals in protocol B appeared to have a somewhat higher mean response than the OVX animals in protocol C and even in protocol D with the extended dosing period. The results for the same MX dose in the dose-response and coded single-dose studies are shown in Table 5. The mean uterine responses at the selected MX doses relative to controls were 2 or greater for most laboratories, and often exceed 3 in protocols A and B. All laboratories in their respective response distributions achieved statistical significance in each protocol. In the case of MX, the immature animals in protocol B appeared to have a somewhat higher mean response than the OVX animals in protocol C and even protocol D with the extended dosing period. The results for the same NP dose in the dose-response and coded single-dose studies are shown in Table 6. In protocol A, 13 of 14 studies achieved statistical significance at a dose of 250 mg NP/kg/day. This is at first surprising, given that 11 of these laboratories experienced animal mortalities that reduced their power of the already small group size of six. However, the mean relative increase in uterine weights was no lower than 1.71 in any study, and the only laboratory that did not reach statistical significance had only two surviving animals and a mean relative increase of 1.97. In the sc protocols, the mean relative increases in uterine weight at the selected dose of 80 mg NP/kg/day were more modest, and greater than 2 in only 6 of 42 studies. In protocol B, 17 of 24 studies combined from the coded single-dose and the close-response sets achieved statistical significance. In protocol C, 8 of 12 studies achieved statistical significance. In protocol D, all NP coded samples achieved statistical significance with the extended dosing period. The results for the same DDT dose in the dose-response and coded single-dose studies are shown in Table 7. In protocol A, all 13 studies achieved statistical significance at a dose of 300 mg DDT/kg/day, as the minimum mean relative increase in uterine weight was 2.67. In the sc protocols at a dose of 100 mg DDT/kg/day, the relative increase in uterine weights was considerably lower, with only 4 of 36 studies greater than 1.5. As a result, only 6 of 19 studies achieved statistical significance in protocol B, 5 of 11 in protocol C, and 4 of 6 studies in protocol D with the extended dosing period. Dibutylphthalate studies. The results for the DBP studies are shown in Table 8. In protocols A and D, none of the 15 DBP-treated groups were statistically significant versus the vehicle controls. However, in protocol B, the results of 4 of 14 studies, and in protocol C, the results of 1 of 7 studies, achieved statistical significance. Of these five studies, three had significantly increased blotted uterine weights when treated with DBP, whereas the other two had significantly decreased blotted uterine weights when treated with DBP. Discussion and Conclusions The OECD is composed of over 20 nations, and OECD protocols such as the uterotrophic bioassay are intended for use in all of the member nations. As such, this validation study was carried out in 21 laboratories in nine nations. Funding for the study came primarily from national regulatory agencies regulatory agency Independent government commission charged by the legislature with setting and enforcing standards for specific industries in the private sector. The concept was invented by the U.S. and industry associations, but several laboratories freely contributed their time and effort to the study. This large, international nature of the program, however, increased the organizational and logistical lo·gis·tic also lo·gis·ti·cal adj. 1. Of or relating to symbolic logic. 2. Of or relating to logistics. [Medieval Latin logisticus, of calculation workload The term workload can refer to a number of different yet related entities. An amount of labor While a precise definition of a workload is elusive, a commonly accepted definition is the hypothetical relationship between a group or individual human operator and task demands. . For example, the protocol bad to be clearly understood by speakers of a variety of languages for the procedures to be performed in a similar manner in all laboratories. Data had to be recorded in the different laboratories and provided to an independent statistician in a accurate, timely, and efficient manner. The animal husbandry animal husbandry, aspect of agriculture concerned with the care and breeding of domestic animals such as cattle, goats, sheep, hogs, and horses. Domestication of wild animal species was a crucial achievement in the prehistoric transition of human civilization from supplies, vehicles, and reagents, as well as the laboratory animal themselves, also bad to be widely and readily available. Finally, the central repository had to deal with international shipments with different customs regulations and laboratory safety regulations. The Validation Management Group addressed these challenges with several efforts. Both the ovariectomy ovariectomy /ovar·i·ec·to·my/ (o-var?e-ek´tah-me) oophorectomy. o·var·i·ec·to·my n. The surgical removal of one ovary or both ovaries. Also called oophorectomy. and uterine dissection procedures were videotaped, and the videotape videotape Magnetic tape used to record visual images and sound, or the recording itself. There are two types of videotape recorders, the transverse (or quad) and the helical. was distributed to the technical staff of all the participating laboratories. The draft protocols were distributed to all national authorities and participating laboratories for comments and inquiries for any ambiguities. A common electronic spreadsheet spreadsheet Computer software that allows the user to enter columns and rows of numbers in a ledgerlike format. Any cell of the ledger may contain either data or a formula that describes the value that should be inserted therein based on the values in other cells. was constructed and distributed for comment so the data could be recorded and electronically transmitted to the independent statistician. Despite these efforts and preparations, some laboratories encountered difficulty with certain dose-preparation instructions, two errors in the spreadsheet itself were later discovered, and the breakage of some vials during shipment required their rapid replacement because of the imminent delivery of immature animals whose births were timed for protocols A and B. Given the number of laboratories and individual studies, these were minor problems that did not affect the quality or the success of the results. It should also be recognized that the protocols allowed variations in a number of experimental conditions. These variables include the choice of rat strain, the laboratory diet, housing and husbandry practices such as the use of cage bedding, the administration vehicle, and to a modest degree, the age of both immature and OVX animals. The judgment was that rigorous and detailed standardization standardization In industry, the development and application of standards that make it possible to manufacture a large volume of interchangeable parts. Standardization may focus on engineering standards, such as properties of materials, fits and tolerances, and drafting of all of these variables would constrain con·strain tr.v. con·strained, con·strain·ing, con·strains 1. To compel by physical, moral, or circumstantial force; oblige: felt constrained to object. See Synonyms at force. 2. the ability to widely and easily practice the uterotrophic bioassay in many of the OECD member nations, where the intended purpose is as a rapid screening bioassay for a large number of chemicals. The laboratory specifics for most laboratories have been described previously (Table 1 in Kanno et al. 2001; Table 7 in Kanno et al. 2003) or can be found for the remaining laboratories in Table 9. The coded nature of the study also introduced some difficulties. To avoid giving very specific information that could be used to identify the coded test substances, broad general advice was given about dose preparation. Unfortunately, estrogen receptor agonists and antagonists antagonists, n muscles that counterbalance agonists during specific movements. opioid Neurology A pain-attenuating peptide that occurs naturally in the brain, which induces analgesia by mimicking endogenous opioids at opioid tend to be hydrophobic hydrophobic /hy·dro·pho·bic/ (-fo´bik) 1. pertaining to hydrophobia (rabies). 2. not readily absorbing water, or being adversely affected by water. 3. and to have limited solubility solubility Degree to which a substance dissolves in a solvent to make a solution (usually expressed as grams of solute per litre of solvent). Solubility of one fluid (liquid or gas) in another may be complete (totally miscible; e.g. . As noted, some laboratories encountered difficulty in solubilizing the test substances, and two laboratories decided to halt administration of particular preparations rather than administer apparent suspensions. This experience also suggests another source of variation in administered doses among the participating laboratories. As with the dose-response studies, there was a consistent association in the coded single-dose studies between the occurrence of mortalities, reduced body weight gain, and clinical signs with the weak agonists DDT and NP in protocol A, and for reduced body weight gain with the EE high dose, BPA, and MX in protocol D. The 10% and greater differences in body weights between vehicle and treated animals occurred within just 4 days (protocols A, B, and C) or 8 days (protocol D) of treatment initiation, indicating a rapid onset of systemic systemic /sys·tem·ic/ (sis-tem´ik) pertaining to or affecting the body as a whole. sys·tem·ic adj. 1. Of or relating to a system. 2. toxicity toxicity /tox·ic·i·ty/ (tok-sis´i-te) the quality of being poisonous, especially the degree of virulence of a toxic microbe or of a poison. at those doses. Despite the apparent magnitude apparent magnitude: see magnitude. of these insults, the uterine response appeared to remain undiminished, confirming the underlying robustness of this biological response for estrogen-screening programs. Overall, for each protocol, the mean relative increase in uterine weight was reproducible within and among laboratories for both the dose-response and coded single-dose studies with each test substance. The dose-response results for each protocol and test substance are in the accompanying paper (Kanno et al. 2003). It is important to distinguish between when the results for a given test substance have been consistently reproduced within and across laboratories over time from whether statistical significance was consistently achieved in all or none of the laboratories. The objective here is the former, the reproducibility of the bioassay. The results here should be interpreted by taking into account the following considerations. First, several of selected doses were in the lower regions of a substance's dose-response curve (Kanno et al. 2001, 2003). Second, the lower region of the dose-response curve implies a distribution of statistically positive and negative responses, with the ratio between positive and negative results depending upon the precise dose employed in the dose response of that particular substance. That is, the rate of studies lacking statistical significance should rise as the doses move further down the dose-response curve for a substance, particularly in the case of weak agonists when the slope of the dose response is shallow. Several doses herein were at or near maximum uterine responses, for example, the high EE po and sc doses, the GN and MX po and sc doses, and the DDT po dose, and these doses consistently achieved statistical significance. Where the selected doses were increasingly in the lower portion of the dose-response curve, although the numerical numerical expressed in numbers, i.e. Arabic numerals of 0 to 9 inclusive. numerical nomenclature a numerical code is used to indicate the words, or other alphabetical signals, intended. results were reproducible within and across laboratories, an increasing number of studies failed to achieve a statistically significant difference, for example, the BPA po dose, the NP sc dose for adult OVX animals, and certainly the DDT sc dose. To assess reproducibility, the mean relative uterine weight increases were calculated in an overall global analysis (Table 10). The uterotrophic responses were consistent and reproducible between the dose-response and the coded single-dose studies without exception for every test substance and every protocol. The global analysis in Table 10 also shows subtle test substance-specific differences in the protocols that were consistent in both the dose-response and coded single-dose studies. Comparing the intact, immature, and adult OVX versions as protocols B and C, respectively, the adult OVX version appears to be more responsive with BPA, whereas the intact, immature version appears to be more responsive with GN and MX. More than doubling the time of treatment with extended dosing (protocol D), did increase the response with BPA, and marginally with GN, MX, and NP. The global analysis includes the results of all laboratories, regardless of mortalities in protocol A or the possible issues with laboratories 19 and 20 that are discussed below. Except for the lower means in the coded single-dose, high-dose EE studies for protocols B and C, no overall impact of their inclusion was observed. The data were analyzed an·a·lyze tr.v. an·a·lyzed, an·a·lyz·ing, an·a·lyz·es 1. To examine methodically by separating into parts and studying their interrelations. 2. Chemistry To make a chemical analysis of. 3. for an association between uterine weights and body weights and for the variability and power of the wet and the blotted weights. Although there was no consistent correlation between uterine weight and body weight, the data suggest that body weight is more strongly correlated cor·re·late v. cor·re·lat·ed, cor·re·lat·ing, cor·re·lates v.tr. 1. To put or bring into causal, complementary, parallel, or reciprocal relation. 2. with uterine weight in the immature animals than in the adult OVX animals. As with phase 1 and the dose-response studies, wet uterine weights were more variable than blotted weights (Kanno et al. 2001, 2003). The blotted uterine weights in phase 2, again, showed slightly less interlaboratory and intragroup Adj. 1. intragroup - occurring within an institution or community; "intragroup squabbling within the corporation" internal intramural - carried on within the bounds of an institution or community; "most of the students participated actively in the college's variability than wet weights with imbibed fluid, suggesting that blotted uterine weight will provide slightly better power for detecting uterotrophic effects than the wet weight. In 5 of 36 studies, the uterine weights after DBP treatment were statistically different from controls, indicating a certain rate of false positives and negatives will occur. Three sets of results were statistically higher than the vehicle groups, a false positive rate of about 8%, and two were statistically lower. This nearly even division into higher and lower differences supports random chance due to variability about the baseline The horizontal line to which the bottoms of lowercase characters (without descenders) are aligned. See typeface. baseline - released version . In further support, the margins by which the respective upper and lower 95% confidence intervals confidence interval, n a statistical device used to determine the range within which an acceptable datum would fall. Confidence intervals are usually expressed in percentages, typically 95% or 99%. achieved statistical significance were minimal (Table 8). In absolute terms (Alg.) such as are known, or which do not contain the unknown quantity. See also: Absolute , the mean relative increase in uterine weight in these three incidents was just under 40% and suggests a source of variability in the uterine weight from one group to another. When the raw body weight and uterine weight data were in these laboratories were examined, there were no obvious anomalies or inconsistencies such as outliers or high standard deviations In statistics, the average amount a number varies from the average number in a series of numbers. (statistics) standard deviation - (SD) A measure of the range of values in a set of numbers. when compared with other laboratories. When the overall patterns of these laboratories were assessed, one (laboratory 20) had the minimum response with five of six test chemicals and was below average for the two EE doses, consistent with its statistically decreased result. A second (laboratory 14) had responses that were the maximum with two test substances and above average for the remainder and the EE doses, consistent with its statistically increased result. The patterns of the other laboratories were unremarkable. Four of the five incidents occurred with immature animals. Although body weights were randomized ran·dom·ize tr.v. ran·dom·ized, ran·dom·iz·ing, ran·dom·iz·es To make random in arrangement, especially in order to control the variables in an experiment. , there is the possibility of group-to-group variations based on a litter-related effect. The animals used would have been born on the same day, meaning that the animals were likely from a limited number of litters. In fact, some investigators have taken the precaution to also randomize ran·dom·ize tr.v. ran·dom·ized, ran·dom·iz·ing, ran·dom·iz·es To make random in arrangement, especially in order to control the variables in an experiment. their groups by litter litter /lit·ter/ (lit´er) stretcher. lit·ter n. 1. A flat supporting framework, such as a piece of canvas stretched between parallel shafts, for carrying a disabled or dead person; a (Christian Christian flees the City of Destruction. [Br. Lit.: Pilgrim’s Progress] See : Escape Christian travels to Celestial City with cumbrous burden on back. [Br. Lit. et al. 1998). As the litter of origin for each individual was not recorded, this possibility cannot be assessed here. It is clear, however, that borderline borderline /bor·der·line/ (-lin) of a phenomenon, straddling the dividing line between two categories. borderline false positives can occur with the present protocols, and that a weight-of-the-evidence integration of the uterotrophic results with other structural, in vitro, and in vivo data may be necessary for interpretation. Similarly, false negatives may also occur, and data to qualify the performing laboratory and criteria to accept the results may be necessary (Owens Owens, river, c.120 mi (190 km) long, rising in the Sierra Nevada, E Calif., SE of Yosemite National Park and flowing SE, to enter Owens Lake, near Mt. Whitney. Since 1913, at a point c. et al. 2003). The results in three laboratories deserve comment. These laboratories displayed a trend toward lower responsiveness to both the EE and to several weak agonists when compared with other laboratories. The performance of laboratory 6 with its high vehicle control weights and limited responsiveness in some cases has been previously noted (Kanno et at. 2003). Here, we also note the lower general response in this laboratory to the EE doses in protocol B (Tables 1 and 2). Laboratory 19 observed no statistically significant uterotrophic responses for the test substances it could formulate formulate /for·mu·late/ (for´mu-lat) 1. to state in the form of a formula. 2. to prepare in accordance with a prescribed or specified method. or either of the two EE doses in protocol B (Tables 1, 2, 6, and 7). The pattern of responses in this laboratory in protocol C, however, was unremarkable when compared with other laboratories. A close examination of the data, including dietary analyses, has not revealed any apparent reasons for this lack of responsiveness. Laboratory 20 observed statistical significance with both EE doses, but the relative increases in weight were somewhat lower than other labs at the low EE dose and among the lowest at the high EE dose (Tables 1 and 2). Although statistical significance was observed with GN and MX, the increases in the uterine weights were the lowest observed in any laboratory (Tables 4 and 5). Statistical significance was not observed in either of the dose-response or the coded single-dose studies with either BPA or NP, and again, the increase in the uterine weights were the lowest observed in any laboratory (Tables 3 and 6). A review of the data and laboratory variables first indicated that the vehicle control uterine weights were > 50 mg, which was well above the 20-to 40-mg range in most other laboratories. Then, an analysis of laboratory diets for phytoestrogens Phytoestrogens Compounds found in plants that can mimic the effects of estrogen in the body. Mentioned in: Premenstrual Syndrome phytoestrogens, n.pl plant-derived estrogen analogs. found that laboratory 20's diet had the highest combined total GN and daidzein daidzein an estrogenic plant isoflavone. levels of > 500 [micro]g/g diet. This leads to the suspicion that the dynamic range of the bioassay in this particular case may have been impaired by the high phytoestrogen phytoestrogen /phy·to·es·tro·gen/ (-es´tro-jen) any of a group of weakly estrogenic, nonsteroidal compounds widely occurring in plants. phy·to·es·tro·gen n. content of the diet (Owens et al. 2003). Collectively with other observations in the dose-response studies, these data suggest the need to monitor the uterine weights of vehicle control animals, to specify that laboratory diets have low to modest phytoestrogen levels (< 350 [micro]g/g diet) (Owens et al. 2003), and to qualify laboratories with both reference and weak agonists before performing tests of unknown substances. In addition, care should be taken not to exceed the maximum tolerated dose, to reduce animal pain, suffering, and mortalities. The results in the current coded dose study provide additional evidence that a strong uterine response occurs even in the presence of severe systemic toxicity. The robustness of the uterine response in turn supports its use in a screening assay. In conclusion, the uterotrophic bioassay yields reproducible results within the same laboratory and across the participating laboratories over time with a range of test substances including the EE positive reference substance, the five weak agonist substances (BPA, GN, MX, NP, and DDT), and the negative substance (DBP). The results of the dose-response and coded single-dose studies are in agreement. No substantive performance differences were found between the different versions or their protocols that would support one version being consistently superior to another. Therefore, both the intact immature and OVX versions of the uterotrophic bioassay and the protocols herein are judged to be qualitatively equivalent to one another. Low rates of false negatives and false positives were observed. The false negatives occurred with very weak agonists (BPA, DDT, and NP) in the lowest portions of the their dose-response curves. The false-positive false-positive /false-pos·i·tive/ (pos´it-iv) 1. denoting a test result that wrongly assigns an individual to a category. 2. an individual so categorized. 3. an instance of a false-positive result. rate with DBP was just over 8%, with mean relative weight increases of 30-40%, suggesting the importance of controlling group-to-group variations in the baseline and using a weight-of-the-evidence approach in interpreting very modest responses. These and other results from the dose-response studies and the dietary analyses will be used to develop the draft OECD test guideline guideline Medtalk A series of recommendations by a body of experts in a particular discipline. See Cancer screening guidelines, Cardiac profile guidelines, Gatekeeper guidelines, Harvard guidelines, Transfusion guidelines. for the uterotrophic bioassay. These results will be submitted along with other data for independent peer review to provide support for the validation of the uterotrophic bioassay.
Table 1. Relative increase in uterine weights versus vehicle controls
with replicate low EE doses.
Protocol
A
1 [micro]g/kg/day
Lab Coded studies (a) Dose response (b)
1 -- 1.10 (0.92, 1.32) (c,d)
-- 0.99 (0.77, 1.26) (a,d)
2 2.78 (2.19, 3.53) * 3.17 (2.52, 3.99) *
3 1.52 (1.24, 1.88) * 2.25 (1.77, 2.86) *
1.53 (1.20, 1.96) *
4 2.81 (2.18, 3.61) * 3.20 (2.36, 4.35) *
5 1.36 (1.07, 1.74) * 1.40 (1.04, 1.90) *
6 -- --
7 -- 2.16 (1.73, 2.69) * (f)
-- 3.15 (2.57, 3.85) * (f)
8 3.31 (2.73, 4.00) * 3.09 (2.55, 3.73) *
9 -- 2.19 (1.72, 2.79) *
11 2.88 (2.26, 3.68) * 3.04 (2.42, 3.83) *
12 2.98 (1.47, 6.03) * 2.85 (2.21, 3.67) *
13 4.74 (3.88, 5.80) * 1.44 (1.06, 1.95) *
14 2.44 (1.61, 3.70) * 3.11 (2.44, 3.98) *
15 -- --
16 -- --
17 -- --
18 -- --
19 -- --
20 -- --
9/9 13/15
Protocol
B
0.3 [micro]g/kg/day
Lab Coded studies Dose response
1 -- 2.17 (1.80, 2.62) *
-- 2.49 (2.12. 2.93) *
2 2.25 (1.80, 2.82) * 2.11 (1.70, 2.62) *
3 2.42 (1.96, 3.00) * 2.00 (1.68, 2.38) *
2.31 (1.89, 2.81) *
4 1.79 (1.34, 2.39) * 2.75 (1.96, 4.06) *
5 2.07 (1.68, 2.55) * See note (e)
6 -- 1.59 (1.15, 2.18) *
7 -- 1.73 (1.48, 2.01) *
-- 1.79 (1.52, 2.10) *
8 2.99 (2.40, 3.72) * 2.65 (2.37, 2.97) *
9 -- 4.22 (3.63, 4.91) *
11 3.24 (2.49, 4.24) * 3.50 (2.83, 4.34) *
12 1.32 (0.91, 1.90) (d) 1.68 (1.11, 2.53) *
13 5.04 (3.67, 6.90) * 1.61 (1.08, 2.42)
14 2.69 (1.97, 3.68) * 2.61 (2.05, 3.32) *
15 -- 4.45 (3.46, 5.71) *
16 1.58 (1.02, 2.46) * --
17 1.83 (1.29, 2.61) * --
18 3.51 (2.86, 4.32) * 3.81 (323, 4.50) *
19 0.95 (0.60, 1.50) (d) --
20 1.78 (1 36, 2.33) * 1.83 (1.45, 2.31) *
12/14 18/18
Protocol
C
0.3 [micro]g/kg/day
Lab Coded studies Dose response
1 -- 1.98 (1.70, 2.30) *
-- 2.14 (1.88, 2.43) *
2 2.45 (1.87, 3.19) * 2.41 (2.06, 2.81) *
3 2.35 (1.84, 3.02) * 2.43 (2.14, 2.77) *
-- 2.96 (2.36, 3.71) *
4 -- --
5 -- --
6 -- 2.43 (1.55. 3.82) *
7 -- 1.78 (1.47, 2.16) *
-- 2.71 (2.27, 3.24) *
8 2.72 (2.03, 3.63) * 2.16 (1.91, 2.43) *
9 -- --
11 2.92 (2.47, 3.46) * 3.04 (2.63, 3.51) *
12 2.00 (1.44, 2.77) * 1.95 (1.52, 2.49) *
13 -- --
14 -- --
15 -- --
16 -- --
17 -- --
18 -- --
19 1.97 (1.62, 2.40) * --
20 -- --
6/6 11/11
Protocol
D
0.3 [micro]g/kg/day
Lab Coded studies Dose response
1 -- 2.93 (2.38, 3.60) *
-- 2.75 (2.30, 3.30) *
2 3.31 (2.74, 4.01) * 3.71 (2.91, 4.74) *
3 3.05 (2.40, 3.88) * 2.77 (2.44, 3.14) *
-- 2.85 (2.50, 3.26) *
4 -- --
5 -- --
6 -- --
7 -- 2.45 (1.97, 3.05) *
-- 3.28 (2.48, 4.35) *
8 -- --
9 -- 4.14 (3.34, 5.13) *
11 4.82 (3.61, 6.42) * 5.16 (3.65, 7.28) *
12 -- --
13 -- --
14 -- --
15 -- --
16 -- --
17 -- --
18 -- --
19 -- --
20 -- --
3/3 9/9
--, laboratory did not perform this particular study. (a) Results
from studies with coded or blinded doses for each substance. (b)
Results from the dose--response studies reported in the accompanying
paper (Kanno et al. 2003). (c) Ratio of geometric means of treated
blotted uterine weights to the vehicle control blotted uterine
weights after adjusting for the body weights at necropsy as a
covariable (lower 95% confidence limit, upper 95% confidence limit).
(d) This study did not achieve statistical significance. (e) This
laboratory used po dilution instructions to use doses of 1 and 3
[micro]g/kg/day. Therefore, no 0.3-[micro]g/kg/day dose was available.
(d) This laboratory used sc dilution instructions to use doses of 0.3
and 1 [micro]g/kg/day. The 1-[micro]g/kg/day dose was the actual two
EE dose and is reported here. * Level of significance, p < 0.05.
Table 2. Relative increase in uterine weights versus vehicle controls
with replicate high EE doses.
Protocol
A
3 [micro]g/kg/day
Lab Coded studies Dose response
1 -- 1,50 (1.25, 1.79) * (a)
-- 1.64 (1.29, 2.10) *
2 4.41 (3.47, 5.60) * 4.13 (3.27, 5.22) *
3 2.79 (2.27, 3.44) * 2.55 (2.00, 3.25) *
2.80 (2.20, 3.58) *
4 3.70 (2.88, 4.76) * 4.04 (2.97, 5.48) *
5 1.80 (1.40, 2.31) * 1.91 (1.41, 2.59) *
6 -- --
7 -- See note (c)
-- --
8 5.02 (4.15, 6.08) * 4.59 (3.88, 5.66) *
9 -- 5.19 (4.10, 6.58)
11 4.29 (3.36, 5.48) * 4.52 (3.54, 5.78) *
12 See note (d) 4.68 (3.63, 6.02) *
13 4.66 (3.83, 5.66) * 2.55 (2.05, 3.17) *
14 4.20 (2.73, 6.47) * 4.69 (3.74, 5.89) *
15 -- --
16 -- --
17 -- --
18 -- --
19 -- --
20 -- --
8/8 13/13
Protocol
B
1 [micro]g/kg/day
Lab Coded studies Dose response
1 -- 4.19 (3.47, 5.05)*
-- 4.07 (3.46, 4.80) *
2 4.42 (3.52, 5.54) * 4.44 (3.60, 5.48) *
3 4.63 (3.74, 5.73) * 3.82 (3.17, 4.60) *
3.79 (3.11, 4.63) *
4 3.41 (2.55, 4.56) * 4.52 (3.06, 6.67) *
5 4.15 (3.37, 5.11) * 3,61 (2.91, 4.46) * (b)
6 -- 2.30 (1.71, 3.10) *
7 -- 4.06 (3.49. 4.72) *
-- 4,15 (3.53, 4.90) *
8 4.76 (3.81, 5.95) * 4.96 (4.43, 5.55) *
9 -- 4.26 (3.64, 4,98) *
11 4.26 (3.17, 5.71) * 4.58 (3.70, 5.69) *
12 See note (d) 3.64 (2.43, 5.45) *
13 5.21 (3.81, 7.12) * 3.44 (2.25, 5.27)
14 4.83 (3.52, 6.63) * 4.55 (3.59, 5.76) *
15 -- 4.95 (3.66, 6.69) *
16 3.29 (2.13, 5.09) * --
17 3.50 (2.45, 5.00) * --
18 5.12 (4.18, 6.29) * 5.62 (4.89, 6.47) *
19 0.80 (0.50, 1.27) * --
20 2.41 (1.87, 3.11) * 2.38 (1.90, 2.99) *
12/13 19/19
Protocol
C
1 [micro]g/kg/day
Lab Coded studies Dose response
1 -- 3.13 (2.66, 3.67) *
-- 3.77 (3.27, 4.35) *
2 3.68 (2.77, 4.88) * 3.19 (2.66, 3.78) *
3 3.54 (2.74, 4.57) * 3.57 (3.06, 4.18) *
-- 3.61 (2.84, 4.59) *
4 -- --
5 -- --
6 -- 3.89 (2.45, 6.17) *
7 -- 3.29 (2.69, 4.01) *
-- 4.32 (3.55, 5.25) *
8 3.31 (2.47, 4.42) * 2.70 (2.39, 3.05) *
9 -- --
11 3.92 (3.28, 4.68) * 3.97 (3.36, 4.69) *
12 See note (d) 3.08 (2.41, 3.94) *
13 -- --
14 -- --
15 -- --
16 -- --
17 -- --
18 -- --
19 0.96 (0.79, 1.16) (e) --
20 -- --
4/5 11/11
Protocol
D
1 [micro]g/kg/day
Lab Coded studies Dose response
1 -- 4.40 (3.45, 5.61) *
-- 4.11 (3.36, 5.04) *
2 4.74 (3.88, 5.81) * 4.86 (3.55, 6.64) *
3 4.41 (3.32, 5.87) * 3.67 (3.13, 4.29) *
-- 4.04 (3.49, 4.69) *
4 -- --
5 -- --
6 -- --
7 -- 4.50 (3.53, 5.73) *
-- 5.67 (4.15, 7.74) *
8 -- --
9 -- 4.68 (3.66, 5.99) *
11 5.47 (4.14, 7.21 ) * 5.85 (4.26, 8.05) *
12 -- --
13 -- --
14 -- --
15 -- --
16 -- --
17 -- --
18 -- --
19 -- --
20 -- --
3/3 9/9
--, laboratory did not perform this particular study. (a) Ratio
of geometric means of treated blotted uterine weights to the
vehicle control blotted uterine weights after adjusting for the
body weights at necropsy as a covariable (lower 95% confidents limit,
upper 95% confidence limit). (b) This laboratory used po dilution
instructions to use doses of 1 and 3 [micro]g/kg/day. The 1
[micro]g/kg/day dose was the actual high EE dose and is reported here.
(c) This laboratory used sc dilution instructions for doses of 0.3 and
1 [micro]g/kg/day. Therefore, no 3 [micro]g/kg/day high EE dose was
performed. (d) This laboratory incorrectly diluted the high EE dose
in all studies. (e) This study did not achieve statistical
significance. * Level of significance, p < 0.05.
Table 3. Relative increase in uterine weights versus vehicle controls
with replicate BPA doses.
Protocol
A
600 mg/kg/day
Lab Coded studies Dose response
1 1.11 (0.90, 1.37) (a,b) --
2 1.45 (1.14, 1.84) * 1.49 (1.25, 1.77) * (c)
3 1.40 (1.14, 1.73) * --
4 1.36 (1.05, 1.74) * --
5 1.23 (0.95, 1.57) (b) --
6 -- --
7 -- 1.31 (1.03, 1.68) * (c)
8 1.91 (1.58, 2.31) * --
11 1.41 (1.11, 1.80) * --
12 1.08 (0.43, 2.71) (b,d) 1.63 (1.29, 2.06) (c)
13 1.25 (1.01, 1.56) * (d) 1.17 (0.79, 1.72) (b,c)
14 1.50 (0.95, 2.37) (b,d) --
15 -- --
16 -- --
17 -- --
18 -- --
19 -- --
20 -- --
6/10 3/4
Protocol
B
300 mg/kg/day
Lab Coded studies Dose response
1 1.58 (1.21, 2.08) * --
2 1.77 (1.40, 2.24) * 2.30 (1.88, 2.81) *
3 2.00 (1.62, 2.48) * --
4 1.45 (1.08, 1.94) * --
5 2.02 (1.64, 2.49) * --
6 -- 1.37 (1.05, 1.79) *
7 -- 1.95 (1.64, 2.32) *
8 1.91 (1.53, 2.39) * 1.91 (1.50, 2.43) *
11 1.82 (1.36, 2.44) * --
12 1.60 (1.12, 2.31) * 1.33 (0.88, 1.99) (b)
13 1.52 (1.11, 2.08) * 1.72 (1.08, 2.76) *
14 2.82 (2.05, 3.87) * --
15 -- 1.37 (1.03, 1.81) *
16 2.11 (1.37, 3.22) * --
17 2.35 (1.64, 3.37) * --
18 2.32 (1.88, 2.86) * 2.12 (1.81, 2.50) *
19 -- --
20 1.11 (0.86, 1.44) * 0.95 (0.69, 1.32) (b)
13/14 7/9
Protocol
C
300 mg/kg/day
Lab Coded studies Dose response
1 2.61 (2.23, 3.06) * --
2 2.61 (1.99, 3.42) * 2.79 (2.28, 3.41) *
3 2.89 (2.24, 3.72) * --
4 -- --
5 -- --
6 -- 2.41 (1.79, 3.23) *
7 -- 3.44 (2.76, 4.30) *
8 2.89 (2.16, 3.88) * 2.65 (2.16, 3.24) *
11 3.39 (2.85, 4.05) * --
12 2.30 (1.67, 3.18) * 2.72 (2.05, 3.61) *
13 -- --
14 -- --
15 -- --
16 -- --
17 -- --
18 -- --
19 2.42 (1.99, 2.95) * --
20 -- --
7/7 5/5
Protocol
D
300 mg/kg/day
Lab Coded studies Dose response
1 3.65 (2.84, 4.68) * --
2 3.91 (3.21, 4.76) * 3.74 (2.89, 4.84) *
3 3.26 (2.51, 4.24) * --
4 -- --
5 -- --
6 -- --
7 -- 3.90 (3.18, 4.78) *
8 -- --
11 4.05 (3.08, 5.33) * --
12 -- --
13 -- --
14 -- --
15 -- --
16 -- --
17 -- --
18 -- --
19 -- --
20 -- --
4/4 2/2
--, laboratory did not perform this particular study. (a) Ratio
of geometric means of treated blotted uterine weights to the vehicle
control blotted uterine weights after adjusting for the body weights
at necropsy as a covariable (lower 95% confidence limit, upper 95%
confidence limit). This study did not achieve statistical
significance. (c) In the dose--response studies at this dose, one
animal died in laboratory 2, one in laboratory 7, one in laboratory
12, and one in laboratory 13. (d) in the coded single-dose studies at
this dose, three animals died in laboratory 12, one in laboratory 13,
and two in laboratory 14. * Level of significance, p < 0.05.
Table 4. Relative increase in uterine weights versus vehicle controls
with replicate GN doses.
Protocol
A
300 mg/kg/day
Lab Coded studies Dose response
1 2.39 (1.94, 2.95) * (a) 2.22 (1.67, 2.95) *
2 2.47 (1.93, 3.17) * (b) --
3 2.73 (2.21, 3,36) * --
4 2.58 (2.01, 3.31) * --
5 1.60 (1.25, 2.05) * --
8 3.20 (2.65, 3.88) * 2.96 (2.42, 3.61) *
9 -- 2.57 (2.03, 3.25) *
11 2.86 (2.25, 3.65) * --
12 3.74 (1.83, 7.62) * 3.47 (2.71, 4.45) *
13 2.64 (2.17, 3,22) * --
14 2.98 (1.93, 4.61) * (b) --
16 -- --
18 -- --
20 -- --
10/10 4/4
Protocol
B
35 mg/kg/day
Lab Coded studies Dose response
1 2.16 (1,64, 2.84) * 2.33 (1.75, 3.10) *
2 2.95 (2.35, 3.70) * --
3 2.69 (2,17, 3.33) * --
4 2.26 (1.69, 3.03) * --
5 2.31 (1.88, 2.85) * --
8 2.53 (2.03, 3.15) * 2.69 (2.19, 3.30) *
9 -- 2.57 (2.19, 3.02) *
11 2.38 (1,78, 3.17) * --
12 2.20 (1.53, 3.17) * 2.28 (1.70, 3.05) *
13 2.25 (1.64, 3.07) * --
14 3.44 (2.50, 4.72) * --
16 3.21 (2.10, 4.89) * --
18 2.53 (2.06, 3.11) * --
20 1.32 (1.02, 1.70) * --
13/13 4/4
Protocol
C
35 mg/kg/day
Lab Coded studies Dose response
1 1.67 (1.42, 1.97) * 1,78 (1.46, 2.18) *
2 2.07 (1.59, 2.70) * --
3 1.66 (1.30, 2.13) * --
4 -- --
5 -- --
8 1.95 (1.46, 2,61) * --
9 -- 1.87 (1.56, 2.23) *
11 1.73 (1.47, 2.05) * --
12 1.57 (1.12, 2.20) * 1.56 (1.09, 2.21) *
13 -- --
14 -- --
16 -- --
18 -- --
20 -- --
6/6 3/3
Protocol
D
35 mg/kg/day
Lab Coded studies Dose response
1 2.11 (1.68, 2.65) * 2.20 (1.78, 2.73) *
2 2.33 (1.93, 2.80) * --
3 1.85 (1.48, 2.32) * --
4 -- --
5 -- --
8 -- --
11 -- 2.54 (2.15, 2.99) *
12 2.46 (1.93, 3.14) * --
13 -- --
14 -- --
16 -- --
17 -- --
18 -- --
20 -- --
4/4 2/2
--, laboratory did not perform this particular study. (a) Ratio of
geometric means of treated blotted uterine weights to the vehicle
control blotted uterine weights after adjusting for the body weights
at necropsy as a covariabre (lower 95% confidence limit, upper 95%
confidence limit). (b) In the coded single-dose studies at this
dose, one animal died in laboratory 2 and one in laboratory 14.
Level of significance, p < 0.05.
Table 5. Relative increase in uterine weights versus vehicle controls
with replicate MX doses.
Protocol
A
300 mg/kg/day
Lab Coded studies Dose response
1 2.97 (2.39, 3.71) * (a) 2.59 (2.13, 3.15) *
2 3.14 (2.47, 4.00) * --
3 2.77 (2.24, 3.41) * 2.94 (2.34, 3.69) * (b)
4 3.01 12.34, 3.86) * --
5 3.10 (2.41, 3.99) * --
8 3.71 (3.07, 4.49) * --
11 3.46 (2.67, 4.48) * --
12 3.20 (1.34, 7.61) * 3.98 (3.07, 5.15) *
13 3.31 (2.72, 4.02) * --
14 2.95 (1.94, 4.48) * (c) 3.46 (2.51, 4.77) *
16 -- --
17 -- --
18 -- --
20 -- --
10/10 4/4
Protocol
B
500 mg/kg/day
Lab Coded studies Dose response
1 2.73 (2.08, 3.58) * 2.47 (1.91, 3.21) *
2 3.01 (2.39, 3.80) * --
3 2.66 (2,15, 3.29) * 2.98 (2.42, 3.65) *
4 3.33 (2,49, 4.45) * --
5 3.61 (2.93, 4.45) * --
8 2.91 (2.33, 3.63) * --
11 2.39 (1.81, 3.16) * --
12 3.14 (2,18, 4.51) * 3.34 (2,53, 4.40) *
13 2.89 (2.11, 3.96) * --
14 4.07 (2.97, 5.56) * 3.76 (2.78, 5.09) *
16 4.29 (2.81, 6.55) * --
17 3.25 (2.28, 4.63) * --
18 3.18 (2.59, 3.90) * --
20 1.76 (1.37, 2.28) * --
14/14 4/4
Protocol
C
500 mg/kg/day
Lab Coded studies Dose response
1 1.96 (1.67, 2.30) * 2,32 (1.86, 2.89) *
2 2.08 (1.58, 2.73) * --
3 2.11 (1.64, 2.71) * 2.42 (1.96, 2.98) *
4 -- --
5 -- --
8 2.08 (1.54, 2.80) * --
11 3.14 (2.63, 3.75) * --
12 1.49 (1.09, 2.03) * 1.95 (1.45, 2.62) *
13 -- --
14 -- --
16 -- --
17 -- --
18 -- --
20 -- --
6/6 3/3
Protocol
D
500 mg/kg/day
Lab Coded studies Dose response
1 2.23 (1.77, 2.80) * 2.38 (1.76, 3.22) *
2 2.71 (2.21, 3.31) * --
3 2.67 (2.03, 3.51) * 2.46 (1.98, 3.06) *
4 -- --
5 -- --
8 -- --
11 3.34 (2.55, 4,36) * --
12 -- --
13 -- --
14 -- --
16 -- --
17 -- --
18 -- --
20 -- --
4/4 2/2
--, laboratory did not perform this particular study. (a) Ratio of
geometric means of treated blotted uterine weights to the vehicle
control blotted uterine weights after adjusting for the body weights
at necropsy as a covariable (lower 95% confidence limit, upper 95%
confidence limit). (b) In the dose--response studies at this dose,
one animal died in laboratory 3. (c) In the coded single-dose studies
at this dose, three animals died in laboratory 14. * Level of
significance, p < 0.05.
Table 6. Relative increase in uterine weights versus vehicle controls
with replicate NP doses.
Protocol
A
250 mg/kg/day
Lab Coded studies Dose response
1 1.71 (1.37, 2.14) * (a) --
2 2.03 (1.48, 2.77) * (b) --
3 1.80 (1.43, 2.27) * --
4 1.89 (1.24, 2.88) * (b) 2.61 (1.69, 4.04) * (d)
5 1.74 (1.28, 2.35) * (b) --
6 -- --
7 -- 2.17 (1.72, 2.74) * (d)
8 2.89 (2.33, 3.57) * (b) --
9 -- 2.17 (1.62, 2.90)*
11 2.33 (1.65, 3.28) * (b) --
12 1.97 (0.73, 5.33) (b,c) 2.95 (2.02, 4.32) * (d)
13 2.24 (1.81, 2.78) * (b) --
14 2.05 (1.17, 3.59) * (b) --
15 -- --
16 -- --
17 -- --
18 -- --
19 -- --
20 -- --
9/10 4/4
Protocol
B
80 mg/kg/day
Lab Coded studies Dose response
1 1.65 (1.26, 2.17) * --
2 1.34 (1.06, 1.68) * --
3 1.81 (1.46, 2,24) * --
4 1.45 (1.08, 1.94) * 2.05 (1.44, 2.92) *
5 1.64 (1.30, 2.07) * --
6 -- 1.24 (0.91, 1.88) (c)
7 -- 1.68 (1.36, 2.08) *
8 1.32 (1.06, 1,65) * 1.44 (1.15, 1.80) *
9 -- 1.86 (1.47, 2,36) *
11 2.05 (1.54, 2.73) * --
12 1.71 (1.19, 2.47) * 2,02 (1.49, 2.75) *
13 1.08 (0.79, 1.48) (c) --
14 1.72 (126, 2,35) * --
15 -- 1.22 (0.91, 1.65) (c)
16 1.30 (0.85, 1.99) --
17 2.49 (1.24, 3.55) * --
18 1.73 (1.40, 2.14) * 1.93 (1.56, 2.39) *
19 1.22 (0.76, 1.96) (c) --
20 1.07 (0.83, 1.38) (c) 0.75 (0.51, 1.11) (c)
11/15 6/9
Protocol
C
80 mg/kg/day
Lab Coded studies Dose response
1 1.43 (1.22, 1.68) * --
2 1.24 (0.95, 1.62) (c) --
3 1.37 (1.07, 1.76) * --
4 -- --
5 -- --
6 -- 1.16 (0.90, 1.48) (c)
7 -- 1.64 (1.32, 2.03) *
8 1.59 (1.19, 2.13) * 1.17 (0.98, 1.39) (c)
9 -- 1.23 (0.99, 1.52) (c)
11 1.38 (1.16, 1.63) * --
12 1.38 (1.01, 1.90) * 1.33 (1.02, 1.73) *
13 -- --
14 -- --
15 -- --
16 -- --
17 -- --
18 -- --
19 1.40 (1.15, 1.70) * --
20 -- --
6/7 2/5
Protocol
D
80 mg/kg/day
Lab Coded studies Dose response
1 1.54 (1.23, 1.93) * --
2 1.86 (1.55, 2.24) * --
3 1.73 (1.37, 2.18) * --
4 -- --
5 -- --
6 -- 2.11 (1.73, 2.58) *
7 -- --
8 -- --
9 -- 1.83 (1.51, 2.21) *
11 2.02 (1.57, 2.60) * --
12 -- --
13 -- --
14 -- --
15 -- --
16 -- --
17 -- --
18 -- --
19 -- --
20 -- --
4/4 2/2
--, laboratory did not perform this particular study. (a) Ratio of
geometric means of treated blotted uterine weights to the vehicle
control blotted uterine weights after adjusting for the body weights
at necropsy as a covariable (lower 95% confidence limit, upper 95%
confidence limit). (b) In the coded single-dose studies, one animal
died in laboratory 2, four animals died in laboratory 4, two animals
died in laboratory 5, one animal died in laboratory 8, two animals
died in laboratory 11, four animals died in laboratory 12, one animal
died in laboratory 13, and four animals died in laboratory 14. (c)
This study did not achieve statistical significance. (d) In the
dose--response studies at this dose, two animals died in laboratory
4, one animal died in laboratory 7, and three animals died in
laboratory 12. * Level of significance p < 0.05.
Table 7. Relative increase in uterine weights versus vehicle controls
with replicate DDT doses.
Protocol
A
300 mg/kg/day
Lab Coded studies Dose response
1 2.70 (2.15, 3.39) * (a) --
2 3.68 (2.88, 4.71) * (c) --
3 3.05 (2.45, 3.81) * 2.67 (1.99, 3.59) *
4 3.76 (2.91, 4.87) * --
5 2.92 (2.23, 3.83) * 2.71 (1.92, 3.24) *
8 3.87 (3.18, 4.71) * --
11 3.58 (2.79, 4.60) * 3.43 (2.96, 3.98) *
12 See note (b) 3.45 (2.41, 4.94) * (e)
13 4.12 (3.32, 5.12) * --
14 4.26 (2.65, 6.83) * (c) --
16 -- --
17 -- --
18 -- --
19 -- --
20 -- --
9/9 4/4
Protocol
B
100 mg/kg/day
Lab Coded studies Dose response
1 2.05 (1.57, 2.70) * --
2 1.13 (0.90, 1,42) (b) --
3 1.18 (0.95, 1.46) (b) 1.01 (0.83, 1.23) (b)
4 1.57 (1.17, 2.10) * --
5 0.95 (0.78, 1.17) (b) 1.18 (0.91, 1.54) (b)
8 1.06 (0.85, 1.32) (b) --
11 1.03 (0.78, 1,37) (b) 1.08 (0.87, 1.34) (b)
12 1.50 (1.04, 2.16) * 1.47 (1.11, 1.94) *
13 1.79 (1.31, 2.45) * --
14 1.17 (0.85, 1.60) (b) --
16 1.29 (0.84, 1.97) (b) --
17 1.46 (1.02, 2.08) * --
18 0.98 (0.80, 1.21) (b) --
19 1.07 (0.67, 1.69) (b) --
20 0.79 (0.61, 1.02) (b) --
5/15 1/4
Protocol
C
100 mg/kg/day
Lab Coded studies Dose response
1 1.65 (1.41, 1.93) * --
2 1.43 (1.09, 1.86) * --
3 1.29 (1.0035 (d), 1.65) * 1.43 (1.21, 1.69) *
4 -- --
5 -- --
8 1.17 (0.87, 1.57) (b) 1.25 (0.98, 1.59) (b)
11 1.24 (1.05, 1.47) * --
12 0.96 (0.71, 1.31) (b) 1.31 (0.96, 1.78) (b)
13 -- --
14 -- --
16 -- --
17 -- --
18 -- --
19 1.08 (0.87, 1.29) (b) --
20 -- --
4/8 1/3
Protocol
D
100 mg/kg/day
Lab Coded studies Dose response
1 1.05 (0.83, 1.31) (b) --
2 1.21 (1.01, 1.46) * --
3 1.14 (0.92, 1.43) (b) 1.18 (1.03, 1.36) *
4 -- --
5 -- --
8 -- --
11 1.31 (1.03, 1.68) * 1.48 (1.17, 1.87) *
12 -- --
13 -- --
14 -- --
16 -- --
17 -- --
18 -- --
19 -- --
20 -- --
2/4 2/2
--, laboratory did not perform this particular study. (a) Ratio of
geometric means of treated blotted uterine weights to the vehicle
control blotted uterine weights after adjusting for the body weights
at necropsy as a covariable (lower 95% confidence limit, upper 95%
confidence limit). (b) This study did not achieve statistical
significance. (c) In the coded single-dose studies, all six animals
died in laboratory 12, and two animals died in laboratory 14. (d)
With the lower confidence level number > 1.00, the result is
statistically significant. (e) In the dose--response studies at this
dose, one animal died in laboratory 12. * Level of significance,
p < 0.05.
Table 8. Relative increase in uterine weights versus vehicle
controls with replicate DBP doses.
Protocol
A B
Lab 1000 mg/kg/day 500 mg/kg/day
1 0.91 (0.74, 1.13) (a) 0.97 (0.74, 1.28)
2 0.99 (0.78, 1.26) 1.04 (0.83, 1.31)
3 1.00 (0.81, 1.23) 1.01 (0.81, 1.25)
4 0.99 (0.77, 1.28) 0.85 (0.64, 1.14)
5 1.03 (0.81, 1.32) 1.06 (0.86, 1.31)
8 0.98 (0.81, 1.18) 1.00 (0.80, 1.24)
11 0.95 (0.75, 1.22) 1.39 (1.06, 1.82) * (b)
12 0.91 (0.38, 2.20) --
13 0.86 (0.69, 1.07) 0.98 (0.72, 1.34)
14 0.91 (0.60, 1.38) 1.38 (1.01, 1.89) * (b)
16 -- 0.80 (0.59, 1.38)
17 -- 0.88 (0.62, 1.26)
18 -- 0.74 (0.6, 0.91) * (b)
19 -- 0.75 (0.47, 1.20)
20 -- 0.73 (0.56, 0.96) * (b)
0/10 4/14
Protocol
C D
Lab 500 mg/kg/day 500 mg/kg/day
1 1.37 (1.17, 1.61) * (b) 0.91 (0.73, 1.15)
2 0.99 (0.76, 1.28) 1.03 (0.86, 1.24)
3 0.90 (0.70, 1.15) 1.01 (0.81, 1.26)
4 -- --
5 -- --
8 1.24 (0.92, 1.65) --
11 0.99 (0.83, 1.16) 1.00 (0.78, 1.28)
12 0.97 (0.67, 1.40) 0.93 (0.68, 1.26)
13 -- --
14 -- --
16 -- --
17 -- --
18 -- --
19 0.84 (0.69, 1.02) --
20 -- --
1/7 0/5
--, laboratory did not perform this particular study. (a) Ratio of
geometric means of treated blotted uterine weights to the vehicle
control blotted uterine weights after adjusting for the body
weights at necropsy as a covariable (lower 95% confidence limit,
upper 95% confidence limit). (b) This study did not achieve
statistical significance. * Level of significance p < 0.05.
Table 9. Details for animals, diet, vehicles, and bedding in
laboratories participating only in coded single-dose studies. (a)
Strain of rat (b) Animal diet (b)
Lab Immature rats 0VX rats Immature 0VX
16 Wistar NA Altromin 1324 NA
FORTII
17 Wistar/Han NA Altromin 1324 NA
19 CD Sprague- CD Sprague- RM1(E) SQC RM1(E) SQC
Dawley Dawley expanded pellet expanded pellet
For oral gavage (b) For sc injection (b)
Lab Vehicle 1 Vehicle 2 Vehicle 1 Vehicle 2
16 NA NA Peanut oil NA
17 NA NA Peanut oil NA
19 NA NA Corn oil Ethanol/corn oil
Lab Bedding
16 Woodchip--low dust
17 Tapvei bedding
19 Lignocel grade 4/4
woodflakes (immature)/
none for 0VX
(a) The details for laboratories participating in the dose--response
studies and that may have participated in the coded single-dose studies
herein can be found in Table 7 in Kanno et al. (2003). (b) Detailed
information is available from the corresponding author of this article.
Table 10. Global analysis of results.
Protocol
Substance/dose A B
BPA mg/kg/day 600 300
DR 1.41 (1.07, 185) (a) 1.61 (1.00, 2.58)
CSD 1.34 (1.09, 1.66) 1.85 (1.58, 2.16)
DDT mg/kg/day 300 100
DR 3.13 (2.38, 4.12) 1.16 (0.94, 1.44)
CSD 3.60 (2.94, 4.41) 1.23 (0.97, 1.58)
GN mg/kg/day 300 35
DR 2.75 (1.98, 3.80) 2.47 (1.82, 3.37)
CSD 2.65 (2.21, 3.18) 2.42 (2.05, 2.86)
MX mg/kg/day 300 500
DR 3.16 (2.09, 4.79) 3.13 (1.70, 5.75)
CSD 3.21 (2.58, 3.99) 3.03 (2.54, 3.62)
NP mg/kg/day 250 80
DR 2.40 (1.90, 3.04) 1.51 (1.05, 2.16)
CSD 2.12 (1.72, 2.61) 1.53 (1.26, 1.88)
EE [micro]g/kg/day 1 0.3
DR 2.27 (1.71, 3.02) 2.42 (1.86, 3.13)
CSD 2.57 (1.88, 3.51) 2.18 (1.64, 2.90)
EE [micro]g/kg/day 3 1
DR 3.42 (2.56, 4.57) 5.09 (2.44, 10.62)
CSD 3.78 (2.83, 5.05) 3.56 (2.61, 4.85)
DBP [micro]g/kg/day 1,000 500
CSD 0.95 (0.77, 1.18) 0.97 (0.80, 1.17)
Protocol
Substance/dose C D
BPA mg/kg/day 300 300
DR 2.73 (2.07, 3.61) 3.78 (2.98, 4.79)
CSD 2.68 (2.36, 3.04) 3.84 (3.39, 4.35)
DDT mg/kg/day 100 100
DR 1.33 (1.04, 1.69) 1.31 (1.08, 1.59)
CSD 1.24 (1.00, 1.52) 1.17 (1.06, 1.30)
GN mg/kg/day 35 35
DR 1.73 (1.45, 2.07) 2.36 (1.61, 3.46)
CSD 1.77 (1.58, 2.00) 2.18 (1.91, 2.49)
MX mg/kg/day 500 500
DR 2.25 (1.79, 2.83) 2.43 (1.55, 3.83)
CSD 2.07 (1.72, 2.48) 2.62 (2.28, 3.00)
NP mg/kg/day 80 86
DR 1.29 (1.06, 1.58) 1.96 (1.59, 2.42)
CSD 1.40 (1.24, 1.57) 1.77 (1.58, 1.98)
EE [micro]g/kg/day 0.3 0.3
DR 2.33 (1.97, 2.76) 3.34 (2.79, 4.01)
CSD 2.30 (2.02, 2.62) 3.50 (2.80, 4.37)
EE [micro]g/kg/day 1 1
DR 3.40 (2.87, 4.03) 4.51 (3.75, 5.43)
CSD 2.67 (1.60, 4.43) 4.87 (4.34, 5.45)
DBP [micro]g/kg/day 500 500
CSD 1.02 (0.84, 1.24) 0.99 (0.91, 1.07)
Abbreviations: CSD, coded single-dose studies; DR, dose--response
studies.
(a) Ratio of geometric means of treated blotted uterine weights
to the vehicle control blotted uterine weights after adjusting
for the body weights at necropsy as a covariable (lower 95%
confidence limit, upper 95% confidence limit).
We acknowledge the dedicated efforts and work of the participating labs in generating these data, namely, the Institute of Environmental Toxicology toxicology, study of poisons, or toxins, from the standpoint of detection, isolation, identification, and determination of their effects on the human body. Toxicology may be considered the branch of pharmacology devoted to the study of the poisonous effects of drugs. , Japan; Mitsubishi Mitsubishi: see zaibatsu. Chemical Safety Institute, Japan; Japan Bioassay Research Centre, Japan; Sumitomo Chemical Company, Japan; Syngenta Syngenta AG is a large global agribusiness which markets seeds and crop protection products (pesticides). Syngenta is involved in biotechnology and genomic research. The company is a leader in crop protection, and ranks third in total sales in the commercial agricultural seeds Central Toxicology Laboratory, United Kingdom; WIL See WinBatch. Research Laboratories, United States United States, officially United States of America, republic (2005 est. pop. 295,734,000), 3,539,227 sq mi (9,166,598 sq km), North America. The United States is the world's third largest country in population and the fourth largest country in area. ; BASF BASF Bar Association of San Francisco (since 1872; San Francisco, California) BASF Badische Anilin und Soda Fabrik (German chemical products company) BASF Builders Association of South Florida , Germany Germany (jûr`mənē), Ger. Deutschland, officially Federal Republic of Germany, republic (2005 est. pop. 82,431,000), 137,699 sq mi (356,733 sq km). ; Bayer AG Bayer AG German chemical and pharmaceutical company. Founded in 1863 by Friedrich Bayer (1825–1880), it now operates plants in more than 30 countries. Bayer has originated scores of pharmaceuticals, chemicals, and synthetic materials; it was the first developer and , Germany; Aventis Aventis Is a pharmaceutical and lab assay testing company. It was formed in 1999 when Rhône-Poulenc S.A. merged with Hoechst AG. The merged company was based in Strasbourg, France. Sanofi-Aventis was formed in 2004 when Sanofi-Synthélabo purchased Aventis. Crop Sciences, France; Exxon Biomedical Sciences Noun 1. biomedical science - the application of the principles of the natural sciences to medicine bioscience, life science - any of the branches of natural science dealing with the structure and behavior of living organisms Inc., United States; Free University of Berlin, Germany; National Institute of Toxicological Research, Korea Korea (kôrē`ə, kə–), Korean Hanguk or Choson, region and historic country (85,049 sq mi/220,277 sq km), E Asia. ; Huntingdon
emanating from or pertaining to Europe. European bat lyssavirus see lyssavirus. European beech tree fagussylvaticus. European blastomycosis see cryptococcosis. Chemical Industry Council and the Japanese Japanese (jăp'ənēz`), language of uncertain origin that is spoken by more than 125 million people, most of whom live in Japan. There are also many speakers of Japanese in the Ryukyu Islands, Korea, Taiwan, parts of the United States, and Chemical Industry Association. Nonylphenol was kindly donated do·nate v. do·nat·ed, do·nat·ing, do·nates v.tr. To present as a gift to a fund or cause; contribute. v.intr. To make a contribution to a fund or cause. by Schenectady Schenectady (skənĕk`tədē), city (1990 pop. 65,566), seat of Schenectady co., E central N.Y., on the Mohawk River and Erie Canal; founded 1661 by Arent Van Curler, inc. 1798. International Inc., (Schenectady, NY, USA) and bisphenol A was kindly donated by Bayer AG. We thank E. Zeiger and H. Koeter for reviewing the manuscript manuscript, a handwritten work as distinguished from printing. The oldest manuscripts, those found in Egyptian tombs, were written on papyrus; the earliest dates from c.3500 B.C. . REFERENCES Blair R, Fang H, Branham WS, Hess Hess , Walter Rudolf 1881-1973. Swiss physiologist. He shared a 1949 Nobel Prize for his research on the brain's control of the body. B, Dial SL, Moland Moland is a former municipality in Aust-Agder county, Norway. It was created on January 1 1962, when a merger took place between the municipalities of Stokken, Austre Moland and Flosta as well as Strengereid district in Tvedestrand. CL, et al. 2000. Estrogen receptor relative binding affinities of 188 natural and xenochemicals: structural diversity of ligands. Toxicol Sci 54:138-153. Christian MS, Hoberman AM, Bachmann S Bachmann may refer to: People
1. estrus-producing; having the properties of, or similar to, an estrogen. 2. pertaining to, having the effects of, or similar to an estrogen. response assay) in untreated control and positive control (DES-DP, 2.5 Pc/kg, BID) Wistar Wistar can refer to:
Ema M, Miyawaki E. 2001. Adverse effects on development of the reproductive system The development of the reproductive system is a part of the prenatal development, and concerns the sex organs. It is a part of the stages of sexual differentiation. Because its location to a large extent overlaps the urinary system, the development of them can also be described together in male offspring off·spring n. 1. The progeny or descendants of a person, animal, or plant considered as a group. 2. A child of particular parentage. of rats given monobutyl phthalate Phthal´ate n. 1. (Chem.) A salt of phthalic acid. , a metabolite metabolite, organic compound that is a starting material in, an intermediate in, or an end product of metabolism. Starting materials are substances, usually small and of simple structure, absorbed by the organism as food. of dibutyl phthalate Dibutyl phthalate (DBP) is a commonly used plasticizer. It is also used as an additive to adhesives or printing inks. It is soulble in various organic solvents, e.g. in alcohol, ether and benzene. , during late pregnancy. Reprod Toxicol 15:189-194. Ema M, Miyawaki E, Kawashima Kawashima is a common Japanese name. It may refer to: Towns
n. A member of a Baltic people constituting the main population of Latvia. [German Lette, from Latvian Latvi.] 111:271 278. ICCVAM (Intraagency Coordinating Committee on the Validation of Alternative Methods). 1997. Validation and regulatory acceptance of toxicological test methods. A report of the Ad Hoc For this purpose. Meaning "to this" in Latin, it refers to dealing with special situations as they occur rather than functions that are repeated on a regular basis. See ad hoc query and ad hoc mode. Interagency in·ter·a·gen·cy adj. Involving or representing two or more agencies, especially government agencies. Coordinating Committee on the Validation of Alternative Methods, NIH "Not invented here." See digispeak. NIH - The United States National Institutes of Health. Report No. 97-3981. Research Triangle Park Research Triangle Park, research, business, medical, and educational complex situated in central North Carolina. It has an area of 6,900 acres (2,795 hectares) and is 8 × 2 mi (13 × 3 km) in size. Named for the triangle formed by Duke Univ. . NC:National Institute of Environmental Health Sciences The National Institute of Environmental Health Sciences (NIEHS) is one of 27 Institutes and Centers of the National Institutes of Health (NIH),which is a component of the Department of Health and Human Services (DHHS). The Director of the NIEHS is Dr. David A. Schwartz. . Kanno J, Onyon L, Haseman J, Fenner-Crisp P, Ashby Ashby may refer to: Surname
For example, data entry validity checking determines whether the data make sense (numbers fall within a range, numeric data the rat uterotrophic bioassay to screen compounds for in vivo estrogenic responses: phase I, Environ Health Perspect 109:785-794. Kanno J, Onyon L, Peddada S, Ashby J, Owens W. 2003. The OECD program to validate the rat uterotrophic bioassay. Phase 2: dose-response studies. Environ Health Perspect 111:1530-1549. Mylchreest E, Cattley RC, Foster PMD (Polarization Mode Dispersion) The type of dispersion that occurs in singlemode fiber due to a lack of perfect symmetry in the fiber and from external pressures on the cable. Light travels over singlemode fiber in two polarization states. . 1998. Male reproductive re·pro·duc·tive adj. 1. Of or relating to reproduction. 2. Tending to reproduce. reproductive subserving or pertaining to reproduction. tract malformations in rats following gestational gestational pertaining to or emanating from gestation. gestational age the age of the fetus in terms of time lapse, e.g. three month fetus, or in terms of proportion of total gestational duration, e.g. first trimester fetus. and lactational exposure to di(c-butyl) phthalate: an antiandrogenic mechanism? Toxicol Sci 43:474-60. Mylchreest E, Sar M, Cattley RC, Foster PMD. 1999. Disruption of androgen-regulated male reproductive development by di(n-butyl) phthalate during late gestation GESTATION, med. jur. The time during which a female, who has conceived, carries the embryo or foetus in her uterus. By the common consent of mankind, the term of gestation is considered to be ten lunar months, or forty weeks, equal to nine calendar months and a week. in rats is different from flutamide flutamide /flu·ta·mide/ (floo´tah-mid) a nonsteroidal antiandrogen, used in the treatment of metastatic prostatic carcinoma. flutamide (floo´t . Toxicol Appl Pharmacol 158:81-95. OECD (Organisation for Economic Co-operation and Development). 1998. The Validation of Test Methods Considered for Adoption as OECD Test Guidelines. ENV/MC/CHEM(g8)6, Paris:OECD. --.2000. Guidance Document on the Recognition, Assessment, and Use of Clinical Signs as Humane humane pertaining to the avoidance of infliction of pain, discomfort and harassment; used especially with regard to animals. humane considerations Endpoints for Experimental Animals Used in Safety Evaluation. OECD Environmental Health and Safety Publications. Series on Testing and Assessment, No. 19. ENV/JM/MOMO(2000)7, Paris:OECD. --.2002. OECD Series on Principles of Good Laboratory Practice and Compliance Monitoring. Twelve documents are available for downloading downloading - download , including "OECD principles of Good Laboratory Practice." Available: http:// webnet1.oecd.org/EN/doeument/O,,EN-document-526-14-no-24-6553-0,00.html [accessed 1 August 2002. Owens W, Ashby J, Odum Odum may refer to: In places:
A trademark used for a screw with a head having two intersecting perpendicular slots and for a screwdriver with a tip shaped to fit into these slots. , Sar M, Foster PMD, Gaido KW. 2001. Altered gene profiles in fetal fetal /fe·tal/ (fe´tal) of or pertaining to a fetus or the period of its development. fe·tal adj. Of, relating to, or being a fetus. rat testes testes or testicles Male reproductive organs (see reproductive system). Humans have two oval-shaped testes 1.5–2 in. (4–5 cm) long that produce sperm and androgens (mainly testosterone), contained in a sac (scrotum) behind the penis. after in utero in utero (in u´ter-o) [L.] within the uterus. in u·ter·o adj. In the uterus. in utero adv. exposure to di(n-butyl)phthalate. Toxicol Sci 64:233-242, Zacharewski TR, Meek meek adj. meek·er, meek·est 1. Showing patience and humility; gentle. 2. Easily imposed on; submissive. MO, Clemens JH, Wu ZF, Fielden Field´en a. 1. Consisting of fields. The fielden country also and plains. - Holland. MR, Matthews Matthews may refer to: In places:
n.pl organic compounds synthesized from acids and alcohols, typically possessing fruity aromas. , Toxicol Sci 46:282-293. Jun Kanno, (1) Lesley Lesley (Scottish, from the grey fort) can refer to any of the following: Places
The following people bear the first name Lesley:
(1) National Institute of Health Sciences, Tokyo Tokyo (tō`kēō), city (1990 pop. 8,163,573), capital of Japan and of Tokyo prefecture, E central Honshu, at the head of Tokyo Bay. , Japan; (2) Environmental Health and Safety Division, Organisation for Economic Co-operation and Development, Paris, France; (3) National institute of Environmental Health Sciences, Research Triangle Park, North Carolina North Carolina, state in the SE United States. It is bordered by the Atlantic Ocean (E), South Carolina and Georgia (S), Tennessee (W), and Virginia (N). Facts and Figures Area, 52,586 sq mi (136,198 sq km). Pop. , USA; (4) Syngenta Central Toxicology Laboratory, Macclesfield Macclesfield (măk`əlzfēld), town (1991 pop. 46,832), Cheshire, W England. Silk manufacture, of which Macclesfield is the principal center in England, was introduced in the town in 1756. , Cheshire Cheshire, county, England Cheshire (chĕsh`ər), county (1991 pop. 937,300), 901 sq mi (2,334 sq km), W central England. The county seat is Chester. The terrain is generally low, flat, and fertile. , United Kingdom; (5) BASF Aktiengesellschaft Aktiengesellschaft (IPA: ['aktsiəngəzεlʃaft]; abbreviated AG) is a German term that refers to a corporation that is limited by shares, i.e., owned by shareholders. , Ludwigshafen Lud·wigs·ha·fen A city of southwest Germany on the Rhine River opposite Mannheim. Founded as a fortress in the early 17th century, it is now a leading center of the country's chemical industry. Population: 162,000. , Germany; (6) The Procter
Cincinnati is a city in the U.S. state of Ohio and the county seat of Hamilton County. , USA This article is part of the mini-monograph "The OECD Validation of the Uterotrophic Bioassay." Address correspondence to J.W. Owens, Central Product Safety, The Procter & Gamble Company, 11810 East Miami River Miami River or Great Miami River A river rising in western Ohio and flowing about 257 km (160 mi) southwest to the Ohio River. Rd., Cincinnati Cincinnati (sĭnsənăt`ē, –năt`ə), city (1990 pop. 364,040), seat of Hamilton co., extreme SW Ohio, on the Ohio River opposite Newport and Covington, Ky.; inc. as a city 1819. , OH 45252 USA. Telephone: (513) 627-1385. Fax: (513) 627-1208. E-mail owens.jw@pg.com Address any correspondence or questions about the OECD Programme and OECD documents to Herman Herman only goal in life becomes winning at cards. [Russ. Opera: Tchaikovsky, Queen of Spades, Westerman, 401] See : Obsessiveness Koeter, OECD Environmental Health and Safety Division, OECD, 2 rue Andre An·dré , John 1751-1780. British army officer hanged as a spy in the American Revolution for conspiring with Benedict Arnold. Pascal, 75775 Paris Cedex CEDEX Centro de Estudios y Experimentación de Obras Públicas CEDEX Courrier d'Entreprise à Distribution Exceptionnelle (French) CEDEX Centre des Expositions 16 France. Telephone: 33 1 45 24 9849. Fax: 33 1 45 24 1675. E-mail:herman.koeter@ oecd.org See .org. (networking) org - The top-level domain for organisations or individuals that don't fit any other top-level domain (national, com, edu, or gov). Though many have .org domains, it was never intended to be limited to non-profit organisations. RFC 1591. This paper represents the opinions of the authors and may not reflect the official positions and recommendations of the OECD. The authors declare they have no conflict of interest. Received 1 July July: see month. 2002; accepted 7 October October: see month. 2002. |
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