The preclinical toxicology of salmeterol hydroxynaphthoate.

An extensive toxicology programme on salmeterol hydroxynaphthoate (Serevent), a marketed long-acting beta(2)-adrenoceptor agonist, has been carried out. The studies evaluated both the local (respiratory tract) and systemic tolerance to single and repeated dosing, effects on all stages of reproduction, as well as the genotoxic and oncogenic potential. High acute doses were well tolerated and caused no specific target organ toxicity. In repeat dose studies, animals tolerated salmeterol very well both locally and systemically. No significant effects on the respiratory tract of dogs were seen and only minor laryngeal changes, typical of those occurring with many inhaled medicines, were noted in rats. The high systemic concentrations achieved resulted in a number of changes that are considered to be the result of excessive and prolonged beta( 2)-adrenoceptor stimulation. These included tachycardia, skeletal muscle hypertrophy and minor haematological and blood biochemical changes in general toxicity studies, foetal effects in rabbit organogenesis studies and increased incidences of smooth muscle tumours of the mesovarium in the rat and of the uterus in the mouse oncogenicity studies. Salmeterol showed no evidence of any genotoxic potential. Results of the extensive toxicology programme provide good assurance of the safety for the inhaled use of salmeterol in patients; this has ben confirmed by many years of clinical experience during its development and marketing.

Determinants of functional performance in long-term survivors of allogeneic hematopoietic stem cell transplantation with chronic graft-versus-host disease (cGVHD).

This study examined factors accounting for functional performance limitations in 100 long-term survivors of allogeneic hematopoietic stem cell transplantation with chronic graft-versus-host disease (cGVHD). Functional performance, measured by the SF-36 physical component summary score, was substantially lower (mean=36.8+/-10.7) than the US population norm of 50 (P<0.001). The most severe decrements were in physical function (mean=38.8+/-10.9) and physical role function (mean=37.88+/-11.88); 68% of respondents exceeded the five-point threshold of minimum clinically important difference below the norm on these subscales. Controlling for age and gender, six variables explained 56% of the variance in functional performance: time since cGVHD diagnosis, cGVHD severity, intensity of immunosuppression, comorbidity, functional capacity (distance walked in 2 min, grip strength, and range of motion), and cGVHD symptom bother (F=11.26; P<0.001). Significant independent predictors of impaired performance were intensive systemic immunosuppression, reduced capacity for ambulation, and greater cGVHD symptom bother (P<0.05). Symptom bother had a direct effect on functional performance, as well as an indirect effect partially mediated by functional capacity (Sobel test, P=0.004). Results suggest two possible mechanisms underlying impaired functional performance in survivors with cGVHD and underscore the importance of testing interventions to enhance functional capacity and reduce symptom bother.

Does genomic imprinting contribute to valproic acid teratogenicity?

Reciprocal outcrosses and backcrosses were made between strains of mice with different susceptibilities to valproic acid (VPA) teratogenicity. Relatively resistant C57BL/6J (C) and more susceptible SWV (S) strains of mice produced F1 hybrids in which the female parent was C and the male parent was S (CS-F1) as well as the reciprocal with S dams and C sires (SC-F1). Each was backcrossed to each strain, producing 8 types of backcross matings: CS x C, SC x C, CS x S, SC x S; C x CS, C x SC, S x CS, S x SC (for all matings dams are listed first). At 8d:12 +/- 5h of gestation, a teratogenic dose, 600 mg/kg, of aqueous VPA was injected ip into the dams. Fetuses were examined on gestation day (gd) 18 for abnormality, mortality, litter size, and weight. Genomic imprinting (imprinting) is a phenomenon at least in part involving hyper- or hypomethylation of bases in DNA, which is believed to determine whether or not the imprinted gene will be expressed. Imprinting has been reported to occur differentially in the male and female for a number of gene loci. Thus, in crosses between strains with differing susceptibility to VPA, if imprinting is occurring, the susceptibility of a fetus might be predicted to be disproportionately influenced by susceptibility of its grandparents. Significant differences in frequency (%) of occurrence of exencephaly in progeny of all backcrosses with F1 dams consistent with those expected for imprinting were found in the present study (CS-F1x C = 21.8 +/- 3.9%, SC-F1x C = 10.8 +/- 3.2%, P < 0.03; CS-F1x S = 14.8 +/- 3.1%, SC-F1x S = 6.3 +/- 2.3%, P < 0.03). SWV dams revealed the same pattern (S x SC-F1 = 50.0 +/- 8.3%, S x CS-F1 = 37.1 +/- 4.7%, P < 0.04). Differences in prenatal mortality also consistent with genomic imprinting occurred in backcrosses with pure-line SWV dams (S x SC = 64.4 +/- 8.0%, S x CS = 30.5 +/- 4.5%, P < 0.001). Fetal weight was reduced in a manner consistent with imprinting in backcrosses involving SWV (S x SC = 0.50 +/- 0.18 g, S x CS = 0.96 +/- 0.05, P < 0.01). Three of four of the parameters investigated showed differences in some of the backcrosses of reciprocal F1's consistent with those expected if genomic imprinting were occurring.

Caffeine decreases the occurrence of cadmium-induced forelimb ectrodactyly in C57BL/6J mice.

BACKGROUND: Cadmium is a well-known animal teratogen. Caffeine is an alkaloid widely consumed by humans. Interactions between teratogens and nonteratogenic doses of other agents are becoming widely studied, as they may shed light on understanding mechanisms of teratogenicity or possible prevention of teratogenic effects. METHODS: C57BL/6JBK mice were injected intraperitoneally (ip) with cadmium sulfate (Cd) at 0, 1.00 (LDCd), 2.50 (MDCd), or 5.00 (HDCd) mg/kg, immediately followed by subcutaneous (sc) administration of 0 or 50 mg/kg caffeine (CAFF) on gestation day (GD) 9. Fetuses were examined on GD 18 for ectrodactyly and other gross morphological malformations. RESULTS: Amelioration of cadmium-induced forelimb ectrodactyly by CAFF was seen in both the high-dose cadmium (HDCd = 65.4%, HDCd+CAFF = 39.2%) and medium-dose cadmium (MDCd = 46.2%, MDCd+ CAFF = 20.8%) treatment groups (P < 0.025). Bilateral expression of ectrodactyly was also decreased in the presence of caffeine. A statistically significant reduction in Cd-induced abnormalities, including: eye, abdominal, and other skeletal defects, was not seen with caffeine addition, although they did trend downward in the caffeine-supplemented groups. Litter size, fetal weight, fetal mortality, and dam weight also were not affected by co-treatment with caffeine. CONCLUSIONS: This study provides evidence that a subteratogenic dose of caffeine can ameliorate cadmium-induced forelimb ectrodactyly in the Cd-sensitive C57BL/6J inbred mouse strain.

Psychometric testing of fatigue instruments for use with cancer patients.

BACKGROUND: Cancer treatment-related fatigue (CRF) is a common side effect of cancer treatment. A problem identified in most reviews of CRF is lack of sound approaches to measurement that are congruent with the conceptualization of CRF as a self-perceived state. The diversity of instruments available to measure fatigue and the lack of comprehensive testing of several promising instruments with cancer patients undergoing treatment provided the rationale for this study. The purpose of this article is to report the results of psychometric testing of several fatigue instruments in patients undergoing cancer treatment. OBJECTIVES: The aims of this study were to determine the reliability, validity, and responsiveness of each instrument and to determine the ability of each instrument to capture CRF. METHODS: Existing fatigue instruments with published psychometric information that indicated suitability for further testing were selected and included the Profile of Mood States Short Form fatigue subscale (F_POMS-sf), Multidimensional Assessment of Fatigue (MAF), Lee Fatigue Scale (LFS), and the Multidimensional Fatigue Inventory (MFI). Data were collected at a university-based clinical cancer center and a freestanding comprehensive cancer center. Subjects completed all study instruments, which were presented in random order, at a time when CRF was expected to be high and again when it was expected to be low. A subset of subjects completed the instruments within 48 hours of one of the data collection points when CRF was expected to be relatively unchanged to provide stability data. RESULTS: Reliability estimates using Cronbach's alpha indicated that all instruments examined had good internal consistency. Test-retest correlations showed good stability for total scores on all the instruments, but some subscales of the LFS and MFI had marginal stability. Factor analysis of all instruments indicated that only the LFS and the F_POMS-sf fully supported their construct validity. All of the instruments showed responsiveness to changes in CRF related to treatment. CONCLUSIONS: The results of the study provide researchers and clinicians with detailed comparisons of the performance of established fatigue measures in cancer patients undergoing treatment to use when selecting measures of CRF.

An ethnographic study of factors influencing cancer pain management in South Africa.

Although the knowledge exists to relieve cancer pain, inadequate pain relief persists as an international health problem. The World Health Organization has recommended a threefold strategy to improve cancer pain management: health policy, drug availability, and education. Yet major barriers to effective cancer pain management exist in every country of the world. Effective strategies to improve pain management must be based on an understanding of the issues in individual countries. This report evaluates cultural and other factors influencing cancer pain management in the Republic of South Africa. This ethnographic field study included multiple methods of data collection: analysis of documents, participant observation, focused interviews, and in-depth interviews of informants (n = 33) who represented multiple stakeholders in a variety of settings. Cultural beliefs and practices form the context for understanding cancer pain and how it is managed. Cultural variability exists regarding cancer as a disease, pain expectations, pain tolerance, pain expression, and health care practices. Key factors that influence how pain is managed included standards, knowledge, resources, communication and the patient-provider relationship, and teamwork and professional relationships. The existence of these factors promotes effective pain management, whereas their absence creates a barrier to achieving this aim.

Folinic acid reduces cleft lip [CL(P)] in A/WySn mice.

The A/WySnBk strain of mice displays 25-35% spontaneous CL(P). Pregnant mice were treated with folinic acid continuously delivered via osmotic minipumps at the rate of 7.6 +/- 0.2 microl/ hr (12 mg/ 24 hr) for the period covering gestation day (gd) 8.5-9.5, early in the critical period for formation of the face. Untreated and osmotic minipump-delivered, saline-treated groups served as controls. Individual fetuses were examined for CL(P) and other abnormalities on gd 18. The treatment resulted in a decrease in the frequency of CL(P) from 40.0+/-7.0% among untreated to 10.2+/-3.3% in the folinic acid group (P<.001). The difference between the folinic-exposed group and the saline-filled minipump and surgical control was also significant (P<.029). With respect to mortality, litter size, and fetal weight, the two minipump groups did not differ significantly, nor did they differ from the untreated group. Thus the reduction in CL(P) frequency was due to the presence of folinic acid and not to effects of the surgical procedures. This study provides evidence that administration of folinic acid during pregnancy has an important ameliorating effect on genetically predisposed CL(P).

Contributions of dam and conceptus to differences in sensitivity to valproic acid among C57 black and SWV mice.

To ascertain the relative contributions of genotypes of conceptus and dam to developmental toxicity occasioned by valproic acid (VPA), crosses were established between resistant C57BL/6JBk (C, C57) and susceptible SWV/Bk (S, SWV) strains of mice. These included matings of pure lines, reciprocal outcrosses, and reciprocal backcrosses with F1 hybrids. At 8 d:12 h +/- 5 h, for each mating, 0, 500, or 600 mg/kg aqueous VPA was injected ip. Fetuses were examined on gestation day (gd) 18 for exencephaly (the paradigmatic anomaly), other abnormalities, mortality, litter size, and fetal weight. At 600 mg/kg, sensitivity to exencephaly induction in all cases was that of the dam, regardless of sire. Thus exencephaly here seems to be largely a function of the uterine environment produced by the maternal genotype. This inference is confirmed in backcrosses where F1-dams x S-sires and F1-dams x C-sires produced-identical outcomes, and S-dams x F1-sires produced much higher frequencies of exencephaly than C-dams x F1-sires. For prenatal mortality, the genotypes of both dam and conceptus appear to be important determinants. Fetal contribution is inferred from the observations that S-dam x S-sire matings produced a much higher frequency of mortality than S-dams x C-sires, and C-dams x C-sires produced higher mortality than C-dams x S-sires. Therefore, heterozygosity of the conceptus was protective. Among backcrosses, fetal determination of sensitivity to mortality is also seen by the observation that F1-dams x C-sires produces the same fetal mortality as C-dams x F1-sires. The contribution of uterine environment is seen in the observation that matings of S-dams x C-sires resulted in higher fetal mortality than did those with C-dams x S-sires. Therefore, identical conceptuses in different dams showed different levels of fetal loss. Thus exencephaly response appears to be largely controlled by genes active in the dam, and mortality as a result of a multigenic outcome with contributing genes active in both conceptus and dam. The data also suggest that SWV pure-line dams make a contribution to prenatal mortality not seen in C57 or F1 dams. Mean litter size among VPA-exposed litters showed high variability in pure lines and outcrosses. In backcrosses, F1 dams produced larger litters than pure line dams, arguing for heterosis as a contributor to this parameter. Reduction in litter size occasioned by VPA exposure was great in pure line dams and nonexistent in F1 dams. The SWV dams crossed with F1 sires were the only group among the backcrosses to show reduction of litter size, providing further confirmation of the increased sensitivity of pure-line (i.e., homozygous) SWV dams to VPA exposure. Fetal weight seems to be a function of uterine environment because female SWV produced conceptuses with lower fetal weight in all crosses, and produced a greater reduction in fetal weight attributable to VPA exposure than C57 or F1 dams. Fetal weight did not correlate closely with litter size, suggesting that a lower fetal weight may be a strain characteristic, as are exencephaly induction and prenatal mortality in response to VPA. Differences in sensitivity to VPA insult are seen for all parameters investigated with SWV dams being the most sensitive, but mechanisms seem to differ for a number of the endpoints.

Health policy, health services, and cancer pain management in the New South Africa.

In 1982, the World Health Organization (WHO) identified inadequate relief from cancer pain as an international health problem. WHO recommended that governments develop and implement national policies and programs for cancer pain relief. This report evaluates national health policy and the systems of health care delivery in relation to cancer pain management in the new South Africa. This field study included multiple methods of data collection: analysis of documents, field trips with participant observation in sites of care delivery, focused interviews, and in-depth interviews of key informants. The purposive sample of key informants (n = 33) represented multiple stakeholders in a variety of settings. Strengths of the developing health policy include specific recommendations related to palliative care; the shift to universal primary care; policies to support drug availability; the inclusion of morphine and codeine as essential drug at the primary health care level; and the development of a national standard related to cancer pain management. Health services are characterized by two parallel systems of care (private and public) with numerous vestiges of the inequities of apartheid. The management of pain varies by provider and setting; major problems with access exist in the rural areas. Health services in South Africa have been plagued by inequity and inadequate resources. New health policies have set a path to ensure universal access to health care including palliative care for cancer. Their successful implementation is the next necessary step toward improving health services and alleviating the suffering of increasing numbers of individuals with cancer.

Planning and conducting a multi-institutional project on fatigue.

PURPOSE/OBJECTIVES: To describe the process used in proposal development and study implementation for a complex multisite project on cancer treatment-related fatigue (CRF), identify strategies used to manage the project, and provide recommendations for teams planning multisite research. DATA SOURCES: Information derived from project team meeting records, correspondence, proposals, and personal recollection. DATA SYNTHESIS: The project was built on preexisting relationships among the three site investigators who then built a team including faculty, research coordinators, staff nurses, and students. Study sites had a range of organizational models, and the proposal was designed to capitalize on the organizational and resource strengths of each setting. Three team members drawn from outside oncology nursing provided expertise in measurement and experience with fatigue in other populations. Planning meetings were critical to the success of the project. Conference calls, fax technology, and electronic mail were used for communication. Flexibility was important in managing crises and shifting responsibility for specific components of the work. The team documented and evaluated the process used for multisite research, completed a major instrumentation study, and developed a cognitive-behavioral intervention for CRF. CONCLUSIONS: Accomplishments during the one-year planning grant exceeded initial expectations. The process of conducting multisite research is complex, especially when the starting point is a planning grant with specific research protocols to be developed and implemented over one year. Explicit planning for decision-making processes to be used throughout the project, acknowledging the differences among the study settings and planning the protocols to capitalize upon those differences, and recruiting a strong research team that included a member with planning grant and team-building expertise were essential elements for success. IMPLICATIONS FOR NURSING PRACTICE: Specific recommendations for others planning multisite research are related to team-building, team membership, communication, behavioral norms, role flexibility, resources, feedback, problem management, and shared recognition.

Terminology of developmental abnormalities in common laboratory mammals (version 1).

This paper presents the first version of an internationally-developed glossary of terms for structural developmental abnormalities in common laboratory animals. The glossary is put forward by the International Federation of Teratology Societies (IFTS) Committee on International Harmonization of Nomenclature in Developmental Toxicology, and represents considerable progress toward harmonization of terminology in this area. The purpose of this effort is to provide a common vocabulary that will reduce confusion and ambiguity in the description of developmental effects, particularly in submissions to regulatory agencies worldwide. The glossary contains a primary term or phrase, a definition of the abnormality, and notes, where appropriate. Selected synonyms or related terms, which reflect a similar or closely related concept, are noted. Nonpreferred terms are indicated where their usage may be incorrect. Modifying terms used repeatedly in the glossary (e.g., absent, branched) are listed and defined separately, instead of repeating their definitions for each observation. Syndrome names are generally excluded from the glossary, but are listed separately in an appendix. The glossary is organized into broad sections for external, visceral, and skeletal observations, then subdivided into regions, structures, or organs in a general overall head to tail sequence. Numbering is sequential, and not in any regional or hierarchical order. Uses and misuses of the glossary are discussed. Comments, questions, suggestions, and additions from practitioners in the field of developmental toxicology are welcomed on the organization of the glossary as well as on the specific terms and definitions. Updates of the glossary are planned based on the comments received.

Evaluation of scheduled J-pouch irrigations on decreasing stool frequency after ileoanal pull-through and ileostomy closure.

This study was intended to determine whether J-pouch irrigations through the efferent limb of the protective ileostomy stoma after ileoanal pull-through are effective in decreasing high stool frequency after ileostomy closure. Patients undergoing ileoanal pull-through may have high stool frequency after ileostomy closure. J-pouch irrigations through the efferent ileostomy stoma may decrease stool frequency by increasing J-pouch volume, improving storage capacity. The study used a randomized, prospective design in a university hospital outpatient setting. Participants (N = 58) were randomly assigned to control and experimental groups. Effectiveness of irrigation was determined by stool frequency. Both groups were taught Kegel exercises (anal muscle strengthening exercises). The experimental group was taught how and when to irrigate the J-pouch daily; the control group was not. Forty-seven subjects, 25 men and 22 women ranging in age from 15 to 65 years, completed the study. Results of MANOVA indicated no significant between-group difference in the average number of times that subjects performed Kegel exercises; however, there was a significant decrease during the 4-week study period (p < 0.001). There was no significant difference between groups in stool frequency, which decreased with time. There also was no significant effect on nocturnal leakage or satisfaction with surgical outcome. Additional clinical variables that were measured but had no significant effect included eating late, pouch size, and intake of sugar, fiber, bulk-forming products, and antidiarrheal agents. The study did not support the effectiveness of J-pouch irrigation in decreasing stool frequency after ileostomy closure. The cost, time commitment, and burden of performing daily irrigations are not warranted in this patient group.

Using the journal club as a component of the research utilization process.

Although journal clubs are recommended for research utilization, the various ways in which journal clubs are conducted do not always lead to research utilization. Major institutional changes in practice must be preceded by a comprehensive literature search and a complete review. An example is provided of how a series of journal clubs using the comprehensive search approach led to research utilization.

Rupture characteristics of the deep bolivian earthquake of 9 june 1994 and the mechanism of deep-focus earthquakes.

The M(w) = 8.3 deep (636 kilometers) Bolivian earthquake of 9 June 1994 was the largest deep-focus earthquake ever recorded. Seismic data from permanent stations plus portable instruments in South America show that rupture occurred on a horizontal plane and extended at least 30 by 50 kilometers. Rupture proceeded at 1 to 3 kilometers per second along the down-dip azimuth of the slab and penetrated through more than a third of the slab thickness. This extent is more than three times that expected for a metastable wedge of olivine at the core of the slab, and thus appears to be incompatible with an origin by transformational faulting. These large events may instead represent slip on preserved zones of weakness established in oceanic lithosphere at the Earth's surface.

Using patient focus groups for new patient services.

BACKGROUND: This article is intended to illustrate the usefulness of patient input in the strategic planning process and to demonstrate in particular the use of focus groups in concept development, concept testing, and program evaluation. INTERACTIVE PLANNING: Three areas of patient services were designed partly on the basis of patient input. "Service Teams with Appropriate Resources" (STARs) were conceived as basic organizational units to deliver interdisciplinary care to meet the needs of specific groups of patients. A patient services "menu" was envisioned to allow the patient or caregiver to decide which service would most appropriately and efficiently meet patient needs. The "Service Expectation Program" was formulated to ease entry into the hospital by providing information on what patients should expect from the hospital experience. CONCEPT TESTING: Focus groups were used again to test and refine the concepts developed during the interactive planning stage. General themes included the need for improved communication, the desire to be treated with respect and dignity (personhood), the need for coordination across the continuum of care, and the desire for more personal choice and control. ONGOING EVALUATION: Sources of patient feedback used in the ongoing evaluation process included a patient satisfaction survey and a telephone survey. Additional focus groups and telephone surveys are planned. MANAGEMENT ISSUES: The focus group discussions with patients introduced useful data into the quality improvement and interactive planning process. Findings were disseminated to all levels of hospital management and program staff through newsletters, reports, and in-service training sessions. Data were useful in interactive planning, program concept testing, and the development and implementation of new services.

Additional endpoints and overview of a mouse skeletal variant assay for detecting exposure to teratogens.

CD-1 mice were exposed in utero to one of 14 treatment regimes, several of them being replicated, with close agreement between series. Prenatal exposure to a teratogenic dose at a sensitive time enabled detection of 10 of 14 teratogen regimes by alterations in frequency or severity of a substantial number of the 88 variants in the Skeletal Variant Assay System (SVAS) screen when examined at 60-65 days post natal (DPN). These included 2,4,5-T (245T), Trifluralin (TFL), Maneb (MNB), Decamethrin (DMT), Acetazolamide (ACZM) either at 8 days post-coitus (DPC) or days 9-11 PC, trypan blue (TB), or 5' Bromodeoxyuridine (BUDR) on either 7 DPC, 8 DPC, or 9 DPC. Most of these observations have been reported elsewhere. All of the treatment regimes mentioned above, and another group of treatments, could be detected in the exposed CD-1 cohorts when additional endpoints were employed. One such endpoint was "frequently responding variants." These were: Interfrontals (IF), Parted Frontals (PF), Preoptic Sutures (PS), Foramina Transversaria Imperfecta of the first cervical (C) vertebra (FTI C1), FTI of the axis (C2), Accessory (Acc) Transverse Foramina (TF) of C3-C6, malformations of C3-C7, Fourteen (14) Ribs, Carpal Fusions (Fus), Lumbar Fus, 27-Presacral Vertebrae (PSV), and Sacral Fus. This endpoint revealed significant differences in the initial group of 10, plus Captan (CAPT) and Phenytoin (DPH). Yet another useful endpoint reported here was the existence of high magnitude effects (i.e., dramatic alterations in frequency of occurrence of a variant). These included IF in TB and ACZM; PF in ACZM; PS in BUDR; FTI-C1 in TB and 245T; FTI-C2 in 245T; 14 Ribs in ACZM, BUDR, and TFL; Carpal Fus in TB; 27-PSV in ACZM; Fewer than (<) 30 Caudal Vertebrae (Vert) in 245T, TFL; Caudal Fus in TB, ACZM-D9. Eight treatment regimes in all could be detected by the existence of 3 or more high magnitude effects (245T, MNB, TB, ACZM8, ACZM9-11, phenytoin, and possibly BUDR on days 7 or 8, each seen in one of two series only). Clusters of related variants were affected in 9 of the 14 groups: Frontal (F) bones and C Vert in 245T; F bones in ACZM-D8; Fus in Posterior Vert Column in ACZM-D9-11; C Vert and Fus in Vert and articular skeleton in TB; Thoracic (Th) Vert and rib-cage effects in BUDR.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetazolamide with caffeine causes exencephaly in "resistant" SWV mice.

Pregnant SWV mice were treated on day 9 of gestation (PC) with 50 mg/kg of caffeine (CAFF), 200 mg/kg (LD) or 1000 mg/kg (HD) of acetazolamide (ACZM), or a combination of both agents, or on day 8 PC with both agents (ACZM + CAFF). Untreated (UNTD) and vehicle-treated (VEH) groups served as controls. The SWV strain is widely reported to be resistant to ACZM; it was resistant to ACZM or CAFF + ACZM when treated on day 9 of gestation, but a significant frequency of malformations, primarily exencephaly, was produced by ACZM + CAFF on day 8 PC. This study provides evidence that ACZM, coupled with a subteratogenic dose of caffeine can produce abnormalities in the "resistant" SWV mice, using the endpoint of exencephaly on day 8 of gestation. The mean number of ossified caudal vertebrae in day-9 treatments and ossified cervical vertebral centra in day-8 treatments were reduced. The frequency of ossification of the first cervical vertebra (C1) was reduced from 93% in UNTD to 39% in HD-ACZM day 9 PC and 69% in HD-ACZM + CAFF day 9 PC groups, and was also significantly reduced in the HD-ACZM + CAFF day-8 treated group.