Environmental applications of magnetic measurements.

A wide range of examples of the application of magnetic measurements to environmental studies illustrate the advantages of magnetic techniques over conventional methods. Magnetic measurements, in both the field and the laboratory, are particularly useful for reconnaissance work because of their spee and flexibility, Quantification as well as simple diagnosis of the transformation and movement of magnetic minerals within and between the atmosphere, lithosphere, and hydrosphere is practical. Techniques of investigating intrinsic and mineral magnetic properties, in addition to paleomagnetic remanence, are described in subjects as diverse as meteorology, hydrology, sedimentology, geophysics, and ecology.

Carcinogenic effects of acrylamide in Sencar and A/J mice.

Acrylamide structurally resembles vinyl carbamate, a proposed proximate carcinogenic form of ethyl carbamate. To test the hypothesis that acrylamide should possess carcinogenic properties, it was tested in the Salmonella-microsome assay for point mutation, as a skin tumor initiator in the Sencar mouse, and for its ability to induce lung adenomas in the A/J mouse. Acrylamide was found to be without activity as a mutagen in Salmonella strains TA 1535, TA 1537, TA 98, and TA 100 both in the presence and absence of rat liver microsomes using both the plate and liquid suspension assays. However, acrylamide was found to approximate ethyl carbamate in potency as a tumor initiator in the skin of the female Sencar mice. As with ethyl carbamate, acrylamide was more potent by systemic routes of administration relative to topical application. Acrylamide was also found to induce lung adenomas in male and female A/J mice using both the p.o. and i.p. routes of administration. Acrylamide was approximately one-seventh as potent as ethyl carbamate in the induction of lung adenomas. These data confirm the hypothesis that acrylamide possesses carcinogenic properties similar to ethyl carbamate.

Absence of a relationship between smoking and diabetic microangiopathy.

The relationship between cigarette smoking and retinopathy and proteinuria was examined in a group of 973 subjects with diabetes. Other variables known to influence the risk of microangiopathy were also measured in a standardized fashion (e.g., duration of diabetes, blood glucose, blood pressure). The characteristics of smokers and nonsmokers were studied in detail. It was therefore possible to take into account the effects of confounding variables on the relationship between smoking and risk of microangiopathy. Associations between smoking and risk of microangiopathies, previously reported in some of the smaller studies, were not confirmed in this larger study.

Carcinogenic activity of acrylamide in the skin and lung of Swiss-ICR mice.

Doses of acrylamide ranging from 12.5 to 50 mg/kg were administered orally to female ICR-Swiss mice over 3 days for each of 2 weeks (total doses of 75, 150 and 300 mg/kg). Two weeks later some of the animals were started on a promotion schedule involving the application of 2.5 micrograms TPA/mouse 3 times weekly. Development of tumors was observed weekly in the skin, and in the lungs at 1 year. Acrylamide was found to initiate squamous cell adenoma and carcinomas in the skin and increased the yield of adenomas and carcinomas in the lung. Skin tumor development was dependent upon 12-O-tetradecanoylphorbol-13-acetate (TPA) promotion whereas lung tumor induction was not. These data extend previous observations of carcinogenic activity of acrylamide in the skin of SENCAR mice and lungs of strain A/J mice to a third strain of mouse, the ICR-Swiss.

Carcinogenic effects in A/J mice of particulate of a coal tar paint used in potable water systems.

Coal tar paints are among the products used as inside coatings for water pipes and storage tanks to retard corrosion in potable water supply systems. Four different formulations of these paints were tested in earlier work by this laboratory in the Ames mutagenesis and the mouse skin carcinogenesis bioassays. The paint most active in these assays were then tested in a particulate form in the lung adenoma assay with A/J mice. The paint was applied to clean glass plates, cured, collected and homogenized in 2% Emulphor. Doses of this coal tar suspension were administered by gavage at 1.0, 10.0 and 55.0 mg in 0.2 ml per mouse 3X weekly for 8 weeks. The total doses of coal tar paint were 24, 240, and 1320 mg/mouse. Benzo[a]pyrene (BaP), administered in a parallel schedule to a total dose of 6 mg/mouse, served as positive control. A negative control group received an equivalent volume of 2% Emulphor. Animals were killed at 9 months of age (8 months after first dose) and lung adenomas counted. A dose-related response, in the average number of lung tumors per mouse, was observed with the coal tar particulate. There were also squamous cell tumors of the forestomach in 42% of the mice receiving 55.0 mg coal tar paint per application.

Carcinogenic activity associated with halogenated acetones and acroleins in the mouse skin assay.

Several chlorinated acetones have been identified in drinking water and these, as well as a number of chlorinated acroleins, are produced by chlorination of humic acid solutions. Many of these chlorinated compounds and the brominated acrolein analog were positive in the Ames Assay in the laboratory. To determine if carcinogenic activity was associated with these chemicals the following acetone derivatives: monochloro (MCA); 1,1-dichloro (1,1-DCA), 1,3-dichloro (1,3-DCA), 1,1,1-trichloro (1,1,1-TCA), 1,1,3-trichloro (1,1,3-TCA), and substituted acroleins: 2-chloro (CAC), 3,3-dichloro (DCAC), 2,3,3-trichloro (TCAC) and 2-bromo (BAC), were applied topically to SENCAR mice (25, 30, or 40/group) at the following dose levels: 50 mg/kg (MCA and 1,1,3-TCA); 50, 75 and 100 mg/kg (1,3-DCA); 100, 200 and 400 mg/kg (CAC, DCAC, and TCAC); 200 and 300 mg/kg (BAC); and 400, 600, and 800 mg/kg (1,1-DCA, and 1,1,1-TCA). Doses were applied six times over a 2-week period in 0.2 ml ethanol per application. 1,3-DCA was also tested with single doses of 37.5, 75, 150 and 300 mg/kg in 0.2 ml ethanol. Control animals received 0.2 ml ethanol per application as a single dose or multiple doses to match corresponding studies. Two weeks after the final dose, 1.0 microgram TPA in 0.2 ml acetone was applied three times weekly for 20 weeks. After 24 weeks the percentage of animals with tumors for dose groups above were: MCA (8); 1,1,3-TCA (10); 1,3-DCA, multiple doses (48, 45, 32); CAC (30, 28, 38); DCAC (3, 0, 0); TCAC (10, 5, 0); BAC (54, 43); 1,1-DCA (0, 5, 0); 1,1,1-TCA (10, 5, 0); 1,3-DCA, single doses (47, 47, 63, 20); controls (12--Table 3, 9--Table 4 average). These data show that 1,3-DCA, CAC and BAC, when applied topically, initiate tumors in the mouse skin. These chemicals administered orally in a 2% emulphor solution, at doses described in Table 3, did not initiate tumors in the mouse skin.

Induction of nuclear anomalies in the gastrointestinal tract by polycyclic aromatic hydrocarbons.

A selective list of polycyclic aromatic hydrocarbons (PAH) with varied carcinogenic and mutagenic potencies, which are identified as common contaminants at industrial sites and which often contaminate the neighboring ground water, are investigated for their ability to induce nuclear anomalies (NA) in the mouse gastrointestinal (G.I.) tract. These studies examined the hypothesis that a relationship between NA induction and carcinogenic potency of these PAH exists. Among the PAH tested, 7,12-dimethylbenzanthrene (DMBA) was most effective inducer of NA in all G.I. tract tissues examined, with the relative potency in duodenum of DMBA much much greater than benzo[a]pyrene (B[a]P) much greater than benzo[b]fluoranthene (B[b]F). The induction of NA by benzo[a]anthracene (B[a]A), pyrene (PY) and benzo[e]pyrene (B[e]P) was not different from that elicited by vehicle controls. MNU, a known potent inducer of NA in the mouse G.I. tract, yielded a high level of NA in duodenum and proximal colon but was less effective than DMBA in the forestomach. The data suggest that induction of NA by DMBA and B[a]P PAH are in approximate accordance with their relative carcinogenic potency in the gastrointestinal tract. When binary mixtures of some PAH were administered the yield of NA was less than that expected by simple additivity and closer to that expected by averaging the activities of the two PAH comprising the mixture. Thus, this short-term in vivo assay may be useful as a predictor of the genotoxic or carcinogenic strength of individual PAH and/or mixtures of these compounds.

Methodology for statistical analysis of SENCAR mouse skin assay data.

Various response measures and statistical methods appropriate for the analysis of data collected in the SENCAR mouse skin assay are examined. The characteristics of the tumor response data do not readily lend themselves to the classical methods for hypothesis testing. The advantages and limitations of conventional methods of analysis and methods recommended in the literature are discussed. Several alternative response measures that were developed specifically to answer the problems inherent in the data collected in the SENCAR bioassay system are described. These measures take into account animal survival, tumor multiplicity, and tumor regression. Statistical methods for the analysis of these measures to test for a positive dose response and a dose-response relationship are discussed. Sample data from representative initiation/promotion studies are used to compare the response measures and methods of analysis.

Asbestos in water supplies of the United States.

The review of available data on the concentrations of asbestos in U.S. water supplies suggests that the majority of water consumers are not exposed to asbestos concentrations over 1 million fibers/Liter. A few populations, however, may be exposed to concentrations over 1 billion fibers/L. Of the 538 water supplies for which waterborne asbestos data are available, 8% have concentrations of fibers over 10 million fibers/L. The vast majority of asbestos fibers found in U.S. water supplies are under 5 micron in length.

Use of biological assay systems to assess the relative carcinogenic hazards of disinfection by-products.

Other workers have clearly shown that most, if not all, drinking water in the U.S. contains chemicals that possess mutagenic and/or carcinogenic activity by using bacterial and in vitro methods. In the present work, increased numbers of tumors were observed with samples of organic material isolated from 5 U.S. cities administered as tumor initiators in mouse skin initiation/promotion studies. Only in one case was the result significantly different from control. In studies designed to test whether disinfection practice contributes significantly to the tumor initiating activity found in drinking water mixed results have been obtained. In one experiment, water disinfected by chlorination, ozonation or combined chlorine resulted in a significantly greater number of papillomas when compared to nondisinfected water. In two subsequent experiments, where water was obtained from the Ohio River at different times of the year, no evidence of increased initiating activity was observed with any disinfectant. Analysis of water obtained at the comparable times of the year for total organic halogen, and trihalomethane formation revealed a substantial variation in the formation of these products. Considering the problems such variability poses for estimating risks associated with disinfection by-products, a model system which makes use of commercially obtained humic acid as a substrate for chlorination was investigated using the Ames test. Humic and fulvic acids obtained from two surface waters as well as the commercially obtained humic acid were without activity in TA 1535, TA 1537, TA 1538, TA 98 or TA 100 strains of S. typhimurium. Following treatment with a 0.8 molar ratio of chlorine (based on carbon) significant mutagenic activity was observed with all humic and fulvic acid samples. Comparisons of the specific mutagenic activity of the chlorinated products suggests that the commercial material might provide a useful model for studying health hazards associated with disinfection reactions by-products.

A combined carcinogen bioassay utilizing both the lung adenoma and skin papilloma protocols.

To test the feasibility of employing a combined lung adenoma/skin papilloma assay for broader detection of chemical carcinogenesis than that realized with either bioassay done separately, four strains of mice, SENCAR, BALB/c, A/J, and ICR-Swiss, were administered carcinogens either by the oral or intraperitoneal (IP) routes. The carcinogens administered were ethyl carbamate (EC), benzo(a)pyrene [B(a)P], N-[4-(5-nitro-2-furyl)thiazolyl]formamide (FANFT), and acrylamide (ACR). Starting 2 weeks later, 1 to 5 micrograms (depending on strain) of 12-O-tetradecanoylphorbol-13-acetate (TPA) in 0.2 mL acetone/mouse was applied three times weekly to the shaved back for 20 weeks. All strains displayed increases in the yield of lung adenomas in response to EC at 32 weeks. B(a)P increased lung adenomas in only the SENCAR and A/J strain. Only the SENCAR and ICR-Swiss mice gave positive responses in the skin. In the SENCAR mice, positive response was seen with all four chemicals, however, FANFT gave an inconsistent response. The ICR-Swiss mice responded with an increased skin papilloma yield only to EC. In a separate experiment involving only SENCAR mice, animals were treated with a single oral dose of diethylnitrosamine (DEN) followed by triweekly application of 1.0 microgram TPA. This treatment resulted in 51/57 animals developing lung adenomas vs. 5/57 in the control animals. No treatment-related skin tumors resulted with DEN. Histopathologically confirmed lesions indicate that the spectrum of chemicals detected in the SENCAR mouse may be broadened using a combined bioassay that examines both lung and skin responses.

Enhancement of the hepatotoxicity of chloroform in B6C3F1 mice by corn oil: implications for chloroform carcinogenesis.

A recent study of the ability of chloroform in drinking water to produce cancer reported that male Osborne-Mendel rats developed renal tumors, but that female B6C3F1 mice failed to develop hepatocellular carcinomas. The results obtained in the male Osborne-Mendel rats were comparable to those observed in an earlier study sponsored by the National Cancer Institute (NCI). On the other hand, the lack of an increased incidence of hepatocellular carcinomas in female B6C3F1 mice was in sharp contrast to previously reported results. The doses of chloroform used were comparable to that which produced an 85% incidence in the NCI study. We have investigated the extent to which the vehicle might be responsible for the different results in these two studies by examining the differential effects of chloroform when it was administered by gavage using corn oil versus a 2% Emulphor suspension as the vehicle. Male and female B6C3F1 mice were administered chloroform at 60, 130, and 270 mg/kg per day for 90 days. At sacrifice, body and organ weights were measured, and blood was recovered to perform the following serum chemistry measurements (in order of priority): glutamate oxalacetate transaminase (SGOT), lactate dehydrogenase (LDH), blood urea nitrogen (BUN), and triglyceride (TG) levels. The liver was sectioned for histopathological examination. Chloroform increased SGOT levels significantly only when administered in corn oil at a dose of 270 mg/kg in both male and female mice. It had no effect on LDH activity. There was a small increase in BUN when chloroform was administered in corn oil, but not when administered in 2% Emulphor. When administered in corn oil, chloroform significantly decreased serum TG levels but was without effect on this parameter when administered in 2% Emulphor. Chloroform decreased body weight and increased liver weight with both vehicles, but the effects were significantly greater when it was administered in corn oil. Mice administered chloroform in corn oil displayed a significant degree of diffuse parenchymal degeneration (5 of 10 males and 1 of 10 females) and mild to moderate early cirrhosis (5 of 10 males and 9 of 10 females); significant pathological lesions were not observed in the animals administered corn oil without chloroform nor in mice receiving chloroform in 2% Emulphor. These data indicate that administration of chloroform by corn oil gavage results in more marked hepatotoxic effects than observed when it is provided in an aqueous suspension.(ABSTRACT TRUNCATED AT 400 WORDS)

Epidemiology study of the use of asbestos-cement pipe for the distribution of drinking water in Escambia County, Florida.

Cancer mortality for the population census tracts of Escambia County, FL, which use asbestos-cement (AC) pipe for public potable water distribution, was compared with cancer mortality data collected from census tracts in the same county where other types of piping materials are used. An analysis of covariance was run to test for differences in standard mortality ratios for seven cancer sites among three potential asbestos exposure groups based on AC pipe usage. Twelve variables representing nonexposure-related influences on disease rates were combined in four independent factors and used as covariates in these analyses. No evidence for an association between the use of AC pipe for carrying drinking water and deaths due to gastrointestinal and related cancers was found. The limitations on the sensitivity of the analysis are discussed.

Induction of gastrointestinal tract nuclear anomalies in B6C3F1 mice by 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone and 3,4-(dichloro)-5-hydroxy-2[5H]-furanone, mutagenic byproducts of chlorine disinfection.

Two chlorinated hydroxylated furanones, 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX) and 3,4-(dichloro)-5-hydroxy-2[5H]-furanone (MA), are bacterial mutagens and they are also byproducts of chlorine disinfection, and frequent contaminants of drinking water. In this work MX is shown to induce nuclear anomalies in the gastrointestinal tract of the B6C3F1 mouse. The other chlorohydroxy-furanone, MA, gives suggestive evidence of activity. In this bioassay MX was approximately equivalent in potency to epichlorohydrin (ECH) but was much less potent than methylnitrosourea (MNU). The latter two chemicals are confirmed rodent gastrointestinal tract carcinogens. The duodenum was the most sensitive tissue responding with both increased numbers of nuclear anomalies per mouse and increased incidence of animals presenting the nuclear aberrations 24 hr after a single oral dose of 0.37 mmol/kg-1 of MX. MA also induced a significant increase in duodenal nuclear anomalies, but only at the highest dose (0.46 mmol/kg-1). The proximal colon and forestomach responded to MX but not MA. This is the first study demonstrating that chlorohydroxyfuranones are capable of inducing nuclear toxicity in vivo. However, it is clear, for MX at least, that its potency in the gastrointestinal tract nuclear anomalies assay is not commensurate with its extreme bacterial mutagenicity. Since the gastrointestinal tract tissues are directly exposed to orally administered genotoxins, one possible explanation for the weak response observed in this study could be that mammalian cells can effectively detoxify chlorohydroxyfuranones.

The automated analysis of rat sperm motility following subchronic epichlorohydrin administration: methodologic and statistical considerations.

The automated analysis of sperm motion endpoints is potentially useful in identifying male reproductive toxicants and ultimately in predicting fertility in humans. The present study was designed to evaluate the automated analysis of rat sperm motility characteristics following subchronic administration of epichlorohydrin. This type of validation is a prerequisite for inclusion of sperm motion measurements in the process of reproductive risk assessment. In the present studies videotapes were made of cauda epididymal spermatozoa from Long-Evans rats, both untreated and treated with epichlorohydrin. From analysis of videotapes of control epididymal spermatozoa, the relationship of various sperm motion endpoints and settings of the CellSoft computer-assisted sperm motion analysis system (Cryo Resources, Ltd., New York, NY) is described. Optimal settings of the system for analysis of rat spermatozoa are detailed. Employing data from both control and epichlorohydrin-treated animals, a statistical methodology is described that evaluates: (1) the distributions of CellSoft generated sperm motion endpoints, (2) the correlations between these endpoints, and (3) techniques for detection of dose-related effects.

Correlation of sperm motion parameters with fertility in rats treated subchronically with epichlorohydrin.

We investigated the relationship between fertility and sperm motin endpoints in rats treated subchronically with the male reproductive toxicant, epichlorohydrin (ECH). Male rats were given ECH orally for 23 days at dosages of 0, 6.25, 12.5, or 25 mg/kg/day. They were mated twice (at 19 and 22 days) to estimate fertility by (1) detection of fertilized ova (presence of sperm head and tail or two pronuclei) 18 hours after mating and by (2) counting implants on day 14 of gestation. Both indices showed dose-related reductions (P less than 0.001). Motion parameters of cauda epididymal sperm were assessed using the CellSoft computer-assisted sperm motion analysis (CASA) system after the rats were asphyxiated on day 25. Curvilinear velocity, straight-line velocity, linearity, and amplitude of lateral head displacement were reduced in a dose-related manner. The fertility indices, percent fertilized ova, and percent implantation on day 14 of gestation were correlated significantly (r = 0.68; P = 0.0001). The following motion parameters were also correlated significantly with fertility (P less than 0.0003; r1 = percent fertilized ova and r2 = percent implantation): linearity (r1 = 0.42; r2 = 0.40), amplitude of lateral head displacement (r1 = 0.54; r2 = 0.48), curvilinear velocity (r1 = 0.53; r2 = 0.50), straight-line velocity (r1 = 0.55; r2 = 0.50), and percent motile sperm (r1 = 0.42; r2 = 0.32). These results suggest a relationship between toxicant-induced reductions in sperm motion and fertility.

Ninety-day toxicity study of sodium monochloroacetate in Sprague-Dawley rats.

Male and female Sprague-Dawley rats were administered the sodium salt of monochloroacetic acid (SMCA) by oral gavage for a period of 90 consecutive days. Dosage levels of 15, 30, 60 or 120 mg/kg per day were employed. SMCA clearly induced toxicity in both females and males, with the greatest severity in the male animals. Both the liver and kidneys were identified as target organs. At 120 mg/kg per day, 30% of females and 80% of the males died, most within the first 2 days of treatment. Hemorrhagic and congested lungs (possibly a postmortem change) were seen in the early deaths (1-3 days) whereas liver lesions were observed in later deaths. In addition, there was nephrotoxicity as evidenced by elevated creatinine, blood calcium (BCAL), and blood urea nitrogen (BUN) levels. Hepatotoxicity was indicated by increases in the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Both organs showed increased organ-to-body weight ratios. Microscopic examination revealed a significant (P less than or equal to 0.001) increase in chronic renal nephropathy and increased splenic pigmentation at 60 mg/kg per day in the males. Based on the observation of toxicity at all treatment levels in males, a lowest observed adverse effect level (LOAEL) of 15 mg/kg per day is proposed for a 90-day exposure to SMCA by oral gavage to the Sprague--Dawley rat.

Developmental toxicity of halogenated acetonitriles: drinking water by-products of chlorine disinfection.

The developmental toxicity of acetonitrile and 5 halogenated derivatives was examined with an in vivo teratology screen adapted for use in the Long-Evans rat. The screen was extended to an evaluation of growth till postnatal Days 41-42, and weight of several organs at sacrifice. Acetonitrile was without developmental effects even at doses toxic to the dam. Of the halogenated compounds, treatment with trichloroacetonitrile (TCAN) and dichloroacetonitrile (DCAN) resulted in reduced fertility and increased early implantation failure. There was no effect on litter size in females bearing live litters, but pup birth weight was reduced in all litters exposed to halogenated compounds. Perinatal survival of the pups was adversely impacted by DCAN and TCAN. Postnatal growth till Day 4 was reduced by DCAN and bromochloroacetonitrile (BCAN) while growth till Day 42 was consistently affected only by TCAN. Some general observations were made on the usefulness of the criteria used in the screen, and TCAN, the most toxic of the halogenated compounds, was selected for further in-depth evaluation.

Site-specific modulation of carcinogen-induced gastrointestinal tract nuclear anomalies in B6C3F1 mice by chloroform.

Chloroform (CHCl3) is an established rodent carcinogen and a prevalent contaminant of chlorine-disinfected drinking water. Thus in the United States CHCl3, along with other trihalomethanes, is regulated not to exceed 100 ppb in potable water. Recently, several studies have shown that CHCl3 also has anti-cancer properties as it inhibits tumor growth in mouse liver and in the gastrointestinal tract of the rat. In this paper we show that CHCl3 also inhibits the propensity for three gastrointestinal tract carcinogens, benzo(a)pyrene (BAP), 1,2-dimethylhydrazine (DMH) and methylnitrosourea (MNU), to induce nuclear anomalies in the proximal colon of the B6C3F1 mouse. For example, in mice pre-adapted to 1800 ppm CHCl3 for 30 days prior to the carcinogen administration the level of nuclear anomalies induced in the proximal colon by BAP was reduced by four-fold (0.9 +/- 0.7 v. 3.6 +/- 1.0 anomalies/10 crypts; p less than 0.001) and two-fold for MNU (2.4 +/- 1.0 v. 4.6 +/- 1.6; p less than 0.001) and DMH (0.9 +/- 0.9 v. 1.7 +/- 0.8; p = 0.03). In the duodenum CHCl3 was effective at inhibiting unclear anomalies only for MNU (45.3 +/- 4.6 v. 30.4 +/- 3.5; p = 0.02). The inhibitory effect of CHCl3 does not extend to nuclear anomalies of the forestomach. The anti-cancer properties of CHCl3 are discussed in light of its cancer causing potential and possible application to human risk assessment.

Sources of variation in the computer-assisted motion analysis of rat epididymal sperm.

Random and nonrandom factors associated with sample preparation and the automated analysis (CellSoft) of rat cauda epididymal sperm motion were studied. Random factors included inherent system variation at both the individual cell level and at the multiple cell level. Repeated analyses of identical tracks across grey level revealed a statistical interaction between grey settings and curvilinear velocity. However, in multiple track analyses, grey level was seen to be a factor only at higher settings. Nonrandom factors included time after sample preparation, dilution medium, and sample preparation procedures. Using a nicked preparation of the entire cauda epididymis from Long-Evans rats, the effects of time were studied on sperm suspended in 1) phosphate-buffered saline + 10 mg BSA/mL, 2) TEST yolk buffer, and 3) Medium 199. In PBS/BSA, the percent motile sperm estimate decreased (50% to 30%) over an hour, while the curvilinear velocity increased (127 to 142 microns/sec). Both sperm motion parameters were maintained in the TEST yolk buffer and in the Medium 199, although at lower values for the latter. Evaluation of the relative contribution of several factors, nested within sample, to the overall variance of three separate motion endpoints revealed that there was a large variation from field to field, negligible variation between overall CellSoft analyses of 200 cells or more, low variation at the preparation aliquot level, and moderate variation at the animal level. In planning experiments to test for effects on sperm motion endpoints, consideration of the relative contribution of the individual study factors to the overall variance of the parameter estimates will result in more sensitive experimental designs.