Animal metrics: Tracking contributions of new approach methods to reduced animal use.

Many companies and global regulatory programs have expressed the intent to move away from in vivo animal testing to new approach methods (NAMs) as part of product safety assessments. NAMs, which include non-animal approaches for testing and assessment ­ from computer-based modeling to in chemico or in vitro models ­ allow faster data gener­ation with potentially greater relevance to humans while avoiding animal use. To monitor progress implementing NAMs, each organization first must define what is in scope, starting with the definition of "animal" (e.g., mammals, vertebrates) and applicable studies (e.g., animals used for "in-house" experiments, at contract research organizations, as part of envi­ronmental monitoring). Next, organizations must establish baseline animal use, including defined rules for inclusion/ exclusion of animals that ensure consistency in future assessments. Lastly, organizations must establish metrics for animal savings based on the utility of NAM data. This paper presents one approach to establish "animal use" metrics in a toxi­cology program at The Dow Chemical Company. The premise of our program is that most NAM information has value for animal savings, but the value depends on how data are used (e.g., research and development, screening, or regulatory requirements) and the level of certainty for internal decision-making. This manuscript provides metrics on the impact of NAMs, allowing a quantitative assessment of animal use numbers over time, accountability for resources spent on NAM development, and identification of areas where NAM development is still needed. This approach can be refined for use at other organizations.

Pronamide: Human relevance of liver-mediated rat leydig cell tumors.

Dietary exposure to pronamide resulted in higher incidences of Leydig cell tumors (LCT) at 1000ppm in a 2-year cancer bioassay, but there were no testes effects at 40 or 200ppm, and no testes effects at 12-months at any concentration. A 90-day mode-of-action (MoA) study was conducted at concentrations of 0, 200, 1000 and 2000ppm. Standard parameters and stereological and proliferation analyses of LCs, targeted testis and liver gene expression, in vitro metabolism of testosterone by liver microsomes, and quantification of serum hormones and testosterone metabolites were evaluated. Increased testosterone metabolism due to increases in hepatic microsomal activity, alterations in serum hormone levels, and other data suggest that LCTs were mediated through a perturbation of the HPG-axis. Data suggest that this occurs after a threshold of exposure is reached, indicating a nonlinear/threshold dose-response. Pronamide-induced rat LCTs mediated by alterations to the HPG-axis have low relevance to humans due to quantitative differences in sensitivity between rats and humans to LCTs. Pronamide displayed no genotoxicity or direct endocrine effects. A margin of exposure approach for risk assessment and derivation of the chronic reference dose based on a point of departure of 200ppm is most appropriate and protective of human health.

An F1-extended one-generation reproductive toxicity study in Crl:CD(SD) rats with 2,4-dichlorophenoxyacetic acid.

2,4-Dichlorophenoxyacetic acid (2,4-D) was assessed for systemic toxicity, reproductive toxicity, developmental neurotoxicity (DNT), developmental immunotoxicity (DIT), and endocrine toxicity. CD rats (27/sex/dose) were exposed to 0, 100, 300, 600 (female), or 800 (male) ppm 2,4-D in diet. Nonlinear toxicokinetic behavior was shown at high doses; the renal clearance saturation threshold for 2,4-D was exceeded markedly in females and slightly exceeded in males. Exposure was 4 weeks premating, 7 weeks postmating for P1 males and through lactation for P1 females. F1 offspring were examined for survival and development, and at weaning, pups were divided in cohorts, by sex and dose, and by systemic toxicity (10), DNT (10), DIT (20), and reproductive toxicity (≥ 23). Remaining weanlings were evaluated for systemic toxicity and neuropathology (10-12). Body weight decreased during lactation in high-dose P1 females and in F1 pups. Kidney was the primary target organ, with slight degeneration of proximal convoluted tubules observed in high-dose P1 males and in high-dose F1 males and females. A slight intergenerational difference in kidney toxicity was attributed to increased intake of 2,4-D in F1 offspring. Decreased weanling testes weights and delayed preputial separation in F1 males were attributed to decreased body weights. Endocrine-related effects were limited to slight thyroid hormone changes and adaptive histopathology in high-dose GD 17 dams seen only at a nonlinear toxicokinetic dose. 2,4-D did not cause reproductive toxicity, DNT, or DIT. The "No Observed Adverse Effect Level" for systemic toxicity was 300 ppm in both males (16.6 mg/kg/day) and females (20.6 mg/kg/day), which is approximately 6700- to 93 000-fold higher than that reported for 2,4-D exposures in human biomonitoring studies.

Effects of intensity and type of prepulse stimulus on prepulse inhibition in scopolamine treated rats.

Prepulse inhibition (PPI) of the auditory startle response (ASR) is a behavioral test that has been used to measure auditory thresholds, to assess sensory-motor integration functions, and its use has been recommended in the United States Environmental Protection Agency Developmental Neurotoxicity Guideline (OPPTS 870.6300). The purpose of the present study was to determine to what extent the intensity and/or type of prepulse stimuli modulate PPI in scopolamine-treated rats. The PPI of the ASR peak amplitude was measured when the intensity of a 10-kHz prepulse tone was varied (69-, 80-, and 90 dB[A]; Experiment 1) and when both the intensity and type of auditory prepulse (a 10-kHz tone vs. a white noise burst) were varied (Experiment 2). Scopolamine treatment attenuated PPI in both experiments and interacted significantly with the prepulse stimulus intensity in Experiment 1. In Experiment 2, the percent of PPI was linearly related to prepulse stimulus intensity for trials using a tone, but was biphasic on trials using a white-noise prepulse stimulus. Prepulse stimuli of certain intensities elicited a response, and this response was greater when the prepulse stimulus was a white noise burst versus a tone of the same intensity. Further, the response to the prepulse altered the amount of inhibition and, therefore, confounded the overall measure of PPI at the higher prepulse stimulus intensity levels. Overall, these results indicate that careful consideration of the intensity and type of prepulse stimuli be taken in the context of their potential to induce a prepulse-elicited response, as well as providing the appropriate measures of such a response, when designing and interpreting PPI experiments.

Factors affecting grip strength testing.

The rodent grip strength test was developed decades ago and is a putative measure of muscular strength. This test has been included in the functional observational battery (FOB) to screen for neurobehavioral toxicity, and changes in grip strength have been interpreted as evidence of motor neurotoxicity. Despite its widespread use, questions remain about what the grip strength test actually measures. In this study, potential confounders of the grip strength test were identified and tested, including operational parameters, disruption of peripheral sensory function and changes in body weight. Operational parameters (sampling rate, system type and trial angle but not trial speed) had dramatic effects on grip strength data. Doxorubicin (DX, 10 mg/kg iv) was used to cause sensory impairment. It decreased forelimb and hindlimb grip strength (by 27% and 32%, respectively, compared with controls), an effect that was correlated with degeneration of peripheral and central sensory components (distal tibial and sural nerves, dorsal funiculus of the spinal cord and dorsal, but not ventral, spinal roots). Feed restriction-induced loss of body weight (26% compared with controls) and muscle mass (20% compared with controls) reversibly decreased both forelimb and hindlimb grip strength (18% and 17%, respectively, compared with controls). Ignoring these confounding factors could potentially lead to increased data variability and inconsistency within single studies, across studies and in historical control data sets. To assist in data interpretation and evaluation of grip strength results, it is suggested that exact conditions of application of the test be reported in greater detail. Furthermore, given that the grip strength test can be influenced by factors other than true muscular strength, use of the term grip performance is proposed to better reflect the apical nature of this test.