In vitro preclinical testing of nonoxynol-9 as potential anti-human immunodeficiency virus microbicide: a retrospective analysis of results from five laboratories

Abstract

The first product to be clinically evaluated as a microbicide contained the nonionic surfactant nonoxynol-9 (nonylphenoxypolyethoxyethanol; N-9). Many laboratories have used N-9 as a control compound for microbicide assays. However, no published comparisons of the results among laboratories or attempts to establish standardized protocols for preclinical testing of microbicides have been performed. In this study, we compared results from 127 N-9 toxicity and 72 efficacy assays that were generated in five different laboratories over the last six years and were performed with 14 different cell lines or tissues. Intra-assay reproducibility was measured at two-, three-, and fivefold differences using standard deviations. Interassay reproducibility was assessed using general linear models, and interaction between variables was studied using step-wise regression. The intra-assay reproducibility within the same N-9 concentration, cell type, assay duration, and laboratory was consistent at the twofold level of standard deviations. For interassay reproducibility, cell line, duration of assay, and N-9 concentration were all significant sources of variability (P < 0.01). Half-maximal toxicity concentrations for N-9 were similar between laboratories for assays of similar exposure durations, but these similarities decreased with lower test concentrations of N-9. Results for both long (>24 h) and short (<2 h) exposures of cells to N-9 showed variability, while assays with 4 to 8 h of N-9 exposure gave results that were not significantly different. This is the first analysis to compare preclinical N-9 toxicity levels that were obtained by different laboratories using various protocols. This comparative work can be used to develop standardized microbicide testing protocols that will help advance potential microbicides to clinical trials.

FIG. 1.

Intra-assay reproducibility across assay durations for all cell lines. The intra-assay reproducibility was assessed using SD (log₁₀). All replicates within one concentration were compared. The two-, three-, and fivefold levels of SD log₁₀ are indicated from bottom to top, respectively, by horizontal lines. P < 0.05 indicates the SD (log₁₀) at which there is 90% confidence to find a fivefold difference (4).

FIG. 2.

Interassay reproducibility across assay duration and cell lines. Interassay reproducibility was assessed using a regression analysis with general linear models, taking into account all N-9 concentrations. Low reproducibility was indicated where the variability between replicates was higher than the variability within replicates and was significant (P < 0.05) when the F ratio was greater than approximately 3.0 (PROC GLM, SAS 2004). *, F ratio equals the variability in experimental measures between replicates divided by the variability in experimental measures within replicates. The horizontal line indicates the approximate F value that would result from a significant replicate effect when P was <0.05.

FIG. 3.

TC₅₀ (μg/ml) for N-9 toxicity assays for the various exposure times. Toxicity increased with longer compound exposure.

FIG. 4.

Means and SDs of TC₅₀ values (μg/ml) per N-9 exposure time. The mean from 5 to 10 min of exposure was obtained from 52 data points, 1 to 2 h of exposure from 8 data points, 4 to 8 h of exposure from 20 data points, and >24 h of exposure from 32 data points. The following outliers were excluded: explant, 10 min (GH); explant, 1 h (GH); and explant, 1 h, 35 min (GH).

FIG. 5.

Comparison of calculation of TC₅₀ values by four-parameter curve-fitting and point-to-point linear regression. The data points are listed in ascending order. A total of 127 assays were compared.