Ames test study designs for nitrosamine mutagenicity testing: qualitative and quantitative analysis of key assay parameters

Abstract The robust control of genotoxic N-nitrosamine (NA) impurities is an important safety consideration for the pharmaceutical industry, especially considering recent drug product withdrawals. NAs belong to the ‘cohort of concern’ list of genotoxic impurities (ICH M7) because of the mutagenic and carcinogenic potency of this chemical class. In addition, regulatory concerns exist regarding the capacity of the Ames test to predict the carcinogenic potential of NAs because of historically discordant results. The reasons postulated to explain these discordant data generally point to aspects of Ames test study design. These include vehicle solvent choice, liver S9 species, bacterial strain, compound concentration, and use of pre-incubation versus plate incorporation methods. Many of these concerns have their roots in historical data generated prior to the harmonization of Ames test guidelines. Therefore, we investigated various Ames test assay parameters and used qualitative analysis and quantitative benchmark dose modelling to identify which combinations provided the most sensitive conditions in terms of mutagenic potency. Two alkyl-nitrosamines, N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) were studied. NDMA and NDEA mutagenicity was readily detected in the Ames test and key assay parameters were identified that contributed to assay sensitivity rankings. The pre-incubation method (30-min incubation), appropriate vehicle (water or methanol), and hamster-induced liver S9, alongside Salmonella typhimurium strains TA100 and TA1535 and Escherichia coli strain WP2uvrA(pKM101) provide the most sensitive combination of assay parameters in terms of NDMA and NDEA mutagenic potency in the Ames test. Using these parameters and further quantitative benchmark dose modelling, we show that N-nitrosomethylethylamine (NMEA) is positive in Ames test and therefore should no longer be considered a historically discordant NA. The results presented herein define a sensitive Ames test design that can be deployed for the assessment of NAs to support robust impurity qualifications.

4-Nitroquinoline-1oxide (4NQO) TA100 TA1535 TA1537 TA98 WP2 uvrA (pKM101) NDMA (µg/plate) Figure 1 (Suppl.)Bacterial reverse mutation plate incorporation mean revertant ratio treated/vehicle control data with NDMA, using solvent vehicles DMSO (top), methanol (middle) and purified water (bottom), in the absence of S9-mix (Y axis representative of mean revertant ratio treated/vehicle, X axis representative of test article concentration (µg/plate) per bacterial strain).Blue bars refer to concentrations where mean revertant ratio treated/vehicle is lower than 2-fold for TA100, TA98 and WP2uvrA (pKM101) and 3-fold for TA1535 and TA1537.The maximum concentration tested was 5000 ug/plate, the maximum concentration in accordance with current guidelines.Figure 2 (Suppl.)Bacterial reverse mutation plate incorporation mean revertant ratio treated/vehicle control data with NDMA, using solvent vehicles DMSO (top), methanol (middle) and purified water (bottom), in the presence of Rat liver S9mix (Y axis representative of mean revertant ratio treated/vehicle, X axis representative of test article concentration (µg/plate) per bacterial strain).Bars represent concentrations where mean revertant ratio treated/vehicle is less than (blue) or exceed (red) the 2-fold for TA100, TA98 and WP2uvrA (pKM101) and 3-fold for TA1535 and TA1537.The maximum concentration tested was 5000 ug/plate, the maximum concentration in accordance with current guidelines.Figure 3 (Suppl.)Bacterial reverse mutation plate incorporation mean revertant ratio treated/vehicle control data with NDMA, using solvent vehicles DMSO (top), methanol (middle) and purified water (bottom), in the presence of Hamster liver S9-mix (Y axis representative of mean revertant ratio treated/vehicle, X axis representative of test article concentration (µg/plate) per bacterial strain).Bars represent concentrations where mean revertant ratio treated/vehicle is less than (blue) or exceed (red) the 2-fold for TA100, TA98 and WP2uvrA (pKM101) and 3-fold for TA1535 and TA1537.The maximum concentration tested was 5000 ug/plate, the maximum concentration in accordance with current guidelines.) Bacterial reverse mutation pre-incubation mean revertant ratio treated/vehicle control data with NDMA, using solvent vehicles DMSO (A), Methanol (B), Water (C), Acetone (D), Acetonitrile (E), NMP (F), DHF (G) and DMF (H), in the presence of Rat liver S9-mix (Y axis representative of mean revertant ratio treated/vehicle, X axis representative of test article concentration (µg/plate) per bacterial strain).Bars represent concentrations where mean revertant ratio treated/vehicle is less than (blue) or exceed (red) the 2-fold for TA100, TA98 and WP2uvrA (pKM101) and 3-fold for TA1535 and TA1537.The maximum concentration tested was 5000 ug/plate, the maximum concentration in accordance with current guidelines.Bars represent concentrations where mean revertant ratio treated/vehicle is less than (blue) or exceed (red) the 2-fold for TA100, TA98 and WP2uvrA (pKM101) and 3-fold for TA1535 and TA1537.The maximum concentration tested was 5000 ug/plate, the maximum concentration in accordance with current guidelines.N.b Due to toxicity, no data available for TA98 or TA1537 using acetonitrile as a vehicle.and WP2uvrA (pKM101) and 3-fold for TA1535 and TA1537 Figure 13 (Suppl.)Bacterial reverse mutation plate incorporation mean revertant ratio treated/vehicle control data with NMEA, using solvent vehicles DMSO (top), methanol (middle) and water (bottom), in the presence of Rat liver S9-mix (Y axis representative of mean revertant ratio treated/vehicle, X axis representative of test article concentration (µg/plate) per bacterial strain).Bars represent concentrations where mean revertant ratio treated/vehicle is less than (blue) or exceed (red) the 2-fold for TA100, TA98 and WP2uvrA (pKM101) and 3-fold for TA1535 and TA1537.The maximum concentration tested was 5000 ug/plate, the maximum concentration in accordance with current guidelines.) Bacterial reverse mutation pre-incubation mean revertant ratio treated/vehicle control data with NMEA, using solvent vehicles DMSO (top), methanol (middle) and purified water (bottom), in the presence of Rat liver S9mix (Y axis representative of mean revertant ratio treated/vehicle, X axis representative of test article concentration (µg/plate) per bacterial strain).Bars represent concentrations where mean revertant ratio treated/vehicle is less than (blue) or exceed (red) the 2-fold for TA100, TA98 and WP2uvrA (pKM101) and 3-fold for TA1535 and TA1537.The maximum concentration tested was 5000 ug/plate, the maximum concentration in accordance with current guidelines ) Bacterial reverse mutation pre-incubation mean revertant ratio treated/vehicle control data with NMEA, using solvent vehicles methanol (top) and purified water (bottom), in the presence of Hamster liver S9-mix (Y axis representative of mean revertant ratio treated/vehicle, X axis representative of test article concentration (µg/plate) per bacterial strain).Bars represent concentrations where mean revertant ratio treated/vehicle is less than (blue) or exceed (red) the 2-fold for TA100, TA98 and WP2uvrA (pKM101) and 3-fold for TA1535 and TA1537.The maximum concentration tested was 5000 ug/plate, the maximum concentration in accordance with current guidelines.

FFigure 9 (
Figure 9 (Suppl.)Bacterial reverse mutation pre-incubation mean revertant ratio treated/vehicle control data with NDEA, using solvent vehicles DMSO (A), Methanol (B), Water (C), Acetone (D), Acetonitrile (E), NMP (F), DHF (G) and DMF (H), in the presence of Hamster liver S9-mix (Y axis representative of mean revertant ratio treated/vehicle, X axis representative of test article concentration (µg/plate) per bacterial strain).Bars represent concentrations where mean revertant ratio treated/vehicle is less than (blue) or exceed (red) the 2-fold for TA100, TA98 and WP2uvrA (pKM101) and 3-fold for TA1535 and TA1537.The maximum concentration tested was 5000 ug/plate, the maximum concentration in accordance with current guidelines.N.b Due to toxicity, no data available for TA98 or TA1537 using acetonitrile as a vehicle.

Figure 10 (
Figure 10 (Suppl.)-BMD-derived sensitivity ranking for positive NDMA studies.A, Sensitivity ranking using 90% confidence intervals of the BMD 100 (i.e., 90% confidence interval of the dose estimated to cause a two-fold increase in response relative to vehicle control.).For each confidence interval, test conditions (i.e., vehicle, incubation method, strain and S9 source) are indicated in the Table on the right-side of the plot.B/C, quantile-quantile and residuals against dose (respectively) for the fitted dose-response data showing approximate normality and variance homogeneity on log 10 scale.D, Exponential model fits to the dose-response data underlying the BMD confidence intervals shown in A. Horizontal and vertical dashed lines indicate interpolation at the benchmark response of 100% to define the BMD 100 (respectively).

Figure 11 (Figure 12 (
Figure 11 (Suppl.)-BMD-derived sensitivity ranking for positive NDEA studies.A, Sensitivity ranking using 90% confidence intervals of the BMD 100 (i.e., 90% confidence interval of the dose estimated to cause a two-fold increase in response relative to vehicle control.).For each confidence interval, test conditions (i.e., vehicle, incubation method, strain and S9 source) are indicated in the Table on the right-side of the plot.B/C, quantile-quantile and residuals against dose (respectively) for the fitted dose-response data showing approximate normality and variance homogeneity on log 10 scale.D, Exponential model fits to the dose-response data underlying the BMD confidence intervals shown in A. Horizontal and vertical dashed lines indicate interpolation at the benchmark response of 100% to define the BMD 100 (respectively).

Figure 14 (
Figure 14 (Suppl.)Bacterial reverse mutation pre-incubation mean revertant ratio treated/vehicle control data with NMEA, using solvent vehicles DMSO (top), methanol (middle) and purified water (bottom), in the presence of Rat liver S9mix (Y axis representative of mean revertant ratio treated/vehicle, X axis representative of test article concentration (µg/plate) per bacterial strain).Bars represent concentrations where mean revertant ratio treated/vehicle is less than (blue) or exceed (red) the 2-fold for TA100, TA98 and WP2uvrA (pKM101) and 3-fold for TA1535 and TA1537.The maximum concentration tested was 5000 ug/plate, the maximum concentration in accordance with current guidelines Figure 15 (Suppl.)Bacterial reverse mutation pre-incubation mean revertant ratio treated/vehicle control data with NMEA, using solvent vehicles methanol (top) and purified water (bottom), in the presence of Hamster liver S9-mix (Y axis representative of mean revertant ratio treated/vehicle, X axis representative of test article concentration (µg/plate) per bacterial strain).Bars represent concentrations where mean revertant ratio treated/vehicle is less than (blue) or exceed (red) the 2-fold for TA100, TA98 and WP2uvrA (pKM101) and 3-fold for TA1535 and TA1537.The maximum concentration tested was 5000 ug/plate, the maximum concentration in accordance with current guidelines.
Figure16(Suppl.) -BMD-derived sensitivity ranking for positive NMEA studies.A, Sensitivity ranking using 90% confidence intervals of the BMD 100 (i.e., 90% confidence interval of the dose estimated to cause a two-fold increase in response relative to vehicle control.).For each confidence interval, test conditions (i.e., vehicle, incubation method, strain and S9 source) are indicated in the Table on the right-side of the plot.B/C, quantile-quantile and residuals against dose (respectively) for the fitted dose-response data showing approximate normality and variance homogeneity on log 10 scale.D, Exponential model fits to the dose-response data underlying the BMD confidence intervals shown in A. Horizontal and vertical dashed lines indicate interpolation at the benchmark response of 100% to define the BMD 100 (respectively).

Figure 6 (Suppl.
representative of test article concentration (µg/plate) per bacterial strain).Bars represent concentrations where mean revertant ratios treated/vehicle is less than (blue) or exceed (red) the 2-fold for TA100, TA98 and WP2uvrA (pKM101) and 3-fold for TA1535 and TA1537.The maximum concentration tested was 5000 ug/plate, the maximum concentration in accordance with current guidelines.
Figure 7 (Suppl.)Bacterial reverse mutation pre-incubation mean revertant ratio treated/vehicle control data with NDMA, using solvent vehicles DMSO (A), Methanol (B), Water (C), Acetone (D), Acetonitrile (E), NMP (F), DHF (G) and DMF (H), in the presence of Hamster liver S9-mix (Y axis representative of mean revertant ratio treated/vehicle, X axis Figure 8 (Suppl.)Bacterial reverse mutation pre-incubation mean revertant ratio treated/vehicle control data with NDEA, using solvent vehicles DMSO (A), Methanol (B), Water (C), Acetone (D), Acetonitrile (E), NMP (F), DHF (G) and DMF (H), in the presence of Rat liver S9-mix (Y axis representative of mean revertant ratio treated/vehicle, X axis representative of test article concentration (µg/plate) per bacterial strain).Bars represent concentrations where mean revertant ratio treated/vehicle is less than (blue) or exceed (red) the . The maximum concentration tested was 5000 ug/plate, the maximum concentration in accordance with current guidelines.