The Toxicologist - Society of Toxicology

vated only in high-dose-treated animals (max. 7.5×10-6 vs 2.7×10-6 control) and
only when measured at 6, 8 and 9 weeks post-treatment. The Hprt mutant frequency
was elevated in all high-dose-treated animals at all measured time-points
(max, 41×10-6 vs 4.7×10-6 control) and in mid-dose-treated animals at 16 weeks
(the only time measured). The results suggest that the two gene mutation assays differ
in their ability to detect the genotoxicity of CP. Potential explanations for such a
differential response will be discussed.
1412 INTERNATIONAL, INTERLABORATORY PIG-A
MUTATION ASSAY TRIAL: EVALUATION OF
TRANSFERABILITY.
S. Dertinger 1 , S. Phonethepswath 1 , P. Weller 1 , L. Stankowski 2 , D. Roberts 2 , J.
Shi 3 , L. Krsmanovic 3 , H. Vohr 4 , L. Custer 5 , C. Gleason 5 , A. Henwood 5 , K.
Sweder 5 , A. Giddings 6 , A. Lynch 6 , W. Gunther 7 , C. Thiffeault 7 , T. Shutsky 7 ,
R. Fiedler 7 , J. Bhalli 8 , R. Heflich 8 , J. Nicolette 9 , P. Sonders 9 , J. Murray 9 , T.
Kimoto 10 and J. MacGregor 11 . 1 Litron Laboratories, Rochester, NY, 2 Covance
Laboratories, Vienna, VA, 3 BioReliance, Rockville, MD, 4 Bayer Schering
Pharmacology AG, Wuppertal, Germany, 5 Bristol-Myers Squibb, Syracuse, NY,
6 GlaxoSmithKline, Ware, United Kingdom, 7 Pfizer Global R&D, Groton, CT, 8 U.S.
FDA-NCTR, Jefferson, AR, 9 Abbott Laboratories, Abbott Park, IL, 10 Teijin
Pharmacology Tokyo, Japan and 11 Toxicology Consulting Services, Arnold, MD.
An in vivo mutation assay has been developed based on the enumeration of CD59negative
erythrocytes (indicative of Pig-a gene mutation). In this system, anti-
CD59-PE and SYTO 13 dye are used to label leukocyte-depleted blood samples,
and the frequency of CD59-Neg erythrocytes (RBCs) and reticulocytes (RETs) are
determined via flow cytometry. To evaluate transferability, ten labs determined the
effect of N-ethyl-N-nitrosourea (ENU) administered to male rats via oral gavage
for 3 consecutive days. All labs studied 0, 20 and 40 mg/kg/day (n = 5/group), and
two also studied ENU at 4 mg/kg/day. Serial blood samples were collected on Days
-1, 15, & 30, with some labs also sampling on Days 45 & 90. Samples were
processed according to standardized methods and data acquisition protocols, and 3
endpoints were measured: %RETs and CD59-Neg RET & RBC frequencies. Each
lab observed dose-related increases in the frequency of CD59-Neg RETs and
CD59-Neg RBCs on Day 15. Maximal CD59-Neg RET responses tended to occur
by Day 15, whereas peak CD59-Neg RBC responses were achieved by approximately
Day 45. Elevated CD59-Neg RET and RBC frequencies were maintained
through the latest time-point studied (Day 90). These data demonstrate close correspondence
among labs, both in terms of the timing and magnitude of the responses,
and illustrate that this assay is robust and readily transferable across sites.
Additional interlab studies are in progress that will increase the number of chemicals
evaluated in order to systematically characterize assay performance.
1413 TOXICOLOGICAL CHARACTERIZATION OF DIESEL
ENGINE EMISSIONS USING BIODIESEL AND A
CLOSED SOOT FILTER.
I. M. Kooter, M. A. van Vugt, A. D. Jedynska, R. P. Verbeek and C. A. Krul.
TNO, Utrecht, Netherlands. Sponsor: R. Woutersen.
This study was designed to determine the toxicity (oxidative stress, cytotoxicity,
genotoxicity) in extracts of combustion aerosols. A typical Euro III heavy truck engine
was tested over the European Transient Cycle with three different fuels: conventional
diesel EN590, biodiesel EN14214 as B100 and blends with conventional
diesel (B5, B10, and B20) and pure plant oil DIN51605 (PPO). In addition application
of a (wall flow) diesel particulate filter (DPF) with conventional diesel
EN590 was tested. The use of B100 or PPO as a fuel or the DPF reduced PM mass
and numbers over 80%. Similarly, significant reduction in the emission of chemical
constituents (EC 90%, (oxy)-PAH 70%) were achieved. No significant changes in
nitro-PAH were observed. The use of B100 or PPO led to a NOx increase of about
30%, but no increase is found for DPF application. The effects of B100, PPO and
the DPF on the biological test results vary strongly from positive to negative depending
on the biological end point. The oxidative potential, measured via the
DTT assay, of the B100 and PPO or DPF emissions is reduced by 95%. The cytotoxicity
is increased for B100 by 200%. The measured mutagenicity, using the
Ames assay test with TA98 and YG1024 strains of Salmonella typhimurium indicate
a dose response for the nitroarene sensitive YG1024 strain for B100 and PPO
(fold induction: 1.6). In summary B100 and PPO have good potential for the use
as a second generation biofuel resulting in lower mass exhaust gas, similar to application
of DPF, but caution should be made due to potential increased toxicity.
Besides regulation via mass, the biological reactivity of exhaust emissions of new
(bio)fuels and application of new technologies, needs attention. The different responses
of different biological tests as well as differences in results between test laboratories
underline the need for harmonization of test methods and international
cooperation.
1414 IN VITRO PREDICTIONS OF IN VIVO GENOTOXICITY
ARE BETTER THAN PREDICTIONS OF
CARCINOGENICITY.
R. Walmsley 1, 2 . 1 Life Sciences, University of Manchester, Manchester, United
Kingdom and 2 Gentronix Ltd., Manchester, United Kingdom.
The sensitivity and specificity of regulatory in vitro assays in the prediction of carcinogenicity
were calculated by Kirkland et al (2005), using the carcinogenicity potency
database (CPDB). in vitro mammalian tests had higher sensitivities (70-90%)
than Ames (60%), but produced positive results for more non-carcinogens (~50%)
than Ames (77%). Hence Ames positive pharmaceutical candidates rarely proceed
to animal testing, whereas Ames negative in vitro mammalian test positives often
proceed. Genotoxicity assays are not expected to predict carcinogenicity where tumours
arise by a non-genotoxic mode of action. However, in vivo genotoxicity prediction
avoids the need for this distinction, and its accuracy is equally important in
making decisions regarding the fate of candidate pharmaceuticals. This study presents
in vivo sensitivity and specificity figures for 4 commonly used in vitro assays in
a collection of 155 compounds.
Ames predictions for in vivo genotoxicity and carcinogenicity (Kirkland’s in brackets)
were similar in sensitivity, 58% (59-60%), though specificity was higher, 86%
(74-77%). The GADD45a-GFP ‘GreenScreen HC’ assay predictions were similar
for sensitivity, 85% (88% Hastwell, 2009) and specificity, 92% (95%, Hastwell
2009). MLA showed slightly better in vivo sensitivity, 86% (73– 81%), and considerably
increased specificity, 79% (39–48%). MNT/CA showed similar improvements
in sensitivity, 90% (79– 81%), though specificity remains low 65%
(31–55%).
The generally higher sensitivity to in vivo genotoxins (cf carcinogens) probably reflects
the expectation that in vitro assays will produce negative results for non-genotoxic
carcinogens. The higher in vivo specificity figures might reflect the greater
prevalence of pharmaceuticals in this collection (cf CPDB), which are generally less
reactive. The GADD45a-GFP assay has the highest specificity, and hence the most
reliable positive results. It is already used in screening, but might usefully be applied
to compounds uniquely positive in MLA/MNT/CA, to identify those most likely
to be in vivo positive.
1415 CYTOTOXICITY AND GENE EXPRESSION
ALTERATIONS FOLLOWING TREATMENT OF TK6
CELLS WITH ETOPOSIDE AND SODIUM CHLORIDE.
Y. Chen 1 , M. M. Moore 1 and J. C. Fuscoe 2 . 1 Division of Genetic and Molecular
Toxicology, U.S. FDA/NCTR, Jefferson, AR and 2 Division of Systems Biology, U.S.
FDA/NCTR, Jefferson, AR.
The use of gene expression data in determining the relevance of genotoxic damage
is gaining momentum. The dose at which gene expression is evaluated is a key question.
Using TK6 cells and 4 genes involved in genotoxic stress response (p21,
GADD45, ATF3, and DDB2) we are investigating whether gene expression alterations
can be observed at chemical concentrations inducing levels of cytotoxicity
that would be considered acceptable (biologically relevant) in the standard gene
mutation assays. Cells were treated with etoposide (0.1, 0.4, 1.1, 3.3 and 10 ug/ml)
and sodium chloride (0.1, 0.4, 1.1, 3.3 and 10 mg/ml). Both MTS and plating efficiency
were used to assess cytotoxicity. Based on MTS, the top concentrations of
the two chemicals induced approximately 50% cytotoxicity. Plating efficiency assessment
revealed that the top two concentration of etoposide resulted in greater
than 95% cytotoxicity and the top concentration of sodium chloride, substantially
higher than the recommended concentration for in vitro mammalian cell assays, resulted
in greater than 90% cytotoxicity. For etoposide treated cells there were small
increases in the expression of all 4 genes at the first 3 concentrations with dramatic
increases seen only at concentrations causing very high cytotoxicity. For sodium
chloride, significant up- regulation of the 4 genes only occurred at the 10 mg/ml
concentration. Etoposide is an extremely potent clastogen while sodium chloride
only induces genetic damage at high concentrations causing alterations in osmolality.
Given the small magnitude of gene expression changes seen with etoposide, it is
important that a large number of additional chemicals be assessed before conclusions
can be drawn concerning the utility of this gene expression approach to detecting
genotoxicants.
SOT 2011 ANNUAL MEETING 303