The Toxicologist - Society of Toxicology

years old should give a high probability of sexual maturity (1) but histopathology
data at this laboratory has shown otherwise. This review of animal data from toxicology
studies using sexually mature animals examines the parameters and factors
involved in the determination of sexual maturation onset and completion.
Discussions with suppliers, delivery, acclimatisation and pre-treatment investigations
are included. The parameters reviewed include age, weight, physical characteristics
of secondary development (e.g. descent and dimensions of testes, appearance
of adult dentures), positive vaginal swabs and confirmed semen production
and viability. Regular menstrual cycling, repeat viable semen sampling and assessment
of hormone levels provide essential baseline data for intra-study/animal comparison,
facilitating identification of toxicity and natural variability or progression
of parameters. The results show that in isolation most of the parameters examined
were open to interpretation as they were measured qualitatively. There was evidence
that long-distance transport of the animals led to disruption of parameters measured
at source. No individual parameter gave a clear determination of sexual maturity
but repeated production of viable semen for males and a regular menstrual cycle
for females provided the strongest evidence, accompanied by other parameters.
Determination of sexual maturity in both sexes is a complex issue and many factors
are involved. In conclusion, it is preferable to employ a weight of evidence approach
using all available parameters to confirm sexual maturity in cynomolgus
macaques.(1) Boyd Group Papers on the use of non-human primates in research
and testing 2002 Paper 4.
1924 SERIAL COLLECTION OF CEREBROSPINAL FLUID VIA
A SURGICALLY OR PERCUTANEOUSLY IMPLANTED
CATHETER IN CYNOMOLGUS MONKEYS.
J. K. Herman, R. Avery, M. Taschwer, P. Love, J. Fohey, M. Abdelhameed and
W. Meier. Nonclinical Safety Assessment, Covance Laboratories, Madison, WI.
Serial Cerebral Spinal Fluid (CSF) collection in primates was evaluated for studies
involving test materials that are active in the central nervous system (CNS).
Although exposure can be confirmed with a single cisternal CSF collection, this single
point evaluation does not allow assessment of time profiles of exposure and
must be conducted in anesthetized animals. The purpose of this study was to evaluated
two procedures for placing an indwelling catheter in the lumbar intrathecal
space for collecting serial CSF samples from unanesthetized animals. The two models
evaluated were 1) a surgically implanted catheter placed in the lumbar intrathecal
space via hemilaminectomy technique and 2) a percutaneously implanted
catheter placed in the lumbar intrathecal space via a “through the needle” catheterization
technique. Animals with the hemilaminectomy were allowed at least 1 week
post surgery for recovery while animals with the percutaneous catheter were allowed
24 to 48 hours for recovery. Animals were then given either a single dose of
vehicle or a commercially available glycine transport inhibitor via oral gavage at a
dose level of 2 mg/kg. Blood and CSF were collected predose and 1, 2, 4, 8 and 24
hours post dose. There were no clinical signs or changes in body weight or body
temperature associated with test article administration or the surgically implantation
of the catheters. The time to maximum levels (Tmax) for the test article in the
CSF was similar to the plasma Tmax although exposure (AUC) was approximately
10 fold lower in the CSF than in the plasma. In general, exposure assessments between
the two catheterization techniques were similar, although the percutaneous
approach tended to yield slightly higher values. In conclusion, serial collection of
CSF from conscious, restrained primates is a valid method for determining test article
exposure in the CNS and allows for comparisons between plasma exposure,
CNS exposure and potential clinical signs or pathology changes.
1925 TEMPERATURE REGULATION AND THE ASSESSMENT
OF TOXICITY.
K. L. Hastings 1 and M. D. Green 2 . 1 Corporate Regulatory Affairs, sanofi-aventis,
Bethesda, MD and 2 OVRR, DVRPA, U.S. FDA, Bethesda, MD.
Temperature regulation is an important part of homeostatic regulation that influences
many physiological and pharmacological systems. Factors involving body
temperature involve both peripheral and centrally mediated physiological systems
reflective of various classes of products making them potential responsive to a wide
variety of drugs and biologicals. Changes in body temperature can be an important
endpoint itself for example with certain classes of investigational products such as
experimental vaccines. Additionally, body temperature may be a factor in the assessment
of other endpoints of toxicological interest such as in vivo mutagenicity
and exert on influence on the pharmacokinetics and pharmacodynamics of various
drugs. Despite the potential value of measuring body temperature, this endpoint is
infrequently determined in spite of the availability of sophisticated systems for its
measurement. This presentation reviews the uses and tools presently available to examine
body temperature for the assessment of toxicity.
412 SOT 2011 ANNUAL MEETING
1926 APPLICATION OF THE DRIED BLOOD SPOT
TECHNIQUE IN THE BIOANALYSIS OF SULPIRIDE.
J. Legrand, T. Ameller, R. Forster, R. Michaud and S. Laurent. CIT, EVREUX
Cedex, France.
There has been much recent interest in the application of the Dried Blood Spot
(DBS) technique in pharmacokinetics and toxicokinetics. In our facility, we have
validated a bioanalytical method using the DBS approach for the determination of
sulpiride in dog whole blood. Sulpiride is a second-generation antipsychotic molecule
exhibiting bipolar functional groups. The DBS collection technique consisted
of spotting 15.0μL droplets of whole blood samples containing sulpiride directly
onto treated collection cards (GE-Whatman, DMPK cards type A®). After an appropriate
drying time (at least 2 hours at room temperature), a small circular disc (3
mm wide) was punched out of the blood spot and immersed in methanol containing
the Internal Standard (safinamide 0.20 μg/mL, 150 μL per sample). The samples
were vortexed for 20 minutes at a moderate speed, the paper was removed from
the solvent, and the extracts were evaporated (+35°C, 5 minutes) under a gentle
stream of nitrogen. After reconstitution in organic solvent, the resulting mixture
was transferred to LC-MS/MS for analysis (ESI positive mode). All the obtained results
for sensitivity, linearity, reproducibility, selectivity and stability at room temperature
fulfil the acceptance criteria described in current international guidelines.
Specifically, no significant matrix effect or carry-over phenomenon was detected
and precision and accuracy results were within ± 15%. Short-term stability was
demonstrated after 69 hours of storage at room temperature and 114 hours at
+4°C. A cross-validation between freshly prepared and overnight drying calibration
curves and QC samples was also performed. Taking into account the practical advantages
of the DBS technique (decreased number of required animals; lower collected
blood volume; easier handling, shipping and storage), this method has been
implemented for routine use in our facility for pre-clinical toxicocinetic/pharmacokinetic
studies.
1927 INTRAVITREOUS ADMINISTRATION IN THE RABBIT
AND MINIPIG.
R. Forster, V. Haag, J. Legrand and B. Palate. CIT, EVREUX Cedex, France.
Intravitreous administration to the eye brings drugs into direct access with the
retina, and there has been much recent interest in the clinical use of this route of administration
in the therapy of macular degeneration and other diseases. For the preclinical
development of these approaches, intravitreous administration to laboratory
animals is required. Over recent years, we have performed several hundred
intravitreous administrations to rabbits, minipigs and other species. The formulations
to be administered must have appropriate characteristics, including attention
to physico-chemical properties such as pH and tonicity. Critical points in the administration
technique concern adequate preparation of the animals, the volume
and rate of administration, needle gauge and sterile technique. Attention should be
given to the frequency of the administration in order to avoid problems of tolerability.
Finally, in a small proportion of administrations, adverse findings such as inflammation
may result in clinical and histopathological findings.
Electroretinography may be used to provide an indication that administration or
reactions to administration do not have an adverse impact on functionality. In this
poster we present the technique of administration that we have used in the rabbit
and minipig, and data on background findings and their frequency. Overall, when
appropriate precautions are taken, this route of administration can be used routinely
for preclinical studies in the rabbit and the minipig.
1928 ASSESSMENT OF COMMON PRECLINICAL VEHICLE
FORMULATIONS USED IN DRUG DISCOVERY BY
MAGNETIC RESONANCE SPECTROSCOPY (MRS).
R. Sriram and S. Liachenko. Pfizer Inc., Groton, CT. Sponsor: M. Paule.
To evaluate the effect of excipients in drug formulations MRS was utilized to monitor
acute changes in creatine(Cr) levels in rats, which under normal physiological
conditions is constant. By monitoring the level of total creatine(tCr) in vivo the potential
untoward effects of seemingly inert vehicles can be measured. MRS was conducted
on male SD rats under isoflurane using a 7T magnet. MRS signal was obtained
from the hippocampus (64 cu mm voxel) by PRESS sequence with VAPOR
water suppression (TE = 16.2 ms, TR = 3 s, NS = 512). After the baseline MRS
scan, one of four different vehicles (2ml/kg) - saline (N = 4), or 1-2-97 (1% 1N
HCl, 2% cremophor, 97% saline, N = 4), or DMA+PG (10% dimethyl acetamide
(DMA), 45% propylene glycol (PG) and 45% saline, N = 9) or nanosuspension
(0.025% sodium laurel sulfate and 2% poly-vinyl pyrrolidone in saline, N=4) was
given subcutaneously, followed immediately by dynamic MRS scans. LCModel was
used for tCr estimation. tCr (sum of phosphocreatine(PCr) and Cr) levels show a