ReproTox-CaF 14_SBR

Reproductive and Developmental Toxicology Assessment
of Oral Dietary Calcium Formate in Yucatan Miniature Swine
Timothy J. Madsen1, David W. Hobson2, Catherine Selby1, G.P. Georges4, Robert P. Hanzlik3, Derek Brocksmith1, Alain Stricker-Krongrad1, Jason Liu1, Guy F. Bouchard1*
1Sinclair Research Center, LLC; 2LoneStar Pharm Tox, LLC; 3University of Kansas, Lawrence, KS, 4#Nephro-Tech 1, LLC, Shawnee, KS *SOT Member Sponsor; #Study Sponsor
ABSTRACT
OBJECTIVE/RATIONALE
RESULTS (CONTINUED)
DISCUSSION
Rationale: Calcium Formate (CaF) is being considered as a
dietary calcium supplement.
Assess the reproductive/developmental toxicity of CaF using the
miniature pig model when administered orally via food
consumption. .
Figure 1. Piglet Body Weight by Day (kg)
Decreased food intake (P≤0.01) was noted in both genders of
breeding mates for high dose animals during premating but not
gestational periods. The palatability of CaF diets might be lower
than control diet in pigs. Consumption in control animals was not
affected.
Experimental Procedures: Sixty (30 male and 30 female) sexually
mature Yucatan miniature swine were gender paired for breeding
and randomized into 3 groups of 10 pairs receiving untreated
control, low dose (2.25% CaF), and high dose (4.5% CaF) in the
daily diet. Standard reproductive and developmental variables
were assessed, including clinical, gross, and microscopic
pathology (parents and piglets).
Results: Female fertility index/group ranged from 80-100%. Total
fetuses delivered were 174 with 170 livebirths. Piglet viability was
good. Length of gestation, birth weights and body weight change
were comparable. Litter size was robust (range 5.4-7.1) for all
groups.
CaF supplementation was associated with decreased food intake
in both parents in the high dose group and with decreased weight
gain in both parents during the premating phase of the study.
These between group differences persisted through the gestational
phase for the females, but there was not a statistically significant
increase in the magnitude of these differences over time.
CaF supplementation was also associated with increased serum
calcium levels in both parents. Although statistically significant, the
magnitudes of these differences were small, and well within the
normal physiologic ranges.
There were no significant findings of developmental/reproductive
toxicity related to dietary exposure to CaF.
Conclusion(s): Decreased food intake, decreased weight gain
during premating phase, and increased serum calcium were
associated with oral dietary CaF administration. These findings
were consistent with previous research, and/or represented
expected responses to the administration of oral CaF
supplementation.
INTRODUCTION
Jorgensen (1998) published one of the first accounts on use of
minipigs in reproductive toxicology assessments. van der Laan et
al. (2010) recently reviewed the subject of regulatory acceptability
of minipigs for a wide range of toxicology studies, including
reproductive/fertility, teratology, and developmental toxicology.
Miniswine use in reproductive toxicology studies has many years of
favorable history. Miniswine are useful alternatives to the most
commonly used nonrodent, the rabbit. The Yucatan miniswine is a
popular, docile lineage of miniswine which is easily hand mated.
Calcium Formate (CaF) is being
considered as a dietary calcium
supplement and is under regulatory
development. Because CaF metabolism
by miniature swine is more similar to the
human than other species commonly used
for reproductive/developmental studies
(rat and rabbit), the miniature swine was
the model of choice for this study.
1.800
EXPERIMENTAL PROCEDURES
1.600
Sixty (30 male and 30 female) sexually mature Yucatan miniature
swine were gender paired for breeding (hand mating) and
randomized into 3 groups of 10 pairs receiving untreated control,
low dose (2.25% CaF), and high dose (4.5% CaF) in the daily diet.
Doses were selected based on the results of the 14-day MTD
study. CaF was placed in the powdered/granular diet (HarlanTeklad Certified Miniswine Diet, 3% body weight/d) for up to 182
days from prior to mating until necropsy. Age range at mating was
9.6-12.7 months. This study was up to 214 days in length.
Standard reproductive and developmental variables were
assessed, including clinical, gross, and microscopic pathology
(parents and piglets). Plasma and urine samples were collected
and analyzed by the Megazyme Formate Microplate Kit (K-Form,
Megazyme International, Ireland) with appropriate matrix
standards. The limit of quantitation of this assay is stated as 1 mM
formate.
1.400
Body Weight (kg)
Objective: Assess the reproductive/developmental toxicity of CaF
using the miniature pig model when administered orally via food
consumption.
2.000
Control F
1.200
Control M
1.000
Low Dose F
0.800
Low Dose M
0.600
High Dose F
High Dose M
0.400
0.200
0.000
Day 0
Day 1
Day 7
Time Point (Day of Birth = Day 0)
Table 1. Group Mean (±SD) Piglet Body Weight by Gender,
Day 0, Day 1, Day 7
RESULTS
Female fertility index/group ranged from 80-100%. Offspring
delivered naturally totaled 174 piglets with 170 livebirths. Viability
was good. Length of gestation, birth weights and body weight
change were comparable. Litter size was robust (range 5.4-7.1) for
all groups, mirroring the normal litter size (range 4.9-7.4, avg 5.8)
for this lineage.
Over the course of the pre-mating phase, there was a statistically
significant main effect of dose found for male animals, seen as a
decrease in body weight in the high dose group (4.50%) relative to
the corresponding untreated controls (P < 0.05). For female
animals during the premating period, there was a statistically
significant dose group by time interaction (P < 0.01). No maternal
dose by time interaction at P < 0.05 from RMANCOVA analysis
was noted for piglet body weights. Decreases in parental body
weights were significant in the low dose group (2.25%) during preconception weeks P3, P5, P6 and P7, and in the high dose group
for weeks P2 –P8 when compared with controls. There were no
statistically significant differences found between treated and
control females during the gestation phase.
Decreased weight gain during mating phase was recorded for high
dose animals. This is an expected outcome in the face of
decreased food intake.
The increased serum calcium and lowered inorganic phosphorus
observed in this study is expected since the dietary intake of
calcium was elevated (Reinhart & Mahan, 1986). Despite lowered
serum phosphorus in high dose adults, no evidence of negative
bone changes by either histology or radiology were recorded.
Residual formate was noted in urine of 3 of 8 high dose females
but no residual formate was found in plasma of parents during this
study.
For adult/parental animals, the no observed effect level (NOEL) for
oral calcium formate dietary supplementation was not determined.
The no observed adverse effect level (NOAEL) was determined to
be greater than 4.5%. For offspring, the NOEL related maternal
exposure to dietary calcium formate was determined to be greater
than 4.5% through up to 7 days of life.
CONCLUSION(S)
Decreased food intake, decreased weight gain during premating
phase, and increased serum calcium were associated with oral
dietary CaF administration. These findings were consistent with
previous research, and/or represented expected responses to the
administration of oral CaF supplementation.
REFERENCES
Table 2. Litter Size and Offspring Viability Summary
CaF supplementation was associated with decreased food intake
in both parents in the high dose group during the premating phase
of the study. These between group differences persisted through
the gestational phase for the females, but there was not a
statistically significant increase in the magnitude of these
differences over time.
1. Jorgensen KD (1998). Minipig in reproductive toxicology.
Scand J. of Lab An Sci 25 (Suppl. 1), 63-75.
2. van der Laan J-W, Brightwell J, McAnulty P, Ratky J, and Stark
C (2010). Regulatory acceptability of the minipig in the
development of pharmaceuticals, chemicals and other products,
Journal of Pharmacological and Toxicological Methods. 62: 184195.
3. Reinhart G.A. and Mahan D.C. (1986). Effect of Various
Calcium:Phosphorus Ratios at Low and High Dietary
Phosphorus for Starter, Grower and Finishing Swine. J ANIM
SCI Vol 63:457-466.
http://www.journalofanimalscience.org/content/63/2/457
CaF supplementation was associated with increased serum
calcium levels in both parents. Although statistically significant, the
magnitudes of these differences were small, and well within the
normal physiologic ranges.
There were no significant findings of developmental/reproductive
toxicity related to dietary exposure to CaF.
There were no findings of toxicity in the offspring related to
maternal exposure to dietary CaF after many detailed
assessments.
www.sinclairbioresources.com
aFor
calculation purposes, “Day 1” indicates animals that survived at least 24 hours