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| Review of Zentek paper |
Posted: Thursday, November 20, 2008 2:11 pm
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Review of Zantek paper
At the request of Monsanto, I, James C. Lamb, IV, Ph.D., DABT, ATS,
have completed a preliminary review of the report “Biological effects
of transgenic maize NK603xMON810 fed in long term reproduction studies
in mice” by Dr. A Velimirov, Dr. C. Binter, and Univ. Prof. Dr. J.
Zentek. I have focused my efforts on the multigeneration (MG) study and
the reproductive assessment by continuous breeding (RACB) study. The
RACB study design was developed by me at the U.S. National Toxicology
Program (NTP) in the early 1980s to improve the assessment of fertility
in rodent toxicology studies. Work on this protocol was continued by
Dr. Richard Morrissey and then by Dr. Robert Chapin of the NTP. The
RACB has the potential to test certain biological indices that the MG
study cannot address. The authors of this report made modifications to
the RACB study design, but the basic elements remain the same. Male and
female mice were divided into control and test groups and were house as
breeding pairs for about 20 weeks. Each male and female pairing
produced litters continuously through the cohabitation period, which
allowed the pairs to produce as many as 4 litters per pair. As we
originally developed this protocol at NTP, an effect on reproduction
would be followed by a cross-over mating to help determine whether the
effects might be attributed to males, to females or to both genders.
That cross-over mating was not performed in this study.
The study report describes the design, conduct and interpretation of
the study. My review is limited to the information provided in the
report. The design is appropriate for the hypothesis that is being
tested. The RACB study design can generate a large amount of data and
analyses, but the report is not sufficiently detailed to interpret
fully the study. Nevertheless, there are some significant issues raised
by the results as presented and the interpretation that must be
discussed. I have found already some significant errors in the data
calculations that led to the important misinterpretations of the
findings by the authors. Unfortunately, the study does not describe a
quality assurance process that should have prevented these errors.
Errors are clear in two of the most important tables in the report
(Table 36 and Table 59). These errors directly impact the
interpretation of the MG and the RACB studies.
The authors analyzed the MG study in an unconventional manner. The
report is not clear whether the statistical tests and evaluation
methods were defined before or after the data were developed. The
methods were not consistent with the methods that we applied at the
NTP. One important point is that the litter size was analyzed using the
breeding pair as the denominator rather than the delivering breeding
pair. The number of deliveries in the F3 (3rd generation) matings
dropped off for both control and GM test groups and there was no
explanation for this decline (see second row of Table 36). This should
have been addressed. The decline in fertility resulted in an apparent
decrease in litter size that is an artifact of the denominator that
they used (see fourth row Table 36). This might best be illustrated by
a fictitious example. If one cohabited 20 pairs and only one pair
delivered a litter of 10 animals, the mean litter size would be 10; not
0.5 as would have been reported by the authors. If the proper
denominator was used, the litter size did not change for either group
compared to the other generations. The authors did not report the
number of litters analyzed at weaning, so we cannot determine whether
or not a similar error was made in the number of weanings/pair. Without
the individual animal data, we cannot test the statistical findings
either. In the end, the authors did not see a treatment effect in this
study, but the computational errors raise concern about other
examinations in such an important data set.
The authors also divided the MG study litters into less than 8 and
greater than 8 pups per litter. I have never seen this approach to
litter analysis and wonder why and when they chose to make this
comparison. No rationale is provided for this approach. These data are
very difficult to interpret and have dubious scientific meaning. The
various charts lead to a good deal of discussion, but do not really aid
in the data interpretation.
The RACB study has similar issues that do affect the authors’
interpretation of their study. In Table 59 they report data similar to
the MG study. Instead of multiple generations, the table shows multiple
litters across the study. One odd point is that they had one pair that
never delivered any litters. There is no explanation for this, and that
is inconsistent with the MG study. They probably should have analyzed
the data with and without this pair in the study because it is not
likely related to the treatment. This amplifies the error of using the
wrong denominator because the litter size is understated for the
treated group throughout the study because they again divided by the
pairs cohabited without considering the actual litter size for those
mated pairs that actually delivered litters. They have made the same
mistake on the number of pups at birth per pair and on number of pups
at weaning per pair.
The authors’ computational approach results in some key misstatements
in Table 59. In 3rd litter for the GM group the mean number of pups per
litter is incorrect and should be 9.41, not 9.06. For the 4th litter,
the GM group should be 8.65 pups per litter, not 7.21. Similar mistakes
are presented for the pups weaned and the numbers for the GM are
consistently understated. For example, the 4th litter had an average of
9.11 weaned pups per litter, not 7.21 and the 3rd litter had 12.18
weaned pups per litter not 9.06.
These two tables illustrate that the computational errors resulted in
an overstatement of the differences between the GM and the ISO groups.
No historical control data have been presented or discussed. Such data
could be very important in the interpretation of such differences, to
the extent that any significant differences remain. The specific
details of how computations were made should have been included in the
report. It is not possible to reconstruct all of the data, but the ones
that we have investigated indicted errors in the authors’ methods and
therefore interpretations. The statistical methods and presentation
should have been designed prior to the conduct of the experiments, and
it is not clear whether or not this was done. The statistics cannot be
tested appropriately without the individual animal data. The
computational errors in such critical tables (Tables 36 and 59) raise
serious questions about the other data in the report and the quality
assurance methods that were or should have been applied before the
conclusions were drafted and the report was released. When properly
analyzed, these data do not appear to support an effect on fertility or
reproduction from consumption of GM corn.
James C. Lamb, IV, Ph.D., DABT, ATS
Executive Vice President
The Weinberg Group
November 19, 2008
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