The Open Plant Science Journal, 2009, 3, 49-53

49Open AccessLongterm Biosafety Assessment of A Genetically Modified (GM) Plant:
The Genetically Modified (GM) Insect-Resistant Bt11 Corn Does Not
Affect the Performance of Multi-Generations or Life Span of Mice
Yoshihiko Haryu1, Yoko Taguchi1, Eisui Itakura1, Osamu Mikami2, Katsuhiro Miura2,
Takakiyo Saeki2 and Yasuyuki Nakajima2,*1

Animal Care, National Institute of Animal Health, Kannondai, Tsukuba 305-0856, Japan

2

Team for Safety Research, National Institute of Animal Health, Kannondai, Tsukuba 305-0856, Japan
Abstract: The longterm performance of multi-generations or life span was assessed using genetically modified (GM)insect-resistant Bt11 corn. Diet containing 68% of GM Bt11 or non-Bt isoline with sufficient nutrient composition
was fed to male and female ICR mice through 5 generations. The results of growth, mating, gestation, milking periods,reproduction and life span were not different between the GM Bt11 and non-Bt fed groups. The percentage of embryonic
death, litter size, newborn sex ratio and body weight (21-60 days after birth) were not different between these groups. Thelife span of the third-generation mice did not differ over 1,072 days of observation. In addition, there was a tendency for aweight decrease among each group as the generations progressed, but there was no significant difference in performance
among each group in each generation of mice.

Key Words: Genetically modified, corn, biosafety, longterm performance.
INTRODUCTION
Genetically modified (GM) plant products are being
widely used now in the farm industry, and thus by consumers. One of the keys to acceptance of use of GM is “substantial equivalence” of the products [1-11]. This means that
there is no change of the products except for GM gene andits subsequent effects, while most of consumers use GM
products daily and through subsequent generations without
making any sure reliable information about their bio-safety.
The consumers still have vague fairs of unexpected effects
of using GM products, and want to get conformation about
the safety of GM products from scientific journals. However, most of the animal performance were done in shortterm and case by case manner. There have been numberof papers on animal experiments read in academic meetingsor conferences, also many reports including reviews on
bio-safety assessment of GM products have been publishedin scientific journals [1-8, 12]. However, there still havebeen a few longterm biosafety assessments conducted using
multi-generations or life span of animals. The present study,
therefore, was a report on longterm biosafety assessment
through five generations or whole life span of mice.

basic diet used was a commercially available rodent dietfor breeding “CE-2, CLEA Jpn” containing soybean,
wheat, grain sorghum, fish meal and corn. The GM Bt11(Bt) corn (N58-D1 Lot #2608611) or non-Bt isoline(NX5768 Lot #2608612) purchased from Novartis Seed, Inc.
(Greensboro, NC) was substituted for corn ingredients. The
applied diets were composed of 12.8% of crude protein,
5.1% crude lipid, 67.6% nitrogen-free extract, 0.9% calcium
and 0.75% phosphate (Table 1).
Table 1.

Components of the Diet Fed to Mice. Corn Starch
Used was Derived from non-Bt Corn
Materials

Amount (%)

Corn (Bt11 or non-Bt)

67.85

Milk casein

8.4DL-methionine

0.05Corn oil (Bt11 or non-Bt)

3.0

Corn starch*

10.0

Cellulose powder

5.0

AIN-76 mixed mineral

1.0

LONGTERM BIOSAFETY ASSESSMENT

AIN-76A vitamins

0.2

DietCholine bitartrate

1.0

A suitable diet for longterm performance assessment was
based on AIN-93 composed of 67.85% of maize [13]. The

Calcium carbonate

3.5

Mice and Sampling*Address correspondence to this author at the Team for Safety Research,
National Institute of Animal Health, Kannondai, Tsukuba, 305-0856, Japan;
E-mail: yasunk@affrc.go.jp
1874-2947/09

To observe the clinical performance of the mice, a dietcontaining GM Bt or non-Bt were fed up to 5 generations[9-11]. Body weight, temp, feed intake, feces check, and2009 Bentham Open

50 The Open Plant Science Journal, 2009, Volume 3

Haryu et al.

clinical performance were observed during the experiments.
The mice were divided into GM Bt fed female or male andnon-Bt fed female or male groups. Four mice were placed in
one cage, and the room temp was maintained at 25 C, with a
12 hr light/dark cycle. The bedding was changed twice aweek. Water was available ad libitum. The experiments wereconducted under the Guideline for the Use of Laboratory
Animals, National Institute of Animal Health.
Generation 0 (F0) consisted of 4 week-old ICR mice of 4groups (GM Bt and non Bt; 31 females and 16 males each).
They were fed either the GM Bt11 or non-Bt. Between 60
and 70 days old, they were cross-bred, and the rate ofvaginal plug formation, delivery rate and period, number offetuses, rate of male and female fetuses born, and weaning
period were checked. After the end of the weaning, the F0
mice were sacrificed for autopsy (Table 2).
The derived fetuses were labeled as F1, and similar
subchronic performance from breeding to breeding of next
Table 2.

generation was done in F1 through F3 (Table 2). To examine
the effects of GM in pregnant mice and their fetuses, the F4
generation derived from F3 (16 pregnant mice in the GM Btgroup and 22 pregnant mice in the non-Bt group) wascrossed and the pregnant mice were sacrificed after 18 daysof plug formation (1 day before delivery). The numberof fetuses, and presence of abnormalities in the fetuses werechecked, and the sizes and weights of the placenta andovaries were measured to compare between the GM Bt group
and non-Bt (Table 3). In addition, performance during
life span in the F3 was assessed, and a pathological examination conducted at the end of the span (Table 4, Fig.4). The
significance was assessed using the Student’s t-test.
Feed Intake and Body Weight
In the F0 and F1 generations, preference for the GM Bt
feed did not differ with that of non-Bt, as suggested by feedintake. There also were no significant differences in gain ofbody weight (Figs. 1, 2). Feed intake varied individually, but

Reproduction in F0 and F1Group

Selected

WeanedF0CrossedPlugBirthTotalFemale

MaleTotal

FemaleMale

non-Bt312927216

99117

207

98

109

GM Bt

3123

27216

108

108

212

106

106

Selected

WeanedF1

CrossedPlugBirthTotalFemale

MaleTotal

FemaleMale

non-Bt46

10538

211736

2115

GM Bt

49

2117107

52

55

10651

55

Table 3.

Table 4.

Weight of Fetus, Ovary, Placenta, Uterus and Number of Fetuses in 18 Days of Pregnancy (F4)F4 mice

Gm Bt (n=16)

non-Bt (n=22)

Body weight (g56.53  6.33

54.24  4.16

Uterus (g)19.60  4.63

19.66  3.16

Fetus (g)1.22  0.18

1.26  0.13

Placenta (mg)75.70  15.07

72.99  15.54

Ovary (mg)10.87  4.64

11.50  3.36

Live fetus (n)12.75  3.09

12.45  2.24

Died (n)0.31  0.60

0.80  1.20

Absorbed (n)0.75  0.93

0.70  1.03

Lifespan in Days of the F3 Generation Mice. (f): Female, (m): Male
NumberMean

S.D.

LongestShortestMedianVariance

non GM (f)

44694

154

981

345

687

23688

GM (f)

45677

188

1072

269

653

35181

non GM (m)

45

675

158

1014

252

673

23688

GM (m)

45656

148

1011

351

681

35181

Longterm Biosafety Assessment of A Genetically Modified (GM) Plant

The Open Plant Science Journal, 2009, Volume 3

51

Feed intake g/day/head

76
5
non-Bt (f)4Bt (f)3

non-Bt (m)Bt (m)

2105

6

7

8

Age in weeks

Fig. (1). Feed intake in the F1 mice.

the difference was not significant between the GM Bt andnon-Bt groups. In addition, the mean body weight of the F3
mice in the GM Bt group from 1 to 9 weeks was higher thanin the non-Bt with significance (p<0.05), while feed intake
was not significantly different in either of the two groups.

Fig. (3) compares the body weight of the 8 week-old
mice in each generation. There was a tendency for a weightdecrease as the generations progressed, but in each generation, also there were no significant difference among eachgroup of mice. The decrease would be due to feeding ahigher contents of corn, 68%, to multi-generations rather
than the feeding of GM materials. In addition, the bodyweight of the mice significantly increased in the GM Btgroup in F3; however no conclusion was made from thisbecause similar results were not oBserved in F1 and F2.
Breeding Performance
The results of pregnancy and birthing such as the numberof delivered fetuses or rate of males and females did not differ between the GM Bt and non-Bt groups (Table 2).
In the F0, there were 27/31 delivery mice in both groups,
and the delivered fetuses, labeled as F1, consisted of 176females and 202 males in the GM Bt group, and 172 females
and 175 males in the non-Bt, without significant difference
in mean number of fetuses per mother and rates of males andfemales between the two groups. Of them, total of 212 in theGM Bt, and 207 in the non-Bt were weaned (Table 2). In theF1, the rate of plug formation and birthrate varied, with 17
delivery mice in the GM Bt group and 5 in the non-Bt. TheFig. (2). Body weight in the F1 mice.

Body weight (g)50403020

F1F2F3100
non-Bt (f) Bt (f) non-Bt (m) Bt (m)Fig. (3). Body weight of 8 week-old mice.

52 The Open Plant Science Journal, 2009, Volume 3Haryu et al.

Life span (Suvival rate)

（%）
100

Bt♀

80C ♀
Bt♂

60C ♂

40
2001591317Month21252933Fig. (4). Life span in months of the F3 mice.

fetuses, labeled as F2, consisted of 51 females and 55 males
in the GM Bt group and 21 females and 25 males in the nonBt, without significant difference in mean number and rate ofmale and female of fetuses between the two groups. In theF3, there were 52 mice which delivered in the GM Bt and 21
in the non-Bt, while of the delivered fetuses, 199 werefemale and 231 male in the GM Bt, and 93 were female and81 male in the non-Bt, without significant difference in mean
number and rate of female and male fetuses between bothgroups.
The breeding performance in the F4 generation, assuggested by rates of pregnancy and plug formation between
the GM Bt and non-Bt groups, was not significantly different. Total vaginal weight, mean weight of fetuses, mean
placental weight, mean ovarian weight, numbers of live
fetuses, and dead/absorbed fetuses per pregnant mice werenot significantly different between the 18 day pregnant miceof the GM Bt and non-Bt groups (Table 3).
Lifespan Assessment
With regard to the lifespan performance of the F3, thelongest was survival time was 1,072 days (37 months) (Table4). There was no significant difference by statistical analysis
with Student’s t test. The number of weeks in the GM Bt andnon-Bt groups (males and females) survived are also shownin Fig. (4). Cause of death include senility, tumor, malnutrition, internal bleeding, anemia, uremia or ileus. Tumors weredetected in 19/45 females and 13/45 males in the GM Btgroup, and 15/44 females and 15/45 males in the non-Bt
with no significant difference. Histopathological diagnosesof these tumors included lung adenocarcinoma, hepatocytetumor, malignant lymphoma, histiocytic sarcoma, and interstitial cell tumor. No significant difference was shown
between the occurrence of the causes of death, including
tumors, and each group. These results showed that thefeeding of GM Bt11 did not affect life span of the F3 andbreeding performance of the F4 mice.
CONCLUSIONSThe present results of longterm biosafety assessment
showed no significant difference between the GM Bt andnon-Bt fed mice in whole life span, and breeding, birthing
and growth of mice through multi-generation. Pathologicalexamination after the death of these mice supported nodifference between the GM Bt and non-Bt groups. These
results were one of reinforcement on biosafety of GM
products for consumers who had vague fair for unexpected
effect during the use of GM products and want to havecertification. In addition, a weight decrease was detected ineach group of mice as the generations progressed, which
would be due to feeding a higher contents of corn, 68%, tomulti-generations.
Certification of the biosafety of GM products needs toappear in scientific journals. Number of animal performances have been done in short term and case by case manneras depending on its cost performance to maintain animals
and institutional facilities combined with guidelines of safetyassessments [1-11, 13]. Such papers have been read in
academic meetings or conferences. Though the sections ofintroduction, materials and methods, results and discussion
are nearly the same except for name of used GM products,
such information has become to publish in many scientificjournals [1-6, 7, 8]. However longterm assessments throughthe animal life or multi-generations still have been a small[12].
Ubiquitous plant and enterobacterial gene fragments
were ingested then transferred into the bodies through GI
contents [14-17], then to the livers of suckling mice throughtheir mother’s milk [18], while the GM cry1Ab genefragment was not detected due to a few number of copy
using PCR. Combined with the present results we did not
find out any deleterious effects from the chronic ingestionof the GM Bt11 corn. No evidence has been suggested thatplant-derived genes are expressed in the mammalian genes.
Therefore, routine use of GM Bt11 corn would not be amatter of concern.
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Received: March 19, 2009

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Accepted: April 22, 2009

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