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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15/10/1997 - 08/06/1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP, Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
(1981)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPP 83-3 (Prenatal Developmental Toxicity Study)
Version / remarks:
(1984)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: JMAFF 59 NohSan no. 4200 (1985)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
1-methyl-2-nitro-3-(tetrahydrofuran-3-ylmethyl)guanidine
EC Number:
605-399-0
Cas Number:
165252-70-0
Molecular formula:
C7H14N4O3
IUPAC Name:
1-methyl-2-nitro-3-(tetrahydrofuran-3-ylmethyl)guanidine
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Purity 92.9% + 6.9% water, purity of dried material 99.1%
Batch No. 2200210

Test animals

Species:
rat
Strain:
other: Crj:CD(SD) IGS (SPF)
Details on test animals or test system and environmental conditions:
Source: Charles River Japan, Inc., Kawasaki, Japan
Age/weight at study initiation: 10-12 weeks old, weighing 212.22-269.37 g for females
Number of animals per group: 24 mated females per group. See Table 1
Mating period: 12 – 13 days

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5%
Details on exposure:
Total volume applied: 10 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical results for homogeneity showed concentration being 98.2 to 101%.
Analytical method not reported.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1:1
- Length of cohabitation:
- Proof of pregnancy: sperm in vaginal smear
Duration of treatment / exposure:
Duration of exposure: Rat, day 6-15, post-mating
Post-exposure period: 5 days
Frequency of treatment:
Daily
Duration of test:
Approximately 30 days
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
100 mg/kg/day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
300 mg/kg/day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
1000 mg/kg/day
Basis:
nominal conc.
No. of animals per sex per dose:
24 mated females per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: to find appropriate dose levels for the present study, "a dose finding teratogenicity study of MTI-446 given orally to rats (report no. H-97162)" was performed (dose levels: 30, 100, 300 and 1000 mg/kg). Dams in the 1000 mg/kg group showed decreased bodyweight gains and food consumption in the early period of treatment. In the other groups, there were no treatment-related changes. Therefore, the 1000 mg/kg group was selected as the high dose. Other dose levels were 300 and 100 mg/kg with a common decremntal ratio of about 3.

Examinations

Maternal examinations:
Body weight: Yes, on days 0 and 3 of gestation and then daily from day 5 of gestation until sacrifice.
Food consumption: Yes, on days 0 and 3 of gestation and then daily from day 6 of gestation until sacrifice.
Clinical signs: Yes, at least once daily on non-treatment days and at least twice daily during the treatment period.

Ovaries and uterine content:
Examination of uterine content: Yes, the uterine tract and ovaries were removed and pregnancy was confirmed. If implantations were not visible macroscopically, the uterus was immersed in ammonium sulphate to aid visualisation. Maternal organs of the cranial, thoracic and abdominal cavities, and ovaries (including corpora lutea count) and uteri (implantation site count) were examined macroscopically. The uterine contents were classified as live fetuses, embryo/fetal deaths, placental remnants, early or late resorptions, or macerated fetuses.
Fetal examinations:
General: Fetuses were sexed, examined for external malformations, and weighed.
Skelet: Yes, approximately half of the foetuses from each litter were subjected to skeletal evaluation using a dual staining technique for cartilage and bone and examined for skeletal malformations and variations including counting the number of ossification centers in vertebrae, metacarpals, metatarsals, proximal and medial phalanges.
Soft tissue: Yes, approximately half of the foetuses were examined for soft-tissue malformations and variations by fixation in Bouin’s solution and subsequent micro-dissection of the cranial and abdominal cavities by Wilson’s method and of the thoracic cavity by the method of Nishimura .
Statistics:
Where appropriate, data were analysed for homogeneity of variance using Bartlett’s test followed by one-way ANOVA for homogeneous data. If significant, Dunnett’s test was performed. Non-homogeneous data and percentage/dam data were analysed using the Kruskal-Wallis H-test followed by Dunnett’s test if significant.
Indices:
Not applicable
Historical control data:
Not applicable

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
There were no deaths during the study. With the exception of a single animal treated at 1000mg/kg bw/day that showed transient hypoactivity on days 8 to 10 of gestation, there were no clinical signs of toxicity at any doe level. The body weight gain from day 6 to 11 of the group treated at 1000mg/kg bw/day was significantly reduced by 21% (Table 2). Thereafter, weight gain was not significantly different from the controls and on day 20 of gestation the group mean body weights of all treated groups were not significantly different from control values. The mean food consumption of the group treated at 1000mg/kg bw/day was significantly reduced by 10.5 to 13.0% on days 6, 7 and 9 of gestation. The mean water consumption of this group was significantly increased by 19.8 to 23.8% on days 10 to 12 of gestation. On other occasions during the treatment period the food and water consumption at 1000mg/kg bw/day were comparable to control values. There were no treatment-related effects on food and water consumption in the groups treated at 100 or 300mg/kg bw/day.

Effect levels (maternal animals)

Dose descriptor:
NOEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There were no treatment-related macroscopic findings in the maternal animals at any dose level. Litter parameters as assessed by pregnancy incidence, numbers of corpora lutea, implantations and live fetuses, post-implantation loss, external anomalies, fetal weights and sex ratio, were unaffected by treatment at all dose levels (Table 3). Although pre-implantation loss in the group treated at 1000mg/kg bw/day was high (24.0%) in relation to the control group (9.2%), it was not significantly different from the control value and is considered incidental to treatment with dinotefuran since implantation was complete at the initiation of treatment. The mean number of implantations in the 1000mg/kg bw/day group was slightly lower than, but not significantly different from, the control group as a consequence of higher pre-implantation loss.
There were no external fetal abnormalities in any of the treated or control groups. There were no treatment-related or statistically significant differences between treated and control groups on the incidence and nature of skeletal and visceral abnormalities and variations. No skeletal abnormalities occurred in any group and the incidences of visceral abnormalities, thymic remnant, microphthalmia, ectopic ovary, pyeloectasia, ureteroectasia and left umbilical artery, were similar in all groups (Table 4). Delayed ossification, as assessed by the number of vertebral and phalangeal ossification centers, was not apparent at any dose level.

Effect levels (fetuses)

Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: embryotoxicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Table 2: Group mean body weights and weight gains of pregnant animals

Treatment group

Mean body

Mean body weight gain (g) on days:

Mean body weight

(mg/kg bw/day)

weight on day 6 (g)

6 -11

6 -15

6 -20

on day 20 (g)

0

280

21.9

41.2

107

386

100

281

22.0

40.3

110

390

300

281

22.2

43.0

109

390

1000

276

17.3*

37.3

97

373

* p < 0.05

 

Table 3: Group mean caesarean data

Parameter

0 mg/kg

100mg/kg

300mg/kg

1000mg/kg

No. pregnant / no. mated

22 / 24

22 / 24

20 / 24

20 / 24

No. corpora lutea±SD (mean/dam)

15.6±2.0

16.2±1.6

15.3±2.7

15.7±2.1

No. implantations±SD (mean/dam)

14.2±3.0

14.8±1.6

14.1±3.5

12.2±5.3

Pre-implantation loss (%)

9.2

8.3

10.1

24.0

Total embryofetal loss (%)

5.1

5.1

3.4

3.6

- Implant remnant (%)

0.0

0.0

0.0

0.0

- Retained placenta (%)

4.3

3.7

3.4

2.7

- Early death (%)

0.0

1.1

0.0

0.9

- Late death (%)

0.8

0.0

0.0

0.0

- Macerated fetuses (%)

0.0

0.3b

0.0

0.0

No. live fetuses±SD (mean/dam)

13.5±3.1

14.0±2.1

13.6±3.4

11.8±5.3

Sex ratio (% males)a

53.0

55.7

50.4

42.8

Mean body weight±SD (g) - males

3.73±0.29

3.72±0.19

3.83±0.23

3.71±0.25

Mean body weight±SD (g) - females

3.55±0.23

3.51±0.22

3.65±0.23

3.47±0.35

Live fetuses with external abnormality (%)

0.0

0.0

0.0

0.0

are-calculated by reviewer;

bconjoined twin macerated fetuses

 


Table 4: Group mean skeletal and visceral examination data

Parameter

0 mg/kg

100mg/kg

300mg/kg

1000mg/kg

No. litters examined

22

22

19

20

No. fetuses examined (skeletal)

143

150

132

114

Total no. abnormal fetuses (skeletal):

0

0

0

0

Skeletal variations (mean %)±SD:

Total variations

- cervical rib

- 14thrib

- shortened 13thrib

 

18.4±17.5

1.2±4.0

13.6±16.6

3.6±11.2

 

7.6±12.4

0.0±0.0

7.6±12.4

0.0±0.0

 

10.5±14.9

0.7±2.9

9.1±14.6

0.8±3.3

 

12.1±23.3

0.6±2.5

11.5±23.5

0.0±0.0

Mean no. ossification centres±SD:

- caudal centra

- caudal arches

- forelimb phalanges

- hindlimb phalanges

 

2.7±1.0

0.8±0.5

2.7±1.0

2.5±1.2

 

2.6±0.7

0.8±0.4

2.8±0.5

2.4±1.1

 

2.8±0.8

0.9±0.4

2.8±0.6

2.5±1.2

 

2.7±0.8

0.8±0.3

2.5±1.1

1.9±1.4

No. fetuses examined (visceral)

155

159

140

122

No. abnormal fetuses (visceral):

Total abnormal fetuses (mean %)±SD

- thymic remnant

- microphthalmia

- ectopic ovary

- pyeloectasia

- ureteroectasia

- left umbilical artery

 

5.0±9.0

3.1±7.3

0.6±2.7

0.8±3.6

0.0±0.0

0.0±0.0

1.3±4.3

 

3.5±11.1

3.5±11.1

0.0±0.0

0.0±0.0

0.0±0.0

0.0±0.0

0.0±0.0

 

8.4±22.9

2.7±7.2

0.0±0.0

0.0±0.0

5.7±22.4

5.0±22.4

0.0±0.0

 

5.1±11.9

4.6±11.9

0.0±0.0

0.0±0.0

0.0±0.0

0.0±0.0

0.6±2.5

 

Applicant's summary and conclusion

Conclusions:
The no adverse effect level of dinotefuran was estimated to be 300 mg/kg/d in pregnant females in terms of general toxicological effects and 1000 mg/kg/d in foetuses in terms of embryonic development and teratogenicity.
Executive summary:

There were no premature deaths during the study. With the exception of a single animal treated at 1000 mg/kg bw/day that showed transient hypoactivity on days 8 to 10 of gestation, there were no clinical signs of toxicity at any dose level.

The body weight gain from day 6 to 11 of the group treated at 1000mg/kg bw/day was significantly reduced by 21%. Thereafter, weight gain was not significantly different from the controls. The mean food consumption of the group treated at 1000 mg/kg bw/day was reduced up to day 9 of gestation. The mean water consumption of this group was increased on days 10 to 12 of gestation. There were no treatment-related effects on food and water consumption in the groups treated at 100 or 300mg/kg bw/day.

There were no treatment-related macroscopic findings in the maternal animals at any dose level. Litter parameters were unaffected by treatment at all dose levels. The mean number of implantations in the 1000mg/kg bw/day group was slightly reduced as a consequence of a non-treatment-related higher pre-implantation loss.

There were no external fetal abnormalities in any of the treated or control groups. There were no treatment-related or statistically significant differences between treated and control groups in the incidence and nature of skeletal and visceral abnormalities and variations. Delayed ossification, as assessed by the number of vertebral and phalangeal ossification centers, was not apparent at any dose level.