Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 April 2006 - 06 Aug 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
other: MAFF Japan, 12-Nousan No. 8147, 2-1-18
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent

Test animals

Species:
rabbit
Strain:
other: Japanese White rabbits (Kbl:JW)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Minowa Breeding Centre, KITAYAMA LABES Co., Ltd. (Nagano, Japan)
- Age at study initiation: females: 18 weeks, males: 10-38 months
- Weight at study initiation: 3.00-3.56 kg
- Fasting period before study: No
- Housing: Individually in aluminium cages with wire-mesh floors.
- Diet: Certified solid feed (LRC4, Oriental Yeast Co.,Ltd., Tokyo, Japan), ad libitum
- Water: Filtered and sterilised well water, ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23±2
- Humidity (%): 55±15
- Air changes (per hr): at least 10/hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2006-05-15 To: 2006-06-12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
1%
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
For each dose level, dosing solution was prepared by suspending a specified amount of the test substance in an aqueous solution of 1% sodium carboxymethylcellulose (CMC). When the amount of the test substance required for each dose level was calculated, correction by the purity of the test substance was excluded.
Dosing solutions were prepared 4 times at weekly intervals during the study period based on the result of a stability study for the test substance in the 1% CMC (lET 05-5013). A volume of 4000 mL of the dosing solution was prepared for each dose group at every preparation.

VEHICLE
- Amount of vehicle (if gavage): 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dosing solutions prepared for each dose level were analyzed for concentration of the test substance before use and were verified that the test substance has been presented at the target concentrations in each dosing solution. At the first preparation, dosing solutions for the low- and high-dose groups were further analyzed for homogeneity of the test substance.

The samples were analyzed by the methods that demonstrated analytical validity by a validation study for analytical method of the test substance in 1% CMC solution (IET 05-5015).
The standard solutions of active ingredient were prepared as follows: An aliquot (50.25 mg, corrected for purity) of analytical standard was accurately weighed into a 50 mL volumetric flask and dissolved in acetonitrile to make a 1000 mg/L solution. A 50 mg/L solution was prepared by diluting the 1000 mg/L solution with acetonitrile-water-formic acid (80:20:0.1, v/v/v). The standard solutions of 0.04, 0.2, 0.5, 1 and 2 mg/L concentrations were then prepared by diluting the 50 mg/L solution with acetonitrile-water-formic acid (80:20:0.1, v/v/v).
The each standard solution was injected into HPLC (LC-2000 Plus (Jasco Corporation, Tokyo, Japan).
Details on mating procedure:
- Impregnation procedure: artificial insemination
Duration of treatment / exposure:
22 days; from Day 6-27 of gestation.
Frequency of treatment:
Daily
Duration of test:
Females were euthanized on Day 28 of gestation.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: A dose-range finding study (IET 04-0148) was performed with 8 rabbits/group, administered 0, 100, 300 and 1000 mg/kg bw/d during Day 6-27 of gestation.
As for maternal animals, body weight gains and food consumption in the 1000 mg/kg group were slightly but not statistically significant lower on and after Day 24 of gestation. Necropsy revealed hydrothorax and pale in colour of the heart in two maternal rabbits out of seven in the 1000 mg/kg group. In the 100 and 300 mg/kg groups, no treatment-related effects were observed in maternal rabbits.
As for fetuses, mean fetal weights of both sexes in the 1000 mg/kg group were slightly but not statistically significant lower than those in the control group. No treatment-related fetal effects were observed in the 100 and 300 mg/kg groups.
The changes noted in the preliminary study were slight, and were not determined to be the treatment-related effects. Therefore, a dose level of 1000 mg/kg/day was selected for the high-dose level of this study. Dose levels of 300 and 100 mg/kg/day were selected for the middle- and low-dose levels, respectively, to set the common ratio between dosages to approximately 3.



Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily during dosing period, otherwise daily
- Cage side observations included: Clinical signs, mortality

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: The body weights were recorded on Days 0, 6, 9, 12, 15, 18, 21, 24, 27 and 28 of gestation.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: The amount of food supplied or unconsumed was determined on Days 0, 3, 6, 9, 12, 15, 18, 21, 24, 27 and 28 of gestation.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #28
- Organs examined: Ovaries and uteri.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes [all per litter]
- Soft tissue examinations: Yes [all per litter]
- Skeletal examinations: Yes [all per litter]
- Head examinations: Yes [all per litter]
Statistics:
The following statistical tests were used to estimate significance of differences between the control group and the treated groups. The data on body weights, adjusted body weights, body weight gains, food consumption, weights of gravid uteri of maternal rabbits, numbers of corpora lutea, implants, and live fetuses, and weights of fetuses and placentas were evaluated as follows: Equality of variances was first evaluated by Bartlett’s test (alpha = 0.05). When group variances were homogeneous, Dunnett’s multiple comparison test (alpha = 0.05 or 0.01) was performed to detect any statistically significant differences between the treated groups and their corresponding controls. When Bartlett’s test indicated that the variances were not homogeneous, Dunnett-type nonparametric multiple comparison test (alpha = 0.05 or 0.01) was performed to detect any statistically significant differences between the treated groups and their corresponding controls. As for the data on the percent incidences of preimplantation losses and percent incidences of resorptions and fetal deaths, Dunnett-type nonparametric multiple comparison tests (alpha = 0.05 or 0.01) were performed to detect any statistically significant differences between the treated groups and theft corresponding controls. As for the data on the incidences of clinical and gross pathological findings in maternal rabbits, incidences of maternal rabbits having fetuses with malformations or variations, incidences of fetal malformations or variations, and fetal sex ratio, chi-square test for independence (alpha = 0.05, 0.01, or 0.001) was used when all expected values of control and treated groups were 5 or more, and if not, Fisher’s exact probability test (alpha = 0.05, 0.01, or 0.001) was used.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
Adverse effects on maternal rabbits were observed in the 1000 mg/kg bw/d group, where statistically significant decreases in food consumption on and after Day 15 of gestation, body weight on Day 21 of gestation, and body weight gain during Days 6-21 of gestation were noted (see Table M1). Moreover, some females whose decrease in food consumption was found on relatively earlier gestation day (Day 9 through 15) than the others in the same group showed signs of abortion, suggesting that abortion is a secondary effect of treatment-related decrease in food consumption.
In addition, necropsy of females survived to the day of cesarean section revealed enlargement, pale in colour, and accentuated lobular pattern of the liver in the group with incidences that were significantly higher than those in the control group. As liver enlargement was as well seen in the two generation study and the oral repeated dose toxicity study where these gross pathological findings were not accompanied by histopathological effects, these liver effects can be considered to be an adaptive response to the test substance. No treatment-related adverse effects on maternal animals were noted in the 100 and 300 mg/kg bw/d groups.
Examination of the ovary and uterus revealed no treatment-related effects in any of the treated groups. No statistically significant differences were noted in mean gravid uterine weights, mean numbers of corpora lutea and implants, and percent incidences of pre-implantation losses between the control group and the treated groups.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
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:
In the 1000 mg/ kg bw/d group, a statistically significant decrease in fetal body weight in the presence of severe maternal toxicity was observed (see Table F1), compared to that in the control group. The mean placental weight was decreased in the 1000 mg/kg bw/d group with statistical significance (see Table F1).
Both these effects were considered to be treatment-related alterations. No treatment-related effects on fetal body weights and placental weights were noted in the 100 and 300 mg/kg bw/d groups. Numbers of live fetuses, percent incidences of resorptions and fetal deaths, and sex ratio in all the treated groups were not affected by administration of the test substance. External, visceral, and skeletal examination of fetuses revealed no treatment-related increases in incidences of malformations and variations in any of the treated groups.

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: teratogenicity
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: fetotoxicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Table M1: Group mean body weight gains (g) of maternal rabbits

Dose level

(mg/kg bw/d)

Body weight gain during gestation days:

6-9

6-12

6-15

6-18

6-21

6-24

6-27

6-28

0

0

30

102

123

170

212

250

265

100

10

48

111

125

166

212

234

242

300

-3

32

90

104

131

181

222

246

1000

1

21

65

38

18*

135

140

144

* p< 0.05

Table F1: Fetal and Placental weights

Dose level

(mg/kg bw/d)

No. of females with live fetuses

Fetal weight (g; mean ± SD)

Placental weight

(mg; mean ± SD)

male

female

0

21

39.3 ±

4.8

39.2 ±

5.1

5360 ±

586

100

23

36.0 ±

4.9

35.8 ±

4.6

5096 ±

800

300

25

37.3 ±

5.7

36.1 ±

4.9

5246 ±

923

1000

19

32.0 ±

5.9**

31.3 ±

6.1**

4727 ±

682*

* p< 0.05; ** p<0.01

Applicant's summary and conclusion