Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April to October 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted in accordance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
(2001)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
(1998)
Qualifier:
according to
Guideline:
other: Japanese Guidelines (Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Teratology (2-1-18), 2000)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: Liquid
- Storage condition of test material: In the dark at room temperature (20 ± 5 °C)

Test animals

Species:
rabbit
Strain:
Himalayan
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Germany, Niederlassung Kisslegg, Germany
- Age at study initiation: 25 - 36 weeks
- Weight at study initiation: 2265 to 3375 g
- Housing: individually in stainless steel cages equipped with an automatic cleaning system; a piece of wood and a haystick were also provided for environmental enrichment.
- Diet (e.g. ad libitum): pelleted standard Kliba Nafag 3418 (batch no. 70/07) rabbit maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst/Switzerland) was available ad libitum
- Water (e.g. ad libitum): Community tap-water from Füllinsdorf was available ad libitum in water bottles
- Acclimation period: 7 days, under test conditions and after health examination

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3 °C
- Humidity (%): 30 - 70%
- Temperature and humidity values outside of the ranges given above occasionally occurred, usually following room cleaning; they were considered not to have any influence on the study.
- Air changes (per hr): 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hrs/ 12 hrs

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% CMC and 0.1% Tween 80 in highly purified water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS
The dose formulations were prepared weekly. The test material was weighed into a glass beaker on a tarred precision balance and approximately 80% of the vehicle (0.5% CMC and highly purified water) were added (w/v). Using an appropriate homogenizer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle (0.5% CMC and highly purified water) was added. Separate formulations were prepared for each concentration. The correct amount of 0.1% Tween 80 was added each morning to the daily portions of the dose formulations. Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer. Dose formulations in glass beakers were stored in the refrigerator (2 - 8 °C) where they were stable for 8 days.

VEHICLE
- Identification: 0.5% CMC, 0.1% Tween 80, highly purified water
- Source: Fluka Chemie GmbH, 9471 Buchs / Switzerland
- Batch Number: 1367388 (CMC), 34707017 (Tween 80)
- Storage Conditions: Room temperature (20 ± 5 °C)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical monitoring:
On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 2 g of each concentration were taken from the middle only to confirm stability (8 days). During the last week of the treatment, samples were taken again as above. The aliquots for analysis of dose formulations were frozen and stored at -20 ± 5 °C until analysis.
The samples were analyzed by gas chromatography coupled to a flame ionization detector following an analytical procedure provided by the Sponsor and adapted at the testing facilities. The test item was used as the analytical standard.

Results of the analytical monitoring:
The identity of the test item was confirmed by its retention time. The test item content of 13 out of 18 samples was found to be within the accepted range of ±20% of the nominal content. In addition, the homogenous distribution of the test item in 0.5% CMC/0.1% Tween 80 was demonstrated for 4 out of 6 groups. It is indicated that homogeneity was easier to achieve for the lower content levels of dose groups 2 and 3 and that correct sampling was difficult due to formulation properties. This had an impact on results of storage stability as well. 2 out of 6 results met the limit of ±10% of nominal only. Deviating results were predominantly above nominal content which is not a sign of test item degradation. Therefore, the application formulations were considered to be stable for at least 8 days when kept at 2-8 °C. In conclusion, the results obtained within this part confirm the correct preparation of application formulations during the conduct of this study.
Details on mating procedure:
After acclimatization, females were placed in cages with sexually mature males (1:1) until copulation had been observed. After mating, the females were removed and placed in individual cages. The day of mating was designated day 0 post coitum.
The male rabbits were from the same source and strain as the females and were used for the mating only; their fertility had been proven and was continuously monitored.
Duration of treatment / exposure:
22 days (from day 6 to 27 post coitum)
Frequency of treatment:
once daily
Duration of test:
27 days (sacrifice was done on day 28)
Doses / concentrations
Remarks:
Doses / Concentrations:
20, 40, 80 mg/kg bw/day
Basis:
actual ingested
(the application volume was 4 mL/kg bw)
No. of animals per sex per dose:
20 mated females per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were selected based on a previous non-GLP dose range finding toxicity study (RCC B55170, 2008)

Examinations

Maternal examinations:
MORTALITY AND CLINICAL SIGNS
The dams were examined for mortality twice daily. Cage-side clinical observations were made once daily up to the day of necropsy for assessment of clinical symptoms.

BODY WEIGHTS
Body weights were recorded daily for day 0 to day 28 post coitum.

FOOD CONSUMPTION
Food Consumption was recorded for the following periods: days 0-3, 3-6, 6-9, 9-12, 12-15, 15-18, 18-21, 21-24 and 24-28 post-coitum.

BLOOD SAMPLING
Blood samples (approximately 1 mL) were collected from the ear vein from 10 females each in groups 0, 20, 40 and 80 mg/kg bw/day. The samples were taken 24 h before the first administration (day 5 post coitum) and 24 h after the last administration (day 28 post coitum, directly before the necropsy). Plasma samples were prepared from the blood and frozen (-80 °C) for possible examination of the level of T3, T4 and TSH. However, based on the results of this study, these further investigations were not considered necessary.

NECROPSY
At the scheduled necropsy on day 28 post coitum, females were sacrificed by an intravenous injection of sodium pentobarbital and the fetuses removed by Caesarean section. Any female sacrificed or found dead during the study was subjected to macroscopic examination with emphasis on the uterus and its contents.
Gross macroscopic examination of all internal organs was performed, and particular attention was given to the uterus, uterine contents, position of fetuses in the uterus and the number of corpora lutea. The uteri (and contents) of all females with live fetuses were weighed during necropsy on day 28 post coitum to enable the calculation of the corrected body weight gain.
The following organs from all sacrificed females were weighed: liver, adrenal glands, thyroid gland, pituitary gland.
The above organs (but only half of the liver) were preserved in neutral phosphate buffered 4% formaldehyde solution for possible histopathological examination. The other half of the liver was placed immediately in a bag in pieces of 4 - 5 g and put in liquid nitrogen for storage at -80 °C for possible examination of necessary parameters (for example enzyme induction). However, based on the results of this study, these further investigations were not considered necessary.
Fetal examinations:
Fetuses were removed from the uterus, sexed, weighed individually, examined for gross external abnormalities, sacrificed by a subcutaneous injection of sodium pentobarbital and allocated to one of the following procedures:
1. The fetuses were dissected, the organs examined and any abnormal findings were recorded. The sex of each fetus was noted.
2. After the skin had been removed, the cranium was examined for the degree of ossification.
3. From half of the fetuses the heads were separated from the trunks and fixed in Bouin's fixative. They were serially sectioned and examined (evaluation of the internal structures of the heads, including the eyes, brain, nasal passages and tongue) according to the method of Wilson JG (In: Teratology: Principles and Techniques. Eds., J.G. Wilson and J. Warkany, University of Chicago Press, 265-277,1965). Descriptions of any abnormal findings were recorded. After examination, the sections were preserved in a solution of ethyl alcohol and glycerin (one head per container). From the other half of the fetuses the heads were not separated but processed and stained as described in the next paragraph.
4. From all fetuses the skin with the exception of over the paws and the dorsal-cervical fat pads were removed and discarded. The trunks of the fetuses without heads and the fetuses with heads were processed through solutions of ethanol, glacial acetic with Alcian blue (for cartilage staining), potassium hydroxide with Alizarin red S (for clearing and staining ossified bone) and aqueous glycerin for preservation and storage; the procedure were based on the method of Inouye M (Differential staining of cartilage and bone in fetal mouse skeleton by Alcian blue and Alizarin red-S. Congenital Anomalies 16, 171-173, 1976). The skeletons were examined and all abnormal findings and variations were recorded
Statistics:
The following statistical methods were used to analyze food consumption, body weights and reproduction data:
- Means and standard deviations of various data were calculated
- The Dunnett-test [Dunnett CW, J. Amer. Statist. Assoc. 50, 1096-1121, 1955] (many to one t-test) based on a pooled variance estimate was applied if the variables could not be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test [Miller RG, Simultaneous Statistical Inference, Springer Verlag, New York,1981] (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
-Fisher's exact-test [Fisher RA, Statistical Methods for Research Workers, Oliver and Boyd, Edinburgh, 1950] was applied for the macroscopical findings.
Historical control data:
yes

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
MORTALITY AND CLINICAL SIGNS
All dams survived until the scheduled necropsy. No clinical symptoms related to treatment with the test item were noted during the study.
One dam of the high dose group (80 mg/kg bw/day) aborted on day 27 post coitum. This female was necropsied on day 28 post coitum, as scheduled. No other clinical signs or symptoms were noted for any dam in any group.

BODY WEIGHTS
In the high dose group (80 mg/kg bw/day), mean percentage body weight gain was generally statistically significantly reduced from day 9 post coitum onwards. Over days 6-28, mean body weight increased by 3.3% compared to 6.3% in the control group. Although mean body weight also became reduced in comparison to the control group as the study progressed, it was at no time statistically significantly reduced. Mean corrected body weight gain was slightly reduced (-5.9% compared to -4.7% in the control group). This was considered to be a sign of maternal toxicity.
In the mid and low dose groups (40 and 20 mg/kg bw/day) mean body weight, body weight gain and corrected body weight gain were not affected by treatment with the test item.

FOOD CONSUMPTION
In the high dose group (80 mg/kg bw/day), mean food consumption was reduced, at times statistically significantly, throughout the treatment period (-24.9% over days 6-28 post coitum, compared to the control group). This was considered to be a result of treatment with the test item. In the 40 mg/kg bw/day group, there was a slight transient reduction in food consumption over days 9-12 (-9.2%) and 21-24 post coitum (-21.6%) but this was not statistically significant and did not have an effect on the mean body weight in this dose group. In the low dose group treated with 20 mg/kg bw/day, mean food consumption was not affected by treatment.

REPRODUCTION PARAMETERS (for details, see table below)
All females in all groups were mated.
One female each in the control and the mid dose groups (0 and 40 mg/kg bw/day) were not pregnant as well as 2 in the high dose group (80 mg/kg bw/day). These occurrences were considered to be incidental. Post-implantation loss was statistically significantly increased in the high dose group (27.5% in 12 out of 17 dams compared to 10.0% in 9 out of 19 dams in the control group). As a result, the total number of fetuses was statistically significantly reduced (4.6 fetuses per dam compared to 6.2 in the control group). This was considered to be a test item-related effect. One female in the high dose group aborted on day 27 post coitum. This was considered likely to be a further post-implantation loss and therefore may also be test item-related. In the low and mid dose groups, no test item-related effects were noted in the reproduction data.

NECROPSY
Gross examination of the dams after necropsy revealed several abnormalities in the high dose group (80 mg/kg bw/day) when compared to the remaining groups; the findings consisted of raised crateriform area in the stomach containing hair, pale kidneys, and enlarged gall bladder. Due to the increase in the incidence of these findings in this
dose group, they were considered likely to be related to treatment with the test item.
Findings in the stomach were noted in one dam each in the low (20 mg/kg bw/day) and mid (40 mg/kg bw/day) dose groups. The low dose dam had a raised area on the pylorus and the mid dose dam had a red area in the stomach. Since these were the only findings in the stomach in these dose groups, they were considered likely to be incidental.
Referring to the organ weights, the weights of the left and right thyroid and adrenal glands were increased in the high dose group. These increases were considered to be probably test item-related but since they were not statistically significant, they were not considered to be adverse. In the low and mid dose groups, the organ weights were similar to those of the control group.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
40 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
40 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Dose descriptor:
LOAEL
Effect level:
80 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
LOAEL
Effect level:
80 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes. Remark: at the same dose as maternal toxicity

Details on embryotoxic / teratogenic effects:
SEX RATIO
No test item-related effects on the sex ratio of the fetuses were noted in any group.

FETAL WEIGHTS
In the high dose group (80 mg/kg bw/day), mean fetal weights were statistically significantly reduced (30.5 g versus 32.7 g in the control group; p<0.01). This was considered to be an effect of the observed maternal toxicity in this dose group.
Mean fetal weights in the low and mid dose groups (20 and 40 mg/kg bw/day) were within control range (31.7 and 32.4 g, respectively) and thus were not affected by treatment with the test item. The statistically significant reduction of body weight of the males on an individual basis in the low dose group (31.7 g versus 33.4 g in the control group; p>0.05; see table below) was within the range of the historical control data for litters of this size and therefore was incidental.

EXAMINATION OF SECTIONED HEADS
Examination of fixed sectioned fetal heads did not reveal any test item-related effects. In fact, in the high dose, one fetuse of 41 (i.e. 2% of the fetuses) examined showed dilation of the lateral brain ventricles. The finding was incidental.

EXTERNAL AND VISCERAL EXAMINATIONS (for details, see the attached tables)
The main findings were summarized in a table (see below). During visceral examination of the fetuses, findings were noted in:
63% examined fetuses (in 100% litters) in the control group;
58% examined fetuses (in 100% litters) in the low dose group (20 mg/kg bw/day);
59% examined fetuses (in 94% litters) in the mid dose group (40 mg/kg bw/day);
66% examined fetuses (in 94% litters) in the high dose group (80 mg/kg bw/day).
Abnormalities were noted in 2% of fetuses in the control group, 3% in the low and mid dose groups, and 9% in the high dose group. Whereas the findings in the low and mid dose groups were considered to be incidental, in the high dose group, the higher incidence of abnormalities was considered to be test item-related, since there was also a higher incidence of post-implantation loss at the same dose level. Since maternal toxicity was noted in this dose group and there was no clear pattern in the nature of the findings, a direct effect of the test item on the fetuses is not suspected. None of the variations noted were considered to be test item-related. Although there was an increased incidence of an additional artery arising from the aortic arch, this was within the range of the historical control data.

SKELETAL EXAMINATIONS (for details, see the attached tables)
In the high dose group 4, the number of supernumerary ribs was marginally increased on a fetus basis compared to the concurrent control, but was not statistically significant on a litter basis. There was a statistically significant increase in incompletely ossified limbs/digits in all 3 treated groups (20, 40 and 80 mg/kg bw/day). The level in the low dose group was within the range of the historical control data and was therefore considered unlikely to be of toxicological relevance. The level in the mid dose group was within the historical control data on a litter level but outside on a fetus level. This was considered to be a transient and non-adverse effect of the test item. The level in the high dose group was outside the historical control range and was considered to be related to the maternal toxicity at this dose level.
During cartilage examination of the fetuses, abnormal findings were noted in:
6% examined fetuses (in 16% litters) in group 1
4% examined fetuses (in 20% litters) in group 2
7% examined fetuses (in 33% litters) in group 3
10% examined fetuses (in 41% litters) in group 4
In the high dose group, there was a decreased incidence of short costal cartilage 10 as well as a decreased incidence of costal cartilage 7 not reaching the sternum. These findings, together with the increased incidence of supernumerary costal cartilages, suggest a possible minor disturbance in the development of the axial skeleton. This would not be unexpected in the presence of the observed maternal toxicity at this dose level. In the mid dose group, these effects only attained statistical significance on a fetal level and therefore the toxicological relevance is unclear.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Summary of reproduction data:

 

Reproduction Parameters

 

Dose groups (mg/kg bw/day)

0

20

40

80

NUMBER OF DAMS

19

20

18

17

CORPORA LUTEA

 

 

 

 

 

Total

154

148

135

130

 

Mean/Dam

8.1

7.4

7.5

7.6

PRE-IMPLANTATION LOSS

24

24

30

21

 

% OF CORP. LUTEA

15.6

16.2

22.2

16.2

 

MEAN

1.3

1.2

1.7

1.2

 

NUMBER OF DAMS AFFECTED

11

12

11

10

IMPLANTATION SITES

130

124

105

109

 

% OF CORP. LUTEA

84.4

83.8

77.8

83.8

 

MEAN

6.8

6.2

5.8

6.4

POST-IMPLANTATION LOSS

13

5

11

30

 

% OF IMPLANT. SITES

10

4

10.5

27.5**

 

MEAN

0.7

0.3

0.6

1.8

 

NUMBER OF DAMS AFFECTED

9

4

6

12

EMBRYONIC/FETAL DEATHS TOTAL

13

5

11

30

EMBRYONIC RESORPTIONS

12

5

10

23

 

% OF IMPLANT. SITES

9.2

4

9.5

21.1**

 

MEAN

0.6

0.3

0.6

1.4

 

NUMBER OF DAMS AFFECTED

9

4

6

8

FETAL RESORPTIONS

1

0

1

7

 

% OF IMPLANT. SITES

0.8

 

1

6.4*

 

MEAN

0.1

 

0.1

0.4

 

NUMBER OF DAMS AFFECTED

1

 

1

4

FETUSES

 

 

 

 

 

TOTAL

117

119

94

79

 

% OF IMPLANT. SITES

90

96

89.5

72.5**

 

MEAN

6.2

6

5.2

4.6

 

LIVE FETUSES

117

119

94

79

 

DEAD FETUSES

0

0

0

0

 

ABNORMAL FETUSES

0

0

0

0

SEX OF THE FETUSES

 

 

 

 

TOTAL MALES

57

53

46

33

 

% OF FETUSES

48.7

44.5

48.9

41.8

 

MEAN

3

2.7

2.6

1.9

TOTAL FEMALES

60

66

48

46

 

% OF FETUSES

51.3

55.5

51.1

58.2

 

MEAN

3.2

3.3

2.7

2.7

LIVE MALES

57

53

46

33

LIVE FEMALES

60

66

48

46

WEIGHTS OF LIVE FETUSES (g)

(LITTER BASIS)

 

 

 

 

TOTAL FETUSES

 

 

 

 

 

N (LITTERS)

19

20

18

17

 

MEAN

33.3

32.4

33.3

30.4*

MALES

 

 

 

 

 

N (LITTERS)

19

19

17

16

 

MEAN

34

32.5

33.6

31.2*

FEMALES

 

 

 

 

 

N (LITTERS)

17

20

16

16

 

MEAN

32.6

32.3

32.1

30.8

WEIGHTS OF LIVE FETUSES (g)

(INDIV. BASIS)

 

 

 

 

TOTAL FETUSES

 

 

 

 

 

N (LITTERS)

117

119

94

79

 

MEAN

32.7

31.7

32.4

30.5**

MALES

 

 

 

 

 

N (LITTERS)

57

53

46

33

 

MEAN

33.4

31.7*

33.3

30.9**

FEMALES

 

 

 

 

 

N (LITTERS)

60

66

48

46

 

MEAN

32

31.7

31.6

30.2*

*, p<0.05; **, p<0.01

Summary of fetal data: see attached document.

Applicant's summary and conclusion