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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:
July 24, - July 31, 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study; GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
(January 22, 2001)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Analytical Purity: 98.1% (confirmed by reanalysis)
- Physical state: liquid/colourless
- Lot No.: 000STD77L0
- Stability: Confirmed by reanalysis
- Test Substace No.: 01/0369-2
- Storage: RT and under nitrogen

Test animals

Species:
rat
Strain:
other: Wistar (CrIGIxBrIHan:Wl)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Germany
- Age at study initiation: 70-80 days
- Weight at study initiation: 144.8 - 189.0 g
- Housing: singly (day 0 - 20 post mating)
- Diet: ad libitum (ground Kliba diet mouse/rat (PROVIMI KLIBA SA)), Fed. Reg. Vol. 44, No. 91 of May 9, 1979, p. 27354 (EPA) served as a guideline for maximum tolerable chemical contaminants.
- Water: ad libitum [German Drinking Water Regulation (December 05, 1990) served as a guideline for maximum tolerable contaminants].
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20- 24
- Humidity (%): 30- 70
- Photoperiod (hrs dark / hrs light): 12/12
- Room disinfection: before study begin (AUTEX). Room was cleaned weekly with water

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- Time schedule: at the beginning of the administration period and thereafter at intervals, taking into account the stability.
- Procedure: An appropriate amount of the test substance was weighed in a graduated measuring flask depending on the dose group, topped up with olive oil Ph.Eur./DAB and subsequently thoroughly mixed.

VEHICLE
- Concentration in vehicle: 0, 100, 300 and 900 mg/100 ml for doses 0, 5, 15 and 45 mg/kg bw/d respectively
- Amount of vehicle (if gavage): 5 ml/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
CONCENTRATION CONTROL
- Sample: test substance in vehicle (olive oil)
- Method of detection: GC-FID (external and internal calibration), solvent was acetone
- Results: Stability in vehicle over 12 day period was confirmed (storage at ambient temperature). 77.5% - 91.7% of the nominal concentrations were recovered making the mean adjusted effective dose levels 4.1; 12.7 and 40.70 mg/kg body weight/day.
Details on mating procedure:
Animals were supplied from breeders. The animals were time-mated and supply occured on the day that vaginal plug and/or sperm was detected in the vagina (Day 0).
Duration of treatment / exposure:
from Day 6 - Day 20 post mating
Frequency of treatment:
daily
Duration of test:
-
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
(target doses) 0; 5; 15; 45 mg/kg bw/d
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
(Effective doses (Determined by reanalysis)) 0; 4.1; 12.7; 40.7 mg/kg bw/d
Basis:
actual ingested
The authors stated in the report that the deviations between the targeted and the actual concentrations of about 10 - 20% were not considered to impair the overall validity of the study.
No. of animals per sex per dose:
25 females/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for animal selection: The strain was selected since extensive experience was available on Wistar rats and because the strain was proven to be sensitive to substances with a teratogenic potential.

OTHER
- The study was carried out in 3 replicates due to technical reasons. Each dose group was represented in each replicate

Examinations

Maternal examinations:
MORTALITY: Yes
- Time schedule: twice a day on working days or once a day (saturday, sunday or on public holidays)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once a day, and more often in case of manifestation of clinical signs of toxicity

BODY WEIGHT: Yes
- Time schedule for examinations: 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20 post mating
- Corrected body weight gain: Calculated after terminal sacrifice (body weight on day 20 post mating minus weight of the unopened uterus minus body weight on day 6 post mating)

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule for examinations: 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20 post mating

CLINICAL CHEMISTRY:
- No. of animals: 25
- Time schedule: day 20 post mating, 1 hour after application
- Fasting: No
- Anesthesia: Yes, Isofurane
- Sampling: Blood from the retroorbital venous plexus
- Parameters measured: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, serum-gamma-glutamyltransferase, glutamate dehydrogenase, serum cholinesterase, erythrocyte cholinesterase, brain cholinesterase, protein in brain homogenate

HAEMATOLOGY: Yes
- No. of animals: 25
- Time schedule: day 20 post mating, 1 hour after application
- Fasting: No
- Anesthesia: Yes, Isofurane
- Sampling: Blood from the retroorbital venous plexus
- Parameters measured: Hematocrit

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice: gestation day 20 by decapitation
- Examination: Gross pathology, liver, uterus and ovaries
- Organ parameter examined: Weight
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 and distribution of implantations: Yes, (classified as live fetuses and dead implantations)
- Number of early resorptions: Yes (only decidual or placental tissues visible or utero from non pregnant animals and the empty uterus horn in the case of single-horn pregnancy)
- Number of late resorptions: Yes (embryonic or fetal tissue in addition to placental tissue visible)
- Number of dead fetuses: Yes (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened)
Fetal examinations:
- Viability of fetuses: Yes
- Determination of weight, sex and macroscopical examination: Yes
- Soft tissue examinations: Yes, half per litter
- Skeletal examinations: Yes, half per litter

In the present study the glossary of WISE et al. (Wise et al., 1997) was used as much as possible to describe findings in fetal morphology. Classification of these findings was based on the terms and definitions proposed by CHAHOUD et al. (Chahoud et al., 1999; Solecki et al., 2001; Solecki et al., 2003). A malfomation was defined as permanent structural change that is likely to adversely affect the survival or health and a variation was defined as change that occurs also in fetuses of control animals and is unlikely to adversely affect the survival or health. This includes delays in growth or morphogenesis that have otherwise followed a normal pattern of development. "Unclassified observation" or "unclassified cartilage observation" were used for those fetal findings, which could not be classified as malformations or variations (e.g. focal liver necrosis in fetuses, isolated cartilage findings without any impact on the respective bony structure).
Indices:
- The conception rate (in %): number of pregnant animals divided by number of fertilized animals multiplied by 100
- The preimplantation loss (in %): (number of corpora lutea - number of implantations) divided number of corpora lutea multiplied by 100
- The postimplantation loss (in %): (number of implantations - number of live fetuses) divided by number of implantations multiplied by 100

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
MATERNAL TOXIC EFFECTS
MORTALITY
- There were no substance-related nor spontaneous mortalities in any of the test groups.

CLINICAL SYMPTOMS
- No substance dependent adverse nor toxic effect was seen the test animals of all groups
- High dose animals: 5/25 animals; Salivation that lasted about 30 min after treatment on Days 13-20 of study. Salivation was judged by the authors to be substance related but not as an adverse or toxic effect. Most probably cause of salivation was bad taste of test substance or local affection of the upper digestive tract.

FOOD CONSUMPTION
- Low dose animals: no adverse effect on food consumption seen
- Mid dose animals: no adverse effect on food consumption seen
- High dose animals: Slight but statistically significant substance related reduction (ca. 18%) of food uptake in comparison to control animals in the first two days of treatment. By termination food consumption was normalised and comparable to that of control animals.

BODY WEIGHT GAIN
see table below

CORRECTED BODY WEIGHT GAIN
- Low- and mid dose animals: Similar to control values
- High dose animals: Significantly lower than weight gain in control animals with approximately 32% difference.

CLINICAL CHEMISTRY
- Mid dose animals: In comparison to control animals, the modulation of the activities of following enzymes were statistically significant; Serum and erythrocyte cholinesterase (17% and 9% below control, respectively), serum alanine aminotransferase (19% above control).
- High dose animals: In comparison to control animals, the modulation of activities of following enzymes were statistically significant; Serum and erythrocyte cholinesterase (43% and 16% below control, respectively), serum alanine aminotransferase (29% above control), glutamate dehydrogenase (79% above control).

ORGAN WEIGHTS
- Low dose animals: Statistically significant increase in absolute (10 %) and relative (9 %) mean liver weight (not considered by authors as an adverse effect because the effect was not accompanied by clinical pathology effects as in the mid and high doses)
- Mid dose animals: Increased absolute (13%) and relative (11%) mean liver weight (statistically significant, compared to control values, dose dependent)
- High dose animals: 20% reduction in mean uterus weight (statistically significant, compared to control values) and increased mean absolute (11%) and relative (19%) mean liver weight (statistically significant, compared to control values, dose dependent)

GROSS PATHOLOGY
- There were no substance-related observations at necropsy in any of the dams.

REPRODUCTION DATA:
There were no substance-related and/or biologically relevant differences between the test groups in the conception rate (88 -92%), in the mean number of corpora lutea, implantation sites and the preimplantation loss values

Low and mid dose animals: Postimplantation loss values, the number of early and late resorptions, and the mean number of live fetuses/dam were similar to the respective control values.

High dose animals: The number of total resorptions was elevated as indicated by the statistically significant increased postimplantation loss value (15.1% vs 4.4% (control group) and outside the historical control range: 6.8% (3.4 - 11.3%). The vast majority of these postimplantation losses were early resorptions. The mean number of live fetuses per dam was decreased (8.1/ 8.2/ 8.8/7.4 at control, low dose, mid dose, and high dose) and hence a statistically significant lower mean percentage of live fetuses was obtained

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
4.1 mg/kg bw/day (actual dose received)
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
SEX DISTRIBUTION OF FETUSES
- comparable with that of the control fetuses for all dose groups

WEIGHT OF PLACENTAE
- comparable with that of the control fetuses with no clear dose relation for all dose groups

WEIGHT OF FETUSES
see table below

EXTERNAL MALFORMATIONS
- Control: 0/186 fetuses (from 23 litters)
- Low dose: 0/180 fetuses (from 22 litters)
- Middle dose: 0/203 fetuses (from 23 litters)
- High dose: 2/170 fetuses (1.2%) (in 2 out of 23 litters (8.7%). These included anasarca and small spleen (female), polydactyly confirmed as supernumerary phalanx. The mean percentages of affected fetuses/litter with total malformations amounted to 0.0, 0.0, 0.0, and 1.5%* (* p<= 0.05) at 0; 5; 15 or 45 mg/kg body weight/day, respectively.

EXTERNAL VARIATIONS: no external variations were observed

UNCLASIFIED EXTERNAL OBSERVATION
- Blood coagulum around the placenta occured in the progeny of one control dam.

SKELETAL MALFORMATIONS
- Control animals: 0/96 (from 23 litters),
- Low dose animals: 0/94 fetuses (from 23 litters)
- Middle dose animals: 0/110 fetuses (from 23 litters)
- High dose animals: 2/92 (2.2%) fetuses (from 2/23 litters [8.7%]); these included supernumerary phalanx (male) and cervical hemivertabra (female)
The mean percentages of affected fetuses/litter with skeletal malformation amounted to 0, 0, 0, 2.3%, respectively in test groups 0, 5, 15 and 45 mg/kg bw/d

SKELETAL VARIATIONS
see table below

SOFT TISSUE MALFORMATION
- Control: 0/90 (from 23 litters)
- Low dose: 0/86 (from 22 litters)
- Middle dose: 0/93 (from 23 liiters)
- High dose: 1/78 (1.3%) [from 1/22 litters (4.5%)]
The mean percentages of affected fetuses/ litter with soft tissue malformation amounted to 0, 0, 0 and 2.3% respectively in test groups 0, 5, 15, 45 mg/kg bw/d. The animal (female) with soft tissue malformation displayed a small spleen. Same animal already displayed anarsaca

SOFT TISSUE VARIATIONS
- Control: 7.8%
- Low dose: 7.7%
- Middle dose: 3.6%
- High dose: 5.3%
Soft tissue variations consisted of uni- or bilateral dilation of the renal pelvis and/or ureter. These were detected in each group including the controls in 2 - 8 fetuses from 2 - 5 litters without any dose-response relationship. Moreover, a slightly dilated cerebral ventricle occurred in high dose female fetus, which showed already anasarca and small spleen (see above)

UNCLASSIFIED SOFT TISSUE FINDINGS
- Control: 0%
- Low dose: 0%
- Middle dose: 1.7%
- High dose: 15.5%
Discoloured fetal livers were observed. Since the liver of the dams seemed to represent a target organ of the test substance, it was assumed by the authors of the study that the liver of the fetuses most likely will be affected. Hence liver discolouration in the fetuses was defined as substance induced.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
4.1 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
other: Corresponds to 5 mg/kg bw/d (nominal dose).

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

COMMENTS TO FETAL FINDINGS

The increased occurrence of skeletal variations at the mid and the high dose was described by the authors to represent common findings on fetal morphology due to the observable fetal growth retardations and/or due to maternal stress. The authors based their arguments on the thesis of WICKRAMARATNE who states that an increase in supernumerary ribs "should be considered not as indicative of fetal dysmorphogenesis but as a manifestation of a non-specific stress the dam is placed under in such (i.e. prenatal developmental toxicity) studies . . .". In support, KIMMEL and WILSON state, that "when an increase in either rudimentary or extra ribs occurs during teratogenicity testing, it may be assumed that the maternal animal is being stressed sufficiently to express the developmental instability inherent in the species. The expectation would be, if other teratogenic effects did not occur at that dose level, that at a higher dose, frank embryotoxicity would become evident".

WICKRAMARATNE, G.A. Journal of Applied Toxicology 8 (2), 91 - 94 (1988)

KIMMEL, C.A. and WILSON, J.G. Teratology 8, 309 - 316 (1973)

Maternal systemic toxicity based on body weight/ changes and food consumption. Effects on mean fetal body weights on a litter basis and occurrence of statistically significantly increased fetal skeletal variations (expressed as mean percentage of affected fetuses/litter). All statistically significant differences, which showed a dose-response relationship and/or were outside historical control ranges (at date of study) were marked in bold types. * = p ≤ 0.05; ** = p ≤ 0.01; mg/kg bw/d (mg/kg body weight/day); HCD (Historical control data).

Finding

 

0 mg/kg bw/d

5 mg/kg bw/d

15 mg/kg bw/d

45 mg/kg bw/d

HCD Mean % (range) report

HCD Mean % (range) 2012

Mean maternal food consumption              (d6-8 p.c.)

grams/ animal/ day 

15.4           

15.0           

14.7           

12.7**           

-

-

Mean maternal body weight change         (d6-8 p.c.)

grams                   

5.0           

4.2           

2.2**           

- 5.5**           

-

-

Mean maternal body weight change         (d6-20 p.c.)

grams

 75.3           

75.7          

77.0          

56.8**           

-

-

Mean maternal body weights                            (d20 p.c.)

grams                  

 264.4  

265.2   

268.1   

247.2*        

-

-

Mean net body weight change from day 6

grams               

 30.0         

30.6     

30.6          

20.5**  

-

-

Mean fetal weights

grams                 (on a litter basis)

3.6           

3.5           

3.3**           

2.9**           

3.5            (2.8-4.2)

3.6           (2.6-5.5)

Misshapen sacral vertebra

Fetal incidence %

2.1

0

2.7

12

0.7

(0-3.5)

1.8

(0-7.1)

Litter incidence %

8.7

0

13

39*

3.4

(0-16.7)

7.6

(0-28)

Affected Fetuses/litter

1.7

0

3

11.9**

0.8

(0-3.8)

1.9

(0-7.5)

Supernumerary thoracic vertebra

Fetal incidence %

1

2.1

10

14

1.9

(0-4.7)

2.9

(0-10.1)

Litter incidence %

4.3

9.1

26*

30*

7.3

(0-16.7)

10.7

(0-30)

Affected Fetuses/litter

1.1

2.7

9.8*

13.6**

1.9

(0-5.3)

3.0

(0-11.5)

Supernumerary rib (14th), cartilage present

Fetal incidence %

1

2.1

10

8.7

4

(0-8.7)

6.2

(0-19.2)

Litter incidence %

4.3

9.1

26*

22

13.4

(0-29.2)

20

(0-62.5)

Affected Fetuses/litter

1.1

2.3

9.8*

8.4*

4.1

(0-10.2)

6.2

(0-18.3)

Supernumerary rib (14th), cartilage not present

Fetal incidence %

41

31

51

39

41.9

(31.3-58.1)

48.8

(31.2-72)

Litter incidence %

78

64

91

65

81.2

(65.2-95)

85.7

(65.2-100)

Affected Fetuses/litter

38.5

30.7

51.1*

36.7

41.7

(32.4-58.8)

48.6

(32.4-73.1)

Incomplete ossification of supraoccipital, unchanged cartilage

Fetal incidence %

13

19

17

22

14

(5.8-21.4)

17.9

(5.8-46.2)

Litter incidence %

39

55

43

61

41.5

(22.2-60)

48.1

(22.2-92)

Affected Fetuses/litter

12.8

20.1

17.2

24.6*

14

(6.2-21.2)

17.8

(6.2-45.1)

Supraoccipital hole(s)

Fetal incidence %

23

17

28

42

31.3

(8-59.3)

29.2

(4-59.3)

Litter incidence %

52

50

70

87*

70.7

(33.3-100)

67.4

(19-100)

Affected Fetuses/litter

23.4

17.3

28.4

39.8*

31.7

(8.6-60.5)

29.4

(3.6-60.5)

Incomplete ossification of skull, unchanged cartilage

Fetal incidence %

0

4.3

2.7

7.6

5.4

(0-10.8)

5.5

(0-15.2)

Litter incidence %

0

14

13

26*

17.3

(0-30.4)

18.1 (0-44.0)

Affected Fetuses/litter

0

5.1*

2.8*

6.7**

5.4

(0-11)

5.4

(0-15.8)

Unossified sternebra, unchanged cartilage

Fetal incidence %

3.1

8.5

13

46

10.6

(3.4-35.7)

7.9

(0.9-35.7)

Litter incidence %

13

32

26

83**

32.2

(13-70.8)

24.9     (4.2-70.8)

Affected Fetuses/litter

3.3

9.4

12.8

49.9**

11.1

(3.4-39)

8.0

(0.8-39)

Incomplete ossification of sternebra, unchanged cartilage

Fetal incidence %

47

61

76

77

56

(38.5-73.7)

64.3

(38.5-87.3)

Litter incidence %

78

91

100*

96

88.5

(70-100)

91.6

(70-100)

Affected Fetuses/litter

46.1

60.1

76.7**

76.6**

55.9

(38.4-74.6)

63.6

(38.4-87.2)

Bipartite ossification of sternebra, unchanged cartilage

Fetal incidence %

0

0

2.7

0

0.8

(0-4.9)

0.4

(0-4.9)

Litter incidence %

0

0

13

0

2.9

(0-13)

1.6

(0-13)

Affected Fetuses/litter

0

0

2.8*

0

0.8

(0-4.6)

0.4

(0-4.6)

Incomplete ossification of sacral arch, cartilage present

Fetal incidence %

1

2.1

0

12

12.5

(0-46.2)

6.9

(0-46.2)

Litter incidence %

4.3

9.1

0

39**

31

(0-87)

18.8

(0-87)

Affected Fetuses/litter

1.1

2.4

0

16.2**

12.2

(0-46.1)

6.8

(0-46.1)

Incomplete ossification of pubis, cartilage present

Fetal incidence %

0

0

1.8

5.4

0.4

(0-1.2)

0.3

(0-1.9)

Litter incidence %

0

0

8.7

22*

2

(0-5.6)

1.6

(0-8.3)

Affected Fetuses/litter

0

0

2

8**

0.4

(0-1.2)

0.4

(0-2.1)

Incomplete ossification of interparietal, unchanged cartilage

Fetal incidence %

21

36

21

11

21.6

(13.6-33.3)

23.4

(12.7-36.1)

Litter incidence %

52

82*

65

35

58.5

 (37.5-81.8)

60.4

 (37.5-82.6)

Affected Fetuses/litter

20.7

36*

20.9

9.8

21.5

(12.5-33.3)

23.3

(12.4-35.3)

Incomplete ossification of parietal, unchanged cartilage

Fetal incidence %

11

30

14

14

15.2

(3.2-29.9)

15.0

(3.2-29.9)

Litter incidence %

39

68*

52

48

43.4

(12.5-68.2)

43.7

(12.5-68.2)

Affected Fetuses/litter

11.7

29.8**

13.6

13.4

15.4

(3.1-27.6)

15.3

(3.1-27.6)

Total fetal skeletal variations

Fetal incidence %

91

91

99

98

94.6

(88-99.2)

96.6

(88-100)

Litter incidence %

100

100

100

100

100

(100-100)

100

(100-100)

Affected Fetuses/litter

89.1

92

99.1**

98.3*

94.7

(87-99.2)

96.6     (87-100)

 

Applicant's summary and conclusion