Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2013

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:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
other: Japanese MAFF guidelines: Guideline on the Compiling of Test Results on Toxicity “Teratology Study”, 12-Nousan No. 8147 of November 24, 2000, amended June 26, 2001
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Stability under test conditions: confirmed in analytical testings conducted under GLP (confirmed for at least 8 days)

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Nederlands, 5960 AD Horst, The Netherlands
- Age at study initiation: 11 - 17 weeks
- Weight at study initiation: males 434 - 587 g, females 208 - 258 g
- Housing: Starting from gestation day 0 individually in Type IIIh Makrolon cages on low-dust wood shavings.
- Diet and water: ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): approximately 55 %
- Air changes (per hr): > 10 per hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Administration volume: 2 mL/kg bw

VEHICLE
- Justification for use and choice of vehicle (if other than water): The vehicle was suitable as analytical investigations revealed that the test substance was stable in the vehicle for at least 8 days.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The results of the homogeneity tests and content checks in samples with concentrations of 50, 150 (content checks, only), and 500 mg/mL during the study showed no meaningful deviation of the active ingredient content from the nominal value.
Details on mating procedure:
The animals were mated by placing one female overnight into a Type IIIh cage together with one male rat. If sperm was detected in the vaginal smear taken on the morning following mating, this day was regarded as day 0 of gestation.
Duration of treatment / exposure:
Day 6 - 20 p. c.
Frequency of treatment:
once daily (between 06:00 and 12:00 CET)
Duration of test:
From experimental starting to end of in-life-Phase 38 days.
No. of animals per sex per dose:
25 female rats
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on the results of a previous pilot prenatal developmental toxicity study in rats with the test substance (T9081874).

Examinations

Maternal examinations:
CLINICAL EXAMINATIONS: Yes
- Time schedule: The females were inspected once daily from day 0 to 21 p.c. (once daily only on weekends, on public holidays, and on day 21 p.c.), and all findings were recorded. Attention was paid to disturbances in the general condition of the rats (appearance, behavior), and any alterations concerning their excretory products.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weights of the females were determined on day 0 p.c. and daily from days 6 to 21 p.c. Corrected body weight gain was determined by subtracting the uterus weight on day 21 p.c. from the body weight gain from days 0 to 21 p.c.

FOOD AND WATER CONSUMPTION: Yes
- The food intake of the animals was determined from the difference in weight between the food offered and the food not consumed for the following days of gestation: Days 0 - 3, 3 - 6, 6 - 9, 9 - 12, 12 - 15, 15 - 18, and 18 -21.
Water intake was assessed daily by visual estimation of the quantities left over and reported together with clinical findings.

POST-MORTEM EXAMINATIONS: Yes
- The females were subjected to gross pathological evaluation at cesarean section on day 21 p.c. Necropsy was performed without knowledge of treatment groups.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Necropsies/cesarean sections were performed on day 21 p.c. without knowledge of treatment groups.
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
- Number of dead and live fetuses: Yes
- Other: individual weight and appearance of the placentas
Fetal examinations:
- Sex of live fetuses
- Individual weights of live fetuses
- External examinations: Yes, findings in alive and dead fetuses are included
- Soft tissue and head examinations: Yes, evaluation of about half of alive fetuses per litter
- Skeletal and cartilage abnormalities: Yes, evaluation in about half of alive fetuses per litter
Statistics:
Differences between the control and test item treated groups were considered to be significant when p < 0.05. Significant differences from the control group are indicated with * for p < 0.05 and ** for p < 0.01.
Statistical evaluation was performed on an Alpha 800 5/500 computer using the following methods:
- Analysis of Variance (ANOVA); in case of significance Dunnett's test for feed intakes, body weights, body weight gains, and corrected body weight gains, uterine weights, number of corpora lutea per female, number of implantations per female, number of live fetuses per female and as percentage of implantations per female, placental weights per female, fetal weights per female.
- 2 by N CHI2 test; in case of significant differences Fisher's exact test with Bonferroni correction for fertility rate, gestation rate, number of implantations per group, number of preimplantation losses per group, number of postimplantation losses, early resorptions, late resorptions or dead fetuses per group, number of live fetuses per group as percentage of implantations per group, number of male or female fetuses or fetuses with indeterminable sex per group, number of placentas with findings or litters with placental findings per group, number of fetuses or litters with external, visceral or skeletal findings, with malformations or with external or visceral deviations per group.
- Kruskal-Wallis test and in case of significant differences Dunn's test for number of preimplantation losses per female, number of postimplantation losses, early resorptions, late resorptions or dead fetuses per female, number of male or female fetuses or fetuses with indeterminable sex per female, number of placentas with findings per female, number of fetuses with external or visceral findings, with malformations or with external or visceral deviations per female.
- CHI2 test (correction according to yates) for number of fetuses or litters with cartilaginous tissue observations.
Indices:
gestation rate

Results and discussion

Results: maternal animals

Maternal developmental toxicity

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

Details on maternal toxic effects:
Salivation after administration occurred in all dose groups, which is most likely related to the bad taste of the test item and considered as not adverse.
Thus, appearance, behavior, mortality, absolute and corrected body weight gains, food intake, water intake, and fecal and urinary excretions were unaffected at dose levels up to 1000 mg/kg.

No treatment related gross pathological findings occurred at dose levels up to 1000 mg/kg.

Fertility rate (percentage of inseminated females with implantations), the mean numbers of corpora lutea, preimplantation losses, and implantation sites in the dose groups did not differ to a meaningful extent from the control group values indicating a homogeneous distribution regarding these parameters.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Basis for effect level:
other: developmental toxicity

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
The gestation rate, appearance and weights of placentas, postimplantation loss and correspondingly the number of fetuses, fetal sex distribution, and fetal weights were unaffected by treatment at dose levels up to 1000 mg/kg.

A treatment related effect on malformations, external, visceral, and skeletal (including cartilage) deviations was not evident at dose levels up to 1000 mg/kg.

The overall incidences of fetuses or litters with malformations lay within the range of historical control data, revealed no statistical significance, and were thus unaffected by treatment at dose levels up to 1000 mg/kg. The types of malformations observed in this study are generally a representative sample of spontaneous malformations in the rat strain used (e.g. multiple malformations, microphthalmia, situs inversus, retina folded, ventricular septal defect, vertebral malformations with pelvis shift, dysplastic limb bones) and comparable with spontaneous findings in the current control group and/or historical control groups. Malformations of the eyes (eye ball reduced in size) occurred in all dose groups and the control group in comparable incidences of affected fetuses and litters without a dose dependency and were thus unaffected by treatment at dose levels up to 1000 mg/kg. At the 1000 mg/kg and 300 mg/kg levels, one fetus each revealed a cleft palate (at the 300 mg/kg level in combination with a multiple malformation), which is a rare finding, but also known as a spontaneous finding in the rat strain used (for example 2 fetuses out of 1 litter in the unaffected low dose group of a prenatal developmental toxicity study, T7062991, conducted in the same laboratory in 2002). Furthermore, a concurrent study with a structural similar substance (Aspartic acid, N,N'-[methylenebis(2-methyl-4,1-cyclohexanediyl)]bis-, 1,1',4,4'-tetraethyl ester, CAS No. 136210-32-7, T5081691, performed in the same laboratory) showed no comparable finding so that a treatment related effect is not assumed. Furthermore, one fetus of the 1000 mg/kg group showed a finding of the vertebral column (3rd lumbar vertebral arch has the shape of a 4th lumbar vertebral arch bilateral, cartilaginous part of 6th lumbar vertebral arch fused with iliac bone, 1st sacral vertebrae missing, bilateral, pelvis shift to cranial, bilateral), which is comparable with spontaneous findings in the rat strain used. Therefore, a treatment related effect is not assumed for this finding. All remaining findings revealed no dose dependency and were thus considered as incidental. Thus, a treatment related effect on malformations (incidence or type) was not evident at dose levels up to 1000 mg/kg.

The overall incidences of fetuses or litters with external and visceral deviations were unaffected at dose levels up to 1000 mg/kg and revealed the highest value on a fetal and litter basis in the control group. The external and visceral deviations observed in this study were of a common type and comparable with spontaneous findings in the current control group and/or historical control groups and represented the normal range of scattering in the rat strain used. Histopathological evaluation of additional circumscribed hard whitish tissue in the nasopharynx was performed in a prenatal developmental toxicity study with the same rat strain (T0076746) and revealed calcium concrements without connection to the underlying tissue in the affected localizations. Calcium might have been dissolved from the fetal bones by the Wilson fixative and precipitated in the nasopharyngeal duct so that these findings were regarded as artifacts. Thus, a treatment related effect on external and visceral deviations was not evident at dose levels up to 1000 mg/kg.

At the 1000 mg/kg level, fetal examinations for skeletal retardations and variations revealed statistically significantly retarded ossification of several localizations (3rd distal phalanges of digits left, 3rd proximal phalanges of digits bilateral, 4th proximal phalanges of digits left, 2nd and 5th cervical vertebral bodies, 6th-8th caudal vertebral bodies, parietal bones bilateral), when calculation was done on a fetal basis, for which a treatment related effect is not assumed, as these values were comparable with the control values of the concurrent study with a structural similar substance (Aspartic acid, N,N'-[methylenebis(2-methyl-4,1-cyclohexanediyl)]bis-, 1,1',4,4'-tetraethyl ester, CAS No. 136210-32-7, T5081691, performed in the same laboratory), and since statistical significance was lacking in all but one case, when calculation was done on a litter basis. At the 300 mg/kg level, fetal examinations for skeletal retardations and variations revealed statistically significantly retarded ossification of a single localization (8th caudal vertebral bodies), when calculation was done on a fetal basis, for which a treatment related effect is not assumed, because statistical significance was lacking, when calculation was done on a litter basis, and due to its single occurrence. At the 100 mg/kg level, fetal examinations for skeletal retardations and variations revealed statistically significantly retarded ossification of two localization (9th caudal vertebral bodies, parietal bones bilateral), when calculation was done on a fetal basis, for which a treatment related effect is not assumed, because statistical significance was lacking, when calculation was done on a litter basis, and since a dose dependency was lacking.
Evaluation of fetal cartilaginous tissue revealed no treatment related findings at dose levels up to 1000 mg/kg. Thus, a treatment related effect on skeletal deviations (retardations, variations, including cartilaginous tissue findings) was not evident at dose levels up to 1000 mg/kg.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

General Reproduction Data:

Dose (mg/kg b.w./day)

0

100

300

1000

inseminated females

25

25

25

25

inseminated females evaluated

25

25

25

25

females with implantations

24

21

23

22

in % of those inseminated

96.0

84.0

92.0

88.0

mean values per female with implantation sites

corpora lutea

15.7

15.6

15.4

15.5

preimplantation loss

2.0

1.6

1.5

2.2

implantations

13.6

14.0

13.9

13.4

Gestation Rate:

Dose

Females with

 

viable fetuses on day 21 p.c.

total resorption

mg/kg b.w./day

n

in % of females with implantations

n

0

24

100.0

0

100

21

100.0

0

300

23

100.0

0

1000

22

100.0

0

Mean Values of the Parameters of Intrauterine Development:

Dose (mg/kg b.w./day)

0

100

300

1000

number of females

with implantations (a)

24

21

23

22

with viable fetuses (b)

24

21

23

22

mean values per female

placental weight in gb

0.57

0.59

0.57

0.61

number of fetusesb

13.1

13.4

13.4

12.8

postimplantation lossa, b

0.5, 0.5

0.6, 0.6

0.5, 0.5

0.6, 0.6

males in %b

55.9

50.7

50.4

52.4

fetal weight in gb

4.96

4.92

4.88

4.93

Fetal Malformations:

Malformation

Dose (mg/kg bw/day)

 

0

100

300

1000

umbilical cord shortened, generalized edema, domed head, skull bones partially missing, mandible shortened, ears missing, eye rudiment flat right, limbs reduced in size, abdominal hernia with protruding organs, absent genital tubercle, anal atresia, tail shortened and kinked

-

-

1

-

cleft palate

-

-

-

1

eye ball reduced in size with/without eye rudiment flat

2 (2)

2 (2)

1

1

situs inversus

-

1

-

-

retina folded

-

1

-

-

ventricular septal defect of the heart

-

1

-

-

malformation of 1stsacral vertebral arch with/without cartilage, pelvis shift

-

1

3 (2)

1

3rdlumbar vertebral arch has the shape of a 4thlumbar vertebral arch bilateral, cartilaginous part of 6thlumbar vertebral arch fused with iliac bone, 1stsacral vertebrae missing, bilateral, pelvis shift to cranial, bilateral

-

-

-

1

multiple skeletal malformation of the whole body, dysplasia of forelimbs and hind limbs, nearly completely missing ossification of head and sternebrae, cleft palate

-

-

1

-

number of fetuses per group

314

281

308

281

number of fetuses with malformations

2

6

5

4

malformed fetuses per group (%)

0.6

2.1

1.6

1.4

number of litters per group

24

21

23

22

number of litters with malformations

2

6

4

3

malformed litters per group (%)

8.3

28.6

17.4

13.6

() number of litters affected 

Fetal External and Visceral Deviations

Deviation

Dose (mg/kg b.w./day)

 

0

100

300

1000

eye rudiment flat

-

1

-

1

retina slightly folded

1

-

1

-

slight dilation of lateral brain ventricle(s)

2 (1)

2 (1)

-

1

slight dilation of 3rdbrain ventricle

-

2 (1)

-

1

thyroid gland reduced in size

2 (1)

4 (4)

3 (3)

4 (4)

thyroid gland enlarged

-

-

2 (2)

-

membranous part of trachea slightly folded, lying in tracheal lumen

1

1

2 (2)

-

thymus extended cranially

14 (10)

13 (9)

11 (7)

13 (8)

origin of left carotid artery displaced towards left subclavian artery on aortic arch

-

1

-

1

innominate artery and left carotid artery arise from aortic arch, left subclavian artery displaced and arises from descending aorta

1

-

-

-

thoracic cavity filled with brown mass

-

-

-

1

pericard filled with brown mass

1

-

-

-

brown mass in abdominal cavity

8 (6)

2 (2)

7 (6)

5 (5)

brown spot(s) in the liver

10 (7)

7 (6)

11 (9)

13 (8)

slight dilation of renal pelvis

10 (6)

7 (3)

6 (5)

7 (5)

dilation of renal pelvis

1

-

-

-

kidney flat and stretched

1

-

-

-

slight dilation of ureters

5 (4)

5 (2)

1

2 (2)

testi(e)s lying on bladder

5 (4)

4 (3)

6 (6)

2 (2)

testi(e)s lying slightly more cranially

6 (5)

1

7 (7)

4 (4)

right testis lying on the left side

-

1

-

-

circumscribed hard whitish area at choane, nasopharynx, larynx, nasal sinus, and/or palate

6 (4)

2 (2)

3 (3)

5 (3)

number of fetuses per group

314

281

308

281

number of fetuses with deviations

52

39

48

41

fetuses with deviat. per group (%)

16.6

13.9

15.6

14.6

number of litters per group

24

21

23

22

number of litters with deviations

23

17

23

19

litters with deviat. per group (%)

95.8

81.0

100.0

86.4

() number of litters affected

Applicant's summary and conclusion

Executive summary:

A developmental toxicity study according to OECD TG 414 was performed with the substance. Twenty-five inseminated female Wistar rats received daily doses of 0 (vehicle control), 100, 300 and 1000 mg/kg bw by gavage from day 6 to day 20 p.c. The fetuses were delivered by cesarean section on day 21 of gestation. Investigation was performed on general tolerance of the test substance by the females as well as on its effect on intrauterine development.

Appearance, behavior, mortality, absolute and corrected body weight gains, food intake, water intake, and fecal and urinary excretions were unaffected at dose levels up to 1000 mg/kg bw.

No treatment related gross pathological findings occurred at dose levels up to 1000 mg/kg.

The gestation rate, appearance and weights of placentas, postimplantation loss and correspondingly the number of fetuses, fetal sex distribution, and fetal weights were unaffected by treatment at dose levels up to 1000 mg/kg.

A treatment related effect on malformations, external, visceral, and skeletal (including cartilage) deviations was not evident at dose levels up to 1000 mg/kg.

Summarizing and evaluating all data investigated the following NOAELs were determined: for maternal toxicity 1000 mg/kg bw, for developmental toxicity 1000 mg/kg bw.