Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011/2012
Reliability:
1 (reliable without restriction)

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Principles of method if other than guideline:
Twenty-five inseminated female Wistar rats each were treated daily orally by gavage with Vulcuren (purity 96.9 %) in corn oil from day 6 to day 20 p.c. in the following doses: 0, 100, 300, and 1000 mg/kg body weight (bw)/day, respectively. 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.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Name used in Raw Data, Study Plan, and Report: Vulcuren
CAS No.: 151900-44-6
Purity: 96.9 %

Test animals

Species:
rat
Strain:
Wistar

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Analytical verification of doses or concentrations:
yes
Details on mating procedure:
The animals were mated by placing two females 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:
From days 6 to 20 p.c.
Frequency of treatment:
Daily
Duration of test:
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.
No. of animals per sex per dose:
Twenty-five inseminated females per dose
Control animals:
yes, concurrent vehicle

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Gross pathological findings:
no effects observed

Effect levels (maternal animals)

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

Results (fetuses)

Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Basis for effect level:
other: embryotoxicity

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

Analysis of Test Item in the Formulation:

Stability was confirmed analytically.

Appearance, Behavior, and Mortality:

No remarkable clinical observations occurred at dose levels up to 1000 mg/kg. Thus, appearance, behavior, and mortality were unaffected by treatment at dose levels up to 1000 mg/kg bw/day.

Body Weights:

Mean Body Weight Gain

Dose (mg/kg b.w./day):      

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

0 100 300 1000 mg/kg

absolute body weight gain (g) days 6 - 20 p.c.

103.6 109.3 103.0 99.0

absolute body weight gain (g) days 0 - 21 p.c.

142.2 149.5 142.7 139.6

corrected body weight gain (g) days 0 - 21 p.c.

50.4 51.5 50.8 44.7

Overall: absolute and corrected body weight gains were unaffected by treatment at dose levels up to 1000 mg/kg.

Food and Water Intake, Excretory Products:

Food intake, water intake, and fecal and urinary excretions were unaffected by treatment at dose levels up to 1000 mg/kg.

Necropsy:

One female of the 1000 mg/kg group showed a dilation of renal pelvis unilateral, for which a treatment related effect is not assumed, as two females of the current control group also revealed this finding, and because this finding is known as a spontaneous finding in the rat strain used. One female each of the 300 mg/kg and 100 mg/kg groups revealed a uterus filled with brownish or clear fluid, for which a treatment related effect is not assumed due to a lacking dose dependency.

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

General Reproduction Data

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

0 100 300 1000

inseminated females

25 25 25 25

inseminated females evaluated

25 25 24 25

females with implantations

24 24 21 22

in % of those inseminated

96.0 96.0 87.5 88.0

corpora lutea

15.8 15.4 15.7 15.9

preimplantation loss

1.5 1.3 1.8 1.1

implantations

14.3 14.1 14.0 14.7

Overall: 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.

Gestation Rate:

 Dose

mg/kg b.w./day

 Number of females with viable fetuses on day 21 p.c.  n % of females with implantations  number of total resorption
 0  24  100.0  0
 100  24  95.8  1
 300  21  100.0  0
 1000  22  100.0  0

The gestation rate was decreased at the 100 mg/kg group by one female with a total resorption, for which a treatment related effect is excluded due to a lacking dose dependency, and since single total resorptions are known as spontaneous findings in the rat strain used. Thus, the gestation rate was unaffected by treatment at dose levels up to 1000 mg/kg.

Mean Values of the Parameters of Intrauterine Development

 Dose (mg/kg b.w./day)  0  100   300  1000 
number of females        
  with implantations  24  24  21  22
with viable fetuses  24  23  21 22
mean values per female        
  placental weight in g  0.59  0.56 0.59 0.59 
  number of fetuses  13.3  14.5  13.4  13.8
  postimplantation loss  0.9,  0.3  0.5 1.0
  males in %  48.4  41.3  54.9  55.7
  fetal weight in g  4.86  4.83  4.87  4.83

Overall: there are no effects on the above mentioned parameters of intrauterine development. The appearance of placentas revealed 10 engorged placentas out of 1 litter at the 1000 mg/kg, level, for which a treatment related effect cannot be excluded, because this value lay outside the range of historical control data. The mean placental weights were unaffected by treatment at dose levels up to 1000 mg/kg.

Fetal Malformations:

 Dose (mg/kg b.w./day)  0  100  300  1000
 cleft palate  -  -  -  1
 eye ball(s) reduced in size with/without eyehole reduced in size  1  2 (2)  1  -
 generalized edema  -  -  -  1
 hairline ventricular septal defect of the heart  -  -  1  -
 malformation of 1st sacral vertebral arch with/without cartilage, pelvis shift  3 (2)  2 (2)  1  1
 7th cervical vertebral arch fused with 1st thoracic vertebral arch right, head of 1st rib missing right  -  -  1  -
 multiple skeletal malformation of ribs, sternebrae, and vertebrae  -  1  -  -
 humerus bent  -  -  -  !
 clavicle bent  1 -  -
 number of fetuses per group  320  333  282 303 
 number of fetuses with malformations  4  5  4  4
 malformed fetuses per group (%)  1.3  1.5  1.4  1.3
 number of litters per group  24  23  21  22
 number of litters with malformations 3  5  4  4 
 malformed litters per group (%) 12.5  21.7 19.0    18.2 

() number of litters affected

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 (microphthalmia, generalized edema, ventricular septal defect, vertebral malformations with pelvis shift, malformation of ribs and sternebrae, dysplastic forelimb bones and comparable with spontaneous findings in the current control group and/or historical control groups.

At the 1000 mg/kg level, one fetus revealed a cleft palate, 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 2002 in the same laboratory). Thus, a treatment related effect is not assumed for this isolated finding. Furthermore, each one fetus of the 1000 mg/kg group showed a generalized edema or a bent humerus, which are also known as spontaneous findings in the rat strain used. Therefore, a treatment related effect is not assumed for these findings.

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.

Fetal External and Visceral Deviations.

 Dose (mg/kg b.w./day)  0  100  300  1000
 slight edema -  -  -  1 
 skin pale  - -  -  1 
 brown mass in nuchal fatty tissue  1  -  1  -
duct of sublingual gland slightly enlarged  -  1  -  -
 circumscribed hard whitish area in nasal cavities  2 (1)  4 (2) -  3 (2) 
 retina slightly folded  -  2 (2)  -  -
 slight dilation of 3rd brain ventricle  -  -  1  -
 thyroid gland reduced in size  2 (2)  2 (1)  3 (3)  3 (2)
 membranous part of trachea slightly folded, lying in tracheal lumen  -  -  1  -
 thymus extended cranially  12 (10)  13 (11)  18 (14)  17 (10)
 innominate artery reduce in length  1  -  -  1
 innominate artery missing  -  1  -  -
 pericard filled with dark brown mass  1  -  1  -
 brown mass in abdominal cavity  7 (4)  12 (9)  14 (6)  7 (4)
 brown spot(s) in the liver  8 (6)  15 (10)  15 (8)  10 (6)
 dilation of stomach  -  1  -  -
slight dilation of renal pelvis  12 (6)  6 (5)  2* (2)  4 (4) 
 kidney flat and stretched  1  -  -  -
 slight dilation of ureters  8 (4)  2 (2)  0*  2 (2)
 testi(e)s lying on bladder  3 (3) 3 (2)    6 (6)  5 (5)
 testi(e)s lying slightly more cranially  5 (3)  5 (5)  8 (8)  5 (5)
 testi(e)s lying more cranially  -  1  1  1
 right testis lying on the left side  -  2 (2)  -  -
 number of fetuses per group  320  333  282  303
 number of fetuses with deviations  39  54  50  41
 fetuses with deviat. per group (%)  12.2 16.2  17.7  13.5 
 number of litters per group  24  23  21  22
 number of litters with deviations  18  20  20  18
 litters with deviat. per group (%) 75.0  87.0  95.2  81.8 

() number of litters affected

Statistically significant difference to control * = p < 0.05

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 basis in the 300 mg/kg 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 (see Annex, Section 8.9.12) 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 another prenatal developmental toxicity study with the same rat strain 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.

Fetal Skeletal Deviations Including Cartilaginous Deviations:

At the 1000 mg/kg level, fetal examinations for skeletal retardations and variations revealed statistically significantly retarded ossification of isolated localizations (1st distal phalanges of digits right, 5th proximal phalanges of toes bilateral), when calculation was done on a fetal basis, for which a treatment related effect was not evident, because statistical significance was lacking, when calculation was done on a litter basis, and since all values lay within the range of historical control data.

At the 300 mg/kg level, fetal examinations for skeletal retardations and variations revealed statistically significantly progressed ossification of a single localization (5th distal phalanges of toes left), when calculation was done on a fetal basis, for which a treatment related effect was not evident, because statistical significance was lacking, when calculation was done on a litter basis, and since a dose dependency was lacking.

At the 100 mg/kg level, fetal examinations for skeletal retardations and variations revealed statistically significantly retarded ossification of isolated localization (1st distal phalanges of digits right, calcaneus bilateral), when calculation was done on a fetal basis, for which a treatment related effect was not evident, because statistical significance was lacking, when calculation was done on a litter basis, and since a dose dependency was lacking. Furthermore statistically significantly progresses ossification occurred at this dose level at isolated localizations (1st distal phalanges of digits left, 2nd distal phalanges of digits right, 5th distal phalanges of digits bilateral, 1st distal phalanges of toes bilateral, 5th distal phalanges of toes right), when calculation was done on a fetal basis, for which a treatment related effect was not evident due to a lacking dose dependency, and because statistical significance was lacking, when calculation was done on a litter basis.

Evaluation of fetal cartilaginous tissue revealed no treatment related findings at dose levels up to 1000 mg/kg. Histopathological examination of several fetuses at all dose levels with additional alcian blue stained tissue between vertebral bodies of the spine most likely confirmed a staining artifact.

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.

Applicant's summary and conclusion

Conclusions:
NOAEL maternal toxicity: 1000 mg/kg bw/day.
NOEL appearance of placentas: 300 mg/kg bw/day. An increased incidence of engorged placentas occurred at the 1000 mg/kg level in one litter, for which a treatment related effect cannot be excluded.
NOAEL remaining developmental toxicity: 1000 mg/kg bw/day.
Executive summary:

In this OECD TG 414 guideline study twenty-five inseminated female Wistar rats each were treated daily orally by gavage with Vulcuren (purity 96.9 %) in corn oil from day 6 to day 20 p.c. in the following doses: 0, 100, 300, and 1000 mg/kg body weight (bw)/day, respectively. 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.

Toxicity to the Females:

Appearance, behavior, and mortality were unaffected by treatment at dose levels up to 1000 mg/kg bw/day. Absolute and corrected body weight gains were unaffected by treatment at dose levels up to 1000 mg/kg. Food intake, water intake, and fecal and urinary excretions were unaffected by treatment at dose levels up to 1000 mg/kg. No treatment related gross pathological findings occurred at dose levels up to 1000 mg/kg.

Toxicity to Intrauterine Development:

The gestation rate was unaffected by treatment at dose levels up to 1000 mg/kg. An increased incidence of engorged placentas occurred at the 1000 mg/kg level, for which a treatment related effect cannot be excluded, whereas weights of placentas were unaffected by treatment at dose levels up to 1000 mg/kg. Postimplantation loss and correspondingly the number of fetuses were unaffected by treatment at dose levels up to 1000 mg/kg. Fetal sex distribution was unaffected by treatment at dose levels up to 1000 mg/kg. The fetal weights were unaffected by treatment at dose levels up to 1000 mg/kg. A treatment related effect on malformations was not evident at dose levels up to 1000 mg/kg. A treatment related effect on external and visceral deviations was not evident at dose levels up to 1000 mg/kg. A treatment related effect on skeletal deviations (retardations, variations, including cartilaginous tissue findings) was not evident at dose levels up to 1000 mg/kg.

Overall:

NOAEL maternal toxicity: 1000 mg/kg bw/day

NOEL appearance of placentas: 300 mg/kg bw/day. An increased incidence of engorged placentas occurred at the 1000 mg/kg level in one litter, for which a treatment related effect cannot be excluded.

NOAEL remaining developmental toxicity: 1000 mg/kg bw/day