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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Jan 2009 - March 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
(2001)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2-benzofuran-1,3-dione, addition product with 2-(2-hydroxyethoxy)ethanol, ethoxylated
EC Number:
701-218-5
Molecular formula:
Not applicable (UVCB substance)
IUPAC Name:
2-benzofuran-1,3-dione, addition product with 2-(2-hydroxyethoxy)ethanol, ethoxylated
Test material form:
liquid
Specific details on test material used for the study:
Stability in the solvent was analytically proven.

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Strain: Hsd Cpb:WU (SPF-bred)
- Source: Harlan-Nederlands, 5960 AD Horst, The Netherlands
- Age at study initiation: 12-14 weeks
- Weight at study initiation: 204 - 261 g (females); 421 - 545 g (males)
- 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
- Humidity (%): approximately 55
- Air changes (per hr): > 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The formulations were prepared as needed taking into account the analytically determined stability.

VEHICLE
- Lutrol® (PEG 400).
- Justification for use and choice of vehicle (if other than water): The test item in the vehicle gave a solution which was analytically confirmed to be stable for at least 8 days.
- Amount of vehicle (if gavage): 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Content checks of the active ingredient in samples with concentrations of 20, 60, and 200 mg/mL were carried out twice during the study.
For this purpose representative samples were dissolved with a mixture of acetonitrile/ aqueous 0.05 % formic acid (4:1) and subsequently quantified by reversed phase (C18) HPLC with UV-detection (DAD, wavelength 200 nm). Standard solutions of the authentic test item were used for calibration. The results of the content checks in samples with concentrations of 20, 60, and 200 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 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:
Days 6 - 20 p.c.
Frequency of treatment:
once daily (between 06:00 and 12:00 CET)
Duration of test:
From study initiation date to end of in-life-phase 35 days.
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
25 females
Control animals:
yes, concurrent vehicle
Details on study design:
The dose levels were selected based on the results of a previous dose toleration study in rats with 1000 mg/kg, which revealed no treatment related findings.

Examinations

Maternal examinations:
CLINICAL EXAMINATIONS: Yes
- Time schedule: The females were inspected from days 0 to 21 p.c. twice daily (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 weight of the females was determined on day 0 p.c. and daily from day 6 to day 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 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 CONSUMPTION: Yes
- Water intake was assessed daily by visual estimation of the quantities left over and reported together with clinical findings.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
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]
Furthermore, the eyes of fetuses No. 533, 582, 1136, and 1175 exemplarily underwent histopathological examination after investigation according to the modified WILSON technique to clarify unusual findings.

Findings in abdominal, pelvic, and thoracic organs as well as skeletal and cartilage findings by the modified DA WSON technique with the addition of cartilage staining [method described by INOUYE, modified]: evisceration, cartilage staining with alcian blue GX, clearing of the fetuses with diluted potassium hydroxide solution, staining of the skeletal system with alizarin red S, and evaluation of the skeletal system including cartilaginous findings (processing and evaluation after cesarean section without knowledge of treatment group ). Every other fetus within a litter was prepared for either skeletal or visceral evaluation with generally the first fetus of each litter assigned to skeletal evaluation.
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:
Body Weight Gains: Calculated against the body weight on Day 6 post-coitum.
Corrected Body Weight Gains: Body weight determined on Day 21 post-coitum minus the body weight on Day 0 to 21 post-coitum and the weight of gravid uterus on day 21 post-coitum.
Relative Food Consumption: Calculated against the body weight for scheduled intervals.
Fertility rate: percentage of inseminated females with implantations
Pre-implantation loss (%): number of corpora lutea minus number of implantation sites
Gestation rate: percentage of females with viable fetuses on day 21 pc of those with implantation sites
Post-implantation loss (%): ((number of implantation sites - number of live fetuses)/(number of implantation sites)) x 100
Historical control data:
historical control data

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
One female of the 1000 mg/kg level revealed respiratory sounds and transient piloerection, and one further female ofthis dose group also revealed piloerection for one single day. A treatment related effect is not assumed due to the isolated occurrence, and since these fmdings
may occur spontaneously in the rat strain used (see Annex, Section 590). Furthermore, 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.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Absolute and corrected body weight gains were unaffected by treatment at dose levels up to and including 1000 mg/kg.
Food efficiency:
no effects observed
Description (incidence and severity):
The placental weights were unaffected by treatment, see table 1.
Description (incidence and severity):
One pale placenta occurred at the 1000 mg/kg bw level, for which a treatment related effect is not assumed due to its single occurrence, and as this finding is known as a spontaneous findin in the rat strain used. Thus, the appearance of placentas was unaffected by treatment.
Other effects:
no effects observed
Description (incidence and severity):
The fertility rate (percentage of inseminated females with implantations) was unaffected by treatment see table 1.

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
see table 1
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
see table 1
Early or late resorptions:
no effects observed
Description (incidence and severity):
see table 1
Dead fetuses:
no effects observed
Description (incidence and severity):
see table 1
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
see table 1
Details on maternal toxic effects:
Maternal toxic effects:no effects

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

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Remarks:
Maternal toxicity
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No adverse findings in maternal animals up to and incl. 1000 mg/kg

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
see table 1
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
see table 1
Changes in sex ratio:
no effects observed
Description (incidence and severity):
fetal sex distribution was unaffected by treatment.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
see table 1
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
see Attachments 1 (fetal malformations), 2 (historical control data for spontaneous malformations), 3 (external and visceral malformations) and 4 (historical control data for spontaneous external and visceral deviations):
The criteria for classifying the observed external or visceral findings as deviation or malformation are shown in the Attachment 6, Section 8.5. If fetuses revealed more than one external or visceral deviation, the number of deviations is higher than the number of affected fetuses in these groups.

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 and including 1000 mg/kg.
The types of malformations observed in this study are generally a representative sample of spontaneous malformations in the rat strain used (meningoencephalocele, abdominal hernia, microphthalmia/anophthalmia, malposition of forelimb, cardiovascular malformations, sternal malformations, vertebral malformations with pelvis shift, dysplastic forelimb bones or skull bones (see historical control data in Attachment 2).
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 ( each two fetuses out of two litters) and were thus unaffected by treatment at dose levels up to and including l000mg/kg.
At the 1000 mg/kg level, fetal examinations for malformations revealed some further findings (malposition of forelimb, ventricular septal defect of the heart, atrial septal defect and ventricular septal defect of the heart with pulmonary trunk arisen from left ventricle,
dysplastic forelimb bones with malformation of sternum and ribs and one supernumerary lumbar vertebra, one supernumerary lumbar vertebra with misshapen 7th lumbar vertebral
arches and pelvis shift, and dysplastic humerus ), for which a treatment related effect is not assumed, as these findings also occurred in the current control group (ventricular septal defect of the heart as part of a multiple thoracic malformation) and/or historical control groups (see Attachment 2, Section 8.8.11: dysplasia of forelimb bones: 4.31 % affected fetuses and 20.00 % affected litters in study T5068551; atrial septal defects of the heart: 2.12 % affected fetuses and 18.18 % affected litters in study Tl067765; ventricular septal defects of the heart: 2.17 % affected fetuses and 26.32 % affected litters in study T0073280; malposition of limbs (malrotation of hind limb(s)): 0.5 % affected fetuses and 5.6 % affected litters in study T0076746, and remaining findings).
At the 300 mg/kg level, fetal examinations for malformations revealed two fetuses out of two litters with folded retina, each without a dose dependency, and two further isolated findings (multiple malformation including meningoencephalocele and abdominal hernia, ventricular septal defect of the heart), for which a treatment related effect is not assumed due to the single occurrences, and as these findings also occurred in the current control group (multiple malformation, folded retina, ventricular septal defect of the heart as part of a multiple thoracic malformation) and/or historical control groups (see Attachment 2, Section 8.8.11: multiple malformation, meningoencephalocele, abdominal hernia (umbilical hernia), folded retina, ventricular septal defect of the heart).
At the 100 mg/kg level, fetal examinations for malformations revealed some further isolated findings (folded retina, malformation of vertebrae, sternum, ribs, and skull bones), for which a treatment related effect is not assumed, as these findings also occurred in the current control group (folded retina) and/or historical control groups (see Attechment 2, Section 8.8.11: folded retina, malformation of vertebrae, sternum, ribs, and skull bones).
Thus, a treatment related effect on malformations (incidence or type) was not evident at dose levels up to and including 1000 mg/kg.
Description (incidence and severity):
see Attachments 5 (historical control data for spontaneous skeletal findings (section 8.8.13).
The criteria for classifying the observed external or visceral findings as deviation or malformation are shown in Attachment 6, Section 8.5. If fetuses revealed more than one external or visceral deviation, the number of deviations is higher than the number of affected fetuses in these groups.

Fetal Skeletal Deviations Including Cartilaginous Deviations
At the 100 mg/kg and 300 mg/kg levels, fetal examinations for skeletal retardations and variations revealed statistically significantly retarded ossification of a few localizations (300 mg/kg: 1st distal phalanges of digits left; 100 mg/kg: 1st distal phalanges of digits left, 2nd proximal phalanges of digits left, 3rd proximal phalanges of digits left, 4th proximal phalanges of digits right, 5th proximal phalanges of digits right, 5th distal phalanges of toes right, parietal bones 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.
At the 100 mg/kg and 300 mg/kg, and 1000 mg levels, statistically significantly progressed ossification occurred (1000 mg/kg and 300 mg/kg: calcaneus bilateral; 300 mg/kg: 8th caudal vertebral bodies; 100 mg/kg: 2nd sternebrae), when calculation was done on a fetal basis, for which a treatment related effect was not evident due to a lacking dose dependency except for calcaneus, because statistical significance was lacking, when calculation was done on a litter basis, and since the values lay inside the range of historical control data except for 8th caudal vertebral bodies (see Attachment 5, Section 8.8.13).
A statistically significantly increased incidence of wavy ribs (10th right, 11th right and sum) and a statistically significantly decreased incidence of punctiform 14th ribs right occurred at the 100 mg/kg level, when calculation was done on a fetal basis. A treatment related effect for these findings was not evident, because statistical significance is lacking, when calculation was done on a litter basis (except for wavy ribs sum), and due to a lacking dose dependency.
A statistically significantly decreased incidence of dumbbell shaped 11th thoracic vertebral bodies occurred at the 1000 mg/kg level, when calculation was done on a fetal basis. A treatment related effect for this finding was not evident, because statistical significance is lacking, when calculation was done on a litter basis.
Evaluation of fetal cartilaginous tissue revealed a statistically significantly increased incidence of unciformed cartilaginous ends of 8th ribs at the 300 mg/kg level, for which a treatment related effect was not evident due to a lacking dose dependency.

Thus, a treatment related effect on skeletal deviations (retardations, variations, including cartilaginous tissue findings) was not evident at dose levels up to and including 1000 mg/kg.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
see Attachments 1 (fetal malformations), 2 (historical control data for spontaneous malformations), 3 (external and visceral malformations) and 4 (historical control data for external and visceral deviations):
The criteria for classifying the observed external or visceral findings as deviation or malformation are shown in Attachment 6, Section 8.5. If fetuses revealed more than one external or visceral deviation, the number of deviations is higher than the number of affected fetuses in these groups.

Attachment 3 (Table 5-7) gives an overview on external and visceral deviations (findings other than malformations) in live fetuses.
The overall incidences of fetuses or litters with external and visceral deviations were
unaffected at dose levels up to and including 1000 mg/kg and revealed the highest value on a fetal basis in the 100 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 Attachment 4, 8.8.12) and represented the normal range of scattering in the rat strain used.
Exemplarily histopathological evaluation of fetuses no. 533, 582, 1136, and 1175 (whitish
mass between lens and cornea) revealed lens material in the anterior eye chamber, which is regarded as artifacts, so that remaining fetuses with this finding were judged equally.
Histopathological evaluation of additional circumscribed hard whitish tissue in the nasopharynx was performed in a recently conducted 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.
The finding of a small hollow structure in different locations of the head is also considered as a fixation artifact, which also occurred in all dose groups of a recent prenatal developmental toxicity study (T7078264; control group: two fetuses out of two litters; low dose group: one fetus; medium dose group: two fetuses out of two litters; high dose group: one fetus) with the same rat strain used, without showing a dose dependency.
A statistically significantly increased incidence of slight dilation of renal pelvis occurred at the 100 mg/kg level, for which a treatment related effect is not assumed due to a lacking dose dependency, and as the incidence lay inside the range of historical control data (see Attachment 4, Section 8.8.12).
Slight dilation of lateral brain ventricle(s) occurred in all dose groups, for which a treatment related e:ffect is not assumed due to a lacking dose dependency, and as the incidences lay inside the range of historical control data (see Attachment 4, Section 8.8.12: 5.16 % affected fetuses and 47.37 % affected litters in study T6062954).
The remaining findings of the dose groups showed no dose dependency or were isolated findings.
Thus, a treatment related effect on external and visceral deviations was not evident at dose levels up to and including 1000 mg/kg.
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
The gestation rate was unaffected by treatment at dose levels up to and including 1000 mg/kg.
Appearance and weights of placentas were unaffected by treatment at dose levels up to and including 1000 mg/kg.
Postimplantation loss and correspondingly the number of fetuses were unaffected by treatment at dose levels up to and including 1000 mg/kg.
Fetal sex distribution was unaffected by treatment at dose levels up to and including 1000 mg/kg.
The fetal weights were unaffected by treatment at dose levels up to and including 1000 mg/kg.
A treatment related effect on malformations was not evident at dose levels up to and including 1000 mg/kg.
A treatment related effect on external and visceral deviations was not evident at dose levels up to and including 1000 mg/kg.
A treatment related effect on skeletal deviations (retardations, variations, including cartilaginous tissue findings) was not evident at dose levels up to and including 1000 mg/kg.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no treatment related effects at any dose

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Table 1: general reproduction data

 dose (mg/kg bw/day)  0  100  300  1000
 inseminated females  25  25  25  25
 inseminated females evaluated  25  25  25  25
 females with implantations  23  25  22  24
 in % of those inseminated  92.0  100  88.0  96.0
 mean values per female with implantation sites        
 corpora lutea  14.6  15.6  15.4  14.8
 preimplantation loss  1.3  1.5  1.4  1.9
 implantations  13.3  14.1  14.0  12.9
 gestation rate        
 females with viable fetuses on day 21 pc  23  25  22  24
 in % of meles with implantations  100  100  100  100
 total resorptions  0  0  0  0
 intrauterine development; mean value per female        
 placental with in g  0.59  0.60  0.60  0.60
 number of fetuses  12.8  13.7  13.7  12.6
 postimplantation loss  0.5, 0.5  0.4, 0.4  0.2, 0.2  0.3, 0.3
 male fetuses in %  48.5  49.7  51.9  53.5
 fetal weight in g  4.98  5.00  5.03  5.02
         

Applicant's summary and conclusion

Executive summary:

A developmental toxicity study with the substance was performed according to OECD TG 414. Twenty-five inseminated female Wistar rats per dose group were administered (gavage) daily from day 6 to 20 p.c. the test substance at doses of 0, 100, 300, and 1000 mg/kg bw in Lutrol®(PEG 400). 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 and including 1000 mg/kg bw.


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


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 the treatment at dose levels up to and including 1000 mg/kg bw.


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


Summarizing and evaluating all data investigated revealed a NOAEL of 1000 mg/kg bw/day for each maternal toxicity and developmental toxicity.