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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline study. Read across was performed with butyl methacrylate.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid

Test animals

Species:
rabbit
Strain:
Himalayan
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany
- Age at study initiation: 18-23 weeks
- Weight at study initiation: 1901-2589 g
- Fasting period before study: no
- Housing: the rabbits were housed singly in type 12.2395.C stainless steel wire mesh cages supplied by Draht-Bremer GmbH, Marktheidenfeld, Germany (floor area about 3,000 cm²)
- Diet: pelleted “Kliba maintenance diet for rabbits & guinea pigs, GLP, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, ad libitum
- Water: tap water ad libitum
- Acclimation period: at least 4 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12 / 12 (6.00 p.m. to 6.00 a.m. dark, 6.00 a.m. to 6.00 p.m. light)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The aqueous test substance preparations were prepared at the beginning of the administration period and thereafter at daily intervals, which took into account the analytical results of the stability verification. For the test substance preparation, an specific amount of test substance was weighed depending on the dose group, into a graduated flask, topped up (shortly under the marking) with 1% Carboxymethylcellulose suspension in drinking water and a few drops Cremophor EL and one drop of 32% hydrochloric acid. Afterwards the preparation was filled up with 1% Carboxymethylcellulose suspension in drinking water. The graduated flask was sealed and the preparation was intensely mixed with a magnetic stirrer. During administration, the
preparations were kept homogeneous with a magnetic stirrer.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the test substance preparations were sent to the analytical laboratory twice during the study period (at the beginning and towards the end) for verification of the concentrations. Samples were analyzed by GC with external calibration.
Details on mating procedure:
The animals were paired by the breeder (time-mated animals), the day of pairing was designated as gestation day (GD) 0. Presumed pregnant animals were supplied one day after mating; this day was referred to as GD 1 and the following day as GD 2.
Duration of treatment / exposure:
from implantation to one day prior to the expected day of parturition (GD 6-28)
Frequency of treatment:
daily
Duration of test:
until GD 29
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg/day
Basis:
nominal conc.
No. of animals per sex per dose:
25 time-mated females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
The dose volume was 10 mL/kg body weight. The calculation of the volume administered was based on the most recent individual body weight.

Examinations

Maternal examinations:
CLINICAL EXAMINATIONS
- Mortality: Mortality was checked in the females twice a day on working days or once a day on Saturdays, Sundays or on public holidays (GD 1-29).
- Clinical symptoms: A clinical examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. If such signs occurred, the animals were examined several times daily (GD 1-29).
- Food consumption: The food consumption was determined daily on GD 2–29.
- Body weight data: All animals were weighed on GD 1, 4, 6, 9, 11, 14, 16, 19, 21, 23, 25, 28 and 29. The body weight change of the animals was calculated.
- Corrected (net) body weight gain: The corrected body weight gain was calculated after terminal sacrifice (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6).

TERMINAL EXAMINATIONS OF THE DOES
After the does had been sacrificed on GD 29, they were necropsied and assessed by gross pathology in randomized order.
Ovaries and uterine content:
On GD 29, the surviving does were sacrificed in randomized order by an intravenous injection of pentobarbital (Narcoren; dose: 2 mL/animal). All prematurely dead or sacrificed females were examined following the same procedures as for females sacrificed on schedule with the exception that no uterine weights were determined.
After the does had been sacrificed, they were necropsied and assessed by gross pathology in randomized order. The uterus and the ovaries were removed and the following data were recorded:
- Weight of the unopened uterus
- Number of corpora lutea
- Number and distribution of implantation sites classified as:
1) live fetuses
2) dead implantations: a) early resorptions (only decidual or placental tissues visible or according to SALEWSKI (Salewski, 1964) from uteri from apparently non-pregnant animals and the empty uterus horn in the case of single-horn pregnancy); b) late resorptions (embryonic or fetal tissue in addition to placental tissue visible); c) dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened).
After the weight of the uterus had been determined, all subsequent evaluations of the does and the gestational parameters were conducted by technicians unaware of treatment group in order to minimize bias. For this purpose the animal numbers were encoded.
Furthermore, calculations of conception rate and pre- and postimplantation losses were carried out:
The conception rate (in %) was calculated according to the following formula: (number of pregnant animals)/(number of fertilized animals) x 100.
The preimplantation loss (in %) was calculated based on each individual pregnant animal with scheduled sacrifice according to the following formula: (number of corpora lutea – number of implantations)/(number of corpora lutea) x 100.
The postimplantation loss (in %) was calculated based on each individual pregnant animal with scheduled sacrifice from the following formula: (number of implantations – number of live fetuses)/(number of implantations) x 100.
Fetal examinations:
All fetal analyses were conducted by technicians unaware of the treatment group, in order to minimize bias.
- Examination of the fetuses after dissection from the uterus: Each fetus was weighed and examined macroscopically for any external findings.
Furthermore, the viability of the fetuses and the condition of the placentae, the umbilical cords, the fetal membranes, and fluids were examined. Individual placental weights were recorded. Thereafter, the fetuses were sacrificed by a subcutaneous injection of pentobarbital (Narcoren; 0.2 mL/fetus).
- Soft tissue examination of the fetuses: After the fetuses had been sacrificed, the abdomen and the thorax were opened in order to examine the organs in situ before they were removed. The heart and the kidneys were sectioned in order to evaluate the internal structure. The sex of the fetuses was determined by examination of the gonads in situ. After these examinations, the heads of approximately one half of the fetuses per litter and the heads of those fetuses, which revealed severe findings during the external examination (e.g. anophthalmia, microphthalmia or hydrocephalus) were severed from the trunk. These heads were fixed in BOUIN's solution and were, after fixation, processed and evaluated according to WILSON's method (Wilson and Warkany, 1965). About 10 transverse sections were prepared per head. After the examination these heads were discarded. All fetuses (partly without heads) were skinned and fixed in ethyl alcohol. After fixation for approx. 1-5 days, the fetuses were removed from the fixative for awhile. With a scalpel, a transversal incision was made into the frontal / parietal bone in the heads of the intact fetuses. The two halves of the calvarium were then cauteously bent outward and the brain was thoroughly examined. Subsequently, the fetuses were placed back into the fixative for further fixation.
- Skeletal examination of the fetuses: After fixation in ethyl alcohol the skeletons were stained according to a modified method of KIMMEL and TRAMMELL (Kimmel, C.A., and Trammell, C., 1981). Thereafter, the stained skeleton of each fetus was examined. After the examination the stained skeletons were retained individually.
Statistics:
- DUNNETT-test (two-sided) for food consumption, body weight, body weight change, corrected body weight gain (net maternal body weight change), carcass weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportions of preimplantation loss, proportions of postimplantation loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight.
- FISHER'S EXACT test (one-sided) for female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings.
- WILCOXON-test (one-sided) for proportions of fetuses with malformations, variations and/or unclassified observations in each litter.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Details on maternal toxic effects:
- Mortality: Seven high-dose females had to be sacrificed after abortion on several days towards the end of the treatment period (GD 24-28). Before abortion, the food consumption in all affected individuals was distinctly reduced and they showed adverse clinical symptoms like reduced and no defecation. These 7 premature sacrifices because of abortions are considered to be a consequence of test substance-induced maternal toxicity. There were no test substance-related or spontaneous mortalities in any other group.
- Clinical symptoms: Test substance-associated clinical findings such as reduced/no defecation and abortions were observed in the high-dose group. Although such findings are not uncommon in rabbits, they were, in these particular cases, most likely a consequence of the massively reduced food consumption in these rabbits. Taking this fact and the high incidence of findings in the high-dose group into account they are thus considered to be related to the treatment. By contrast the low frequency of findings like reduced defecation or blood in bedding does not suggest a relationship to the treatment in the low- and mid-dose groups.
- Food consumption: The food consumption in the high-dose females (1000 mg/kg bw/d) was distinctly and statistically significantly reduced during the almost entire treatment period (GD 6-27 and 28-29). During the treatment period (GD 6-28) the total average food consumption of the highdose
rabbits was about 44% below controls. Additionally, a particularly massive decrease of individual food consumption was noted for all high-dose does which aborted. During the days prior to abortion the food consumption was almost disrupted in these animals. The females of the mid-dose group (300 mg/kg bw/d) also had a statistically significant lower food consumption in a number of intervals during the treatment (GD 8-9, 13-16, 17-18, 19-20, 21-22, and 26-29). During the treatment period (GD 6-28) the total average food consumption of the mid-dose rabbits was about 18% below controls. The food consumption of the low-dose does (100 mg/kg bw/d) did not show test substance-related impairments. The reduced food consumption at the 300 and 1000 mg/kg bw/d levels is considered to be related to the treatment.
- Body weight data: The mean body weights of the high-dose rabbits (1000 mg/kg bw/d) were statistically significantly decreased on GD 21, 23, 25 and 29. At termination (GD 29) the body weight of the surviving pregnant high-dose rabbits was about 5% below the concurrent control. The average body weight gain of these rabbits was also statistically significantly reduced at a number of treatment intervals (GD 6-9, 16-19, 19-21, 25-28). The mean body weight gain of the high-dose rabbits was significantly reduced by about 47% during the treatment period. The mean body weights and the mean body weight gain of the low- and mid-dose rabbits (100 and 300 mg/kg bw/d) were not significantly different from the concurrent controls throughout the study.
- Corrected (net) body weight gain: Mean carcass weights were comparable among all groups. The corrected (net) body weight gain (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6) was statistically significant (about 54 %) below the concurrent control at the high-dose (1000 mg/kg bw/d) level. Although not statistically significant the net body weight gain was still 37% below control at the mid-dose (300 mg/kg bw/d) level. These reductions in net body weight gain arer considered to be related to the treatment. No adverse effect on net body weight gain was noted in the low-dose group.
- Uterus weight: The mean gravid uterus weight in the high-dose group (1000 mg/kg bw/d) was statistically significantly reduced (about 21% below the control). The mean gravid uterus weights of test groups 1 and 2 (100 or 300 mg/kg bw/d) did not show statistically significant differences in comparison to the control group.
- Necropsy findings: At necropsy, 7/25 high-dose does had stomach erosions, 5/25 high-dose does had no feces in the small intestine and 3/25 high-dose does had watery feces in the intestines. These findings are related to the markedly reduced food consumption and are considered to be treatment-related. In the mid- and low-dose groups, only incidental, not test substance-related findings were noted in single females of each of these test groups.
- Reproduction data of does: The conception rate was 100% in all test groups including controls (0; 100; 300 or 1000 mg/kg bw/d). Importantly, a sufficient number of pregnant females was available for the purpose of the study, as 18-25 pregnant rabbits per group had implantation sites in the uterus, at terminal sacrifice. The resorption rate (and hence the postimplantation loss) was statistically significant higher in the high-dose group (1000 mg/kg bw/d). This apparent effect was mainly caused by two does of this dose group which had only resorptions but no viable fetuses in the uterus and contributed to the average with 100% resorption rates. The litter losses in these two does are considered to be a direct consequence of maternal toxicity. In particular the markedly reduced food consumption after mid-gestation affected these animals in the same manner as the animals with abortions. All other does of the high-dose group did not show any changes of gestational parameters which were outside the normal range for this strain. There were no test substance-related and/or biologically relevant differences between the low- and mid-dose groups in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and the postimplantation losses, the number of resorptions and viable fetuses. Gestational parameters were within the normal range for animals of this strain and age; see also Part III (SUPPLEMENT) for historical control data.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
- Sex distribution of fetuses: The sex distribution of the fetuses in test groups 1-3 (100; 300 and 1000 mg/kg bw/d) was comparable to the control fetuses. Observable differences were without biological relevance.
- Weight of placentae: The mean placental weights in test groups 1, 2 and 3 (100; 300 and 1000 mg/kg bw/d) were comparable to the controls.
- Weight of fetuses: The mean fetal weight of the high-dose group (1000 mg/kg bw/d) was statistically significant below the corresponding control value (-10%). The mean fetal weights of the low- and mid-dose groups (100 or 300 mg/kg bw/d) were not influenced by the test substance. Neither female nor male weights showed statistically significant or biologically relevant differences between these groups and the controls.
- Fetal external malformations: External malformations, some associated with corresponding soft tissue and skeletal malformations, were recorded for single fetuses of all test groups including controls (0; 100; 300 and 1000 mg/kg bw/d). With the exception of cleft palate all external malformations were present in the historical control data. Each of the findings affected individual fetuses and neither statistically significant differences between the test groups nor a dose-response relationship were observed. No malformation pattern was evident. Thus an association of these individual findings to the treatment is not assumed. The total incidences of external malformations were comparable to the historical control data.
- Fetal external variations: One external variation (paw hyperflexion) occurred in single fetuses of the mid- and high-dose groups and the control. The incidences did not demonstrate a dose-response relationship and were comparable to the historical control data. Thus an association of this finding to the treatment is not assumed.
- Fetal external unclassified observations: No unclassified external observation was recorded in this study.
- Fetal soft tissue malformations: The examination of the soft tissues revealed a variety of malformations in fetuses of all test groups including the controls (0; 100; 300 and 1000 mg/kg bw/d). All individual soft tissue malformations were present in the historical control data at comparable frequencies. Neither statistically significant differences between the test groups nor a dose-response relationship were observed. No malformation pattern was evident. Thus an association of these findings to the treatment is not assumed.
- Fetal soft tissue variations: A number of soft tissue variations such as absent lung lobe (lobus inferior medialis) and malpositioned carotid branch was detected in each test group including the controls. Incidences were without a relation to dosing. Neither statistically significant differences between the test groups nor differences to the historical control data were noted.
- Fetal soft tissue unclassified observations: Unclassified soft tissue observations, such as infarct of liver, discolored kidney, hemorrhagic ovary and blood coagulum around urinary bladder, were recorded for some fetuses of test groups 0, 1 and 2 (0; 100 and 300 mg/kg bw/d). A relation to dosing is not present. Therefore, a test substance induced effect is not assumed.
- Fetal skeletal malformations: Malformations of the fetal skeletons were noted in all test groups including controls (0; 100; 300 and 1000 mg/kg bw/d). All individual skeletal malformations were present in the historical control data. With the exception of severely fused sternebrae (bony plate), which was observed at a slightly higher incidence, the frequencies of those malformations were comparable to the historical control data. With regard to the total malformation incidences, in particular the litter incidence, neither statistically significant differences between treated groups and control nor a dose-response relationship were observed.
- Fetal skeletal variations: For all test groups, variations in different skeletal structures were detected with or without effects on the corresponding cartilages. The observed skeletal variations were related to various parts of the fetal skeletons and were statistically significant higher exclusively in the
high dose group on a fetus per litter basis. Several specific skeletal variations were statistically significant higher than the concurrent and outside the historical control in the high dose group (on a fetus per litter basis). These findings are delays or minor disturbances of ossification which are reversible or do not considerably affect the integrity of the underlying structures. Such slight changes of the ossification process occur very frequently in gestation day 29 rabbit fetuses of this strain, but considering the overall higher rate of skeletal variations in the top-dose group they may be correlated to the treatment.
- Fetal skeletal unclassified cartilage observations: Additionally, isolated cartilage findings without impact on the respective bone structures, which were designated as unclassified cartilage observations, occurred in all groups including the control. The observed unclassified cartilage findings did not show a relation to dosing and were considered to be spontaneous in nature.
- Abstract of all classified fetal external, soft tissue and skeletal observations: Various external, soft tissue and skeletal malformations occurred throughout all test groups including the control. The most salient of these malformations affected the head/skull (malformed skull bones, cleft palate), the heart/great vessels (various defects), gall bladder (absent), kidneys (absent, small) and the sternebrae (fused to a bone plate). All malformations are present in the historical control data, with the exception of cleft palate. No dose-response relationship is evident for the individual malformations. At the low- and mid-dose levels (100 and 300 mg/kg bw/d) the overall incidences are comparable to the concurrent and historical controls, whereas the overall incidence is slightly above the controls at the high-dose level. Importantly, as a number of morphological structures of different ontogenic origin is affected these various fetal findings do not form a distinct and consistent malformation pattern. One external (paw hyperflexion), two soft tissue (absent lobus inferior medialis and malpositioned carotid branch) and a broad range of skeletal variations occurred in all test groups including the controls. At the low- and mid-dose levels (100 and 300 mg/kg bw/d) all fetal and litter incidences for these variations and the corresponding rates of affected fetuses/litter were comparable to the control whereas at the high-dose level a slightly higher rate of affected fetuses per litter was noted. However, all incidences including the high-dose incidence can be found at a comparable frequency in the historical control data. A spontaneous origin is assumed for soft tissue and unclassified skeletal cartilage observations, which were observed in several fetuses of test groups 0, 1, 2 and 3 (0; 100; 300 and 1000 mg/kg bw/d) and the control. Distribution and type of these findings did not suggest relation to treatment.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Table: Postimplantation loss and resorption data

 

 

Group 0

0 mg/kg/d

Group 1

100 mg/kg/d

Group 2

300 mg/kg/d

Group 3

1000 mg/kg/d

HCD

(range)

Postimplantation loss

Mean %

8.9

8.2

7.1

23.1*

12.1

(4.6 – 50.0)

Total resorptions

Mean

0.6

0.6

0.5

1.6*

0.7

(0.4 – 3.1)

Mean %

8.9

8.2

7.1

23.1*

12.0

(4.6 – 50.0)

Late resorptions

Mean

0.2

0.3

0.3

0.9*

---

Mean %

2.0

4.5

3.9

14.2*

---

HCD = Historical control data; * = p ≤ 0.05 (DUNNETT-test [two-sided])

 

 

Table: Occurence of statistically significantly increased fetal skeletal variation (expressed as mean percentage of affected fetuses/litter)

Finding

Group 0

0 mg/kg/d

Group 1

100 mg/kg/d

Group 2

300 mg/kg/d

Group 3

1000 mg/kg/d

HCD

(range)

Incomplete ossification of parietal; unchanged cartilage

0.0

1.1

1.1

4.2*

0.2

(0.0 – 1.0)

Splitting of skull bone

1.2

1.2

2.6

8.1**

2.8

(0.0 – 7.1)

Supemumerary 13th rib; cartilage present

3.8

5.3

2.5

13.1*

4.0

(0.0 – 8.7)

Supemumerary 13th rib; cartilage not present

10.7

10.3

7.6

33.5**

6.2

(2.1 – 13.6)

Total fetal skeletal variations

64.8

63.3

66.4

79.3*

63.1

(46.3 – 78.9)

HCD = Historical control data; * = p ≤ 0.05, ** = p ≤ 0.01 (Wilcoxon-Test [one-sided])

 

 

Table: Total fetal malformations

 

 

Group 0

0 mg/kg/d

Group 1

100 mg/kg/d

Group 2

300 mg/kg/d

Group 3

1000 mg/kg/d

Litter

Fetuses

N

N

25

172

25

165

25

176

16

100

Fetal incidence

N (%)

6 (3.5%)

8 (4.8%)

6 (3.4%)

11 (11%)

Litter incidence

N (%)

6 (24%)

6 (24%)

6 (24%)

7 (44%)

Affected fetuses/litter

Mean%

3.9

4.7

3.2

14.3*

* = p ≤ 0.05 (Wilcoxon-Test [one-sided])

 

 

Table: Total fetal variations

 

 

Group 0

0 mg/kg/d

Group 1

100 mg/kg/d

Group 2

300 mg/kg/d

Group 3

1000 mg/kg/d

Litter

Fetuses

N

N

25

172

25

165

25

176

16

100

Fetal incidence

N (%)

122 (71%)

113 (68%)

125 (71%)

82 (82%)

Litter incidence

N (%)

25 (100%)

24 (96 %)

25 (100 %)

16 (100%)

Affected fetuses/litter

Mean%

71.9

69.7

72.5

83.1*

* = p ≤ 0.05 (Wilcoxon-Test [one-sided]

Applicant's summary and conclusion