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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:
2017-08-14 to 2017-09-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
22 Jan 2001
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
Aug 1998
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No 440/2008 of 30 May 2008, Part B: Methods for the determination of toxicity and other health effects: Prenatal Developmental Toxicity Study; Official Journal of the European Union, No. L 142
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: 010545EDA0
- Expiration date of the lot/batch: Jan 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: refrigerator, avoid temperatures > 35 °C

Test animals

Species:
rabbit
Strain:
New Zealand White
Remarks:
Crl:KBL(NZW)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH/ Charles River Laboratories, France
- Age at study initiation: 29-38 days
- Weight at study initiation: 3046 – 4305 g
- Fasting period before study: no
- Housing: single, Cage System, Type 4X03B700CP, with Wooden gnawing blocks (Typ KNH E-041), Abedd®, Lab. and Vet. Service GmbH, Vienna, Austria
- Diet: ad libitum, Pelleted Kliba maintenance diet rabbit and guinea pig “GLP”, Provimi Kliba SA, Kaiseraugst/Switzerland
- Water: ad libitum, tap water
- Acclimation period: at least 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-22
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
drinking water (with 10 mg/ 100 mL Cremophor EL)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
For the test substance preparations, the specific amount of test substance was weighed, topped up with drinking water (with 10 mg/ 100 mL Cremophor EL) in an Erlenmeyer flask and intensely mixed with a homogenizer.

- Rate of preparation:The test substance preparations were prepared at the beginning of the administration period and thereafter at intervals, which took into account the period of established stability.
- Storage temperature: Room temperature

VEHICLE
- Justification for use and choice of vehicle: ??Cremophor EL was added to enhance water solubility. ??
- Concentration in vehicle: 0.5, 1.5, 5.0 g/100 mL
- Amount of vehicle: 10 mL/kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance in drinking water (10 mg/100 mL Cremophor EL) over a period of 7 days at room temperature had been verified prior to the start of the study in a similar batch.
Samples of the test substance preparations were sent to the analytical laboratory once during the study period (at the beginning of administration) for verification of the concentrations. HPLC was used for the analysis. The samples taken for the concentration control analyses were also used to verify the homogeneity of the samples of the low- and high-concentrations each (50 and 500 mg/kg bw/d). Three samples (one from the top, middle and bottom) were taken for each of these preparations from the preparation vessel with a magnetic stirrer running.
The mean measured concentrations of the samples (mean values of the samples 3-5 and 7-9 as well as the single value of the sample 6) of treatment groups corresponded to the expected values within the limits of the analytical method, i.e. were above 90 % and below 110 % of the nominal concentrations.
Details on mating procedure:
- Impregnation procedure: artificial insemination
A synthetic hormone (0.2 mL), which stimulates release of LH and FSH from the anterior pituitary lobe (Receptal) was injected intramuscularly to the female rabbits about 1 hour before insemination.
The ejaculate samples used for the artificial insemination were obtained from male New Zealand White rabbits of the same breed as the females.
Each female was inseminated with the sperm of a defined male donor as documented in the raw data. The male donors were kept under conditions (air conditioning, diet, water) comparable to those of the females participating in this study.
The day of insemination was designated as GD 0 (beginning of the study) and the following day as GD 1.
Duration of treatment / exposure:
23 days (GD 6 - 28)
Frequency of treatment:
once daily
Duration of test:
30 days (GD 0 - 29)
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
25 females per group, no males
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The high dose was selected based on signs of toxicity noted at dose levels of 300 and 1000 mg/kg bw/d in a previously conducted maternal toxicity range-finding study which preceded this definitive prenatal developmental toxicity study.
In the maternal toxicity range‐finding study, 5 pregnant New Zealand White rabbits per dose were administered the test substance by oral gavage from gestational day (GD) 6 through GD 28.
At 1000 mg/kg bw/d food consumption was reduced by an average of 40 % during treatment, in mid-gestation food consumption was actually less than half the normal intake. At 300 mg/kg bw/d food consumption was still decreased by about 20 %. Body weights were lower as well in both treatment groups, final weights were 10 % and 7 % below control, respectively. Corrected (net) body weight change was 4 times lower at 1000 mg/kg bw/d than in controls. Necropsy revealed abnormal content (whitish plastic-like streaks intermingled in food) in the stomach in 4 of 5 high-dose does. From the appearance of the stomach content it seems likely that this high concentration of test substance polymerizes under acidic conditions in the stomach and in/or the presence of food ingredients. Based on these data dose levels of 50, 150, and 500 mg/kg bw/d were considered to be appropriate for the present definitive study. The oral route was selected since this has proven to be suitable for the detection of a toxicological hazard.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily, during the administration period (GD 6 - 28) before the administration as well as within 5 hours after the administration
- Cage side observations: morbidity, pertinent behavioral changes and signs of overt toxicity

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 2, 4, 6, 9, 11, 14, 16, 19, 21, 23, 25, 28 and 29
- 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).

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes, recorded daily

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 29
- Organs examined: uteri and ovaries
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 of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Site of implantations
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter

In the present study the internationally harmonized glossary of WISE et al. (1997) and the updated version MAKRIS et al. (2009) was essentially used to describe findings in fetal morphology. Classification of these findings was based on the terms and definitions proposed by CHAHOUD and SOLECKI.
Statistics:
- Simultaneous comparison of all dose groups with the control group using the DUNNETTtest (two-sided) for the hypothesis of equal means:
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

- Pairwise comparison of each dose group with the control group using FISHER'S EXACT test (one-sided) for the hypothesis of equal proportions:
Female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings

- Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians:
Proportions of fetuses with malformations, variations and/or unclassified observations in each litter

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 low-dose group (50 mg/kg bw/d) and two females of the mid-dose group (150 mg/kg bw/d) had blood in bedding before and/or after treatment: doe No. 29 on GD 18, doe No. 54 on GD 20 and doe No. 67 on GD 18 (this animal died on the following day).

In total, reduced defecation was observed in eleven control, seven low-dose, nine mid-dose and thirteen high-dose females (0, 50, 150 and 500 mg/kg bw/d). No defecation was observed in six control females, five low-dose females, four mid-dose females and ten high-dose females.

Incidence and distribution of these findings do not indicate a relationship to the test substance.

There were no further clinical findings in the other does in this study.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
150 mg/kg bw/d : One female (No. 67) was found dead during the administration period (GD 19), after showing blood in bedding and reduced defecation on the days before. The gross pathological examination revealed multiple erosions in the stomach and no feces in rectum.

500 mg/kg bw/d: One female (No. 92) was sacrificed after abortion ahead of schedule (GD 27). Spontaneous abortions in single does are not uncommon findings in the strain of rabbits used for this study. Thus, this was considered to be a spontaneous incident.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean body weights and the average body weight gain of the low-, mid- and high-dose groups (50, 150 and 500 mg/kg bw/d) were generally comparable to the concurrent control group throughout the entire study period.

Mean carcass weights and corrected body weight gain (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6) were not significantly different between all test groups including controls (0, 50, 150 or 500 mg/kg bw/d).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In comparison to the control group the mean food consumption of the does in test group 3 (500 mg/kg bw/d) was statistically significantly reduced at the beginning of the treatment period (GD 6-7), but recovered afterwards and was comparable again towards the end of the treatment period. During the treatment period (GD 6-28) the high-dose does consumed 6 % less food than the concurrent control does.

The food consumption of the low- and mid-dose rabbits (50 and 150 mg/kg bw/d) was comparable to the concurrent control (0 mg/kg bw/d) throughout the entire study period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The mean gravid uterus weight of the rabbits of test groups 1-3 (50, 150 or 500 mg/kg bw/d) was not influenced by the test substance. The differences between these groups and the control group showed no dose-dependency and were assessed to be without biological relevance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Some spontaneous findings were noted in individual females of test groups 0-3 (0, 50, 150 and 500 mg/kg bw/d). These gross findings were:
- Single erosion in stomach in two control (Nos. 10 and 15), one low-dose (No. 35 - 50 mg/kg bw/d), one mid-dose (No. 61 - 150 mg/kg bw/d) and one high-dose doe (No. 100 - 500 mg/kg bw/d).
- Abnormal content in stomach in one high-dose doe (No. 86 - 500 mg/kg bw/d).
- Malpositioned kidney in one high-dose doe (No. 91 - 500 mg/kg bw/d).
- Absence of uterine horn(s) in one mid-dose doe (No. 69, not pregnant - 150 mg/kg bw/d).

Additional findings were noted in test groups 2 and 3. Both were associated with unscheduled maternal death or sacrifice:
- Multiple erosions in stomach and no feces in rectum in mid-dose doe No. 67 (died on GD 19).
- A stomach filled with very dry, hard feed, no feces in small intestine and rectum and a large intestine filled with fluid in high-dose doe No. 92 (sacrificed after abortion on GD 27).
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
One high-dose female (No. 92 - 500 mg/kg bw/d) was sacrificed after abortion ahead of schedule (GD 27). Spontaneous abortions in single does are not uncommon findings in the strain of rabbits used for this study this was considered to be a spontaneous incident.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There were no test substance-related and/or biologically relevant differences between the different test groups in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and the postimplantation losses.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
There were no test substance-related and/or biologically relevant differences between the different test groups in the number of resorptions.
Early or late resorptions:
no effects observed
Description (incidence and severity):
There were no test substance-related and/or biologically relevant differences between the different test groups in the number of resorptions.
Dead fetuses:
no effects observed
Description (incidence and severity):
There were no test substance-related and/or biologically relevant differences between the different test groups in the number of viable fetuses.
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
There were no test substance-related and/or biologically relevant differences between the different test groups in conception rate.
The conception rate was 92 % in the mid-dose group (150 mg/kg bw/d), 96% in the control and low-dose groups (0 and 50 mg/kg bw/d) and 100% in the high-dose group (500 mg/kg bw/d).
A sufficient number (approximately 20, but not fewer than 16 females with implantation sites) of pregnant females was available for the purpose of the study.
Other effects:
not examined

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: No test substance related systemic effects were observed.

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
The mean fetal weights of test groups 1, 2 and 3 were not influenced by the test substance and did not show any biologically relevant differences in comparison to the control group.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There were no test substance-related and/or biologically relevant differences between the different test groups in the number of viable fetuses.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex distribution of the fetuses in test groups 1-3 (50, 150 and 500 mg/kg bw/d) was comparable to the control fetuses. Any observable differences were without biological relevance.
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The distribution of external malformations about the dose groups does not indicate an association to the treatment, no statistically significant differences between the groups were noted (table 1).
0 mg/kg bw/d: 1 fetus with open eye, 1 fetus with umbilical hernia
50 mg/kg bw/d: no findings
150 mg/kg bw/d: 2 fetuses with umbilical hernia
500 mg/kg bw/d: 1 fetus with malrotated limb, 1 fetus with cleft palate
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Skeletal malformations were detected in single fetuses of the test groups 1, 2 and 3 (50, 150, 500 mg/kg bw/d). Male high-dose fetus No. 82-09 had associated external and soft tissue malformations. The addition of those individual findings resulted in a slightly increased affected fetuses per litter-incidence of skeletal malformations in test group 3 (500 mg/kg bw/d), which became statistically significant as the control incidence was zero (table 7). However, as these were individual findings without any ontogenetic coherence and the high-dose incidence is even below the historical control mean (HCD: 1.4 mean% [0.0 - 4.4]), these findings were considered to be spontaneous in origin and not treatment-related.
0 mg/kg bw/d: no findings
50 mg/kg bw/d: 1 fetuses with fused skull bone
150 mg/kg bw/d: 1 fetuses with additional bony structure
500 mg/kg bw/d: 1 fetus with branched rib, 1 fetus with small palatine bone, 1 fetus with thoracic hemivertebra, misshapen thoracic vertebra, branched rib
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Soft tissue malformations occurred in single fetuses of the test groups 2 and 3 (150 or 500 mg/kg bw/d). Male high-dose fetus No. 82-09 had additional external and skeletal malformations. No statistically significant differences between the groups were noted (table 4).
0 mg/kg bw/d: no findings
50 mg/kg bw/d: no findings
150 mg/kg bw/d: 1 fetuses with persistent truncus arteriosus, heart: muscular ventricular septum defect
500 mg/kg bw/d: 1 fetus with malpositioned lens, 1 fetus with small cerebrum
Other effects:
no effects observed
Description (incidence and severity):
- External:
One external variation was recorded in two fetuses of the same high-dose litter (500 mg/kg bw/d), i.e. paw hyperflexion (table 2). This finding can be found in the historical control data at comparable incidences, thus it is considered to be incidental.

Three unclassified external observations, i.e. amniotic fluid discolored, placentae fused and placentae necrobiotic, were recorded in one fetus, each, of test groups 0, 1 and 2 (0, 50 or 150 mg/kg bw/d) (table 3). These findings are not considered to be related to treatment.

- Soft tissue:
The examinations of the soft tissues revealed cystic dilatation of the brain, dilated cerebral ventricle and malpositioned carotid branches in individual fetuses of test groups 0, 1 and 3 (0, 50 or 500 mg/kg bw/d) (table 5). An absent lung lobe (Lobus inferior medialis) was noted in all substance-treated groups. The incidences of these variations were neither statistically significantly different from control nor dose-dependent and, therefore, not considered
biologically relevant. All of them can be found in the historical control data of the test facility at comparable incidences.

One unclassified soft tissue observation was recorded in two control fetuses, six low-dose, four mid-dose and three high-dose fetuses: a blood coagulum around urinary bladder (table 6). This finding is not considered to be treatment-related.

- Skeletal:
For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeletons and appeared without a relation to dosing (table 8 + 9). The overall incidences of skeletal variations were comparable to the historical control data.

Some isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in all test groups (tabel 10). The observed unclassified cartilage findings were related to the sternum and the ribs and did not show any relation to dosing. Therefore, they were assessed as not treatment-related.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No toxicologically relevant adverse effects of the test substance were observed.

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: Total external malformations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

50 mg/kg bw/d

Test group 2

150 mg/kg bw/d

Test group 3

500 mg/kg bw/d

Litter

Fetuses

N

N

24

227

24

232

22

212

24

201

Fetal incidence

N (%)

2 (0.9)

0

2 (0.9)

2 (1.0)

Litter incidence

N (%)

2 (8.3)

0

2 (9.1)

1 (4.2)

Affected fetuses/litter

Mean (%)

0.8

0

1.0

1.0

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Table 2: Total external variations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

50 mg/kg bw/d

Test group 2

150 mg/kg bw/d

Test group 3

500 mg/kg bw/d

Litter

Fetuses

N

N

24

227

24

232

22

212

24

201

Fetal incidence

N (%)

0

0

0

2 (1.0)

Litter incidence

N (%)

0

0

0

1 (4.2)

Affected fetuses/litter

Mean (%)

0

0

0

1.0

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Table 3: Total external unclassified observations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

50 mg/kg bw/d

Test group 2

150 mg/kg bw/d

Test group 3

500 mg/kg bw/d

Litter

Fetuses

N

N

24

227

24

232

22

212

24

201

Fetal incidence

N (%)

1 (0.4)

1 (0.4)

1 (0.5)

0

Litter incidence

N (%)

1 (4.2)

1 (4.2)

1 (4.5)

0

Affected fetuses/litter

Mean (%)

0.4

0.5

0.5

0

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Table 4: Total soft tissue malformations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

50 mg/kg bw/d

Test group 2

150 mg/kg bw/d

Test group 3

500 mg/kg bw/d

Litter

Fetuses

N

N

24

227

24

232

22

212

24

201

Fetal incidence

N (%)

0

0

1 (0.5)

2 (1.0)

Litter incidence

N (%)

0

0

1 (4.5)

2 (8.3)

Affected fetuses/litter

Mean (%)

0

0

0.6

0.9

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Table 5: Total soft tissue variations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

50 mg/kg bw/d

Test group 2

150 mg/kg bw/d

Test group 3

500 mg/kg bw/d

Litter

Fetuses

N

N

24

227

24

232

22

212

24

201

Fetal incidence

N (%)

3 (1.3)

3 (1.3)

5 (2.4)

5 (2.5)

Litter incidence

N (%)

3 (13)

3 (13)

3 (14)

4 (17)

Affected fetuses/litter

Mean (%)

1.4

1.6

2.6

2.2

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Table 6: Total soft tissue unclassified observations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

50 mg/kg bw/d

Test group 2

150 mg/kg bw/d

Test group 3

500 mg/kg bw/d

Litter

Fetuses

N

N

24

227

24

232

22

212

24

201

Fetal incidence

N (%)

2 (0.9)

6 (2.6)

4 (1.9)

3 (1.5)

Litter incidence

N (%)

2 (8.3)

4 (17)

3 (14)

2 (8.3)

Affected fetuses/litter

Mean (%)

1.9

3.2

2.2

1.4

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Table 7: Total skeletal malformations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

50 mg/kg bw/d

Test group 2

150 mg/kg bw/d

Test group 3

500 mg/kg bw/d

Litter

Fetuses

N

N

24

227

24

232

22

212

24

201

Fetal incidence

N (%)

0

1 (0.4)

1 (0.5)

3 (1.5)

Litter incidence

N (%)

0

1 (4.2)

1 (4.5)

3 (13)

Affected fetuses/litter

Mean (%)

0

0.3

0.6

1.2*

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent * = p0.05 (Wilcoxon-test [one-sided])

 

Table 8: Total fetal skeletal variations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

50 mg/kg bw/d

Test group 2

150 mg/kg bw/d

Test group 3

500 mg/kg bw/d

Litter

Fetuses

N

N

24

227

24

232

22

212

24

201

Fetal incidence

N (%)

207 (91)

200 (86)

201 (95)

183 (91)

Litter incidence

N (%)

24 (100)

24 (100)

22 (100)

24 (100)

Affected fetuses/litter

Mean (%)

91.9

87.1

94.5

92.5

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Table 9: Occurrence of statistically significantly increased fetal skeletal variations

(expressed as mean percentage of affected fetuses/litter)

Finding

Test group 0

0 mg/kg bw/d

Test group 1

50 mg/kg bw/d

Test group 2

150 mg/kg bw/d

Test group 3

500 mg/kg bw/d

HCD

Mean % (range)

Incomplete ossification of hyoid; cartilage present

17.8

19.1

28.0*

16.0

31.1

(11.3-47.3)

Unossified talus; cartilage present

0

1.8*

1.7

0.7

1.0

(0.0-2.6)

Incomplete ossification of pubis; cartilage present

0

0

0.5

1.6*

0.7

(0.0-1.9)

 

Table 10: Total unclassified cartilage observations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

50 mg/kg bw/d

Test group 2

150 mg/kg bw/d

Test group 3

500 mg/kg bw/d

Litter

Fetuses

N

N

24

227

24

232

22

212

24

201

Fetal incidence

N (%)

18 (7.9)

14 (6.0)

21 (9.9)

15 (7.5)

Litter incidence

N (%)

8 (33)

7 (29)

7 (32)

8 (33)

Affected fetuses/litter

Mean (%)

6.8

5.3

10.2

6.2

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Table 11: Total fetal malformations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

50 mg/kg bw/d

Test group 2

150 mg/kg bw/d

Test group 3

500 mg/kg bw/d

Litter

Fetuses

N

N

24

227

24

232

22

212

24

201

Fetal incidence

N (%)

2 (0.9)

1 (0.4)

4 (1.9)

5 (2.5)

Litter incidence

N (%)

2 (8.3)

1 (4.2)

4 (18)

4 (17)

Affected fetuses/litter

Mean (%)

0.8

0.3

2.3

2.2

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Table 12: Total fetal variations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

50 mg/kg bw/d

Test group 2

150 mg/kg bw/d

Test group 3

500 mg/kg bw/d

Litter

Fetuses

N

N

24

227

24

232

22

212

24

201

Fetal incidence

N (%)

207 (91)

200 (86)

201 (95)

184 (92)

Litter incidence

N (%)

24 (100)

24 (100)

22 (100)

24 (100)

Affected fetuses/litter

Mean (%)

91.9

87.1

84.5

92.9

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Table 13: Mean maternal body weights during gestation – grams

 

 

 

 

Test group 0

0 mg/kg bw/d

Test group 1

50 mg/kg bw/d

Test group 2

150 mg/kg bw/d

Test group 3

500 mg/kg bw/d

Day 0

Mean

S.D.

N

3710 D

280.4

24

3739

256.3

24

3701

304.7

23

3736

248.0

25

Day 2

Mean

S.D.

N

3793 D

273.4

24

3815

260.5

24

3776

322.0

23

3801

253.7

25

Day 4

Mean

S.D.

N

3851 D

280.5

24

3875

269.3

24

3830

331.3

23

3847

257.3

25

Day 6

Mean

S.D.

N

3882 D

296.1

24

3916

279.5

24

3876

336.9

23

3885

285.4

25

Day 9

Mean

S.D.

N

3964 D

284.8

24

3993

264.7

24

3944

349.2

23

3942

286.3

25

Day 11

Mean

S.D.

N

4009 D

294.4

24

4038

276.7

24

3991

371.9

23

3988

280.2

25

Day 14

Mean

S.D.

N

4032 D

251.4

24

4050

240.5

24

4015

342.2

23

3971

266.6

25

Day 16

Mean

S.D.

N

4050 D

270.7

24

4090

286.9

24

4049

331.3

23

4009

291.3

25

Day 19

Mean

S.D.

N

4079 D

313.9

24

4106

323.2

24

4091

368.7

22

4036

308.3

25

Day 21

Mean

S.D.

N

4070 D

316.6

24

4102

325.8

24

4092

371.6

22

4030

324.9

25

Day 23

Mean

S.D.

N

4063 D

313.5

24

4095

279.6

24

4089

370.8

22

4022

333.6

25

Day 25

Mean

S.D.

N

4059 D

278.8

24

4078

270.3

24

4080

338.1

22

4004

334.0

25

Day 28

Mean

S.D.

N

4056 D

253.8

24

4094

275.6

24

4096

323.6

22

4048

300.1

24

Day 29

Mean

S.D.

N

4075 D

261.5

24

4105

275.2

24

4116

335.8

22

4060

294.9

24

 

D=Dunnett-test (two-sided)

 

Table 14: Mean maternal body weight change during gestation – grams

 

 

 

Test group 0

0 mg/kg bw/d

Test group 1

50 mg/kg bw/d

Test group 2

150 mg/kg bw/d

Test group 3

500 mg/kg bw/d

Days 0 to 2

Mean

S.D.

N

83.2 D

46.80

24

76.3

42.51

24

75.0

41.52

23

64.5

45.09

25

Day 2 to 4

Mean

S.D.

N

58.6 D

34.98

24

59.5

26.74

24

54.3

23.97

23

46.3

52.87

25

Day 4 to 6

Mean

S.D.

N

30.6 D

37.49

24

41.0

71.01

24

46.0

32.71

23

38.1

76.64

25

Day 6 to 9

Mean

S.D.

N

82.4 D

52.25

24

76.7

63.04

24

67.5

78.12

23

56.8

58.10

25

Day 9 to 11

Mean

S.D.

N

44.6 D

34.67

24

45.5

44.56

24

47.5

40.96

23

45.7

54.88

25

Day 11 to 14

Mean

S.D.

N

22.9 D

89.41

24

12.1

95.44

24

24.1

104.33

23

-16.6

130.69

25

Day 14 to 16

Mean

S.D.

N

17.8 D

102.44

24

40.2

88.11

24

34.0

107.80

23

38.1

77.44

25

Day 16 to 19

Mean

S.D.

N

29.1 D

95.36

24

15.3

106.80

24

31.3

111.41

22

26.5

150.35

25

Day 19 to 21

Mean

S.D.

N

-9.0 D

56.80

24

-3.4

40.07

24

0.7

58.95

22

-5.3

50.45

25

Day 21 to 23

Mean

S.D.

N

-6.5 D

59.44

24

-6.8

61.58

24

-2.5

44.88

22

-8.7

41.68

25

Day 23 to 25

Mean

S.D.

N

-4.1 D

75.53

24

-17.0

42.96

24

-9.0

63.80

22

-17.7

60.66

25

Day 25 to 28

Mean

S.D.

N

-3.1 D

76.19

24

16.0

67.83

24

16.0

73.00

22

19.0

94.14

24

Day 28 to 29

Mean

S.D.

N

18.5 D

43.84

24

10.9

34.81

24

19.6

35.10

22

12.8

39.47

24

D=Dunnett-test (two-sided)

 

Table 15: Summary of mean maternal body weight change during gestation – grams

 

 

Test group 0

0 mg/kg bw/d

Test group 1

50 mg/kg bw/d

Test group 2

150 mg/kg bw/d

Test group 3

500 mg/kg bw/d

Days 0 to 6

Mean

S.D.

N

172.4 D

80.18

24

176.7

85.03

24

175.3

69.82

23

149.0

79.67

25

Day 6 to 28

Mean

S.D.

N

174.1 D

146.69

24

178.5

182.42

24

210.9

180.61

22

162.5

212.08

24

Day 0 to 29

Mean

S.D.

N

365.0 D

164.25

24

366.1

232.16

24

410.0

207.24

22

321.5

205.70

24

D=Dunnett-test (two-sided)

 

Table 16: Mean gravid uterine weights and net maternal body weight change – grams

 

 

Test group 0

0 mg/kg bw/d

Test group 1

50 mg/kg bw/d

Test group 2

150 mg/kg bw/d

Test group 3

500 mg/kg bw/d

Gravid uterus

Mean

S.D.

N

464.4 D

123.44

24

492.2

88.75

24

488.3

95.12

22

420.5

153.43

24

Carcass

Mean

S.D.

N

3610.2 D

282.67

24

3613.0

252.95

24

3627.3

288.58

22

3639.9

334.38

24

Net weight change from day 6

Mean

S.D.

N

-271.8 D

172.77

24

-302.8

167.46

24

-257.8

182.94

22

-245.3

236.39

24

D=Dunnett-test (two-sided)

Carcass weight = terminal body weight minus uterine weight

Net weight change from day 6 = carcass weight minus day 6 body weight

 

Table 17: Summary Reproductive data

 

 

Test group 0

Test group 1

Test group 2

Test group 3

 

 

0 mg/kg bw/d

50 mg/kg bw/d

150 mg/kg bw/d

500 mg/kg bw/d

Females Mated [N]

 

25

25

25

25

 

Pregnant [N]

24

24

23

25

 

Conception rate [%]

96

96

92

100

 

Aborted [N]

0

0

0

1

 

Premature Births [N]

0

0

0

0

 

Dams with viable fetuses [N]

24

24

22

24

 

Dams with all resorptions [N]

0

0

0

0

Female mortality [N (%)]

 

0 Fi (0)

0 (0)

1 (4.0)

1 (4.0)

Pregnant at terminal sacrifice [N (%)]

 

24 Fi (96)

24 (96)

22 (88)

24 (96)

Corpora Lutea [mean ± S.D. (total)]

 

10.9 D ± 2.41 (262)

11.3 ± 1.87 (270)

10.8 ± 2.17 (238)

10.0 ± 3.13 (240)

Implantation Sites [mean ± S.D. (total)]

 

10.0 D ± 3.21 (239)

10.5 ± 2.15 (251)

10.3 ± 2.06 (227)

9.3 ± 3.91 (222)

Preimplantation loss [mean ± S.D.]

 

10.4 D ± 20.98

7.3 ± 10.13

4.4 ± 2.83

12.3 ± 24.21

Postimplantation loss [mean ± S.D.]

 

4.5 D ± 8.21

7.2 ± 8.02

6.8 ± 8.37

6.9 ± 11.49

Resportions

 

 

 

 

 

 

Total [mean ± S.D. (total)]

0.5 D ± 0.93 (12)

0.8 ± 0.83 (19)

0.7 ± 0.78 (15)

0.9 ± 1.68 (21)

 

Total [mean % ± S.D.]

4.5 D ± 8.21

7.2 ± 8.02

6.8 ± 8.37

6.9 ± 11.49

 

Early [mean ± S.D. (total)]

0.4 D ± 0.58(9)

0.6 ± 0.77(15)

0.4 ± 0.73(8)

0.6 ± 0.97(15)

 

Early [mean % ± S.D.]

3.4 D ± 5.22

5.8 ± 7.73

3.6 ± 7.7

5.3 ± 7.92

 

Late [mean ± S.D. (total)]

0.1 D ± 0.45(3)

0.2 ± 0.38(4)

0.3 ± 0.57(7)

0.3 ± 0.9(6)

 

Late [mean % ± S.D.]

1.1 D ± 3.89

1.4 ± 3.19

3.1 ± 5.88

1.6 ± 5.55

Dead fetuses [N]

 

0

0

0

0

Live fetuses [mean ± S.D. (total)]

 

9.5 D ± 3.11 (227)

9.7 ± 1.97 (232)

9.6 ± 2.13(212)

8.4 ± 3.24(201)

Live fetuses [mean % ± S.D.]

 

95.5 D ± 8.21

92.8 ± 8.02

93.2 ± 8.37

93.1 ± 11.49

 

Females [mean ± S.D. (total)]

4.8 D ± 2.21(115)

4.9 ± 2.33(117)

4.9 ± 2.04(108)

4.3 ± 2.18(103)

 

Females [mean % ± S.D.]

45.1 D ± 19.09

45.2 ± 19.38

47.4 ± 15.48

46.3 ± 23.78

 

Males [mean ± S.D. (total)]

4.7 D ± 1.93(112)

4.8 ± 1.89(115)

4.7 ± 1.67(104)

4.1 ± 2.26(98)

 

Males [mean % ± S.D.]

50.4 D ± 19.94

47.6 ± 20.17

45.8 ± 12.06

46.7 ± 25.63

% live females

 

50.7

50.4

50.9

51.2

% live males

 

49.3

49.6

49.1

48.8

D=Dunnett-test (two-sided)

Fi=Fisher’s exact test (one-sided)

 

Table 18: Fetal weights

 

 

Test group 0

Test group 1

Test group 2

Test group 3

 

 

0 mg/kg bw/d

50 mg/kg bw/d

150 mg/kg bw/d

500 mg/kg bw/d

Fetal weights [unit: g]

 

 

 

 

 

 

All viable fetuses [mean ± S.D. (N)]

36 D ± 7.33(24)

35.6 ± 5.67(24)

35.6 ± 4.74(22)

36.6 ± 7.71

 

Females [mean ± S.D. (N)]

36.3 D ± 7.33(24)

35.3 ± 5.97(24)

36.2 ± 5.27(22)

35.6 ± 7.11(23)

 

Males [mean ± S.D. (N)]

34.1 D ± 5.59(22)

35.1 ± 5.06(23)

35.3 ± 4.83(22)

35.2 ± 6.32(22)

D=Dunnett-test (two-sided)

DISCUSSION

In a prenatal developmental toxicity study, cyclohexyl methacrylate was administered to pregnant New Zealand White rabbits daily by stomach tube from implantation to one day prior to the expected day of parturition (GD 6-28).

Analyses confirmed the correctness of the prepared concentrations, their homogeneous distribution and the stability of the test substance in the vehicle.

Clinical observations revealed no toxicologically relevant difference between the animals receiving 50, 150 or 500 mg/kg bw/d cyclohexyl methacrylate and the controls.

The mean food consumption of the high-dose dams (500 mg/kg bw/d) was significantly below the concurrent control at the beginning of the treatment and recovered afterwards, however, overall these animals consumed about 6 % less food than the control group. There was no treatment related effect on body weights/body weight gain, carcass weights or corrected (net) body weight gain at all dose levels. The minor effect on food consumption with no consequence for the well-being of the animals is indicative of a slight local effect in the gastrointestinal tract of the animals rather than a sign of systemic toxicity.

No differences of toxicological relevance between the control and the treated groups (50, 150 or 500 mg/kg bw/d) were determined for any reproductive parameters, such as conception rate, mean number of corpora lutea, mean number of implantations, as well as pre- and postimplantation loss. All differences observed are considered to reflect the normal range of fluctuations for animals of this strain and age.

Similarly, no influence of the test substance on uterine weight, placental weight, fetal weight and sex distribution of the fetuses was noted at any dose.

Fetal examinations revealed no toxicologically relevant adverse effect of the compound on embryofetal development.

 

CONCLUSION

Under the conditions of this prenatal developmental toxicity study, the oral administration of cyclohexyl methacrylate to pregnant New Zealand White rabbits from implantation to one day prior to the expected day of parturition (GD 6-28) caused no evidence of systemic maternal toxicity up to the high- dose level of 500 mg/kg bw/d. The minor effect on food consumption with no consequence for the well-being of the animals at this dose is indicative of a slight local effect in the gastrointestinal tract of the animals rather than a sign of systemic toxicity. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 500 mg/kg bw/d.

Since there was no evidence for toxicologically relevant adverse effects of the test substance on fetal morphology at any dose, the no observed adverse effect level (NOAEL) for prenatal developmental toxicity is the highest dose of 500 mg/kg bw/d.

Applicant's summary and conclusion