Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

No adverse effects on reproductive organs were observed in the subchronic toxicity study in rats. A screening study in rats is ongoing.

Effect on fertility: via oral route
Endpoint conclusion:
no study available (further information necessary)
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information

Two prenatal developmental toxicity studies according to OECD 414 were performed in rats and rabbits. No direct developmental toxicity, teratogenic or embryotoxic effects were observed in the studies.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD 414 and GLP compliant study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy
- Age at study initiation: (age at delivery 10 weeks)
- Weight at study initiation: (weight range at delivery 177-196 g)
- Fasting period before study: none
- Housing: individual cages (during gestation)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 18 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2 °C
- Humidity (%): 55% +/- 15%
- Air changes (per hr): 15 -20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2014-05-20 To: 2014-06-16
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The formulations were prepared daily and the concentrations were calculated and expressed in terms of test item as supplied.


VEHICLE
- Justification for use and choice of vehicle: The substance is miscible in corn oil and insoluble in water.
- Concentration in vehicle: 12.5, 37.5 and 125 mg/mL
- Amount of vehicle: 4 ml/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The proposed formulation procedure for the test item was checked in the range from 12.5 to 125 mg/mL by chemical analysis (concentration and
homogeneity) during the pre-treatment period to confirm that the method was suitable. Final results for all levels were within the acceptability limits
for concentration (90-110%). Stability after 24 hours at room temperature was verified in the range from 1 to 300 mg/mL in the validation study.
Samples of the formulations prepared on week 1 and Last Week were analysed to check the homogeneity and concentration.
Details on mating procedure:
Females were paired one to one in the home cage of the male and left overnight. Vaginal smears were taken daily in the morning from the day after pairing until a positive identification of matingwas made. The day of mating, as judged by the presence of sperm in the vaginal smear or by the presence of a copulation plug, was considered as Day 0 of gestation (or Day 0 post coitum).
Duration of treatment / exposure:
Day 6 through Day 19 post coitum
Frequency of treatment:
daily
Duration of test:
Day 6 through Day 20 post coitum
Remarks:
Doses / Concentrations:
50, 150 and 500 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Range-finding study with pregnant rats. The highest dose group of 500 mg/kg was expected to cause maternal toxicity as indicated by adverse effects on body weights and evidence on liver toxicity as indicated by clinical chemistry parameters. For details it is referred to the robust study summary of the maternal toxicity study.
- Rationale for animal assignment: Females were allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations: mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 3, 6, 9, 12, 15, 18 and 20 post coitum

FOOD CONSUMPTION : Yes
- Time schedule for examinations: Days 3, 6, 9, 12, 15, 18 and 20 post coitum starting from Day 0 post coitum


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: ovaries and uteri

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: gross evaluation of placentae, number of intra-uterine deaths,. Uteri or individual uterine horns without visible implantations were immersed
in a 20% solution of ammonium sulphide to reveal evidence of embryonic death at very early stages of implantation.
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
Statistics:
For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t-test, depending on the
homogeneity of data. Statistical analysis of non-continuous variables was carried out by means of the Kruskal-Wallis test and intergroup differences
between the control and treated groups assessed by a non-parametric version of the Williams test. The mean values, standard deviations and statistical
analysis were calculated from actual values in the computer without rounding off.
Indices:
Preimplantation loss
Postimplantation loss
Total implantation loss
Sex ratios
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
A slight decrease in body weight was noted in all treated females when compared to controls, reaching a statistical significance in females receiving
500 mg/kg bw/day (up to 7%), starting from Day 9 post coitum until the end of the study.
Statistically significant decrease was also recorded in body weight gain of females of the same group on Day 9 post coitum (109%; body weight loss) and Day 12 post coitum (22%). Starting from Day 15 post coitum the mean values of body weight gain were comparable between control and high dose group.

Statistically significant decrease (up to 22%) in food consumption was observed in treated females receiving 500 mg/kg bw/day, starting from Day 9 post coitum until the end of the study.

A slight trend to decrease was observed in terminal body weight of all treated females with respect to the control. This change was about -6% in the high
dose group, without statistical significance. A statistically significant decrease in corrected body weight (up to 6%) and corrected body weight gain (up
to 50%) was noted in treated females receiving 150 and 500 mg/kg bw/day. Gravid uterus weight was similar between control and treated groups.

There were no adverse findings at the macroscopic examination at necropsy.
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Litter data, mean foetal weight and sex ratio were unaffected by treatment.

A total of 15 small foetuses (foetal weight < 2.7 g) were detected: 2 out of 269 in the control group, 2 out of 214 in the low dose group, 1 out of 269 in the mid-dose group and 10 out of 254 in the high dose group. One foetus in the high dose group showed malrotation of the hindlimb, considered incidental. Of the ten small fetuses in the high dose group, 7 were from the dam which suffered most strongly from maternal toxicity as indicated by the lowest corrected body weight gain of minus 9.8g. This dam was also the only dam showing hunched posture and piloerection on gestation day 20. The higher incidence of small foetuses is therefore considered to be related to the lower maternal body weight gain.

No relevant findings that could be considered treatment-related were observed at visceral examination of foetuses in the treated groups, compared to controls.
The alterations recorded at skeletal examinations of foetuses were noted both in control and treated groups with a similar incidence.
Dose descriptor:
NOAEL
Effect level:
>= 500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No effects observed
Abnormalities:
no effects observed
Developmental effects observed:
no

Table 1: TERMINAL BODY WEIGHT, UTERUS WEIGHT, CORRECTED BODY WEIGHT AND CORRECTED BODY WEIGHT GAIN OF FEMALES - GROUP MEAN DATA

Group   Terminal body weight (g) Gravid uterus weight (g) Body weight at necropsy minus gravid uterus weight (corrected body weight) (g)  Body weight at necropsy minus gravid uterus weight, minus body weight on GD6 (corrected body weight gain) (g) 
1 (control) Mean 336.38 66.40 269.98 29.87
  SD 15.84 8.80 14.79 7.67
  (n) 23 23 23 23
2 Me 323.9 60.42 263.5 29.45
  SD 23 18 18 16
  (n) 20 20 20 20
3 Me 323.8 66.20 257.6* 20.5*
  SD 28 12 18 9.
  (n) 23 23 23 23
4 Me 315.9 62.53 253.3* 14.83*
  SD 24 14 23 16
  (n) 22 22 22  22

* = Statistically significantly different from control group value at p< 0.05

Table 2: FOOD CONSUMPTION (g/animal/day) - GROUP MEAN

Group   gestation day 3 gestation day 6 gestation day 9 gestation day 12 gestation day 15 gestation day 18 gestation day 20
1 (control) n  23 23 23 23 23 23 23
  Mean 18.13 20.66 18.72 20.99 21.78 23.68 23.06
  SD 2.37 2.54 2.05 1.98 2.10 2.20 1.99
2 n  20 20 20 20 20 20 20
  Mean 17.99 20.97 18.32 20.64 21.04 23.43 22.83
  SD 2.3 3.03 2.46 2.19 2.68 2.99 4.33
3 n  23 23 23 23 23 23 23
  Mean 18.7 20.23 17.36 19.28 20.90 22.32 21.56
  SD 1.99 2.53 2.59 2.90 2.40 2.91 3.14
4 n  22 22.00 22 22.00 22.00 22.00 22.00
  Mean 18.24 20.97 15.33** 17.1** 19.01** 20.12** 18.02**
  SD 2.62 2.55 1.98 2.76 2.68 3.02 2.96

** = mean value of group is significantly different from control at p < 0.01

Table 3: Pregnancy status overview

Group 1 (control) 2 3 4
Initial group size (n) 24 24 24 24
Not pregnant (n) 1 4 1 2
Unilateral implantation (n) 0 1 0 0
With live foetuses at gestation Day 20 (n) 23 20 23 22

Table 4: Litter data and sex ratios - group mean data

    Corpus lutea Implantations Early Uterine Deaths Late Uterine Deaths Total Uterine Deaths Viable Young (total) Viable males Viable females % Males Preimplantation loss (%) Postimplantation loss Total implantation loss(%) Litter weight (g) Mean fetal weight (g)
1 Mean 12.87 12.26 0.52 0.04 0.57 11.7 6 5.7 50.96 4.68 4.15 8.52 43.1 3.69
  SD 1.63 1.63 1.47 0.21 1.5 1.77 2.24 2.05 17.27 5.13 10.27 11.78 6.71 0.34
  (n) 23 23 23 23 23 23 23 23 23 23 23 23 23 23
2 Mean 12.26 11.47 0.21 0.05 0.26 11.21 5.67 5.84 49.82 6.56 4.12 10.05 39.88 3.63
  SD 2.58 2.44 0.54 0.23 0.56 2.84 2.22 2.29 14.18 6.13 11.67 13.68 9.32 0.4
  (n) 19 19 19 19 19 19 18 19 18 19 19 19 19 19
3 Mean 13.09 12.22 0.52 0 0.52 11.7 5.78 5.91 449.19 6.28 4.73 10.86 42.41 3.65
  SD 2.7 2.35 0.73 0 0.73 2.55 1.93 1.98 12.79 6.68 6.74 7.39 8.85 0.28
  (n) 23 23 23 23 23 23 23 23 23 23 23 23 23 23
4 Mean 12.27 11.73 0.14 0 0.14 11.59 5.36 6.23 46.95 6.11 1.18 7.23 40.11 3.51
  SD 2.66 3.06 0.35 0 0.35 3.08 2.06 2.25 12.97 10.6 3.07 10.96 9.86 0.39
  (n) 22 22 22 22 22 22 22 22 22 22 22 22 22 22
Conclusions:
The substance is not teratogenic and not embryotoxic in rats. It causes maternal toxicity at a dose level of 500 mg/kg bw.
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-03-28 until
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
22 Jan 2001
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt fuer Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Kaiser-Friedrich-Strasse 7, 55116 Mainz, Germany
Species:
rabbit
Strain:
New Zealand White
Remarks:
Crl:KBL(NZW)
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% CMC suspension in deionized water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- specific amount of the test substance was weighed, topped up with vehicle (with 10 mg/100 mL Cremophor EL) in a calibrated beaker and intensely mixed with a magnetic stirrer
- test substance preparations were warmed up to approximately 30 degrees Celsius
- before and during administration, the preparations were kept homogeneous with a magnetic stirrer and kept at approximately 30 degrees Celsius with a heating sleeve or with a magnetic stirrer with heating plate

VEHICLE
- Justification for use and choice of vehicle: stability and homogenity could be proven in the vehicle
- Amount of vehicle: the preparation was administered as a 10 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
HPLC method with UV/VIS detection
Details on mating procedure:
- Impregnation procedure: artificial insemination
- GD0: day of insemination
Duration of treatment / exposure:
gestation days 6 through 28
Frequency of treatment:
daily
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: futher details are given in the dose-range finder in the the supporting study record.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily and if effects were observed, several times daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: (during GD 6-28) daily check, as well as within 5 hours after the administration

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: uteri, 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: Dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened)
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
Statistics:
- Simultaneous comparison of all dose groups with the control group using the DUNNETT-test (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
Indices:
conception rate = number of pregnant animals / number of fertilized animals * 100
preimplantation loss, for each individual pregnant animal which underwent scheduled sacrifice = (number of corpora lutea – number of implantations) / number of corpora lutea * 100
postimplantation loss, for each individual pregnant animal which underwent scheduled sacrifice = (number of implantations – number of live fetuses) / number of implantations * 100
Historical control data:
- mean maternal bw during gestation, reproduction data, placenta weights, mean maternal weights, fetal external malformations/variations, fetal soft tissue malformations/variations, fetal skeletal malformations/variations
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
For details, see to table 1.
- 0/10/30/100 mg/kg bw/d dose group: 4/2/11/20 females showed reduced defecation, respectively
- no defecation was observed in 2/0/4/4 females, respectively
- exceptionally high incidence of reduced defecation in the high dose group, along with reduced food consumption indicates a treatment-related effect

For details, see to table 5.
- high-dose (100 mg/kg bw/d) group: mean food consumption distinctly and statistically significantly reduced from GD 7-23 (up to -59% in comparison to the control)
- high-dose does: 31% less food consumption compared to the concurrent control (GD 6-28)
- low- and mid-dose groups (10 and 30 mg/kg bw/d): comparable food consumption to control throughout the entire study period
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female of the control group (No.21) died after a gavage error.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
For details, see to table 3 and 4 and the attached figure
- mean body weights (BW); average body weight gain (BWC)
- high-dose (100 mg/kg bw/d) group: BW statistically significant reduced on GD 14-25 and BWC statistically significant reduced on GD 9-11
- high-dose rabbits: lost weight overall (-24.0 g vs. +104.4 g in control) during the treatment period (GD 6-28)
- no effects observed in the low- and mid-dose groups (10 and 30 mg/kg bw/d)
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry 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):
- no effects observed for the mean gravid uterus compared to the control group
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
For details, see to table 6.
spontaneous effects were observed in the control and the test group:
- watery feces or no feces in rectum: one control doe, one mid-dose doe and one high-dose doe
- rudimentary appearance of uterus and ovaries in one control doe
- granulated surface of liver in one low-dose doe
- malpositioned kidney in combination with a short ureter in one low-dose and one mid-dose doe
- absence of uterine horn(s) in one low-dose doe
- findings after gavage error (thoracic cavity filled with blood) in one control doe
Description (incidence and severity):
Two control (Nos. 7 [GD 20] and 11 [GD 28]) and four high-dose females (Nos. 78 [GD 24], 79 [GD 26], 84 [GD 28] and 94 [GD 27] - 100 mg/kg bw/d) were sacrificed after abortion ahead of schedule. Although spontaneous abortions in single does are not uncommon findings in the strain of rabbits used for this study, the high-dose cases may already represent exaggerated maternal toxicity, given also the distinct drop in food consumption and body weight gain as well as an exceptionally high number of does showing reduced defecation.

For details, see to table 2.
Number of abortions:
effects observed, treatment-related
Description (incidence and severity):
For details, see to table 2.
- abortions were observed in two control females and four high-dose females
- the females were sacrificed and excluded from calculations of mean maternal food consumption, body weight and body weight change, mean gravid uterine weights, mean organ weights, corrected (net) body weight gain and summary of reproduction data
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
For details, see to table 2.
- no test substance-related and/or biologically relevant differences between the different test groups observed
Early or late resorptions:
no effects observed
Description (incidence and severity):
For details, see to table 7.
- no test substance-related and/or biologically relevant differences between the different test groups in the numbers of resorptions observed
Dead fetuses:
no effects observed
Description (incidence and severity):
For details, see to table 7.
- no test substance-related and/or biologically relevant differences between the different test groups in the numbers of viable fetuses
One dead fetus was found at cesarean section of one low-dose doe which may occur spontaneously in this rabbit strain.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
For details, see to table 2.
- conception rate was 92% in the control group and 96% in the low-, mid- and high-dose groups
- no test substance-related and/or biologically relevant differences between the different test groups in the conception rate observed
Other effects:
no effects observed
Description (incidence and severity):
All differences observed are considered to reflect the normal range of fluctuations for animals of this strain and age for historical control data.
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
30 mg/kg bw/day (nominal)
Basis for effect level:
clinical signs
body weight and weight gain
number of abortions
other: distinct decrease of food consumption
Dose descriptor:
NOEL
Remarks:
developmental toxicity
Effect level:
> 100 mg/kg bw/day (nominal)
Basis for effect level:
other: no test substance-related and/or biologically relevant differences for conception rate, mean numbers of corpora lutea and implantation sites or for pre- and post-implantation losses, the numbers of resorptions and viable fetuses
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
For details, see to table 8.
- high-dose (100 mg/kg bw/d) group: mean fetal weight statistically significantly lower than control in male fetuses and -12% for both sexes in comparison to the concurrent control
- mean weight of the female high-dose fetuses was also slightly lower (not statistically significanct)
- low- and mid-dose group: not influenced by the test substance and did not show any biologically relevant differences in comparison to the control group
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex distribution of the fetuses in all three test groups was comparable to the control fetuses. Any observable differences were without biological relevance.
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
- no statistically significant differences of overall incidences were noted between the groups
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
- detected in single fetuses of all test groups including the control
All findings were considered to be spontaneous in origin and not treatment-related.
No statistically significant differences between the groups were noted. The overall incidences were well within the historical control range of the test facility.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
- soft tissue malformations occurred in all test groups including the control
- distribution of the findings about the test groups does not indicate an association to the treatment and no statistically significant differences between the groups were noted
- total incidence of soft tissue malformations in treated animals did not differ significantly from the control group
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
VARIATIONS/UNCLASSIFIED OBSERVATIONS:

EXTERNAL (refer to table 9)
- paw hyperflexion: recorded in four fetuses of one high-dose litter, occurred together with multiple external malformations
Paw hyperflexion can be found in the historical control data at comparable incidences.
- discolored placentae: recorded in one fetus of the low-dose test group
This finding is not considered to be related to treatment.

SOFT TISSUE (refer to table 10)
- absent lung lobe (Lobus inferior medialis) in all test groups including the control observed
- for the high-dose group: not statistically significant, however, it was outside the historical control range (historical control data [HCD]: mean% 0.9, range 0.0 - 2.0); these findings were clustered in only 3 litters = litter incidence of 15% which is inside the historical control range (HCD: mean% 7.0, range 0.0 – 17.4)
Overall, this finding is a common anatomical variant in this rabbit strain. Thus, the slightly higher high-dose incidence it is not considered to be of toxicological relevance.
- other variations (e.g. cerebral ventricle, malpositioned carotid branches narrowed pulmonary trunk, dilated aorta and dilated renal pelvis) occurred in individual fetuses of low- and/or high-dose test animals as well as in control groups
The incidences of these variations were neither statistically significantly different from control nor dose-dependent and, therefore, not considered biologically relevant.
- blood coagulum around urinary bladder: in four mid-dose and three high-dose fetuses
This finding can be found in the historical control data at comparable incidences, therefore, it was neither assessed as treatment-related nor as adverse.
- empty stomach (devoid of amniotic fluid): in four fetuses of the same high-dose litter
These fetuses had multiple other findings, discussed in the section for details.

SKELETAL (refer to table 11)
- skeletal variations of different bone structures: observed in all test groups with or without effects on corresponding cartilages
The observed skeletal variations were related to several parts of fetal skeletons and appeared in the majority of cases without a relation to dosing.
- irregular ossification of interparietal: in the mid- and high-dose groups increased and outside the historical control range
This finding represents small irregularities in the shape of the ossification nuclei in the interparietal. As desmal ossification of the neurocranium continues during later development and interparietal membrane as well as surrounding bones were intact, a completely regular ossification of this bone can be expected to occur postnatally. Thus, this finding was considered to be of little, if any toxicological relevance.
- ‘unossified talus (with present cartilage)’: statistically significantly increased and outside the historical control range in the high-dose group
This finding may represent slight delays of ossification which did not affect morphology, as the underlying cartilage model was completely intact in all these cases. This assessment is supported by the fact, that the mean fetal weight of all 10 fetuses showing this finding (i.e. 18.8 g) was clearly below the mean fetal weight of all fetuses in test group 3 (33.6 g), which indicates a delay in overall development going along with the delay in ossification.
The other increased incidences of skeletal variations were either not related to dose and/or inside the historical control range. Thus, they are not considered to be associated with treatment.
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.
Details on embryotoxic / teratogenic effects:
ASSESSMENT OF ALL OBSERVATIONS
- distribution of total malformations about the groups was not related to dose
One fetus of the control, two fetuses of the low-dose, four fetuses of the mid-dose and four fetuses of the high-dose group had more than one malformation or were multiple-malformed across the different examination areas.

For details, see table 12.
- a cluster of 4 fetuses in litter No.76 showed a spectrum of malformations and a number of less severe findings, e.g. paw hyperflexion and empty stomach (devoid of amniotic fluid)
All these findings contributed to apparently higher high-dose rates of external malformations and variations as well as unclassified soft tissue observations.
The clustered appearance in one litter and the almost identical spectrum of ontogenetically different findings in all those fetuses strongly suggests an origin of these anomalies which is unrelated to treatment. Consequently, the higher incidence of high-dose findings in their respective sections is also considered to be unrelated to treatment.

- other malformations appeared in individual fetuses of test groups 1 or 3
- no ontogenetic pattern is recognizable for the individual malformations, nor was there any cluster of any of these individual malformations
- malformations were not dose-related and all of them can be found in the historical control data at comparable or higher frequency
Overall, an association of all these findings to the treatment is not assumed.

- spontaneous origin is assumed for the external variations, soft tissue variations and the skeletal variations as observed in all test groups and the control group
If all different types of variations are summarized, none of the incidences showed a relation to dosing and can be found in the historical control data at a comparable frequency.

- spontaneous origin is assumed for the unclassified external, unclassified soft tissue and unclassified skeletal cartilage observations, observed in several fetuses of all groups
The distribution and type of these findings do not suggest any relation to treatment.
Finally, fetal examinations revealed that there is no adverse effect of the compound on the respective morphological structures up to the highest dose tested (100 mg/kg bw/d).
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
Abnormalities:
no effects observed
Description (incidence and severity):
Four fetuses in one litter showed malformations and a number of less severe findings such as (among others) paw hyperflexion and empty stomach (devoid of amniotic fluid). These findings contributed to apparently higher high-dose rates of external malformations and variations as well as unclassified soft tissue observations.
The clustered appearance in one litter and the almost identical spectrum of ontogenetically different findings in all those fetuses strongly suggests an origin of these anomalies which is unrelated to treatment. Consequently, the higher incidence of high-dose findings in their respective sections is also considered to be unrelated to treatment.
Developmental effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
no
Relevant for humans:
no

Table 1: Excerpt of the results of maternal clinical signs during gestation

 

 

Day of gestation

 

 

 

Group#

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

18

19

20

21

22

23

24

25

26

27

28

TOTAL

# OF FEMALES EXAMINED

0

25

25

25

25

25

25

25

25

25

25

24

24

24

24

24

24

24

24

24

24

24

23

23

23

23

23

23

23

23

22

 

 

1

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

 

 

2

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

 

 

3

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

25

24

24

23

22

21

 

 

BLOOD IN BEDDING BEFORE

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

1

 

1

 

1

 

0

 

1

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

1

TREATMENT

1

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

2

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

3

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

ABORTION AFTER TREATMENT

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

0

0

0

0

0

0

0

0

0

1

 

1

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

2

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

3

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

0

0

0

0

0

1

ABORTION BEFORE TREATMENT

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

0

1

 

1

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

2

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

3

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

1

1

0

3

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Day of gestation

 

Group#

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

18

19

20

21

22

23

24

25

26

27

28

TOTAL

STOOL/URINE

 

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

1

2

1

0

0

0

0

0

0

0

1

1

2

1

0

4

REDUCED DEFECATION BEFORE

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

1

2

1

0

0

0

0

0

0

0

1

1

2

1

0

4

TREATMENT

1

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

1

0

0

0

0

0

0

0

1

1

0

0

0

0

2

 

2

0

0

0

0

0

0

0

0

0

0

0

0

0

1

2

3

2

2

2

3

3

1

0

1

2

5

3

1

3

0

11

 

3

0

0

0

0

0

0

0

0

0

1

0

0

1

4

7

17

10

9

6

9

8

8

5

6

6

5

3

5

5

0

20

REDUCED DEFECATION AFTER

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

1

2

1

0

0

0

0

0

0

0

1

1

2

1

0

4

TREATMENT

1

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

1

0

0

0

0

0

0

0

1

1

0

0

0

0

2

 

2

0

0

0

0

0

0

0

0

0

0

0

0

0

1

2

3

2

2

2

3

3

1

0

1

2

5

3

1

3

0

11

 

3

0

0

0

0

0

0

0

0

0

1

0

0

1

4

7

17

10

9

6

9

8

8

5

6

6

5

3

5

5

0

20

NO DEFECATION BEFORE

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

2

0

2

TREATMENT

1

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

2

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

1

1

1

1

2

3

1

0

4

 

3

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

1

3

4

3

3

3

2

2

2

1

0

0

0

0

0

4

NO DEFECATION AFTER

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

1

0

2

TREATMENT

1

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

 

2

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

1

1

1

1

2

3

1

0

4

 

3

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

1

3

4

3

3

3

2

2

2

1

0

0

0

0

0

4

Table 2: Summary of reproduction data

 

TEST GROUP 0

0 mg/kgbw/d

TEST GROUP 1

10 mg/kgbw/d

TEST GROUP 2

30 mg/kgbw/d

TEST GROUP 3

100 mg/kgbw/d

Females Mated (N)

25

25

25

25

Pregnant (N)

23

24

24

24

Conception Rate (%)

92

96

96

96

Aborted (N)

2

0

0

4

Premature Births (N)

0

0

0

0

Dams with Viable Fetuses (N)

20

24

24

20

Dams with all Resorptions (N)

0

0

0

0

Female Mortality (N)

3 Fi

0

0

4

(%)

12

0.0

0.0

16

Pregnant at Terminal Sacrifice (N)

20 Fi

24

24

20

(%)

80

96

96

80

Corpora Lutea MEAN

9.8 D

9.8

10.8

11.0

S.D.

1.74

2.45

2.50

2.66

TOTAL

195

235

259

220

Implantation Sites MEAN

9.1 D

8.4

9.2

9.6

S.D.

2.24

2.93

2.69

3.35

TOTAL

181

201

221

192

Preimplantation Loss MEAN (%)

7.9 D

15.0

14.6

14.8

S.D.

13.88

22.83

19.55

18.32

Postimplantation Loss MEAN (%)

3.7 D

6.6

6.8

6.2

S.D.

8.47

9.64

8.66

11.52

Statistics: D = Dunnett-test (two-sided), * : p<=0.05 ** : p<=0.01; Fi =Fisher's exact test (one-sided)

Table 3: Mean maternal body weights during gestation (g)

 

 

 

TEST GROUP 0

0 mg/kg bw/d

TEST GROUP 1

10 mg/kg bw/d

TEST GROUP 2

30 mg/kg bw/d

TEST GROUP 3

100 mg/kg bw/d

DAY

0

MEAN

3755

D

3735

3738

3722

 

 

S.D.

161.8

 

174.4

165.0

163.5

 

 

N

22

 

24

24

24

DAY

2

MEAN

3820

D

3803

3813

3804

 

 

S.D.

163.3

 

174.3

159.5

157.9

 

 

N

22

 

24

24

24

DAY

4

MEAN

3869

D

3851

3847

3845

 

 

S.D.

177.5

 

176.5

167.6

156.0

 

 

N

22

 

24

24

24

DAY

6

MEAN

3932

D

3914

3908

3910

 

 

S.D.

175.9

 

192.7

170.9

157.3

 

 

N

22

 

24

24

24

DAY

9

MEAN

3969

D

3939

3944

3911

 

 

S.D.

191.9

 

186.3

194.7

179.9

 

 

N

22

 

24

24

24

DAY

11

MEAN

3982

D

3967

3965

3885

 

 

S.D.

174.7

 

191.0

191.4

196.7

 

 

N

22

 

24

24

24

DAY

14

MEAN

4036

D

4022

4020

3888*

 

 

S.D.

167.4

 

215.5

206.3

189.2

 

 

N

22

 

24

24

24

DAY

16

MEAN

4060

D

4062

4043

3875*

 

 

S.D.

167.9

 

245.2

209.3

211.7

 

 

N

22

 

24

24

24

DAY

19

MEAN

4012

D

4034

4006

3824**

 

 

S.D.

154.6

 

216.7

183.5

219.0

 

 

N

22

 

24

24

24

DAY

21

MEAN

4020

D

4006

3969

3808**

 

 

S.D.

142.0

 

233.8

185.8

228.7

 

 

N

21

 

24

24

24

 

 

 

 

 

 

 

 

DAY

23

MEAN

4026

D

4036

3994

3816**

 

 

S.D.

153.5

 

222.1

208.6

248.6

 

 

N

21

 

24

24

24

DAY

25

MEAN

4016

D

4030

4002

3848**

 

 

S.D.

163.9

 

197.0

232.9

244.3

 

 

N

21

 

24

24

23

DAY

28

MEAN

4030

D

4071

4018

3864

 

 

S.D.

202.8

 

216.6

255.6

291.3

 

 

N

21

 

24

24

20

 

 

 

 

 

 

 

 

DAY

29

MEAN

4066

D

4082

4038

3897

 

 

S.D.

179.2

 

213.0

273.2

289.9

 

 

N

20

 

24

24

20

Statistics: D=Dunnett-test (two-sided), * : p<=0.05 ** : p<=0.01

Table 4: Mean maternal body weight change during gestation (g)

 

 

 

 

 

TEST GROUP 0

0 mg/kg bw/d

TEST GROUP 1

10 mg/kg bw/d

TEST GROUP 2

30 mg/kg bw/d

TEST GROUP 3

100 mg/kg bw/d

DAYS

0

TO

2

MEAN

65.6 D

67.5

75.1

81.9

 

 

 

 

S.D.

32.00

40.53

36.07

33.40

 

 

 

 

N

22

24

24

24

DAYS

2

TO

4

MEAN

48.1 D

47.7

33.8

40.5

 

 

 

 

S.D.

36.94

36.09

36.35

33.23

 

 

 

 

N

22

24

24

24

DAYS

4

TO

6

MEAN

63.2 D

63.0

60.8

65.0

 

 

 

 

S.D.

23.22

46.02

36.95

45.39

 

 

 

 

N

22

24

24

24

DAYS

6

TO

9

MEAN

37.7 D

25.7

35.8

1.7

 

 

 

 

S.D.

56.02

79.94

67.63

68.06

 

 

 

 

N

22

24

24

24

DAYS

9

TO

11

MEAN

12.9 D

27.3

21.5

-26.6*

 

 

 

 

S.D.

53.91

27.89

50.28

48.89

 

 

 

 

N

22

24

24

24

DAYS

11

TO

14

MEAN

53.6 D

55.8

54.3

3.0

 

 

 

 

S.D.

54.88

60.38

90.43

80.20

 

 

 

 

N

22

24

24

24

DAYS

14

TO

16

MEAN

23.7 D

39.6

23.9

-13.4

 

 

 

 

S.D.

69.71

60.25

58.82

51.46

 

 

 

 

N

22

24

24

24

DAYS

16

TO

19

MEAN

-47.8 D

-28.4

-37.8

-50.1

 

 

 

 

S.D.

65.23

70.23

91.54

75.09

 

 

 

 

N

22

24

24

24

DAYS

19

TO

21

MEAN

-0.3 D

-27.5

-36.7*

-16.4

 

 

 

 

S.D.

34.96

50.66

38.55

61.69

 

 

 

 

N

21

24

24

24

DAYS

21

TO

23

MEAN

6.0 D

29.7

25.0

7.7

 

 

 

 

S.D.

40.54

56.58

62.58

67.35

 

 

 

 

N

21

24

24

24

DAYS

23

TO

25

MEAN

-10.6 D

-5.5

8.4

20.3

 

 

 

 

S.D.

73.17

57.69

68.65

72.82

 

 

 

 

N

21

24

24

23

DAYS

25

TO

28

MEAN

13.7 D

40.1

15.8

-4.2

 

 

 

 

S.D.

72.97

66.74

66.45

81.05

 

 

 

 

N

21

24

24

20

DAYS

28

TO

29

MEAN

11.4 D

11.4

19.9

32.8

 

 

 

 

S.D.

33.17

36.99

47.77

55.59

 

 

 

 

N

20

24

24

20

Statistics: D=Dunnett-test (two-sided), * : p<=0.05 ** : p<=0.01

Table 5: Mean maternal food consumption during gestation

 

 

 

TEST GROUP 0

TEST GROUP 1

TEST GROUP 2

TEST GROUP 3

 

 

 

0 mg/kg bw/d

10 mg/kg bw/d

30 mg/kg bw/d

100 mg/kg bw/d

DAYS

0 TO 6

MEAN OF MEANS

176.2

171.4

173.7

175.3

 

 

S.D.

4.60

6.55

5.07

7.45

 

 

N (days)

6

6

6

6

DAYS

6 TO 28

MEAN OF MEANS

118.2

123.0

114.1

81.6

 

 

S.D.

29.57

25.04

28.78

31.98

 

 

N (days)

22

22

22

22

DAYS

0 TO 29

MEAN OF MEANS

129.6

132.1

125.9

101.5

 

 

S.D.

35.47

30.24

35.35

47.50

 

 

N (days)

29

29

29

29

Table 6: Summary of maternal necropsy observations

 

FEMALES EXAMINED

 

N

TEST GROUP 0

0 mg/kg bw/d

25

TEST GROUP 1

10 mg/kg bw/d

25

TEST GROUP 2

30 mg/kg bw/d

25

TEST GROUP 3

100 mg/kg bw/d

25

NOTHING ABNORMAL DETECTED

N

20

22

22

20

 

%

80

88

88

80

FINDINGS AFTER GAVAGE ERROR

N

1

0

0

0

 

%

4.0

0.0

0.0

0.0

LUNGS: ACUTE FIBRINOUS -

N

1

0

0

0

PURULENT PNEUMONIA

%

4.0

0.0

0.0

0.0

LIVER: GRANULATED SURFACE

N

0

1

0

0

 

%

0.0

4.0

0.0

0.0

KIDNEY: MALPOSITIONED

N

0

1

1

0

 

%

0.0

4.0

4.0

0.0

RECTUM: NO FECES

N

1

0

2

1

 

%

4.0

0.0

8.0

4.0

WATERY FECES

N

2

0

1

1

 

%

8.0

0.0

4.0

4.0

URETER: SHORT

N

0

1

1

0

 

%

0.0

4.0

4.0

0.0

OVARIES: RUDIMENTARY

N

1

0

0

0

 

%

4.0

0.0

0.0

0.0

Table 7: Summary of reproduction data of the females

 

TEST GROUP 0

0 mg/kg bw/d

TEST GROUP 1

10 mg/kg bw/d

TEST GROUP 2

30 mg/kg bw/d

TEST GROUP 3

100 mg/kg bw/d

Pregnant at Terminal Sacrifice (N)

20

24

24

20

Resorptions: Total MEAN

0.3 D

0.5

0.7

0.6

S.D.

0.81

0.78

0.82

1.23

TOTAL

7

12

16

13

MEAN (%)

3.7 D

6.2

6.8

6.2

S.D.

8.47

9.70

8.66

11.52

Early MEAN

0.3 D

0.3

0.3

0.4

S.D.

0.80

0.69

0.48

1.14

TOTAL

6

7

8

8

MEAN (%)

3.2 D

3.8

3.6

3.7

S.D.

8.32

8.94

5.42

10.47

Late MEAN

0.1 D

0.2

0.3

0.3

S.D.

0.22

0.51

0.56

0.64

TOTAL

1

5

8

5

MEAN (%)

0.6 D

2.3

3.3

2.5

S.D.

2.48

5.72

5.80

6.52

Dead Fetuses (N)

0

1

0

0

Statistics: D=Dunnett-test (two-sided), *: p<=0.05 ** : p<=0.01

Table 8: Mean placental and fetal body weights (based on litter)

 

 

TEST GROUP 0

0 mg/kg bw/d

TEST GROUP 1

10 mg/kg bw/d

TEST GROUP 2

30 mg/kg bw/d

TEST GROUP 3

100 mg/kg bw/d

PLACENTAL WEIGHTS UNITS; GRAMS:

 

 

 

 

 

 

MEAN

5.0 D

5.1

4.8

4.7

 

S.D.

0.80

0.94

0.77

0.87

 

N

20

24

24

20

of Male Fetuses

MEAN

5.1 D

5.2

4.9

4.8

 

S.D.

0.83

0.85

0.75

1.00

 

N

20

23

23

20

of Female Fetuses

MEAN

4.9 D

5.0

4.7

4.7

 

S.D.

0.80

0.93

0.89

0.86

 

N

20

23

24

20

FETAL WEIGHTS, UNITS:

GRAMS

 

 

 

 

of all Viable Fetuses

MEAN

38.3 D

38.6

36.3

33.6*

 

S.D.

5.23

6.72

5.07

5.93

 

N

20

24

24

20

of Male Fetuses

MEAN

39.1 D

39.2

36.0

33.9*

 

S.D.

5.76

5.48

5.31

5.91

 

N

20

23

23

20

of Female Fetuses

MEAN

37.6 D

37.3

36.0

33.6

 

S.D.

5.15

6.83

5.49

6.30

 

N

20

23

24

20

Statistics: D=Dunnett-test (two-sided), * : p<=0.05 ** : p<=0.01

Table 9: Total fetal external malformations

 

 

Test group 0

0 mg/kgbw/d

Test group 1

10 mg/kg bw/d

Test group 2

30 mg/kg bw/d

Test group 3

100 mg/kg bw/d

Litter Fetuses

N N

20

174

24

189

24

205

20

179

Fetal incidence

 

N (%)

 

1 (0.6)

 

1 (0.5)

 

0.0

 

5 (2.8)

Litter incidence

 

N (%)

 

1 (5.0)

 

1 (4.2)

 

0.0

 

2 (10)

Affected fetuses/litter

 

Mean %

 

0.5

 

0.4

 

0.0

 

2.4

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

Table 10: Total soft tissue malformations

 

 

Test group 0

0 mg/kgbw/d

Test group 1

10 mg/kg bw/d

Test group 2

30 mg/kg bw/d

Test group 3

100 mg/kg bw/d

Litter Fetuses

N N

20

174

24

189

24

205

20

179

Fetal incidence

 

N (%)

 

2 (1.1)

 

3 (1.6)

 

3 (1.5)

 

1 (0.6)

Litter incidence

 

N (%)

 

1 (5.0)

 

3 (13)

 

2 (8.3)

 

1 (5.0)

Affected fetuses/litter

 

Mean %

 

0.9

 

1.6

 

1.4

 

0.4

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

Table 11: Total skeletal malformations

 

 

Test group 0

0 mg/kgbw/d

Test group 1

10 mg/kg bw/d

Test group 2

30 mg/kg bw/d

Test group 3

100 mg/kg bw/d

Litter Fetuses

N N

20

174

24

189

24

205

20

179

Fetal incidence

 

N (%)

 

1 (0.6)

 

2 (1.1)

 

2 (1.0)

 

1 (0.6)

Litter incidence

 

N (%)

 

1 (5.0)

 

2 (8.3)

 

2 (8.3)

 

1 (5.0)

Affected fetuses/litter

 

Mean%

 

0.6

 

1.2

 

1.1

 

0.4

Table 12: Fetuses with more than one malformation

Test group

Doe No.-Fetus No., Sex

Finding

0 (0 mg/kg bw/d)

25-10 F

domed head, hydrocephaly

1 (10 mg/kg bw/d)

26-10 M

thoracic hemivertebra, misshapen thoracic vertebra

44-01 F

malpositioned kidney, short ureter

2 (30 mg/kg bw/d)

68-07 F

exoccipital fused with 1st cervical arch, cervical hemivertebra

69-03 M

multiple malformations of the great vessels (persistent truncus arteriosus, aortic arch atresia, malpositioned subclavian origin)

75-02 F

aortic arch atresia, malpositioned kidney

75-06 F

thoracic hemivertebra, branched rib

3 (100 mg/kg bw/d)

76-04 M

multiple external malformations (domed head, cleft palate, small tongue), hydrocephaly

76-06 F

multiple external malformations (domed head, cleft

palate, small tongue)

76-11 F

multiple external malformations (domed head, cleft

palate, small tongue), severely malformed skull bones

76-12 M

multiple external malformations (domed head, cleft

palate, small tongue)

mg/kg bw/d = milligram per kilogram body weight per day; No.= number; M = male; F = female

The stability of the test substance in 0.5% CMC suspension in deionized water (with 10 mg/100 mL Cremophor EL) over a maximum of 7 days and warmed up to approximately 30 degrees Celsius was demonstrated. The homogeneous distribution of the test substance in the vehicle was shown. The correctness of the prepared concentrations was shown.

Conclusions:
The oral administration of the test substance to pregnant New Zealand White rabbits from implantation to one day prior to the expected day of parturition (GD 6-28) caused evidence of systemic maternal toxicity at the high-dose level of 100 mg/kg bw/d, such as a slightly higher incidence of abortions and reduced defecation in almost all females of this group, along with a distinct decrease of food consumption as well as body weight/body weight gain.
In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 30 mg/kg bw/d.
The no observed adverse effect level (NOAEL) for prenatal developmental toxicity is also set at 30 mg/kg bw/d. The mean fetal body weights were significantly lower in the 100 mg/kg bw/d group compared to the control group (33.6 g versus 38.6 g). Considering the immediate effect of dosing on maternal food consumption and body weight loss during GD 6-28, the reduced fetal body weights are a result of the poor nutritional condition of the does. The substance has no direct developmental toxicity. No teratogenicity was observed.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

A developmental toxicity study in rats (OECD 414) is available. Dose levels were 50, 150 and 500 mg/kg bw and corn oil was used as vehicle. Dose levels were chosen based on a range-finding study in pregnant rats with 300 and 1000 mg/kg bw. In the maternal toxicity study, alterations of some liver/metabolic parameters were found specially in the high dose group and included increased values of alkaline phosphatase, alanine aminotransferase, cholesterol, triglycerides, glucose and decreases of creatinine, protein, albumin, globulin, calcium and bile acids. Relative liver weights were increased. Increased absolute and relative adrenals weights and decreased absolute spleen and kidneys weights were found in the high dose group when compared to controls. Values of alkaline phosphatase, triglycerides and bile acids were also different to controls at 300 mg/kg bw/day. Pale discoloration of liver was observed in four dams of the high dose group. Terminal body weight, corrected body weight and corrected body weight gain were significantly reduced in the high dose group. Uterus weight did not differ between groups.

The doses chosen for the main study are sufficient to ensure some but not excessive maternal toxicity. In the main study, statistically significant decrease in body weight (up to 7%) and body weight gain was observed in treated females receiving 500 mg/kg bw/day, starting from Day 9 post coitum. Statistically significant decrease in food consumption (up to 22%) was observed in treated females receiving 500 mg/kg bw/day, starting from Day 9 post coitum. Statistically significant decrease in corrected body weight and corrected body weight gain was noted in treated females receiving 150 and 500 mg/kg bw/day. The difference in corrected body weight at 150 mg/kg bw/day was minor (258 versus 270 g) and therefore not considered adverse. There were no macroscopic findings at necropsy. Considering the effects on food consumption, corrected body weight and corrected body weight gain and the knowledge on clinical chemistry from the range-finding study, the high dose group caused liver toxicity and the NOAEL for maternal toxicity was 150 mg/kg. Litter data, mean foetal weight and sex ratio were unaffected by treatment. No treatment-related differences were seen at visceral examination of foetuses between the control and treated groups. No relevant changes were recorded at the skeletal examination of foetuses in treated groups, compared to controls. An increase in the presence of small foetuses was recorded in the high dose group. This higher number of small foetuses was due to one litter, and the corresponding dam had the smallest corrected body weight gain and was most strongly affected by maternal toxicity.

A prenatal developmental toxicity in New Zealand White rabbits was performed according to OECD 414. The test substance was administered as an aqueous suspension to groups of 25 inseminated female New Zealand White rabbits orally by gavage in doses of 10, 30 and 100 mg/kg body weight/day (mg/kg bw/d) on gestation days (GD) 6 through 28. The vehicle control group, consisting of 25 females, was dosed with the vehicle (0.5% Sodium carboxymethyl cellulose [CMC] suspension in deionized water (with 10 mg/100 mL Cremophor EL) in parallel. A standard dose volume of 10 mL/kg body weight was used for each test group.

At terminal sacrifice on GD 29, 20-24 females per group had implantation sites. Food consumption and body weight of the animals were recorded regularly throughout the study period. The state of health of the animals was checked each day.

On GD 29, all females were sacrificed and assessed by gross pathology (including weight determinations of the unopened uterus and placentas). For each doe, corpora lutea were counted and number and distribution of implantation sites (differentiated between resorp-tions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed and further investigated for any external, soft tissue and skeletal (inclusive cartilage) findings.

The stability of the test substance in 0.5% CMC suspension in deionized water (with 10 mg/100 mL Cremophor EL) over a maximum of 7 days and warmed up to approximately 30 degrees Celsius was demonstrated. The homogeneous distribution of the test substance in the vehicle was shown. The correctness of the prepared concentrations was shown.

The following test substance-related adverse effects/findings were noted:

Test group 3 (100 mg/kg bw/d):

Dams: Increased number of abortions (4 vs. 2 in control), reduced food consumption, overall 31% less food than the concurrent control does during GD 6-28, reduced mean body weights (BW) and average body weight gain (BWC), weight loss during the treatment period (-24.0 g vs. +104.4 g in control), lower net weight gain ( 421.0g) in comparison to the concurrent control (-322.2 g).

Fetuses: No test substance-related, independent adverse effects on fetuses.

Test group 2 (30 mg/kg bw/d):

No test substance-related adverse effects on does, gestational parameters or fetuses. The mean fetal body weights were significantly lower in the 100 mg/kg bw/d group compared to the control group (33.6 g versus 38.6 g). Considering the immediate effect of dosing on maternal food consumption and body weight loss during GD 6-28, the reduced fetal body weights are a result of the poor nutritional condition of the does. The substance has no direct developmental toxicity. No teratogenicity was observed.

Test group 1 (10 mg/kg bw/d):       

No test substance-related adverse effects on does, gestational parameters or fetuses.

Under the conditions of this prenatal developmental toxicity study, the oral administration of the test substance to pregnant New Zealand White rabbits from implantation to one day prior to the expected day of parturition (GD 6-28) caused evidence of systemic maternal toxicity at the high- dose level of 100 mg/kg bw/d, such as a slightly higher incidence of abortions and reduced defecation in almost all females of this group, along with a distinct decrease of food consumption as well as body weight/body weight gain. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 30 mg/kg bw/d. Since there was no evidence for independent, 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 100 mg/kg bw/d.

Justification for classification or non-classification

The available data on reproductive organs from the subchronic toxicity study does not give rise of concern for adverse effects on fertility. Further testing on fertility is currently ongoing.

The developmental toxicity studies (OECD 414) did not give rise of concern for developmental toxicity. No need for classification and labelling can be derived from the studies.