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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:
Feb - Aug 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
25 Jun 2018
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt Rheinland-Pfalz
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: DEB2228074 ex Clariant
- Expiration date of the Batch: 01 May 2028
- Purity: 90.0 % area
- Physical state / appearance: solid / red

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: The stability of the test substance in deionized water over a period of 7 days at room temperature waas demonstrated before the start of the study.
- Solubility and stability of the test substance in the solvent/vehicle: Before and during administration, the preparations were kept homogeneous with a magnetic stirrer.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: 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. The preparations were kept at room temperature.
- Final preparation of a solid: For the test substance preparations, the specific amount of test substance was weighed, topped up with 0.5 % CMC suspension in deionized water in a calibrated breaker and intensely mixed with a magnetic stirrer.

FORM AS APPLIED IN THE TEST (if different from that of starting material) : suspension

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: about 10 - 12 weeks
- Weight at study initiation: varied between 162.1 - 218.6 g
- Housing: individually in Polycarbonate cages type III
- Diet (e.g. ad libitum): ground Kliba maintenance diet mouse / rat "GLP", meal, supplied by Granovit AG, Kaiseraugst, Switzerland; ad libitum
- Water (e.g. ad libitum): potable tap water in water bottles; ad libitum
- Acclimation period: start of the study until first administration on GD 6

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 45 - 65 %
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5 % suspension in deionized water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The specific amount of test substance was weighed, topped up with 0.5 % CMC suspension in deionized water in a calibrated beaker and intensely mixed with a magnetic stirrer.

VEHICLE
- Concentration in vehicle: 0, 0.03, 0.10, and 0.30 g/100 mL
- Amount of vehicle (if gavage): 10 mL/kg bw/d
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the test substance preparations were sent three times (at the beginning (samples 1-9) and after the end of administration (reserve samples 1R-9R and samples 11-18)) to the analytical laboratory for verification of the concentrations. The samples taken for the concentrations control analyses were also used to verify the homogeneity of the samples of the low- and high-concentrations each (3 and 30 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.
Details on mating procedure:
The animals were paired by the breeder ("time-mated"); the day of evidence of mating (= detection of vaginal plug / sperm) was referred to as GD 0.
Duration of treatment / exposure:
The test substance preparations were administered to the animals once a day orally by gavage, from implantation to one day prior to the expected day of parturition (GD 6 to GD 19).
Frequency of treatment:
once daily
Duration of test:
20 days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
vehicle control
Dose / conc.:
3 mg/kg bw/day (nominal)
Remarks:
low-dose level
Dose / conc.:
10 mg/kg bw/day (nominal)
Remarks:
mid-dose level
Dose / conc.:
30 mg/kg bw/day (nominal)
Remarks:
high-dose level
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were chosen at the request of the sponsor.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily before and after treatment period (GD 0 - 5 and 20). During treatment period (GD 6 - 19) all rats were checked daily.
- Cage side observations checked: signs of morbidity, pertinent behavioral changes and / or signs of overt toxicity before administration as well as within 2 hours and within 5 hours after administration

DETAILED CLINICAL OBSERVATIONS: No
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule: The consumption of food was recorded for the intervals GD 0-1, 1-3, 3-6, 6-8, 8-10, 10-13, 13-15, 15-17, 17-19 and 19-20.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations:

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: thyroid glands, uteri, ovaries, spleen

OTHER: hematology, clinical chemistry, thyroid hormones
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: live fetuses, dead fetuses
Fetal examinations:
- External examinations: Yes
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
- Head examinations: No
Statistics:
- Simultaneous comparison of all dose groups with the control group using the DUNNETT-test (two-sided) for the hypothesis of equal means
- Pairwise comparison of each dose group with the control group using FISHER'S EXACT test (one-sided) for the hypothesis of equal proportions
- Pairwise comparison of each dose group with the control group using the WILCOXON test (one-sided) for the hypothesis of equal medians
- Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (twosided) for the hypothesis of equal medians
- Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians
- Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians
Indices:
- conception rate
- preimplantation loss
- postimplantation loss
- anogenital distance

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any female at dose levels of 3, 10 or 30 mg/kg bw/d during the entire study period.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no test substance-related or spontaneous mortalities in any females of all test groups (0, 3, 10 or 30 mg/kg bw/d).
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 dams (3, 10 and 30 mg/kg bw/d) were generally comparable to the concurrent control group throughout the entire study period.
The corrected body weight gain of test groups 1, 2 and 3 (3, 10 and 30 mg/kg bw/d) revealed no difference of any biological relevance to the corresponding control group. Moreover, mean carcass weights of all test groups remained unaffected by the treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The mean food consumption of the high-, mid- and low-dose dams (30, 10 and 3 mg/kg bw/d) was generally comparable to the concurrent control group 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:
effects observed, treatment-related
Description (incidence and severity):
At gestation day 20, in dams of test groups 2 and 3 (10 and 30 mg/kg bw/d) red blood cell (RBC) counts, hemoglobin and hematocrit values were significantly decreased whereas absolute reticulocyte counts were significantly increased. Although each parameter was only slightly changed, the sum of altered red blood cell parameters was regarded as treatment-related and adverse.
Mean corpuscular hemoglobin concentration (MCHC) was significantly decreased in dams of test group 3 (30 mg/kg bw/d), but the mean was within the historical control range (MCHC 21.16-22.65 mmol/L). The same was true for significantly increased absolute and relative monocyte counts in dams of test groups 1, 2 and 3 (3, 10 and 30 mg/kg bw/d; test group 3 no significant increase) as well as decreased relative eosinophil counts in dams of test group 3 (absolute monocytes 0.08-0.18 Giga/L; relative monocytes 2.0-3.1 %, relative eosinophils 0.8 - 2.0 %). Therefore, these changes were regarded as incidental and not treatment-related.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At gestation day 20, in dams of test groups 2 and 3 (10 and 30 mg/kg bw/d) total bilirubin values were significantly increased. In combination with the changed red blood cell parameters, this alteration was regarded as treatment-related and adverse.
In dams of test group 2 (10 mg/kg bw/d) albumin values were significantly decreased, but the change was not dose-dependent. Therefore, it was regarded as incidental and not treatmentrelated.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Absolute weights:
When compared to control group 0 (= 100 %), the mean absolute weight of the spleen was significantly increased in test group 3. All other mean absolute weight parameters did not show significant differences when compared to the control group 0.

Relative organ weights
When compared to control group 0 (= 100 %), the mean relative weights of the spleen were significantly increased in test groups 2 and 3. All other mean relative weight parameters did not show significant differences when compared to the control group 0.

In test group 3, the significant increases in absolute (+20 %) and relative (+21 %) spleen weights were assessed as treatment-related.
In test group 2, the significantly increased relative spleen weight (0.224%) was within historical control range values (0.193 % - 0.227 %) and showed no histopathological correlate. Therefore, these changes were considered not treatment-related.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The spleen of 5 out of 25 animals in test group 3 and 1 out of 25 animals in test group 2 was enlarged.
All other findings occurred individually. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related findings were observed in the spleen of females of test group 3.
All other findings were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
Other effects:
no effects observed
Description (incidence and severity):
In dams of test groups 1, 2 and 3 (3,10 and 30 mg/kg bw/d) no treatment-related alterations of T3, T4 and TSH levels were observed.

Maternal developmental toxicity

Pre- and post-implantation loss:
effects observed, non-treatment-related
Total litter losses by resorption:
no effects observed
Early or late resorptions:
effects observed, non-treatment-related
Dead fetuses:
no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
The conception rate was 92% in the mid-dose group (10 mg/kg bw/d) and 100% in the control and the low- and high-dose groups (0, 3 and 30 mg/kg bw/d). With these rates, a sufficient number of pregnant females were available for the purpose of this study.
Details on maternal toxic effects:
There were no test substance-related and/or biologically relevant differences between the test groups in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and post-implantation losses, the number of resorptions and viable fetuses. This includes the statistically significant lower number of resorptions/ early resorptions, and lower value for postimplantation loss, as well as the statistically significant higher number of live fetuses in test group 2 (10 mg/kg bw/d) which was assessed as incidental. All observed differences are considered to reflect the normal range of fluctuations for animals of this strain and age.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
3 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
haematology

Maternal abnormalities

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.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex distribution of the fetuses in test groups 1-3 (3, 10 and 30 mg/kg bw/d) was comparable to the control fetuses.
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No external malformations were recorded.
No external variations were recorded.
One unclassified external observation was recorded. Fused placentae were seen in one litter, each, of test groups 1 and 2 (3 and 10 mg/kg bw/d). This finding was not considered biologically relevant, since it was a single event in the respective dose group.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Skeletal malformations were recorded for one fetus, each, in test groups 0, 2 and 3 . All findings were single events in individual fetuses and no ontogenetic pattern was obvious, therefore, the observed malformations were not assessed as treatment-related and adverse. The mean values of total incidences of skeletal malformations did not differ significantly and were within the historical control data.
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 dose. The overall incidences of skeletal variations were comparable to the historical control data.
The increased incidences of skeletal variations were not related to the dose and they were inside the historical control range. Therefore, they were not considered as treatment-related.
Additionally, some isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in all test groups. The observed unclassified cartilage findings were related to the skull, the ribs and the sternum and did not show any relation to dosing. However, the overall incidence of unclassified cartilage observations was statistically significantly increased in test group 3 (30 mg/kg bw/d) nevertheless well within the historical control range.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Soft tissue malformations occurred in two control and one fetus, each, in test group 1 and 3 (0, 3 and 30 mg/kg bw/d). These findings were not related to dose and single events in individual fetuses. The overall incidences of soft tissue malformations were comparable to those found in the historical control data.
Four soft tissue variations were detected, i.e. malpositioned carotid branch in the control group, elongated innominate in test group 1, dilated renal pelvis in all test groups and dilated ureter in test groups 0-2. The incidences of these variations were neither statistically significantly nor
dose-dependently increased in the treated groups. Except ‘elongated innominate’, which occurred once in test group 1, all of them can be found in the historical control data at comparable incidences. Therefore, they were not assessed as treatment-related.
No soft tissue unclassified observations were recorded.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: absence of adverse effects

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

Table 2: Absolute organ weights of maternal animals

Absolute weights

Females

Test group [mg/kg bw/d]

1 [3]

2 [10]

3 [30]

Spleen

99 %

106 %

120 % **

* : p 0.05, **: p 0.01

Table 3: Relative organ weights of maternal animals

Relative weights

Females

Test group [mg/kg bw/d]

1 [3]

2 [10]

3 [30]

Spleen

98 %

108 % *

121 % **

* : p 0.05, **: p 0.01

Table 4: Treatment-related findings in the spleen with incidences and gradings

 

Female animals

Test group [mg/kg bw/day]

0 [0]

1 [3]

2 [10]

3 [30]

No. of dams

25

25

23

25

Spleen

25

25

23

25

Hematopoiesis, extramedullary

25

25

23

25

-         Grade 1

5

7

3

0

-         Grade 2

17

16

14

3

-         Grade 3

3

2

6

19

-         Grade 4

0

0

0

3

Table 5: Total external unclassified observations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

3 mg/kg bw/d

Test group 2

10 mg/kg bw/d

Test group 3

30 mg/kg bw/d

Litter

Fetuses

N

N

25

265

25

282

23

265

25

261

Fetal incidence

 

 

N [%]

 

0.0

 

1 [0.4]

 

1 [0.4]

 

0.0

Litter incidence

 

 

N [%]

 

0.0

 

1 [4.0]

 

1 [4.3]

 

0.0

Affected fetuses/litter

Mean%

0.0

0.3

0.3

0.0

N = number

Table 6: Total soft tissue malformations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

3 mg/kg bw/d

Test group 2

10 mg/kg bw/d

Test group 3

30 mg/kg bw/d

Litter

Fetuses

N

N

25

128

25

134

23

128

25

123

Fetal incidence

 

 

N [%]

 

2 [1.6]

 

1 [0.7]

 

0.0

 

1 [0.8]

Litter incidence

 

 

N [%]

 

2 [8.0]

 

1 [4.0]

 

0.0

 

1 [4.2]

Affected fetuses/litter

Mean%

1.6

0.8

0.0

0.6

N = number

Table 7: Total soft tissue variations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

3 mg/kg bw/d

Test group 2

10 mg/kg bw/d

Test group 3

30 mg/kg bw/d

Litter

Fetuses

N

N

25

128

25

134

23

128

25

123

Fetal incidence

 

 

N [%]

 

4 [3.1]

 

9 [6.7]

 

4 [3.1]

 

2 [1.6]

Litter incidence

 

 

N [%]

 

2 [16]

 

6 [24]

 

4 [17]

 

2 [8.3]

Affected fetuses/litter

Mean%

3.4

6.2

3.4

1.9

N = number

Table 8: Total skeletal malformations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

3 mg/kg bw/d

Test group 2

10 mg/kg bw/d

Test group 3

30 mg/kg bw/d

Litter

Fetuses

N

N

25

137

25

148

23

137

25

138

Fetal incidence

 

 

N [%]

 

1 [0.7]

 

0.0

 

1 [0.7]

 

1 [0.7]

Litter incidence

 

 

N [%]

 

1 [4.0]

 

0.0

 

1 [4.3]

 

1 [4.0]

Affected fetuses/litter

Mean%

0.8

0.0

0.9

0.8

N = number

Table 9: Total skeletal variations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

3 mg/kg bw/d

Test group 2

10 mg/kg bw/d

Test group 3

30 mg/kg bw/d

Litter

Fetuses

N

N

25

137

25

148

23

137

25

138

Fetal incidence

 

 

N [%]

 

125 [91]

 

143 [97]

 

120 [88]

 

130 [94]

Litter incidence

 

 

N [%]

 

25 [100]

 

25 [100]

 

23 [100]

 

25 [100]

Affected fetuses/litter

Mean%

91.4

96.9

88.2

94.1

N = number

Table 10: Occurence of statistically significantly increased fetal skeletal variations

Finding

Test group 0

[0 mg/kg bw/d]

Test group 1

[3 mg/kg bw/d]

Test group 2

[10 mg/kg bw/d]

Test group 3

[30 mg/kg bw/d]

HCD Mean %

[range]

Unossified sternebra;

Unchanged cartilage

0.0

4.3 **

3.8 *

1.5

3.8

[0.0 – 9.6]

Unilateral ossification of sternebra;

Unchanged cartilage

0.0

2.5 *

1.4

1.6

1.0

[0.0 – 4.6]

Wavy rib

0.7

2.7

4.9 *

2.0

4.6

[0.0 – 13.3]

HCD: Historical control data

*p ≤ 0.05 (Wilcoxon-test [one-sided])

**p ≤ 0.01 (Wilcoxon-test [one-sided])

Table 11: Total unclassified cartilage observations

 

 

Test group 0

[0 mg/kg bw/d]

Test group 1

[3 mg/kg bw/d]

Test group 2

[10 mg/kg bw/d]

Test group 3

[30 mg/kg bw/d]

Litter

Fetuses

N

N

25

137

25

148

23

137

25

138

Fetal incidence

 

 

N [%]

 

88 [64]

 

109 [74]

 

103 [75]

 

112 [81]

Litter incidence

 

 

N [%]

 

25 [100]

 

24 [96]

 

23 [100]

 

25 [100]

Affected fetuses/litter

Mean%

64.8

73.5

73.7

81.3*

N = number

*p ≤ 0.05 (Wilcoxon-test [one-sided])

Table 12: Total fetal malformations

 

 

Test group 0

[0 mg/kg bw/d]

Test group 1

[3 mg/kg bw/d]

Test group 2

[10 mg/kg bw/d]

Test group 3

[30 mg/kg bw/d]

Litter

Fetuses

N

N

25

265

25

282

23

265

25

261

Fetal incidence

 

 

N [%]

 

3 [1.1]

 

1 [0.4]

 

1 [0.4]

 

2 [0.8]

Litter incidence

 

 

N [%]

 

2 [8.0]

 

1 [4.0]

 

1 [4.3]

 

2 [0.8]

Affected fetuses/litter

Mean%

1.2

0.4

0.4

0.7

N = number

Table 13: Total fetal variations

 

 

Test group 0

[0 mg/kg bw/d]

Test group 1

[3 mg/kg bw/d]

Test group 2

[10 mg/kg bw/d]

Test group 3

[30 mg/kg bw/d]

Litter

Fetuses

N

N

25

265

25

282

23

265

25

261

Fetal incidence

 

 

N [%]

 

129 [49]

 

152 [54]

 

124 [47]

 

132 [51]

Litter incidence

 

 

N [%]

 

25 [100]

 

25 [100]

 

23 [100]

 

25 [100]

Affected fetuses/litter

Mean%

48.9

54.0*

47.4

52.5

N = number

*p ≤ 0.05 (Wilcoxon-test [one-sided])

Applicant's summary and conclusion

Conclusions:
Under the conditions of this prenatal developmental toxicity study, the oral administration of the test substance to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6 - 19) caused evidence of maternal toxicity, such as a regenerative normocytic, normochromic, hemolytic anemia at doses of 10 mg/kg bw/d and above.
In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 3 mg/kg bw/d.
There were no toxicologically relevant adverse fetal findings evident. Thus, the no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 30 mg/kg bw/d, the highest tested dose.
The test substance is not teratogenic in rats.
Executive summary:

The test substance was tested for its prenatal developmental toxicity in Wistar rats. The test substance was administered as an aqueous preparation to groups of 25 time-mated female Wistar rats by gavage at doses of 3, 10 and 30 mg/kg body weight/day (mg/kg bw/d) on gestation days (GD) 6 through 19. The control group, consisting of 25 females, was dosed with

the vehicle (0.5 % Sodium carboxymethyl cellulose (CMC) suspension in deionized water) in parallel. A standard dose volume of 10 mL/kg body weight was used for each test group. At terminal sacrifice on GD 20, 23 - 25 females per group had implantation sites.

Food consumption and body weights of the animals were recorded regularly throughout the study period. The state of health of the animals was checked each day.

On GD 20, blood samples were obtained from all females by retrobulbar venous puncture following isoflurane anesthesia. After blood sampling all females were sacrificed by decapitation (under isoflurane anesthesia) and assessed by gross pathology (including organ sampling of the spleen, the thyroid glands (including parathyroid glands) as well as weight

determinations of the spleen, thyroid glands (with parathyroid glands), the unopened uterus and placentas). For each dam, corpora lutea were counted and number and distribution of implantation sites (differentiated between resorptions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed and further investigated for

external findings. Anogenital distance measurements were conducted on all liveborn fetuses. Thereafter, one half of the fetuses of each litter were examined for soft tissue findings and the remaining fetuses for skeletal (inclusive cartilage) findings.

Results

Analytics

• The stability of the test substance in deionized water over a period of 7 days at room temperature was demonstrated.

• The homogeneous distribution of the test substance in the vehicle was shown.

• The overall correctness of the prepared concentrations was shown.

Effects

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

Test group 3 (30 mg/kg bw/d):

Dams

• Decreased red blood cell (RBC), hematocrit and hemoglobin values.

• Increased absolute reticulocyte counts and total bilirubin values.

• Statistically significant increase of absolute (+20%) and relative (+21%) spleen weight.

• Spleen: Hematopoiesis, extramedullary: 22 out of 25 animals (moderate to severe).

Fetuses

• No test substance-related adverse effects on fetuses.

Test group 2 (10 mg/kg bw/d):

Dams

• Decreased red blood cell (RBC), hematocrit and hemoglobin values.

• Increased absolute reticulocyte counts and total bilirubin values.

Fetuses

• No test substance-related adverse effects fetuses.

Test group 1 (3 mg/kg bw/d):

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

Under the conditions of this prenatal developmental toxicity study, the oral administration of the test substance to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) caused evidence of maternal toxicity, such as a regenerative normocytic, normochromic, hemolytic anemia at doses of 10 mg/kg bw/d and above.

In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 3 mg/kg bw/d.

There were no toxicologically relevant adverse fetal findings evident. Thus, the no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 30 mg/kg bw/d, the highest tested dose.

The test substance is not teratogenic in rats.