Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
22 January 2001
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
August 1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: 2015-03-24

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

Test animals

Species:
rabbit
Strain:
New Zealand White
Remarks:
(Crl:KBL(NZW))
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Supplied by Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 16-17 weeks
- Weight at study initiation: 3347 - 4302 g
- Fasting period before study: None
- Housing: Singly in Type 4X03B700CP cages (floor space 4264 mm2, internal height 450 mm). For enrichment, wooden gnawing blocks were added (Typ KNH E-041, supplied by Abedd® Lab. and Vet. Service GmbH, Vienna, Austria).
- Diet: Pelleted Kliba maintenance diet rabbit and guinea pig “GLP”, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland; ad libitum
- Water: Portable tap water in water bottles; ad libitum
- Acclimation period: Yes

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20±2
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs light / hrs dark): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% suspension in drinking water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

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. For the test substance preparation, the specific amount of the test substance was weighed, topped up with 0.5% Carboxymethylcellulose suspension in drinking water in a calibrated beaker and intensely mixed with a homogenizer. During administration, the preparations were kept homogeneous with a magnetic stirrer.



Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
The analyses of homogeneous distribution of the test substance in the vehicle as well as correctness of the prepared concentrations are performed in a separate GLP study which was not completed by the end of January 2017. The present study report is being finalized without the results of these analyses because of urgent authority submission requirements of the toxicology results. This has no impact on the outcome of the present toxicology study. The results of these analyses will be covered in a GLP-compliant Amendment to the Report for submission to sponsor/authority as soon as they become available.

The concentrations in the endpoint study record and the endpoint summary are thus currently the nominal concentrations.
Details on mating procedure:
- Impregnation procedure: artificial insemination (day of insemination was designated as GD 0 (beginning of the study) and the following day as GD 1)
Duration of treatment / exposure:
Gestation days (GD) 6 through 28
Frequency of treatment:
Daily
Duration of test:
Terminal sacrifice on GD 29
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
25 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for animal assignment: Random

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily (GD 0-29)
- Cage side observations checked: morbidity, pertinent behavioral changes and signs of overt toxicity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily (GD 6-28)

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

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule: Daily during GD 0-29

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: heart, kidneys
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
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:
Statistical analyses were performed according to the following:
- 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 preg-nant 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.
Historical control data:
Yes

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
One female, each, of the control (No. 21 - 0 mg/kg bw/d, GD 22), mid-dose (No. 57 - 300 mg/kg bw/d, GD 29) and high-dose (No. 91 - 1000 mg/kg bw/d, GD 28) groups had blood in bedding.
In total, reduced defecation was observed in four control, three low-dose, two mid-dose and nine high-dose females (0, 100, 300 and 1000 mg/kg bw/d). No defecation was observed in two females of test group 3. Incidence and distribution of these findings do not indicate a relationship to the test substance.
There were no clinical findings in the other does in the study.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One control female (No. 3 - 0 mg/kg bw/d), one low-dose female (No. 37 - 100 mg/kg bw/d) and two high-dose females (Nos. 83 and 84 - 1000 mg/kg bw/d) were sacrificed after abortion ahead of schedule. Spontaneous abortions in single does are not uncommon findings in the strain of rabbits used for this study. Thus, these were considered to be spontaneous incidents.
Low-dose female No. 28 (100 mg/kg bw/d) was found dead on GD 21. The gross pathological examination of this animal revealed findings indicative of gavage error.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight:
No statistically significant difference was observed for the mean body weights (BW) of the high-dose females (1000 mg/kg bw/d) when compared to the concurrent control group. However, from GD 19 onwards the high-dose body weights were distinctly lower than the body weights of the other groups including the control. Also, the mean body weight gain (BWC) of the high-dose rabbits was statistically significantly below control on GD 25-28. During the treatment period (GD 6-28) the does of the high-dose group gained 69% less body weight than the control does.
Though not statistically significant, the body weight gain during treatment (GD 6-28) was also reduced at the mid-dose level by 33%, which indicates beginning systemic toxicity at 300 mg/kg bw/d.
The mean BW and the average BWC of the low-dose group (100 mg/kg bw/d) were generally comparable to the concurrent control group throughout the entire study period.

Corrected (net) body weight gain:
The corrected body weight gain (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6) was distinctly and statistically significantly lower in test group 3 (1000 mg/kg bw/d). Furthermore, the carcass weight of the high-dose does was also reduced, but without attaining statistical significance (about 5% below controls). Though not statistically significant, the corrected body weight gain during treatment (GD 6-28) was also reduced in the mid-dose does. These effects are assessed as test substance-related signs of maternal toxicity.
Mean carcass weights and the corrected body weight gain were not significantly different in test groups 0 and 1 (0, 100 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 (1000 mg/kg bw/d) was reduced during major parts of the treatment period, attaining statistical significance on GD 14-17 and GD 27-29. During the treatment period (GD 6-28) the high-dose does consumed 16% less food than the concurrent control does.
Though not statistically significant, the food consumption during treatment (GD 6-28) was also reduced at the mid-dose level by 8%.
The food consumption of the low-dose rabbits (100 mg/kg bw/d) was comparable to the concurrent control (0 mg/kg bw/d) throughout the entire study period.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Uterus weights:
The mean gravid uterus weights of the rabbits of test groups 1-3 (100, 300 and 1000 mg/kg bw/d) were 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.

Weight of the placentae:
The mean placental weights in test groups 2 and 3 (300 and 1000 mg/kg bw/d) were lower than in the concurrent control group, however, the difference was small and did not gain statistical significance. The mean placental weights in test group 1 (100 mg/kg bw/d) were similar to the control value.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Petechiae in lungs of one high-dose doe (No. 84 - 1000 mg/kg bw/d).
Stomach filled with very dry, hard feed in one high-dose doe (No. 78 - 1000 mg/kg bw/d).
Watery feces in two mid-dose does (Nos. 52 and 67 - 300 mg/kg bw/d) and eight high-dose does (Nos. 77, 78, 79, 83, 84, 85, 90, 97 - 1000 mg/kg bw/d).
Findings indicative of misgavaging, i.e. thoracic cavity filled with blood and test substance, in low-dose doe No. 28 (100 mg/kg bw/d - died on GD 21).

Maternal developmental toxicity

Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
One control female (No. 3 - 0 mg/kg bw/d), one low-dose female (No. 37 - 100 mg/kg bw/d) and two high-dose females (Nos. 83 and 84 - 1000 mg/kg bw/d) were sacrificed after abortion ahead of schedule. Spontaneous abortions in single does are not uncommon findings in the strain of rabbits used for this study. Thus, these were considered to be spontaneous incidents.
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 values calculated for the pre- and the post-implantation losses. All differences observed are considered to reflect the normal range of fluctuations for animals of this strain and age.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
In the mid-dose group (300 mg/kg bw/d) the mean number of early resorptions was slightly, but statistically significantly, increased. This is assessed as incidental, as the total number of resorptions (10.1 mean%) was well within the historical control range (HCD Re-sorptions: mean% 7.2 [2.4 - 12.6]) and there is no dose-response.
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
One dead fetus was found at cesarean section of mid-dose doe No. 52. Alike abortions this is considered as an expression of maternal toxicity in rabbits, in particular as this animal suffered from markedly reduced food consumption and body weight loss near term.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Female rabbits were placed into the study in four cohorts. The conception rate was 84% in the mid-dose group (300 mg/kg bw/d), 88% in the low- and high-dose groups (100 and 1000 mg/kg bw/d) and 92% in the control group (0 mg/kg bw/d). There were no test substance-related biologically relevant differences between the different test groups in conception rate.
There were no test substance-related and/or biologically relevant differences between the different test groups in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and the postimplantation losses, the number of resorptions and viable fetuses. All differences observed are considered to reflect the normal range of fluctuations for animals of this strain and age.
Details on maternal toxic effects:
Test group 1 (100 mg/kg bw/d):
- No test substance-related adverse effects observed.

Test group 2 (300 mg/kg bw/d):
- Reduced mean food consumption, 8% below control on GD 6-28
- Reduced mean body weight gain, 33% below control on GD 6-28
- Corrected body weight gain below control (-267.2 g vs. -209.4 g in control)

Test group 3 (1000 mg/kg bw/d):
- Reduced mean food consumption, 16% below control on GD 6-28
- Reduced mean body weight gain, 69% below control on GD 6-28
- Corrected body weight gain distinctly below control (-410.6 g vs. -209.4 g in control), lower carcass weight (about 5% below controls).

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Weight of fetuses:
The mean fetal weights of test groups 2 and 3 (300 and 1000 mg/kg bw/d) were statistically significantly reduced (about 11% and 13% below control - both sexes combined). The mean fetal weights of test group 1 (100 mg/kg bw/d) were 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 test groups 1-3 (100, 300 and 1000 mg/kg bw/d) was comparable to the control fetuses. Any observable differences were without biological relevance.
External malformations:
no effects observed
Description (incidence and severity):
External malformations did not occur in any fetuses in this study and no external variations were recorded.
Two unclassified external observations, i.e. necrobiotic placentae and discolored placentae, were recorded in one litter of the mid-dose group (300 mg/kg bw/d) and in two individual litters of the high-dose group (1000 mg/kg bw/d).
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Skeletal malformations were detected in test groups 1 and 2 (100 or 300 mg/kg bw/d). No statistically significant differences between the test substance-treated groups and the control were noted and no dose-response relationship was observed.

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 skeleton. Generally, all skeletal variations are equally distributed about the different test groups including controls, if normal biological variation is taken into account. The changes which were significantly different from the concurrent control were either not related to dose or can be found in the historical control data at comparable or higher frequency.
One skeletal variation (incomplete ossification of talus; cartilage present) was noted at an incidence above the concurrent and historical control in the high-dose group (1000 mg/kg bw/d). The other increased incidences of skeletal variations were either not related to dose or they were inside the historical control range. Therefore, these minor changes are not considered as treatment-related adverse events.
As can be seen from the historical background data, increased incidences of such incomplete or non-ossifications of skeletal elements are routinely quantified and are among the most frequently noted skeletal variants in control populations of this New Zealand White rabbit strain. This indicates that these findings reflect species-specific anatomic variation at the time around birth without any detrimental effects on further development. They are most likely related to the lower pup body weights which were considered to be a consequence of a distinctly lower body weight gain of the dams in the high-dose group, particularly in the last week of pregnancy. Notably, the marginal increase of these changes did not affect the overall rate of variations.

Some isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in test groups 0-3. The observed unclassified cartilage findings did not show any relation to dosing and were considered to be spontaneous in nature.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
A number of soft tissue malformations occurred in all test groups including the control (0, 100, 300 or 1000 mg/kg bw/d). Nine cases of small spleen were observed in one litter of the high-dose group - dam No. 94. No findings in organs of related ontogenetic origin is seen in the other offspring of this test group. Thus the clustering of this observation in just one litter suggests a non-treatment related origin of the spleen findings. Other malformations, such as absent subclavian, absent gallbladder and misshapen thoracic vertebra were scattered observations in individual fetuses of test groups 0-2. No statistically significant differences between the groups were noted. They were not dose-related and some of them can be found in the historical control data. An association of these findings to the treatment is not assumed.

Soft tissue variations occurred in all test groups including the control. The examinations of the organs revealed dilated cerebral ventricle, malpositioned carotid branch, malpositioned carotid origin and a small gallbladder in individual fetuses of test groups 1 and 2 (100 and 300 mg/kg bw/d). An absent lung lobe (lobus inferior medialis) was noted in all test groups and showed a slightly but statistically significantly increased incidence in the high-dose group (2.8% affected fetuses/ litter vs. 0.6% in control); also slightly above the historical control range. (0.0 - 2.7%). As this is a commom variation in this rabbit strain which is unlikely to adversely affect health of the developing offspring, and the incidence was just above the historical range, no toxicological relevance is attributed to this finding. Although the total incidence of soft tissue variations were statistically significantly increased in test groups 2 and 3, these incidences are well covered by the historical control range (HCD: 2.2% [0.4 - 5.0]). Thus these increases are also not considered to be of toxicological relevance.

Two unclassified soft tissue observations were recorded: a (reddish) discolored thymus in one mid-dose and three high-dose fetuses in three different litters (300 and 1000 mg/kg bw/d), and a blood coagulum around urinary bladder in one control fetus (0 mg/kg bw/d). The affected fetuses/litter incidence of discolored thymus was statistically significantly increased in test group 3. However, this is considered to be a mere notice which is not supposed to have any adverse effect on the developing offspring. In terms of the overall rates of soft tissue unclassified observations no statistically significant differences between the groups were noted.
Details on embryotoxic / teratogenic effects:
Test group 1 (100 mg/kg bw/day):
- No test substance-related adverse effects observed.

Test group 2 (300 mg/kg bw/day):
- Reduced fetal weight (about 11% below control)

Test group 3 (1000 mg/kg bw/day):
- Reduced fetal weight (about 13% below control)
- Increased incidence of one fetal variation indicating developmental delay

Additional information on fetal external, soft tissue and skeletal observations:
External malformations did not occur in any fetuses in this study. There were noted soft tissue and skeletal malformations in all test groups (0, 100, 300 or 1000 mg/kg bw/d) which are described in detail in the respective sections obove.
Four fetuses were multiple-malformed. Female low-dose fetus No. 27-07 (100 mg/kg bw/d) had a malpositioned, small kidney and an absent subclavian. For male mid-dose fetus No. 55-08 (300 mg/kg bw/d) a thoracic hemivertebra, a misshapen thoracic vertebra and a fused rib were recorded, while the findings in male fetus No. 9 of the same litter (No. 55-09 - 300 mg/kg bw/d) consisted of a thoracic hemivertebra, a misshapen thoracic vertebra and a branched rib. Furthermore, female high-dose fetus No. 94-07 (1000 mg/kg bw/d) had a small spleen and multiple malformations of the great vessels, i.e. narrowed pulmonary trunk, dilat-ed aorta, dilated carotid (right side) and absent carotid (left side).

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
300 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:
yes

Any other information on results incl. tables

Table 1: Mean maternal body weight change during gestation

 

 

0 mg/kg bw/d

100 mg/kg bw/d

300 mg/kg bw/d

1000 mg/kg bw/d

Days 0 to 6

Mean

S.D.

N

190.4 D

78.16

23

170.9

105.47

21

187.0

68.53

21

163.7

66.91

22

Days 6 to 28

Mean

S.D.

N

222.3 D

129.20

22

272.5

189.21

20

172.8

165.01

21

66.9*

202.66

20

Days 0 to 29

Mean

S.D.

N

438.6 D

162.11

22

477.3

207.14

20

366.4

205.74

21

263.0*

225.45

20

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

Table 2: Individual fetal soft tissue malformations

Test group

Doe No.-Fetus No., Sex

Finding

0 (0 mg/kg bw/d)

24-01 M

absent subclavian

1 (100 mg/kg bw/d)

27-07 F

 

34-05 M, 34-09 M

absent subclavian, malpositioned kidney, small kidney

absent gallbladder

2 (300 mg/kg bw/d)

66-06 F

72-11 M

absent subclavian

absent gallbladder

3 (1000 mg/kg bw/d)

94-07 F

 

94-03 M, 94-04 M, 94-05 M, 94-06 M, 94-08 F, 94-09 F, 94-10 F, 94-11 F

multiple malformations of the great vessels, small spleen

small spleen

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

Table 3: Total soft tissue malformations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

Litter
Fetuses

N
N

22
180

20
189

21
194

20
192

Fetal incidence

 

N (%)

 

1 (0.6)

 

3 (1.6)

 

2 (1.0)

 

9 (4.7)

Litter incidence

 

N (%)

 

1 (4.5)

 

2 (10)

 

2 (9.5)

 

1 (5.0)

Affected fetuses/litter

 

Mean%

 

0.6

 

1.4

 

1.0

 

4.1

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

Table 4: Total soft tissue variations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

Litter
Fetuses

N
N

22
180

20
189

21
194

20
192

Fetal incidence

 

N (%)

 

1 (0.6)

 

6 (3.2)

 

7 (3.6)

 

8 (4.2)

Litter incidence

 

N (%)

 

1 (4.5)

 

3 (15)

 

5 (24)

 

6 (30)*Fi

Affected fetuses/litter

 

Mean%

 

0.6

 

2.8

 

3.5*Wi

 

3.7*Wi

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = percent
*Fi = p ≤ 0.05 (Fisher’s exact test [one-sided])        ** Fi= p ≤ 0.01 (Fisher’s exact test [one-sided])
*Wi = p ≤ 0.05 (Wilcoxon-test [one-sided])              ** Wi= p ≤ 0.01 (Wilcoxon-test [one-sided])

Table 5: Individual fetal skeletal malformations

Test group

Doe No.-Fetus No., Sex

Finding

0 (0 mg/kg bw/d)

none

 

1 (100 mg/kg bw/d)

40-02 M

misshapen thoracic vertebra

2 (300 mg/kg bw/d)

55-08 M

misshapen thoracic vertebra, thoracic hemivertebra, fused rib

 

55-09 M

misshapen thoracic vertebra, thoracic hemivertebra, branched rib

3 (1000 mg/kg bw/d)

none

 

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

Table 6: Total sekeletal malformations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

Litter
Fetuses

N
N

22
180

20
189

21
194

20
192

Fetal incidence

 

N (%)

 

0.0

 

1 (0.5)

 

2 (1.0)

 

0.0

Litter incidence

 

N (%)

 

0.0

 

1 (5.0)

 

1 (4.8)

 

0.0

Affected fetuses/litter

 

Mean%

 

0.0

 

0.6

 

1.0

 

0.0

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

Table 7: Total fetal skeletal variations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

Litter Fetuses

N

N

22

180

20

189

21

194

20

192

Fetal incidence

N (%)

167 (93)

169 (89)

170 (88)

178 (93)

Litter incidence

N (%)

22 (100)

20 (100)

21 (100)

20 (100)

Affected fetuses/litter

Mean%

92.6

89.5

89.5

92.6

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

Table 8: 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

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

HCD

Mean% (range)

Incomplete ossification of cervical centrum; unchanged cartilage

4.5

6.0

7.5

11.9*

17.5

(1.9 - 33.4)

Supernumerary thoracic vertebra

9.5

18.7

24.8**

14.2

20.7

(12.6 - 29.9)

Misshapen sacral vertebra

1.9

4.7*

3.2

3.3

6.4

(2.6 - 12.1)

Incomplete ossification of talus; cartilage present

4.8

3.9

8.5

10.8*

1.1

(0.0 - 2.2)

mg/kg bw/d = milligram per kilogram body weight per day; HCD = Historical control data; % = percent

*= p ≤0.05 (Wilcoxon-test [one-sided]); ** = p0.01 (Wilcoxon-test [one-sided])

Table 9: Total fetal malformations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

Litter Fetuses

N

N

22

180

20

189

21

194

20

192

Fetal incidence

N (%)

1 (0.6)

4 (2.1)

4 (2.1)

9 (4.7)

Litter incidence

N (%)

1 (4.5)

3 (15)

3 (14)

1 (5.0)

Affected fetuses/litter

Mean%

0.6

2.0

1.9

4.1

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

Table 10: Total fetal variations

 

 

Test group 0

0 mg/kg bw/d

Test group 1

100 mg/kg bw/d

Test group 2

300 mg/kg bw/d

Test group 3

1000 mg/kg bw/d

Litter Fetuses

N

N

22

180

20

189

21

194

20

192

Fetal incidence

N (%)

167 (93)

196 (89)

171 (88)

178 (93)

Litter incidence

N (%)

22 (100)

20 (100)

21 (100)

20 (100)

Affected fetuses/litter

Mean%

92.6

89.5

88.4

92.6

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

 

Applicant's summary and conclusion

Conclusions:
Under the conditions of this prenatal developmental toxicity study, the oral administration of N,N’-(isobutylidene)diurea to pregnant New Zealand White rabbits from implantation to one day prior to the expected day of parturition (GD 6-28) caused evidence of maternal toxicity at dose levels of 300 mg/kg bw/d and above, such as reduced food consumption and body weight gain.
In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 100 mg/kg bw/d.
The no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 100 mg/kg bw/d. However, reduced fetal body weights (at 300 and 1000 mg/kg) as well as a temporary delay of prenatal development of offspring (at 1000 mg/kg only) were associated with congenerous effects in their mothers and thus not considered as an independent effect.
There is no evidence for selective developmental toxicity of N,N’-(isobutylidene)diurea. The test substance is not teratogenic in rabbits at the tested dose levels.
Executive summary:

N,N’-(isobutylidene)diurea 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) at dose levels of 100, 300 and 1000 mg/kg bw/day.

No test substance related adverse clinical observations were noted in any test group.

The mean food consumption of the high-dose dams (1000 mg/kg bw/d) was reduced during major parts of the treatment period, during the treatment period (GD 6-28) the high-dose does consumed 16% less food than the concurrent control does. Though not statistically significant, the food consumption during treatment (GD 6-28) was also reduced at the mid-dose level by 8%. This effect is regarded to be treatment-related.

The high-dose of the test substance distinctly affected the gross and corrected (net) body weight gain of the does in the respective test group. These does gained about 69% less gross weight or lost twice as much net weight than the concurrent control during the treat-ment period (GD 6-28). In the mid-dose group the gross weight gain was 33% below control and the corrected (net) body weight change was also reduced compared to control, which indicates beginning systemic toxicity at 300 mg/kg bw/d. These effects are assessed as test substance-related signs of maternal toxicity.

The mean fetal weights of test groups 2 and 3 (300 and 1000 mg/kg bw/d) were slightly but statistically significantly reduced (about 11% and 13% below control - both sexes combined). These slight reductions are considered to be subsequent to an overall lower body weight gain of the does in the mid- and high-dose groups.

Fetal examinations revealed that there is no effect of the compound on the respective mor-phological structures up to the highest dose tested (1000 mg/kg bw/d). Incidences of incom-plete ossifications of skeletal elements at the tips of the lower limbs represent temporary de-lays in development which have no permanent effect on morphology and function of the af-fected structures.

Thus, the test item is not teratogenic in rabbits.