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Diss Factsheets

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 Mar 2016 - 25.4.2017
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
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2001
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Commission Regulation (EC) No 440/2008 of 30 May 2008 Part B: Methods for the determination of toxicity and other health effects: Prenatal Developmental Toxicity Study; Official Journal of the European Union, No. L 142
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
1998
GLP compliance:
yes (incl. QA statement)

Test material

1
Reference substance name:
A mixture of: triphenylthiophosphate and tertiary butylated phenyl derivatives
EC Number:
421-820-9
EC Name:
A mixture of: triphenylthiophosphate and tertiary butylated phenyl derivatives
Cas Number:
192268-65-8
Molecular formula:
Unspecified
IUPAC Name:
O,O,O-triphenyl phosphorothioate; O-2,4-di-tert-butylphenyl O,O-diphenyl phosphorothioate; O-2-tert-butylphenyl O,O-diphenyl phosphorothioate; O-2-tert-butylphenyl O-4-tert-butylphenyl O-phenyl phosphorothioate; O-4-tert-butylphenyl O,O-diphenyl phosphorothioate

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 10-12 weeks
- Weight at study initiation: 155.3 – 195.2 g
- Fasting period before study: no
- Housing: individually in Polycarbonate cages type III
- Diet: ground Kliba maintenance diet mouse/rat “GLP”, meal, ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
PEG 400
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 preparations, the specific amount of test substance was weighed, topped up with Polyethylene Glycol, PEG 400 in a calibrated beaker and intensely mixed with a magnetic stirrer. During administration, the preparations were kept homogeneous with a magnetic stirrer.

VEHICLE
- Concentration in vehicle: 1.25, 3.75 and 12.5 g/100 ml
- Amount of vehicle (if gavage): 4 ml/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the test substance preparations were sent to the analytical laboratory at the beginning of administration for verification of the concentrations. The samples were also used to verify the homogeneity of the low and the high concentrations (50 and 500 mg/kg bw/d). Three samples (one from the top, middle and bottom in each case) were taken from the beaker with a magnetic stirrer running.
The stability of the test substance in Polyethylene Glycol, PEG 400 over a period of 7 days at room temperature was demonstrated. The homogeneous distribution of the test substance in the vehicle (Polyethylene Glycol, PEG 400) was demonstrated. The results of the analysis of the test substance preparations confirmed the correctness of the prepared concentrations. The measured concentrations of the samples corresponded to the expected values within the limits of the analytical method, i.e. were alway above 90% and below 110% of the nominal concentrations.
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. The animals arrived on the same day (GD 0) at the experimental laboratory.
Duration of treatment / exposure:
From implantation to one day prior to the expected day of parturition (GD 6-19)
Frequency of treatment:
daily
Duration of test:
On GD 20, all surviving dams were sacrificed and examined. The fetuses were removed from the uterus and investigated.
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on a range finder study

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
A check was made twice a day on working days or once a day on Saturdays, Sundays or on public holidays.

DETAILED CLINICAL OBSERVATIONS: Yes
A cage-side examination was conducted 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 for any 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.

BODY WEIGHT: Yes
All animals were weighed on GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20. The body weight change of the animals was calculated based on the obtained results. Furthermore, the corrected body weight gain was calculated after terminal sacrifice (terminal body weight on GD 20 minus weight of the unopened uterus minus body weight on GD 6).

FOOD CONSUMPTION: Yes
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: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: All gross lesions, Liver

OTHER:
The following weights were determined in all animals sacrificed on schedule: Liver
The carcass weights (GROSSE-System) were transferred to the ACOPAT-System to calculate the relative organ weights.
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
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes: [half per litter]
Statistics:
- DUNNETT-test (twosided): Food consumption), body weight, body weight change, corrected body weight gain (net maternal body weight change), carcass weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportions of preimplantation loss, proportions of postimplantation loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight
- FISHER'S EXACT test (onesided): Female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings
- WILCOXON-test (onesided): Proportions of fetuses with malformations, variations and/or unclassified observations in each litter
- 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): Weight parameters
Indices:
conception rate, preimplantation loss, postimplantation loss

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
All 25 high-dose females (500 mg/kg bw/d) and 8 mid-dose females (150 mg/kg bw/d) showed transient salivation during the treatment period. Salivation occurred in the respective animals shortly after treatment (i.e. within 2 hours post-dosing) and was initially observed on GD 11. After 2 hours and up to 5 hours post-dosing no clinical signs or changes of general behavior were detected in any female of all test groups (50, 150 or 500 mg/kg bw/d), nor were before dosing in the morning.
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, 50, 150 or 500 mg/kg bw/d).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weights of the dams in test group 3 (500 mg/kg bw/d) were in general comparable to the controls throughout the entire study period. Corresponding to reduced food consumption the mean body weight change of the high-dose dams was statistically significantly reduced on GD 6-8 (approx. 54% below control), but recovered afterwards and was comparable or even exceeded the concurrent control (attaining statistical significance on GD 15-17). This temporary decrease of body weight gain in the high-dose group is considered as treatment-related. The mean body weights and the average body weight gain of the low- and mid-dose dams
(50 and 150 mg/kg bw/d) were generally comparable to the concurrent control group throughout the entire study.
The corrected body weight gain (terminal body weight on GD 20 minus weight of the unopened uterus minus body weight on GD 6) was statistically significantly lower in test group 2 (150 mg/kg bw/d – about 13% below the concurrent control value). The corrected body weight gain of test groups 1 and 3 (50 and 500 mg/kg bw/d) revealed no difference of any biological relevance to the corresponding control group. Mean carcass weights of all test groups were comparable to the carcass weights in the control group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The mean food consumption of the dams in test groups 2 and 3 (150 and 500 mg/kg bw/d) was statistically significantly reduced from GD 6-10 (maximum 10 and 16%, respectively), but recovered afterwards. Overall, during the treatment period (GD 6-19) the mid- and high-dose dams consumed about 4% less food in comparison to the concurrent control group. This temporary reduction of food consumption was considered as treatment-related. The marginal but statistically significantly reduced mean food consumption value in test group 2 on GD 19-20 was assessed incidental, because there was no dose-response relationship. The mean food consumption of the dams in test group 1 (50 mg/kg bw/d) was generally comparable to the concurrent control group throughout the whole study period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The mean gravid uterus weights of the animals in test groups 2 and 3 (150 and 500 mg/kg bw/d) were statistically significantly increased (+14% / +16%) in comparison to the concurrent control group. The mean gravid uterus weights of the animals of test group 1 (50 mg/kg bw/d) were comparable to the control.
When compared to control group 0 (set to 100%), the mean absolute and mean relative weights of the livers were significantly increased in all test groups. The liver weights in all test groups lay above the range of historical control data and were regarded as treatment related.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The livers of 4/25 animals in test group 3 (500 mg/kg bw/d) were enlarged when compared to control group 0. The enlarged livers were regarded as treatment related and correlated to increased liver weights in these animals.
All other findings occurred either individually or 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.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
The conception rate was 96% in the control, mid- and high-dose groups (0, 150 and 500 mg/kg bw/d) and 100% in the low-dose group (50 mg/kg bw/d). With these rates, a sufficient number of pregnant females were available for the purpose of this study. There were no test substance-related and/or biologically relevant differences between test groups 0, 1, 2 and 3 (0, 50, 150 and 500 mg/kg bw/d) in conception rate, in the mean number of corpora lutea or in the values calculated for the postimplantation loss and the number of resorptions. The mean number of implantation sites was statistically significantly higher in the mid- and high-dose groups (10.0/11.0/11.7**[p<=0.01]/11.3*[p<=0.05] in test groups 0-3). Consequently, the mean number of live fetuses was also higher (9.2/10.0/10.8**/10.7** [p<=0.01]). The preimplantation loss was significantly lower in all treatment groups (8.3%/3.2%*/1.1%**/2.5%* in test groups 0-3). However, these apparent increases/ decreases came from comparatively low/high control values for these parameters rather than being real treatment-related effects. In addition, these are not at all adverse findings of any toxicological relevance.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
The mean fetal weights of test groups 1, 2 and 3 were not influenced by the test substance and did not show any biologically relevant differences in comparison to the control group.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
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 (50, 150 and 500 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:
no effects observed
Description (incidence and severity):
No external malformations were recorded, no external variations were recorded and no external unclassified observations were recorded.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Shortened scapula along with shortened humerus were observed in one mid-dose fetus and in 3 individual high-dose fetuses from 3 different litters, in comparison to one shortened humerus in the control group. For both findings, the 3 high-dose cases add up to increased rates of affected fetuses per litter in test group 3 which became statistically significant for shortened scapula (0.0/0.0/0.8/2.3* [p<=0.05] at 0, 50, 150 or 500 mg/kg bw/d). The affected
fetuses/litter incidence of both findings exceeds the historical control range of the test facility (HCD: mean 0.1 (range 0.0 - 0.8)). All other skeletal malformations were scattered over single fetuses of all test groups (0, 50,
150 or 500 mg/kg bw/d). The overall skeletal malformation rate was not statistically significantly different from control.
For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeletons and appeared without a relation to dosing. The overall incidences of skeletal variations were comparable to the historical control data.
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.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No soft tissue malformations were recorded. Two soft tissue variations, i.e. dilated renal pelvis and dilated ureter, were detected in all test groups including the controls. The incidences of these variations were neither statistically significantly nor dose-dependently increased in the treated groups and therefore, not considered biologically relevant. All of them can be found in the historical control data at comparable incidences. No soft tissue unclassified observations were recorded.
Description (incidence and severity):
The mean placental weights of the low-, mid- and high-dose groups were comparable to the corresponding control group.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
skeletal malformations

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

ORGAN WEIGHTS

Regarding pathology, the target organ was the liver. The livers showed significantly increased absolute and relative weights in test groups 1 to 3. The liver weights in all test groups lay above the range of historical control data (absolute: 10.808g - 11.455g; relative: 4.549% - 4.729%) and correlated to macroscopically enlarged livers in 4/25 animals in the high-dose group. The increased absolute and relative liver weights in all test groups were regarded as treatment related. However, the weight increase was only slight in the low- and mid-dose groups (test group 1: 8% absolute, 6%relative; test group 2: 14% absolute, 16% relative) and was regarded as non-adverse. Animals of test group 3 showed considerably increased liver weights (35% absolute, 34% relative) which were interpreted as treatment related and adverse.

Table 1: Absolute organ weights

Absolute weights Females
Test group
(mg/kg bw/d)		 1
(50)		 2
(150)		 3
(500)
Liver 108%** 114%** 135%**

* : p <= 0.05, **: p <= 0.01

Table 2: Relative organ weights

Relative weights Females
Test group
(mg/kg bw/d)		 1
(50)		 2
(150)		 3
(500)
Liver 106%** 116%** 134%**

* : p <= 0.05, **: p <= 0.01

SOFT TISSUE MALFORMATIONS AND VARIATIONS

Soft tissue malformations did not occur in any of the fetuses in this study. Two soft tissue variations, i.e. dilated renal pelvis and dilated ureter, were detected in all test groups including the controls. The incidences of these variations were neither statistically significantly nor dose-dependently increased in the treated groups and therefore, not considered biologically relevant. All of them can be found in the historical control data at comparable incidences.

Table 3: Total soft tissue variations

    Test group 0
0 mg/kg bw/d		 Test group 1
50 mg/kg bw/d		 Test group 2
150 mg/kg bw/d		 Test group 3
500 mg/kg bw/d
Litter
Fetuses

N

N

 24
104		 25
118		 24
122		 24
122
Litter incidence
 N (%) 4 (3.8) 5 (4.2) 2 (1.6) 7 (5.7)
Litter
incidence		 N (%) 2 (8.3) 4 (16) 2 (8.3) 7 (29)
Affected fetuses/litter Mean% 3.9 5.3 1.7 5.5

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

SKELETAL MALFORMATIONS & VARIATIONS

There were noted skeletal malformations in one control, three low-dose, two mid-dose and four high-dose fetuses. Male mid-dose fetus 74-11 (150 mg/kg bw/d) as well as female high-dose fetus No. 81-11, male high-dose fetus No. 95-05 and female high-dose fetus No. 98-01 (500 mg/kg bw/d respectively) had a shortened scapula combined with a shortened humerus (uni- or bilateral), respectively. One shortened humerus was noted in control fetus No. 19-03. For both findings, the 3 high-dose cases add up to increased rates of affected fetuses per litter in test group 3 which became statistically significant for shortened scapula (0.0/0.0/0.8/2.3* [p<=0.05] at 0, 50, 150 or 500 mg/kg bw/d). The affected fetuses/litter incidence of both findings exceeds the historical control range of the test facility (HCD: mean 0.1 (range 0.0 - 0.8)). Both findings are not uncommon in the used Crl:WI(Han) rat strain and there is no pattern recognizable of any specific alterations of other skeletal elements in the shoulder girdle or the thorax. However, the dose-relationship and the exceeding of the historical control range in the high-dose group may indicate that the test substance affects the development of shoulder girdle in Wistar rats at high dose levels. Thus, a relationship to the treatment cannot be excluded. The single case of shortened scapula/humerus at 150 mg/kg bw/d is comparable to both concurrent and historical control and thus considered as an accidental finding. Further malformations, i.e., malpositioned and bipartite sternebra, branched rib and misshapen tuberositas deltoidea were not related to dose and most of them can be found in the historical control data. An association of these findings to the treatment is also not assumed.

Table 4: Individual fetal skeletal malformations

Test group Dam No.-Fetus No., Sex Finding
0 (0 mg/kg bw/d) 19-03 M shortened humerus
1 (50 mg/kg bw/d) 40-06F malpositioned and bipartite sternebra
47-07 M branched rib
50-06 M misshapen tuberositas deltoidea
2 (150 mg/kg bw/d) 67-05 M malpositioned and bipartite sternebra
74-11 M shortened scapula, shortened humerus
3 (500 mg/kg bw/d) 81-11 F shortened scapula, shortened humerus
95-05 M shortened scapula, shortened humerus
98-01 F shortened scapula, shortened humerus
98-08 M malpositioned and bipartite sternebra

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

Table 5: Total skeletal malformations

    Test group 0
0 mg/kg bw/d		 Test group 1
50 mg/kg bw/d		 Test group 2
150 mg/kg bw/d		 Test group 3
500 mg/kg bw/d
Litter
Fetuses

N

N

 24
117		 25
131		 24
138		 24
134
Fetal incidence N (%) 1 (0.9) 3 (2.3) 2 (1.4) 4 (3.0)
Litter incidence N (%) 1 (4.2) 3 (12) 2 (8.3) 3 (13)
Affected fetuses/litter Mean% 1.0 2.3 1.5 3.6

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

A broad range of skeletal variations occurred in all test groups including the control. The majority of the skeletal variations are equally distributed among the different test groups including controls, if normal biological variation is taken into account, and can be found in the historical control data at similar frequencies. Two variations - incomplete ossification of skull (unchanged cartilage) and wavy ribs - were statistically significantly increased in test group 3 (500 mg/kg bw/d) and outside the historical control range. The incomplete ossification of skull represents a slight delay of ossification which did not affect morphology, as the underlying cartilage/membrane model was completely intact. Wavy ribs emerge from minor disturbances of the chondrification and ossification process of the ribs and are known to be completely reversible postnatally (Hofmann et al., 2016). In the historical background data, increased incidences of such minor modifications of skeletal elements are routinely quantified and are among the most frequently noted skeletal variants in control populations of this Crl:WI(Han) rat strain. This indicates that these findings reflect species-specific anatomic variation at the time around birth without any detrimental effects on further development. Notably, the marginal increase of these changes did not affect the overall rate of variations, as can be seen from Table 10. Concerning all other statistically significant findings, no dose dependency was observed and/or all values were clearly inside the historical control range, thus, an association to the test substance and a toxicological relevance is not assumed.

Table 6: Total fetal skeletal variations

    Test group 0
0 mg/kg bw/d		 Test group 1
50 mg/kg bw/d		 Test group 2
150 mg/kg bw/d		 Test group 3
500 mg/kg bw/d
Litter Fetuses

N

N

 24
117		 25
131		 24
138		 24
134
Fetal incidence N (%) 111 (95) 125 (95) 136 (99) 131 (98)
Litter incidence N (%) 24 (100) 25 (100) 24 (100) 24 (100)
Affected fetuses/litter Mean% 95.0 95.3 98.8 97.9

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

Table 7: Occurrence of statistically significantly increased fetal skeletal variations (expressed as mean percentage of affected fetuses/litter)

Finding Test group 0
0 mg/kg bw/d		 Test group 1
50 mg/kg bw/d		 Test group 2
150 mg/kg bw/d		 Test group 3
500 mg/kg bw/d		 HCD
Mean % (range)
Incomplete ossification of parietal;
unchanged cartilage		 5.8 6.5 17.1* 13.3* 10.4
(2.0 – 17.1)
Incomplete ossification of supraoccipital; unchanged cartilage 16.3 35.4** 23.7 27.0 40.5
(15.4 – 64.3)
Incomplete ossification of skull;
unchanged cartilage		 11.1 18.6* 15.0 26.5** 8.0
(0.8 – 21.4)
Incomplete ossification of temporal 0.0 1.6 3.5* 2.1 0.2
(0.0 – 2.0)
Misshapen sacral vertebra 0.0 3.1* 2.8* 4.6** 4.2
(1.5 – 8.8)
Unossified sternebra; un- changed cartilage 1.7 3.1 6.9* 3.5 5.2
(0.0 – 10.3)
Incomplete ossification of sternebra;
unchanged cartilage		 72.1 86.1* 87.9* 83.4 88.4
(72.8 – 95.5)
Misshapen sternebra; un- changed cartilage 28.7 30.0 41.7* 32.9 48.0
(20.0 – 76.9)
Wavy rib 8.8 7.1 10.9 31.7** 4.6
(0.8 – 8.7)

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

* = p ≤ 0.05 (Wilcoxon-test [one-sided]) ** = p ≤ 0.01 (Wilcoxon-test [one-sided])

Additionally, some isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in all test groups (Tab. 8). The observed unclassified cartilage findings were related to the skull, the ribs and the sternum. A spontaneous origin is assumed for these observations which were observed in several fetuses of test groups 0, 1, 2 and 3 (0, 50, 150 and 500 mg/kg bw/d). The distribution and type of these findings do not suggest any relation to treatment.

Table 8: Total unclassified cartilage observations

    Test group 0
0 mg/kg bw/d		 Test group 1
50 mg/kg bw/d		 Test group 2
150 mg/kg bw/d		 Test group 3
500 mg/kg bw/d

Litter

Fetuses

N

N

 24
117		 25
131		 24
138		 24
134
Fetal incidence N (%) 89 (76) 90 (69) 99 (72) 92 (69)
Litter incidence N (%) 24 (100) 24 (96) 23 (96) 24 (100)
Affected fetuses/litter Mean% 76.5 68.7 71.1 68.4

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

Table 9: Total fetal malformations

    Test group 0
0 mg/kg bw/d		 Test group 1
50 mg/kg bw/d		 Test group 2
150 mg/kg bw/d		 Test group 3
500 mg/kg bw/d

Litter

Fetuses

N

N

 24
221		 25
249		 24
260		 24
256
Fetal incidence N (%) 1 (0.5) 3 (1.2) 2 (0.8) 4 (1.6)
Litter incidence N (%) 1 (4.2) 3 (12) 2 (8.3) 3 (13)
Affected fetuses/litter Mean% 0.6 1.2 0.8 2.0

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

Table 10: Total fetal variations

    Test group 0
0 mg/kg bw/d		 Test group 1
50 mg/kg bw/d		 Test group 2
150 mg/kg bw/d		 Test group 3
500 mg/kg bw/d

Litter

Fetuses

N

N

 24
221		 25
249		 24
260		 24
256
Fetal incidence N (%) 115 (52) 130 (52) 138 (53) 138 (54)
Litter incidence N (%) 24 (100) 25 (100) 24 (100) 24 (100)
Affected fetuses/litter Mean% 52.5 52.6 53.3 54.0

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

Applicant's summary and conclusion