Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03-03-2016 until 10/10/2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Proviplast 0142 (bis(2-butoxyethyl)adipate)
- Chemical Name: Bis(2-butoxyethyl)adipate
- Substance type: liquid
- Physical state: clear liquid with a very faint odour
- Analytical purity:98,83%
- Composition of test material, percentage of components: see Condifential details
- Purity test date: 2011-07-11
- Lot/batch No.:11701913 SA
- Expiration date of the lot/batch: 2012-12-14
- Storage condition of test material: store in tightly closed containers between -20 and +40°C

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633, from Sulzfeld , Germany.
- Age at study initiation: Young adult female rats, nulliparous and non-pregnant, approximately 11-12 weeks old
- Housing: Standard laboratory conditions; individual housing. Type III H, polysulphone cages on Altromin saw fibre bedding(lot no 02102151120) (expect during the pre-mating period when females were kept in groups of two animals and during mating period when two females where paired with one male).During the pre-mating period and after mating,males were housed in groups ( up to 5 animals/cage) in type IV cages.
- Diet (e.g. ad libitum): The animals have access to Altron 1324 maintenance diet for rats and mice ( lot nr 0922)
- Water (e.g. ad libitum): The animals were provided with tap water as for human consumption, in water bottles, ad libitum. The quality control analysis of the water is performed and microbiological assessment is performed at regualr intervals.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/- 3°C
- Humidity (%):55 +/-10%
- Air changes (per hr): 10 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 13 May 2014 To: 6 June 2014

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
The test item formulation or vehicle was administered at a single dose to the animals by oral gavage. The application volume for all groups was 4 mL/kg body weight (Table 1).
For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.

Table 1: Dose, Application Volume and Concentration
Group No. Group
Name Dose
[mg/kg bw] Application volume
[mL/kg bw] Concentration
[mg/mL]
1 C 0 4 0
2 LD 100 4 25
3 MD 200 4 50
4 HD 375 4 93.75
C = control, LD = low dose, MD = medium dose, HD = high dose
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The assessment of homogeneity as well as a determination of the nominal concentration of the test item in the vehicle was performed at various intervals.
Samples for analysis of concentration of the test item in the vehicle were taken in the first and last week of the study for all doses (8 samples in total).
Samples for homogeneity were taken from the top, middle and bottom of the high dose and low dose preparation. Samples were taken in the first and last week of the study (12 samples in total).
All samples were analysed within 10 days after sampling and until then stored under appropriate conditions based on available stability data (study no. 156144).
Each sample was retained in duplicate (sample A, sample B, each of at least 5 mL). The A-samples were analysed at Eurofins Munich. The procedures followed for the study sample analysis are mentioned in a phase plan (study no. 156148) that was amended to the study plan. The B-samples were retained at -15 to -35°C at BSL Munich and discarded after completion of the final study report.
Analytical results are reported in the appendix of the final report
Details on mating procedure:
Mating was performed using a ratio of 1:2 (male to female). Females were paired for cohabitation in batches in order to control the number of animals for terminal sacrifice on a particular day. At the subsequent mornings, the vaginal smear of the female was checked to confirm the pregnancy. The day on which sperms were observed in the vaginal smear was considered as gestation day ‘0’. Mated females were assigned in an unbiased manner to the control and treatment groups ensuring that group mean body weights were comparable with each other. Each animal was assigned a unique identification number. After getting 97 sperm positive females, the remaining females and males were discarded without any observations
Duration of treatment / exposure:
From gestation day 5 to gestation day 19
Frequency of treatment:
daily on a 7 days/week basis
Duration of test:
Caesarean section and necropsy at gestation day 20
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
200 mg/kg bw/day
Dose / conc.:
375 mg/kg bw/day
No. of animals per sex per dose:
156 animals (52 males and 104 females) were included in the study.
Control animals:
yes, concurrent no treatment
Details on study design:
According to the results of the dose range finding study (BSL Munich study no. 156141) and in consultation with the sponsor the following doses (0,100,200,375 mg/kg/bw) were selected for the 3 dose groups (LD = low dose, MD = medium dose, HD = high dose) and 1 control group (C). The animals were treated with the test item formulation or vehicle on 7 days per week between gestation day 5 until gestation day 19.The highest dose level was chosen with the aim of inducing toxic effects, but not death or severe suffering. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dose-related response and a NOAEL.
The animals in the control group were handled in an identical manner to the test group subjects and received the vehicle using the same volume as used for the treatment groups.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
Time schedule: Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day). Cage-side clinical observations were made at least daily after the treatment.
Any clinical observations (including spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size) were recorded if observed. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded if observed.
BODY WEIGHT: Yes
All animals were weighed once before initiation of pairing to ensure that the body weights were within + 20% variation
Time schedule for examinations: The body weight of sperm positive females was recorded on GD 0, 5, 8, 11, 14, 17, and 20.
FOOD CONSUMPTION: Yes
Food consumption of sperm positive females was measured on GD 0, 5, 8, 11, 14, 17 and 20.
Food consumption was not measured for males during the entire study or for both male and females during the mating period
POST-MORTEM EXAMINATIONS: Yes
On gestation day 20, sperm positive (presumed pregnant females) were subjected to a caesarean section after sacrificing the animals using an overdose of sodium pentobarbital injected intraperitoneally at a dosage of approximately 8 mL/kg bw.
Females showing signs of abortion or premature delivery prior to scheduled termination were sacrificed and subjected to a thorough macroscopic examination.
At the time of termination or death during the study, the dam (presumed pregnant female) was examined macroscopically for any structural abnormalities or pathological changes which may have influenced the pregnancy. Any macroscopic findings were preserved in 10 % neutral buffered formalin.
Immediately after the termination or as soon as possible after death, the uteri were removed and the pregnancy status of the dams was confirmed. Uteri that appear non-gravid were further examined by staining with 10 % ammonium sulphide solution to confirm the non-pregnant status.
Each gravid uterus with the cervix was weighed. However, the gravid uterus obtained from dead animals was not weighed.
The number of corpora lutea was counted for pregnant animals. The uterine contents were examined for embryonic or foetal deaths as well as the number of viable foetuses. The degree of resorption (late and early) was confirmed in order to help estimate the relative time of death of the conceptus. The position and number of foetuses in each uterine horn was also recorded.
Males were sacrificed without any observations at any time after the completion of the mating or were used for other studies
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: The degree of resorption was described in order to estimate the relative time of death of the conceptus.
Fetal examinations:
- External examinations: Yes: all per litter (live foetuses)
- Soft tissue examinations: Yes: half per litter (visceral examination)
- Skeletal examinations: Yes: half per litter (other half of the litter)
- Head examinations: No data
Statistics:
The findings of this study were evaluated in terms of the observed effects.
Maternal and foetal test results, including an evaluation of the relationship between the exposure of the animals to the test substance and the incidence and severity of all findings as well as findings of foetal external, soft tissue and skeletal anomalies are reported.
Food consumption is presented for intervals during gestation days 0-5, 5-8, 8-11, 11-14, 14-17 and 17-20.
All results were reported in tabular form (summarised in mean or summary tables and/or listed in individual data tables).
Analytical results were presented in a separate phase report (Appendix 3).
Toxicology and pathology data were captured either on paper according to appropriate SOPs or using the validated data capture function of the computerised system Ascentos® System (version 1.1.3, Pathology Data Systems Ltd.).
A statistical assessment of the results of the body weight and food consumption was performed by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Foetal evaluation parameters like external, visceral, craniofacial and skeletal parameters were analysed using Fisher’s exact test. The statistics were performed with GraphPad Prism V.6.01 software or Ascentos 1.1.3 software (p<0.05 is considered as statistically significant).

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Pale skin was observed in 11/24 females of the MD group and in 23/25 females of the HD group. This remarkable clinical sign was noted after few days of treatment and mainly persisted until sacrifice. It was related to the treatment with the test item. In the 90-day toxicity study, reduced blood cell mass, regenerative processes and other associated changes to red blood cell parameters were observed as test item related, adverse effects at the same dose levels of 200 and 350 mg/kg body weight/day. The pregnant rats in current study were more affected, which was evident from the pale skin which was noted after few days of treatment.
Mortality:
mortality observed, treatment-related
Description (incidence):
7/25 females of the HD group were either found dead or euthanised in a moribund condition in the course of the study. Females no. 90, 93 and 96 were found dead on the respective gestation day 9, 10 and 15 and females no. 89, 94, 98 and 100 were euthanised on day 12, 10, 13 and 6.On the day of sacrifice they were observed with clinical signs such as piloerection, pale skin, kyphosis, hypothermia, reduced spontaneous activity and/or half eyelid closure.
These animals’ morbidity and death was considered to be related to the treatment with the test item.
Female no. 96 of the HD group was found dead directly after dose application on gestation day 15. At necropsy the thorax was noted to be filled with white jelly and fatty fluid. Due to its sudden death and the macroscopic finding, it was considered to be related to a gavage error.
All remaining animals survived until the end of the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weight increased in all groups before start of the dose administration. After initiation of treatment a dose dependent loss of mean body weight was noted in the LD (-0.09 g), the MD (-8.30 g) and the HD group (-19.86 g) compared to a body weight gain of 4.39 g in the control group. This reached statistical significance in the HD group (p < 0.001). Slightly but statistically significantly lower body weight gain was also observed on gestation day (GD) 8-11 in the HD group (33 % of controls, p < 0.05). Thus, a moderately lower body weight gain was observed over the entire study period (GD 0-20) in the HD group (61 % of controls, p < 0.001). Differences in body weight gain compared to controls resulted in a slightly and statistically significantly reduced mean body weight of the MD group on GD 8 (95 % of controls, p < 0.05) and of the HD group during the entire treatment period with the greatest effect on GD 20 (89 % of controls, p < 0.001). Effects on body weight gain in the dose groups were considered as test item related. However, as overall body weight gain from GD 0-20 in the LD and MD group was comparable to controls, slight transient body weight loss after initiation of treatment was not considered as adverse.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In correlation to the body weight loss after initiation of treatment, food consumption in the MD group was slightly and statistically significantly lower compared to the control group (GD 5-8: 75 % of controls, p < 0.01). No biologically or statistically significant differences were observed in the remaining study period. Furthermore, food consumption in the HD group was noted to be moderately and statistically significantly lower compared to the control group during the entire treatment period (except for the interval of GD 14-17). This effect was most pronounced directly after initiation of treatment (GD 5-8: 44 % of controls, p < 0.001). Thus, there was a moderate and statistically significantly effect on food consumption in the HD group over the entire study period (GD 0-20) (80 % of controls, p < 0.001) which was considered as test item related.
Mean food consumption of the LD was unaffected by the treatment with the test item.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Haematuria was transiently observed in one single female of the LD group on gestation day 8, in two females of the MD group (gestation days 6-7 and gestation day 11) and in one female of the HD group on gestation day 11. A relation to the treatment with the test item cannot be excluded.
Behaviour (functional findings):
no effects observed
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Gross pathological changes at necropsy were seen in some of the females of the HD group which included enlarged spleen in 2/25, discoloured (dark) kidneys in 4/25 females, discoloured (dark) liver and urinary bladder in 1/25 and bloody urine in 1/25 females. A relation of macroscopic organ findings in the HD group to the treatment with the test item cannot be excluded.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
None of the females showed signs of abortion or premature delivery prior to the scheduled sacrifice.
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
No test item related effects of toxicological relevance were noted for number of corpora lutea, implantation sites, late resorptions, sex ratio, dead foetuses and percent pre-implantation loss. However, early resorptions were noted in 13/17 pregnant females of the HD group at scheduled caesarian section. 2 females of the HD group were seen with 100 % early resorptions. Thus, mean number of early resorptions in the HD group (3.35) was statistically significantly higher compared to the control group (1.17) which resulted in a slightly higher mean post-implantation loss of 29.70 % when compared to 10.29 % in the control group. This was considered as related to the treatment with the test item.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
effects observed, treatment-related
Description (incidence and severity):
Early resorptions were noted in 13/17 pregnant females of the HD group at scheduled caesarian section. 2 females of the HD group were seen with 100 % early resorptions. Thus, mean number of early resorptions in the HD group (3.35) was statistically significantly higher compared to the control group (1.17) which resulted in a slightly higher mean post-implantation loss of 29.70 % when compared to 10.29 % in the control group. This was considered as related to the treatment with the test item.
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
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
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Body weight and food consumption was adversely reduced in females dosed with 375 mg/kg body weight. Furthermore, uterus weight and adjusted maternal weight was significantly lower compared to controls and number of early resorptions was significantly higher,In the MD and HD anaemia was observed in the females. The dose level of 375 mg/kg BW/day was considered excessively toxic as demonstrated by the mortality rate and clinical conditions indicative of bad conditions.
Details on maternal toxic effects:
The dose level of 375 mg/kg body weigth/day was considered excessively toxic as demonstrated by the mortality rate and clminical conditions indicative of bad condition. The dose level of 200 mg/kg body weight was considered maternally toxic as demonstrated by the clinical signs and initial lower body weight gain. In the 90-day toxicity study, reduced blood cell mass, regenerative processes and other associated changes to red blood cell parameters were observed as test item related, adverse effects at the same dose levels of 200 and 350 mg/kg body weight/day. The pregnant rats in current study were more affected, which was evident from the pale skin which was noted after few days of treatment in 11/24 and 23/25 females, respectively, and which mainly persisted until sacrifice

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
behaviour (functional findings)
pre and post implantation loss
early or late resorptions
dead fetuses
necropsy findings
other:
Remarks on result:
other: The pregnant rats at 200 and 375 mg/kg were affected by anemia

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
15.8 % reduction of fetal body weight in the HD.The HD level of 375 mg/kg body weigth/day was considered excessively toxic as demonstrated by the mortality rate and clinical conditions indicative of bad condition.
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
19% reduction in live foetuses in the HD group.The dose level of 375 mg/kg body weigth/day was considered excessively toxic as demonstrated by the mortality rate and clinical conditions indicative of bad condition.
Changes in sex ratio:
no effects observed
Changes in postnatal survival:
effects observed, treatment-related
Description (incidence and severity):
increased post inplantation loss in the HD group (29.70 v 10.29 control).The HD (375 mg/kg body weigth/day) was considered excessively toxic as demonstrated by the mortality rate and clinical conditions indicative of bad condition.
External malformations:
effects observed, treatment-related
Description (incidence and severity):
5/8 foetuses of dam 91 (HD group) were noted with generalised oedema ( anasarca).The HD( of 375 mg/kg body weigth/day) was considered excessively toxic as demonstrated by the mortality rate and clinical conditions indicative of bad condition. The latter findings were seen in one litter of a dam in bad conditions.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
One foetus was noted with split sternum , the malformation of small premaxilla was observed in another foetus. And slight to moderate misshapen humerus was noted in 6 foetuses.Slightly to moderately bent scapula was observed in total in 7/15 litters (46.67 %) of the HD. The dose level of 375 mg/kg body weigth/day was considered excessively toxic as demonstrated by the mortality rate and clminical conditions indicative of bad condition
Reduced ossification ( sternebra, metacarpals) in several bones was noticed in foetuses at 200 and 375 mg/kg.Observed generalised delay in ossification is often seen in rodents and could be related to maternal toxicity due to anaemia which might have led to uterine hypoxia with malnutrition of the foetuses. The foetal findings at both dose levels are considered to be secondary to maternal toxicity.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The dose level of 375 mg/kg body weigth/day was considered excessively toxic as demonstrated by the mortality rate and clminical conditions indicative of bad condition. One foetus of female 91( bad condition) was noted with the malformation of split sternum and misplaced aortic arch at visceral examination. The latter findings were seen in one litter of a dam in bad conditions.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
fetal/pup body weight changes
skeletal malformations
visceral malformations
other: skelertal Variations: unossified and incomplete ossified sternebra, small premaialla,,bent scapula.,wavy ribs
Remarks on result:
other: Anaemia in the pregnant rats have let to uterine hypoxia with malnutrition of the foetuses

Fetal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Description (incidence and severity):
findings seen in animal 91 are considered as secondary due to maternal anaemia and deteriorating bad conditions.Observed generalised delay in ossification is often seen in rodents and could be related to maternal toxicity due to anaemia which might have led to uterine hypoxia with malnutrition of the foetuses. The foetal findings at both dose levels (HD, MD) are considered to be secondary to maternal toxicity.

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
200 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
A key prenatal development toxicity was conducted in Wistar rats at dose levels of 100, 200 and 375 mg/kg body weight/day from gestation days 5 to 19. The dose level of 375 mg/kg body weigth/day was considered excessively toxic as demonstrated by the mortality rate and clminical conditions indicative of bad condition. The dose level of 200 mg/kg body weight was considered maternally toxic as demonstrated by the clinical signs and initial lower body weight gain. The pregnant rats at these dose levels were affected by anemia, which was evident from the pale skin which was noted after few days of treatment in 11/24 and 23/25 females, respectively, and which mainly persisted until sacrifice. This target organ was also demonstrated in the 90-day toxicity study, but resulted in deteriorating condition in the prenatal toxicity study. At a dose level of 375 mg/kg body weight/day mean foetus and litter weight was significantly lower compared to controls. At the same dose an accumulation of foetal abnormalities (variations as well as malformations) was observed. Most of the latter findings were seen in one litter of a dam in bad condition. Dose dependently reduced ossification in several bones was noted in foetuses at 200 and 375 mg/kg body weight/day. Observed generalised delay in ossification is often seen in rodents and could be related to maternal toxicity due to anaemia which might have led to uterine hypoxia with malnutrition of the foetuses. The foetal findings at both dose levels are considered to be secondary to maternal toxicity. No effects on females and foetuses were found at a dose level of 100 mg/kg body weight/day. The NOAEL for both maternal toxicity and foetal toxicity was 100 mg/kg body weight/day.
Executive summary:

The aim of this study was to assess possible adverse effects on pregnant females and embryo-foetal development which could arise from repeated exposure of Bis(2-butoxyethyl) adipate via oral administration (gavage) to female rats during gestation days 5 to 19. Nulliparous and non-pregnant females were mated with males (2:1 ratio) and divided into four groups based on their body weights on the day of sperm positive vaginal smears (GD 0). The 4 groups comprised each 24 female Wistar rats in the control group (C), the low dose group (LD) and the medium dose group (MD) and 25 female Wistar rats in the high dose group (HD). The following doses were evaluated: Control:0mg/kg body weight/day Low Dose:100mg/kg body weight/day Medium Dose:200mg/kg body weight/day High Dose:375mg/kg body weight/day The test item formulation was prepared with corn oil at least once every ten days based on available stability data. The prepared formulation was stored at room temperature. Dose volumes were adjusted individually based on the body weight most recently measured. Animals of the control group were handled identically as the dose groups, but received corn oil, the vehicle used in this study. Sperm positive females were weighed on gestation days 0, 5, 8, 11, 14, 17 and 20. Food consumption of sperm positive females was measured on gestation days 5, 8, 11, 14, 17 and 20. During the period of administration, the animals were observed precisely each day for signs of toxicity and mortality. Animals that died or were euthanised during the study were examined macroscopically. All surviving female animals were sacrificed on the respective gestation day 20. Following the gross necropsy, the uteri and ovaries were removed, weighed and examined for number of implantations, resorptions (early and late), live and dead foetuses. The uteri of the non-pregnant females were processed with 10 % ammonium sulphide solution and checked for early embryonic deaths. Foetuses were identified by using strings with numbered plates, sexed and weighed. All foetuses were observed for external abnormalities, half of the fetuses for visceral and craniofacial abnormalities and the remaining half of the litter was observed for skeletal abnormalities..Summary Results

Maternal Findings .

7/25 females of the HD group were either found dead or euthanised in a moribund condition in the course of the study. Shortly before death or euthanasia they were observed with clinical signs such as piloerection, pale skin, kyphosis, hypothermia, reduced spontaneous activity, prostration and/or half eyelid closure. These animals’ morbidity and death was considered to be related to the treatment with the test item except for female no. 96 of the HD group which was found dead directly after dose application. Due to its sudden death and the macroscopic finding of fatty fluid in the thorax, it was considered to be related to a gavage error. Gross pathological changes at necropsy were seen in some of the females of the HD group which included enlarged spleen in 2/25, discoloured (dark) kidneys in 4/25 females, discoloured (dark) liver and urinary bladder in 1/25 and bloody urine in 1/25 females. A relation of macroscopic organ findings in the HD group to the treatment with the test item cannot be excluded. Remarkable clinical sign related to the treatment with the test item was pale skin which was observed in 23/25 females of the HD group and 11/24 females of the MD group after few days of treatment and mainly persisted until sacrifice. Haematuria was transiently observed in one single female of the LD group, in two females of the MD group and in one female of the HD group on single days of gestation. A relation to the treatment with the test item cannot be excluded, however, due to its transient character it was not considered as adverse. Some females, mostly of the HD group, were noted occasionally with unspecific clinical signs. Kyphosis was noted in 4/25 females of the HD group, nasal discharge in 5/25 females of the HD group, sunken flanks in 4/25 females of the HD group, lacrimation in 1/25 females of the HD group, reduced spontaneous activity in 3/25 females of the HD group and piloerection in 9/24 females of the MD group and 21/25 females of the HD group. These signs were considered to be related to slightly, generally reduced health condition or discomfort of the animals. Few females of the MD group and some females of the HD and were transiently observed with moving their bedding and rarely females of the HD group with salivation. As the clinical signs of moving the bedding and salivation were noted directly after dose administration, these findings were considered to be a sign of discomfort attributed to the oral administration of the test item and not as an adverse systemic effect. None of the females showed signs of abortion or premature delivery prior to the scheduled sacrifice. Dose dependent effects on body weight and/or food consumption were observed in the dose groups. After initiation of treatment a dose dependent loss of mean body weight was noted in the LD (-0.09 g), the MD (-8.30 g) and the HD group (-19.86 g) compared to a body weight gain of 4.39 g in the control group. This reached statistical significance in the HD group. Slightly but statistically significantly lower body weight gain was also observed on gestation day (GD) 8-11 in the HD group (33 % of controls). Thus, a moderately lower body weight gain was observed over the entire study period (GD 0-20) in the HD group (61 % of controls). Differences in body weight gain compared to controls resulted in a slightly and statistically significantly reduced mean body weight of the MD group on GD 8 (95 % of controls, p<0.05) and of the HD group during the entire treatment period with the greatest effect on GD 20 (89 % of controls). In correlation to the body weight loss, food consumption in the MD group and HD group after initiation of treatment was slightly to moderately and statistically significantly lower compared to the control group. Food consumption in the HD group was 80 % of controls over the entire study period (GD 0-20). Effects on body weight and food consumption were considered as adverse in the HD group whereas slight and transient effects in the LD and MD group were not considered as adverse. Further to the test item related slightly lower mean terminal body weight in the HD group, a moderately and statistically significantly lower uterus weight was noted in the HD group (89 % of controls). Thus, slightly and statistically significantly lower mean adjusted maternal weight (maternal weight minus gravid uterus weight) was also noted in the HD group (92 % of controls). This was considered as an effect of the test item. No test item related effects of toxicological relevance were noted for number of corpora lutea, implantation sites, late resorptions, sex ratio, dead foetuses and percent pre-implantation loss. However, early resorptions were noted in 13/17 pregnant females of the HD group at scheduled caesarian section. 2 females of the HD group were seen with 100 % early resorptions. Thus, mean number of early resorptions in the HD group (3.35) was statistically significantly higher compared to the control group (1.17) which resulted in a slightly higher mean post-implantation loss of 29.70 % when compared to 10.29 % in the control group. This was considered as related to the treatment with the test item. Accordingly slightly but not statistically significantly lower mean number of live foetuses was observed in the HD group (8.59 foetuses compared to 10.57 in the control group). Successful mating resulted in 22/24 pregnancies in the LD group, 23/24 in the MD group and 22/25 in the HD group compared to 23/24 pregnancies in the control group which was in the normal range of variation.

Foetal Findings.

Test item related effects were noted for foetal and litter weight data in the HD group. Mean foetus weight of the HD group (3.15 g) was moderately and statistically significantly lower (84 % of controls) when compared to the control group (3.74 g). Likewise, mean litter weight of the HD group was slightly lower compared to controls with reaching statistical significance for female (67 % of controls) and total litter weight (85 % of controls). Mean foetal weight and litter weight of the LD and MD group was unaffected by the test item administration. Overall an accumulation of foetal abnormalities (variations as well as malformations) was observed in the HD group which was considered as an effect of the treatment with the test item. One litter of the HD group (female no. 91) was particularly affected by retardation in terms of markedly reduced ossification of several bones combined with severely reduced foetal weight (mean foetal weight of 1.38 g compared to 3.74 g in controls). Several foetuses of this litter were noted with the external malformation of generalised subcutaneous oedema (anasarca). Furthermore, one foetus of female no. 91 was noted with the malformations of split sternum and misplaced aortic arch at visceral examination. Small pituitary gland was seen without statistical significance in 2 foetuses of the HD group and 1 of the control group. As the litter incidence of 14.29 % in the HD group was slightly above maximum value of historical control data, a relation to the treatment with the test item cannot be excluded. There were no further foetal external, visceral or craniofacial abnormalities considered to be of toxicological relevance in any of the dose groups. Statistical analysis showed no significant differences to the control group. Skeletal examination of the Alizarin red stained foetuses revealed a range of findings in all groups including control. Slightly to moderately bent scapula was observed in total in 7/15 litters (46.67 %) of the HD group which was markedly above maximum litter incidence of historical control data (5.26 %). Bilateral (B) bent scapula was statistically significantly higher in the HD group (26.67 %) compared to 0.00 % in controls. One foetus of the HD group that was affected with left (L) bent scapula also showed bent radius (B). There are some new studies suggesting that bent scapula and bent long bones are transient and not permanent foetal changes that are repaired postnatally. As such, they may be classified as variations and not as malformations . The malformation of slightly to moderately misshapen humerus was noted in 6 foetuses of 4 litters of the HD group. The malformation of a small premaxilla was observed in one single foetus of the HD group. One foetus of the HD group was noted with wide 1st sternebra and split 2nd-3rd sternebra. Furthermore, one foetus was seen with wide 1st-3rd sternebra and another one with split 1st-6th sternebra. In total 3 foetuses from 3 litters were noted with split sternebra (2 at time of skeletal, 1 at time of visceral examination). The finding of wide sternebra can be regarded as precursor to the malformation of split sternebra. Markedly and statistically significantly higher litter incidence of wavy ribs was noted in the HD group (73.33 %) when compared to the concurrent control group (30.43 %). Incidence of the HD group was also marginally above maximum litter incidence of historical control data (72.73 %). This was likely correlated to an observed generalised delayed ossification in foetuses of the HD group resulting also in delayed ossification and bending of parts of the ribs. Wavy ribs are a common finding in rodent studies and are classified as variation as they are likely to be postnatally reversible . Dose dependently reduced ossification in several bones was noted in foetuses of the MD and the HD group when compared to the control group which was considered as an effect of the treatment with the test item. Moderately reduced ossification was noted in some bones of the skull which reached statistical significance for litter incidence of parietals (B) (61.90 % and 66.67 % compared to 26.09 % in controls) and supraoccipital (76.19 % and 73.33 % compared to 26.09 % in controls) in the MD and the HD group, respectively, of interparietal in the MD group (85.71 % compared to 56.52 % in controls) and frontals (B) in the HD group (40.00 % compared to 4.35 % in controls). Dose dependently reduced ossification was also noted for shoulder girdle, pelvic girdle and long bones. Incomplete ossification of scapula (B) was observed at a moderately and statistically significantly higher litter incidence of 26.67 % compared to 0.00 % in the concurrent control and a maximum litter incidence of 4.35 % of historical control data. Incomplete ossification of humerus (B/L) was noted in 1 foetus of the MD group (litter incidence 4.76 %) and 3 foetuses of 3 litters of the HD group (litter incidence 20 %) without statistical significance compared to 0.00 % in concurrent and historical control data. Incomplete ossification of ischium (B) was observed in 10 foetuses of 4 litters in the HD group (litter incidence 26.67 % compared to 0.00 % in the control group) with statistical significance. Furthermore, there was a non-significant trend towards increased incomplete ossification of ilium (B) and pubis (B) in the HD group which was not observed in controls. Dose dependently reduced ossification was also noted for sternebra. Statistical significance compared to controls was reached for litter incidence of unossified sternebra (2nd) in the HD group (26.67 % compared to 0.00 %), of incomplete ossification of sternebra (5th) in the MD (38.10 %) and the HD group (60.00 % compared to 8.70 %), of unossified sternebra (5th) in the HD group (66.67 % compared to 4.35 %), of incomplete ossification of sternebra (6th) in the MD group (90.48 % compared to 60.87 %) and of unossified sternebra (6th) in the HD group (26.67 % compared 0.00 %). There was a trend towards dose dependently higher litter incidence of unossified cervical centrum with all litters being affected in the MD and the HD group compared to 82.61 % in controls without reaching statistical significance. Incomplete ossification of lumbar arches and of sacral arches was noted with a litter incidence of 20.00 % and 13.33 %, respectively, in the HD group compared to 0.00 % in controls. A statistically significantly higher litter incidence of 60.00 % was noted for unossified metacarpals in the HD group compared to 13.04 % in controls. The observed statistically significantly reduced ossification of several bones indicated a generalised delay of ossification in the MD and the HD group. In the HD, but not the MD group, delayed ossification was associated with fetal growth retardation (significantly reduced mean foetal weight). Maternal toxicity in terms of clinical signs in the MD group and reduced body weight gain, food consumption and health condition in the HD group might have mediated the delay in ossification . Pale skin in females of the MD and the HD group in terms of anaemia might have led to uterine hypoxia with malnutrition of foetuses. No further statistically significant differences for skeletal data were noted between the dose groups and the control group. There were no findings of toxicological relevance in the LD group. Observed findings in the LD and the control group were considered to be spontaneous in nature.