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
Study period:
2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to GLP and valid methods so it is reliable and relevant

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2016

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Provichem 0216, Diphenyl sulphone
- Substance type: Organic monoconstituent
- Physical state: White odourless powder (solid)
- Analytical purity: 99.90 %
- Impurities (identity and concentrations): See confidential details
- Isomers composition: -
- Lot/batch No.: 201101190020
- Expiration date of the lot/batch: Not applicable
- Stability under test conditions: Stable
- Storage condition of test material: Room temperature

Test animals

Species:
rat
Strain:
other: Wistar: Crl: WI(Han) (Full Barrier)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: e.g. Charles River, 97633 Sulzfeld, Germany
- Age at study initiation:
Age of females at arrival at BSL BIOSERVICE: approx.. 11-12 weeks old
Age of males at the start of pairing: approx.. 11-12 weeks old
- Weight at allocation of the animals to the experiment al groups:
Males: 291 - 342 g (mean: 314.33 g, ± 20% = 251.46 – 377.19 g)
Females: 188 - 224 g (mean: 202.17 g, ± 20% = 161.74 – 242.61 g)
- Fasting period before study:
- Housing: Full barrier in an air-conditioned room. The females were kept individually in type III H, polysulphone cages on Altromin saw fibre bedding (lot no. 02102150227) (except during the pre-mating period when females were kept in groups of two animals and during mating period when two females were paired with one male). During the pre-mating period and after mating, males were housed in groups (3 animals / cage) in type IV cages.- Diet (e.g. ad libitum): Free acces to Altromin 1324 maintenance diet for rats and mice (lot no. 1239)
- Water (e.g. ad libitum): Free acces to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Acclimation period: At least five days

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

IN-LIFE DATES: From: 9 July 2015 (Acclimatisation) To: 14 September 2015 (End of Necropsies)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was dissolved in corn oil.
The test item was weighed into a tared plastic vial on a suitable precision balance and the vehicle was added to give the appropriate final concentration of the test item and further vortexing and or use of ulta turrax homogeniser for 2-3 minutes.
Homogeneity of the test item in the vehicle was maintained by vortexing the prepared suspension thoroughly before every dose administration.
The test item formulation was prepared at least once every ten days based on available stability data (study no. 151888). The prepared formulation was stored at room temperature. The vehicle was also used as control item.
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
For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The vehicle was selected based on the test item’s characteristics and testing guideline.
- Lot/batch no. (if required): MKBP7039V
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before beginning of the treatment period, formulation samples were prepared and analysed in order to obtain knowledge about stability and homogeneity of the test item in the chosen vehicle at BSL BIOSERVICE Scientific Laboratories GmbH as part of a separate GLP study (study no. 151888).
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 of the study.
Samples for analysis of concentration of test item in the dosing formulations 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).
Each sample was retained in duplicate (sample A, sample B, each of at least 5 mL).
All formulation samples were stored at -15 to -35 °C until analysis. At the end of treatment period or within time period covered by stability data, all A samples were analysed at BSL BIOSERVICE Scientific Laboratories GmbH. The procedures followed for the study sample analysis were mentioned in a phase plan (phase study no. 151887) that was amended to the study plan.
The B samples were retained at BSL BIOSERVICE until the analysis has been performed, and discarded after completion of the final study report.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1/2
- Length of cohabitation: Until sperm was observed in vaginal smear
- 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. Due to increased number of animals from HD group sacrificed due to welfare reasons, additional 4 females were mated and included in the HD group.
- Proof of pregnancy: sperm in vaginal smear referred to as day 0] of pregnancy


Duration of treatment / exposure:
From gestation day 5 to gestation day 19.
Frequency of treatment:
7 days/week
Duration of test:
On gestation day 20, i.e. the day prior to the expected day of delivery, the presumed pregnant females were subjected to a caesarean section.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 62;5, 125 and 250/200 mg/kg bw
Basis:
actual ingested
In the HD group 23 females of the HD group (no. 82-104) were treated with a dose of 200 mg/kg bw/day for the whole treatment period, whereas 6 females (no. 76-81) were partly dosed with 250 mg/kg bw/day.
No. of animals per sex per dose:
52 males and 104 females (25 females in control, low dose and mid dose group and 29 females in high dose group)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: According to the results of the dose range finding study (BSL project no. 151884) and in consultation with the sponsor the following doses were selected for the 3 dose groups (LD: low dose, MS: medium dose, HD= high dose) and 1 control group (C).
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.
6 females from the HD group were treated with 250 mg/kg body weight/day from the start of the study for several treatment days (until 28 July 2015):
-animal no. 76 from GD 5 to GD 12
-animal no. 77 from GD 5 to GD 12 (Euthanised on GD 15)
-animal no. 78 from GD 5 to GD 11 (Euthanised on GD 14)
-animal no. 79 from GD 5 to GD 11 (Euthanised on GD 13)
-animal no. 80 from GD 5 to GD 6 (Euthanised on GD 15)
-animal no. 81 from GD 5 to GD 6.
After marked reduction of the body weight, food intake and test item related clinical signs in those presumed pregnant animals of the HD group, the high dose was reduced to 200 mg/kg body weight/day. Females no. 82-104 of the HD group were treated with a dose of 200 mg/kg body weight/day for the whole treatment period.
The animals in the control group were handled in an identical manner to the test group subjects and received corn oil using the same volume as used for the high dose group.


Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: General clinical observations were made at least once a day, preferably at the same time each day. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.
None of the female showed signs of abortion or premature delivery prior to the scheduled sacrifice.
Clinical observations included 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. 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.

DETAILED CLINICAL OBSERVATIONS: Yes.
- Time schedule: Prior to the start of the mating a detailed clinical observation outside the home cage was made. No animal showed pathological signs before mating.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed once before initiation of pairing to ensure that the body weights are within + 20% variation.
The sperm positive females were weighed on gestations days 0, 5, 8, 11, 14, 17 and 20. Males were not weighed in this study except once before initiation of pairing.

FOOD CONSUMPTION: Yes
- Food consumption of pregnant females was measured on gestations days 5, 8, 11, 14, 17 and 20. Food consumption was measured neither for males during the entire study nor for both male and females during the mating period.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: At the time of termination or death during the study, the dam (presumedly pregnant female) was examined macroscopically for any structural abnormalities or pathological changes which may have influenced the pregnancy. All macroscopic findings were preserved in 10% neutral buffered formalin. Males were sacrificed without any observations any time after the mating.

OTHER:
Animals that died during the study or euthanised due to animal welfare reasons 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 the early embryonic deaths. Each gravid uterus with the cervix was weighed. However, the gravid uterus obtained from animals found dead 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.



Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes, but the gravid uterus obtained from animals found dead was not weighed.
- Number of corpora lutea: Yes for pregnant animals
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other:
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 animals found dead 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.

Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter (microdissection technique)
- Skeletal examinations: Yes: half per litter (Alizarin red staining)
- Head examinations: Yes: half per litter. Craniofacial examination of the heads of the foetuses used for the soft tissue examination was performed for internal structure including the eyes, brain, nasal passage and tongue by razor blade serial sectioning technique.

Statistics:
A statistical assessment of the results of the body weight, 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 a Fisher’s exact test. The statistics were performed with GraphPad Prism V.6.01 software (p<0.05 is considered as statistically significant).

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Seven females (no. 77-80, 95-96 and 101) of the HD group were euthanized in a moribund condition (marked loss of body weight, slight to severe piloerection, reduced spontaneous activity, wasp waist, abnormal breathing and moving the bedding) for animal welfare reasons between gestation day 13-19. Another female of the HD group (no. 83) was found dead on gestation day 20.
There were no clinical signs of toxicological relevance in the LD and MD dose groups when compared to the control group.
In HD group, prior to euthanasia for animal welfare reasons between gestation day 13-19, female no. 77-80, 95-96 and 101 (250/200 mg/kg bw/day) showed slight to severe piloerection, reduced spontaneous activity, wasp waist, abnormal breathing and moving the bedding on various days.
Before female no. 83 of the HD group was found dead on gestation day 20 (after treatment with 200 mg/kg bw/day), no clinical signs were observed except moving the bedding from GD 8-19.
The clinical signs of localised alopecia on various body parts, slight to severe piloerection, salivation, moving the bedding was noted during gestation day 6-19 in several other females of the HD group treated with 250 to 200 mg/kg bw/day.
None of the females showed signs of abortion or premature delivery prior to the scheduled sacrifice.
The mean body weight increased with the progress of the study in the control, the LD and the MD group. There were no test item related effects of toxicological relevance noted for body weight and body weight gain. Throughout the treatment period, body weights were within the normal range of variation for this strain. However, mean body weight of the HD group was statistically significantly lower compared to the control group from gestation day 8 onwards. Body weight gain was also lower in the HD group compared to the control group on various intervals within the study period and achieved statistical significance on day 5-8 (p<0.001) in MD and HD group and on day 17-20 and 0-20 in HD group when compared with the controls.
In correlation to the body weight and body weight gain, food consumption in the HD group was noted to be lower compared to the control group after the beginning of the treatment. Statistical significance was noted over the study period from day 0 to day 20 (p<0.001) in HD group as well as on several treatment intervals in HD ( 5-8, 8-11, 11-14 and 17-20) and during GD 5-8 in LD and MD when compared with the controls.
Prenatal parameters like group mean terminal body weight, gravid uterus weight, adjusted maternal weight, number of corpora lutea, implantation sites, early and late resorptions, number of live foetuses, male and female foetuses, sex ratio, number of foetuses in each uterine horn and percent pre- and post-implantation loss remained unaffected in the dose groups when compared to the control group. There were no statistically significant differences observed for prenatal data.
No gross pathological changes of toxicological relevance were observed during the macroscopic examination of the females of LD and MD group at necropsy.
At necropsy of female no. 77 of the HD group which was euthanised for animal welfare reasons on gestation day 15, red discharge from vagina and thymus was noted to be of a small size. In females 79, 80 and 101 which were euthanised for animal welfare reasons on gestation day 13, 15 and 19, respectively, blood filled vagina was observed.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
99.90% purity
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
There were no test item related effects of toxicological relevance for the total number of foetuses, number of male and female foetuses, total litter weight and male and female litter weight. However, statistically significantly lower mean foetus weight was observed in HD group when comparared with the controls. This dose dependent effect on mean foetus weight in HD group was considered to be test item related.
There were no external abnormalities considered to be of toxicological relevance in any of the dose groups. Statistical analysis showed no significant differences to the control group. Low incidences of haematoma on various body parts were noted in isolated females of the control group and/or the dose groups without dose dependency. This was considered to be incidental in nature.
Internal observation of the foetal viscera by free hand microdissection technique revealed a range of visceral findings in all groups including control.
Visceral findings observed in the dose groups were at frequencies generally comparable to or in some cases slightly higher or lower in frequency compared to controls. As observed findings were either minor variations and/or due to a lack of dose dependency and consistency, no serious toxicological significance can be attributed to these findings and they were considered to be spontaneous in nature. Litter incidences were statistically insignificant except for extra tissue on median liver lobe which was observed at lower frequencies in the HD group in comparison to the control group. Thus, this finding was considered incidental.
Craniofacial examination by razor blade serial sectioning technique revealed few findings in all groups including control. Statistical analysis of litter incidences revealed no statistical significance of any of the findings as compared to the control group. Therefore, these findings are not to be considered to be treatment related and spontaneous in nature.
Skeletal examination of the Alizarin red stained foetuses revealed a range of findings which were of a type or which occurred at an incidence generally comparable to or slightly lower or higher in the dose groups when compared to the control group. A statistically significant decrease in litter incidence for incomplete ossification of interparietal and parietal bone in the MD and HD group and spuraoccipital in MD group compared to the control group was considered to be incidental as frequencies were even less in numbers compared to controls. Therefore, these findings are not to be considered as treatment-related and solely spontaneous in nature .
A statistically significantly higher litter incidence of 14th rudimentary rib (bilateral) in the LD group and ilium (bilateral) caudal offset in MD group was seen without dose dependency when compared to the control group and was not considered as an effect of the test item.
A Statistically significantly higher litter incidence of incomplete ossification of 1st sternebra in HD, 4th sternebra in LD and HD, unossified 4th sternebra in HD and dumbbell shaped ossification of thoracic centrum in MD and HD were observed when compared to the control group. These sternebra findings and dumbbell shaped ossification of thoracic centrum are considered to be variations and considered not to be adverse.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
99.90% purity
Basis for effect level:
other: fetotoxicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
On the basis of this prenatal developmental toxicity study in Wistar pregnant female rats with Provichem 0216 at dose levels of 62.5, 125, and 200/250 mg/kg body weight day administered on gestation days 5 to 19, the following conclusions can be made:
At a dose level of 250/200 mg/kg bw/day seven animals were sacrificed for welfare reasons and mortality was observed in one animal and clinical symptoms like marked loss of body weight, slight to severe piloerection, reduced spontaneous activity, wasp waist, abnormal breathing and moving the bedding were observed. Animals treated with 250 to 200 mg/kg bw/day were noted with a statistically significantly reduced body weight gain and food consumption.
No effect on prenatal data, litter data, foetal external, visceral, skeletal and craniofacial parameters were observed up to the highest dose level except decrease in mean foetal weight.
No effects of Provichem 0216 on females and foetuses were found at dose levels of 125 mg/kg body weight/day. The NOAEL for both maternal toxicity and fetal toxicity of Provichem 0216 in this study is considered to be 125 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 Provichem 0216 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 25 female Wistar rats each in the control, in the LD group, in the MD group and 29 in the HD group. The following doses were evaluated: 0 - 62.5 - 125 - 250/200 mg/kg body weight/day. 6 females from the HD group were treated with 250 mg/kg body weight/day from the start of the study for several treatment days (until 28 July 2015):

-animal no. 76 from GD 5 to GD 12

-animal no. 77 from GD 5 to GD 12 (Euthanised on GD 15)

-animal no. 78 from GD 5 to GD 11 (Euthanised on GD 14)

-animal no. 79 from GD 5 to GD 11 (Euthanised on GD 13)

-animal no. 80 from GD 5 to GD 6 (Euthanised on GD 15)

-animal no. 81 from GD 5 to GD 6.

After marked reduction of the body weight, food intake and test item related clinical signs in those presumed pregnant animals of the HD group, the high dose was reduced to 200 mg/kg body weight/day. Females no. 82-104 of the HD group were treated with a dose of 200 mg/kg body weight/day for the whole treatment period.

The test item formulation was prepared at least once every ten days and was stored at room temperature. The test item was dissolved in corn oil and administered daily during a gestation day 5 and 19 to female animals. Dose volumes were adjusted individually based on weekly body weight measurements.

Animals of the control group were handled identically as the dose groups, but received corn oil, the vehicle used in this study.

During the period of administration, the animals were observed precisely each day for signs of toxicity and mortality. Animals that died during the study or euthanised due to animal welfare reasons 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 the early embryonic deaths.

Foetuses were identified by 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.

The sperm positive females were weighed on gestations days 0, 5, 8, 11, 14, 17 and 20.

Food consumption of pregnant females was measured on gestations days 5, 8, 11, 14, 17 and 20.

Maternal Findings

Seven females (no. 77-80, 95-96 and 101) of the HD group were euthanized in a moribund condition (marked loss of body weight, slight to severe piloerection, reduced spontaneous activity, wasp waist, abnormal breathing and moving the bedding) for animal welfare reasons between gestation day 13-19. Another female of the HD group (no. 83) was found dead on gestation day 20.

There were no clinical signs of toxicological relevance in the LD and MD dose groups when compared to the control group

In HD group, prior to euthanasia for animal welfare reasons between gestation day 13-19, female no. 77-80, 95-96 and 101 (250/200 mg/kg bw/day) showed slight to severe piloerection, reduced spontaneous activity, wasp waist, abnormal breathing and moving the bedding on various days.

Before female no. 83 of the HD group was found dead on gestation day 20 (after treatment with 200 mg/kg bw/day), no clinical signs were observed except moving the bedding from GD 8-19.

The clinical signs of localised alopecia on various body parts, slight to severe piloerection, salivation, moving the bedding was noted during gestation day 6-19 in several other females of the HD group treated with 250 to 200 mg/kg bw/day.

None of the females showed signs of abortion or premature delivery prior to the scheduled sacrifice.

The mean body weight increased with the progress of the study in the control, the LD and the MD group. There were no test item related effects of toxicological relevance noted for body weight and body weight gain. Throughout the treatment period, body weights were within the normal range of variation for this strain. However, mean body weight of the HD group was statistically significantly lower compared to the control group from gestation day 8 onwards. Body weight gain was also lower in the HD group compared to the control group on various intervals within the study period and achieved statistical significance on day 5-8 (p<0.001) in MD and HD group and on day 17-20 and 0-20 in HD group when compared with the controls.

In correlation to the body weight and body weight gain, food consumption in the HD group was noted to be lower compared to the control group after the beginning of the treatment. Statistical significance was noted over the study period from day 0 to day 20 (p<0.001) in HD group as well as on several treatment intervals in HD ( 5-8, 8-11, 11-14 and 17-20) and during GD 5-8 in LD and MD when compared with the controls.

Prenatal parameters like group mean terminal body weight, gravid uterus weight, adjusted maternal weight, number of corpora lutea, implantation sites, early and late resorptions, number of live foetuses, male and female foetuses, sex ratio, number of foetuses in each uterine horn and percent pre- and post-implantation loss remained unaffected in the dose groups when compared to the control group. There were no statistically significant differences observed for prenatal data.

No gross pathological changes of toxicological relevance were observed during the macroscopic examination of the females of LD and MD group at necropsy.

At necropsy of female no. 77 of the HD group which was euthanised for animal welfare reasons on gestation day 15, red discharge from vagina and thymus was noted to be of a small size. In females 79, 80 and 101 which were euthanised for animal welfare reasons on gestation day 13, 15 and 19, respectively, blood filled vagina was observed.

At necropsy of female no. 83 of the HD group which was found dead on gestation day 20, the whole body autolysis was observed. There was a single incidence of a fluid content in stomach was observed in female no. 84 of the HD group. In female 96 which was euthanised for animal welfare reasons on gestation day 19, vaginal discharge and discoloured dark lung was observed.

Foetal Findings

There were no test item related effects of toxicological relevance for the total number of foetuses, number of male and female foetuses, total litter weight and male and female litter weight. However, statistically significantly lower mean foetus weight was observed in HD group when comparared with the controls. This dose dependent effect on mean foetus weight in HD group was considered to be test item related.

There were no external abnormalities considered to be of toxicological relevance in any of the dose groups. Statistical analysis showed no significant differences to the control group. Low incidences of haematoma on various body parts were noted in isolated females of the control group and/or the dose groups without dose dependency. This was considered to be incidental in nature.

Internal observation of the foetal viscera by free hand microdissection technique revealed a range of visceral findings in all groups including control.

Visceral findings observed in the dose groups were at frequencies generally comparable to or in some cases slightly higher or lower in frequency compared to controls. As observed findings were either minor variations and/or due to a lack of dose dependency and consistency, no serious toxicological significance can be attributed to these findings and they were considered to be spontaneous in nature. Litter incidences were statistically insignificant except for extra tissue on median liver lobe which was observed at lower frequencies in the HD group in comparison to the control group. Thus, this finding was considered incidental.

Craniofacial examination by razor blade serial sectioning technique revealed few findings in all groups including control. Statistical analysis of litter incidences revealed no statistical significance of any of the findings as compared to the control group. Therefore, these findings are not to be considered to be treatment related and spontaneous in nature.

Skeletal examination of the Alizarin red stained foetuses revealed a range of findings which were of a type or which occurred at an incidence generally comparable to or slightly lower or higher in the dose groups when compared to the control group. A statistically significant decrease in litter incidence for incomplete ossification of interparietal and parietal bone in the MD and HD group and spuraoccipital in MD group compared to the control group was considered to be incidental as frequencies were even less in numbers compared to controls. Therefore, these findings are not to be considered as treatment-related and solely spontaneous in nature .

A Statistically significantly higher litter incidence of 14th rudimentary rib (bilateral) in the LD group and ilium (bilateral) caudal offset in MD group was seen without dose dependency when compared to the control group and was not considered as an effect of the test item.

A statistically significantly higher litter incidence of incomplete ossification of 1st sternebra in HD, 4th sternebra in LD and HD, unossified 4th sternebra in HD and dumbbell shaped ossification of thoracic centrum in MD and HD were observed when compared to the control group. These sternebra findings and dumbbell shaped ossification of thoracic centrum are considered to be variations and considered not to be adverse.

Dose Formulation Analysis

Formulation analysis for concentration verification and homogeneity was performed on collected samples at various intervals during the study. Nominal concentrations were confirmed for all dose groups, as measured concentration did not differ from nominal concentration by more than 20 %. All samples were homogenous, as COV was below or equal to 20 %.

Conclusion

On the basis of this prenatal developmental toxicity study in Wistar pregnant female rats with Provichem 0216 at dose levels of 62.5, 125, and 200/250 mg/kg body weight day administered on gestation days 5 to 19, the following conclusions can be made:

At a dose level of 250/200 mg/kg bw/day seven animals were sacrificed for welfare reasons and mortality was observed in one animal and clinical symptoms like marked loss of body weight, slight to severe piloerection, reduced spontaneous activity, wasp waist, abnormal breathing and moving the bedding were observed. Animals treated with 250 to 200 mg/kg bw/day were noted with a statistically significantly reduced body weight gain and food consumption.

No effect on prenatal data, litter data, foetal external, visceral, skeletal and craniofacial parameters were observed up to the highest dose level except decrease in mean foetal weight.

No effects of Provichem 0216 on females and foetuses were found at dose levels of 125 mg/kg body weight/day. The NOAEL for both maternal toxicity and fetal toxicity of Provichem 0216 in this study is considered to be 125 mg/kg body weight/day.