Diphenyl sulphone

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

one-generation reproductive toxicity

Remarks:

based on generations indicated in Effect levels (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed under GLP and according to valid methods and is therefore considered reliable, relevant and adequate.

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Wistar (Crl:WI)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Animal house facility of Department of Toxicology, IIBAT, Padappai – 601 301, India.
- Age at study initiation: between 12 and 14 weeks old
- Weight at study initiation: Males: 317 – 358g; Females: 229 – 243g. The weight variation in animals involved in the study was not exceeded ± 20% of the mean weight.
- Fasting period before study:
- Housing: Females were housed in groups in cages, each cage containing 5 animals during pre mating period. Males were housed individually during pre mating and post mating. During mating one male and one female were kept together in a cage until the confirmation of mating. After confirmation of mating females were caged individually. Standard polypropylene rat cages with stainless steel top grill supplied by M/s. Vishnu Traders, UP, India were used to house the animals. For mating, cages with additional bottom grill were used. Gamma irrariated corn cobs supplied by M/s. Ceutics Pharma Pvt. Ltd., Bangalore, India were used as the bedding material. During mating an absorbent paper was laid below the bottom grill.
- Diet (e.g. ad libitum): standard gamma irradiated pelleted feed supplied by M/s. Tetragon Chemie Pvt. Ltd., Bangalore, India, ad libitum
- Water (e.g. ad libitum): reverse osmosis water ad libitum
- Acclimation period: 5 days prior to test

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6-23.4°C
- Humidity (%): 57-64%
- Air changes (per hr): Not provided
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 03.12.2011 To: 21.01.2012

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance was powdered using Pestle and Mortar and the required concentration of the test substance was mixed with corn oil and then stirred in magnetic stirrer for 15 minutes prior to start of dosing and the required amount of the preparation was administered to the rats at the desired dose level. The preparation was stirred continuously in the magnetic stirrer until dosing accomplish.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: until the confirmation of mating, until pregnancy occurs
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.: not applicable (succesful mating)
- Further matings after two unsuccessful attempts: not applicable (succesful mating)
- After successful mating each pregnant female was caged (how): individually

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Dosing of both sexes began 2 weeks prior to mating, after acclimatization. Dosing was continued in both sexes during the mating period. Males were further dosed after the mating until the dosing period of 28 days has been completed and the sacrificed.
Dosing of mating confirmed females was continued throughout gestation until day 3 post partum.
All dams were allowed to litter naturally and the size, weight of litter and sex of litter-mates were recorded at parturition (day 0) and at day 4 post partum.
Dams with offspring were sacrificed day 4 post partum.

Frequency of treatment:

Daily single dose by gavage

Remarks:

Doses / Concentrations:
62.5, 125 and 250 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

Range finding: 3
Main study: 10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Range Finding Study: Prior to the start of main experiment, a range finding study was carried out using one control and three treated groups with 3 males and 3 females in each group. 250, 500 and 1000 mg/kg bw of the test substance was daily administered orally for 7 days and observed for mortality and signs of toxicity daily. Control group animals were treated similarly, but with corn oil alone. The animals in G3 (500 mg/kg bw) and G4 (1000mg/kg bw) showed clinical signs viz. dullness, polyuria, salivation and piloerection onset being day 2 and day 1 respectively in both the sexes. In G3 one female died on day 5 while one male and one female died on day 6. As well three females in G4 were found dead on day 4 and two males on day 6. Macroscopically one male and one female of the high dose group as well as one female of the intermediate group showed reddish discoloration in stomach mucosa. Based on the results of the range finding study, 62.5 mg/ kg bw (low dose), 125 mg/kg bw (intermediate dose) and 250 mg/kg bw (high dose) were selected for the main study.
- Rationale for animal assignment (if not random): Randomization procedure involved assigning serial numbers to animals, generating random numbers from scientific calculator, ranking random numbers and assigning the animals to the groups as per IIBAT SOP (SOP/TOX/001).

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes : changes in skin, fur, eyes and mucous membranes, occurrence of secretions, excretions and autonomic activity- Time schedule: 2x/day for morbidity/mortality; 1x/day for toxicity signs, preferably after dosing in morning; 1x/day general observation.


DETAILED CLINICAL OBSERVATIONS: Yes : changes in gait, mobility, posture, presence of clonic or tonic movements, stereotypes and bizarre behavior.
- Time schedule: 1x/day

BODY WEIGHT: Yes
- Time schedule for examinations: All animals: prior to administration (day 0) and weekly thereafter; Females: during pregnancy on day 0, 7, 14 and 20 of pregnancy and within 24 hours of parturition (day 0 or 1 post-partum) and day 4 post-partum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):Yes
Feed consumption was recorded daily during pre-mating (cage wise), pregnancy and lactation in females. The feed consumption was not recorded during mating period. In males, feed consumption was recorded daily only during pre-mating.
- Food consumption for each animal determined as g food/day: Yes

Sperm parameters (parental animals):

Parameters examined in all male parental generations:
testis weight, epididymis weight
testis and epididymis histopathology with special emphais on stages of spermatogenesis and histopathhology of interstitial testicular cell structure

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, runts, presence of gross anomalies, weight gain (weighed within 24 hours of parturition day 0 post partum) and day 4 post partum), physical or behavioural abnormalities, other: sex ratio (m/f)

GROSS EXAMINATION OF DEAD PUPS:
No. of pups ; possible cause of death was not determined for pups born or found dead.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals 28 days after mating
- Maternal animals: All surviving animals at day 4 post partem

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including recording the numbers of implantation sites and corpora lutea. Special attention was paid to the organs of the reproductive system.

HISTOPATHOLOGY / ORGAN WEIGHTS
The ovaries, testes and epididymis, were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at 4 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external examinations

HISTOPATHOLOGY / ORGAN WEIGTHS
Not performed

Statistics:

Body weight, feed consumption and organ weight, corpora lutea, implantations, litter data of rats belonging to the experimental groups were assured for homogeneity. When the data is homogeneous then it was analysed using ANOVA. (Student’s Newman – Keul’s Test was employed for post-hoc comparison). When the data is not homogeneous it was analysed with Kruskal-Wallis One-Way ANOVA on rank basis.

Reproductive indices:

Pre-implantation loss (corpora lutea minus implantations), Pre-natal / Post-implantation loss (implantations minus live birth), and Post-natal loss (live births minus alive at post-natal day 4) were calculated.

Offspring viability indices:

Each litter was examined as earliest after delivery to establish the numbers and sex of pups, still births, live births, runts and the presence of gross abnormalities. Live pups were counted and sexed, litters were weighed within 24 hours of parturition (day 0 post partum) and day 4 post partum.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs viz. polyuria, dullness, piloerection and lacrimation were observed in animals of both sexes of high dose at various intervals after 7 days of dosing andcontinued till the end of the experiment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Statistical significant decreases in body weight were observed in males of high dose group from second. week till termination, while in female of high dose group during first week till the termination, except week 5 i.e. week 2 of gestation.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Statistical significant decreases in body weight were observed in males of high dose group from second. week till termination, while in female of high dose group during first week till the termination, except week 5 i.e. week 2 of gestation.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Test substance intake: gavage

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

No effects

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Clinical signs (polyuria, dullness, piloerection and lacrimation) were observed in animals of both sexes of high dose at various intervals after 7 days of dosing and continued till the end of the experiment.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Statistical significant decreases in body weight were observed in males of high dose group from second week till termination, while in female high dose group during first week till the termination, except week 5 i.e. week 2 of gestation. (Table 1, 2, 3, 10, 11 and 12)
Feed consumption was decreased significantly during the second week of pre mating in males of high dose group. (Table 4, 5, 13 and 14)

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS): oral gavage (actual doses)

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- Mating period of females in treated groups was 2.2 ± 1.14 days (mean±SD) in G2, 2.3 ± 0.95 days (mean±SD) in G3 and 2.9± 0.99 days (mean±SD) in G4 which was comparable with the mating period in control group animals (2.4 ±1.17 days).
- Gestation length of in the treated groups (G2, G3 and G4) was 21- 24 days which was considered normal and comparable with gestation length in control group animals (21-23 days).
- Implantations: When compared to control group females there was no difference in the mean implantations in any ofthe treated groups (G2, G3 and G4). The mean implantations in G1, G2, G3 and G4 were 12.90, 12.60, 12.20 and 12.30 respectively.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No statistical significant changes were observed in both absolute and relative organ weights in any of the treatment groups (G2,G3 and G4) when compared with control group (G1).

GROSS PATHOLOGY (PARENTAL ANIMALS)
No test substance related gross pathological observations were observed in any of the treated ( G2, G3 & G4) group both in male and female rats, except two females of high dose showed blackish and reddish discoloration in stomach respectively, where as all other macroscopic findings were either related to agonal, spontaneous, and incidental or of the type routinely observed in Wistar rats of this age.

HISTOPATHOLOGY (PARENTAL ANIMALS)
No test substance related histopathological findings were observed in any of the treated groups. All microscopic findings were either related to agonal, spontaneous, and incidental or of the type routinely observed in Wistar rats of this age.

Dose descriptor:

NOAEL

Effect level:

125 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

VIABILITY (OFFSPRING)
No test substance related effect was observed on pre implantation, post implantation and post natal loss in any of the treated groups (G2,G3 and G4) when compared with the control group (G1) of animals.
- Mean Litter Size: Test substance related effect was not observed on mean litter size in any of the treated group (G2, G3 and G4) of animals when compared to control group (G1) of animals at day 0 and day 4 post partum. The mean litter size was 12.40, 12.30, 11.60 and 11.90 on post partum day 0 and 12.00, 12.10, 11.30 and 11.40 on post partum day 4 respectively in G1, G2, G3 and G4.
- Mean Litter Weight: Test substance related effect was not observed in mean litter weight on day 0 and day 4 post partum in any ofthe treated groups (G2, G3 and G4) when compared with control group (G1) of animais. The mean litter weight was 77.82 g, 75.53 g, 71.60 g and 75.31g on post partum day 0 and 108.93 g, 110.18 g, 102.92 g and 108.08 g on post partum day 4 respectively in G1, G2, G3 and G4.
- Dam with Live Pups: No test substance related effect was observed on the number of dams delivered with live pups in any of the treated groups (G2, G3 and G4) when compared to control group.
- Loss of Offspring: No test substance related effect was observed on pre implantation, post implantation and postnatal loss in any ofthe treated groups (G2, G3 and G4) when compared with the control group (Gl) ofanimals.
- Sex ratio of the pups in any of the treated groups (G2, G3 and G4) was not affected when compared with the control group (G1) of animals.

CLINICAL SIGNS (OFFSPRING)
No abnormal behavior of the offspring was recorded.

BODY WEIGHT (OFFSPRING)
Test substance related effect was not observed in mean litter weight on day 0 and day 4 post partum in any of the treated groups (G2, G3 and G4) when compared with control group (G1) of animals. The mean litter weight was 77.82g, 75.53g, 71.60g and 75.31g on post partum day 0 and 108.93g, 110.18g, 102.92g and 108.08g on post partum day 4 respectively in G1, G2, G3 and G4. (Table 7, 8 and 17)

GROSS PATHOLOGY (OFFSPRING)
Gross external examination of live pups sacrificed on day 4 post-partum did not reveal any abnormality that could be attributed to the treatment. ( Table 8 and 25)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

125 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Reproductive effects observed:

not specified

Conclusions:

Based on the above findings it can be concluded that the dose 125 mg/kg bw of Diphenyl Sulphone (Provichem 0216) is non-toxic to Wistar rats with respect to reproduction/developmental toxicity screening test, therefore the NOAEL of the test substance is regarded as 125 mg/kg body weight.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

250 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

High quality (Klimish 1)

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A reproduction toxicity screening study was carried out to evaluate Provichem 0216 for its effect on reproduction and developmental toxicity in Wistar rats (IIBAT, 2012). Eighty rats were randomly distributed in to four groups with 10 animals/group/sex. The test substance was administered daily orally at doses of 0 (control), 62.5, 125 and 250 mg/ kg bw by suspending in corn oil (after powdering with mortar and pestle) to rats belonging to G2 (low), G3 (intermediate) and G4 (high) respectively in males till 28 day (pre mating 14 days and mating 14 days) and in females during premating, mating, gestation and up to post partum day 3. Control group were treated with corn oil alone. All dams were allowed to litter naturally and the size and weight of litters were recorded within 24 hours of parturition (day 0) and on day 4 post partum.

 

Body weight of individual male and female rats were recorded individually in males and cage wise in females, while during gestation and post partum period in the females it was recorded individually. All animals were observed once daily for mortality/morbidity and clinical signs of toxicity.

 

There was no morbidity/mortality in any of the treated groups in males and females throughout the experiment. Clinical signs like polyuria, dullness, piloerection and lacrimations were observed in both the sexes of high dose group. Test substance related decrease in body weight of males was observed after second week till the termination and first week till termination in females of high dose group, while decrease in feed consumption of males from high dose group was observed during second week of pre mating.

 

No test substance related effects were observed on reproduction/developmental performance like fertility, mating period, gestation length, mean litter size, mean litter weight, sex ratio of pups, pre implantation loss, post implantation loss, post natal loss and external malformations in pups in any of the treated groups. Gross pathology findings were unremarkable except discoloration of stomach in two out of ten in high dose females. Also, no test substance related histopathological findings were observed in testis, epididymis and ovaries in treated groups. Weight of the testis and epididymis from all treated groups were not affected when compared with control group.

 

From the study, it is concluded that doses up to 125 mg/kg bw of Provichem 0216 was considered to be non-toxic and safe to sires and dams, therefore the NOAEL of the test substance was arrived at 125 mg/kg bw (maternal and paternal NOAEL), however the reproductive NOAEL can be considered to be 250 mg/kg bw.

Short description of key information:
A reproduction toxicity screening study was carried out in Wistar rats at the doses of 0, 62.5, 125 and 250 mg/ kg bw. NOAEL for general toxicity of the test substance in sires and dams was 125 mg/kg bw, whereas reproductive NOAEL was 250 mg/kg bw.

Justification for selection of Effect on fertility via oral route:
Key reproductive and developmental toxicity screening study

Effects on developmental toxicity

Description of key information

A reproduction toxicity screening study was carried out in Wistar rats at the doses of 0, 62.5, 125 and 250 mg/ kg bw. NOAEL for general toxicity of the test substance in sires and dams  was 125 mg/kg bw, whereas developmental NOAEL was 250 mg/kg bw. 
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 and dumbbell shaped ossification of thoracic centrum.
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 .

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Wistar: Crl: WI(Han) (Full Barrier)

Details on test animals or test system 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)

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.

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.

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).

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.

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

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.

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

Abnormalities:

not specified

Developmental effects observed:

not specified

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.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

125 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

High quality (Klimish 1)

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Screening study

A reproduction toxicity screening study was carried out to evaluate Provichem 0216 for its effect on reproduction and developmental toxicity in Wistar rats (IIBAT, 2012). Eighty rats were randomly distributed in to four groups with 10 animals/group/sex. The test substance was administered daily orally at doses of 0 (control), 62.5, 125 and 250 mg/ kg bw by suspending in corn oil (after powdering with mortar and pestle) to rats belonging to G2 (low), G3 (intermediate) and G4 (high) respectively in males till 28 day (pre mating 14 days and mating 14 days) and in females during premating, mating, gestation and up to post partum day 3. Control group were treated with corn oil alone. All dams were allowed to litter naturally and the size and weight of litters were recorded within 24 hours of parturition (day 0) and on day 4 post partum.

Body weight of individual male and female rats were recorded individually in males and cage wise in females, while during gestation and post partum period in the females it was recorded individually. All animals were observed once daily for mortality/morbidity and clinical signs of toxicity.

There was no morbidity/mortality in any of the treated groups in males and females throughout the experiment. Clinical signs like polyuria, dullness, piloerection and lacrimations were observed in both the sexes of high dose group. Test substance related decrease in body weight of males was observed after second week till the termination and first week till termination in females of high dose group, while decrease in feed consumption of males from high dose group was observed during second week of pre mating.

No test substance related effects were observed on reproduction/developmental performance like fertility, mating period, gestation length, mean litter size, mean litter weight, sex ratio of pups, pre implantation loss, post implantation loss, post natal loss and external malformations in pups in any of the treated groups. Gross pathology findings were unremarkable except discoloration of stomach in two out of ten in high dose females. Also, no test substance related histopathological findings were observed in testis, epididymis and ovaries in treated groups. Weight of the testis and epididymis from all treated groups were not affected when compared with control group.

From the study, it is concluded that doses up to 125 mg/kg bw of Provichem 0216 was considered to be non-toxic and safe to sires and dams, therefore the NOAEL of the test substance was arrived at 125 mg/kg bw (maternal and paternal NOAEL), however the developmental NOAEL can be considered to be 250 mg/kg bw.

Prenatal development toxicity

As the screening study was only supportive, a test for a prenatal developmental study in rats according to OECD 414 was proposed; the study was started and is still ongoing (BSL BIOSERVICE, 2015b). A proof of status is given by the Laboratory (attached in the endpoint record ) and an update with full results and adapted CSR will be provided by Mid February 2016.

A dose-range findings study was conducted as dose range finding and supporting study with Provichem 0216 (BSL BIOSERVICE, 2015a). Three groups of 8 female Wistar rats were dosed by oral gavage at 0 (corn oil as vehicle), 200 and (400 reduced to) 300 mg/kg bw/day during gestation days 5 to 19. All 8 females from the high dose group were treated with a dose of 400 mg/kg bw/day from the start of the study, however due to excessive toxicity this dose was reduced to 300 mg/kg body weight/day. On the basis of this prenatal developmental toxicity study in pregnant female Wistar rats with Provichem 0216 at dose levels of 200 and 300 or 400 mg/kg bw/day administered on gestation days 5 to 19, the following conclusions can be made. Test item-related toxicological effects in terms of clinical signs, morbidity/mortality, body weight and food consumption reduction were observed in the low and high dose group pregnant females when compared with the controls. A significantly lower maternal terminal body weight, gravid uterus weight, adjusted maternal weight and slightly higher number of early resorptions and preimplantation loss was observed in the treatment groups when compared to the controls. Mean foetal weight, male foetal weight and female foetal weight (both on litter and individual basis) and total litter weights, male and female litter weights (litter basis) were marginally lower in treatment groups when compared with the controls. No effect on foetal external and female gross pathological observations was observed up to the highest dose level tested. Based on the data generated from this dose range finding study, dose levels of 62.5, 125 and 250 mg/kg bw/day were proposed for the subsequent main prenatal developmental toxicity study with Provichem 0216.

In the main Key prenatal development toxicity study with Provichem 0216, pregnat female Wistar rats were dosed via oral gavage administration during gestation days 5 to 19 at doses of 0 - 62.5 - 125 - 250/200mg/kg body weight/day. In the high dose group, 6 females were treated with 250 mg/kg body weight/day from the start of the study for several treatment days, but after marked reduction of the body weight, food intake and test item related clinical signs, the high dose was reduced to 200 mg/kg body weight/day. Females no. 82-104 of the high dose group were treated with a dose of 200 mg/kg body weight/day for the whole treatment period.

Maternal Findings: Seven females (no. 77-80, 95-96 and 101) of the high dose 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 low and mid dose groups when compared to the control group. The clinical signs of localised alopecia on various body parts, slight to severe piloerection, salivation, moving the bedding were noted during gestation day 6-19 in several other females of the the high 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 low adn mid dose 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 high dose group was statistically significantly lower compared to the control group from gestation day 8 onwards. Body weight gain was also lower in the high dose 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 the mid and high dose group and on day 17-20 and 0-20 in high dose group when compared with the controls.

In correlation to the body weight and body weight gain, food consumption in the high dose 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 in the high dose group as well as on several treatment intervals in high dose ( 5-8, 8-11, 11-14 and 17-20) and during gestation day 5-8 in low and mid dose groups 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 low and mid dose group at necropsy.

At necropsy of female no. 77 of the high dose 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 high dose 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 high dose group when comparared with the controls, which 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 mid and high dpose group and spuraoccipital in mid dose 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 low dose group and ilium (bilateral) caudal offset in mid dose 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 high dose, 4th sternebra in low and high dose, unossified 4th sternebra in high dose and dumbbell shaped ossification of thoracic centrum in mid and high dose 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: 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.

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 .

Justification for selection of Effect on developmental toxicity: via oral route:
Main prenatal developmental toxicity study

Justification for classification or non-classification

As there were no reproductive or developmental toxicity findings, classification is not warranted according to the EC criteria for classification and labeling requirements for dangerous substances and preparations (Guidelines in Commission Directive 93/21/EEC) and CLP regulation (EC No. 1272/2008 of 16 December 2008).