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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Proviplast 0142; Bis(2-Butoxyethyl) adipate
- Substance type: organic monoconstituent
- Physical state: Clear liquid
- Analytical purity: 98.83%
- Lot/batch No.: 201012140024 ( Sponsor Ref.: 11701913 SA )
- Expiration date of the lot/batch: shelf life 2 years
- Stability under test conditions: Test substance was prepared freshly daily
- Storage condition of test material: the material was stored in tightly close container in a dry, cool and well-ventilated area in the utility room.

Test animals

Species:
rat
Strain:
other: Wistar (Crl:WI)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Procured from Charles River, USA and bred at IIBAT animal house facility.
- Age at study initiation: between 12 and 14 weeks old
- Weight at study initiation: Males: 247-310 g; Females: 208-280 g. The weight variation in animals involved in the study was not exceeded 20 % of the mean weight of each sex
- Fasting period before study: not applicable
- Housing: Females were housed in groups in cages, each cage containing five animals during pre mating period. Males were housed individually during pre mating and post 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 was used to house the animals. For mating, cages with additional bottom grill were used. Gamma irradiated corn cobs supplied by M/s. Ceutics Pharma Pvt. Ltd., Bangalore, Indiawas used as the bedding material. During mating an absorbent paper were laid below bottom grill.
- Diet (e.g. ad libitum): standard gamma irradiated pelleted food supplied by M/s. Tetragon Chemie Pvt. Ltd., Bangalore, India. , ad libitum
- Water (e.g. ad libitum): Reverse osmosis water was provided, ad libitum
- Acclimation period: Six days prior to experiment in the test room.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): between 19.5 and 21.8°C
- Humidity (%): between 55 and 64%
- Air changes (per hr): at least 12 air changes per hour
- Photoperiod (hrs dark / hrs light): 12h light/12h dark

IN-LIFE DATES:
Dose finding study: From 02.02.2012 To 09.02.2012
Main Study:From: 21.02.2012 To: 17.04.2012

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: glycerol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance was mixed with glycerol and thereafter administered to group of rats at the desired dose level. Control group received glycerol alone. The dose volume was maintained at 10 ml/kg b.w. Test substance was prepared freshly daily.
Details on mating procedure:
Females were housed in groups in cages, each cage containing five animals during pre mating period. Males were housed individually during pre mating and post 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.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Dosing of both the sexes began 2 weeks prior to mating, after acclimatization. Dosing was continued for two weeks in both sexes during the mating period. Males were further dosed after the mating period until the minimum dosing period of 28 days has been completed and then sacrificed.
Dosing of mating confirmed females was continued throughout gestation and up to including 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 on day 4 post partum.
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 200 and 400mg/kg b.w.
Basis:
actual ingested
No. of animals per sex per dose:
dose finding study: 3 females + 3 males/dose
main study:
control (glycerol): 10 males + 10 females
low (100 mg/kg b.w.): 10 males + 10 females
intermediate (200 mg/kg b.w.): 10 males + 10 females
high (400 mg/kg b.w.): 10 males + 10 females

Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: range finding study: prior to the start; using one control and three dose groups (500, 1000 and 2000 mg/kg b.w.) of the test substance with 3 males and 3 females of each. Test substance was administrered orally deily for 7 days and observed for morbidity/mortality and signs of toxicity daily. Control groups animals were treated similarity but with glycerol alone. Test substance related, signs of toxicity like dullness, respiratory distress were observed in intermediate (1000 mg/kg b.w.) and high (2000 mg/kg b.w.) dose animals. whereas dullness was observed in low (500 mg/kg b.w.) dose animals. All animals in high dose died before terminal sacrifice from 5th day of dosing. Macroscopically, high dose males (2/3) and females (1/3) showed reddish discoloration in stomach. Based on the results of range finding study, three doses i.e., 100 mg/kg b.w. (Low dose), 200(intermediate) and 400mg/kg b.w. (High dose) were selected for the main study.
- Rationale for animal assignment: 80 animals were randomized into four groups, each consisting 10 animals/sex. Randomization procedure involved assigning serial numbers to animals, generating random numbers from scientific calculator, ranking random numbers and assigning the naimals to the groups as per IIBAT SOP/TOX/001. Each group was sub grouped and sufficed as 'a' and 'b' by randomly selecting five animals/sex from the same group at the end of pre-mating period (befor blood collection).

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes,
- Check for morbidity/mortality
- 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 toxcity signs, preferably after dosing in morning; 1x/day general observation
- Cage side observations were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Changes in skin, fur, eyes, and mucous membranes, occurrence of secretions, excretions and autonomic activity (e.g. lacrimation, piloerection, palpebral closure, palpebral reflex, pupil light response and unusual respiratory pattern). Changes in gait, mobility, arousal, rearing, posture, vocalizations, activity levels and response to handling, approach response, touch response, click response, tail pinch response, toe pinch as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling), bizarre behavior (e.g. self-mutilation, walking backwards). Pertinent behavioral changes, signs of difficult or prolonged parturition and all signs of toxicity, including mortality.
- Time schedule: 1x/week

BODY WEIGHT: Yes
- Time schedule for examinations:
males: PMD (post mating day) 0, 7, MD (mating day) 0, 7 and 13.
females: PMD 0, 7, MD 0, 7 and 14, PD (pregnancy days) 0, 7, 14 and 20, within 24 hrs of parturition (PPD0/1) and on PPD 4.
- Body weight on MD 7 and/or 14 for one female in intermediate and six females in high dose group were recorded for adjusting dose volume but not reported as unavailbility of camparative values from control and low doses.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day.
- Feed consumption was recorded daily during pre-mating, gestation and during lactation till post partum day 4 in females. The feed consumption was not recorded during mating period. In males, feed consumption was recorded only during pre-mating.

OTHER:
- Haematology: see Section 7.5.1
- Clinical chemistry: see Section 7.5.1
- Neurobehavioural examination: see Section 7.5.1

Sperm parameters (parental animals):
Parameters examined in all male parental generations:
testis weight, epididymis weight
testis and epidymis histopathology with special emphasis on stages of spermatofgenesis and histopathology 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: 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. Sex ratio (m/f). Live pups were counted and sexed. Litters were weighed within 24 hours of parturition {post partum day (PPD) 0 or 1} and PPD 4.

Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All surviving animals at day 4 post-partum

GROSS NECROPSY
- Gross necropsy consisted of macroscopically examination for any abnormalities, number of implantation sites and corpora lutea was recorded. Special attention was paid to the organs of the reproductive system.
- For other organs: see Section 7.5.1

HISTOPATHOLOGY / ORGAN WEIGHTS
- The ovaries, testes, epididymis, accessory sex organs were prepared for macroscopic examination and weighed.
- For other organs: see Section 7.5.1
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring are sacrificed at day 4 post-partum
- These animals were subjected to postmortem examinations (macroscopic examination) as follows: All fetuses were examined for external malformation during necropsy.

GROSS NECROPSY
- Gross necropsy consisted of external examinations

HISTOPATHOLOGY / ORGAN WEIGTHS
not performed
Statistics:
Body weight, food consumption, detail signs of toxicity, FOB, hematology, biochemistry and organ weight, corpora lutea, implantations, litter data of rats belonging to the experimental groups 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:
Females showing evidence of copulation
Mating period
Females achieving pregnancy
Gestation length
Dams with live young born
Dams with live young at day 4 pp
Corpora lutea/dam (mean)
Implants/dam (mean)
Number of pups born
pre-implantation loss
post-implantation loss
Offspring viability indices:
Each litter was examined at the earliest after delivery to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted and sexed. Litters were weighed within 24 hours of parturition {post partum day (PPD) 0 or 1} and PPD 4.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
dullness, piloerection, respiratory distress in 400 mg/kg dosed males; three females showing dullness at 400 mg/kg bw
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
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 / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
No morbidity/mortality was observed in any of the animals during the entire observation period. In high dose (G4-400 mg/kg b.w.) males’ dullness, piloerection, respiratory distress, signs of toxicity were observed whereas there were no clinical signs observed in any other group animals during the observation period except few males in intermediate group showing dullness during second and third week which subsequently recovered. None of the females in any of treated groups exhibited clinical signs except 3 females in high dose showing dullness for transient period which recovered subsequently.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS):
Statistical significant changes in body weight was not observed in males and females of any of treated group. No test related effect was observed in feed consumption in treated groups in males and females when compared with control group (G1) animals. Statistical differences observed randomly in both males of G2 and G3 group and females of G2, G3, and G4 group. These changes considered to be biological variations commonly occur in this parameter.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Histopathology evaluation of testis was done with special emphasis on stages of spermatogenesis and interstitial testicular cell structure, however not any test subsrance related effects were observed in any of trealed groups.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS):
Mating: Increase (≥ 6 days) in mating period in six females of high dose groups (G4) was observed as compared to the mating period of control group (G1) females. This change may have been caused among others due to clinical conditions of males of high dose group. These females showed pregnancy after re-pairing with proven males of same group. Hence, increase in mating period in high dose group was considered to be non-adverse findings and secondary to any unknown intrinsic factors. One female from intermediate group found mated only on day 14th; this was not attributed to test substance.
Gestation: Gestation length in females of treated groups (G2, G3 & G4) was comparable with gestation length of the control group (G1) females
Implantations: No test substance related changes were observed on mean Corpora lutea and Implantations in any of treated group when compared with the control group (G1) animals

ORGAN WEIGHTS (PARENTAL ANIMALS):
Statistical significant decrease was observed in relative organ weights of spleen of high dose group males when compared with control group (G1) males. The effect was unisex with lack of correlating gross and histopathology.
Test substance related statistical differences were not observed in mean weight of any of the other organs including testis and epididymis of treated groups when wompared to control group animals, however, in high dose, decrease in individual absolute/relative testis and epididymis weight was observed in 2/10 males.

GROSS PATHOLOGY (PARENTAL ANIMALS):
No test substance related gross pathological observations were observed in any of treated groups however, two males were observed with small size testis and epididymis in high dose group. All 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 treated group however, two males in high dose group showed tubular atrophy in testis and corresponding oligospermia in epididymis. Correlating gross observations/low organ weight was recorded in thes two males. Histopathology evaluation of testis was done with special emphasis on stage of spermatogenesis and interstitial testicular cell structure, however no test substance related effect were observed in any of treated group. All other microscopic findings were eiter related to agonal, spontaneous, and incidental or of the type routinely observed in Wistar rats of the age


OTHER FINDINGS (PARENTAL ANIMALS)
See Section 7.5.1

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: systemic toxicity

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

VIABILITY (OFFSPRING):
-No test substance related changes was observed on mean litter size in any of treated group when compared with the control group animals (G1) at day 0 and day 4 post partum
-No test substance related effect was observed on the number of dams delivered with live pups in any of the treated groups (G1, G2, G3 & G4) of animals
-No loss of offspring (pre implantation, post implantation and post natal) in any of the treated groups (G1, G2, G3 & G4) was observed
-No test substance related effect was observed on sex ratio of the pups in any of the treated groups (G2, G3 & G4) when compared with the control group (G1) of animals

CLINICAL SIGNS (OFFSPRING):
- no abnormal behavior of the offspring was recorded.

BODY WEIGHT (OFFSPRING)
-No test substance related changes was observed on mean litter weight in any of treated group when compared with the control group animals (G1) at day 0 and day 4 post partum

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.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
400 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reproductive toxicity

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the above findings it can be concluded that the dose 400mg/kg b.w. of Proviplast 0142 probably toxic to the males in view of observed clinical signs, mainly dullnes in males and secondary effect on mating period therefore, the NOAEL of the test substance is considered as 200 mg/kg b.w. for repeated dose toxicity whereas NOAEL for reproduction was 400 mg/kg bw.
Executive summary:

A combined repeated dose and reproduction/developmental toxicity screening test in Wistar rats was performed according to OECD TG422 by oral gavage at dose levels of 100, 200 and 400 mg/kg b.w. in glycerol. Males were dosed for 14 days before and 14 days after mating (at least 28 days); females were dosing during premating, mating, gestation and up to day 3 post partum. The control group was treated similarly but with glycerol alone. No morbidity/mortality was observed in any of the treated groups in males and females throughout the experiment. Test substance related signs of toxicity were mainly dullness, along with piloerection, and respiratory distress in all males and dullness in three females of high dose (400mg/kg b.w.) were observed. In reproduction performance, a test substance related increase in mating period of high dose group females was observed, most probably secondary to the clinical signs especially in males. No test substance related effects were observed in parameters like body weight, feed consumption, FOB, hematology, biochemistry and fertility, gestation length, mean corpora lutea, mean implantations, mean litter size, implantation losses, mean litter weight and sex ratio of offspring. Not any adverse gross and histopathological observations were observed that could be attributed to test substance. Test subslance related organ weight changes were not observed in any of treated groups when compared with control group. External observations of pups did not reveal any test substance related findings. From the above observations, it can be concluded that 400 mg/kg b.w. of Proviplast 0142 is toxic to the parents with respect to observed clinical signs, mainly dullness, and secondary effect on mating period therefore, the NOAEL of the of Proviplast 0142 for repeated dose toxicity is considered as 200 mg/kg body weight, whereas the NOAEL of Proviplast 0142 for reproduction and developmental screening is considered as 400 mg/kg body weight.