Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

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.

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 421 (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:
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
- Purity test date: 2011-06-24
- Lot/batch No.:201106140020
- Expiration date of the lot/batch: shelf life 2 years; production date 14/06/2011
- Stability under test conditions: Daily prepared
- Storage condition of test material: in a tightly closed container in dry, cool and well ventilated area in utility room between -20 and 40°C

Test animals

Species:
rat
Strain:
other: Wistar (Crl:WI)
Sex:
male/female
Details on test animals 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

Administration / exposure

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
Doses / concentrations
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).

Examinations

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.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

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
Gross pathological findings:
no effects observed
Description (incidence and severity):
≤125 mg/kg bw.
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:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
No effects

Details on results (P0)

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.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female

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 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)

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female

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 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.