N-(2-hydroxypropyl)benzenesulphonamide

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

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

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N-(2-hydroxypropyl)benzenesulphonamide

EC Number:

252-512-0

EC Name:

N-(2-hydroxypropyl)benzenesulphonamide

Cas Number:

35325-02-1

Molecular formula:

C9H13NO3S

IUPAC Name:

N-(2-hydroxypropyl)benzenesulphonamide

Test material form:

liquid: viscous

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system 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 02.08.2012 To 09.08-2012
Main Study:From: 21.08.2012 To: 16.10.2012

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance was mixed with corn oil and thereafter administered to group of rats at the desired dose level. Control group received corn oil 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 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.

Doses / concentrations

Remarks:

Doses/Concentrations: 150,300,600 mg/kg/B.W.

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 daily for 7 days and observed for morbidity/mortality and signs of toxicity daily. Control groups animals were treated similarity but with corn oil.
Test substance related, signs of toxicity like dullness, respiratory distress and discharge from nostril 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. 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., 150 mg/kg b.w. (Low dose), 300(intermediate) and 600mg/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

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):

In the high dose (600 mg/kg b.w) mals dullness, polyuria, respiration distress, piloerection, signs of toxicity were observed whereras there were no clinical signs observed inany other group males during the observation. None of the females in G1( control) G2( 150) G3(300) and G4(600 mg/kg b.w.) exhibited clinical signs and there was no abnormal behavior observed in any off springs.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Statistical significant decrease ijn body weight og males during day 14 to 28 were observed in high dose ( G4 600 mg/kg bw) when compaired to the control (G1). Infemales statistically significant decrease in the body weight was observed at gestation day 14 at high dose whencompaired with control group.Considering only marginal decrease only during 3rd week of gestation which disappeared subseguently and also ma&ck of dose dependejncy. effect on body weoght in female of high dose group could not attributed to test sunstance

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There was a statistically significant change in feed consumption premating day 11 and 12 in high dose males and on day 5 in intermediate dose males compared with control group. this was not attributed as test sibstance effect

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant increase was observed in biochemistry paeeametrs like ALT and AST in high dose (G4) males and females , and potassium in males of high dose group when compared with control. However the values are well within normal range of this species/historical data and no any correlating organ wieght, gross pathology and histopathological changes were observed expect only 2/5 females with hostopatological findings in liver

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

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:
No morbidity/mortality was observed in any of the animals during the entire observation period.
In high dos (G4-600mg/kg b.w.) males, signs of toxicity like dullness, polyuria,piloerection, respiratory distress were observed, whereas there were no clinical signs observed in any other group during the observation period. None of the females in G1, G2,G3 and G4 exhibited clinical signs and there was no abnormal behavior observed in any off springs.
BODY WEIGHT AND WEIGHT GAIN
Statistical significant decrease in body weight og males during day 14 to 28 were observed in high dose ( G4 600 mg/kg bw) when compared to the control (G1). In females statistically significant decrease in the body weight was observed at gestation day 14 at high dose when compared with control group. Considering only marginal decrease only during 3rd week of gestation which disappeared subsequently and also lack of dose dependency. effect on body weight in female of high dose group could not attributed to test substance.
FOOD CONSUMPTION
There was a statistically significant change in feed consumption premating day 11 and 12 in high dose males and on day 5 in intermediate dose males compared with control group. this was not attributed as test substance effect
HAEMATOLOGY:
No any test substance related statistically significant changes were observed in hematology parameters of G2, G3 and G4 dose groups when compared with control (G1) group, except a slight increase in large unstained cells of G2 female which is well within normal limits and considered having no biological/toxicological significance.

CLINICAL CHEMISTRY:
Statistically significant increase was observed in biochemistry parameters like ALT and AST in high dose (G4) males and females , and potassium in males of high dose group when compared with control. However the values are well within normal range of this species/historical data and no any correlating organ weight, gross pathology and histopathological changes were observed expect only 2/5 females with histopatological findings in liver
NEUROBEHAVIOUR:
No test substance related effects were observed in of males (G1, G2, G3 and G4)

ORGAN WEIGHTS:
Statistical significant decrease was observed in both absolute and relative organ weight of spleen of high dose group females when compared with control group. the change was observed in unisex without corresponding gross and histopathology observations hence decrease in spleen weight was not considered of test substance related.
GROSS PATHOLOGY:
Test substance related gross pathological observations like discoloration red in stomach was observed in high dose males .All macroscopic findings were either related to agonal, spontaneous, and incidental or of the type routinely observed in Wistar rats of this age.

HISTOPATHOLOGY:
No test substance related histopathological findings like marked ulceration ,acute inflammation mild to moderate hyperkeratosis, mild hyperplasia of squamous cell in stomach and hepatocellular hypertrophy in liver of high dose females were observed in high dose group .. All other microscopic findings were either related to agonal, spontaneous, and incidental or of the type routinely observed in Wistar rats of the age.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

3 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: observed clinical signs, decreased body weight, increased AST and ALT

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

Developmental neurotoxicity (F1)

Behaviour (functional 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 to 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 ( G2,G3,&G4) of animals.
-No loss of offspring( pre implantations, post implantations and natal) in any of the treated groups ( G2,G3,&G4) was observed.
- No test substance related effect was observed on sex ratio of the pups in ) in any of the treated groups ( G2,G3,&G4) was observed.
Clinical signs (offspring)
-No abnormal behavior of the offspring was recorded.
Body weight (offspring)
- No test substance related changes was observed on mean litter size in any of treated group when compared to control group animals(G1) at day 0 and day 4 post partum.
Gross pathology (offspring)
Gross external examination of the 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:

ca. 600 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects seen in the high dose

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

Based on the above findings it can be concluded that the dose 600 mg/kg b.w. of N-(2-hydroxypropyl)benzene sulfonamide is probably toxic to the males in view of observed clinical signs, mainly decreased body weights, increased AST and ALT levels, gross histopathological observation in stomach and to females with respect to observed increased AST and ALT levels along with histooathological observation in liver therefore, the NOAEL of the test substance is considered as 300 mg/kg b.w. for repeated dose toxicity whereas NOAEL of N-(2-hydroxypropyl)benzene sulfonamide for reproduction/Development is considered as 600 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 150, 300 and 600 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 corn oil alone. No morbidity/mortality was observed in any of the treated groups in males and females throughout the experiment. Test substance related effects were observed on parents from the high dose groups like signs of toxicity were observed as dullness, polyuria ,along with piloerection, and respiratory distress, decrease in body weight of males of high dose group and in biochemistry parameters increase in levels of AST and ALT mean values of males and females of the high dose group. No test substance related effects were observed in parameters like feed consumption, FOB, hematology and fertility, mating period, gestation length, mean corpora lutea, mean implantation, mean litter size, mean litter/pup weight, implantation losses, and sex ratio of offspring From the above observations, it can be concluded that 600 mg/kg b.w. of Proviplast 0142 is toxic to the parents with respect to observed clinical signs, mainly dullness, polyuria ,along with piloerection, and respiratory distress, decrease in body weight of males of high dose group and in biochemistry parameters increase in levels of AST and ALT mean values of males and females of the high dose group. Therefore, the NOAEL of the of N-(2-hydroxypropyl) benzene sulfonamide for repeated dose toxicity is considered as 300 mg/kg body weight, whereas the NOAEL of N-(2-hydroxypropyl) benzene sulfonamide for reproduction and developmental screening is considered as 600 mg/kg body weight.