Registration Dossier

Diss Factsheets

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
N-butylbenzenesulphonamide
EC Number:
222-823-6
EC Name:
N-butylbenzenesulphonamide
Cas Number:
3622-84-2
Molecular formula:
C10H15NO2S
IUPAC Name:
N-butylbenzenesulfonamide
Details on test material:
- Name of test material (as cited in study report): N-n-butyl benzenesulphonamide (BBSA)
- Substance type: mono constituent substance-organic
- Physical state: clear liquid
- Analytical purity: 99.88%
- Impurities (identity and concentrations): 0.1% Diphenylsulphon/0.1% water/0.1% Butylbenzeensulphate/0.02% Butylamine
- Composition of test material, percentage of components: 99.88% BBSA
- Isomers composition:
- Purity test date: 13/10/2006
- Lot/batch No.: 200610130015
- Expiration date of the lot/batch: 13/10/2008
- Stability under test conditions: stable
- Storage condition of test material: Dry, cool and well-ventilated place
- Other:

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: The Animal house facilities of Department if Toxicology, IIBAT
- Age at study initiation: (P) 12-15 wks; (F1) 0 wks:
- Weight at study initiation: (P) Males: 300-330 g; Females: 200-220
- Housing: Standard polypropylene rat cages with stainless steel top grill. Cleaned, sieved and autoclaved paddy husk was used as bedding material
During mating either sex in ratio of 1:1 were accommodated with special cages meant for mating of later pregnant rats were housed
individually.
- Use of restrainers for preventing ingestion (if dermal): no
- Diet (e.g. ad libitum): Standard rodent pellet feed. routinely analyzed; ad libitum
- Water (e.g. ad libitum): Reverse osmosis water. Routinely analyzed; ad libitum
- Acclimation period: 5 days prior to test in the test room
- Fasting: Feed alone was withdrawn over-night prior to sacrifice.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6 - 21.2
- Humidity (%): 59 - 64
- Photoperiod (12 hrs dark /12 hrs light):


IN-LIFE DATES: From: 02/04/2007 To: 30/05/2007

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:


DIET PREPARATION
- Rate of preparation of diet (frequency):daily
- Mixing appropriate amounts with (Type of food): standard rodent pellet feed
- Storage temperature of food:no data


VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Concentration in vehicle: no data
- Amount of vehicle (if gavage): no data
- Lot/batch no. (if required): no data
- Purity:no data
Details on mating procedure:
- M/F ratio per cage:1:1
- Length of cohabitation: no data
- Proof of pregnancy: vaginal plug / sperm in vaginal smear, referred to as day 0 of pregnancy
- After unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: [yes (explain)]: Until two weeks have elapsed.
- After successful mating each pregnant female was caged (how): no data
- Any other deviations from standard protocol:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
analyzed by Analytical Chemistry, IIBAT
Duration of treatment / exposure:
2 weeks prior to mating, after acclimatisation.
Dosing was continued in both sexes during the mating period.
Males were further dosed after the mating period until the min. dosing period of 28d has been completed.
Dosing of confirmed females was continued throughout the pregnancy and including, day 3 post partum.
Frequency of treatment:
Daily
Details on study schedule:
- F1 parental animals not mated until [...] weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were [...] days of age.
- Age at mating of the mated animals in the study: 12-15 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
0-100-200 and 400 mg/kg b.w.
Basis:
nominal conc.
No. of animals per sex per dose:
12 animals/group/sex
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: a range finding study
- Rationale for animal assignment (if not random): standard species used in reproduction/developmental toxicity studie
- Other:
Positive control:
Control group were treated similary but with corn oil alone. All dams were allowed to litter through the natural birth and the size, weight and sex of litters were recorded at parturition (day 0) and the day 4 of post partum.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: males: observed for 28 days (pre and post mating)
females: observed for 54 days (premating, mating, gestation, parturition and post partum)
- Cage side observations checked in table; yes.


DETAILED CLINICAL OBSERVATIONS: yes



BODY WEIGHT: Yes
- Time schedule for examinations: prior to the administration of the test substance (day 0) and weekly during the entire observation period and at
termination


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as mg food/kg body weight/day: yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: yes


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations:/


TOXICITY SIGNS: All groups of animals twice daily preferably after dosing in morning and later in the evening session.


GESTATION AND ONSET OF DELIVERY: The onset of labour, thelitter size, the sex-ratio and weight of pups at birth (day 0) and day 4 post partem
was recorded. Sex ratio (m/f) and live pups/dam were calculated.


MORTALITY/MORBIDITY: All animals were observed twice daily for mortality/morbidity, those found moribund/severly exhibiting toxic signs were
necropsied and recorded.
CAGE SIDE OBSERVATIONS: Yes / No / No data
- Time schedule:
- Cage side observations checked in table.yes


DETAILED CLINICAL OBSERVATIONS: Yes / No / No data
- Time schedule:


BODY WEIGHT: Yes / No / No data
- Time schedule for examinations:


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data
- Time schedule for examinations:


OTHER:
Oestrous cyclicity (parental animals):
no data
Sperm parameters (parental animals):
Parameters examined in P male parental generations:
[testis weight, epididymis weight, daily sperm production, sperm count in testes, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm motility, sperm morphology, other:]
Test substances related adverse histopahological findings were observed in testis and epididymides of male rats in high dose groups. In testis
these findings consisted of sertolicell vacuolation (8/12), spermatid retention (6/12), multinucleated giant cells (5/12), desquamation of germ cells
(4/12), absence of spermatids (2/12) and atrophy of testis (1/12).
Test substance related adverse findings in the epididymides were desquamated germ cells (7/12) and oligospermia (4/12). These effects were in correspondence to the effects observed in the testis.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: [yes/no]
- If yes, maximum of [...] pups/litter ([...]/sex/litter as nearly as possible); excess pups were killed and discarded.


PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups: Test substance related effect was observed on mean litter size in high dose group at day 0 and day 4 post partum. Mean litter size in high dose group was 2.50 followed by 8.33, 10.08 and 9.75 in intermediate, low an control groups at day 0, while it was 1.08, 7.92, 9.33 and 9.17 in high, intermediate, low and control group respectively at day 4 post partum. No test substance related effect was observed on sex ratio of the pups.
stillbirths: no data
live births: Test substance releated effect was observed on the number of dams delivered with live pups in high and intermediate dose group.
Only 50% females delivered with live pups in high dose group. In intermediate group 82.33% females delivered with live pups. 100% dams delivered with live pups in both control and low dose groups.
postnatal mortality:test substance related effect was observed on loss of offspring in high dose group. There was increased rate of loss of offspring in high dose group.
presence of gross anomalies: in high dose group 1/3 dams delivered abnormal pups. Anasarca in whole litter of dam.
weight gain: Test substance related effect was observed in mean litter weight on day 0 and day 4 post partum in high dose group. Mean litter weight in high dose group at day 0 was 11.6 followed by 46.7, 54.2 and 54.1 in intermediate, low and control groups respectively. Mean litter weight in high dose group day 4 post partum was 55.7 followed by 59.7, 72.0 and 70.6 in intermediate, low and control group respectively.
physical or behavioural abnormalities, no data
other:]


GROSS EXAMINATION OF DEAD PUPS:
yes , for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals [describe when, e.g. as soon as possible after the last litters in each generation were produced.]: males were sacrified after the mating period when until the minimum dosing period of 28 days has been completed.
- Maternal animals: All surviving animals [describe when, e.g. after the last litter of each generation was weaned.]: Dams with offspring were sacrified on day 4 post partum.


GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]: At the time of sacrifice or death during the study, the adult animals were examined macroscopically for any abnormalities or pathological changes. Special attention were paid to the organs of the reproductive system. All gross pathological changes are recorded individually for each animal. The number of implantation sites were recorded. The counting of corpora lutea were done. Dead pups and pups sacrified at day 4 post partum, or shortly thereafter, were carefully examined externally for gross abnormalities. The ovaries, epididymides, accessory sex organs and all organs showing macroscopic lesions of all adult animals were preserved in 10% neutral buffered formalin. Testes were preserved in Bouin's fixative.


HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.:
Weights of following organs of all male adult animals were recorded: testes and the epididymides
Testes were evaluated with special emphasis on stages of spermatogenesis in all dose groups. Seminal vesicle, prostate and ovaries were evaluated from control and high dose groups.
The following grading system was used for histopathological evaluation in the study,
1. minimal
2. mild
3. moderate
4. marked
5. severe
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 carefully examined externally for gross abnormalities


Statistics:
The body weight, feed consumption and organ weight data were statistically analysed using one way ANOVA.
Reproductive indices:
no data
Offspring viability indices:
Dam with live pups.
Loss of offspring
external abnormalities in pups

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
effects observed, treatment-related
Reproductive performance:
effects observed, treatment-related

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
In the high dose treated group, few animals exhibited abnormal gait, respiratory distress, tremor, hunched posture, dullness, salivation, piloerection and lethargy.
2 females were found death dead during mating period in high dose group. Based on gross pathologie, cause of death was uncertain. No mortality was observed in the doses of control, 100, 200 mg/kg b.w. No toxicity signs were observed in dams and pups in the doses of 100 and 200 mg/kg b.w throughout the observation period.

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. There was statistical significant decrease in body weight of female in high dose during late pregnancy (3rd and 4th week) and at terminal sacrifice (day 4 post partem) were also observed. Significant increases in body weight were also observed in low dose females during premating.
Feed consumption was decreased significantly during the first week of premating in both the sexes in dose related pattern in intermediate and high dose which recovered in later period. Feed consumption during gestation in female was also decreased when compared to control.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)


REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)


REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Test substances related adverse histopahological findings were observed in testis and epididymides of male rats in high dose groups. In testis
these findings consisted of sertolicell vacuolation (8/12), spermatid retention (6/12), multinucleated giant cells (5/12), desquamation of germ cells
(4/12), absence of spermatids (2/12) and atrophy of testis (1/12).
Test substance related adverse findings in the epididymides were desquamated germ cells (7/12) and oligospermia (4/12). These effects were in correspondence to the effects observed in the testis.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
9/12 females in the high dose group which mated with high dose males were infertile. Above mentioned microscopic finding in the testis and epididymides of high dose males which mated with high dose females could be attributed to the infertility. However, two mating pairs in the high dose group did not exhibit any histopathological changes in testis, epididymide, prostate, seminal vesicle and ovary which could explain their infertility.
All other changes observed in the testis and epididymides were either does not have dose response relationship or does not effect the fertility or does not have relationship between the lesions observed in the epididymides and testis. Hence, all other findings in the testis and epididymides were spontaneous, incidental and their relation with test substance administration is uncertain.
No test substance related findings were observed in seminal vesicles, prostate and ovaries. All findings observed in these organs were spontaneous or incidental.


ORGAN WEIGHTS (PARENTAL ANIMALS)
Statistically significant decrease in absolute testis and epididymides weight of high dose male rats was observed. This effect was correlated histologically with atrophy of testis, absence and depletion of spermatids in testis and oligospermia in epididymides and considered test substance related adverse effects.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No test substance related gross pathological observations were present in the male and female rats.

HISTOPATHOLOGY (PARENTAL ANIMALS)
All other changes observed in the testis and epididymides were either does not have dose response relationship or does not effect the fertility or does not have relationship between the lesions observed in the epididymides and testis. Hence, all other findings in the testis and epididymides were spontaneous, incidental and their relation with test substance administration is uncertain.
No test substance related findings were observed in seminal vesicles, prostate and ovaries. All findings observed in these organs were spontaneous or incidental.

OTHER FINDINGS (PARENTAL ANIMALS)

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Results: P1 (second parental generation)

Effect levels (P1)

Dose descriptor:
other:
Remarks:
OECD 421 not examined
Based on:
other:
Remarks:
not applicable
Sex:
not specified
Remarks on result:
not determinable because of methodological limitations

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
Test substance related effect was observed on loss of offspring in high dose group. There was increased rate of loss of ofspring in high dose group (pre implantation, post implantation and post partum).

CLINICAL SIGNS (OFFSPRING)
In high dose groups, 1/3 dams delivered abnormal pups. (anasarca in whole litter of dam)

BODY WEIGHT (OFFSPRING)
Test substances related effect was observed in mean pup weight on day 0 and day 4 post partum. Mean pup weight in high group day 0 was 1.6 followed by 5.1, 5.4 and 5.6 in intermediate, low and control group respectively. Mean pup weight in high dose at day 4 post partumm were 0.5 followed by 6.4, 7.2 and 7.8 in low and control group respectively.

SEXUAL MATURATION (OFFSPRING)
no data

ORGAN WEIGHTS (OFFSPRING)
no data

GROSS PATHOLOGY (OFFSPRING)
External abnormalities in pups: Anasarca was observed.

HISTOPATHOLOGY (OFFSPRING)
no data

OTHER FINDINGS (OFFSPRING)

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
200 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable because of methodological limitations

Results: F2 generation

Effect levels (F2)

Dose descriptor:
other:
Remarks:
OECD 421 not examined
Generation:
F2
Based on:
other:
Remarks:
not applicable
Sex:
not specified
Remarks on result:
not determinable because of methodological limitations

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the above findings, toxicity signs in animals treated with high dose (400 mg/kg b.w): hunched posture, abnormal gait, tremor, respiratory distress, dullness, salivation, pilo-erection, test substance related adverse effect on body weight changes and on most of the reproduction parameters, statistically decrease in absolute weight of testis and epididymis in high doses group. It is concluded that the dose 100 and 200 mg/kg b.w of N-n-butylbenzene sulphonamide was considered to be safe and non-toxic, while 400 mg/kg b.w of N-n-butylbenzenesulphonamide was considered toxic to Wistar rats in terms of parental and reproduction effects, therefore the NOAEL of the test substance was regarded as 200 mg/kg b.w.
Executive summary:

A screening for reproductive toxicity study (OECD 421) was performed in Wistar rats dosed by oral gavage at 0,100, 200 and 400 mg/kg b.w. Dosing started 2 weeks prior to mating, and continued in both sexes during the mating period. Males were further dosed after the mating period untill the minimum. dosing period of 28days was completed. Dosing of confirmed females was continued throughout the pregnancy and including day 3 post partum. Based on the above findings, toxicity signs in animals treated with high dose (400 mg/kg b.w): hunched posture, abnormal gait, tremor, respiratory distress, dullness, salivation, pilo-erection, test substance related adverse effect on body weight changes and on most of the reproduction parameters, statistically decrease in absolute weight of testis and epididymis in high doses group. It is concluded that the dose 100 and 200 mg/kg b.w of N-n-butylbenzene sulphonamide was considered to be safe and non-toxic, while 400 mg/kg b.w of N-n-butylbenzenesulphonamide was considered toxic to Wistar rats in terms of parental and reproduction effects, therefore the NOAEL of the test substance was regarded as 200 mg/kg b.w.

Categories Display