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Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
other: Read across from a member of the Hydrotropes category
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
not specified
GLP compliance:
not specified

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium toluene-4-sulphonate
EC Number:
211-522-5
EC Name:
Sodium toluene-4-sulphonate
Cas Number:
657-84-1
Molecular formula:
C7H7O3S.Na
IUPAC Name:
sodium 4-methylbenzenesulfonate

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
SPF Crl:CD
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animals' information
Supplier: Atsugi Breeding Center, Charles River Laboratories Japan, Inc.
The strain was selected since rats are the animal species used in these types of studies and abundant background data available in the test facility.
Fifty-seven males and 57 females were purchased at 8 weeks of age on July 13, 2005. The body weight range of the animals at the time of receipt was 253 to 300 g for males and 161 to 203 g for females.

Quarantine and acclimation
During the 14-day period after the receipt, each animal was observed once a day for general condition and weighed 3 times. For females, estrous cycle was examined for 9 days during the acclimation period. During the quarantine and acclimation period, 2 females showed flesh pillar in the vaginal opening, 2 females showed irregular estrous cycle and 3 females showed body weight decrease.

Animal housing
1) Housing environment
The animals were housed in an animal room (Room No. 308) where the temperature was set at 22 ± 3ºC (actual range: 21 to 25ºC), relative humidity at 50 ± 20% (actual range: 41 to 77%), air ventilation at 10 to 15 times per hour, and artificial lighting for 12 hours a day (8:00 to 20:00).
2) Housing instruments and housing methods
The animals were housed in bracket-type metal cages with wire-mesh floor (260W × 380D × 180H, in mm), in 1 or 2 animals of the same sex per cage during the quarantine/acclimation periods, individually after grouping, 1 male and 1 female during the mating period, one dam individually during the gestation period and one litter during the lactation period. For the females with successful copulation, small plates with bedding for experimental animals (White flake, Charles River Laboratories Japan, Inc.) were used from day 17 of gestation to day 4 of lactation. The cages and feeders were exchanged once at the time of grouping, and once every 2 weeks thereafter. The plates were exchanged with sterilized ones by washing twice a week. Draining of water from the automatic water supplier was done once a week. Cleaning and disinfection of the animal room were done once a day. For the disinfection, chloride disinfectant and iodine disinfectant were used every other week.
3) Feed
Animals were allowed free access to γ-irradiation pelleted diet CRF-1 produced by Oriental Yeast Co., Ltd. using metallic feeders.
The feed of the lot numbers (050204, 050307 and 050406) used in this study was analyzed for possible contaminants or presence/absence of microorganisms that could adversely affect this study. They were analyzed by Japan Food Research Laboratories for possible contaminants, and by each food producer for microbial examinations. The analytical data were received from the feed producer and confirmed that there were no abnormalities

4) Drinking water
Animals were allowed free access to Sapporo-city tap water using an automatic water supplying system.

Grouping
At the end of the quarantine/acclimation period, 48 healthy males and 48 healthy females showing normal estrous cycle were selected and subjected to the study at 10 weeks of age. Based on the body weight of the animals on the final day of quarantine/acclimation (2 days before the start of administration), grouping was done by the body weight-stratified random sampling method so that the group mean body weight was comparable as far as possible. The body weight range at the time of grouping was 341 to 411 g for males and 200 to 255 g for females, and they were within the range of ± 20% of the mean body weight (381.5 g for males and 225.1 g for females). The animals remaining after the selection were excluded from the study and euthanized. It was confirmed that the selected animals had no abnormalities in the general condition or estrous cycle on the day before the start of administration.


Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Considering the possibility that the test article could be exposed orally to humans, the test article was administered orally using a stomach tube and a disposable syringe into the stomach once daily between 9:00 and 14:00 for 46 days from day 14 before the start of mating for males and once daily between 9:00 and 14:00 for 14 days before the start of mating, during the mating period up to the day of successful copulation, and during the gestation period up to day 3 of lactation for the females that had successful copulation in accordance with the OECD study method guideline (421).
The dose volume was set at 10 mL/kg and individual dose volume calculated based on the most recently measured body weight of the animals.
Details on mating procedure:
From the evening of day 14 of administration, a pair of a male and a female in the same study group was placed in the same cage for 14 days at maximum. Copulation was confirmed if a vaginal plug was found in the vagina or on the plate or the presence of sperm in the vaginal smear specimens confirmed, and the day designated as day 0 of gestation. The pregnancy was confirmed by the presence of delivery or by the presence of implantation site in the uterus at the time of necropsy.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
5 hours duration
Frequency of treatment:
Once daily
Details on study schedule:
Once daily by oral gavage for 46 days from day 14 before the start of mating for males and once daily for 14 days before the start of mating, during the mating period up to the day of successful copulation, and during the gestation period up to day 3 of lactation for the females that had successful copulation.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: no-effect dose level in a previous 28-day repeated oral dose toxicity study was 1000 mg/kg.
The high dose level was set at 1000 mg/kg and the middle dose level and the low dose level at 300 and 100 mg/kg, respectively, using a common ratio of approximately 3.

Examinations

Parental animals: Observations and examinations:
Cage side observations
- Time schedule: least twice daily from day 1 of administration, counting the day administration started as day 1 of administration, to the day of necropsy, the day following day 46 of administration. On the day of necropsy, they were observed once in the morning.

Observation of general condition
All animals were observed at least twice daily for mortality, external appearance, behavior, etc. of each animal from day 1 of administration, counting the day administration started as day 1 of administration, to the day of necropsy, the day following day 46 of administration. On the day of necropsy, they were observed once in the morning

Measurement of body weight
Male: All animals were weighed using an electronic even balance (GX-2000, A&D Company Limited) on days 1, 2, 5, 7, 10 and 14 of administration and every 7 days thereafter before dosing of the day, on the last day of administration and the day of necropsy, and recorded in units of 1 gram
Female: Counting the starting day of administration as day 1 of administration, the day copulation was confirmed as day 0 of gestation, and the last day of delivery as day 0 of lactation, each female was weighed using an electronic even balance (GX-2000, A&D Company Limited) on days 1, 2, 5, 7, 10 and 14 of administration, on days 0, 1, 3, 5, 7, 10, 14, 17 and 20 of gestation, before administration on days 0 and 1 of lactation, and on day 4 of lactation (day of necropsy)

Food consumption
For all animals, male and female, food consumption was measured on the same days as for measurement of body weight except the mating period and the day of necropsy. On each day of measurement, the amount of the feed supplied and the amount of feed remaining were measured for each cage using an electronic even balance (GX-2000, A&D Company Limited) and recorded in units of 1 gram.

Observation of delivery and lactation conditions
All the females for which copulation had been confirmed were allowed to deliver spontaneously. The delivery condition was observed at least 3 times (9:00, 13:00 and 17:00) every day from day 21 of gestation to day 25 of gestation at maximum. Completion of delivery was confirmed when the dam gathered their pups under the abdomen in the nest and the day designated as day 0 of lactation. On day 0 of lactation, the numbers of live pups and dead pups were counted for each litter and their lactation condition, sex of the pups born and external appearance observed. The sum of the number of live pups and the number of dead pups was regarded as the number of pups born. The sex of the pups was judged by the anogenital distance. For each female, the number of pregnant corpora lutea in the ovaries and the number of implantation sites in the uterus counted macroscopically and the numbers recorded.
The number of days during the gestation period, from day 0 of gestation (the day copulation was confirmed) to day 0 of lactation (ending day of delivery), was calculated.

Clinical observation and viability index of live pups
All live pups were checked for survival and death and observed for general condition and external appearance once a day from day 0 of lactationto day 4 of lactation.

Measurement of body weight of live pups
All live pups were weighted using an electronic balance on days 0, 1 and 4 of lactationto day 4 of lactation. For each litter, mean body weight was calculated for each sex.












Oestrous cyclicity (parental animals):
Estrous cycle examination

For all females, vaginal smear specimens were prepared by Giemsa staining every day from 10 days before the start of administration to the day of successful copulation, observed under a light microscope for the stage of estrous cycle (proestrus, estrus, metestrus, and diestrus), the females that showed repetition of estruses in the interval of 4 to 6 days at least twice judged to be normal, and the length of the estrous cycle calculated. If females showed absence of the stage or the same stage continued for more than 3 days, they were to be judged to have irregular estrous cycle, and if females showed estrus or diestrus for at least 7 days, they were to be judged to have persistent estrus or persistent non-estrus and thus abnormal. However, there were no abnormal females.
Litter observations:
All live pups were weighed on days 0, 1 and 4 of lactation. All live pups were checked for survival and death and observed for general condition and external appearance once a day from day 0 of lactation to day 4 of lactation.
Postmortem examinations (parental animals):
SACRIFICE All surviving animals were subject to necropsy on the day following day 46 of dosing.

GROSS NECROPSY - Gross necropsy consisted of external and internal examinations including the brain (cerebrum, cerebellum), pituitary, thymus, thyroid glands (including parathyroid glands), adrenals, spleen, heart, thoracic aorta, tongue, esophagus, stomach (forestomach and glandular stomach), liver, pancreas, duodenum, jejunum, ileum (including Peyer’s patches), cecum, colon, rectum, larynx, trachea, lungs (including bronchi), kidneys, urinary bladder, testes, epididymides, prostate, seminal vesicles with coagulating glands, eyeballs and Harderian glands, skin (right abdominal region, in principle), sternal and femoral bones (including bone marrow, right), spinal cord (cervical region), mesenteric lymph node, submandibular lymph node, submandibular gland, sublingual gland, parotid gland, skeletal muscles (gastrocnemius muscle, right), and sciatic nerve (right), ovaries, uterus (uterine horn and uterine cervix), vagina, mammary glands and skin (right abdominal region, in principle).

HISTOPATHOLOGY
For all males, the testes and epididymides. For all animals the following were examined microscopically: males - forestomach, stomach (limiting ridge), glandular stomach, liver and heart. Females - ovaries, lungs and trachea

Measurement of organ weight
For all animals, the following organs were weighed at the time of necropsy using an electronic even balance (ER-180A, A&D Company Limited), and the relative organ weight calculated. For bilateral organs, the left organ and the right organ were weighed in combination.
Brain, heart, liver, kidneys, spleen, adrenals, thymus, testes, and epididymides
Postmortem examinations (offspring):
The pups that died were subjected to necropsy immediately after it was found dead and its whole body fixed and preserved.The pups that survived were euthanized after external observation (including the inside of the oral cavity) on day 4 of lactation, and organs/tissue in the whole body observed macroscopically. For the pups that had abnormalities, the whole body was fixed and preserved.
Reproductive indices:
Copulation index and fertility index for each group
Offspring viability indices:
The gestation index, live birth index, sex ratio, delivery index, implantation index and nursing index on day 4 of lactation

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Males: In the control group, one animal (No. 109) showed hematuria on days 45 and 46 of administration, but other animals showed no abnormalities.
In the 100 mg/kg group, there were no abnormalities in any animal during the administration period.
In the 300 mg/kg group, 1 animal (No. 302) showed diarrhea and soft feces each once during the administration period, but the other animals showed no abnormalities.
In the 1000 mg/kg group, all animals showed diarrhea or soft feces during the administration period twice to 19 times.

Females
In the control group, there were no abnormalities in any animal during the pre-mating period, mating period, gestation period or lactation period.
In the 100 mg/kg group, 2 animals (Nos. 251 and 255) showed soft feces only on day 4 of administration before the start of mating, but there were no abnormalities in any animal during the mating, gestation or lactation period.
On day 13 of gestation, one animal (No. 258) died by misadministration.
In the 300 mg/kg group, there were no abnormalities in any animal during the pre-mating, mating, gestation or lactation period.
In the 1000 mg/kg group, diarrhea or soft feces were observed once to 8 times during the pre-mating, mating or gestation period in 9 animals (Nos. 453, 454, 455, 456, 457, 459, 460, 461 and 462), but no animals showed abnormalities during the lactation period.

Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No dose group showed statistically significant differences from the control group during the administration period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No dose group showed statistically significant differences from the control group during the administration period.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Males: In the 1000 mg/kg group, 1 male (No. 410) showed slight atrophy of the seminiferous tubules in the testis and 3 males (Nos. 401, 402 and 410) showed slight spermatic granuloma in the epididymis. However, 2 animals in the control group (Nos. 104 and 108) showed slight atrophy of the seminiferous tubules in the testis and 1 animal (No. 103) showed slight vacuolar degeneration of the ductal epithelium in the epididymis and there were no statistically significant differences for any change. For the organs with gross pathological findings, 7 animals in the 1000 mg/kg group (Nos. 403, 404, 406, 407, 408, 410 and 411) showed slight inflammatory cell infiltration in the lamina propria in the limiting ridge of the stomach and 9 animals (Nos. 402, 404, 405, 406, 407, 408, 409, 411 and 412) showed slight squamous cell hyperplasia in the limiting ridge of the stomach, and they were both statistically significant in the incidence of their occurrences.
One animal (No. 407) showed slight squamous cell hyperplasia, but there were no abnormal changes in the forestomach or glandular stomach and similar changes were not observed in any other dose group.
In the 2 animals in the 1000 mg/kg group (Nos. 409 and 412) that showed yellowish white patch in the liver, slight massive necrosis (No. 409), slight histiocyte accumulation in the capsule and mineralization (No. 412) were observed in the area of the patch.

Females
In the ovaries, there were no females that showed abnormal changes in any animal in the control group or in the 1000 mg/kg group including the animals that were non-pregnant (No. 151 and 456).
In the dam (No. 258) in the 100 mg/kg group that died on day 13 of gestation showed slight edema and moderate hemorrhage in the lungs.


Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
There were no statistically significant differences in any dose group compared to that of the control group in the incidence of the presence of females that showed normal estrous cycle, in the interval of estruses, in the copulation index, fertility index, gestation index, gestation length or nursing index on day 4 of lactation.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
In the stage classification of spermatogenesis, there were significantly low values in the number of leptotene stage spermatocytes in stages IX to XI in comparison with that of the control group, but there were no statistically significant changes in the numbers of sertoli cells, spermatogonia, spermatocytes, or spermatids in any other stage.
Reproductive performance:
no effects observed
Description (incidence and severity):
In the number of corpora lutea, number of implantation sites, implantation index, total number of pups born, sex ratio of the pups born, number of live pups on day 0 of lactation, live birth index, viability index and the number of live pups on day 4 of lactation, there were no statistically significant differences in any dose group compared to that of the control group.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
gross pathology

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The number of pups that died (or became unclear) from day 0 to day 4 of lactation was 1 male in the control group, 2 males in the 100 mg/kg group, 1 female in the 300 mg/kg group, and 2 females in the 1000 mg/kg group.
Otherwise, there were no abnormalities in any dose group.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
In the 100 mg/kg group, there were no significant differences compared to those of the control group. In the 300 mg/kg group, there were significantly low values or tendencies toward low values on day 1 and 4 of lactation (after birth) in male and female pups. In the 1000 mg/kg group, there were no statistically significant differences in comparison with those in the control group.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no abnormal findings in the pups that died or in the survivors on day 4 of lactation.
Histopathological findings:
not examined
Other effects:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
sexual maturation
clinical signs
gross pathology

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

See attached tables

Applicant's summary and conclusion

Conclusions:
NOAEL parental toxicity: 300 mg/kg bw/day male/female
NOAEL reproductive and developmental toxicity: 1000 mg/kg bw/day male/female
Executive summary:

The potential of Sodium toluene 4-sulphonate to cause effects on the reproductive performance of male and female was assessed following official guideline OECD 421, Reproduction/Developmental Toxicity Screening Test. In this screening study the substance was administered to male and female Sprague-Dawley rats (12 animals/sex/dose) by oral gavage at dose levels of 0, 100, 300 or 1000 mg/kg bw/day. Males and females in the 1000 mg/kg bw/day group showed diarrhea or soft feces, and males showed mild inflammatory cell infiltration of the lamina propria and squamous cell hyperplasia in the limiting ridge of the stomach. There were no effects from administration of the substance in the reproductive function or development and growth of the next generation in any dose group.