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REACH

Sodium toluene-4-sulphonate

EC number: 211-522-5 | CAS number: 657-84-1

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

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Administrative data

Key value for chemical safety assessment

Toxic effect type:: dose-dependent

Effects on fertility

Description of key information

The HYDROTOPES Category comprises the following 6 substances:
STS - Sodium toluene 4-sulphonate (CAS 657-84-1, EC 211-522-5)
SXS - Sodium (xylenes and 4-ethylbenzene) sulfonates (EC 701-037-1)
NH4XS - Ammonium (xylenes and 4-ethylbenzene) sulfonates (EC 943-024-5)
SCS - Sodium p-cumenesulphonate (CAS 15763-76-5, EC 239-854-6)
KCS - Potassium p-cumenesulphonate (CAS 164524-02-1, EC 629-764-9)
NH4CS - Ammonium p-cumenesulphonate (CAS 680972-33-2, EC 811-484-5)
In addition CaXS (Calcium Xylenesulphonate, CAS 28088-63-3, EC 248-829-9) was evaluated for complete the assessment despite it is not registered under REACH.

In a reproductive and developmental toxicity screening study (OECD 421) Sodium p-toluene sulphonate was administered to male and female Sprague-Dawley rats by oral gavage at dose levels of 0, 100, 300 or 1000 mg/kg bw/day on a total of 46 days before the start of mating, during the mating period and after the end of mating to male rats and before the start of mating, during the mating and gestation periods up to day 3 of lactation to female rats. Males and females in the 1000 mg/kg bw/day group showed diarrhoea or soft faeces, 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 on the reproductive function or development and growth of the next generation in any dose group. Therefore, it was considered that the NOAEL for parental toxicity was 300 mg/kg bw/day and the NOAEL for reproductive and developmental toxicity was 1000 mg/kg bw/day.

The 90-day oral rat and oral mouse studies and the 2-year chronic dermal rat and mouse studies (OECD 453) on Sodium (xylenes and 4-ethylbenzene) sulfonates included examination of sex organs of both sexes. No treatment related effects on reproductive organs were reported at doses.

No treatment related effects on reproductive organs were reported in any study. A new OECD 443 was performed on Sodium (xylenes and 4-ethylbenzene) sulfonates which is the most representative member of the hydrotropes category since it contains a significant proportion of both mono-alkyl (ethyl) and di-alkyl (dimethyl) chemical species and thus best represents the substances within the category.

The available results on reproductive and developmental toxicity are:
STS, OECD 421 (2004): NOAEL = 1000 mg a.i./kg bw
SXS, OECD 443 (2021): NOAEL = XX mg a.i./kg/bw

The NOAEL value used for the risk assessment is XX mg/kg bw from the OECD YYYY performed on ZZZ both for reproduction and developmental toxicity

Link to relevant study records

Referenceopen all close all

Reference 1

Endpoint:: screening for reproductive / developmental toxicity
Type of information:: experimental study
Adequacy of study:: key study
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: comparable to guideline study
Qualifier:: equivalent or similar to guideline
Guideline:: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:: not specified
GLP compliance:: not specified
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.
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.
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.
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
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: 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.
: 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
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
Behaviour (functional findings):: not examined
Developmental immunotoxicity:: not examined
: 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
Reproductive effects observed:: no
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.

Reference 2

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Type of information:

other: Read across from another member of the category

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

Dose Range Finding

Deviations:

GLP compliance:

yes (incl. QA statement)

Limit test:

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:

Animal house: SPF animal house
Animal arrival: 07. 10. 2020
Acclimatization period: 5 days

Route of administration:

oral: gavage

Vehicle:

water

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

49 days

Frequency of treatment:

Once daily

Details on study schedule:

The test item (suspended in aqua pro iniectione) was orally administered (by gavage) daily at four dose levels to groups of experimental animals for 49 days. The duration of the DRFE was based on the results of a previous study provided by the sponsor (Reproduction / Developmental Toxicity Screening Test with sodium p-toluenesulfonate).

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

equivalent to 279 mg/kg/day a.i.

Dose / conc.:

600 mg/kg bw/day (nominal)

Remarks:

equivalent to 558 mg/kg/day a.i.

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

equivalent to 930 mg/kg/day a.i.

No. of animals per sex per dose:

24 males and 24 females (6 males + 6 females per group)
The dose-range finding experiment with 49-day application period was performed with 3 groups of treated animals and 1 group of control animals.

Control animals:

yes, concurrent vehicle

Details on study design:

Body weight: before first application and then weekly
Health condition check: daily, before the application of test item
Clinical observation: twice a day (except weekend when clinical observation was performed once a day)
Mortality observation: daily
Haematology examination: basic parameters – 1. 12. 2020
Pathological examination: gross necropsy – 1. 12. 2020

Clinical signs:

no effects observed

Description (incidence and severity):

No changes of animal health status and clinical symptoms of toxicity were observed in all treated animals

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:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Before necropsy of animals, blood samples were collected from the orbital plexus by glass micropipette under light diethyl ether narcosis into PVC test tubes containing anticoagulation systems. Basic blood parameters were determined (total erythrocyte count, total leucocyte count, mean corpuscular volume, haematocrit, haemoglobin concentration, total platelet count).

Males
Haematological examination showed differences in the total leucocyte count (WBC). A dose-dependent decrease was reported in treated groups of males in comparison with the control males. Statistical evaluation of WBC was performed. A statistically significant decrease of WBC value was reported in males at the nominal dose level 1000 mg/kg/day (equivalent to 930 mg/kg/day a.i.) compared to control males

Females
The value of total leucocyte count (WBC) was slightly lower in treated females at the nominal dose level 1000 mg/kg/day (equivalent to 930 mg/kg/day a.i.) in comparison with the control group. Statistical evaluation was performed for value of WBC. A statistically significant decreased value of WBC was reported in females at the nominal dose level 1000 mg/kg/day (equivalent to 930 mg/kg/day a.i.). Other haematological parameters of treated females were comparable to the control females.

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, non-treatment-related

Description (incidence and severity):

Histopathological examination of the stomach revealed small foci of minimal to mild acute inflammation in the submucosa of one control and two treated males (1 male at 600 mg/kg/day nominal; 1 male at 1000 mg/kg/day nominal) and one control female and one treated female (at 1000 mg/kg/day nominal). Minimal to mild focal edema accompanied by minimal to mild focal acute inflammation were found in the submucosa of two control males and three treated males (1 male at 600 mg/kg/day nominal; 2 males at 1000 mg/kg/day nominal). The described changes in the submucosa are of unclear origin, without relation to the test item dose administered.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

930 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

histopathology: non-neoplastic

Food consumption and compound intake (if feeding study):

not examined

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Table No. 1: Body weight (g) – Mean values

Body weight	MALES
	Dose level (nominal)
	Control group (0 mg/kg/day)	300 mg/kg/day	600 mg/kg/day	1000 mg/kg/day
Weight before application ± SD	345.42 ± 9.71	346.29 ± 15.74	348.47 ± 12.09	346.28 ± 23.50
Weight on 7^thday ± SD	385.67 ± 11.96	388.79 ± 20.45	388.04 ± 18.93	389.30 ± 33.09
Weight on 14^thday ± SD	414.39 ± 17.44	414.93 ± 29.91	413.43 ± 28.87	413.81 ± 40.25
Weight on 21^stday ± SD	441.11 ± 16.56	432.15 ± 35.74	437.31 ± 33.67	431.35 ± 44.80
Weight on 28^thday ± SD	459.58 ± 22.35	451.80 ± 31.15	457.04 ± 34.53	452.18 ± 48.58
Weight on 35^thday ± SD	472.03 ± 26.51	471.95 ± 29.27	477.16 ± 37.53	473.59 ± 56.07
Weight on 42^ndday ± SD	489.92 ± 23.84	482.55 ± 30.23	493.54 ± 39.61	489.19 ± 57.77
Weight on 49^thday ± SD	503.93 ± 26.50	497.21 ± 31.60	506.15 ± 42.86	503.32 ± 61.58

Note: SD – standard deviation

Table No. 2: Body weight (g) – Mean values

Body weight	FEMALES
	Dose level (nominal)
	Control group (0 mg/kg/day)	300 mg/kg/day	600 mg/kg/day	1000 mg/kg/day
Weight before application ± SD	229.94 ± 17.58	227.78 ± 12.90	229.67 ± 12.20	227.79 ± 10.87
Weight on 7^thday ± SD	243.31 ± 13.15	242.28 ± 11.62	241.79 ± 11.24	237.48 ± 16.53
Weight on 14^thday ± SD	238.47 ± 11.26	241.50 ± 12.16	239.98 ± 11.64	238.99 ± 17.27
Weight on 21^stday ± SD	249.54 ± 11.62	255.24 ± 12.51	255.54 ± 18.55	249.85 ± 17.56
Weight on 28^thday ± SD	254.73 ± 13.68	264.34 ± 17.24	264.78 ± 20.50	256.92 ± 14.20
Weight on 35^thday ± SD	263.96 ± 14.30	273.57 ± 15.76	272.28 ± 17.70	262.49 ± 14.55
Weight on 42^ndday ± SD	265.27 ± 14.88	273.35 ± 14.82	268.99 ± 17.61	265.91 ± 16.32
Weight on 49^thday ± SD	270.88 ± 9.44	280.74 ± 16.57	278.73 ± 21.49	273.24 ± 17.43

Note:SD – standard deviation

Table No. 3: Haematological examination of males

Haematological examination

MALES

Parameter

Unit

Dose level (nominal)

Control group (0 mg/kg/day)

300 mg/kg/day

600 mg/kg/day

1000 mg/kg/day

Values

WBC-Total leucocyte count

10⁹/L

10.43

±1.53

9.39

±2.58

8.39

±2.02

6.95

±1.61

Mean

+SD

RBC -Total erythrocyte count

10¹²/L

7.89

±0.41

8.10

±0.57

7.49

±0.33

7.19

±0.53

Mean

+SD

Haemoglobin

g/dL

15.03

±0.65

15.42

±0.60

14.60

±0.67

14.65

±1.17

Mean

+SD

HCT

Haematocrite

41.55

±1.81

41.58

±2.38

38.92

±1.86

37.88

±2.74

Mean

+SD

MCV -Mean corpuscular volume

52.77

±1.54

51.47

±1.42

52.07

±1.46

52.82

±1.67

Mean

+SD

Platelet count

10⁹/L

947.50

±51.66

936.67

±115.65

983.33

±87.58

838.67

±201.44

Mean

+SD

Note:grey field= values statistically significant (p ≤ 0.05);SD – standard deviation

Table No. 4: Haematological examination of females

Haematological examination

FEMALES

Parameters

Unit

Dose level (nominal)

Control group (0 mg/kg/day)

300

mg/kg/day

600 mg/kg/day

1000 mg/kg/day

Values

WBC-Total leucocyte count

10⁹/L

5.85

±0.87

6.64

±0.89

5.89

±1.15

4.35

±0.69

Mean

+SD

RBC -Total erythrocyte count

10¹²/L

7.33

±0.45

7.42

±0.49

7.18

±0.38

7.09

±0.30

Mean

+SD

Haemoglobin

g/dL

14.83

±0.93

14.78

±0.62

14.88

±0.51

15.02

±0.50

Mean

+SD

HCT

Haematocrite

39.00

±2.45

39.15

±1.96

39.00

±1.56

39.27

±1.29

Mean

+SD

MCV -Mean corpuscular volume

53.28

±1.45

52.90

±1.26

54.48

±1.30

55.53

±1.51

Mean

+SD

Platelet count

10⁹/L

850.67

±53.78

1030.50

±118.88

951.17

±100.19

1024.83

±218.86

Mean

+SD

Note:grey field= values statistically significant (p ≤ 0.05);SD – standard deviation

Table No. 5: Histopathological findings in males

Histopathological Findings
MALES
Organ/Diagnosis	Control Group (0 mg/kg/day)	300 mg/kg/daynominal	600 mg/kg/daynominal	1000 mg/kg/daynominal
Number of examined animals	6	6	6	6
Number of died animals/Mortality	0	0	0	0
Without pathological findings	3	6	4	3
Stomach: focal acute inflammation in submucosa	1	-	1	1
Stomach: focal edema and focal acute inflammation in submucosa	2	0	1	2

Table No. 6: Histopathological findings in females

Histopathological Findings
FEMALES
Organ/Diagnosis	Control Group (0 mg/kg/day)	300 mg/kg/daynominal	600 mg/kg/daynominal	1000 mg/kg/daynominal
Number of examined animals	6	6	6	6
Number of died animals/Mortality	0	0	0	0
Without pathological findings	5	6	6	5
Stomach: focal acute inflammation in submucosa	1	-	-	1

Conclusions:

Based on the results of DRFE the dose levels of 0, 100, 300 and 1000 a.i. mg/kg bw/day will be used for the main EOGRTS (OECD 443)

Executive summary:

The dose-range finding experiment with 49-day application period was performed with 3 groups of treated animals
and 1 group of control animals.The test item (suspended inaqua pro iniectione) was orally administered (by gavage) daily at four dose levels to groups of
experimental animals for 49 days

No animal died during the application period.

Clinical observations did not detect any effects of test item administration on the health condition of animals
at all dose levels.

Body weight was not affected by treatment with the test item at any dose level; body weights wereadequate for the species, sex and age of animals in the experiment.

No macroscopical changes were observed during the necropsy of treated animals.

Haematological examination revealed effects of the test item administration on animals.
The administration of the test item caused adose-dependent decrease of total leucocyte count in males.
In males at the nominal dose level 1000 mg/kg/day, this decrease was statistically significant in comparison
with the control group of males.

The haematological examination reported lower WBC also in females at the highest dose level 1000 mg/kg/day
nominal. In females at the dose level 1000 mg/kg/day nominal, this decrease was statistically significant
in comparison with the control group of femalesHistopathological examination of the stomach detected changes
in the submucosa of unclear origin without relation to the test item dose administration.
Therefore, our conclusion is that the test item administration did not cause histopathological changes
in the stomach.
Although there was a significant decrease in the total leucocyte count (WBC) of males and females at the
nominal dose level of 1000 mg/kg/day, this dose was chosen for the main study (focusing on reproduction)
due to the absence of clinical signs of toxicity. No histopathological findings in the stomach related
to test item administration were recorded.

Effect on fertility: via oral route

Endpoint conclusion:: no adverse effect observed
Dose descriptor:: NOAEL
: 1 000 mg/kg bw/day
Study duration:: subacute
Species:: rat
Quality of whole database:: Sufficient to meet requirements.

Effect on fertility: via inhalation route

Endpoint conclusion:: no study available

Effect on fertility: via dermal route

Endpoint conclusion:: no study available

Effects on developmental toxicity

Description of key information

The study with Calcium Xylenesulphonate performed under similar condition of OECD 414 guideline showed no adverse effects. In this developmental toxicity study on Calcium Xylenesulphonate, thirty (30) female rats received 0, 150, 1500 or 3000 mg test substance/kg bw/day by oral gavage on days 6 to 15 of gestation. Clinical symptoms were noted daily through day 20. Body weight and food consumption were recorded every three days through day 20. All females were macroscopically examined on day 20. The uteri were removed, weighed and examined for number of corpora lutea, implant sites, and number, and location of foetuses and resorptions. Foetuses were inspected on total number, sex, weight and external, visceral (one-half) and skeletal (one-half) defects. One death occurred at the 1500 mg/kg bw/day dose but it was considered a gavage injury. There were no abnormal clinical observations or necropsy findings. There were no effects on body weight or body weight gain. There was a siginifcant increase in food consumption for the 3000 mg/kg bw/day dose during gestation interval (day) 12 -16, but this was considered normal biological variation and not a direct effect of the substance. All indices were comparable to the corresponding controls. The NOAEL based on active ingredient of the test substance is 936 mg/kg bw/day. The NOAEL for both maternal and foetal toxicity was the highest dose tested and the conclusion of the study was no indications of developmental toxicity including teratogenesis.

The OECD 414 performed on Sodium p-cumenesulphonate confirms the absence of effects on developmental toxicity.

OECD 414 was performed on a second species (rabbits) according OECD Guideline 414 to assess the influence of Sodium (xylenes and 4-ethylbenzene) sulfonates on on embryo-fetal survival and development when administered during the organogenesis and fetal growth phases of pregnancy in the New Zealand White Rabbit. Three groups of 22 females received Sodium Xylene Sulphonate at doses of 100, 300 or1000 mg/kg/day by oral gavage administration at a volume-dose of 5 mL/kg body weight from Day 6 to Day 28 after mating, inclusive. A similarly constituted Control group received the vehicle, purified water, at the same dose volume and for the same duration as those receiving the test item. Based on the results obtained in this study of embryo-fetal development, it was concluded that the dose level of 1000 mg/kg/day represented the No Observed Adverse Effect Level (NOAEL) for maternal toxicity and for embryo-fetal survival, growth and development.

A new OECD 443 was performed on Sodium (xylenes and 4-ethylbenzene) sulfonates which is the most representative member of the hydrotropes category since it contains a significant proportion of both mono-alkyl (ethyl) and di-alkyl (dimethyl) chemical species and thus represents both the mono-alkyl and di-alkyl substances contained within the category.

The available results on developmental toxicity are:

SCS, OECD 414 (2020): NOAEL = 1000 mg a.i./kg bw
SXS, OECD 414 (rabbits, 2017): NOAEL = 1000 mg a.i./kg bw
CaXS, similar to OECD 414 (1994): NOAEL = 936 mg a.i./kg bw
SXS, OECD 443 (2021):NOAEL = XX mg a.i./kg/bw

The NOAEL value used for the risk assessment is XX mg/kg bw from the OECD YYYY performed on ZZZ both for reproduction and developmental toxicity.

Link to relevant study records

Referenceopen all close all

Reference 1

Endpoint:

developmental toxicity

Type of information:

other: Read across from another member of the category

Adequacy of study:

key study

Study period:

June 2016 to March 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

Second species rabbits

Deviations:

GLP compliance:

yes

Limit test:

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Number of animals: 96 females.
Duration of acclimatization: 19 days.
Age of the animals at the start of the study (Day 0 of gestation): Approximately 19 to 23 weeks old.
Weight range of the animals at the start of the study (Day 0 of gestation): 2.64 to 4.28 kg.

Environmental Control
Rabbit facility Partial barrier, with limited access - to minimize entry of external biological and chemical agents.
Air supply Filtered fresh air which was passed to atmosphere and not recirculated.
Temperature and relative humidity Monitored and maintained within the range of 15-21°C and 45-70%.
Although conditions were occasionally outside the indicated ranges, these deviations were minor and/or of short duration and were considered not to have influenced the health of the
animals and/or the outcome of the study. Lighting Artificial lighting, 14 hours light : 10 hours dark.
Alarm systems Activated on ventilation failure and when temperature/humidity limits exceeded.
Electricity supply Public supply with automatic stand-by generators.

Animal Accommodation
Cages Suspended cages fitted with perforated floor panels and mounted in batteries. Undertrays lined with absorbent paper were changed at least three times a week. Cages were also
fitted with a plastic resting platform.
Cage distribution The cages constituting each group were blocked by group and mounted in batteries.
Number of animals per cage:
Acclimatization: one female.
During mating one stock male and one female.
Gestation one female.

Environmental Enrichment
Aspen chew block A soft white untreated wood block; provided to each cage throughout the study and replaced when necessary. Stainless steel key ring Attached to the cage.

Diet Supply
Diet Teklad 2930 pelleted diet. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Availability Restricted (initially 150 g/animal/day during acclimatization up to one week prior to the onset of mating and 200 g/animal/day thereafter).
If an individual showed a significant non-treatment related reduction in food consumption, moistened diet (50 g pelleted diet moistened with up to 50 mL of water) was offered, the consumption was recorded.
In addition to this diet, a small supplement of autoclaved hay was given on a daily basis to promote gastric motility and a small amount of chopped fresh vegetables were given twice
weekly. Consumption of hay and vegetables were monitored qualitatively but not quantitatively.

Water Supply
Supply Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals. Water bowls were also used.
Availability Non-restricted.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

The doses were selected based onthe DRF

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, limits of detection and quantification, linearity of detector response, repeatability, method accuracy and precision.
The homogeneity and stability was confirmed for Sodium Xylene Sulphonate in Vehicle formulations at nominal concentrations of 1 mg/mL and 200 mg/mL during distribution between the bottles, during magnetic stirring for 4 hours, ambient temperature (+15 to +25°C) storage for 1 day and refrigerated storage (+2 to +8°C) for up to 15 days. Stability of discrete samples was confirmed for 15 days refrigerated storage
The mean concentrations of Sodium Xylene Sulphonate in test formulations analyzed for the study were within ±10% of nominal concentrations, confirming accurate formulation.

Details on mating procedure:

Male/female ratio: 1:1 using identified stock New Zealand White bucks.
Checks: Natural mating observed.
After mating Each female was injected intravenously with 25 i.u.
luteinizing hormone.
Day 0 of gestation On the day of mating.
A colony of stud males was maintained specifically for the purpose of mating; these animals were not part of the study and were maintained as stock animals.

After mating: Each female was injected intravenously with 25 i.u. luteinizing hormone.

Day 0 of gestation: On the day of mating.

A colony of stud males was maintained specifically for the purpose of mating; these animals were not part of the study and were maintained as stock animals.

Duration of treatment / exposure:

Females were treated from Day 6 to Day 28 (inclusive) after mating,

Frequency of treatment:

Once daily at approximately the same time each day

Duration of test:

28 days

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:

Three groups of 22 females received Sodium Xylene Sulphonate at doses of 100, 300 or 1000 mg/kg/day by oral gavage administration at a volume-dose of 5 mL/kg body weight from Day 6 to Day 28 after mating, inclusive.

Control animals:

yes, concurrent vehicle

Ovaries and uterine content:

For females surviving to term, the following was recorded:
Uterus: Gravid uterine weight (including cervix and ovaries).
The following were recorded for all animals including those prematurely sacrificed, where possible:
For each ovary/uterine horn: Number of: Corpora lutea, Implantation sites, Resorption sites (classified as early or late), Fetuses (live and dead).
Apparently non-pregnant animals and for apparently empty uterine horns: The absence or number of uterine implantation sites was confirmed.

Fetal examinations:

Examination of all viable fetuses and placentae: Dissected from the uterus, individually weighed and identified within the litter using a coding system based on
their position in the uterus. Examined externally with abnormalities recorded, sampled as appropriate and retained in appropriate fixative. All fetuses were subject to a gross internal examination of the viscera of the neck, thorax and abdominal cavities and the sex of each fetus was also recorded.

Fixation: Nominally one half of eviscerated fetuses were decapitated; heads were initially stored in Bouin’s fluid. Remaining eviscerated fetuses and torsos were fixed in Industrial Methylated Spirit.

Processing: Bouin’s fixed fetal heads were subject to free-hand serial sectioning. Industrial Methylated Spirit fixed fetuses and torsos were processed and stained with Alizarin Red.

Bouin’s fixed heads: Serial sections were examined for soft tissue abnormalities.

Alizarin Red stained fetuses and torsos: Assessed for skeletal development and abnormalities.

Statistics:

The following data types were analyzed at each timepoint separately:
Body weight, using gains over appropriate study periods
Gravid uterine weight and adjusted body weight
Food consumption, over appropriate study periods
Litter size and survival indices
Fetal, placental and litter weight

The following comparisons were performed:
Group 1 vs 2, 3 and 4

A sequence of statistical tests was used for body weight, gravid uterus weight, food consumption, corpora lutea, implantations, live young, fetal, placental and litter weight data.

For litter size and survival indices and fetal, placental and litter weight and gravid uterine weight data, if 75% of the data (across all groups) were the same value, for example c, Fisher’s exact tests (Fisher 1973) were performed. Treatment groups were compared using pairwise comparisons of each dose group against the control both for i) values c, as applicable.

Pre/post implantation loss and sex ratio were analyzed by generalized mixed linear model with binomial errors, a logit link function and litter as a random effect (Lipsitz 1991). Each treated group was compared to control using a Wald chi-square test. For pre implantation loss, the numerator was number of corpora lutea - number of implantations, the denominator was Number of corpora lutea. For post-implantation loss, the numerator was number of implantations - number of live fetuses, the denominator was number of implantations. For sex ratio, the numerator was number of males; the denominator was number of live fetuses.

For resorptions, each treated group was compared to control by exact Wilcoxon rank sum test (Wilcoxon 1945).

Significant differences between the groups compared were expressed at the 5% (p<0.05) or 1% (p<0.01) level.

Clinical signs:

no effects observed

Description (incidence and severity):

Among females surviving to scheduled termination, there were no signs observed at routine physical examination or in relation to dose administration that were clearly attributable to Sodium Xylene Sulphonate administration.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were three premature deaths during the course of the study, none of which were considered to be related to Sodium Xylene Sulphonate administration.
On Day 14 of gestation, Female No. 69 receiving 1000 mg/kg/day was killed for reasons of animal welfare. This animal had shown noisy respiration (rales) since Day 12 of gestation and due to this sign was not able to be dosed on Day 12 or 13 of gestation; the animal was despatched to necropsy due to severe respiratory impairment. Other clinical signs observed between Day 12 and Day 14 of gestation included low hay and water intake, thin build, reduced faecal and urine output and small faecal pellets; no signs had been observed in relation to dose administration. Low food consumption was evident from Day 9 of gestation along with weight loss of 0.21 kg from Day 8 of gestation. Macroscopic examination revealed poorly defined dark areas on the lungs, aerated fluid adjacent to the salivary glands and the capsule above the salivary glands was slightly distended with air; there was no evidence of any trauma to the trachea or oesophagus (i.e. no evidence of mis-dosing). The
uterus contained ten grossly normal embryos.

On Day 19 of gestation, Female No. 54 receiving 300 mg/kg/day was found dead shortly after dose administration. This female had previously shown normal food consumption and body weight performance, no ante-mortem clinical signs had been observed during the study, post-dosing observations were limited to difficulty in achieving intubation of the catheter on Day 12 of gestation and signs at despatch to necropsy were limited to abnormally coloured coat staining on the muzzle. There were no significant abnormalities detected at macroscopic examination; the uterus contained six grossly normal embryos and one early resorption.

On Day 22 of gestation, Control Female No. 8 died shortly after dose administration having experienced a prolonged convulsion. On two occasions (Days 16 and 19 of gestation) difficulty in achieving intubation of the catheter had occurred, and the female had shown clinical signs of noisy/irregular respiration on Days 16-17 of gestation. Normal body weight performance and levels of food consumption had been observed throughout the study.

Macroscopic examination revealed a perforation in the lower section of the trachea, indicating that the female had been mis-dosed; the uterus contained three grossly normal fetuses.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The body weight performance of pregnant females surviving to scheduled termination was unaffected by Sodium Xylene Administration at doses up to and including 1000 mg/kg/day. The mean gravid uterine weight on Day 29 of gestation was similar in all groups. When overall mean body weight gain was adjusted for the contribution of the gravid uterus, net mean body weight loss was recorded in all groups of females and no effect of treatment with Sodium Xylene Sulphonate was inferred.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The body weight performance of pregnant females surviving to scheduled termination was unaffected by Sodium Xylene Administration at doses up to and including 1000 mg/kg/day.

The mean gravid uterine weight on Day 29 of gestation was similar in all groups. When overall mean body weight gain was adjusted for the contribution of the gravid uterus, net mean body weight loss was recorded in all groups of females and no effect of treatment with Sodium Xylene Sulphonate was inferred.

Food efficiency:

no effects observed

Description (incidence and severity):

Mean food consumption during Days 1-29 of gestation was similar in all groups and unaffected by the administration of Sodium Xylene Sulphonate.

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

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related macroscopic abnormalities detected among the females at scheduled termination on Day 29 after mating.

The incidence of major and minor fetal abnormalities and skeletal variants showed no relationship to maternal treatment with Sodium Xylene Sulphonate.

Across the treated groups there was a slightly increased incidence of short supernumerary cervical rib and 7th costal cartilage not connected to sternum compared to concurrent control. There was, however, no dose response apparent and all fetal and litter incidences were within the Historical Control Data range, therefore these slight increases were considered were considered to be fortuitous and not adverse.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Litter Responses: At scheduled termination on Day 29 after mating, three Control females (No’s. 5, 12 and 15), three low dose group females (No’s. 32, 40 and 41), two intermediate dose group females (No’s. 45 and 46) and five high dose females (No’s 70, 71, 73, 75 and 82) were found not to be pregnant. Therefore, a total of 18, 19, 19 and 16 litters were available for assessment at 0, 100, 300 and 1000 mg/kg/day, respectively.
Placental, Litter and Fetal Weights: There was no effect of maternal treatment on mean placental, litter or fetal weights at any dose level investigated.

Number of abortions:

no effects observed

Description (incidence and severity):

There was no evidence that maternal treatment with Sodium Xylene Sulphonate at doses up to and including 1000 mg/kg/day had any adverse effect on litter data, as assessed by the mean numbers of implantations, resorptions, live young and pre- and post-implantation losses, at any of the doses investigated. Sex ratio, as assessed by the percentage of males per litter, was in line with expectations and unaffected by maternal treatment.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

Early or late resorptions:

no effects observed

Description (incidence and severity):

Dead fetuses:

no effects observed

Description (incidence and severity):

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

changes in number of pregnant

changes in pregnancy duration

clinical signs

dead fetuses

early or late resorptions

effects on pregnancy duration

food consumption and compound intake

food efficiency

gross pathology

maternal abnormalities

mortality

necropsy findings

number of abortions

pre and post implantation loss

total litter losses by resorption

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There was no effect of maternal treatment on mean placental, litter or fetal weights at any dose level investigated.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Sex ratio, as assessed by the percentage of males per litter, was in line with expectations and unaffected by maternal treatment.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There was no effect of maternal treatment on mean placental, litter or fetal weights at any dose level investigated.

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Description (incidence and severity):

The incidence of major and minor fetal abnormalities and skeletal variants showed no relationship to maternal treatment with Sodium Xylene Sulphonate.

Skeletal malformations:

no effects observed

Description (incidence and severity):

The incidence of major and minor fetal abnormalities and skeletal variants showed no relationship to maternal treatment with Sodium Xylene Sulphonate.

Visceral malformations:

no effects observed

Description (incidence and severity):

The incidence of major and minor fetal abnormalities and skeletal variants showed no relationship to maternal treatment with Sodium Xylene Sulphonate.

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

fetal/pup body weight changes

changes in litter size and weights

changes in postnatal survival

external malformations

skeletal malformations

visceral malformations

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

Conclusions:

NOAEL is 1000 mg/kg/day for maternal toxicity and for embryo-fetal survival, growth and development.

Executive summary:

The potential of Sodium (xylenes and 4-ethylbenzene) sulfonates to influence the embryo-fetal survival and development when administered during the organogenesis and fetal growth phases of pregnancy in rabbit were assessed followign OECD 414. Treatment of pregnant female rabbits with Sodium (xylenes and 4-ethylbenzene) sulfonates at dose levels up to and including 1000 mg/kg/day was well tolerated and there were no test item-related

unscheduled deaths or changes in clinical condition observed. Body weight performance, gravid uterine weight, adjusted body weight gain and food consumption were unaffected by Sodium (xylenes and 4-ethylbenzene) sulfonates administration at all dose levels investigated, and there were no test item-related macroscopic abnormalities detected at scheduled termination.

There was no effect of maternal treatment with Sodium (xylenes and 4-ethylbenzene) sulfonates on litter data, as assessed by the number of implantations, resorptions, live young, sex ratio and pre- and postimplantation losses. Placental, litter and fetal weights were similar in all groups. The incidence of major and minor fetal abnormalities and skeletal variants showed no relationship to maternal treatment with Sodium (xylenes and 4-ethylbenzene) sulfonates.

Reference 2

Endpoint:: developmental toxicity
Type of information:: other: Read across from another member of the category
Adequacy of study:: key study
Study period:: February 7-24, 1994
Reliability:: 2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:: study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:: equivalent or similar to guideline
Guideline:: OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:: Females were mated with males of the same strain and source. A block design was used to assign 30 mated females to controls and each of three dosing levels. Doses were selected based on a range finding study where the highest dose was 3000 mg/kg/day and no maternal or developmental toxicity was observed. The test material was prepared weekly. Analysis of dosing preparations stored for 10 days at room temperature verified the stability of the dosing preparation for at least a week. Dosing was done by intragastric intubation through needles at a volume of 10 ml/kg. Dosing was done on gestation days 6 through 15. A vehicle control was included in the study design. The concentration of the dosing preparation was confirmed analytically. Animals were observed twice daily for mortality and signs of overt toxicity. Clinical observations were made from gestation days - 20. Individual body weights were recorded on gestation days 0, 6, 9, 12, 16 and 20. Individual food consumption was recorded concurrent with the body weighing days. Food consumption was calculated. On gestation day 20 all surviving animals were euthanized and immediately examined by caesarean section. The uterus and ovaries were exposed and examined. The uterus was weighed. The location of viable and nonviable fetuses, early and late resorptions, and the number of total implantations and corpora lutea were recorded. The abdominal and thoracic cavities and organs were examined for grossly evident morphological changes. Uteri from nongravid females were placed in 10% ammonium sulfide solution for detection of implantations. Individual fetuses were weighed, sexed, and examined for external malformations and variations. Approximately one-half of the fetuses were placed in Bouin's solution for subsequent soft-tissue examination using hte Wilson razor-blade sectioning technique. The remainder of the fetuses were prepared for skeletal examination. All gross, visceral and skeletal alterations observed were classified as malformations or developmental variations. Treatment and control groups were subjected to appropriate statistical comparison.
GLP compliance:: yes (incl. QA statement)
Limit test:: no
Species:: rat
Strain:: other: Charles River Crl:CD VAF/Plus
Details on test animals or test system and environmental conditions:: TEST ANIMALS
The 165 rats received were untreated, sexually mature, virgin females. Upon receipt, the animals were assigned temporuy animal numbers and housed in suspended, stainless steel, wire-mesh cages.
- Source: Charles River Laboratories, Michigan
- Age at study initiation: 12.5 weeks
- Weight at study initiation: 243-312g
- Fasting period before study: no data
- Housing: individually in suspended stainless steel wire mesh cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 71-71 degrees F
Humidity (%): 48-59%
- Air changes (per hr): controlled
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: February 7 To: February 24
Route of administration:: oral: gavage
Vehicle:: not specified
Details on exposure:: PREPARATION OF DOSING SOLUTIONS:DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): Purina Certified Rodent Chow #5002
- Storage temperature of food: room temperature
Analytical verification of doses or concentrations:: yes
Details on analytical verification of doses or concentrations:: no data
Details on mating procedure:: At the end of the aeelimation period, all females were weillhed and subjected to a detailed phfSical examination. At this time, females consiilered suitable for study
were cohabited with stock males used exclusively for this purpose.
One female and one male rat of the same source and strain were placed together for mating. The occurrence of copulation was determined by daily inspection for a copulatory plug. The dayevideilce of mating was detected was designated
gestation day 0, and the female was returned to an individual cage, assigned a permanent animal nnmber and properly identified by ear tag.
Duration of treatment / exposure:: On gestation days 6-15
Frequency of treatment:: Daily
Duration of test:: Until gestation day 20
Dose / conc.:: 0 mg/kg bw/day (nominal)
Dose / conc.:: 150 mg/kg bw/day (nominal)
Remarks:: corresponding to 46.8 mg/kg bw based on active ingredient (Purity 31.2 %)
Dose / conc.:: 1 500 mg/kg bw/day (nominal)
Remarks:: corresponding to 468 mg/kg bw based on active ingredient (Purity 31.2 %)
Dose / conc.:: 3 000 mg/kg bw/day (nominal)
Remarks:: corresponding to 936 mg/kg bw based on active ingredient (Purity 31.2 %)
No. of animals per sex per dose:: 30 females per dose
Control animals:: yes, concurrent vehicle
Details on study design:: - Dose selection rationale: prior range finding study (study 715-001)
In that study, the highest dosage lever was 3,000 mg/kg/day. No indIcations of maternal or developmental toxicity were seen at any oosage level tested in the DRF study
Maternal examinations:: Animals were observed twice daily for mortality and signs of overt toxicity. Clinical observations were made from gestation days - 20. Individual body weights were recorded on gestation days 0, 6, 9, 12, 16 and 20. Individual food consumption was recorded concurrent with the body weighing days. Food consumption was calculated. On gestation day 20 all surviving animals were euthanized and immediately examined by cesarean section.
Ovaries and uterine content:: The uterus and overies were exposed and examined. The uterus was weighed. The location of viable and nonviable fetuses, early and late resorptions, and the number of total implantations and corpora lutea were recorded. The abdominal and thoracic cavities and organs were examined for grossly evident morphological changes. Uteri from nongravid females were placed in 10% ammonium sulfide solution for detection of implantations.
Fetal examinations:: Individual fetuses were weighed, sexed, and examined for external malformations and variations. Approximately one-half of the fetuses were placed in Bouin's solution for subsequent soft-tissue examination using hte Wilson razor-blade sectioning technique. The remainder of the fetuses were prepared for skeletal examination. All gross, visceral and skeletal alterations observed were classified as malformations or developmental variations.
Statistics:: Treatment and control groups were subjected to appropriate statistical comparison. Dunnett t-test for body wight, food consumption, numbers of corpora lutea, total implantations, live fetuses and mean fetal body weights. Chi-square or Fishers test for male to female sex ratios and proportion of litters with malformations and developmental variations. Kruskal-Wallis test for the proportion of resorbed and dead fetuses and postimplantation losses
Clinical signs:: no effects observed
Body weight and weight changes:: no effects observed
Food consumption and compound intake (if feeding study):: effects observed, non-treatment-related
Description (incidence and severity):: There was a significant increase in food consumption for the 3000 mg/kg/day during gestation interval 12-16 but this was considered normal biological variation and not a direct effect of the test substance.
Details on maternal toxic effects:: Maternal toxic effects:no effects
Details on maternal toxic effects: One death occurred at the 1500 mg/kg/day dose but it was considered a gavage injury.
No clinical observations or necropsy findings.
No effects on body weight or body weight gain.
: Key result
Dose descriptor:: NOAEL
Effect level:: > 3 000 mg/kg bw/day
Based on:: test mat.
Basis for effect level:: clinical signs; histopathology: neoplastic; mortality
Remarks on result:: other: No other remarks
: Key result
Dose descriptor:: NOAEL
Effect level:: > 936 mg/kg bw/day
Based on:: act. ingr.
Basis for effect level:: clinical signs; histopathology: non-neoplastic; mortality
Remarks on result:: other: No other remarks
Abnormalities:: no effects observed
Fetal body weight changes:: no effects observed
Reduction in number of live offspring:: no effects observed
Changes in sex ratio:: no effects observed
Changes in litter size and weights:: no effects observed
Changes in postnatal survival:: no effects observed
External malformations:: effects observed, non-treatment-related
Skeletal malformations:: no effects observed
Description (incidence and severity):: No skeletal variations were observed in increased incidence among fetuses of treated dams when compared with fetuses of control dams.
Visceral malformations:: no effects observed
Description (incidence and severity):: VISCeral variation were limited to hydronephrosis (grade 0) and distended ureter; the incidence of these was comparable between treated and control groups.
Details on embryotoxic / teratogenic effects:: Embryotoxic / teratogenic effects:no effectsDetails on embryotoxic / teratogenic effects:No indications of developmental toxicity including teratogenesis. All indices were comaparable to the corresponding controls.
: Key result
Dose descriptor:: NOAEL
Effect level:: > 3 000 mg/kg bw/day (nominal)
Based on:: test mat.
Sex:: male/female
Basis for effect level:: reduction in number of live offspring; changes in sex ratio; fetal/pup body weight changes; changes in litter size and weights; changes in postnatal survival; external malformations; skeletal malformations; visceral malformations
Remarks on result:: other: No other remarks
Abnormalities:: no effects observed
Developmental effects observed:: no
Conclusions:: NOAEL is 3,000 mg/kg bw day for maternal toxicity correspoding to 936 mg/kg active ingredient
NOAEL > 3,000 mg/kg bw day for developmental toxicIty correspoding to > 936 mg/kg active ingredient
Executive summary:: There were no indications of materna1 toxicity during the course of this study following similar method of OECD 414. One death occurred at the 1,500 mg/kg/day but this was considered a gavage injury and not test artide-relited. There were no test artide-related clinical observations or findings. There were no treatment-related effects on body weight or body weight gain in this study. There was a significant (p < 0.05) increase in food consuption during gestation day's interval 12-16. However, this was considered a normal variation and not a direct effect of the test artide. There were no indications of developmetal toxicity of any kind, induding teratogenesis in the course of this study. Developmental indices obtained at cesarean section indicated comparable results for the dose groups and the control group. .

Reference 3

Endpoint:: developmental toxicity
Type of information:: other: Read across from another category member
Adequacy of study:: supporting study
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: guideline study
Qualifier:: according to guideline
Guideline:: OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:: yes
Remarks:: see "principle of methods if other than guideline"
Principles of method if other than guideline:: There was one deviation from the test guideline OECD 414 during the main study. Guideline requires external fetal sex to be compared with internal sex in all foetuses (examined for both skeletal and soft tissue malformations). External fetal sex was compared with internal sex only in foetuses examined for soft tissue malformations, in foetuses examined for skeletal malformations it was not possible. The viscera of these foetuses were carefully removed by a very small incision from the abdominal cavity to stain the entire skeleton. This deviation from the test guideline did not affect the reliability of the study.
GLP compliance:: yes (incl. QA statement)
Limit test:: no
Species:: rat
Strain:: other: Wistar CRL
Details on test animals or test system and environmental conditions:: Species: Albino laboratory rat
Selection of animal species: Laboratory rat has been chosen because our testing laboratory has long experience with this species and therefore the historical data on key parameters are available and because rat is recommended according to the OECD 414 guideline.
Strain: Wistar CRL (SPF quality - guaranteed)
Supplier: Charles River SPF breeding, supplied via VELAZ s.r.o., Lysolajské údolí 15/53, 165 00 Prague 6, Czech Republic, RČH CZ 11760500
Sex: Sexually adult females and males
Age at start of the study: Dose-range finding experiment: 12 weeks, Main study: 12 weeks
Acclimatization: Dose-range finding experiment: 6 days, Main study: 6 days
Total number of animals: Dose-range finding experiment: total number of animals before mating: 24 females and 12 males (6 probably pregnant females per group)
Main study: total number of animals before mating: 100 females and 25 males (23 probably pregnant females per group)
Housing conditions: Dose-range finding experiment and main study: SPF conditions according to internal SOP No. 12
Light cycle: 12 hour light/12 hour dark
Microclimate: 22  3 °C, relative humidity 30 – 70 %
Animal per cage: Before mating 2 rats of the same sex in one cage, during mating period – one male and two females in one cage were housed. Pregnant females were then placed individually.
Bedding: Sterilized clean shavings of soft wood or sterilized LIGNOCEL (raw material - spruce; producer: J.Rettenmaier & söhne, Germany).
Food: Complete pelleted diet for rats and mice in SPF breeding (Altromin Spezialfutter) was used.
Manufacturer: Altromin Spezialfutter GmbH & Co. KG, Germany. Diet was sterilized before using.
Water: Free access to drinking water (water ad libitum). Water quality corresponded to Regulation No. 252/2004 Czech Coll. of Law, Health Ministry. Quality standard ČSN 757 111.
Selection of animals: Pregnant females were randomly assigned to the experimental groups after determination of pregnancy.
Identification of animals: The animals were identified by the colour marks (colour for veterinary usage) on their fur, each cage was marked with the number of animals, sex, cage number, name of the test item and dose level of the a.i..
Route of administration:: oral: gavage
Vehicle:: other: Aqua pro iniectione
Details on exposure:: The test item has been supplied as ca 40 % w/w Sodium 4-isopropylbenzenesulfonate aqueous solution. Therefore, a dose of 1000 mg of a.i./kg body weight is equivalent to approximately 2500 mg of the test item replenished with aqua pro iniectione to volume 10 mL/kg body weight.
Analytical verification of doses or concentrations:: yes
Details on analytical verification of doses or concentrations:: Stability and homogeneity of the test item application forms were determined by measuring of a peak area of the test item by a high-performance liquid chromatography based on a method developed at the test facility.
Measuring was performed on two concentrations of application form: 10 mg of a.i./10 mL and 1000 mg of a.i./10 mL.

Homogeneity
The samples were taken after 20 minutes of mixing by magnetic stirrer (500 rpm) from 3 given places - the bottom, the middle and the surface of the beaker content for the determination of homogeneity of both application forms. Two samples were taken from each place.

Stability
The samples were taken from the middle of the beaker content at required time intervals (0, 30, 60, 90 and 120 minutes) for the determination of stability of both application forms. Two samples were taken at all time intervals.
Time interval 0 min represents for both concentration levels the time after 20 minutes of mixing by magnetic stirrer at 500 rpm.
Details on mating procedure:: After acclimatization females were mated with males (1 male and 2 females). Vaginal smears were carried out daily in the morning to control fertilization (first time: 24 hours after the first removing to male). Presence of sperm was examined. Day 0 of pregnancy was the day on which sperm in vaginal smears were observed. Pregnant females were randomly distributed to experimental groups.
Duration of treatment / exposure:: Exposition lasted from implantation (the 5th day after fertilization) to one day prior to the day of scheduled euthanasia (the 19th day after fertilization). Male rats serve only for mating (they were not administered the test item or examined)
The test item was administered in graduated dose levels of a.i. to pregnant females from implantation (gestation day 5) to one day prior to the day of scheduled euthanasia (gestation day 19). The application form (test item in aqua pro iniectione) was administered to the stomach by gavage. The vehicle control group was administered by aqua pro iniectione in the same volume by gavage. The a.i. concentration at single dose level was adjusted so that the administered volume was constant at all dose levels – 1 ml/100 g body weight.
Frequency of treatment:: The animals were treated 7 days per week at the same time (8.00 – 10.00 am).
Duration of test:: 05.10. –21.10.2020
Dose / conc.:: 1 000 mg/kg bw/day (nominal)
Remarks:: based on active ingredient
Dose / conc.:: 500 mg/kg bw/day (nominal)
Remarks:: based on active ingredient
Dose / conc.:: 250 mg/kg bw/day (nominal)
Remarks:: based on active ingredient
Dose / conc.:: 0 mg/kg bw/day (nominal)
No. of animals per sex per dose:: Total number of animals: 24 females + 12 males (6 females and 3 males per dose-group)
Control animals:: yes, concurrent vehicle
Details on study design:: Vaginal smears: daily in mating period. These smears were stained and examined microscopically for presence of spermatozoa. Day 0 of pregnancy was the day when sperm were observed.
Body weight: on the 1st, 5th, 8th, 11th, 14th, 17th and 20th day of pregnancy
Food consumption: on the 5th, 8th, 11th, 14th, 17th and 20th day of pregnancy
Mortality control: daily - during the acclimatization, mating and pregnancy
Health condition control: daily - during the acclimatization, mating and pregnancy
General clinical observations: daily - during the administration period
Laboratory examinations:
- biometry of organs: on the 20th day of pregnancy
- biometry of thyroid gland: after fixation
- pathological examination of females: on the 20th day of pregnancy
- pathological examination of foetuses and AGD measuring: on the 20th day of pregnancy
- microscopical examination: after processing of selected foetuses
- histopathological examination of thyroid gland: after processing
- determination of thyroid hormones: after sampling of all samples
Maternal examinations:: The body weight of pregnant females was recorded on automatic balances with group mean computing module. First weighing was performed on the 1st day of pregnancy and then on the 5th, 8th, 11th, 14th, 17th and 20th day of pregnancy.
Food consumption was determined at three-day intervals; it coincided with the terms of body weight recording.

Mortality of females
All rats were examined for vitality or mortality changes daily during the acclimatization, mating and pregnancy.

Health condition control of females
The health condition was controlled daily during the acclimatization period, during the mating period and during pregnancy. Pre-experimental control of all females was performed to ensure that only the females exhibiting normal behavioral activity would be entered into the study. In administration period this observation was performed before application and immediately after application.

Clinical observations of the females
During the administration period clinical observation was made in order to record possible clinical effects of the test item application and all changes in behaviour of females. Females were observed in natural conditions in their cages after application, once a day 3 hours after test item application each day.

Thyroid Hormones
Blood samples from the pregnant females were assessed for serum levels of thyroid hormones (T3 - Triiodothyronine, T4 - Thyroxine, TSH- Thyroid Stimulating Hormone) by ELISA kits (manufacturer BioVendor, Brno, Czech Republic).

Biometry and histopathological examination of thyroid gland
During macroscopic examination the thyroid glands were removed from all pregnant females and were preserved in fixation medium. The thyroid weights were determined after fixation. For histopathological processing the routine histological paraffin technique with synoptic haematoxylin-eosin staining was used. The histopathology of thyroid glands was performed in all groups
Ovaries and uterine content:: Biometry of uterus and macroscopical examination of females
On the 20th day of pregnancy the females were euthanized. The revision of the external surface of the body was performed. During macroscopy, all orifices, the cranial, thoracic and abdominal cavities were examined and uterus (incl. the cervix) was removed and weighed. In gravid uteri, the number of viable foetuses, number of dead foetuses, number of early resorption (implantation without recognizable embryo/foetus) and number of late resorption (dead embryo or foetus with external degenerative changes) were recorded. The numbers of corpora lutea of both ovaries were recorded.
Uteri of non-pregnant females were examined to confirm the non-pregnant status (by the help of ammonium sulphide staining according to the internal SOP M/6 - Prenatal Developmental Toxicity).

Reproduction parameters
In uteri, the number of viable foetuses, number of dead foetuses and number of resorptions (implantation without recognizable embryo/foetus or dead embryo or foetus with external degenerative changes) were recorded. Number of corpora lutea on ovaries was also recorded. Preimplantation and postimplantation losses were calculated from number of implantations (number of foetuses plus number of resorptions), corpora lutea and resorptions.
Fetal examinations:: Pathological examination of foetuses and anogenital distance (AGD) measurement
Sex, individual body weights and anogenital distance (AGD) of foetuses were recorded. A digital calliper was used for AGD measurements. Corrected AGD was calculated according to the formula: AGD divided by the cube root of body weight.
Each foetus was examined for external alterations: symmetry of fore and hind limbs, number of fingers, closing or opening of eye fissures and external auditory canal, symmetry of head, integrity of superior palatum, status of umbilicus and genital papilla were observed.
One half of each litter (one half of female and male foetuses) was examined for soft tissue alterations using careful gross dissection.
Second half of each litter was processed and microscopically examined for skeletal alterations according to the internal SOP M/6 - Prenatal Developmental Toxicity. Single staining displayed only ossified skeletal structures was used: the foetuses were fixed in ethanol, macerated in potassium hydroxide solution, stained with Alizarin red and placed in glycerine-based solution.
The skeletal examination was performed using a stereomicroscope and included examination of skull, clavicle, scapula, sternebra and sternum, ribs, vertebrae, pelvic girdle, fore limb/hind limb.
Statistics:: For statistical evaluation the software Statgraphic® Centurion (version XV, USA) was used. The data from control group were compared with data from treated groups.
Indices:: number of corpora lutea, number of implantations, number of resorptions
number of live foetuses (males, females, both sex)
number of dead foetuses
Clinical signs:: no effects observed
Description (incidence and severity):: Clinical examinations of treated maternal animals detected no clinical symptoms of toxicity related to treatment with the test item. The behaviour, health condition and clinical status of treated maternal animals were similar compared to the control animals.
Mortality:: no mortality observed
Body weight and weight changes:: no effects observed
Description (incidence and severity):: The test item had no adverse effect on the growth of maternal animals. The body weight of treated maternal animals increased comparably with controls during pregnancy, and individual variability in body weight increments was normal for the species, sex and age of animals used in the study.
Food consumption and compound intake (if feeding study):: no effects observed
Description (incidence and severity):: Food consumption of treated mothers was balanced with controls except the statistically significant increase of food consumption in females at the middle dose level from the 11th day up to 14th day of pregnancy. This dose-independent increase in food consumption was not considered to be of toxicological significance.
Haematological findings:: no effects observed
Description (incidence and severity):: Haematological examination did not show significant differences between dosed and control group animals.
Behaviour (functional findings):: no effects observed
Gross pathological findings:: no effects observed
Description (incidence and severity):: Pathological examination of females revealed no pathological finding related to the test item treatment.
Evaluation of uterus weights did not demonstrate a negative effect of the test item treatment. The statistically significantly increased absolute and relative uterine weights were observed in females at the lowest and highest dose levels of a.i.. This increase was noted in relation to higher litter size at the treated groups. The corrected body weight of treated females was very slightly decreased at the dose level 1000 mg of a.i./kg bw/day.
Histopathological findings: non-neoplastic:: effects observed, non-treatment-related
Description (incidence and severity):: Absolute and relative weight of thyroid glands, histological examination of thyroid glands and serum levels of thyroid hormones, did not reveal any changes associated with the application of the test item. Histological examination of thyroid glands revealed only unilateral squamous cell cyst in one female at the dose level 250 mg of a.i./kg bw/day and in one female at the dose level 500 mg of a.i./kg bw/day. These findings were not treatment related and were probably of spontaneous origin
Histopathological findings: neoplastic:: no effects observed
Number of abortions:: no effects observed
Pre- and post-implantation loss:: no effects observed
Total litter losses by resorption:: no effects observed
Early or late resorptions:: no effects observed
Dead fetuses:: no effects observed
Changes in pregnancy duration:: no effects observed
Changes in number of pregnant:: no effects observed
: Key result
Dose descriptor:: NOAEL
Effect level:: > 1 000 mg/kg bw/day (nominal)
Based on:: act. ingr.
Basis for effect level:: body weight and weight gain; changes in number of pregnant; changes in pregnancy duration; clinical biochemistry; clinical signs; dead fetuses; early or late resorptions; effects on pregnancy duration; gross pathology; haematology; histopathology: non-neoplastic; maternal abnormalities; mortality; necropsy findings; number of abortions; ophthalmological examination; organ weights and organ / body weight ratios; pre and post implantation loss; total litter losses by resorption
Remarks on result:: other: No other remarks
Abnormalities:: no effects observed
Fetal body weight changes:: no effects observed
Description (incidence and severity):: Based on a statistical evaluation of mean values of foetal body weight no significant growth retardation was detected in treated groups.
Reduction in number of live offspring:: no effects observed
Description (incidence and severity):: The number of live foetuses, early and late intrauterine death were evaluated on the basis of examination of uterus content.
The reproductive parameters (numbers of implantations, corpora lutea and resorptions, preimplantation losses and postimplantation losses) were not adversely affected by the treatment with the test item.
No dead foetus was found in the treated groups. The number of live foetuses was not adversely affected by the treatment with the test item. The total numbers of live foetuses and the average number of foetuses per litter were increased in all treated groups in comparison with control. The average numbers of live male foetuses per litter were statistically significantly increased at dosed groups in comparison with control, but the average numbers of live female foetuses were comparable in all treated groups with control group.
Changes in sex ratio:: no effects observed
Changes in litter size and weights:: no effects observed
External malformations:: no effects observed
Description (incidence and severity):: No macroscopic changes of soft tissues and external alteration were found during the pathological examination of the foetuses at all dose levels of a.i..
The mean AGD and corrected AGD of male and female foetuses was comparable between treated and control groups, and therefore, was not affected by the test item application.
Skeletal malformations:: effects observed, non-treatment-related
Description (incidence and severity):: Examination of foetal skeletons indicated mainly delayed development of the skeleton at all dose levels of a.i. as well as in the control group. According to the OECD 414 the litter as the unit for data analysis should be used for statistical evaluation. The statistical evaluation was performed for the number of foetuses with skeletal findings and also for number of litters with skeletal findings in this study. No statistically significant differences with toxicological significance were recorded during the statistical evaluation of the number of foetuses/litters with skeletal findings.
Visceral malformations:: no effects observed
Description (incidence and severity):: Treatment with the test item was not associated with the occurrence of visceral variations and malformations.
Dose descriptor:: NOAEL
Effect level:: > 1 000 mg/kg bw/day (nominal)
Based on:: act. ingr.
Sex:: male/female
Basis for effect level:: reduction in number of live offspring; changes in sex ratio; fetal/pup body weight changes; changes in litter size and weights; changes in postnatal survival; external malformations; skeletal malformations; visceral malformations
Remarks on result:: other: No other remarks
Abnormalities:: no effects observed
Developmental effects observed:: no
Conclusions:: The NOAEL (No Observed Adverse Effect Level) for TOXICITY in pregnant females is 1000 mg of a.i./kg bw/day.
The NOAEL (No Observed Adverse Effect Level) for PRENATAL DEVELOPMENT is 1000 mg of a.i./kg bw/day.
Executive summary:: The potential of Sodium p-cumenesulphonate to cause effects on the reproductive performance of rats was assessed following OECD 414, Prenatal Developmental Toxicity Study. There were no deaths of females during the study at any dose of a.i. No adverse changes in health condition and no clinical symptoms of intoxication were observed in maternal animals following administration of the test item at any dose. Nor were there any toxicologically significant treatment-related effects on body weight or food consumption in maternal animals. Evaluation of uterus weights did not demonstrate a negative effect of the test item treatment. The statistically significantly increased absolute and relative uterine weights were observed in females at the lowest and highest dose levels of a.i.. This increase was noted in relation to higher litter size at the treated groups. The corrected body weight of treated females was very slightly decreased at the dose level 1000 mg of a.i./kg bw/day. Macroscopic structure of examined organs of pregnant females and reproduction parameters (number of females with live foetuses, number of live and dead foetuses, early and late resorptions)were unaffected by treatment with the test item. Examination of the thyroid glands (absolute and relative weight of thyroid gland, histological examination of thyroid gland and serum levels of thyroid hormones)did not reveal any changes associated with the application of the test item. Test item-related foetal mortality was not evident at any dose level. Detailed necropsy of foetuses did not reveal an increase of external and visceral variations and malformations at any dose of a.i.. Based on statistical evaluation of mean values of foetal body weight, no significant growth retardation was detected in treated groups. The mean AGD and corrected AGD of male and female foetuses in treated groups was not statistically significantly different from the control group. No statistically significant differences with toxicological significance were recorded in number of foetuses and litters during the statistical evaluation of skeletal findings.

Effect on developmental toxicity: via oral route

Endpoint conclusion:: no adverse effect observed
Dose descriptor:: NOAEL
: 936 mg/kg bw/day
Study duration:: subacute
Species:: rat
Quality of whole database:: The available developmental study was fully documented and conducted in accordance with GLP. A study in a second species (New Zealand white rabbit) confirms this result with a NOEL of 1000 mg/kg bw/day

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:: no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:: no study available

Justification for classification or non-classification

From the available studies of hydrotropes, there is no concerns for reprodution and developmenatl toxicity.

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Substance	CaXS
Doses (mg/kg/bw)	0, 150, 1500 and 3000 mg/kg/bw corresponding to: 0; 45,8; 458 and 936 mg/kg (a..i)
Species	CRL rats
Number of females	30 for each group
Number of died females	1 female at 1500 mg/kg/bw
Number of non-pregnant females	Control: 2 150 mg/kg: 1 1500 mg/kg: 5 3000 mg/kg: 5
Number of dams with abortions	0
Number of dams with early deliveries	0
Number of dams with stillbirths	0
Number of dams with resorptions	0
Total number of live foetuses	Control: 435 150 mg/kg: 465 1500 mg/kg: 408 3000 mg/kg: 471
Number of live foetuses – males	Control: 217 150 mg/kg: 222 1500 mg/kg: 203 3000 mg/kg: 200
Number of live foetuses – females	Control: 218 150 mg/kg: 243 1500 mg/kg: 205 3000 mg/kg: 217
Number of dead foetuses	Control: 0 150 mg/kg: 0 1500 mg/kg: 0 3000 mg/kg: 0
Number of implantations per female	Control: 17,22 ± 3,02 150 mg/kg: 17,00 ± 3,47 1500 mg/kg: 16,88 ± 2,51 3000 mg/kg: 17,36 ± 1,66
Number of resorptions per female	Control: 1,11 ± 1,09 150 mg/kg: 8,39 ± 18,91 1500 mg/kg: 3,20 ± 3,70 3000 mg/kg: 3,90 ± 4,97
Number of corpora lutea per female	Control: 18,96 ± 3,58 150 mg/kg: 19,43 ± 2,3 1500 mg/kg: 18,84 ± 3,77 3000 mg/kg: 18,72 ± 1,62
Pre-implantation loss, percentage per female (IUDE)	Control: 8,97 ± 8,60 150 mg/kg: 9,60 ± 9,45 1500 mg/kg: 9,61 ± 7,55 3000 mg/kg: 7,21 ± 4,80
Post-implanation loss, percentage per female (IUDL)	Control: 6,78 ± 6,43 150 mg/kg: 8,39 ± 18,91 1500 mg/kg: 3,20 ± 3,70 3000 mg/kg: 3,90 ± 4,97
Pre-implantation loss, number	not repoted
Post-implanation loss, number	Control: 30 150 mg/kg: 28 1500 mg/kg: 14 3000 mg/kg: 17
Body weight at 1st and 20th day of pregnancy (grams for rat, kg for rabbits)	Control: 275,4 ± 9,1, 452,6 ± 31,1 150 mg/kg: 275,8 ± 14.5, 448,3 ± 341,3 1500 mg/kg: 276,9 ± 15,4, 454,7 ± 28,5 3000 mg/kg: 273,0 ± 13,9, 453,0 ± 27,3
Body weight increment, grams	Control: 177.1 ± 27.3 150 mg/kg: 172.5 ± 33.4 1500 mg/kg: 178.7± 18.6 3000 mg/kg: 180.0 ± 20.3
Absolute weight and relative (%) weight of the thyroid	not examined
Histopathology of the thyroid	not examined
Results of the thyroid hormone (T4, T3 and TSH)	not examined
Mean number of live offspring, litter	Control: 16.11± 3.08 150 mg/kg: 16.03 ± 3.94 1500 mg/kg: 16.32 ± 2.39 3000 mg/kg: 16.68 ± 1.82
Percent of live offspring, litter	Control: 93.22 ± 6.43 150 mg/kg: 91.61 ± 18.91 1500 mg/kg: 96.80 ± 3.70 3000 mg/kg: 96.10 ± 4.97
Mean foetal/pup body weight by sexes combined	Control: 3,64 ± 80,19 150 mg/kg: 3,65 ± 0,35 1500 mg/kg: 3,75 ± 0,23 3000 mg/kg: 3,70 ± 0,14
Mean foetal/pup body weight female	Control: 3,54 ± 0,22 150 mg/kg: 3,56 ± 0,33 1500 mg/kg: 3,66 ± 0,22 3000 mg/kg: 3,62 ± 0,14
Mean foetal/pup body weight male	Control: 3,75 ± 0,20 150 mg/kg: 3,74 ± 0,38 1500 mg/kg: 3,84 ± 0,27 3000 mg/kg: 3,79 ± 0,15
AGD (female) and corrected AGD (female), mm	not reported
AGD (male) and corrected AGD (male), mm	not reported
Number (and percent) of foetuses and litters with malformation (including runts) and/or variations, external	Control: 0 fetal; 0 litter 150 mg/kg: 4 (0.9%) fetal; 3 (10.7%) litter 1500 mg/kg: 1 (0.2%) fetal; 1 (4%) litter 3000 mg/kg: 1 (0.2%) fetal; 1 (4%) litter
Description and incidences of malformations and main variations	Occasional effects, not treatment related
Macroscopic changes of soft tissues – individual foetuses
Total number of examined foetuses	Control: 215 150 mg/kg: 231 1500 mg/kg: 202 3000 mg/kg: 206
Total number of examined litters	Control: 27 150 mg/kg: 28 1500 mg/kg: 25 3000 mg/kg: 25
With pathological findings - total (number of affected foetuses)	Not available. The number is alteration specific
Number of examined foetuses in litter (mean ± SD)	Control: 7.96 150 mg/kg: 8.25 1500 mg/kg: 8.08 3000 mg/kg: 8.24
Total number of foetuses with alteration	Not available. The number is alteration specific
Number of foetuses with alteration in litter (mean ± SD)	not reported
Portion of foetuses with alteration in litter(% mean ± SD)	not reported
Skeletal findings, litter	Occasional effects, not treatment related

Pregnancy results
Group code	Number of females with foetuses	Number of non-pregnant females*	Number of females without live foetuses but with implantations and resorptions
0	21	2 (113, 117)	0
250	22	1 (141)	0
500	23	0	0
1000	22	1 (192)	0

Body weight in grams (mean ± standard deviation)
Day of pregnancy	Group code
Day of pregnancy	0	250	500	1000
1^stday	269.78± 14.61	275.00± 14.33	274.08 ± 12.99	265.71 ± 14.04
5^thday	286.39 ± 16.09	290.47± 13.83	290.17 ± 15.27	282.35 ± 15.25
8^thday	296.65 ± 17.43	301.50 ± 16.15	301.64 ± 16.15	293.96 ± 16.96
11^thday	311.69 ± 17.84	316.35 ± 17.83	318.73 ± 18.10	309.68 ± 19.02
14^thday	326.05 ± 20.79	331.74 ± 21.00	336.07 ± 20.05	324.26 ± 17.15
17^thday	358.78 ± 23.21	369.71 ± 25.87	373.11 ± 27.78	362.45 ± 21.16
20^thday	400.87 ± 30.94	413.99 ± 35.76	414.95 ± 36.48	403.36 ± 25.17
Mean increment	131.09	138.98	140.87	137.65

Food consumption/animal/day (grams)
Day of pregnancy	Group code
Day of pregnancy	0	250	500	1000
5^thday	22.18	22.81	22.33	22.33
8^thday	24.19	25.07	26.17	24.58
11^thday	25.47	26.51	28.15	26.89
14^thday	27.43	27.37	29.42	27.72
17^thday	28.64	29.27	30.68	30.08
20^thday	30.47	30.71	32.29	32.07

Health condition control
Week of pregnancy	Group code
Week of pregnancy	0	250	500	1000
1^stweek	1	1	1	1
2^ndweek	1	1	1	1
3^rdweek	1	1	1	1

Clinical observation
Week of pregnancy	Group code
Week of pregnancy	0	250	500	1000
1^stweek	1	1	1	1
2^ndweek	1	1	1	1
3^rdweek	1	1	1	1

Biometry of uterus (mean± standard deviation)
Parameter	Group code
Parameter	0	250	500	1000
Mean necropsy body weight of females (g)	400.87 ± 30.94	413.99 ± 35.76	414.95 ± 36.48	403.36 ± 25.17
Mean absolute weight of uterus (g)	79.72 ± 21.52	93.42 ± 21.81	87.15 ± 23.89	89.26± 11.75
Mean relative weight of uterus (%)	19.64 ± 4.31	22.32 ± 4.45	20.74 ± 5.16	22.09 ± 2.31

Body weight - corrected* (mean ± standard deviation)
Parameter	Group code
Parameter	0	250	500	1000
Body weight(g)	321.15 ± 15.74	320.57 ± 20.78	327.80 ± 23.58	314.10 ± 19.55

Macroscopic findings (number of females with pathological findings)
Parameter	Group code
Parameter	0	250	500	1000
Number of examined females	23	23	23	23
Number of died females	0	0	0	0
Without pathological findings	23	23	23	23

Parameters of reproduction (number per female,mean ± standard deviation)
Parameter	Group code
Parameter	0	250	500	1000
Implantations	14.14± 3.77	16.23± 3.61	14.96± 4.05	16.00± 2.16
Resorptions	0.57± 0.93	0.45± 0.67	0.35± 0.49	0.50± 0.80
Corpora lutea	14.67± 3.34	16.73± 2.91	15.87± 3.57	16.50± 2.13

I U D E and I U D L (% per female,mean ± standard deviation)
Parameter	Group Code
Parameter	0	250	500	1000
IUDE	5.14± 14.85	4.88± 14.17	6.93± 13.14	3.06± 4.17
IUDL	4.28± 7.15	2.63± 3.87	3.99± 10.41	3.38± 6.01

Weight of thyroid gland (group mean±standard deviation)
Thyroid gland	Group code
Thyroid gland	0	250	500	1000
Absolute weight (g)	0.0312 ± 0.0019	0.0307 ± 0.0020	0.0311 ± 0.0015	0.0317 ± 0.0017
Relative weight (%)	0.0078 ± 0.0009	0.0075 ± 0.0008	0.0075 ± 0.0008	0.0079 ± 0.0006

Thyroid hormones (mean concentration ±standard deviation)
Hormone	Group code
Hormone	0	250	500	1000
T3(ng/mL)	0.701 ± 0.079	0.679 ± 0.095	0.689 ± 0.068	0.683 ± 0.092
T4(µg%)	2.997 ± 0.641	2.852 ± 0.517	2.825 ± 0.574	2.844 ± 0.387
TSH(ng/mL)	0.773 ± 0.285	0.766 ± 0.384	0.663 ± 0.257	0.844 ± 0.426

Number of foetuses (total in group)
Parameter	Group code
Parameter	0	250	500	1000
Total number of live foetuses	285	347	345	341
Number of live foetuses – males	127	191	173	190
Number of live foetuses – females	158	156	172	151
Number of dead foetuses	1	0	0	0

Number of foetuses (average per litter; mean ±standard deviation)
Parameter	Group code
Parameter	0	250	500	1000
Total number of live foetuses	13.57 ± 3.92	15.77 ± 3.53	15.00 ± 4.15	15.50 ± 2.50
Number of live foetuses – males	6.05 ± 3.28	8.68 ± 2.85	7.52 ± 2.59	8.64 ± 2.56
Number of live foetuses – females	7.52 ± 2.80	7.09 ± 2.65	7.48 ± 2.84	6.86 ± 2.47
Number of dead foetuses	0.05 ± 0.22	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00

Body weight of foetuses (grams, mean ±standard deviation)
Parameter	Group code
Parameter	0	250	500	1000
weight of all foetues	3.89± 0.83	3.97± 0.74	3.94± 0.74	3.72± 0.41
weight of male foetus	4.01± 0.90	4.05± 0.77	4.02 ± 0.77	3.80± 0.43
weight of female foetus	3.80± 0.83	3.87± 0.71	3.86 ± 0.73	3.60± 0.34

Mean anogenital distance of foetuses (mm)
Group code	0		250		500		1000
Sex	Male	Female	Male	Female	Male	Female	Male	Female
Mean AGD (mm)	3.59	2.27	3.67	2.29	3.68	2.25	3.63	2.22
Corrected AGD (mm)	2.27	1.47	2.31	1.47	2.33	1.44	2.33	1.45

External alterations - foetuses
Alteration	Group code
Alteration	0	250	500	1000
Total number of examined foetuses	285	347	345	341
Total number of examined litters	21	22	23	22
Dead foetus	1	0	0	0
Foetus without tail, absent end part of the spine	0	0	0	0
Number of examined foetuses in litter (mean ± SD)	13.57 ±3.92	15.77 ±3.53	15.00 ±4.15	15.50 ±2.50
Total number of foetuses with alteration	1*	0	0	0
Number of foetuses with alteration in litter(mean ± SD)	0.05 ±0.22	0.00 ±0.00	0.00 ±0.00	0.00 ±0.00
Portion of foetuses with alteration in litter (% mean ± SD)	0.60 ±2.73	0.00 ±0.00	0.00 ±0.00	0.00 ±0.00

Registration Dossier

Registration Dossier

Data platform availability banner - registered substances factsheets

Diss Factsheets

Sodium toluene-4-sulphonate

Endpoint summary

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Link to relevant study records

Referenceopen allclose all

Reference 1

Reference 2

Table No. 1: Body weight (g) – Mean values

Table No. 2: Body weight (g) – Mean values

Haematological examination

MALES

Parameter

Unit

Dose level (nominal)

Haematological examination

FEMALES

Parameters

Unit

Effect on fertility: via oral route

Effect on fertility: via inhalation route

Effect on fertility: via dermal route

Effects on developmental toxicity

Description of key information

Link to relevant study records

Referenceopen allclose all

Reference 1

Reference 2

Reference 3

Pregnancy results

Table 3

Body weight in grams

(mean ± standard deviation)

Group code

0

250

500

1000

Table 4

Food consumption/animal/day

(grams)

Group code

0

250

500

1000

Health condition control

Group code

0

250

500

1000

Clinical observation

Group code

0

250

500

1000

Biometry of uterus

(mean± standard deviation)

Parameter

Group code

0

250

500

1000

Body weight - corrected*

(mean ± standard deviation)

Parameter

Group code

0

250

500

1000

Referenceopen all close all

Referenceopen all close all