Reaction mass of sodium (methylbutyl and pentyl) sulfonate and sodium 1,2-bis(pentyloxycarbonyl)ethanesulphonate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Reproductive toxicity screening was performed with the registered substance in Wistar rats by oral gavage at 0 (propylene glycol), 100, 300 and 1000 mg/kg bw/day in key combined repeated dose/reproductive toxicity study (OECD TG No. 422). The NOAEL for reproductive effects of the parental generation was considered to be 1000 mg/kg bw/day (based on no significant findings). The NOAEL for pup development and survival was considered to be 1000 mg/kg bw/day (based on no significant findings).

Based on the absence of reproductive findings in the repeated dose toxicity studies and the combined repeated dose/reproductive toxicity study, no further testing is needed.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 September 2020 to 21 February 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Qualifier:

according to guideline

Guideline:

other: OECD No. 43 Guidance Document on Mammalian Reproductive Toxicity Testing and Assessment

Version / remarks:

2008

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI (Wistar) rats

Details on species / strain selection:

The test system and the number of animals used in the study were in compliance with the relevant OECD No. 422 guideline. The guideline is designed for use with the rat, which is the preferred rodent species for reproduction toxicity testing. Wistar rat was selected due to experience of the Test Facility with this strain of rat in toxicity and reproduction toxicity studies and its known fertility.
The minimum number of animals was used, corresponding to the regulatory guidelines being followed, but taking into consideration the scientific reliability of the collected information

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH (Address: Sandhofer Weg 7, D-97633 Sulzfeld, Germany) from SPF colony
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Young adult rats, 10/11 weeks old (females/males) at start of the treatment and 12/13 weeks old (females/males) at mating.
- Weight at study initiation: Males: 382-462 g, females: 232-276 g (at the start of the treatment). The body weights did not exceed ± 20% of the mean weight for each sex at start of treatment.
- Fasting period before study: Not specified
- Housing: Rodents were group-housed, up to 2 animals of the same sex and dose group/cage, with the exception of the mating and gestation, delivery, lactation period, when they were paired or individually housed (with pups), respectively. Rodents were housed in Type II, III and/or IV polycarbonate cages. SAFE 3/4-S Hygienic Animal Bedding (Batch number: 03027200710, Expiry date: 10 July 2023) and SAFE Crinklets Natural nesting material (Batch number: 05072200405, Expiry date: 05 April 2023) produced by J. Rettenmaier & Söhne GmbH+Co.KG (Address: Holzmühle 1, D-73494 Rosenberg, Germany) were used in the study. Group housing allowed social interaction. Deep wood sawdust bedding allowed digging and other normal rodent activities, while nesting material allowed normal nesting behaviour. Certified cardboard hiding tunnels (GLP Mini Fun Tunnels, Batch number: A123) produced by LBS (Serving Biotechnology) Ltd. (Address: Unit 20, Gatwick Business Park, Kennel Lane, Hookwood, Surrey, RH6 0AH UK) were also provided to the animals.
- Diet (e.g. ad libitum): Animals received ssniff® SM R/M "Autoclavable complete diet for rats and mice – breeding and maintenance" (Batch number: 560 65984 / 713 70882, Expiry date: 30 November 2020 / 30 April 2021) produced by ssniff Spezialdiäten GmbH (Address: Ferdinand Gabriel Weg 16, D-59494 Soest, Germany) ad libitum.
- Water (e.g. ad libitum): Animals received tap water from municipal supply, as for human consumption from a 400- or 500-mL bottle, ad libitum.
- Acclimation period: 8 days.

DETAILS OF FOOD AND WATER QUALITY: A sample (approximately 100 g) of batch of diet used in the study was retained and kept under appropriate environmental conditions until the finalization of the study report.
The food was routinely analysed and considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
The quality control analysis of the water was performed once every three months and microbiological assessment was performed monthly, by Veszprém County Institute of State Public Health and Medical Officer Service (H-8200 Veszprém, József Attila u. 36., Hungary). Copies of the relevant Certificates of Analysis are included in the raw data and are archived at the Test Facility.
The water was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.8-25.0℃ (target range: 19-25°C)
- Humidity (%): 28-66% (target range: 30-70%)
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12/12 (from 6.00 a.m. to 6.00 p.m.)

IN-LIFE DATES:
From: Start of in life phase: 25 September 2020 (first vaginal smear sampling)
To: End of in life phase: 02 December 2020 (last necropsy)

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
As agreed with the Sponsor, no correction for purity of the test item was applied during formulation.
The test item was formulated in the selected vehicle (propylene glycol), as a visibly stable homogenous solution at the appropriate concentrations according to the dose level and volume selected in the Pharmacy of the Test Facility. The formulations were stirred with manual shaking and a magnetic stirrer from the preparation until completion of each treatment.
Formulations were prepared fresh every day prior to administration to animals according to stability assessment results of the analytical method validation study (Study code: 20/031-316AN).
Formulations were prepared in clean glass containers. The appropriate amount test item was weighed into a clean, calibrated glass container and then mixed properly (with manual shaking and magnetic stirring) with the needed amount of vehicle to reach homogeneity by visual observation. During the formulation, approximately 1-hour ultrasound sonication was applied for proper homogenisation. Formulations were stored surrounded with warming-blocks in a closed container with the intention of keeping formulations above ~25°C until use.

VEHICLE: propylene glycol
- Justification for use and choice of vehicle (if other than water): Based on the available information provided by the Sponsor as well as results of a trial formulation performed at the Test Facility, distilled water was selected as vehicle for the Dose Range Finding (DRF) study (Study code: 20/031-220PE). It was noted during the DRF study that the surfactant type test substance (foamy aqueous formulations) caused local respiratory irritation (by reflux or test item present around the upper respiratory tract) which was considered as a cause of the deaths. These clinical findings were considered not to be related to systemic toxicity, rather the characteristic of the formulations with local effects. Based on these data, as well as results of an additional formulation trial with different vehicles, propylene glycol (abbreviated as PG) was selected as vehicle for this study in agreement with the Sponsor, to reduce the foaming of the formulations and irritation effects. PG is considered as being an acceptable vehicle based on the scientific literature and practice of the Test Facility.
- Concentration in vehicle: 0, 20, 60 and 200 mg/mL (dose formulation concentration)
- Amount of vehicle (if gavage): 5 mL/kg bw (dose formulation volume)
- Lot/batch no. (if required): 1920944
- Purity: 99.99%

Details on mating procedure:

M/F ratio per cage: 1/1
- Length of cohabitation: Females remained with the same male until copulation occurred, for up to 10 days.
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy.
A vaginal smear was prepared daily during the mating period and stained with 1% (w/v) aqueous methylene blue solution. The smear was examined with a light microscope, the presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation.
- Successful coitus (sperm positive vaginal smears and/or vaginal plugs) occurred mostly within 4 days of pairing (cohabitation).
- After successful mating each pregnant female was caged (how): Sperm positive females were caged individually.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Sample collection was performed at a total of three occasions (during the first and last weeks and midway during the treatment period). Samples were collected immediately after formulation preparation in the Pharmacy of the Test Facility by a responsible member of the Analytical Department.
On each sampling occasion, top, middle and bottom duplicate samples were taken from the dedicated test item formulation(s) for concentration and homogeneity measurement, one set to analyse (which was collected in replicates as practical) and one set as a back-up, if required for any confirmatory analyses. Similarly, duplicate samples were taken on three occasions from the middle of the vehicle control formulation for concentration measurement.
After the analytical sampling, the collected formulation samples were stored at room temperature until measurement.
Analysis of control (vehicle) and test item formulations for concentration and/or homogeneity was performed in the Analytical Laboratory of the Test Facility. Representative samples of control (vehicle) and/or test item formulations were analysed three times during the study (during the first and last weeks and approximately midway during the treatment period).
Any samples not required for analysis were discarded following acceptance of the results of the formulation analysis by the Contributing Scientist #1 (Analyst) and Study Director.
The formulation analysis was conducted within the determined stability period under the control of the responsible Contributing Scientist #1 (Analyst) in compliance with the analytical method validation and the relevant SOPs of the Test Facility.
Analysis of the formulations for concentration and/or homogeneity of test item was performed using a validated analytical HPLC-UV method (High Performance Liquid Chromatography with ultraviolet detection) in the Analytical Department of the Test Facility by using a validated analytical method (Study code: 20/031-316AN). The density of the formulations was determined at the first analytical sampling by using three parallels in the Analytical Department of the Test Facility, as it was deemed necessary by the Contributing Scientist #1 (Analyst) and Study Director.
Acceptance criteria of the concentration analysis was 100 ± 10% of the nominal concentration.
Acceptance criteria of the homogeneity was that the CV (coefficient of variation) of replicates (top, middle and bottom of test item formulations) must be less than 10%.
The measured concentrations of the test item in the different formulations varied between 98% and 102% of the nominal concentrations.
All test item formulations were shown to be homogeneous. The relative standard deviation (RSD) was below 10% in each case.
Formulations were considered to be adequately stable under the study conditions.
Overall, the formulations were considered adequate for the study.

Duration of treatment / exposure:

Dosing of both sexes began after the acclimatisation (8 days) and pre-exposure period (14 days), and it was performed 2 weeks before mating, during the mating, and was continued up to and including the day before the necropsy.
The first day of dosing of each animal was regarded as Day 0.
Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating period), then were euthanized and subjected to necropsy examination.
Females were dosed for 14 days pre-mating, for up to 14 days mating period, through gestation and up to and including the day before necropsy (13 days post-partum dosing). The day of birth (when parturition was complete) was defined as Day 0 post-partum. Females not delivering were sacrificed as practical (on the first day of dam’s necropsy).

Frequency of treatment:

daily on a 7 days/week basis, in each morning

Details on study schedule:

Screening study (only Parental generation was mated)
- Age at mating of the mated animals in the study: 12/13 weeks old (females/males) at mating

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected by the Sponsor in consultation with the Study Director based on the results of a Dose Range Finding (DRF) study. Based on those results, 1000 mg/kg bw/day was selected as the High dose for this study
- Rationale for animal assignment (if not random):
All adult/parental (P) male and female animals were sorted according to body weight by computer and divided into weight ranges. There were an equal number of animals from each weight group randomly assigned to each dose group to ensure that animals of all test groups were as nearly as practicable of a uniform weight.
This process was controlled by the software PROVANTIS v.9, to verify the homogeneity/variability between/within the groups. Males and females were randomised separately to the dose groups on the day of the first treatment (prior to the start of the treatment).
Any unused, spare animals were moved back to the stock colony after they were not needed for the study (after the experiment was ended).
Fifty-five male and 65 female Wistar rats were used in the pre-exposure period. At the end of the pre-exposure period, only 48 females showing regular oestrus cycles and 48 males were allocated to treatment groups (12 animals/sex/groups, 4 groups). Animals were assigned to groups before the start of the treatment
- Fasting period before blood sampling for clinical biochemistry: yes, overnight period of food deprivation, in case of females this happened after the litter has been culled.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: General (routine) clinical observations were made once a day*, during the pre-treatment and treatment period in the afternoon (pm).
*Note: No general clinical observations were made on the day of necropsy.
Animals were inspected for signs of morbidity and mortality once per day in the pre-treatment period and twice daily in the treatment period (at the beginning and end of each working day). Any animal (including also all premature decedents) which showed clinical signs considered severe was sacrificed to prevent suffering, cannibalism and/or autolysis, and was processed in the same way as the animals subjected to terminal necropsy

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made at the start of the pre-exposure period and once before the first exposure on Day 0 (to allow for within-subject comparisons), then weekly (in the morning (am), before treatment) and on the day of necropsy. These observations were made outside the home cage in a standard arena, at similar times as practical. Signs evaluated included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self- mutilation, walking backwards) were also recorded. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
Pertinent behavioural changes and all signs of toxicity including mortality were recorded including onset, degree and duration of signs as applicable.
On Gestation Day (GD) 13 and/or 14 the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat).
Furthermore, mated females were examined carefully around the time of expected delivery for any signs of difficult or prolonged parturition. The delivery time was recorded as when it was considered that all pups had been born; checking of cages was made during each day up until 16:00.

BODY WEIGHT: Yes
- Time schedule for examinations: All adult animals were weighed with accuracy of 1 g weekly during the pre-exposure period, then on Day 0, and afterwards weekly, and at termination.
Parent females were weighed on Gestation Day (GD) 0, 3, 7, 10, 14, 17 and 20, on PPD (Post-partum Day) 0, 4, 7, 10 and 13, and at termination. The body weight of the female animals measured on GD3, GD10 and GD17 as well as PPD10 were only additional measurements as aid for the calculation of accurate treatment volumes, but these data was not evaluated statistically.

FOOD CONSUMPTION AND COMPOUND INTAKE (no feeding study):
Animal food consumption was determined by weighing the non-consumed diet with a precision of 1 g at least weekly (on a body weight measurements day). No food consumption was measured during mating. Food consumption was measured more frequently during the lactation period (at least on PPD0, 4, 7, 10 and 13).
Main daily food consumption was calculated for each interval.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (no drinking water study): No
No water consumption was measured in the study.

Oestrous cyclicity (parental animals):

Oestrus cycles was monitored by vaginal smears daily during the pre-exposure period before the treatments started. Any females that fail to show a 4-5 days cycles were not included in the study. Vaginal smears were also checked daily from the beginning of the treatment period until evidence of mating (during the pre-mating and mating periods).
Additionally, vaginal smears were prepared and examined for each surviving female on the day of necropsy to determine the stage of oestrus cycle and allow correlation with histopathology of the reproductive organs.

Sperm parameters (parental animals):

Parameters examined in P male parental generations:
-All surviving adult animals: with a precision of 0.01 g organ weights of testes, epididymides, prostate, seminal vesicles with coagulating glands.
Testes and epididymides were weighed individually. Individual and/or paired absolute organ weight was reported for each animal and adjusted for the body and brain weights. Paired organ weights as applicable were summarised. Relative organ weight (to body and brain weight) was calculated and reported.
-For the adult animals, detailed histological examination was performed on the retained reproductive organs (testes, epididymides, prostate gland, seminal vesicles with coagulation gland for males) of all animals of the Control and High dose groups
Parameters examined in F1 male parental generations:
-No histopathological examination was performed on pups (F1 generation).
-Dead pups and pups killed on PND4 and/or PND13 were carefully examined externally for gross abnormalities. After the external observation, the sex determined at birth was confirmed by observation of the internal reproductive organs, if possible. Presence of nipples/areolae in the PND13 male pups was also recorded.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, 5/sex/litter as nearly as possible.
Pups to be culled within each litter were selected randomly. In litters of insufficient size where the number of males or female pups was less than 5, adjustment of the selection process was made to assure 10 pups were retained. Culling was not performed on litter sizes less (or equal) than 10. All culled pups were subjected to necropsy with detailed macroscopic external and internal examination for any abnormalities.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, runts, postnatal mortality, presence of gross anomalies, individual weight, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD (PND0), presence of nipples/areolae in male pups.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities; possible cause of death was determined for pups born or found dead

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals: Terminally (one day after the last treatment (14 days mating/post-mating period))
- Maternal animals: All surviving animals: Terminally (one day after the last treatment (13 days post-partum dosing))

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities was opened, and the appearance of the tissues and organs were observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate. Special attention was paid to the organs of the reproductive system.
Vaginal smears were prepared and examined for each female on the day of necropsy to determine the stage of oestrus cycle and allow correlation with histopathology of the reproductive organs.
The number of implantation sites and of corpora lutea was recorded in the females as applicable.

HISTOPATHOLOGY / ORGAN WEIGHTS
At the time of termination, body weight and weight of the following organs of all surviving adult animals was determined:
•With a precision of 0.01 g: uterus (including cervix), testes, epididymides, prostate, seminal vesicles with coagulating glands, brain, heart, kidneys, liver, spleen and thymus
•With a precision of 0.001 g: adrenals, ovaries, thyroids with parathyroids
Testes and epididymides were weighed individually. Individual and/or paired absolute organ weight was reported for each animal and adjusted for the body and brain weights. Paired organ weights as applicable were summarised. Relative organ weight (to body and brain weight) was calculated and reported.
The weighed organs and all organs showing macroscopic lesions of all adult animals were preserved. The eyes with the optic nerve, testes and epididymides were retained in modified Davidson’s fixative, all other organs in 10% buffered formalin solution.
In addition, on completion of the macroscopic examination the following tissues and organs were retained from all surviving animals:
Gross findings
Adrenal gland
Animal identification (Fixation and preservation only)
Aorta (Aorta thoracic and abdominal)
Brain (7 section according to the NTP recommendations)
Epididymis
Eye with the optic nerve (If applicable, parathyroids and optic nerves was examined histologically only if present in routine sections)
Oesophagus
Femur with marrow
Heart (Section including both ventricles and atria, septum with papillary muscle)
Kidney
Large intestine (Caecum, colon and rectum)
Extraorbital lachrymal gland
Harderian gland
Liver (Liver, 3 lobes, left lateral, right medial, caudate)
Lungs with bronchi (Lungs of euthanized animals was infused with formalin; 3 lobes, left, right cranial, right caudal)
Lymph node ( Mandibular and mesenteric)
Ovary
Oviduct
Pancreas
Pituitary
Prostate
Salivary gland (including mandibular, sublingual and parotid glands)
Sciatic nerve
Seminal vesicle with coagulating gland
Skin, subcutis with mammary gland (inguinal)
Skeletal muscle (quadriceps)
Small intestine (Duodenum, ileum and jejunum with Peyer’s patches)
Spinal cord (Transverse sections, 3 levels: cervical, thoracic and lumbar)
Spleen
Sternum with marrow
Stomach
Testis
Thymus
Thyroid with parathyroid gland (If applicable, parathyroids and optic nerves was examined histologically only if present in routine sections)
Tongue
Trachea
Urinary bladder
Uterus (Horns, body and cervix)
Vagina
Additionally, thyroid glands from one male and one female PND13 pup from each litter were preserved in 10% buffered formalin solution. In this case, the thyroid weight (pooled) was determined after fixation. Trimming was done very carefully and only after fixation to avoid tissue damage.
The retained tissues and organs required for histopathology (below) were embedded in paraffin wax; sections were cut at 4-6 μm by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope.
For the adult animals, detailed histological examination was performed as follows:
• on the selected list of retained tissues and organs (as above) in the Control and High
dose groups (at least 5 animals/sex/group),
• any animals found dead or euthanized pre-terminally during the study in all groups,
• all macroscopic findings (abnormalities), except of minor order from all animals,
• on the retained reproductive organs (testes, epididymides, prostate gland, seminal vesicles with coagulation gland for males and uterus, cervix, ovary, oviduct and vagina for females) of all animals of the Control and High dose groups, and all females that failed to deliver healthy pups (documented by a memo in the raw data),
• on the spleen in all Control and High Dose animals, as a special request of study pathologist (described in MEMO),
• the additional histology on the non-glandular region of the stomach of the Control, Low and Mid dose animals to determine the appropriate dose level of the NOAEL according to the Amendment 2 to the Study Plan.
Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.
Special attention was paid to the organ weight, appearance and histopathology of immune-system tissues for any evidence of immunotoxicity (spleen, thymus, lymph nodes, bone marrow).
Special attention was paid to the central and peripheral nervous system tissues for any evidence of neurotoxicity.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring: Dead pups and pups killed on PND4 and/or PND13 were carefully examined externally for gross abnormalities. After the external observation, the sex determined at birth was confirmed by observation of the internal reproductive organs, if possible. Presence of nipples/areolae in the PND13 male pups was also recorded.

GROSS NECROPSY
- Dead pups and pups killed on PND4 and/or PND13 were carefully examined externally for gross abnormalities. After the external observation, the sex determined at birth was confirmed by observation of the internal reproductive organs, if possible. Presence of nipples/areolae in the PND13 male pups was also recorded.
Additionally, thyroid glands from one male and one female PND13 pup from each litter were preserved in 10% buffered formalin solution. In this case, the thyroid weight (pooled) was determined after fixation. Trimming was done very carefully and only after fixation to avoid tissue damage.

HISTOPATHOLOGY / ORGAN WEIGTHS
No histopathological examination was performed on pups (F1 generation).

Statistics:

See under "Any other information on material and methods, incl. tables"

Reproductive indices:

Male Mating Index = (Number of males with confirmed mating / Total Number of males cohabited) x 100: Measure of male’s ability to mate

Female Mating Index = (Number of sperm-positive females / Total Number of females cohabited) x 100: Measure of female’s ability to mate

Male Fertility Index = (Number of males impregnating a female / Total Number of males cohabited) x 100: Measure of male’s ability to produce sperm that can fertilise eggs

Female Fertility Index = (Number of pregnant females / Number of sperm-positive females) x 100: Measure of female’s ability to become pregnant

Gestation Index = (Number of females with live born pups / Number of pregnant females) x 100: Measure of pregnancy that provides at least one live pup

Offspring viability indices:

Survival Index % = (Number of live pups (at designated time) / Number of pups born) x 100
Survival index on PND13 was calculated from number of pups after culling on PND4 instead of number of pups born

Pre-implantation mortality % = [(Number of Corpora lutea – Number of Implantations) / Number of Corpora lutea] x 100

Intrauterine mortality % = [(Number of Implantations – Number of liveborns) / Number of Implantations] x 100

Total mortality % = [(Number of implantations – Number of viable pups (PND0/4/13)) / Number of Implantations] x 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test item related noisy respiration was observed sporadic cases in a few animals ascribed to reflux or minor exposure to test item in the region of the upper respiratory tract.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No test item related mortality was observed in the study.
One Mid dose female (#3501) was preterminally euthanised on Day 39 (on the day of delivery). Moderately decreased activity, piloerection, paleness both eyes and both pinna and red liquid was observed on the day of death. The symptoms were not related to the test item administration.
One High dose female (#4502) was found dead on Day 10. Hunched back, piloerection and noisy respiration were recorded before death, gavage error was the cause of death for this animal.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The bodyweight and body weight gain of the test item treated groups did not show any test item related effect.
A sporadic, statistically significantly increased body weight gain (p<0.05) was observed in the Low dose female group in PPD 0-4 period and in the Mid dose male group between Day 0 and Day 27. These were considered incidental, not related to the test item administration.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The food consumption of the test item treated groups did not show any test item related effect.
In case of Mid dose males, statistically significantly increased food consumption was observed between Day 14-21 and between Day 14-27, but the data were within the historical control range, hence these findings are not considered to be test item related effect.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No test item-related findings were seen in the clinical pathology parameters.
No test item-related adverse changes were detected in the test item treated animals (males and females) when comparing haematology parameters to the relevant Control data.
Statistically significantly decreased haematocrit% value (p<0.01) and statistically significantly increased red cell distribution width% (p<0.01), platelet count (p<0.05) and reticulocyte relative% (p<0.05) was detected in the High dose male group. Statistically significantly decreased MCHC (p<0.05), mean platelet volume (p<0.01) was detected in the High dose female group.
Statistically significantly increased basophils relative% (p<0.01) was observed in the Mid dose male group.
Statistically significantly decreased mean platelet volume (p<0.05), basophils relative% (p<0.05) and statistically significantly increased lymphocytes relative% (p<0.05) was detected in the Low dose female group.
All the values were within the historical control range but red cell distribution width in the High dose male group. There is no dose dependency therefore all the findings considered as not test item related adverse effect.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No test item-related findings were seen in the clinical pathology parameters.
There were no significant changes or biologically relevant effects on the serum chemistry that could be ascribed to the test item administration.
Urea was statistically significantly decreased in the High dose males (p<0.01) and glucose was significantly increased in the Low dose females (p<0.01) when compared to control animals. The observed values were within the historical control range, therefore the findings are considered as not being test item related adverse effects.

Endocrine findings:

no effects observed

Description (incidence and severity):

Under the experimental conditions of this study and based on the results of thyroid hormone measurement, thyroid weights, nipple retention, anogenital distance and external reproductive organs analysis, histopathology and reproductive performance, there was no evidence for any endocrine effects.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No test item-related changes were observed in the urinalysis parameters in male and female animals of any dose groups when compared to control.
Statistically significantly increased urine volume (p<0.05) and statistically significantly decreased protein (p<0.05) was observed in the High dose male group. Both data were within the historical control range hence those are not considered as test item related effect.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

At the functional observation battery (FOB), locomotor activity measurement, grip strength and foot splay, there were no effects. There were no changes in animal behaviour or general physical condition in the control or test groups.

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):

Minimal to marked multifocal squamous cell hyperplasia and minimal to mild erosion/ulcer, diffuse minimal to mild infiltrates in non-glandular stomach were observed in the High dose males and females. No other adverse test item related systemic microscopic changes were recorded at histopathology evaluation of routine organs/tissues or in any reproductive organs.

Histopathological findings: neoplastic:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

•Pre-exposure period
Each female selected for the study showed acceptable cycles (mean cycle length of 4.00 days was observed in the different groups) before starting the treatment period.
•Exposure period (pre-mating and mating periods)
No indication of test item related effect was seen in the oestrus cycle data, collected during the pre-mating and mating periods (mean cycle length was 4.02, 4.00, 4.01 and 4.06 days in the Control, Low dose, Mid dose and High dose groups, respectively).
Prolonged oestrus was recorded once in each group (#1509, #2504, #3501 and #4509), but these were considered as not being a test item related effect.
Prolonged dioestrus was noted for one High dose female (#4502), but this frequency (1/12) was in line with the normal, expected range and did not affect the mating or pregnancy.

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no significant differences between the control and test item treated groups with regard to reproductive ability, mating or gestation indices, and no effects considered adverse or toxicologically significant in correlation with the administration of the test item. The mating index was 100% in treated groups (males and females) and fertility index was 92%, 75%, 67% and 91% in the Control Low, Mid and High dose groups.

-Clinical signs:
No test item systemic toxicity related clinical signs were observed in the study.
Noisy respiration was observed in three Mid dose females (#3502, #3509, #3511) and one High dose male (#4007) on Day 6, on Day 4, from Day 22 to Day 23 and from Day 1 to Day 4, respectively. The same symptom was observed in four High dose females (#4507, #4508, #4509, #4512) from Day 15 to Day 50. It was observed up to 9 consecutive days.
Gasping respiration was observed in one High dose female (#4507) from Day 32 to Day 36.
Piloerection was recorded from Day 32 to Day 36 in one High dose female (#4507).
The above findings were probably related to the very irritant effect of small amounts of test item reaching the upper respiratory tract via reflux or contamination during dosing (as seen during the preliminary study). This is considered as a local effect of the test item.
Red liquid around vulva was observed for one High dose female (#4501) on Day 53. These findings, but red liquid around vulva were considered incidental, related to the test item administration.
-Mortality and morbidity:
No test item related mortality was observed in the study.
One Mid dose female (#3501) was preterminally euthanised on Day 39 (on the day of delivery).
Moderately decreased activity, piloerection, paleness both eyes and both pinna and red liquid was observed on the day of death. The symptoms were not related to the test item administration.
One High dose female (#4502) was found dead on Day 10. Hunched back, piloerection and noisy respiration were recorded before death, gavage error was the cause of death for this animal.
-Body weight and weight changes:
No test item related adverse effect was observed on body weight or body weight gain parameters in any dose groups for either sex.
A sporadic, statistically significantly increased body weight gain (p<0.05) was observed in the Low dose female group in PPD 0-4 period and in the Mid dose male group between Day 0 and Day 27. These were considered incidental, not related to the test item administration.
-Food consumption:
No test item related adverse effect on food consumption was observed in any test item treated groups, either sex.
In case of Mid dose males, statistically significantly increased food consumption was observed between Day 14-21 and between Day 14-27, but the data were within the historical control range, hence these findings are not considered to be test item related effect.
-Haematological findings:
No test item-related adverse changes were detected in the test item treated animals (males and females) when comparing haematology parameters to the relevant Control data.
Statistically significantly decreased haematocrit% value (p<0.01) and statistically significantly increased red cell distribution width% (p<0.01), platelet count (p<0.05) and reticulocyte relative% (p<0.05) was detected in the High dose male group. Statistically significantly decreased MCHC (p<0.05), mean platelet volume (p<0.01) was detected in the High dose female group.
Statistically significantly increased basophils relative% (p<0.01) was observed in the Mid dose male group.
Statistically significantly decreased mean platelet volume (p<0.05), basophils relative% (p<0.05) and statistically significantly increased lymphocytes relative% (p<0.05) was detected in the Low dose female group.
All the values were within the historical control range but red cell distribution width in the High dose male group. There is no dose dependency therefore all the findings considered as not test item related adverse effect.
-Clinical biochemistry findings:
There were no significant changes or biologically relevant effects on the serum chemistry that could be ascribed to the test item administration.
Urea was statistically significantly decreased in the High dose males (p<0.01) and glucose was significantly increased in the Low dose females (p<0.01) when compared to control animals.
The observed values were within the historical control range, therefore the findings are considered as not being test item related adverse effects.
-Endocrine findings:
Thyroid Hormone Analysis: Compared to the control, there were no statistically significant thyroid hormone concentration levels recorded in any of the male adults and of the PND13 pup dose groups.
The thyroid gland weights of the PND13 pups were also statistically not different from the Control group. In summary, there were no effects on the thyroid hormone levels or on the thyroid gland weights in the PND13 pups that were ascribed to the test item.
The measurement of the thyroid hormone levels in the PND4 pups and adult females was not performed as it was not deemed necessary by the Study Director.
Under the experimental conditions of this study and based on the results of thyroid hormone measurement, thyroid weights, nipple retention, anogenital distance and external reproductive organs analysis, histopathology and reproductive performance, there was no evidence for any endocrine effects.
-Urinalysis:
No test item-related changes were observed in the urinalysis parameters in male and female animals of any dose groups when compared to control.
Statistically significantly increased urine volume (p<0.05) and statistically significantly decreased protein (p<0.05) was observed in the High dose male group. Both data were within the historical control range hence those are not considered as test item related effect.
-Behaviour (functional findings):
There were no changes in animal behaviour, general physical condition or in the reactions to different type of stimuli in the control or test groups.
There was no effect of treatment noted in the Irwin test or during the assessment of grip strength and landing foot splay.
All dose groups of males and females had a normal locomotor activity. In all cases, the initial activity was high, with reduced activity in each 5-minute period to an approximate plateau by about 20-30 minutes. There was no statistical significance between the test item treated animals (males and females) and the Control when evaluating the overall total travelled distance (0-60 minutes). The test item did not increase or decrease the normal locomotor activity, all treated groups had a profile of activity the same as historical control data.
-Organ weight findings including organ/body weight ratios
•Parental males
No test item-related effects were observed in the organ weights of the test item treated male animals compared to controls.
There were no statistically significant differences in the terminal body weight of the test item treated males compared to control males. The body-related weight of the adrenals was decreased statistically significantly (by 14.8%) in the Mid dose group compared to Control, but based on the lack of dose response and as there were no similar change in case of the absolute or brain-related weights, this difference was considered as incidental.
•Parental females
No test item-related effects were observed in the organ weights of the test item treated female animals compared to controls.
Terminal body weights of test item treated females were not significantly different from control females. The absolute weight of the liver was increased statistically significantly (by 13.9% and by 11.2%) in the Mid and High dose group compared to Controls, without histological change.
Furthermore, the absolute and relative to brain weights of thyroid/parathyroid were statistically significantly higher than Control in the Mid dose group by 27.2% (p<0.05) and 29.4% (p<0.05), but the data showed no dose response and were within the historical control range.
The relative to brain weight of ovaries was statistically significantly higher than Control in the Mid dose group by 17.3% (p<0.05) but the data were within the historical control range.
There were no other statistically significant or biologically relevant differences among groups in the weights of organs measured when compared to Controls in females.
-Gross pathological findings:
NON-PREGNANT FEMALES / Parental Generation
One female in the Control group (#1511), three females in the Low dose group (#2507, #2508 and #2509), four females in the Mid dose group (#3505, #3506, #3509 and #3511) and one female in the High dose group (#4507) were non-pregnant in the study.
Necropsy examination did not show any test item related change in the non-pregnant females or their male mating pairs.
FOUND DEAD ANIMAL / Parental Generation
One High dose female (#4502) was found dead on Day 10 because of gavage error.
No test item-related macroscopic changes were observed at necropsy, but the perforation of thoracic oesophagus and small thymus were major findings.
PRE-TERMINAL EUTHANASIA / Parental Generation
One Mid Dose female (3501) was preterminally euthanized on Day 39, on the day of delivery.
No test item-related changes were observed at necropsy. Necropsy revealed diffuse pale discolouration of the liver, enlarged ovaries and enlargement/red multifocal discoloration of uterus+cervix-horn.
TERMINAL EUTHANASIA / Parental Generation
Test item-related diffuse/focal/multifocal thickness of the non-glandular stomach mucosa were observed at a dose level of 1000 mg/kg bw/day. Affected animals included 12/12 males and 10/10 females from the High Dose groups.
All other changes were incidental or a common background.
-Histopathological findings: non-neoplastic:
NON-PREGNANT FEMALES / Parental Generation
One female in the Control group (#1511), three females in the Low dose group (#2507, #2508 and #2509), four females in the Mid dose group (#3505, #3506, #3509 and #3511) and one female in the High dose group (#4507) were non-pregnant in the study.
No microscopic changes were observed in the sex or accessory organs/tissues of non-pregnant females or their male mating pairs.
FOUND DEAD ANIMAL / Parental Generation
One High dose female (#4502) was found dead on Day 10 because of gavage error.
Histopathology revealed correlates in the oesophagus including minimal focal perforation and degeneration/necrosis of tunica muscularis, and moderate decrease of cortical lymphocytes in thymus. Other noteworthy microscopic changes included mild decrease of white pulp in the spleen and diffuse minimal hypertrophy of zona fasciculata in adrenals. Thymic splenic and adrenal changes were considered as typical responses to stress. Other microscopic changes were incidental or agonal/post mortal.
PRE-TERMINAL EUTHANASIA / Parental Generation
One Mid Dose female (3501) was preterminally euthanized on Day 39, on the day of delivery.
Microscopic examination showed focal minimal erosion/ulcer of the glandular stomach. Since similar test item-related lesions were observed in terminal High Dose animals, this microscopic change was regarded as test item-related effect. Due to minimal focal distribution, this lesion had probably minor attribution to the clinical conditions of this animal. Histopathological correlates for the macroscopic changes were observed in the liver including minimal centrilobular necrosis, minimal bilateral hypertrophy of corpora lutea in ovaries and mild luminal dilatation of the uterus+cervix-horn. In addition, inflammatory infiltrate in the oviduct, mild increased mucification of the vagina, moderate extramedullary haematopoiesis of the spleen and minimal decrease of cortical lymphocyte in thymus, were seen. Minimal liver necrosis could contribute to the decreased activity of this animal.
TERMINAL EUTHANASIA / Parental Generation
Test item-related findings were observed in the non-glandular stomach at a dose level of 1000 mg/kg bw/day. These findings correlated with changes observed at necropsy.
Minimal to marked multifocal squamous cell hyperplasia and minimal to mild erosion/ulcer (superficial erosions confined to the epithelial surface mostly seen), diffuse minimal to mild infiltrates (mixed cell/mixed mononuclear/eosinophilic/neutrophilic) in non-glandular stomach were observed. A total incidence of these findings appeared proportionally between terminal High Dose males and females.
Other microscopic changes were considered incidental or a common background.
The spleen was histologically examined in all Control and High Dose animals due to statistical differences in relative reticulocytes (%) for the High Dose males seen in haematology. No histopathological correlates were observed in the extramedullary haematopoiesis (EMH). The severity and incidence of splenic EMH seen in Control and High Dose males and females, did not indicate any effect of administered test item.
- Reproductive function: oestrus cycle:
•Pre-exposure period
Each female selected for the study showed acceptable cycles (mean cycle length of 4.00 days was observed in the different groups) before starting the treatment period.
•Exposure period (pre-mating and mating periods)
No indication of test item related effect was seen in the oestrus cycle data, collected during the pre-mating and mating periods (mean cycle length was 4.02, 4.00, 4.01 and 4.06 days in the Control, Low dose, Mid dose and High dose groups, respectively).
Prolonged oestrus was recorded once in each group (#1509, #2504, #3501 and #4509), but these were considered as not being a test item related effect.
Prolonged dioestrus was noted for one High dose female (#4502), but this frequency (1/12) was in line with the normal, expected range and did not affect the mating or pregnancy.
-Reproductive performance:
There were no significant differences between the control and test item treated groups with regard to reproductive ability, mating or gestation indices, and no effects considered adverse or toxicologically significant in correlation with the administration of the test item. The mating index was 100% in treated groups (males and females) and fertility index was 92%, 75%, 67% and 91% in the Control Low, Mid and High dose groups.

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive effects

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Clinical signs:

no effects observed

Description (incidence and severity):

Based on the external evaluation, no clinical signs or abnormalities were recorded for any pups.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

There was no test item effect on mortality or survival of the pups (F1 generation).
The number of viable pups on PND0, PND4 and PND13 as well as pup survival indices on PND0, PND4 and PND13 in the Low and High dose groups were comparable to control values in each dose group.
There were no significant differences or effects that could be ascribed to treatment on the pre-natal, post-natal or total mortality values (litter mean and %) in any of the dose groups

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were statistically significantly decreased mean litter pup body weight values in the High dose group on Day 0, but the measured values were within the historical control range and the High dose had more pups per litter (hence it is normal that individual weights are slightly lower with bigger litters).

Food consumption and compound intake (if feeding study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

No test item effect was observed on anogenital distance during the study.
No statistically significant changes in the anogenital distance measured on PND 0 were noted for test item treated male and female pups when litter mean values were compared to control.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No test item effect was observed on nipple retention during the study.
There was no nipples/areolae presence seen in any of the male pups on PND13.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The thyroid gland weights of the PND13 pups were also statistically not different from the Control group. In summary, there were no effects on the thyroid hormone levels or on the thyroid gland weights in the PND13 pups that were ascribed to the test item.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test item-related macroscopic findings were observed in pups up to the dose level of 1000 mg/kg bw/day

Histopathological findings:

not examined

Description (incidence and severity):

No histopathological examination was performed on pups (F1 generation).

There was no test item effect on mortality or survival of the pups (F1 generation).
The number of viable pups on PND0, PND4 and PND13 as well as pup survival indices on PND0, PND4 and PND13 in the Low and High dose groups were comparable to control values in each dose group.
There were no significant differences or effects that could be ascribed to treatment on the pre-natal, post-natal or total mortality values (litter mean and %) in any of the dose groups.
Evidence of suckling was recorded for all live born pups in the study.
There were statistically significantly decreased mean litter pup body weight values in the High dose group on Day 0, but the measured values were within the historical control range and the High dose had more pups per litter (hence it is normal that individual weights are slightly lower with bigger litters).
No test item effect was observed on anogenital distance or nipple retention during the study.
No statistically significant changes in the anogenital distance measured on PND 0 were noted for test item treated male and female pups when litter mean values were compared to control.
No test item-related macroscopic findings were observed in pups up to the dose level of 1000 mg/kg bw/day.
No histopathological examination was performed on pups (F1 generation).
Compared to the control, there were no statistically significant thyroid hormone concentration levels recorded in any of the male adults and of the PND13 pup dose groups.
The thyroid gland weights of the PND13 pups were also statistically not different from the Control group. In summary, there were no effects on the thyroid hormone levels or on the thyroid gland weights in the PND13 pups that were ascribed to the test item.
The measurement of the thyroid hormone levels in the PND4 pups and adult females was not performed as it was not deemed necessary by the Study Director.

Key result

Dose descriptor:

NOAEL

Remarks:

pup development

Generation:

F1

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Key result

Reproductive effects observed:

no

Conclusions:

Under the experimental conditions of this study and based on the results of thyroid hormone measurement, thyroid weights, nipple retention, anogenital distance and external reproductive organs analysis, histopathology and reproductive performance, there was no evidence for any endocrine effects.
The NOAEL for reproductive effects of the parental generation: 1000 mg/kg bw/day. (based on no significant findings).
The NOAEL for Pup development and survival: 1000 mg/kg bw/day. (based on no significant findings).

Executive summary:

The purpose of this OECD No. 422 study was to obtain information on the possible toxic effects of reaction mass of sodium (methylbutyl and pentyl) sulfonate and sodium-1,2-bis(pentyloxycarbonyl)ethanesulphonate (CAS 922-80-5, EC 941-224-7) test item following repeated (daily) administration by oral gavage to Wistar (Crl:WI) rats at 3 dose levels. A control group received the vehicle only (propylene glycol).

The study also comprised a reproductive/developmental toxicity screening test, intended to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition and also on the development of the F1 offspring from conception to Day 13 post-partum.

The dose levels were selected by the Sponsor in consultation with the Study Director based on the results of a Dose Range Finding (DRF) study. Based on those results, 1000 mg/kg bw/day was selected as the High dose for this study.

 

Experimental design:

Group Number	Group designation	Dose level (mg/kg bw/day)	Dose formulation concentration (mg/mL)	Dose formulation volume (mL/kg bw)	Number of animals
					Male	Female
1	Control	0	0	5	12	12
2	Low dose	100	20		12	12
3	Mid dose	300	60		12	12
4	High dose	1000	200		12	12

Results

In summary, under the conditions of this study the daily administration of reaction mass of sodium (methylbutyl and pentyl) sulfonate and sodium-1,2-bis(pentyloxycarbonyl)ethanesulphonate (CAS 922-80-5, EC 941-224-7) by oral gavage to Wistar rats at dose levels of 100, 300 or 1000 mg/kg bw/day (Low, Mid and High dose groups, respectively) did not result in test item related mortality.

Test item related noisy respiration was observed sporadic cases in a few animals ascribed to reflux or minor exposure to test item in the region of the upper respiratory tract.

The bodyweight, body weight gain, and food consumption of the test item treated groups did not show any test item related effect.

At the functional observation battery (FOB), locomotor activity measurement, grip strength and foot splay, there were no effects. There were no changes in animal behaviour or general physical condition in the control or test groups.

No test item-related findings were seen in the clinical pathology parameters.

No test item effect on oestrus cycle of parental females was noted.

No test item related changes were noted in the reproductive parameters during mating and gestation, delivery and post-partum/lactation period until PPD14.

There were no adverse effects on the F1 offspring viability, clinical signs, physical or sexual development. No test item related macroscopic findings were recorded for F1 pups at necropsy.

Minimal to marked multifocal squamous cell hyperplasia and minimal to mild erosion/ulcer, diffuse minimal to mild infiltrates in non-glandular stomach were observed in the High dose males and females. No other adverse test item related systemic macroscopic or microscopic changes were recorded at necropsy or at histopathology evaluation of routine organs/tissues or in any reproductive organs.

Under the experimental conditions of this study and based on the results of thyroid hormone measurement, thyroid weights, nipple retention, anogenital distance and external reproductive organs analysis, histopathology and reproductive performance, there was no evidence for any endocrine effects.

The NOAEL for local toxicity of the parental generation: 300 mg/kg bw/day.
(based on local gastric effects).

The NOAEL for systemic toxicity of the parental generation: 1000 mg/kg bw/day.

(based on no significant findings).

The NOAEL for reproductive effects of the parental generation: 1000 mg/kg bw/day.

(based on no significant findings).

The NOAEL for Pup development and survival: 1000 mg/kg bw/day.

(based on no significant findings).

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:

Klimisch 1

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A key OECD No. 422 study was conducted with the registered substance in Wistar rats (12/sex/group by oral gavage at 0 (propylene glycol), 100, 300 and 1000 mg solid/kg bw/day (Szalóki, 2022). Based on the results of the DRF study, 1000 mg/kg bw/day was selected as the High dose for this study. Male and female Wistar rats were treated for 2 weeks pre-mating and then during the mating/post-mating periods. This was 28 days in total for males. Females were treated throughout gestation and up to and including postpartum/lactation day (PPD) 13. The systemic toxicity parameters are reported under the Section 7.5. The reproductive performance, pregnancy, parturition and postpartum/lactation period were monitored in the adult animals, and viability, clinical signs and development were evaluated in their F1 offspring until post-natal day (PND13). At termination, necropsy with macroscopic examination was performed. Weights of selected organs were recorded, and representative tissues/organs were sampled and preserved in appropriate fixatives from the adult animals and/or offspring. The thyroxine (T4) levels in the Day 13 (PPD13) pups and adult males were also assessed.

No test item effect on oestrus cycle of parental females was noted. No test item related changes were noted in the reproductive parameters during mating and gestation, delivery and post-partum/lactation period until PPD14. There were no adverse effects on the F1 offspring viability, clinical signs, physical or sexual development. No test item related macroscopic findings were recorded for F1 pups at necropsy. Minimal to marked multifocal squamous cell hyperplasia and minimal to mild erosion/ulcer, diffuse minimal to mild infiltrates in non-glandular stomach were observed in the High dose males and females. No other adverse test item related systemic macroscopic or microscopic changes were recorded at necropsy or at histopathology evaluation of routine organs/tissues or in any reproductive organs. Under the experimental conditions of this study and based on the results of thyroid hormone measurement, thyroid weights, nipple retention, anogenital distance and external reproductive organs analysis, histopathology and reproductive performance, there was no evidence for any endocrine effects. The NOAEL for reproductive effects of the parental generation was considered to be 1000 mg/kg bw/day (based on no significant findings). The NOAEL for pup development and survival was considered to be 1000 mg/kg bw/day (based on no significant findings).

Conclusion

Reproductive toxicity screening was performed with the registered substance in Wistar rats by oral gavage at 0 (propylene glycol), 100, 300 and 1000 mg/kg bw/day in key combined repeated dose/reproductive toxicity study (OECD No. 422). The NOAEL for reproductive effects of the parental generation was 1000 mg/kg bw/day and the NOAEL for pups development and survival was 1000 mg/kg bw/day. There were no adverse effects on reproductive organs or tissues or other concerns in relation with reproductive toxicity, therefore further testing data are not required.

Effects on developmental toxicity

Description of key information

Prenatal developmental toxicity was tested by dietary administration of read across substance Docusate sodium (CAS No. 577-11-7) in rats from day 6 to 15 of gestation. 1% in the diet was a maternal and developmental NOAEL, whereas at 2% in the diet visceral and skeletal anomalies were observed, which was secondary to maternal toxicity. This was confirmed in a similar study with Docusate calcium given at subtoxic and toxic dose levels, where the same could be observed.

New Prenatal developmental toxicity studies with read-across substances CAS No. 2373-38-8 and CAS No. 29857-13-4 are ongoing and will be updated later when results are available (currently waived in the dossier).

Testing in a second species was not considered ethically acceptable as Docusate salts have been used for a long time as pharmaceutical agent and ingredient, and extensive data were available in humans. No increased risk of malformations was concluded in epidemiological studies from more than 800 patients (pregnant women) which were available for Docusate sodium (or other salts) as pharmaceutical, mainly used during the first trimester of pregnancy. In the Adverse Drug Reaction database from EMA (up to May 2021), a total of 933 Adverse Drug reactions (ADRs) were reported, however for pregnancy, puerperium and perinatal conditions, only 26 ADRs were reported of which 5 were considered serious. The serious cases were most likely not due to Docusate sodium, but to other comedication.

Various publications pointed out the same conclusion, and the outcome was considered to be reliable and extensive.

No further testing is considered needed based on these data

Based on the absence of reproductive findings in the repeated dose toxicity studies and the multigeneration studies, no further testing is needed.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

See attached read-across justification

Reason / purpose for cross-reference:

read-across source

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Among rats given dietary levels of 2.0% DSS, there were significant depressions in maternal weight-gains.
Rats fed diets containing 1.0% level of DSS showed no significant maternal effects on the various parameters.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

Rats fed diets containing 1.0% level of DSS) showed no significant maternal effects on the various parameters. Among rats given dietary levels of 2.0% DSS, there were significant depressions in maternal weight gains.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

not specified

Total litter losses by resorption:

effects observed, treatment-related

Description (incidence and severity):

In the 2.0% DSS group 1 pregnancy with total resorptions was observed (No statistical significance). No pregnancy with total resorptions was observed in the control or 1.0% DSS group.

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

Among rats given dietary levels of 2.0% DSS, there were significant increases in the number of resorptions of 13.7% as compared to the control frequency of 5.6%.

Dead fetuses:

no effects observed

Description (incidence and severity):

0.5% occurrence of dead fetuses was seen in the 2.0% DSS group versus 0.7% in the control group. No dead fetuses were observed in the 1.0% DSS group.

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined

Changes in number of pregnant:

not examined

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: 2.0% in the diet

Key result

Dose descriptor:

NOAEC

Effect level:

1 other: %

Based on:

act. ingr.

Remarks:

in the diet

Basis for effect level:

body weight and weight gain

early or late resorptions

Key result

Dose descriptor:

NOAEL

Effect level:

1 074 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Basis for effect level:

body weight and weight gain

early or late resorptions

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): There is no postnatal evaluation in an OECD 414 study.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

There is no significant reduction in viable fetuses in the dosed animals animals compared to control animals.

Changes in sex ratio:

not specified

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

Description (incidence and severity):

There is no postnatal evaluation in an OECD 414 study.

External malformations:

effects observed, treatment-related

Description (incidence and severity):

Near toxic or toxic dietary levels of 2.0% DSS produced significant incidences of gross abnormalities either among litters (25.0%) or fetal populations (20.2%) as compared to none in the controls. These abnormalities consisted of cranial buble, exencephaly, spina bifida (not significant), microphtalmia or anophtalmia (not significant).

Skeletal malformations:

no effects observed

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

The visceral observations confirmed the significance of the exencephalous characteristics and anophtalmia for the group given dietary levels of 2.0% DSS.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

In the 2.0% DSS group, skeletal observations revealed a significant incidence of variations including incomplete ossification to absence of the various cranial bones, a curved or open vertebral column, and a variety of defects of the vertebrae and ribs.

Details on embryotoxic / teratogenic effects:

Details on embryotoxic / teratogenic effects:
See Table 1-4.

Key result

Dose descriptor:

NOAEC

Effect level:

1 other: %

Based on:

act. ingr.

Remarks:

diet

Sex:

male/female

Basis for effect level:

external malformations

visceral malformations

other:

Remarks on result:

other: secondary to high maternally toxic dose

Key result

Dose descriptor:

NOAEL

Effect level:

1 074 mg/kg bw/day

Based on:

act. ingr.

Remarks:

diet

Sex:

male/female

Basis for effect level:

external malformations

visceral malformations

other: skeletal variations

Abnormalities:

effects observed, treatment-related

Localisation:

external: cranium

skeletal: skull

skeletal: rib

visceral/soft tissue: central nervous system

visceral/soft tissue: eye

Description (incidence and severity):

only at 2.0% dietary level.

Developmental effects observed:

yes

Lowest effective dose / conc.:

2 other: %

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

no

Table 1. Maternal and fetal results of pregnant rats given various amounts if DSS in their diets during  gestational days 6 through 15.

Parameter	Control	1.0% DSS	2.0% DSS
Maternal	Group  (I-A)	(II-A)	(II-B)
No. of pregnant rats	43	22	20
No. of pregnancies with total resorptions	0	0	1
No. of pregnancies with viable fetuses	43	22	19
Average weight gain of dams with viable fetuses(g):
Days 6 to 15	78	86	52*
Days 15 to 21	66	67	77
Average, apparent food intake of dams with viable fetuses (g/rat/day):
Days 6 to 15	22.5	24.8	21.4
Days 15 to 21	28.6	32.1	33.4
Calculated compound consumed (mg/kg/day)	--	1074	1988
Litters
Total number of: implantations	411	203	219
Resorptions (% occurence)	23 (5.6)	8 (3.9)	30*^a (13.7)
Dead fetuses (% occurrence)	3 (0.7)	0	1 (0.5)
Viable fetuses (% occurrence)	385 (93.7)	195 (96.1)	188 (85.5)
Fetal weight (g)	4.6	5.2	4.7
Litters size (viable fetuses)	8.9	8.9	9.9
External major malformations^1: No. of litters affected (% occurrence)	0	0	5* (25.0)
No. of fetuses affected (% occurrence)	0	0	36*^a (20.2)

* Significantly different from control (p< 0.05)

^a Significance by Chi-square, but not Mann-Whitney U test

¹ Primarily, exencephaly varying degrees and associated anomalies (See Table 2)

    

Table 2. Morphological observations of fetuses delivered from rats given DSS in their diets on gestational days 6 through 15.

Morphology	Control	1.0% DSS	2.0% DSS
External observations^1:	Group (I-A)	(II-A)	(II-B)
Total number examined	388^a	195	189
Major anomalies:   Adactyly	0	0	0
Hemimelia	0	0	0
Schistocelia	0	0	2
Dome shaped head	0	0	0
Cranial bubble (1-2mm)	0	0	9*
Exencephaly	0	0	18*
Exencephaly (cleft condition)	0	0	7*
Anencephaly	0	0	0
Spina bifida	0	0	6
Macroglossia	0	0	0
Micro- or anophtalmia	0	0	3
Defects:   Hematoma (subcutaneous)	2	0	0
Edamatous abdomen	0	0	0
Tail short & curled	0	0	0
Abducted fifth digit, left    Rear foot	0	0	1

¹ Fetuses may have more than one defect

^a Fifty-four fetuses examined grossly only. (Shipment c valid as controls only)

      *Significantly different from control (p< 0.05) by Chi-square only

 

Table 3. Visceral observations of fetuses delivered from rats given DSS in their diets on gestation days  6 through 15.

Visceral observations	Dose:      Control	1.0 % DSS	2.0% DSS
	Groups:       (I-A)	(II-A)	(II-B)
Total number of fetuses examined	165^a	98	91
Defects^1:   Exencephalous   characteristics	0	0	11*
Dilated lateral ventricles	1	3	5
Microphtalmia	0	1	0
Anolphtalmia	0	0	23*
Retinal foldings	0	0	0
Anotia or microtia	0	0	0
Cleft palate	0	0	1
Situs transversus – aorta, esophagus   & stomach	1	0	0
Intestinal agenesis	0	0	0
Arch of aorta absent or right sided	0	0	0
Diaphragmic hernia	0	0	1
Dilated renal pelves	2	0	3
Ectopic kidneys(s) &/or variation in size	1	0	0
Renal agenesis	0	0	2
Dilated ureters	6	0	3
Adrenal agenesis	0	0	1
Testes – ectopic or enlarged	1	0	1
Hermaphroditism	0	0	3

¹Fetuses may have more than one defect

^aExcludes 1 fetus lost

*Significantly different from control (p<0.05) by Chi-square only

Table 4. Skeletal observations of fetuses delivered from rats given DSS in their diets on gestation days  6 through 15.

 

Skeletal observations	Dose:      Control	1.0 % DSS	2.0% DSS
	Group  (I-A)	(II-A)	(II-B)
Total number of fetuses examined	167^a	97	98
Defects^1:   Cranial bones,   incomplete to lack of ossification :    Nasal	0	0	4
Frontal	1	0	20*
Parietal	1	1	19*
Interparietal	1	2	18*
Supraoccipital	0	0	15*
Exoccipital	0	0	2
Atlas	0	0	1
Zygomatic	0	0	1
Premaxilla	0	0	1
Tympanic bullae	0	0	5
Mandibles	0	0	1
Hyoid	0	0	3
Eye orbit, reduction	0	0	0
Exoccipital, fused to atlas	0	0	0
Vertebrla column, curved &/or open	0	0	5
Vertebrae:
misshapened &/or retarded     development	0	0	5
thoracic, bipartite centra	2	1	5
lumbar, bipartite centra	0	0	2
Sternebrae:
fused	0	0	0
hypoplastic to absent	0	0	1
one or two absent	1	0	0
staircase	0	0	3
bipartite	0	0	2
Rib(s):
accesory	6	5	5
Absent or less developed	0	0	7*
wavy	2	2	0
fused	0	0	2
Pelvic, hypoplastic to absent	0	0	0
Brachydactyly	0	0	0
Syndactyly	0	0	0
Adactyly	0	0	0
Hemimelia & small scapula	0	0	0

¹Fetuses may have more than one defect

^aExcludes 1 fetus destroyed during cleaning process

*Significantly different from control (p<0.05) by Chi-square only

 

Conclusions:

Subtoxic dietary levels of 1.0% read-across substance docusate sodium ingested on gestational days 6 through 15 showed no adverse effects on the various maternal or fetal parameters. Near toxic or toxic dietary levels of 2.0% DSS produced significant incidences of resorptions (13.7%) and gross abnormalities either among litters (25.0%) or fetal populations (20.2%) as compared to controls. Interpretation of the results of the present experiments, in which only maternally toxic dose levels induce teratogenicity, indicates no real hazard with the recommended human use of these surfactants.

Executive summary:

Prenatal developmental toxicity was studied in rats dosed from day 6 to day 15 of gestation by dietary administration of read-across substance docusate sodium at dose levels of 1.0 and 2.0 % in the diet. Subtoxic dietary levels of 1.0% showed no adverse effects on the various maternal or fetal parameters. Near toxic or toxic dietary levels of 2.0% docusate sodium produced significant depressions in maternal weight-gains and increased incidences of resorptions (13.7%) and gross abnormalities either among litters (25.0%) or fetal populations (20.2%) as compared the controls. These abnormalities consisted primarily of exencephaly of varying degrees with, at times, spina bifida, anophtalmia and associated skeletal defects. The visceral observations confirmed the significance of the exencephalous characteristics and anophtalmia for the group given dietary levels of 2.0%. In this group, skeletal observations revealed a significant incidence of incomplete ossification to absence of the various cranial bones, a curved or open vertebral column, and a variety of defects of the vertebrae and ribs. There were significant depressions in maternal weight gains in the 2.0% DSS-group. Interpretation of the results of the present experiment, in which only maternally toxic dose levels induce teratogenicity, indicates no real hazard with the recommended human use of these surfactants.

The concentration of 1% in the diet is considered as maternal and developmental NOAEL. This dose level corresponded with 1074 mg/kg body weight, as calculated in the study.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 074 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Klimisch 2

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

No test data were available for current substance, however read across data were available from Docusate sodium (CAS No. 577-11-7). Justification for read across within the category of Di-ester sulphosuccinates is documented in a separate document attached in Section 13.

 

Teratogenicity testing in first species

-A key study for prenatal developmental toxicity was performed in rats dosed from day 6-15 of gestation with read across substance Docusate sodium dosed at dietary dose levels of 1.0 and 2.0 % in the diet (Roell et al., 1976). The study was conducted according to OECD 414 guideline, and was considered to be reliable, adequate and relevant. Subtoxic dietary levels of 1.0% showed no adverse effects on the various maternal or fetal parameters. Toxic dietary levels of 2.0% Docusate sodium produced significant depressions in maternal weight-gains and increased incidences of resorptions (13.7%) and gross abnormalities either among litters (25.0%) or fetal populations (20.2%) as compared to controls. These abnormalities consisted primarily of exencephaly of varying degrees with, at times, spina bifida, anophthalmia and associated skeletal defects. The visceral observations confirmed the significance of the exencephalous characteristics and anophthalmia for the group given dietary levels of 2.0%. In this group, skeletal observations revealed a significant incidence of incomplete ossification to absence of the various cranial bones, a curved or open vertebral column, and a variety of defects of the vertebrae and ribs. Interpretation of the results of the present experiment, in which only maternally toxic doses induce teratogenicity, indicates no real hazard with the recommended human use of these surfactants. The concentration of 1% in the diet is considered as maternal and developmental NOAEL. This dose level corresponded with a test article intake of 1074 mg/kg body weight, as calculated in the study.

- As supporting information, prenatal developmental toxicity was also studied in rats by dietary administration of Docusate 'calcium' (DCS) at dose levels of 0.5, 1.0, 1.5 and 2.0 % in the diet as well as by oral gavage at 250, 500, 750 and 1000 mg/kg bw (Roell et all., 1976). Subtoxic dietary levels of 0.5 and 1.0% Docusate calcium ingested on gestational days 6 through 15 showed no adverse effects on the various maternal or fetal parameters. Near toxic or toxic dietary levels of 1.5 and 2.0% DCS produced significant incidences of resorptions and gross abnormalities consisting primarily of exencephaly of varying degrees with spina bifida, anophthalmia and associated skeletal defects. However, dietary levels of 2% of DCS fed to pregnant rats for 3 days (days 6-8, 8-10 or 10-12) did not produce teratogenic response. Also, DCS given to pregnant rats by oral intubation at maternally subtoxic doses (250-750 mg/kg) and a slightly toxic dose (1000 mg/kg) did not lead to malformations, however the incidence of resorptions was increased at the 2 toxic doses. Likewise doses of 500 and 750 mg/kg given by gavage from day 6-15 produced an increase in resorptions at the highest dose without a teratogenic effect. Since only maternally toxic doses fed on gestational day 6-15 produced embryotoxic and teratogenic effects, it is concluded that no real hazard exists.  

 

Conclusion

Prenatal developmental toxicity was tested by dietary administration of read across substance Docusate sodium in rats from day 6 to 15 of gestation. 1% in the diet was a maternal and developmental NOAEL corresponding to 1074 mg/kg bw, whereas at 2% in the diet visceral and skeletal anomalies were observed, which were seen at systemic toxic dose level and considered secondary to maternal toxicity. This was confirmed in a similar study with Docusate calcium given at subtoxic and toxic dose levels, where the same could be observed.

New Prenatal developmental toxicity studies with read-across substances CAS No. 2373-38-8 and CAS No. 29857-13-4 are ongoing and will be updated later when results are available (currently waived in the dossier).

---

Teratogenicity testing in second species

Based on ECHA request for prenatal development evaluation in a second species for read-across substance CAS 577-11-7, human data of Docusate (sodium) used during pregnancy were investigated. According to the ECHA Guidance on Information Requirements (Chapter R.7a: Endpoint specific guidance Version 6.0 - July 2017), human data on reproductive and developmental toxicity can be used, either based on epidemiological data/studies, case reports and clinical data. As Docusate (sodium) has been used extensively as pharmaceutical agent or ingredient, testing in a second species (e.g. mouse as proposed by ECHA as an alternative to rabbit) was considered not ethically feasible. Therefore a waiver for a second species was justified based on available data that have been entered under Section 7.10 Health surveillance data, epidemiological data and exposure related observations in humans. A summary is provided below, whereas the more detailed endpoints are available in Sections 7.10 (1/2/5).

Various epidemiological data are available in literature for Docusate sodium use by pregnant women, of which a smaller group was exposed anytime (N = 116) during pregnancy, whereas most were exposed in the first trimester of pregnancy. However, two publications used the same data, one including pregnancies with life births only and the other all pregnancies. A corrected total of 821 pregnant women (705 during first trimester) exposed to Docusate (sodium) was derived. In total, the first trimester was therefore studied in a group of >700, for which no increased risk of malformations was reported (incidence = 0.2 - 3.9%). An overview of literature studies and No. of pregnant women is provided below. The studies summarised were considered to be reliable (Klimisch 2).

• N = 116 anytime during pregnancy (Prospective): No increased risk of malformations (Heinonen et al., 1977)


• N = 473 during first trimester (Surveillance): No increased risk of malformations (1/473 = 2%) (Jick et al., 1981) #, *


• N = 319 during first trimester (Surveillance): No increased risk of malformations (3/319 = 0.9%) (Aselton et al., 1985) #, **


• N = 232 during first trimester (Surveillance): no increased risk of malformations (9/232 = 3.9%) (Briggs et al., 2011) §
  Total = 1140 (1024 during first trimester) Corrected = 821 (705 during first trimester)

# Studied Based on GHC (Group Health Cooperative) of 6,837 pregnant women

* Drugs Prescribed During the First Trimester of Pregnancy to at Least 200 of 6,837 Pregnant Women

** Drugs Prescribed During the First Trimester of Pregnancy to at Least 200 of 6,509 Women Having Live Births Studied

 

• In a surveillance study of Michigan Medicaid recipients involving 229,101 completed pregnancies conducted between 1985 and 1992, 232 newborns had been exposed to a docusate salt during the 1st trimester (F. Rosa, personal communication, FDA, 1993). Nine (3.9%) major birth defects were observed (nine expected), including one cardiovascular defect (two expected) and one polydactyly (one expected). No anomalies were observed in four other categories of defects (oral clefts, spina bifida, limb reduction defects, and hypospadias) for which specific data were available. These data do not support an association between the drug and congenital defects.

Docusate (sodium) still seems amongst the most commonly used over-the-counter medication components (Drugs.com; Shafe et al., 2011). Doses in pregnant women vary from 50 to 500 mg/day orally in one to four divided doses or 0.12 g rectally as active docusate sodium in a 10 g enema gel. Most frequently used as docusate salts, sodium docusate and calcium docusate, although other forms are available (Rungsiprakarn et al., 2015). Docusate has not been formally assigned to a pregnancy category by the FDA (Drugs.com).

Docusate sodium is available under multiple brand names. In order to prevent and treat chronic constipation or as an adjunct in abdominal radiological procedures, Docusate sodium should be taken up by adults (p.o.) up to 500 mg daily in divided doses. Treatment should be commenced with large doses, which should be decreased as the condition of the patient improves. According to the FDA inactive ingredient list (2021), Docusate sodium is listed up to a maximum daily exposure of 50 mg for oral use. In the Adverse Drug Reaction database from EMA (2021), a total of 933 ADRs were reported (May/2021). However, for pregnancy, puerperium and perinatal conditions, only 26 ADRs were reported of which 5 were considered serious. The serious cases were most likely not due to Docusate sodium, but to other comedication.

Conclusion

No increased risk of malformations was concluded in epidemiological studies from more than 800 patients (pregnant women) which were available for Docusate sodium (or other salts) as pharmaceutical, mainly used during the first trimester of pregnancy.

Further exploration of literature and databases (e.g. FDA Inactive Ingredient List, EMA Adverse Drug Reaction database) confirmed that Docusate sodium is available as active ingredient under multiple brand names up to 500 mg daily by oral application, and as inactive ingredient up to a maximum daily exposure of 50 mg for oral use. In the Adverse Drug Reaction database from EMA (2021), a total of 933 Adverse Drug Reactions (ADRs) were reported, however for pregnancy, puerperium and perinatal conditions, only 26 ADRs were reported of which 5 were considered serious. The serious cases were most likely not due to Docusate sodium, but to other comedication.

Based on this information from read-across substance Docusate sodium, no further 2nd species testing is considered needed for the registered substance.

Justification for classification or non-classification

Based on these results and according to CLP (No. 1272/2008 of 16 December 2008), the test substance does not have to be classified and has no obligatory labelling requirement for reproductive and developmental toxicity.