Sodium 1,2-diisobutoxycarbonylethanesulphonate

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 (distilled water), 100, 300 and 1000 mg solid/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 1000 mg solid/kg bw/day and the NOAEL for pups’ (F1 generation) development and survival was 1000 mg solid/kg bw/day.

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:

reproductive toxicity, other

Remarks:

Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 July 2020 to 28 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:

Paris, 2016

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Paris, 2008

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI

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.

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, 8/9 weeks old (females/males) at start of the experiment and 12/13 weeks old (females/males) at mating.
- Weight at study initiation: Males: 392-472 g, females: 218-259 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.
- 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. Animals were housed in animal room 506 in type II, III and/or IV polycarbonate cages. SAFE 3/4-S Hygienic Animal Bedding (Batch number: 03027200428 / 03027200710, Expiry date: 28 April 2023 / 10 July 2023) and SAFE Crinklets Natural nesting material (Batch number: 05072191028 / 05072200405, Expiry date: 28 October 2022 / 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: A/123) 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): The animals received ssniff® SM R/M “Autoclavable complete diet for rats and mice –breeding and maintenance” (Batch number: 560 65984, Expiry date: 31 October 2020) produced by ssniff Spezialdiäten GmbH (Address: Ferdinand-Gabriel Weg 16, D-59494 Soest, Germany), ad libitum.
- Water (e.g. ad libitum): The animals received tap water from the municipal supply, as for human consumption from a 400- or 500-mL bottle, ad libitum.
- Acclimation period: Environmental acclimation period for the study was 5 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 (ÁNTSZ, 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): 19.5 -24.5°C (target range: 19-25°C)
- Humidity (%):26-67% (target range: 30-70%)
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES:
From: Start of in life phase: 21 July 2020 (first vaginal smear sampling)
To: End of in-life phase: 28 September 2020 (last necropsy)

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

distilled

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: As agreed with the Sponsor, total solid content (test item) of the supplied product was taken into consideration during formulation (a conversion factor of 2.17 was applied used for this purpose). Concentrations and all dose levels in the study (including raw data and study report) were expressed as solid matter as requested by the Sponsor.
The test item was formulated in the selected vehicle (distilled water), as a visibly stable homogenous formulation (Mid or High dose formulation was opalescent in some cases) at the appropriate concentrations according to the dose levels and volume selected in the Pharmacy of the Test Facility. For the formulation step (to measure the amount needed), the bulk test item was heated up to 40°C for a short period. The formulations were stirred with manual shaking and/or a magnetic stirrer from the preparation until completion of each treatment.
Formulations were prepared a maximum of 4 days prior to administration to animals according to stability assessment results of the analytical method development and method validation studies (Test Facility Study codes: 20/028-316ANE and 20/028-316AN).
The calculated amount test item was weighed into a clean, calibrated glass container and then mixed properly (with manual shaking and/or magnetic stirring) with the needed amount of vehicle to reach homogeneity by visual observation. During the formulation sonication was applied as necessary to aid dissolution. Formulations were stored in a closed container at room temperature until use.
After filling the syringe with the calculated amount to be given to each individual rat, the outside of the gavage tube was cleaned with a wetted tissue first (using the vehicle of the study) and then with a dry tissue, to reduce any potential surface contamination to an absolute minimum. A constant volume of 5 mL/kg bw was administered to all animals. The actual volume administered was calculated and adjusted based on each animal’s most recent body weight.

VEHICLE
- Concentration in vehicle: 0, 20, 60 and 200 mg solid/mL
- Amount of vehicle (if gavage): Dose formulation volume = 5 mL/kg bw
- Lot/batch no. (if required): 202007068 / 202003032

Details on mating procedure:

- M/F ratio per cage: 1:1 mating
- Length of cohabitation: Females remained with the same male until copulation occurred, for up to 5 days.
- Proof of pregnancy: The presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (Day 0 of pregnancy as defined by the relevant guidelines).
- 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 on three occasions (on the first day* and last week and approximately 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.
*Note: At the first sampling, opalescence was noticed in the test item formulations by visual inspection (which was not detected in the DRF study), therefore a second set of test item formulations was additionally prepared and measured to avoid any potential preparation error. Both sets showed acceptable results, the second set was used for dosing.
On each sampling occasion, top, middle and bottom duplicate samples were taken from test item formulations 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 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 at three times during the study (on the first day* and last week and once approximately midway during the treatment period).
*Note: Due to a dilution error on the first day, the diluted sample of the Mid dose formulations did not fit into the calibrated range (the concentration was higher than the highest concentration calibrator). Therefore, the back-up samples were diluted properly and analysed on the subsequent day.
Any sample not required for analysis was 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: 20/028-316AN). 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) had to be less than 10%.
The measured concentrations of the test item in the different formulations varied between 93% and 101% of the nominal concentrations. The results were considered to be acceptable.
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.

Duration of treatment / exposure:

Dosing of both sexes began after the acclimatisation (5 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.
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).

Frequency of treatment:

daily on a 7 days/week basis

Details on study schedule:

- F1 parental animals: not applicable: screening study.
- Selection of parents from F1 generation: not applicable: screening study
- Age at mating of the mated animals in the study: 12/13 weeks old (females/males) at mating (Parental generation (P))

Dose / conc.:

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

Remarks:

solid

Dose / conc.:

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

Remarks:

solid

Dose / conc.:

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

Remarks:

solid

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 (Study code: 20/028-220PE), with the aim of inducing toxic effects but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose.
In the DRF study (after a 14-day treatment period), no clinical signs were recorded and no test item related adverse effects were seen on body weight, food consumption, clinical pathology (including haematology and clinical chemistry), gross necropsy and organ weight determination at the highest examined dose of 1000 mg solid/kg bw/day, thus it was considered as acceptable for the High dose level of this study. Lower doses were spaced with a factor of approximately 3.
- Rationale for animal assignment (if not random): All adult/parental (P) male and female animals will be sorted according to body weight by computer and divided into weight ranges. There are an equal number of animals from each weight group randomly assigned to each dose group to ensure that animals of all test groups are as nearly as practicable of a uniform weight.
This process will be controlled by the software PROVANTIS v.9 (or other appropriate software), to verify the homogeneity/variability between/within the groups. Males and females will be randomised separately to the dose groups at the start of the treatment (Day 0).
- Fasting period before blood sampling for clinical biochemistry: overnight period of food deprivation, in case of females this happened after the litter had 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.
Any clinical sign noted during dosing or at any other occasions was recorded at the time seen.
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).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made at the start of the pre-exposure period and one week later (Day -7) 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, signs of difficult or prolonged parturition 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.

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 (Postpartum Day) 0, 4, 7, 10 and 13, and at termination. The body weight of the female animals measured on GD 3, GD 10 and GD 17 as well as PPD 10 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 (on PPD 0, 4, 7, 10 and 13).
Main daily food consumption was calculated for each interval.

WATER CONSUMPTION AND COMPOUND INTAKE (no drinking water study): No

Oestrous cyclicity (parental animals):

Oestrus cycles was monitored by vaginal smears daily during the pre-exposure period before the treatments started. Any females that failed 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):

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, and of all males that failed to sire.
Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were killed and discarded.
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.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups) and the presence of gross abnormalities. Any abnormal behaviour of the offspring was recorded.
Live pups were counted, sexed, weighed individually within 24 hours of parturition (PND 0), and on PND 4 and PND 13, with accuracy of 0.01 g.
All the litters were checked and recorded daily for the number of viable and dead pups; clinical signs and any abnormal behaviour or appearance of the pups (external abnormalities) were also recorded on each day.
The anogenital distance (AGD) of each pup was measured at the time of the first weighing (PND 0). The anogenital distance was also normalized to a measure of pup size (the cube root of body weight) for statistical analysis.
One male and one female pup per litter (if possible) were previously selected for culling for blood sampling on PND 4.
Number of nipples/areolae in male pups was recorded on PND 13.
All pups were necropsied on PND 13.

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.
All culled pups were subjected to necropsy with detailed macroscopic external and internal examination for any abnormalities.
None of the found dead pups were intact (not cannibalized or autolysed) at the time of detection, therefore they could not be subjected to necropsy for macroscopic examination. All observed abnormalities were recorded.
Dead pups and pups terminated on PND 4 and/or PND 13 were carefully examined externally for gross abnormalities.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals one day after the last treatment
- Maternal animals: All surviving animals one day after the last treatment

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
the external appearance was examined, cranium, thoracic and abdominal avities 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.

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 (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 (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 4
Tongue
Trachea
Urinary bladder
Uterus (Horns, body and cervix)
Vagina
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 (selected 5 animals/sex/group),
• one found dead animal (High dose female of #4503),
• 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 of all males that failed to sire and all females that failed to deliver healthy pups (documented by a memo in the raw data)*.
*Notes: Two non-pregnant females were observed in the Control group (#1507 and #1509), another two in the Low dose group (#2505 and #2506) and one in the High dose group (#4511). One female in the High dose group (#4505) had total intrauterine mortality.
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 immunesystem 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
- Litters were culled on PND 4. All selected F1 offspring were terminated on Post-natal Day (PND) 13. In order to allow the overnight fasting of dams prior necropsy on PPD 14, offspring was euthanized on PPD/PND 13.
- These animals were subjected to postmortem examinations as follows: All culled pups were subjected to necropsy with detailed macroscopic external and internal examination for any abnormalities. No histopathological examination was performed on pups (F1 generation).

GROSS NECROPSY
- Dead pups and pups terminated on PND 4 and/or PND 13 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 PND 13 male pups was also recorded.

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

Statistics:

see under “any information on materials and methods incl. tables”

Reproductive indices:

-Male Mating Index (Measure of male’s ability to mate): (Number of males with confirmed mating / Total Number of males cohabited) x 100
-Female Mating Index (Measure of female’s ability to mate): (Number of sperm-positive females / Total Number of females cohabited) x 100
-Male Fertility Index (Measure of male’s ability to produce sperm that can fertilise eggs): (Number of males impregnating a female / Total Number of males cohabited) x 100
-Female Fertility Index (Measure of female’s ability to become pregnant): (Number of pregnant females / Number of sperm-positive females) x 100
-Gestation Index (Measure of pregnancy that provides at least one live pup): (Number of females with live born pups / Number of pregnant females) x 100

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
-Sex ratio% (females): [Number of female pups (PND0/4/13) / Number of viable pups (PND0/4/13)] x 100

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Daily administration did not result in test item related adverse clinical signs. Slightly noisy respiration was observed for two High dose males and three High dose females; however, the finding was transiently or intermittently noted mostly (not longer than consecutive 7 days), and there were no other observations in those animals. This clinical sign might be related to local irritative effect of the test item but was not considered as a test item related adverse effect.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Daily administration did not result in test item related mortality. One High dose animal died during gestation due to gavage accident.

Description (incidence and severity):

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

Description (incidence and severity):

The food consumption of the test item treated groups did not show any test item related effect.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Description (incidence and severity):

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

Description (incidence and severity):

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

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.

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.

Description (incidence and severity):

At the functional observation battery (FOB) and locomotor activity measurement, there were no changes in animal behaviour, general physical condition, grip strength, motor activity, or in the reactions to different type of stimuli in the control or test groups.

Immunological findings:

not examined

Description (incidence and severity):

No test item-related microscopic findings were observed in evaluated males and females from the High dose group

Description (incidence and severity):

No test item-related microscopic findings were observed in evaluated males and females from the High dose group

Description (incidence and severity):

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

Description (incidence and severity):

No test item related changes were noted in the reproductive parameters during mating and gestation, delivery and post-partum/lactation period until PPD 14

-Mortality and morbility:
No test item related mortality was observed in the study.
One High dose female (#4503) was found dead on Day 29 (GD 13). The animal had no clinical signs previously and based on the necropsy findings a gavage accident was considered as possible cause of death for this animal.
-Clinical observations:
No test item related adverse clinical signs were observed in the study.
Fur thin was observed for two Control males (#1011 on Days 27-28, and #1012 from Day 18 to Day 28) and one Low dose male (#2005 from Day 7 to Day 28). These findings were considered incidental.
Slightly noisy respiration was observed for two High dose males (#4003 on Day 20 and #4012 on Day 23) and three High dose females (#4505 on Days 5-9, #4507 on Days 2-6 and #4509 on Days 5-6 and 36-42). These findings might be related to local irritative effect of the test item but was not considered as a test item related adverse effect.
Tonic convulsions were observed for one High dose female (#4512) on Days 35, 39-40, 45-47 and 49. As these observations were seen near the delivery (Day 39 was the day of delivery) and early lactation, they might be related to a difficult delivery and the loss of pups (all the pups died by PND 2 in this litter). Based on the isolated occurrence this fact was considered as biological variability, not related to the test item administration.
-Body weight and weight changes:
No test item related effect on body weight or body weight gain was detected in the study (males or females).
-Food consumption and compound intake (no feeding study):
No test item related adverse effect on food consumption was seen in any test item treated group (males and females). Statistically significant changes (increases) in the food consumption were recorded for High dose males or Mid dose females in some periods, but the observed values were still within the historical control range for this strain and age of rats, thus no test item related adverse effect was concluded.
-Neurological assessment:
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), any observed statistically significant difference in case of Mid dose males for a short period was without dose response, and had no relevance for the overall total pattern, thus considered as not being related. 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.
-Haematology:
No test item-related changes were detected in the test item treated animals (males and females) when comparing haematology parameters to the relevant Control data.
Statistically significant differences were observed in some cases for Low dose males, but there was no dose dependent relationship and all recorded group mean values were within the historical control range. These differences were considered to not reflect an effect of the test item but being incidental.
-Clinical chemistry:
There were no test item related changes or biologically relevant effects on the serum chemistry that could be ascribed to the test item administration.
Glucose was significantly decreased in the Low dose females (p<0.05) and High dose females (p<0.01) when compared to control animals. However, there was no dose response, the observed values were within the historic control range and no similar trend was seen in males. Thus, those findings were considered incidental, not related to the test item.
-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.
-Oestrus cycle evaluation:
Pre-exposure period
Each female selected for the study showed acceptable cycles (mean cycle length of 4.00-4.07 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 during the pre-mating and mating periods (mean cycle length was 3.98, 4.12, 4.00 and 4.25 days in the Control, Low, Mid and High dose groups, respectively. Although the High dose value showed a statistically significant increase when compared to control, but the observed group mean value was within the historical control range (3.96-4.46), and no clear dose response was seen. Furthermore, the observed group mean value was resulted due to the prolonged oestrus data of two High dose females as detailed below), this frequency (2/12) was also within the historical control range. Thus, no test item effect was concluded.
Prolonged oestrus was recorded in some cases for Low and High dose groups, two Low dose females and two High dose females were affected (#2501, #2511, #4501 and #4510), but as it did not affect mating or pregnancy; these facts were considered as being an occasional finding, not being a test item related effect.
Prolonged dioestrus was noted for two Control females (#1507 and #1511) and one High dose female (#4505), as the incidence was lower in the test item treated groups than in the Control group; this fact was considered as not being a test item related effect.
Pseudo-pregnancy was noted for one High dose female (#4502) but this frequency (1/12) was in line with the normal, expected range and the animal successfully mated and delivered later.
-reproductive ability assessment and indices:
There were no 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 all groups (males and females). The fertility index was 83%, 83%, 100% and 92% in the Control, Low, Mid and High dose groups (males and females), respectively. The gestation index was 100% in Control, Low and Mid dose groups and 90% in the High dose group. However, the single occurrence of one female with total intrauterine mortality was in line with the historical control data, thus it was concluded incidental and not being a test item related effect.
Test item administration was considered to have no impact on the duration of the mating period. Successful coitus (sperm positive vaginal smears and/or vaginal plugs) occurred mostly within 5 days of pairing (cohabitation). The mean duration of mating was 2.83, 2.92, 2.75 and 2.25 days in the Control, Low, Mid and High dose groups, respectively.
Notes: Two non-pregnant females were observed in the Control group (#1507 and #1509), another two in the Low dose group (#2505 and #2506) and one in the High dose group (#4511). One female in the High dose group (#4505) had total intrauterine mortality, but as corpora lutea was present it was counted as fertile. One pregnant High dose female (#4503) was found dead due to gavage accident, that female was excluded from the gestation index calculation.
-Evaluation of the gestation, parturition and post-partum period:
There was no effect of treatment noted during the gestation period, parturition and post-partum period in any of dose groups.
The mean duration of pregnancy was comparable in the Control and test item treated groups.
As far as it could be observed during the study, the parturition was normal for all animals, no abnormal delivery was noted.
The number of implantation sites was comparable to the control mean in all dose groups; no statistically significant differences were noted.
There were no statistically significant differences or effects that could be ascribed to treatment on pre-natal, post-natal or total mortality values (litter mean and %) in any dose group.
-Organ weights:
No test item-related effects were observed in the organ weights of the test item treated male or female animals compared to controls.
Terminal body weights of animals were not statistically significantly different between the groups. There were no treatment-related statistically significant differences among groups of males and females in the weights of organs measured when compared to Controls. Due to the lack of dose response, the occasional statistically significant difference in the adrenal weight (absolute and brain related) of Low dose males compared to Control was considered as animal variability, not related to test item administration.
-Pathology evaluation
NON-PREGNANT OR NOT DELIVERED FEMALES / Parental Generation
Five non-pregnant females were observed in the study (#1507, #1509, #2505, #2506 and #4511). Furthermore, on High dose female (#4505) had total intrauterine mortality.
Macroscopic Findings
Necropsy examination did not show any test item related change in those females or in their male mating pairs.
Microscopic Findings
Organs from the reproductive system were microscopically examined. No microscopic changes were observed in the non-pregnant or not delivered females or in their male mating pairs.
FOUND DEAD ANIMAL / Parental Generation
One High dose female (#4503) was found dead on Day 29. Gavage accident was considered as possible cause of death for this animal.
Macroscopic Findings
No test item-related macroscopic changes were observed at necropsy. Necropsy examination revealed dark red diffuse discolouration of the non-collapsed lungs, 1 mL of clear liquid within the thoracic cavity, dilatation of the stomach with gas, dark red diffuse discolouration of the ovary and dilatation/dark red multifocal discoloration of the uterus.
Microscopic Findings
No test item-related microscopic changes were observed. The microscopic evaluation correlates for the macroscopic changes were observed in following organs including the lungs with mild multifocal haemorrhage, ovary with mild bilateral congestion (considered as postmortem change) and uterus with presence of placenta. In addition, mild extramedullary haematopoiesis in the spleen and mild intrasinusoidal erythrocytes in mandibular lymph nodes were microscopically seen.
TERMINAL EUTHANASIA / Parental Generation
Macroscopic Findings
No test item-related macroscopic findings were observed up to the High dose level (1000 mg solid/kg bw/day).
All changes were incidental or a common background.
Microscopic Findings
No test item-related findings were seen at the High dose level (1000 mg solid/kg bw/day). All changes were incidental or a common background.
-Thyroid hormone analysis:
No test item effect was noted in any dose groups based on the results the T4 hormone measurement, thyroid gland weights and histopathology evaluation.
In parental males, the measured T4 thyroid hormone concentration as well as thyroid weights (absolute and body / brain-related) in the test item treated groups were comparable to Control values. No histopathology (microscopic) findings were detected in the thyroids of any High dose animals except of one male (#4002) where minimal unilateral ectopic tissue (from thymus) was noted and another male (#4012) where minimal unilateral multinucleated giant cells were recorded. Those findings were considered as incidental, not reflecting a test item related effect.
No relevant changes were noted in the absolute or relative (to body / brain) thyroid weights of the parental female animals in any dose groups. No histopathology (microscopic) findings were detected in any High dose females.

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive effects of the parental generation

Effect level:

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

Based on:

other: solid

Sex:

male/female

Basis for effect level:

other: based on the lack of adverse findings

Key result

Critical effects observed:

no

System:

gastrointestinal tract

Description (incidence and severity):

There were no test item effects on the F1 offspring clinical signs.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

There were no test item effects on the F1 offspring viability.

Description (incidence and severity):

There were no test item effects on the F1 offspring physical or sexual development.

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

Description (incidence and severity):

No test item effect was observed on anogenital distance during the study.

Description (incidence and severity):

No test item effect was observed on nipple retention during the study.

Organ weight findings including organ / body weight ratios:

not examined

Description (incidence and severity):

No test item related macroscopic findings were recorded for F1 pups at necropsy.

Histopathological findings:

not examined

Description (incidence and severity):

In case of PND13 pups, there were no statistically significant changes in the T4 thyroid hormone concentration levels when compared to the control. Statistically significant increase compared to control was detected in the absolute and relative (to body) thyroid gland weights in the Low and Mid dose pups on PND13 when compared to the control values. However, there was no dose response, and all the data were within the historic control range. 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.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

-mortality & clinical observations: There was no test item effect on mortality or survival of the pups (F1 generation).
The number of viable pups on PND 0, PND 4 and PND 13 as well as pup survival indices on PND 0, PND 4 and PND 13 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. In case of a High dose litter (#4512), all the pups died by PND 2, but this fact was considered as an incidental finding, not a test item related effect. No clinical signs or abnormalities were recorded for any pups except of two cases.
Developmental abnormality (malformation) was recorded for a Low dose pup (#2510/6). Whole body of a High dose pup (#4512/5) was cold to touch on PND 1, this pup was cannibalized by the next day. Based on the isolated occurrence these facts were considered incidental, not related to the test item administration.
Evidence of suckling was recorded for all live born pups in the study.
Note: There was 11 pregnant females in the High dose group. One High dose female (#4503) was found dead during gestation, another (#4505) did not deliver pups. In case of a High dose litter (#4512), all the pups died by PND2.
-body weight and body weight gain:
There were no test item related differences in the offspring body weights or weight gains in Low, Mid and High dose groups when compared to the controls. The measured values were within the range commonly recorded for this strain and age.
-Anogenital distance, nipple retention:
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.
There was no nipples/areolae presence seen in any of the male pups on PND 13.
-Pathology:
TERMINAL EUTHANASIA / F1 Generation (PND 13)
Macroscopic Findings
No test item-related macroscopic findings were observed up to the High dose (1000 mg solid/kg bw/day). In case of one Mid dose pup (#3510/5), diffuse red discoloration of the lungs (all lobes) was recorded. This finding was considered as minor, incidental finding, not related to the test item treatment.
Microscopic Findings
No histopathological examination was performed on pups (F1 generation).
-Thyroid hormone analysis:
In case of PND13 pups, there were no statistically significant changes in the T4 thyroid hormone concentration levels when compared to the control. Statistically significant increase compared to control was detected in the absolute and relative (to body) thyroid gland weights in the Low and Mid dose pups on PND13 when compared to the control values. However, there was no dose response, and all the data were within the historic control range. 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.

Key result

Dose descriptor:

NOAEL

Remarks:

for pups’ (F1 generation) development and survival

Generation:

F1

Effect level:

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

Based on:

other: solid

Sex:

male/female

Basis for effect level:

other: based on the lack of adverse findings

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

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

Key result

Reproductive effects observed:

no

Conclusions:

The NOAEL for reproductive effects of the parental generation: 1000 mg solid/kg bw/day (based on the lack of adverse findings).
The NOAEL for pups’ (F1 generation) development and survival: 1000 mg solid/kg bw/day (based on the lack of adverse findings).

Executive summary:

The purpose of this OECD No. 422 study was to obtain information on the possible toxic effects of diisobutyl sodium sulfosuccinate (CAS 127-39-9; EC 204-839-5) 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 (distilled water). 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 solid/kg bw/day was selected as the High dose for this study. Concentrations and all dose levels in the study (including raw data and study report) were expressed as solid matter as requested by the Sponsor.

Experimental design:

Group number	Group design	Dose level (mg solid/kg bw/day)	Dose formulation concentration (mg solid/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

 

Parameters measured during the study included twice a day mortality checking, daily routine and weekly detailed observation of clinical signs, weekly body weight and food consumption measurements and clinical pathology evaluation (including haematology, coagulation, clinical chemistry and urinalysis). Neurological assessment (Functional Observation Battery (FOB) including measurements of the landing foot splay, grip strength as well as locomotor activity measurement) was performed during the last week of the treatment for each sex. In addition, 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 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 or F1 animals. The thyroxine (T4) levels in the PND13 pups and parental males were also determined.

 

For the adult animals, a detailed histological examination was performed on the selected list of retained organs of 5 animals/sex in the Control and High dose groups, all found dead animals and all those male / female mating pairs where no liveborn pups were achieved.

Dosing formulation were analysed for concentration and/or homogeneity on three occasions during the study. All test item formulations were shown to be homogeneous. The measured concentrations of the test item in the different formulations varied between 93% and 101% of the nominal concentrations. Overall, the formulations were considered adequate for the study.

 

RESULTS

In summary, under the conditions of this study the daily administration of diisobutyl sodium sulfosuccinate (CAS 127-39-9; EC 204-839-5) by oral gavage to Wistar rats at dose levels of 100, 300 or 1000 mg solid/kg bw/day (Low, Mid and High dose groups, respectively) did not result in test item related mortality or adverse clinical signs.

One High dose animal died during gestation due to gavage accident.

Slightly noisy respiration was observed for two High dose males and three High dose females; however, the finding was transiently or intermittently noted mostly (not longer than consecutive 7 days), and there were no other observations in those animals. This clinical sign might be related to local irritative effect of the test item but was not considered as a test item related adverse effect.

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) and locomotor activity measurement, there were no changes in animal behaviour, general physical condition, grip strength, motor activity, or in the reactions to different type of stimuli 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 PPD 14.

There were no test item 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.

No test item-related macroscopic and microscopic findings were observed in evaluated males and females from the High dose group.

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.

Based on the results of this study, the following No-Observed-Adverse-Effect Levels (NOAELs) were considered:

The NOAEL for systemic toxicity of the parental generation: 1000 mg solid/kg bw/day (based on the lack of adverse findings).

The NOAEL for reproductive effects of the parental generation: 1000 mg solid/kg bw/day (based on the lack of adverse findings).

The NOAEL for pups’ (F1 generation) development and survival: 1000 mg solid/kg bw/day (based on the lack of adverse 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 TG No. 422 study was conducted with the registered substance in Wistar rats (12/sex/group by oral gavage at 0 (distilled water), 100, 300 and 1000 mg solid/kg bw/day (Hargitai, 2022). 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 delivered were sacrificed as practical (27 days after the last day of the mating period). 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 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 or F1 animals. The thyroxine (T4) levels in the PND13 pups and parental males were also determined.
No test item related changes were noted in the reproductive parameters during mating and gestation, delivery and post-partum/lactation period until PPD 14.
There were no test item 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.
No test item-related macroscopic and microscopic findings were observed in evaluated males and females from the High dose group. 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. Based on the results of this study, the following No-Observed-Adverse-Effect Levels (NOAELs) were considered: The NOAEL for reproductive effects of the parental generation: 1000 mg solid/kg bw/day (based on the lack of adverse findings). The NOAEL for pups’ (F1 generation) development and survival: 1000 mg solid/kg bw/day (based on the lack of adverse findings).

 

Conclusion

Reproductive toxicity screening was performed with the registered substance in Wistar rats by oral gavage at 0 (distilled water), 100, 300 and 1000 mg solid/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 solid/kg bw/day and the NOAEL for pups’ (F1 generation) development and survival was 1000 mg solid/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 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 considered to be 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.

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 CAS 577 -117- (Docusate sodium). 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 (CAS No. 577-11-7) 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 was considered to be 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.