Sodium 1,4-diisodecyl sulphonatosuccinate

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 600 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 600 mg /kg bw/day and the NOAEL for pups’ (F1 generation) development and survival was 600 mg/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:

09 December 2020 to 07 March 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:

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, 9-10/10 weeks old (females/males) at start of the experiment and 11-12/12 weeks old (females/males) at mating.
- Weight at study initiation: Males: 354-436 g, females: 206-275 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 Type II, III and/or IV polycarbonate cages. SAFE 3/4-S Hygienic Animal Bedding (Batch number: 03027201022 / 03027201024, Expiry date: 22 October 2023 / 24 October 2023) and SAFE Crinklets Natural nesting material (Batch number: 05072200405 / 05072200824, Expiry date: 05 April 2023 / 24 August 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/B) 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: 713 70882, Expiry date: 28 April 2021) 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 20 days.

DETAILS OF FOOD AND WATER QUALITY:
A sample (approximately 100 g) of batch of diet used in the study was not retained and kept under appropriate environmental conditions until the finalization of the study report however it was indicated in the Study Plan.
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 National Institute of State Public Health and Medical Officer Service. Copies of the relevant Certificates of Analysis were included in the raw data and will be 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): 20.1-24.9℃ (target range: 19-25°C)
- Humidity (%):31-69% (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: 16 December 2020 (first vaginal smear sampling)
To: End of in-life phase: 26 February 2021 (last necropsy)

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
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 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 validation study (Study code: 20/029-901AN). Based on those results, the test item formulation in the 2 and 200 mg/mL concentration range were stable for at least 9 days when stored at room temperature.
The calculated amount test item was weighed into a clean, calibrated glass container and then mixed properly (with ultrasonication and magnetic stirring) with the needed amount of vehicle to reach homogeneity by visual observation. During the formulation sonication was applied to aid dissolution.
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 to be administered was calculated and adjusted based on each animal’s most recent body weight.

VEHICLE
- 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 and the Dose Range Finding (DRF) study (Study code: 20/029-220PE) performed at the Test Facility, propylene glycol (abbreviated as PG in the raw data and study documents) was selected as vehicle for this study in agreement with the Sponsor. Propylene glycol was considered as being an acceptable vehicle based on the scientific literature and practice of the Test Facility.
- Concentration in vehicle: 0, 20, 60 and 120 mg/mL
- Amount of vehicle (if gavage): Dose formulation volume = 5 mL/kg bw
- Lot/batch no. (if required): 1920944

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 (during the first and last weeks 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 Pharmacy Department.
On each sampling occasion, top, middle and bottom duplicate samples was taken from test item formulations for concentration and homogeneity measurement, one set to analyse (which can be 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 the formulations for concentration and homogeneity of test item was performed using a validated analytical HPLC-MS method at the Test Site (Study code for method validation: 20/029-901AN)
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 96.7% and 103.7% of the nominal concentrations.
All test item formulations were shown to be homogeneous. The relative standard deviation (RSD) was below 10%.
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 (20 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 32 days (14 days pre-mating and 18 days mating/post-mating period), then were euthanized and subjected to necropsy examination.
Females were dosed for 14 days pre-mating, for up to 18 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:

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: 11-12/12 weeks old (females/males) at
Mating (Parental generation (P))

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

12
*Note: Because of two dead High dose females, 2 females and two males were used to replace them

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: 20/029-220PE), 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), decreased activity, ataxia and hunched back, piloerection, splayed gait and soft faeces were recorded with very significant body weight loss and low food consumption, without clearly adverse effects on clinical pathology were seen in the High dose groups. Macroscopic findings were observed in the glandular and nonglandular stomach at all dose levels in males and High dose females indicating a dose related local gastric irritation. The changes seen at 1000 mg/kg bw/day dose level were not life threatening but the body weight effect was greater than is acceptable for a longer term study, but no significant body weight effects were seen at 300 mg/kg/day. The High dose level of the main study should be >300 and <1000 mg/kg/day. The dose of 600 mg/kg/day is selected for the High dose. 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 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 randomized separately to the dose groups on the day of the first treatment (prior to the start of the treatment).
- Fasting period before blood sampling for clinical biochemistry: overnight period of food deprivation in case of females this happened after the litter had been necropsied)

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, 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, on Day 0 (randomisation and the first day of exposure), 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 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 and on Day 0. Food consumption was measured during mating. Food consumption was measured more frequently during the lactation period (on Gestation Day (GD) 0, 3, 7, 10, 14, 17 and 20 and on PPD0, 4, 7, 10 and 13 during the lactation period).
Main daily food consumption was calculated for each interval.

WATER CONSUMPTION AND COMPOUND INTAKE (if 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.
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 was selected random. 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) was also recorded on each day. The pups found dead and intact (not cannibalized) were subjected to necropsy with macroscopic examination and the cause of death was identified if possible. All observed abnormalities were recorded.
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.
Number of nipples/areolae in male pups was recorded on PND 13.
One male and one female pup per litter (if possible) were previously selected for culling for blood sampling on PND 4.
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.
The pups found dead and intact (not cannibalized) were subjected to necropsy with macroscopic examination and the cause of death was identified if possible. 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.
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
For 5 adult males and / or females per group standard organ weights (epididymides, heart, kidneys, liver, prostate complex, spleen, testes, thymus, uterus including cervix, adrenals, ovaries and thyroids with parathyroids) was measured. Brain weight was measured for all the animals.
At the time of termination, body weight and weight of the following organs was determined:
• With a precision of 0.01 g: epididymides, levator ani plus bulbocavernosus muscle complex, (all adult males)
• With a precision of 0.001 g: thyroids with parathyroids (all animals), Cowper’s glands and glans penis (all adult males)
Testes and epididymides were weighed individually. Individual and/or paired absolute organ weight is 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
Additionally, thyroid glands from all adult animals and one male and one female PND13 pup from each litter was 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) was 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, for Control females 6 animals were selected),
• 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.
Additional histology on the stomach (glandular and non-glandular), the liver of the Control, Low, Mid and High dose animals (males and females) and on the kidney of the Low and Mid dose males were performed as agreed by the Study Pathologist, the Study Director and the Sponsor.
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, and blood smears if examined).
Special attention was paid to the central and peripheral nervous system tissues for any evidence
of neurotoxicity.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at PND4 and/or PND13.Litters were culled on PND 4. All selected F1 offspring were terminated on Post-natal Day 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 (macroscopic and/or microscopic examination) as follows: 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. Presence of nipples/areolae in the PND13 male pups was also recorded.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations (reproductive organs)

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

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Daily administration of Sodium 1,4-diisodecyl sulphonatosuccinate did not result in test item related clinical signs.
Sporadic cases of noisy respiration in a few animals ascribed to reflux or minor exposure to test item in the region of the upper respiratory tract.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

Daily administration of Sodium 1,4-diisodecyl sulphonatosuccinate did not result in test item related mortality.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Test item related adverse effect was observed on body weight gain parameters in High dose (600 mg/kg bw/day) males and moderate effects were observed on body weight and body weight gain in the gestation and lactation period for High dose females. It is most likely these effects were related to local gastric irritation.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Test item related adverse effect was observed on food consumption in High dose (600 mg/kg bw/day) males

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no adverse changes in clinical pathology in test item treated groups when compared to control.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no adverse changes in clinical pathology in test item treated groups when compared to control.

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.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no adverse changes in animal behaviour, general physical condition or in the reactions to different type of stimuli in test item treated groups when compared to control.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histologically, test item-related changes were observed in the stomach in High and Mid dose animals with squamous hyperplasia, hyperkeratosis and ulcer of the non-glandular gastric mucosa observed at 300 and 600 mg/kg bw/day dose level in females and at 100, 300 and 600 mg/kg bw/day dose level in males, as test item-related adverse local changes.
Also, centrilobular hepatocellular hypertrophy was observed at 600 mg/kg bw/day dose level as a non-adverse adaptive change (associated with liver weigh increase).
Minimal to mild multifocal eosinophilic droplets/globules were seen in the renal tubules in the High dose males and were considered as test item-related but non-adverse.

Histopathological findings: neoplastic:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No indication of test item related effect was seen in the oestrus cycle data, collected during the pre-mating and mating periods.

Reproductive performance:

no effects observed

Description (incidence and severity):

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. Test item administration was considered to have no impact on the duration of the mating period. There was no effect of treatment noted during the gestation period, parturition and post-partum period in any of dose groups.

-Mortality and morbility:
No test item related mortality was observed in the study.
Two deaths of females on days 5 and 6 were considered as accidental and not related to systemic toxicity of the test item, animals were replaced; data for only the replacement animals is given in the report results section.
-Clinical observations:
No test item related systemic clinical signs were observed in the study.
Tonic convulsions were recorded during the lactation period (Day 45, 49 and 51) for a Control female (#1507), on Day 15, 24, 31, 37, 42, 45, 48 and 51 for a Low dose female (#2503), on Day 49 and 51 for one Mid dose female (#3510) and on Day 43 and 50 for one High dose female (#4506) on Day 43 and Day 50.
Red liquid in the oral cavity was observed for one Low dose male (#2002) on Day 14.
Noisy respiration was observed for one Mid dose male (#3008) from Day 16 to 21 and one Mid dose female (#3504) on Day 2 and one High dose female (#4503) on Day 15 and Day 16. These sporadic cases of noisy respiration in a few animals ascribed to reflux or minor exposure to test item in the region of the upper respiratory tract.
Hunched back was observed in one High dose female (#4512) from Day 2 until Day 16.
Piloerection was observed in one High dose female (#4512) from Day 2 until the end of the treatment (there was no symptom between Day 12-34).
Red discharge around nose was observed in one High dose female (#4512) on Day 3.
Alopecia for both fore paws and fur thin on the chest was observed for one Low dose female (#2501) form Day 27 until death. Alopecia for both fore paws was observed for one Mid dose female (#3501) from Day 41 until death.
Broken/missing incisor was observed for one High dose male (#4003) from Day 12 to 18.
Increased respiratory rate was observed in one Control female animal (#1511) on Day 54.
Malocclusion was observed for one Control female animal (#1504) from Day 8 until the end of the treatment.
Scar around the nose / snout was observed for one High dose male (#4012) from Day 14 to 25 and scar on the head (1-2 cm) for one Control female (#1511) was observed on Day 54.
These findings were all considered as being incidental or minor reflux type events, not related to toxicity of the test item administration.
-Body weight and weight changes:
No effect on body weight was observed in the Low, Mid (male and female) and High dose male
groups or body weight gain in the Low and Mid (male and female) dose groups.
Test item related adverse effect was observed on body weight gain in High dose (600 mg/kg bw/day) males. Statistically significantly reduced body weight gain (p< 0.05) was observed in High dose males in the first week of treatment only when compared to control animals. From the end of the first week, the growth rate of High dose males was similar to the other groups (with small statistical differences above and below the control growth rate). At the end of the treatment period the High dose body weight was not significantly different to control (only 1.9% below).
No effects were observed in females during the premating period. Statistically significantly lower than control body weight (by -8.6%, p<0.01) and/or body weight gain (by -20.4%, p<0.01) was observed in the High dose female group during gestation but a higher weight gain was seen during lactation in this group. A similar but lesser difference (-14%), with some statistical differences (p<0.01), was observed in Mid dose females. However, the values of the High and Mid dose groups were within the historical control range, hence the High and Mid dose female body weights were not considered to have been adversely affected by the test item.
-food consumption and compound intake (no feeding study):
A test item related reduction of food consumption was observed in High dose males (600 mg/kg bw/day) during the first 7-14 days only (p<0.01) (reflecting the body weight gain data); Mid and Low dose males were not affected. For High dose females, a similarly reduced food intake was seen in the first 7 days of treatment (p<0.01).
Test item related lower food consumption was observed in High dose females (600 mg/kg bw/day) during the gestation and lactation phases with statistically significant differences (p<0.01), in the Mid dose females a similar trend was observed, with occasional statistical differences. The percentage differences reflected the body weight gain differences reported above, but although the body weight data of the High and Mid groups were in line with the historic control data, the food intake mean values were below the expected range, suggesting a moderate treatment effect.
The food intake effects were considered to be secondary to local gastric irritation caused by the test item and associated with the lower body weight gains.
-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). 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:
Five male and five female animals from each main group (6 females in control group for urine analysis) were selected for clinical pathology sampling and analysis at termination (on Day 33 in males, on PPD 14 in females).
No test item-related adverse changes were detected in the test item treated animals (males and females) in any dosed groups when comparing haematology parameters to the relevant Control data.
Statistically significantly decreased red cell distribution width % was observed in the High dose male (p<0.05) and female (p<0.01) group. Although the RDW parameter was statistically significant higher in High dose male animals and lower in female animals (when compared to Control group) it was considered an incidental finding, as any kind of discrepancies were not observed in values of red blood cells (RBC), haemoglobin and mean cell volume (MCV), neither in High dose males nor females.
-Clinical chemistry:
Five male and five female animals from each main group (6 females in control group for urine analysis) were selected for clinical pathology sampling and analysis at termination (on Day 33 in males, on PPD 14 in females).
There were no significant changes or biologically relevant effects on the serum chemistry that could be ascribed to the test item administration in the Low and Mid dose groups for both sexes.
Glucose was statistically significantly decreased (p<0.05 and p<0.01) in the Mid and High dose male groups. Potassium was statistically significantly increased (p<0.05) in the High dose male group. Alkaline phosphatase activity was statistically significantly increased in the High dose female group. All the above data were inside of the historical control data and in the absence of any associated changes, they are not considered as adverse test item related effects.
Alanine aminotransferase (ALT/GPT) activity was significantly increased in the High dose males (p<0.01) when compared to control animals. The observed value was outside of the historical control range, and a similar but not statistically significant difference was seen in females. However, based on the order of magnitude and the lack of any adverse hepatic histopathological changes, this fact was considered as being a test item related slight effect and not considered as adverse.
-Urinalysis:
Five male and five female animals from each main group (6 females in control group for urine analysis) were selected for clinical pathology sampling and analysis at termination (on Day 33 in males, on PPD 14 in females).
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 was observed in the Mid and High dose female group (p<0.05). The cause of the high volume of the urine is that the animals very often play with the small ball in the water bottle, and the water and the urine will be collected together. The data were outside of the historical control range but because of the abovementioned cause it is not considered as test item related effect.
Statistically significantly decreased specific gravity was observed in the Low (p<0.05) and High dose (p<0.01) female group and decreased protein level was observed in the High dose (p<0.01) female group. The data were inside of the historical control range.
-oestrus cycle evaluation in females:
Pre-exposure period
Each female selected for the study showed acceptable cycles (mean cycle length of 4.03-4.05 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 4.01, 4.08, 4.08 and 4.18 days in the Control, Low dose, Mid dose and High dose groups, respectively).
Prolonged oestrus was recorded in some cases for all dosed groups, a total of 2 Low females, 2 Mid dose females and 1 High dose females were affected (#2508, #2510, #3501, #3509 and #4510), but as it did not affect mating or pregnancy, this fact was considered as being an occasional finding, not being a test item related effect.
Prolonged dioestrus was noted for one Low dose female (#2507) and one High dose female (#4508), but this frequency (1/12) was in line with the normal, expected range and did not affect the mating or pregnancy.
-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. Both the mating and fertility index was 100% in treated groups (males and females). The gestation index was also 100% in all groups.
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 3 days of pairing (cohabitation). The mean duration of mating was 2.67, 2.67, 3.00 and 2.67 days in the Control, Low, Mid and High dose groups, respectively.
-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 related on pre-natal, post-natal or total mortality values (litter mean and %) in any dose group.
-Organ weights:
Parental Males
Test item-related changes were observed in the organ weights of the liver of Mid and High dose male animals compared to controls.
The relative to body weight of the liver was increased statistically significantly by 16.8%, in the Mid Dose group and by 17.8% in the High Dose group, the relative to brain weight increased by 18.8% in the High Dose group compared to Controls, correlated with microscopic findings.
Statistically significantly decreased absolute (p<0.05) and body related (p<0.01) weight of the heart was measured in the High dose male group only, not in females. Both values together with the brain related weight (not statistically significant) were outside of the historical control range hence it is considered as test item related effect. However, in the absence of any histopathological changes, the weight difference is not considered to be adverse.
Statistically significantly decreased absolute, body and brain related spleen weights were measured in the High dose group (p<0.05), but all the data were within the historical control range and no histological changes were seen, therefore it is not considered as test item related effect.

Parental Females
Test item-related changes were observed in the organ weights of the liver of Mid dose female
animals compared to controls.
The relative to body weight of the liver was increased statistically significantly by 12.1%, in the Mid dose group and by 11.3% in the High dose group, the relative to brain weight increased by 21.1% in the Mid Dose group compared to Controls, correlated with microscopic findings. The absolute organ weight of the thymus was decreased (by 37.2%) in the High dose group and was considered as stress induced.
Statistically significantly decreased absolute and brain related (p<0.01) weight of the heart was measured in the High dose group. Both values were outside of the historical control range hence it is considered as test item related effect. However, in the absence of any histopathological changes, the weight difference is not considered to be adverse.
The other statistically significant organ weight changes were without histological relevance and were considered as incidental.
-Pathology evaluation:
NON-PREGNANT FEMALES / Parental Generation
There was no non-pregnant female.
TERMINAL EUTHANASIA / Parental Generation
Macroscopic Findings
Test item-related changes were observed in the stomach, as multifocal, white or brown discoloration and/or multifocal/diffuse thickness of the non-glandular mucosa in High and Mid dose animals. The dark red discoloration of the mandibular lymph nodes was considered as procedure related (due to the sublingual blood collection), the multifocal white discoloration of the gastric glandular mucosa was without histological relevance and was considered as incidental.
All other changes were considered incidental or a common background.
Microscopic Findings
Minimal to marked, multifocal/diffuse squamous hyperplasia and minimal to moderate hyperkeratosis was observed in the non-glandular gastric mucosa, correlated with necropsy. These observations were recorded in High and Mid dose animals of both sexes, and in 3/12 in Low dose males. Additionally, in 1/12 Low and Mid dose males and in 2/12 Mid dose and 1/12 High dose females ulcer at the non-glandular region was seen as well. The gastric changes were considered as test item-related, local irritation, adverse change.
In the liver minimal/mild centrilobular hepatocellular hypertrophy were seen in 7/7 High dose males and in 3/3 High dose females; Mid and Low dose animals were unaffected. These were considered as non-adverse adaptive change.
In the kidney of all examined High dose males minimal/mild multifocal eosinophilic droplets/globules were seen in the renal tubules and were considered as test item-related. The same finding was present in 1/12 Low and 1/12 Mid dose males. These hyaline droplets are most likely to be Alpha 2u globulin which is a low molecular weight protein produced by the liver and excreted through kidney in male rats only; in the context of human safety evaluation, this rat-specific change is considered to be non-adverse.
In the thymus decreased cellularity was seen in 2/6 High dose females (correlated with organ weight changes) and was considered as stress induced.
Other findings were considered as incidental, background or procedural related. Other organs showing statistical differences in weight (heart, spleen) showed no histological changes, hence the weight differences were not considered to represent adverse effect of the test item.

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive effects of the parental generation

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: based on no adverse findings

Key result

Dose descriptor:

NOAEL

Remarks:

local

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Based on the external evaluation, no clinical signs or abnormalities were recorded for any pups except of one Mid dose pup (#3508/3) where cranium, absent, slight was recorded. This finding was considered as minor, incidental finding, not related to the test item treatment.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

There was no test item effect on mortality or survival of the pups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A slightly lower pup growth/weight at the High dose, while within the historic control range, may be related to maternal toxicity (gastric irritation/food intake)

Food consumption and compound intake (if feeding study):

not examined

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

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.

Nipple retention in male pups:

no effects observed

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

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test item-related macroscopic findings were observed up to the dose level of 600 mg/kg bw/day.

Histopathological findings:

not examined

-Mortality and clinical observations:
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 were comparable to control values in each dose group.
Mean pup mortality in the High dose group is relatively high but it is not statistically different from the control. For one High dose female (#4509) all the pups died, leading to large standard deviation and numbers on the mortality.
Evidence of suckling was recorded for all live born pups in the study except for two Low dose females (#2503, #2511), one Mid dose females (#3512) and two High dose females (#4509 and #4510) where there was no evidence of suckling for the minority of the pups.
Based on the external evaluation, no clinical signs or abnormalities were recorded for any pups except of one Mid dose pup (#3508/3) where cranium, absent, slight was recorded. This finding was considered as minor, incidental finding, not related to the test item treatment.
-Body weight and body weight gain:
There were statistically significant alterations observed in the high dose group compared to the Control, but they were within the historical range and therefore they are not considered as an adverse test item related effect.
The observed lower maternal food consumption is considered to result in a lower milk production, which could explain any lower growth of the pups. It is clear that there were maternal toxicity effects (secondary to gastric irritation and consequent lower food intake) hence any slightly lower pup growth/weight, while within the historic control range, may be related to maternal toxicity. There is no indication of a direct adverse effect of the test item on pup development.
-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 PND13.
-Thyroid hormone analysis:
Compared to the control, there were no statistically significant thyroid hormone concentration
levels recorded in any of the PND13 pup dose groups.
The body related weight of the thyroid / parathyroid gland of the male PND13 pups were statistically different from the Control group but the data were within the historical control range. In summary, there were considered to be 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.
-Pathology:
TERMINAL / F1 Generation (PND13)
Macroscopic Findings
No test item-related macroscopic findings were observed up to the dose level of 600 mg/kg bw/day.
Microscopic Findings
No histopathological examination was performed on pups (F1 generation).

Key result

Dose descriptor:

NOAEL

Remarks:

Pup development and survival

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: based on no adverse findings

Key result

Critical effects observed:

no

Reproductive effects observed:

no

Conclusions:

The NOAEL for reproductive effects of the parental generation was considered to be 600 mg/kg bw/day.
The NOAEL for Pup development and survival was considered to be 600 mg/kg bw/day.

Executive summary:

The purpose of this OECD No. 422 study was to obtain information on the possible toxic effects of the test item Sodium 1,4-diisodecyl sulphonatosuccinate 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 Post-natal Day (PND) 13.

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 the results of the DRF study, 600 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)	Concentration (mg/mL)	Dose volume (mL/kg bw)	Animal numbers
					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	600	120		12*	12*

*Note: Two female animals died on Day 5 and 6. These two animals and two males were replaced and the data of these animals were not reported but were documented in the raw data.

RESULTS

In summary, under the conditions of this study the daily administration of Sodium 1,4-diisodecyl sulphonatosuccinate by oral gavage to Wistar rats at dose levels of 100, 300 or 600 mg/kg bw/day (Low, Mid and High dose groups, respectively) did not result in test item related mortality or clinical signs.

Test item related adverse effect was observed on body weight gain parameters and food consumption in High dose (600 mg/kg bw/day) males and moderate effects were observed on body weight and body weight gain in the gestation and lactation period for High dose  females. It is most likely these effects were related to local gastric irritation.

There were no adverse changes in animal behaviour, general physical condition or in the reactions to different type of stimuli or in clinical pathology in test item treated groups when compared to control.

There was no test item effect on mortality or survival of the pups. The litter size of control and treated groups were comparable. A slightly lower pup growth/weight at the High dose, while within the historic control range, may be related to maternal toxicity (gastric irritation/food intake); there is no indication of a direct adverse effect of the test item on pup development.

Test item-related effects were observed in the liver (increased relative organ weights in Mid and High dose males and females) associated with centrilobular hepatocellular hypertrophy in the High dose. Hepatic changes were considered to be adaptive and nonadverse.

 

Test item related effect was observed in the organ weight of the heart (decreased absolute and relative organ weight) of the High dose males (not in females) compared to controls. However, there were no histopathological findings which indicates any adversity.

Histologically, test item-related changes were observed in the stomach in High and Mid dose animals with squamous hyperplasia, hyperkeratosis and ulcer of the non-glandular gastric mucosa observed at 300 and 600 mg/kg bw/day dose level in females and at 100, 300 and 600 mg/kg bw/day dose level in males, as test item-related adverse local changes. Sporadic cases of noisy respiration in a few animals ascribed to reflux or minor exposure to test item in the region of the upper respiratory tract. Also, centrilobular hepatocellular hypertrophy was observed at 600 mg/kg bw/day dose level as a non-adverse adaptive change (associated with liver weigh increase). Minimal to mild multifocal eosinophilic droplets/globules were seen in the renal tubules in the High dose males and were considered as test item-related but non-adverse.

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.

There was no test item effect on mortality or survival of the pups. The litter size of control and treated groups were comparable. A slightly lower pup growth/weight at the High dose, while within the historic control range, may be related to maternal toxicity (gastric irritation/food intake); there is no indication of a direct adverse effect of the test item on pup development.

The NOAEL for local toxicity of the parental generation was considered to be <100 mg/kg bw/day (based on local gastric histological effects in Low, Mid and High dose animals)

The NOAEL for systemic toxicity of the parental generation was considered to be 300 mg/kg bw/day. (based on body weight, body weight gain and food consumption changes at 600 mg/kg bw/day, although these effects were probably secondary to local gastric irritation).

The NOAEL for reproductive effects of the parental generation was considered to be 600 mg/kg bw/day.

The NOAEL for Pup development and survival was considered to be 600 mg/kg bw/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

600 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 theregistered substancein Wistar rats (12/sex/group by oral gavage at 0 (propylene glycol), 100, 300 and 600 mg/kg bw/day (Szalóki, 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. The litter size of control and treated groups were comparable. A slightly lower pup growth/weight at the High dose, while within the historic control range, may be related to maternal toxicity (gastric irritation/food intake); there is no indication of a direct adverse effect of the test item on pup development. 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. There was no test item effect on mortality or survival of the pups. The litter size of control and treated groups were comparable. A slightly lower pup growth/weight at the High dose, while within the historic control range, may be related to maternal toxicity (gastric irritation/food intake); there is no indication of a direct adverse effect of the test item on pup development. The NOAEL for reproductive effects of the parental generation was considered to be 600 mg/kg bw/day. The NOAEL for Pup development and survival was considered to be 600 mg/kg bw/day.

 

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/kg bw/day in key combined repeated dose/reproductive toxicity study (OECD No. 422). The NOAEL for reproductive effects of the parental generation was 600 mg/kg bw/day and the NOAEL for pups’ (F1 generation) development and survival was 600 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 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 Docusate sodium (CAS No. 577-11-7) and Docusate calcium (CAS No. 128-49-4) . 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. 

In conclusion, both a prenatal developmental toxicity with read across substance Docusate sodium and with read across substance Docusate calcium were negative for teratogenicity at non-toxic dose levels.

 

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. Developmental effects were only seen at dose levels where already maternal toxicity was observed. Therefore the developmental effects were considered to be caused by systemic toxicity and were therefore considered secondary findings. This was confirmed in a similar study with Docusate calcium given at subtoxic and toxic dose levels.

Taking into account that same metabolic and toxicological behavior can be expected structural similar substances, no further testing is needed.

---

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 the CLP regulation (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.