Sodium 1,4-bis(1,3-dimethylbutyl) 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 (distilled water), 100, 300 and 1000 (reduced to 600 mg/kg bw/day) 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 600 mg solid/kg bw/day and the NOAEL for pups’ (F1 generation) development and survival was 600 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:

screening for reproductive / developmental toxicity

Remarks:

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 August 2020 – 22 March 2022 (QA audited draft report)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI

Details on species / strain selection:

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

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, (Address: Sandhofer Weg 7, D-97633, Sulzfeld, Germany) from SPF colony
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Young adult rats, 10/11 weeks old (females/males) at start of the experiment and 12/13 weeks old (females/males) at mating.
- Weight at study initiation: Males: 391-441 g, females: 234-282 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 508 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 Maxi Fun Tunnels, Batch number: 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 / 713 70882, Expiry date: 31 October 2020 / 30 April 2021) produced by ssniff Spezialdiäten GmbH (Address: Ferdinand-Gabriel Weg 16, D-59494 Soest, Germany), ad libitum.
- Water (e.g. ad libitum): 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 6 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 the local Public Health and Medical Officer Service (H-8200 Veszprém, József Attila u. 36., Hungary). 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): 20.1-25.0℃ (target range: 19-25°C)
- Humidity (%): 32-68% (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: 19 August 2020 (first vaginal smear sampling)
To: End of in-life phase: 02 November 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 1.255 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 solution at the appropriate concentrations according to the dose levels and volume selected in the Pharmacy of the Test Facility. The formulations were stirred with manual shaking and a magnetic stirrer from the preparation until completion of each treatment.
Formulations were prepared a maximum of 1 or 7 days* prior to administration to animals according to stability assessment results of the analytical method development and method validation studies (Test Facility Study codes: 20/030-316ANE and 20/030-316AN).
*Note: Maximum of 7 days of storage was applied for 20, 60 and 120 mg solid/mL concentration test item formulations, while test item formulation of 200 mg solid/mL concentration was stored for a maximum of 1 day.
Based on those results of the analytical method validation and its extension, the test item formulation in 5-200 mg test item/mL concentration range (equaling 4-159 mg solid/mL range) were stable for at least 7 days when stored at room temperature, while test item formulation at 300 mg test item/mL concentration (equaling 239 mg solid/mL concentration) was proven to be stable for at least 2 days when stored at room temperature.
The appropriate 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. 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 to be administered was calculated and adjusted based on each animal’s most recent body weight.

VEHICLE
- Concentration in vehicle: 0, 20, 60, 120 and 200 mg solid/mL
- Amount of vehicle (if gavage): Dose formulation volume = 5 mL/kg bw
- Lot/batch no. (if required): 202003032 / 202007068

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: Females remained with the same male until copulation occurred, for up to 10 days.
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
-In the High dose group, a dose level of 1000 mg/kg bw/day was used on the first three days. Two High dose males found dead early in the treatment period (#4005 on Day 3 and #4009 on Day 2) were replaced, so 12 males were available for mating. Due to the death of its mating female pair, one Mid dose male (#3001) and three High dose males (#4002, #4006 and #4012) were not used for mating (#4012 was pre-terminally euthanized later on Day 23).
In case of one mating pair (#4007-#4507), the female died before a successful mating, this pair was excluded form evaluation.
One High dose male (#4008) was found dead on Day 18 after a successful mating, it was included in the evaluation. In case of another mating pair (#4005-#4505), the female was found dead during the mating period but had a sperm-positive vaginal smear on the previous day, so they were counted as mated.
- After successful mating each pregnant female was caged (how): individually housed (with pups)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Sample collection was performed at a total of four occasions (during the first* and last weeks and once 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.
*Notes: Sampling was made twice on the first week as the High dose was reduced after three days of treatment: As a formulation with different concentration was used for treatment of the High dose animals from Day 4, additional sampling (sampling #2) was made.
On each sampling occasion, top, middle and bottom duplicate samples were taken from the dedicated 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 four times during the study (twice during the first week, once midway during the treatment period and once on the last week of treatment).
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: 20/030-316AN). No density measurement was needed for these aqueous formulation as confirmed by the Contributing Scientist #1 (Analyst).
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 94% and 102% of the nominal concentrations.
All test item formulations were shown to be homogeneous. The relative standard deviation (RSD) was below 10% in each case.
Formulations were considered to be adequately stable under the study conditions.
Overall, the formulations were considered adequate for the study.

Duration of treatment / exposure:

Dosing of both sexes began after the acclimatisation (6 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.:

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

Remarks:

solid;
The dose level for High dose group was 1000 mg/kg bw/day (using 200 mg/mL formulation) for the first three days (on Days 0-2), then from Day 3 it was reduced to 600 mg/kg bw/day (using 120 mg/mL formulation).

No. of animals per sex per dose:

12 animals/sex/group + 2 males and 6 females for replacement purpose in the high dose group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The oral route was selected as it is one of the possible routes of human exposure as requested by ECHA based on the information provided by the Sponsor (Decision number: CCH-D-2114489558-27-01/F; Helsinki, 14 November 2019). The way of test item and vehicle control item administration was in compliance with the relevant OECD No. 422 guideline.
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: 20/030-220PE), 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 mortality occurred in the Control, Low and Mid dose groups (100 and 300 mg solid/kg bw/day, respectively). Two out of the four males and one out of the four females in the High dose group (1000 mg solid/kg bw/day) were found dead during the study; however, the cause of death was considered not to be related to systemic toxicity, rather than local respiratory irritation from reflux or a very small amount of test item (a known issue with strong surfactants). Noisy respiration, laboured respiration, gasping respiration, (increased) salivation, decreased activity, soft faeces, liquid faeces, hunched back, red discharge and piloerection were observed mainly in some of the High dose animals and in one Mid dose animal for a short period. These changes were not ascribed to systemic toxicity, but probably were secondary to respiratory local effects. No test item related effect on body weight, body weight gain or food intake was observed, indicating the local effects of the test item had no major consequences on normal growth.
Overall, 1000 mg solid/kg bw/day, was considered as acceptable for the High dose level of this study, with the implementation of additional cleaning of gavage tubes and refined gavage procedures, to reduce the risk of respiratory contamination with test item. Lower doses were spaced with a factor of approximately 3.
Based on the early observations of this study, the dose level of the High dose group was reduced to 600 mg solid/kg bw/day from the fourth day of the study.
- Rationale for animal assignment (if not random): All adult/parental (P) male and female animals were sorted according to body weight by computer and divided into weight ranges. There were an equal number of animals from each weight group randomly assigned to each dose group to ensure that animals of all test groups were as nearly as practicable of a uniform weight.
This process was controlled by the software PROVANTIS v.9, to verify the homogeneity/variability between/within the groups. Males and females were randomised separately to the dose groups at start of the treatment (Day 0).
- Fasting period before blood sampling for clinical biochemistry: All animals including the randomly selected animals for blood sampling were fasted (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 (if any) 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). Any animal (including also all premature decedents) which showed clinical signs considered severe was sacrificed to prevent suffering, cannibalism and/or autolysis, and was processed in the same way as the animals subjected to terminal necropsy.

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

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


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

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

Oestrous cyclicity (parental animals):

Oestrus cycles were 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 and uterus, cervix, ovary, oviduct and vagina for females) 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.
On PND 4, litters were culled to yield 5 males and 5 females per litter (or as nearly as possible). Pups to be culled within each litter were selected randomly. In litters of insufficient size where the number of males or female pups was less than 5, adjustment of the selection process was made to assure 10 pups were retained. Culling was not performed on litter sizes less (or equal) than 10.

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. Observations were reported individually for each adult animal. In addition to the observations on parent animals, any abnormal behaviour of the offspring was recorded.
Live pups were counted, sexed, weighed individually within 24 hours of parturition (PND0), and on PND4 and PND13, 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 (PND0). The anogenital distance was also normalized to a measure of pup size (the cube root of body weight) for statistical analysis as considered appropriate by the Study Director.
From the pups dedicated for culling, one male and one female pup per litter (if possible*) were previously selected for blood sampling on PND4.
Number of nipples/areolae in male pups was recorded on PND13.
All pups were necropsied on PND13.

GROSS EXAMINATION OF DEAD PUPS:
Yes: 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.

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.
Gross necropsy was performed on each adult animal irrespective of the date of death. Terminally (one day after the last treatment), animals were sacrificed under anaesthesia by exsanguination; anaesthetic product was diluted for pups’ euthanasia as required.
After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities was opened, and the appearance of the tissues and organs were observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate. Special attention was paid to the organs of the reproductive system.
Vaginal smears were prepared and examined for each female on the day of necropsy to determine the stage of oestrus cycle and allow correlation with histopathology of the reproductive organs.
The number of implantation sites and of corpora lutea was recorded in the females as applicable.

HISTOPATHOLOGY / ORGAN WEIGHTS
At the time of termination, body weight and weight of the following organs of all surviving adult animals and preterminal euthanised 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
Additionally, thyroid glands from one male and one female PND13 pup from each litter were preserved in 10% buffered formalin solution. In this case, the thyroid weight (pooled) was determined after fixation. Trimming was done very carefully and only after fixation to avoid tissue damage.
The retained tissues and organs required for histopathology (below) were embedded in paraffin wax; sections were cut at 4-6 µm by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope.
For the adult animals, detailed histological examination was performed as follows:
•on the selected list of retained tissues and organs (as above) in the Control and High dose groups (selected 5 animals/sex/group),
•any animals found dead or euthanized pre-terminally during the study in all groups,
•all macroscopic findings (abnormalities), except of minor order from all animals,
•stomach samples of 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.
Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.
Special attention was paid to the organ weight, appearance and histopathology of immune-system tissues for any evidence of immunotoxicity (spleen, thymus, lymph nodes, bone marrow).
Special attention was paid to the central and peripheral nervous system tissues for any evidence of neurotoxicity.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring was sacrificed at Post-natal day 13. Litters were culled on Post-natal Day (PND) 4. All selected F1 offspring were terminated on PND 13. In order to allow for overnight fasting of dam prior necropsy on PPD 14, offspring was euthanized on PPD/PND 13, and the dams on PPD/PND 14.
- These animals were subjected to postmortem examinations (macroscopic examination):

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

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, treatment-related

Description (incidence and severity):

The daily administration of Sodium Dihexyl Sulfosuccinate by oral gavage to Wistar rats at a dose level of 100 mg solid/kg bw/day (Low dose group) did not result in test item related clinical signs. In the Mid dose group (300 mg solid/kg bw/day), most animals had minor/transient symptoms of respiratory local effects, related to reflux or by small amounts of test item reaching the upper respiratory tract area in animals; but with no indications of systemic effects of the test item. In the High dose group, the initial dose level of 1000 mg solid/kg bw/day was found to be too high despite very careful gavage procedures to ensure minimal local respiratory effects, so the dose was reduced to 600 mg solid/kg bw/day after three days of treatment.

Mortality:

mortality observed, treatment-related

Description (incidence):

The daily administration of Sodium Dihexyl Sulfosuccinate by oral gavage to Wistar rats at a dose level of 100 mg solid/kg bw/day (Low dose group) did not result in test item related mortality. One Mid dose animal died, related to local effects of the test item. In the High dose group, the initial dose level of 1000 mg solid/kg bw/day was found to be too high despite very careful gavage procedures to ensure minimal local respiratory effects, so the dose was reduced to 600 mg solid/kg bw/day after three days of treatment. A relatively high rate of deaths or euthanasia occurred at the High dose level due to the local effects of the test item; necropsy showed local gastric irritation and respiratory effects.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Test item related adverse effects were observed on body weight parameters in High dose males and females, this was largely transient, in the first week. In High dose females there was a lower weight gain during gestation (~20% less gain than controls).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Test item related adverse effects were observed on food consumption in High dose males and females, this was largely transient, in the first week.

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

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

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No test item-related adverse effects 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.

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):

At the functional observation battery (FOB) and locomotor activity measurement, there were no test-item related 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

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No adverse test item related systemic macroscopic or microscopic changes were recorded at necropsy or at histopathology evaluation of routine organs/tissues or in any reproductive organs.
In animals with respiratory distress, secondary stress effects were recorded (such as organ weight and histological changes in thymus and adrenals).

Histopathological findings: neoplastic:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

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

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure

Reproductive performance:

no effects observed

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 PPD14.

-Mortality and morbidity:
One Mid dose female (#3501 on Day 4) and fourteen High dose animals (#4005 on Day 3, #4008 on Day 18, #4009 on Day 2, #4501 on Day 1, #4502 on Day 5, #4503 on Day 1, #4506 on Day 2, #4507 on Day 15, #4509 on Day 4, #4510 on Day 6, #4512 on Day 12, #4601 on Day 32, #4606 on Day 8 and #4609 on Day 39) were found dead during the study. Three High dose animals (#4012 on Day 23, #4505 on Day 17 and #4602 on Day 9) were pre-terminally euthanized. Thus, a total of 1 Mid dose animal (one female) and 17 High dose animals (4 males and 13 females) were lost during the treatment period.
The indication of gavage associated injury was observed in 3/14 High dose rats (#4506, #4509 and #4512).
No clinical signs were observed in case of #4005, #4009 and #4506.
Gasping and/or noisy respiration or red liquid was observed for 5 High dose females prior to death (#4501, #4502, #4503, #4509, #4512). Laboured, noisy or gasping respiration, piloerection, hunched back and distended abdomen was observed prior to pre-terminal euthanasia in case of #4505 (respiratory signs on Days 4-14, hunched back on Days 5-12, distended abdomen on Day 16) and #4602 animals (respiratory sign on Days 3-8, distended abdomen on Days 7-8). Hunched back, piloerection and noisy or gasping respiration was observed in case of #4507, #4510 and #4609 High dose females prior to death. Noisy respiration and piloerection were observed prior to death for #4601 female. Laboured and noisy respiration, piloerection, hunched back and increased salivation was observed for #4606 female prior to death. Noisy respiration was recorded in one found dead male (#4008) from Day 1 to death (Day 18). In one High dose male (#4012) decreased activity, hunched back, laboured and noisy respiration, piloerection and liquid faeces were recorded, this animal was pre-terminally euthanized due to ethical reason on Day 23.
For one Mid dose female (#3501) gasping and noisy respiration was observed prior to death.
-Clinical observations:
The observed clinical signs for found dead or moribund animals are given in the section on mortality/morbidity. The information below applies the surviving animals.
No clinical signs were observed in the Control male and female and Low dose male animals. Noisy or laboured respiration was recorded for two Low dose females (#2511 on Days 1-2, and #2503 on Day 7).
Laboured respiration was observed in 1/12 Mid dose female (#3510) on Day 38 and Day 39. Noisy respiration was observed occasionally in 8/12 Mid dose males and 10/11 Mid dose females from Day 2 to Day 39 and Day 1 to Day 12 , respectively; longer duration was observed only for one female animal (#3510) in the period of Days 14-31 and on Day 39.
Noisy respiration was observed in 6/10 High dose males and 4/5 High dose females from Day 1 to Day 24 and from Day 0 to Day 59, respectively, the longevity of the observation was 5 and 8 days, respectively. Gasping respiration was observed for one High dose female (#4504) on Days 1-2. Piloerection was observed for two High dose females and two High dose males (#4105 on Day 7 and #4109 on Day 2). Laboured respiration was observed for 3/5 High dose females and for 3/10 High dose males on Day 5 and from Day 29 to Day 34 and on Day 2 and from Day 23 to Day 24, respectively. The longest period of the observation was 3 and 2 days, respectively. Mucoid faeces were observed for one High dose male (#4004) on Day 18. Slightly decreased activity was observed for two High dose males (#4109 from Day 2 to Day 3 and #4010 on Day 21, respectively). Hunched back was observed for two High dose males (#4105 on Day 7 and #4109 on Days 2-3,respectively) and one High dose female (#4508 on Days 17-19).
The above findings were probably related to the very irritant effect of small amounts of test item reaching the upper respiratory tract via reflux or contamination during dosing (as seen during the preliminary study). This was considered as a local effect of the test item.
-Body weight and weight changes:
Decreased body weight gain or body weight loss was observed in the High dose groups in the first week of treatment, with females recovering to near control weights by Day 14 and males by Day 28. During gestation High dose females gained about 20% less than controls (p<0.05) but weight gain in lactation was normal in all groups although the total weight gain (Day 0 to termination) remained lower for High dose females (p<0.01).
No test item related adverse effects on body weight or body weight gain were detected in Low or Mid dose (100 and 300 mg solid/kg bw/day, respectively) animals (males or females).
Taking account of the necropsy findings, reduced body weight growth and food intake, particularly in the first week, was considered to be related to gastric irritation and/or reflux with respiratory irritation effects.
-Food consumption and compound intake (no feeding study):
Test item related adverse effects on food consumption were observed in High dose females (600 mg solid/kg bw/day) during the pre-mating and gestation phases, no effect was observed in the High dose males or Mid and Low dose groups (300 and 100 mg solid/kg bw/day, respectively).
In case of High dose females, reduced values compared to control were recorded during the pre-mating period (by 25%) and gestation period (by 13%), statistical significance was reached in both cases (p<0.05). Based on the effect on body weight values, the observed values were considered as a test item related adverse effect.
-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 statistical significance observed occasionally in any 5-minute segment without dose response was considered as incidental. 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 adverse changes were detected in the test item treated animals (males and females) when comparing haematology parameters to the relevant Control data. Occasional statistically significant changes were within the HC range (and with no dose response in some cases), and were considered as biological variability, not related to the test item treatment.
-Clinical Chemistry:
No test item-related adverse changes were detected in the test item treated animals (males and females) when comparing serum chemistry parameters to the relevant Control data.
The statistically significant changes observed in the High dose group (decreased urea and total bilirubin concentration in High dose males, increased urea and decreased chloride concentration in High dose females), had no similar trend in the other sex and were within the historical control range, so were considered as animal variability and not related to the test item treatment.
-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:
Pre-exposure period:
Each female selected for the study showed acceptable cycles (mean cycle length of 3.97-4.11 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 4.03, 4.21, 4.22 and 4.15 days in the Control, Low dose, Mid dose and High dose groups, respectively).
Prolonged oestrus was recorded in some cases for all test item treated groups, a total of 4 Low dose females, 1 Mid dose female and 1 High dose female were affected (#2501, #2505, #2507, #2508, #3508, and #4505), but as there was no dose related trend and the mating or pregnancy rate was not affected, these values were not considered as a test item related effect.
Prolonged dioestrus was noted for three Mid dose females (#3502, #3506 and #3511) and three High dose females (#4504, #4507 and #4602), but those occurrences did not affect to the mating or pregnancy.
-Reproductive ability assessment and indices:
There were no differences between the control and test item treated groups regarding 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 and fertility index were 100% in all groups (males and females). The gestation index was also 100% in Control, Low, Mid and High dose group.
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 3.17, 2.67, 3.09 and 3.89 days in the Control, Low, Mid and High dose groups, respectively. Longer than usual mating was noted for five animals (6 days for #2507 and #4511, 7 days for #1510, 9 days for #3502 and 10 days for #4603), but they were considered as incidental findings, no test item effect was noted.
-evaluation of 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 group no statistically significant differences were noted.
There were no biologically 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:
There were no test item treatment-related statistically significant differences among groups of males and females in the weights of organs measured when compared to Controls.
Terminal body weights of test item treated males and females were not significantly different from control animals.
The body-related and brain related weight of the liver was statistically significantly increased (by 13.8% and 14.1%) in the High dose males compared to Control, similarly the body related liver weight was statistically significantly increased in the High dose females (by 12.4%) when compared to Control (Table 13). Based on the males and females both having increased liver weight at the High dose with no histological changes, this fact was considered to be a non-adverse adaptive change.
There were no other statistically significant and biologically relevant differences among groups in the weights of organs measured when compared to Controls.
-Pathology evaluation:
FOUND DEAD ANIMALS / Parental Generation
One Mid dose female (3501) and fourteen High dose animals (4005, 4008, 4009, 4501, 4502, 4503, 4506, 4507, 4509, 4510, 4512, 4601, 4606 and 4609) were found dead.
Macroscopic Findings
….
Microscopic Findings
Test item-related multifocal squamous cell hyperplasia, mixed cell submucosal infiltrate (and single occasion of inflammation) and/or erosion/ulcer of the non-glandular stomach mucosa were seen in 10/14 found dead High dose animals. Erosion/ulcer contributed to death in 5/14 High dose animals. A gavage reflux related to the test item induced findings in stomach appeared as possible contributing factors to death in one Mid dose female and six High dose rats. A gavage reflux-related subacute microscopic changes observed in the lungs included aggregation alveolar macrophages, foreign material (bronchiole), mixed/neutrophilic infiltrates and/or granulomatous inflammation (with foreign material) and mixed/neutrophilic infiltrate in trachea. The indication of gavage associated injury (degeneration/necrosis/inflammation of tunica muscularis in the oesophagus) was observed in 3/14 found dead High dose rats. The stomach lesions in unscheduled deaths were similar in distribution as those seen in animals that survived to the end of the study. At necropsy, these stomach lesions were visualized as discoloration and/or thickness. The severity of erosion/ulcer lesions was higher in found dead animals comparing with terminals.
Decreased cellularity of lymphocytes (cortex) in the thymus and diffuse bilateral cortical hypertrophy of zona fasciculata in adrenals microscopically observed in unscheduled deaths, were considered stress-related findings due to treatment and not a direct toxic effect of the test item. Thymic changes were manifested at necropsy as small thymus.
All other changes were incidental or a common background. The results indicated that gastric and respiratory tract irritation were main factors in the deaths.
…
PRE-TERMINAL EUTHANASIA / Parental Generation
Three High dose animals (#4012, #4505 and #4602) were pre-terminally euthanized.
Macroscopic Findings
Test item-related stomach findings contributing to earlier terminations consisted of squamous cell hyperplasia (and mixed cell submucosal infiltrate) and/or erosion/ulcer of the non-glandular mucosa.
Microscopic Findings
Test item-related stomach findings contributing to earlier terminations consisted of squamous cell hyperplasia (and mixed cell submucosal infiltrate) and/or erosion/ulcer of the non-glandular mucosa. This indicated that gastric irritation was a main factor in the moribund conditions.
…
TERMINAL EUTHANASIA / Parental Generation
Macroscopic Findings
Test item-related macroscopic findings were observed in the stomach (non-glandular) of Mid and High dose animas (dose levels of 300 and 600 mg solid/kg bw/day, respectively) and in the thymus of the High dose group. These changes correlated with microscopic findings.
Diffuse/multifocal thickness of the non-glandular mucosa was observed in 1/23 terminal Mid dose and 13/15 terminal High dose animals. White multifocal discoloration of the non-glandular mucosa was seen in 1/23 Mid dose animal. Small thymus correlated with histopathology was observed in 1/23 High dose animal.
All other changes were incidental or a common background.
Microscopic Findings
Treatment-related findings were observed at the High dose level (600 mg solid/kg bw/day). Stomach (non-glandular), thymus and adrenal gland were identified as target organs. It is considered that gastric irritation was a main finding in animals; thymus and adrenal effects were considered as secondary, stress related effects.
-Thyroid hormone analysis:
Compared to the control, there were no statistically significant thyroid hormone concentration levels recorded in any dose groups of parental males or PND13 pups.
No relevant changes were noted in the absolute or relative (to body / brain) thyroid weights of the parental male or female animals in any dose groups. No histopathology (microscopic) findings were detected in any High dose males or females.
The thyroid gland weights of the PND13 pups were also statistically not different from the Control group.
In summary, there were no effects on the thyroid hormone levels or on the thyroid glands in parental males and females and in the PND13 pups that were ascribed to the test item.

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive effects of parental generation

Effect level:

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

Based on:

other: solid

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: No reproductive findings were observed up to highest tested dose.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no adverse effects on the F1 offspring clinical signs.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

There were no adverse effects on the F1 offspring viability.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

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

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 finding was recorded for F1 pups at necropsy.

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

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.
There were no significant differences or effects that could be ascribed to test item treatment on the pre-natal, post-natal or total mortality values (litter mean and %) in any of the dose groups. Statistical differences in sex ratios were due to a high control value, but all data are considered as normal. Data on the number of pups born show differences between groups only when analysed for the whole group, the more relevant data of mean numbers per litter show no treatment related effects.
Based on the external evaluation, no clinical signs or abnormalities were recorded for any pups except for thirteen Low dose pups (#2507 litter), where absent tail /tail tip was recorded, and one Mid dose pup (#3505/18), which was cold to touch on Day 0 (before cannibalized on the next day). These findings were considered as minor, incidental finding, not test item related.
Evidence of suckling was recorded for all live born pups in the study, except three pups of Mid dose group (#3505/18, #3510/14 and #3510/19) which animals died on PND1.
-Body weight and body weight gain:
There were no test item related differences in the offspring body weights or weight gains in all 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 relevant statistically significant changes in the anogenital distance measured on PND 0 were noted for test item treated male and female pups when litter mean values were compared to control.
No nipples/areolae were present in any of the male pups on PND13.
-Pathology evaluation:
TERMINAL / F1 Generation (PND13)
Macroscopic Findings
No test item-related macroscopic findings were observed up to the High dose level (1000/600 mg/kg bw/day).
Microscopic Findings
No histopathological examination was performed on pups (F1 generation).
-Thyroid hormone analysis:
Compared to the control, there were no statistically significant thyroid hormone concentration levels recorded in any dose groups of parental males or PND13 pups. The thyroid gland weights of the PND13 pups were also statistically not different from the Control group. In summary, there were no effects on the thyroid hormone levels or on the thyroid glands in the PND13 pups that were ascribed to the test item.

Key result

Dose descriptor:

NOAEL

Remarks:

pup development and survival

Generation:

F1

Effect level:

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

Based on:

other: solid

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: No reproductive findings were observed up to highest tested dose.

Key result

Reproductive effects observed:

no

Conclusions:

The NOAEL for reproductive effects of the parental generation: 600 mg solid/kg bw/day (based on no significant findings).
The NOAEL for pups (F1 generation) development and survival: 600 mg solid/kg bw/day (based on no significant findings).

Executive summary:

The purpose of this OECD No. 422 study was to obtain information on the possible toxic effects of Sodium Dihexyl Sulfosuccinate (CAS 2373-38-8, EC 219-147-9) 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/kg bw/day was selected as the High dose for this study. Based on the results of the first three days of dosing (mortalities in the High dose group), the dose level of the High dose group was reduced to 600 mg/kg bw/day. 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 designation	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 / 600*	200 / 120*		14	18

*Notes: The dose level for High dose group was 1000 mg/kg bw/day (using 200 mg/mL formulation) for the first three days, then it was reduced to 600 mg/kg bw/day (using 120 mg/mL formulation). Replacement animals started on Day 4 or after (#4602, 4609 and 4610) were treated only at 600 mg/kg bw/day. Dose level of the High dose group is shown as 600 mg/kg bw/day in the report text and tables.

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 or
pre-terminally euthanized animals, and stomach samples of all animals.

Dosing formulation were analysed for concentration and/or homogeneity on four 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 94% and 102% 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 Sodium Dihexyl Sulfosuccinate by oral gavage to Wistar rats at a dose level of 100 mg solid/kg bw/day (Low dose group) did not result in test item related mortality or clinical signs. In the Mid dose group
(300 mg solid/kg bw/day), most animals had minor/transient symptoms of respiratory local effects, related to reflux or by small amounts of test item reaching the upper respiratory tract area in animals; but with no indications of systemic effects of the test item. One Mid dose animal died, related to local effects of the test item. In the High dose group, the initial dose level of 1000 mg solid/kg bw/day was found to be too high despite very careful gavage procedures to ensure minimal local respiratory effects, so the dose was reduced to 600 mg solid/kg bw/day after three days of treatment. A relatively high rate of deaths or euthanasia occurred at the High dose level due to the local effects of the test item; necropsy showed local gastric irritation and respiratory effects.

Test item related adverse effects were observed on body weight parameters and food consumption in High dose males and females, this was largely transient, in the first week. In High dose females there was a lower weight gain during gestation (~20% less gain than controls).

At the functional observation battery (FOB) and locomotor activity measurement, there were no test-item related 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 adverse effects were seen in the clinical pathology parameters.

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

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

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

No adverse test item related systemic macroscopic or microscopic changes were recorded at necropsy or at histopathology evaluation of routine organs/tissues or in any reproductive organs. Local toxicity was observed as gastric irritation in the High and Mid dose groups (with minimal incidence at the Low dose). Gastric reflux resulted in irritation, sometimes severe, in the upper respiratory tract, which was lethal in approximately 50% of the High dose and in one animal in the Mid dose animals. In animals with respiratory distress, secondary stress effects were recorded (such as organ weight and histological changes in thymus and adrenals).

There was an increase in the hepatic weight (12-14%) in both sexes of the High dose group, indicating an adaptive hypertrophy, but below the level that is detectable histologically.

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 local toxicity of the parental generation: 100 mg solid/kg bw/day (based on local gastric effects).

The NOAEL for systemic toxicity of the parental generation: 300 mg solid/kg bw/day (Although body weight/food consumed and mortality in the High dose group were ascribed to secondary effects of the test item from the direct local effects; there could theoretically also be a potential systemic effect. Taking a conservative approach 300 mg solid/kg bw/day was applied here).

The NOAEL for reproductive effects of the parental generation: 600 mg solid/kg bw/day (based on no significant findings).

The NOAEL for pups (F1 generation) development and survival: 600 mg solid/kg bw/day (based on no significant findings).

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:

reliable (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). Based on the results of the DRF study, 1000 mg/kg bw/day was selected as the High dose for this study, however due to the results of the first three days of dosing (mortalities in the High dose group), the dose level of the High dose group was reduced to 600 mg/kg bw/day. Concentrations and all dose levels in the study (including raw data and study report) were expressed as solid matter as requested by the Sponsor. 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 NOAEL for reproductive effects of the parental generation was 600 mg solid/kg bw/day (based on no significant findings); the NOAEL for pups’ (F1 generation) development and survival: 600 mg solid/kg bw/day (based on no significant findings).

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

New Prenatal developmental toxicity studies with CAS No. 2373-38-8 and read-across substance 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:

Read across argumentation is provided in Section 13.

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:

reliable (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 577 -11 -7). Justification for read across within the category of Di-ester sulphosuccinates is documented in a separate document attached in Section 13.

 

Teratogenicity testing in first species

-A key study for prenatal developmental toxicity was performed in rats dosed from day 6-15 of gestation with read across substance Docusate sodium (CAS 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. 

- As doscusate sodium has a long history of pharmaceutical and dietary supplement use, a 3-generation toxicity study in rat was requested by FDA and JECFA, which confirmed that docusate sodium would pose ro reproduction or teratological problems. The data were considered to adequately support safety in combination with information from prenatal developmental toxicity testing of docusate sodium and docusate calcdium, therefore a study in a second species was not considered necessary. In addition, the rabbit was not considered to be an adequate model due to its high gastro-intetinal sensitivity to surfactant properties, as demonstrated by diarrhoea followed by mortality in a repeated dose toxicity study. In conclusion, based on the evaluation of the outcome of the first species and other supporting informatoin, a second species for developmental toxicity was not considered necessary and waived according to REACH Annex IX section 8.7.2 column 2.

Conclusion 

Prenatal developmental toxicity was tested by dietary administration of read across substance Docusate sodium (CAS 577 -11 -7) 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 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 CAS No. 2373-38-8 and read-across substance 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 pecies

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.