Sodium 1,4-diisotridecyl sulphonatosuccinate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

A key combined repeated dose toxicity with reproduction/developmental toxicity screening test in male and female rats by oral gavage at 0, 100, 300 and 1000 mg/kg bw/day, resulted in slight statistically significant decreases in body weight, weight gain and food intake in females at 1000 mg/kg bw in males and females. Test item related non-adverse increases in liver enzymes in the Mid dose males and High dose males and females were considered to be linked to the adaptive response seen in the liver during microscopic examination (hepatocellular hypertrophy and vacuolation which was observed only in High dose animals, but not in Mid or Low dose animals). Hyperkeratosis of the non-glandular gastric mucosa of the stomach was identified at the High dose level (1000 mg/kg bw/day), as test item-related adverse local change. This was related to the irritating properties of the substance. No other relevant parental toxicity findings were observed in the study. No test item related changes were noted in the reproductive parameters during mating and gestation, delivery and post-partum/lactation period until PPD14.The number of implantation or liveborn pups, as well as the pre-natal, post-natal or total mortality values were comparable with the control values in all test item treated dose groups. No test item related macroscopic findings were recorded for F1 pups at necropsy. There were test item-related differences on the offspring body weights or body weight gains in the Mid and High dose groups (300 and 1000 mg/kg bw/day, respectively) when compared to the control values. The difference in the Mid dose group was not considered as an adverse effect of treatment since the values were well within the historic control range, and hence the pup growth was considered to be normal. The measured values of growth and weight were clearly outside the historical control range in case of the High dose group pups, the changes observed in this dose group were considered as a test item related adverse effect (however, significantly lower maternal body weight gain and lower food intake by these lactating dams i.e. maternal toxicity, is considered to be an important factor in, or the direct cause of, the High dose pup effects). No indication of an endocrine disruptor effect was observed in the study based on collected oestrus cycle data, anogenital distance measurement, nipple retention, thyroid hormone measurement, thyroid weight and histopathology and external reproductive organs analysis. Based on the results of this study, the systemic NOAEL for parental toxicity was 300 mg/kg bw/day. The NOAEL for reproductive effects of the parental generation was 1000 mg/kg bw/day (based on the lack of relevant findings). The NOAEL for pups’ (F1 generation) development and survival was 1000 mg/kg bw/day (based on the lack of evidence of a direct effect on the pups at 1000 mg/kg bw/day, where treatment related reduced pup growth and body weight were seen, but were attributed as most probably due to maternal toxicity (~20% lower dam body weight gain and ~20% reduced food intake, associated with local gastric irritation in the lactating dams). No treatment related effect on pup growth was seen at 300 mg/kg/day).

Supporting multigeneration studies were available for read across substance Docusate sodium (CAS 577-11-7) for assessment of reproductive and postnatal developmental function. In a first study, slight maternal/paternal toxicity were observed at 0.5 and 1% in the diet, however this was not confirmed in a second study. The test article was considered to have influenced the taste of the milk, for which adaptations were done in the second study. From both studies, it can be concluded that dosing Docusate sodium up to 1% in the diet did not lead to effects on fertility or postnatal development; this concentration corresponds with 750 mg/kg bw/day.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6 July 2020 to x May 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Paris, 29 July 2016

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

- Premating exposure duration for parental (P0) animals: 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 (first day of dam’s necropsy).
- Basis for dose level selection: 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: 20/027-220PE, with the aim of inducing toxic effects but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose.
In the DRF study (after a 14-day treatment period), no clinical signs were recorded and no test item related adverse effect were seen on body weight, food consumption, clinical pathology (including haematology and clinical chemistry), gross necropsy and organ weight determination, except of significantly increased serum GPT (ALT) level at 1000 and 300 mg/kg/bw/day dose groups.
Overall, 1000 mg/kg bw/day, was considered as acceptable for the High dose level of this study. Lower doses were spaced with a factor of approximately 3.
- Route of administration: The oral route was selected as it is one of the possible routes of human exposure and was requested by ECHA as most appropriate route (ECHA Decision number: CCH-D-2114489557-29-01/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.
- Other considerations, e.g. on choice of species, strain, vehicle and number of animals [if applicable]
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.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL: see confidential details

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL: see confidential details

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was formulated in the selected vehicle (propylene glycol), as a visibly stable homogenous solution at the appropriate concentrations according to the dose level and volume selected in the Pharmacy of the Test Facility. The formulations were stirred with manual shaking and/or a magnetic stirrer from the preparation until completion of each treatment.
Formulations were prepared fresh every day prior to administration to animals or at appropriate intervals to allow their use according to stability assessment results of the analytical method validation study.
- Final dilution of a dissolved solid, stock liquid or gel: Concentration of formulation 0, 20, 60 and 200 mg/mL

OTHER SPECIFICS: see confidential details

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI (Wistar)

Details on species / strain selection:

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 will be used, corresponding to the regulatory guidelines being followed.

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
- Males & Females nulliparous and non-pregnant
- 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: 390-451 g, females: 226-268 g (at the start of the treatment). The body weights did not exceed ± 20% of the mean weight for each sex at start of treatment.
- Fasting period before study: not applicable
- 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. Type II, III and/or IV polycarbonate cages were used. SAFE 3/4-S Hygienic Animal Bedding and SAFE Crinklets Natural (nesting material) produced by J. Rettenmaier & Söhne GmbH+Co.KG (Address: Holzmühle 1, D-73494 Rosenberg, Germany), were used in the study. Group housing allowed social interaction. Deep wood sawdust bedding allowed digging and other normal rodent activities, while nesting material allowed normal nesting behaviour. Certified cardboard hiding tunnels (GLP Mini Fun Tunnels, Batch number: A123) produced by LBS (Serving Biotechnology) Ltd. (Address: Unit 20, Gatwick Business Park, Kennel Lane, Hookwood, Surrey, RH6 0AH UK) were also provided to the animals..
- Diet (e.g. ad libitum): The animals received ssniff® SM R/M “Autoclavable complete diet for rats and mice – breeding and maintenance” (Batch number: 560 65984, Expiry date: 31 October 2020) produced by ssniff Spezialdiäten GmbH (Address: Ferdinand-Gabriel Weg 16, D-59494 Soest, Germany), ad libitum.
- Water (e.g. ad libitum): The animals received tap water from the municipal supply, as for human consumption from a 400- or 500-mL bottle, ad libitum.
- Acclimation period: 5 days and pre-exposure (14 days). Fifty-five male and 65 female Wistar rats were used in the pre-treatment period. At the end of the pre-treatment period, only 48 females showing regular oestrus cycles and 48 males were allocated to treatment groups

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

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.8-25.0℃ (target range: 19-25°C)
- Humidity (%): 31-68% (target range: 30-70%)
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12/12 (12 hours light daily, from 6.00 a.m. to 6.00 p.m)

IN-LIFE DATES:
Start of in life phase: 07 July 2020 (first vaginal smear sampling)
Start of treatment: 21 July 2020
End of treatment: 13 September 2020
End of in life phase: 14 September 2020 (last necropsy)

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: As agreed with the Sponsor, no correction for purity of the test item was applied during formulation.
The test item was formulated in the selected vehicle (propylene glycol), as a visibly stable homogenous solution at the appropriate concentrations according to the dose level and volume selected in the Pharmacy of the Test Facility. The formulations were stirred with manual shaking and a magnetic stirrer from the preparation until completion of each treatment.
Formulations were prepared a maximum of 5 days prior to administration to animals according to stability assessment results of the analytical method validation study (Study code: 20/027-316AN). Based on those results, the test item formulation in the 5 and 250 mg/mL concentration range were stable for at least 7 days when stored at room temperature.


VEHICLE propylene glycol
- Justification for use and choice of vehicle (if other than water): Based on the available information provided by the Sponsor as well as results of a trial formulation and the Dose Rage Finding (DRF) study (Study code: 20/027-220PE) performed at the Test Facility, propylene glycol (abbreviated as PG in the raw data and study documents) was selected as vehicle for this study in agreement with the Sponsor. Propylene glycol was considered as being an acceptable vehicle based on the scientific literature and practice of the Test Facility.
- Concentration in vehicle: Dose formulation concentration 0, 20, 60, 200 mg/mL
- Amount of vehicle (if gavage): A constant dosing 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.
- Lot/batch no. (if required): 1920944 / STBJ4817 (ThermoFisher / Sigma-Aldrich Co.)
- Purity:100%

Details on mating procedure:

- M/F ratio per cage: one female and one male of the same dose group (1:1 mating) in a single cage
- Length of cohabitation: Females remained with the same male until copulation occurred, for up to 8 days.
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: Not applicable
- After successful mating each pregnant female was caged (how): Sperm positive females were caged individually.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

*Sample Collection
Sample collection was performed at a total of four occasions (during the first and last weeks and twice 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: On three occasions (sampling #1, #2 and #4, i.e. during the first and last weeks and approximately midway during the treatment period) samples for all dose formulations were collected. Additional sample was collected for confirmatory purposes from the High dose formulation only near midway of the treatment period (sampling #3).
On each sampling occasion, top, middle and bottom duplicate samples were taken from the dedicated test item formulation(s) for concentration and homogeneity measurement, one set to analyse (which was collected in replicates as practical) and one set as a back-up, if required for any confirmatory analyses. Similarly, duplicate samples were taken on three occasions from the middle of the vehicle control formulation for concentration measurement.
After the analytical sampling, the collected formulation samples were stored at room temperature until measurement.
*Formulation Analysis
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 formulation(s) were analysed at four times during the study (during the first and last weeks and twice approximately midway during the treatment period).
*Analytical Method
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: 20/027-316AN [5]). The density of the formulations was determined at the first analytical sampling by using three parallels in the Analytical Department of the Test Facility, as it was deemed necessary by the Contributing Scientist #1 (Analyst) and Study Director.
Acceptance criteria of the concentration analysis was 100 ± 10% of the nominal concentration.
Acceptance criteria of the homogeneity was that the RSD% (relative standard deviation) of replicates (top, middle and bottom of test item formulations) must be less than 10%.

Duration of treatment / exposure:

Dosing of both sexes began after the acclimatisation (5 days) and pre-exposure period (14 days), and it was performed 2 weeks before mating, during the mating, and was continued up to and including the day before the necropsy.
The first day of dosing of each animal was regarded as Day 0.
Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating period), then were euthanized and subjected to necropsy examination.
Females were dosed for 14 days pre-mating, for up to 14 days mating period, through gestation and up to and including the day before necropsy (13 days post-partum dosing). The day of birth (when parturition was complete) was defined as Day 0 post-partum. Females not delivered were sacrificed as practical (first day of dam’s necropsy).

Frequency of treatment:

The dosing solutions were administered to the test item or vehicle-treated (control) animals daily on a 7 days/week basis, in each morning, by oral gavage using a tipped gavage needle attached to a syringe (the surface of the needle was carefully cleaned before every dosing).

Details on study schedule:

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

Dose / conc.:

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

Remarks:

test material 99.42% purity

Dose / conc.:

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

Remarks:

test material 99.42% purity

Dose / conc.:

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

Remarks:

test material 99.42% purity

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: DRF study
- Rationale for animal assignment (if not random): All adult/parental (P) male and female animals were sorted according to body weight by computer and divided into weight ranges. There were an equal number of animals from each weight group randomly assigned to each dose group to ensure that animals of all test groups were as nearly as practicable of a uniform weight.
This process was controlled by the software PROVANTIS v.9, to verify the homogeneity/variability between/within the groups. Males and females were randomised separately to the dose groups on the day of the first treatment (prior to the start of the treatment).
- Fasting period before blood sampling for clinical biochemistry: Yes (overnight period of food deprivation, in case of females this happened after the litter has 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.
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.
During these processes the principles and criteria summarized in the OECD Humane Endpoints Guidance Document No. 19 was taken into consideration.
Any clinical sign noted during dosing or at any other occasions (if any) was recorded at the time seen.

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.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY: No
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

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

OPHTHALMOSCOPIC EXAMINATION: No
No ophthalmoscopy is conducted in the study.

HAEMATOLOGY: Yes (for results see repeated dose toxicity section)
- Time schedule for collection of blood: immediately prior to scheduled necropsy.
- Anaesthetic used for blood collection: Yes pentobarbital anaesthesia
- Animals fasted: Yes (overnight period of food deprivation, in case of females this happened after the litter has had been culled).
- How many animals: All randomly selected animals (5M and 5F per group)
- Parameters examined:
Red Blood Cell (erythrocyte) count
White Blood Cell (leukocyte) count
Haemoglobin concentration
Haematocrit (relative volume of erythrocytes)
Mean Corpuscular (erythrocyte) Volume
Mean Corpuscular (erythrocyte) Haemoglobin
Mean Corpuscular (erythrocyte) Haemoglobin Concentration
Red Cell (erythrocyte) volume
Platelet (thrombocyte) count
Mean Platelet Thrombocyte volume (fL)
Reticulocyte count (%)
Neutrophil (%)
Lymphocyte (%)
Monocyte (%)
Basophil (%)
Eosinophil (%)
Large Unstained Cells (%)
COAGULATIONS
Activated Partial Thromboplastin Time
Prothrombin Time

CLINICAL CHEMISTRY: Yes (for results see repeated dose toxicity section)
- Time schedule for collection of blood: immediately prior to scheduled necropsy.
- Animals fasted: Yes (overnight period of food deprivation, in case of females this happened after the litter has had been culled).
- How many animals: All randomly selected animals (5M and 5F per group)
- Parameters examined:
Blood sugar concentration
Total Bilirubin concentration
Urea concentration
Cholesterol concentration
Creatinine concentration
Phosphorus concentration
Sodium concentration
Potassium concentration
Calcium concentration
Chloride concentration
Total Protein concentration
Albumin concentration
Alb/glob ration
Aspartate Aminotransferase activity
Alanine Aminotransferase activity
Gamma-Glutamyl transferase activity
Alkaline Phosphatase activity
Bile acids

URINALYSIS: Yes (for results see repeated dose toxicity section)
- Time schedule for collection of urine: prior to necropsy
- Metabolism cages used for collection of urine: Yes (approximately 16 hours)
- Animals fasted: Yes Yes (overnight period of food deprivation, in case of females this happened after the litter has had been culled).
- Parameters examined.:
Leukocyte
Nitrite
pH
Protein
Glucose
Urobilinogen
Bilirubin
Ketones
Blood/Erythrocytes
Specific Gravity
Sediment
Volume
Colour/Appearance

NEUROBEHAVIOURAL EXAMINATION: Yes (for results see repeated dose toxicity section)
Functional Observational Battery and locomotor activity measurement was performed in the study.
- Time schedule for examinations: Assessment of any potential test item related neurotoxicity was performed during the last exposure week (males on Day 23 am, females on PPD13 am). Selected animals were subjected to the functional observation battery, including Irwin test and measurements of the landing foot splay and fore/hind grip strength.
In order to avoid hypothermia of pups, dams were removed from the pups for not more than approximately 30-40 minutes during FOB or 90 minutes during locomotor activity measurement (SMART).
A modified Irwin test was performed when sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity was conducted, and the general physical condition and behaviour of animals was tested. Parameters including body position, locomotor activity, respiration rate, respiration type, piloerection, head searching, compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna reflex, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex, and/or vocalisation were evaluated.
To measure the landing foot splay, the fore/hind paws of the rat were painted with ink and the rat was dropped from a horizontal position onto the appropriate record sheet covering the examination table. This was repeated 3 times for each animal. The distance between the two resulting ink spots of the hind limbs was measured.
Fore/hind grip strength was measured using a grip strength meter (Model GS3, Bioseb, Chaville, France), an instrument designed to quantify objectively rodent muscular strength, in order to identify and assess quantitatively any potential effect of test item. The rats were held appropriately such that the fore limbs were allowed to grip the support bar and gently pulled back until they released the bar; the device measures the maximum grip strength. This was performed 3 times for each animal on each test day. The procedure was repeated with the hind limbs with the appropriate grip support. The results are tabulated with individual and mean data.
Locomotor activity assessment was conducted using Automatic Monitoring System of rat locomotor activity SMART v. 2.5 (Harvard Apparatus, Germany). Locomotor activity was monitored by placing each animal individually into an open-field for 1-hour observation time, when DVD recording of movement was made. Recording was made for a duration of 60 minutes, under dim-light and undisturbed conditions. The DVD was analysed with “SMART” software after all recordings were made to produce the appropriate parameters. Data was evaluated for distance travelled in 5-minute segments. The data from the 5-minute segments was presented graphically with the intention of showing plateau activity in controls and comparing the treatment groups.
- Dose groups that were examined: Five males and five females/group were randomly selected

IMMUNOLOGY: No (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). No additional flow cytometry or other specific examinations were performed additional to the basic histopathological procedure.)

OTHER:
*Observation of the delivery process, offspring and nursing instinct
Females were allowed to litter and rear their offspring. Delivery process was observed as carefully as possible. All observations were recorded. Any evidence of abnormal deliveries was recorded. The duration of gestation was recorded and was calculated from Day 0 of pregnancy.
Dams were observed to record whether they formed a nest from the bedding material and covered their new-borns or not. The efficiency of suckling was observed by the presence of milk in the pups' stomach. All observations were recorded.

*Blood Sampling for Thyroid Hormone Analysis
For thyroid hormone analysis, blood samples were taken by venepuncture (using vena sublingualis in case of adult animals) or decapitation (in case of pups) into tubes containing K3-EDTA as anticoagulant as follows:
• from up to two pups per litter on PND4 (females if possible),
• from all dams* and at least two pups per litter on PPD 14 (females) / PND13 (pups),
• from all non-pregnant adult females at termination,
• from all adult males at termination.
*Note: In case of pre-terminal euthanasia of a Mid dose dam, the blood sampling was performed immediately before termination on PPD0.
The collected pup blood (plasma) samples were pooled by litter.
The timing of the blood collection for thyroid hormone determination was as close as possible between animals and at the same time of the day in case of sampling on different days. The exact time points were documented in the raw data.
Blood samples were kept on ice from sampling until centrifugation (within 30 minutes of collection), then centrifuged rapidly (1600 g / approx. 3000 rpm, 10 minutes, 4°C). The resulting plasma was divided in at least two aliquots (volume target was at least 150 μL for the first aliquot and at least 75 µL for the second aliquot), any remaining sample (if any) was kept as a third aliquot) and stored in an ultra-freezer (-80±10°C) until analysis.

Oestrous cyclicity (parental animals):

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

Sperm parameters (parental animals):

For the adult male animals, detailed histological examination was performed on the retained reproductive organs (testes, epididymides, prostate gland, seminal vesicles with coagulation gland) 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 aree killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1- offspring:
Each litter isexamined 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 will be reported individually for each adult animal. In addition to the observations on parent animals, any abnormal behaviour of the offspring will be recorded.
Live pups are counted, sexed, weighed individually within 24 hours of parturition (PND0) and on PND4, PND7 and PND13, with accuracy of 0.01 g.
All the litters are 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) are also recorded on each day. The pups found dead and intact (not cannibalized) are subjected to necropsy with macroscopic examination and the cause of death will be identified if possible. Dead pups can be preserved in 80% (v/v) 2-Propanol solution, if immediate examination is not possible due to technical reasons. All observed abnormalities will be recorded.
On PND 4, litters are culled to yield, as nearly as possible, 5 males and 5 females per litter. Pups to be culled within each litter are selected at random. In litters of insufficient size where the number of males or female pups is less than 5, adjustment of the selection process is made to assure 10 pups are retained. Culling will not be performed on litter sizes less (or equal) than 10. All culled pups will be subjected at least to necropsy with detailed macroscopic external and internal examination for any abnormalities.
The anogenital distance (AGD) of each pup will be measured at the time of the first weighing (PND0). The anogenital distance may be normalized to a measure of pup size (the cube root of body weight) for statistical analysis if considered appropriate by the Study Director.
Number of nipples/areolae in male pups are recorded on PND13 (individual records are maintained).
One male and one female pup per litter (if possible) is previously selected for culling for blood sampling on PND4.
All pups arenecropsied on PND13.

GROSS EXAMINATION OF DEAD PUPS:
yes,macroscopic examination and the cause of death is identified if possible

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals. Males are dosed for at least 28 days (14 days pre-mating and at least 14 days mating/postmating period plus an optional extended post-mating period), then are euthanized and subjected to necropsy examination or alternatively are retained and continued to be dosed for the possible conduction of a second mating if considered appropriate.
- Maternal animals: All surviving animals. Females are dosed for 14 days pre-mating, for up to 14 days mating period, through gestation and up to and including the day before necropsy (at least 13 days post-partum dosing). The day of birth (when parturition is complete) is defined as Day 0 post-partum. Females showing no-evidence of copulation or not delivered are sacrificed as practical (e. g. 24-26 days after the last day of the mating period; to be documented and reported).

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
Necropsy with organ weight determination (i.e. Day 28 or later, all males)
Necropsy with organ weight determination (i.e. all females)
After exsanguination the external appearance is examined, cranium, thoracic and abdominal cavities are opened and the appearance of the tissues and organs is observed macroscopically. Any abnormality is recorded with details of the location, colour, shape and size, as appropriate. Special attention is paid to the organs of the reproductive system. Vaginal smearsa are 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 will be recorded in the females as applicable.

HISTOPATHOLOGY / ORGAN WEIGHTS
The weighed organs and all organs showing macroscopic lesions of all adult animals are preserved.
The eyes with the optic nerve, testes and epididymides are 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 are retained from all animals:
Gross findings
Adrenal gland
Animal identification
Aorta
Brain
Epididymis
Eye with the optic nerve
Oesophagus
Femur with marrow
Heart
Kidney
Large intestine
Extraorbital lachrymal gland
Harderian gland
Liver
Lungs with bronchi
Lymph node
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
Spinal cord
Spleen
Sternum with marrow
Stomach
Testis
Thymus
Thyroid with parathyroid gland
Tongue
Trachea
Urinary bladder
Uterus
Vagina

Additionally, thyroid glands from one male and one female PND13 pup from each litter are preserved in 10% buffered formalin solution. In this case, the thyroid weight (pooled) can be determined after
fixation. Trimming should be done very carefully and only after fixation to avoid tissue damage.
Additional tissue samples may be taken at the discretion of the attending Pathologist and/or the Study Director to elucidate abnormal findings.
In case microscopic examination is needed for a tissue or organ, the retained tissues and organs required for histopathology (below) are embedded in paraffin wax; sections will be cut at 4-6 µm by microtome and transferred to slides. Tissue sections will be stained with haematoxylin-eosin/phloxine and examined by light microscope. Special stains may be used at the discretion of the Study Pathologist (to be documented and reported).
For the adult animals, detailed histological examination is 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 – the same animals are used for Clinical Pathology
• any animals found dead or euthanized pre-terminally during the study in all groups
• all macroscopic findings (abnormalities), except of minor order from all animals
• on the retained reproductive organs (testes, epididymides, prostate gland, seminal vesicles with coagulation gland for males and uterus, cervix, ovary, oviduct and vagina for females) of all animals
of the Control and High dose groups, and of all males that failed to sire and all females that failed to deliver healthy pups (this will be notified by Memo).
Special attention is 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 covers the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.
Special attention is paid to the organ weight, appearance and histopathology of immunesystem tissues for any evidence of immunotoxicity (spleen, thymus, lymph nodes, bone marrow, and blood smears if examined).
Special attention is paid to the central and peripheral nervous system tissues for any evidence of neurotoxicity.
Examination of tissues from Low and Mid dose, or from additional High dose group animals may be performed where treatment-related changes are found in examined High dose group slides (at additional cost, and following consultation with the Sponsor).
*Organ Weights:
At the time of termination, body weight and weight of the following organs of all adult animals are 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 will be weighed individually. Individual and/or paired absolute organ weight are reported for each animal and adjusted for the body and brain weights. Paired organ weights as applicable are summarised. Relative organ weight (to body and brain weight) are calculated and reported.
Additional organs may be measured as deemed necessary by the Study Director, or to elucidate abnormal findings (to be documented in the raw data and reported).

Postmortem examinations (offspring):

SACRIFICE
- On PND 4, litters are culled to yield, as nearly as possible, 5 males and 5 females per litter.
- The culled pup are subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:
All culled pups will be subjected at least to necropsy with detailed macroscopic external and internal examination for any abnormalities.

GROSS NECROPSY
The pups found dead and intact (not cannibalized) are subjected to necropsy with macroscopic examination and the cause of death will be identified if possible.
All other pups are necropsied on PND13. All F1 offspring is terminated on Post-natal Day 13 or shortly thereafter (F1 offspring selected for blood sampling is terminated on PND4 due to technical reason). In order to allow for overnight fasting of dams prior necropsy on PPD14, offspring is euthanized on PPD/PND13, and the dams on PPD/PND14.
- Gross necropsy consisted of :
The pups found dead and intact (not cannibalized) are subjected to necropsy with macroscopic examination and the cause of death will be identified if possible. All other pups are necropsied on PND13. All F1 offspring will be terminated on Post-natal Day 13 or shortly thereafter (F1 offspring selected for blood sampling will be terminated on PND4 due to technical reason, to be documented and reported). In order to allow for overnight fasting of dams prior necropsy on PPD14, offspring is euthanized on PPD/PND13, and the dams on PPD/PND14.
Dead pups and pups killed on PND4 and/or PND13 are carefully examined externally for gross abnormalities. After the external observation, the sex determined at birth is confirmed by observation of the internal reproductive organs, if possible. Presence of nipples/areolae in the PND13 pups is also recorded.

HISTOPATHOLOGY / ORGAN WEIGTHS
Nor perfomed on the offspring.

Statistics:

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

Reproductive indices:

Male Mating Index
Female Mating Index
Male Fertility Index
Female Fertility Index
Gestation Index
Pre-implantation mortality%
Intrauterine mortality %
Total Mortality %

Offspring viability indices:

Survival Index
Sex ratios%

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test-item related clinical signs were observed in the study.
Tonic convulsions were recorded on the day of delivery (Day 38) and near or at the end of the lactation period (Days 49 and 52) for a Control female (#1503).
A small wound (<1 cm) and later a crust was observed for one Mid dose male (#3010) in the neck dorsal area from Day 3 to Day 22.
These findings were considered incidental, not related to the test item administration.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No test item related mortality was observed in the study. One Mid dose female (#3501) was found dead on Day 43 (on the day of delivery), this fact was most probably related to a difficult delivery process, not related to the test item administration as no test item-related macroscopic or microscopic changes were observed. For another Mid dose female (#3505) hunched back, piloerection and extreme vaginal prolapse were recorded at delivery (GD 22, Day 36), this animal was pre-terminally euthanized due to ethical reason on the same day. No test item-related macroscopic or microscopic changes were observed. These two cases were considered incidental (most probably related to the difficult delivery), not related to the test item administration.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Test item related adverse effect was observed on body weight parameters in High dose (1000 mg/kg bw/day) males and females. No test item related adverse effect on body weight or body weight gain was detected in Mid or Low dose (300 or 100 mg/kg bw/day, respectively) animals (males or females).
Slightly lower body weight (statistically not significant) and reduced body weight gain (statistically significant at p<0.05) was observed in High dose males on the first week of treatment when compared to control animals. This difference still could be observed at the end of the treatment period (body weight lower by 3% and body weight gain lower by 18% compared to control, not statistically significant). This High dose body weight (gain) difference, principally in the first week, was considered as a test item related effect.
No effect on body weight parameters was seen in Mid and Low dose males.
In case of females, the observed body weight or body weight gain values were comparable to the control level at the end of the pre-mating period in all dose groups. But the High dose females showed reduced body weight / body weight gain at the end of the gestation period (body weight was lower by approximately 9%, body weight gain by 20%; the difference to control was statistically significant at p<0.01 in both cases). Reduced body weight of the High dose females compared to control (by approximately 7-8%) was also observed at the beginning of the lactation period (PPD 0, 4 and 7, significant at p<0.01), but the difference was smaller by the end of the lactation period (by approximately 7% lower body weight and by 6% lower body weight gain) and none of them gained statistical difference at that point. However, the body weight gain calculated for the entire treatment period (Day 0 – PPD13) was still statistically significantly lower for High dose females than Control (by 17%, significant at p<0.01). These facts were considered as being a test item related adverse effect.
No effect on body weight parameters was seen in Mid and Low dose females.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Test item related adverse effect on food consumption was observed in High dose females (1000 mg/kg bw/day) during the gestation and lactation phases, no effect was observed in the High dose males or Mid and Low dose groups (300 and 100 mg/kg bw/day, respectively) of both sexes.
No adverse effect on food consumption was seen in male animals in any test item treated group and no effect was observed in test item treated female animals during the pre-mating period.
In case of High dose females, reduced values compared to control were recorded during the gestation period (by 12%) and lactation period (by 21%), statistical significance was reached in both cases (p<0.01). Based on the body weight values, the observed values were considered as a test item related adverse effect.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No test item-related adverse changes were detected in the test item treated animals (males and females) when comparing haematology parameters to the relevant Control data.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

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.
Alanine aminotransferase (ALT/GPT) activity was significantly increased in the Mid dose males (p<0.05) and High dose males (p<0.05) and females (p<0.01) when compared to control animals. Furthermore, increased Alkaline phosphate (AP) activity were recorded in High dose females (by 83%, statistically significant at p<0.01), but the observed mean value was within the historical control range and no similar trend was noted in High dose males. All these increased values were considered as test item related effect; however, in the absence of any indication of adverse histopathology findings in the liver, they were considered as non-adverse.
Reduced cholesterol concentration was observed in High dose females (by 61%, significant at p<0.01), but this fact was most probably related to the significantly reduced food consumption of High dose females during the gestation and lactation periods, not being a direct effect.

Endocrine findings:

no effects observed

Description (incidence and severity):

No indication of an endocrine disruptor effect was observed in the study based on collected oestrus cycle data, anogenital distance measurement, nipple retention, thyroid hormone measurement, thyroid weight and histopathology and external reproductive organs analysis.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No test item-related changes were observed in the urinalysis parameters in male and female animals of any dose groups when compared to control.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no changes in animal behaviour, general physical condition or in the reactions to different type of stimuli in the control or test groups. There was no effect of treatment noted in the Irwin test or during the assessment of grip strength and landing foot splay.
All dose groups of males and females had a normal locomotor activity. In all cases, the initial activity was high, with reduced activity in each 5-minute period to an approximate plateau by about 20-30 minutes. There was no statistical significance between the test item treated animals (males and females) and the Control when evaluating the overall total travelled distance (0-60 minutes). The test item did not increase or decrease the normal locomotor activity, all treated groups had a profile of activity the same as historical control data.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

TERMINAL EUTHANASIA / Parental Generation
Test item-related minimal/mild multifocal hyperkeratosis of the non-glandular gastric mucosa were observed in 9/12 High dose males and 9/12 High dose females. Low and mid doses were also examined but did not reveal histopathological changes.
In the liver, minimal hepatocellular hypertrophy was seen in 4/12 High dose males and in 2/12 High dose females, additionally minimal/mild, periportal or diffuse hepatocellular vacuolation was present in 5/12 High dose females. Those changes were considered as non-adverse adaptive changes. Low and mid doses were also examined but did not reveal histopathological changes.
All other changes are commonly seen in control and/or treated animals, or were without meaningful differences in severity and incidence, therefore were regarded as incidental, procedure-related or a common background.
NON-PREGNANT FEMALES / Parental Generation
Two non-pregnant Control females were observed in the study (#1506 and #1512).
No microscopic changes were observed in the non-pregnant females and their male mating pairs.
FOUND DEAD ANIMAL / Parental Generation
One Mid dose female (#3501) was found dead at delivery.
No test item-related microscopic changes were observed. During the microscopic evaluation haemorrhage in the uterine lumen, lungs and thymus, and moderate extramedullary haematopoiesis in the spleen were observed and were considered as incidental.
PRE-TERMINAL EUTHANASIA / Parental Generation
One Mid dose female (#3505) was pre-terminally euthanized due to extreme vaginal prolapse on the day of delivery.
No test item-related microscopic changes were observed. Microscopically haemorrhage in the uterine lumen and moderate inflammation of the vagina was observed, they were considered as incidental, not related to the test item.

Histopathological findings: neoplastic:

not specified

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

PRE-EXPOSURE PERIOD
Each female selected for the study showed acceptable cycles (mean cycle length of 4.03-4.21 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 4.17, 3.98, 4.19 and 3.97 days in the Control, Low dose, Mid dose and High dose groups, respectively).
Prolonged oestrus was recorded in some cases for all groups, a total of 4 Control females, 2 Mid dose females and 1 High dose female were affected (#1503, #1504, #1507, #1512, #3510, #3512 and #4509), but as it did not affect mating or pregnancy (from these animals only one Control female became non-pregnant) and as the incidence was lower in the test item treated groups than in the Control group; this fact was considered as being an occasional finding, not being a test item related effect.
Prolonged dioestrus was noted for one Low dose female (#2502) and one Mid dose female (#3501), but this frequency (1/12) was in line with the normal, expected range and did not affect the mating or pregnancy.

Reproductive function: sperm measures:

not examined

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.
There were no differences between the control and test item treated groups with regard to reproductive ability, mating or gestation indices, and no effects considered adverse or toxicologically significant in correlation with the administration of the test item. Both the mating and fertility index was 100% in treated groups (males and females). The gestation index was also 100% in the Control, Low and High dose groups, and 83% in Mid dose group. As this value was still within the historical control range and in the absence of microscopic findings or dose response, no test item effect was concluded.
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 8 days of pairing (cohabitation). The mean duration of mating was 2.33, 2.58, 2.92 and 2.92 days in the Control, Low, Mid and High dose groups, respectively. Longer than usual mating was noted for two animals (6 days for #2502 and 8 days for #3501), but they were considered as incidental findings, no test item effect was noted.

Dose descriptor:

NOAEL

Remarks:

Reproductive effects of the parenteral generation

Effect level:

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

Based on:

test mat.

Remarks:

99.42% purity

Sex:

male/female

Basis for effect level:

other: highest dose tested; lack of relevant findings

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Evidence of suckling was recorded for all live born pups in the study, except of the pups of #3501 which dam died at delivery, the litter of #2501 (but pups suckled later and gained weight properly) and two Mid dose pups (#3503/18 and #3506/17).
Based on the external evaluation, most of the pups were normal, clinical signs or abnormalities were recorded for one Low dose male pup (#2501/14), three Mid dose pups (#3503/17 and 18, #3506/17) and two High dose pups (#4509/1, #4511/18), where slight / moderate cyanosis was observed on PND0 and some of those pups were also cold to touch. One of those pups were found dead (#4509/1) on the next day, the others were cannibalized on PND1 (#2501/14, #3503/17 and #3503/18) or PND2 (#4511/18). All these events were considered as minor, incidental findings, not related to the test item treatment.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

There was no test item effect on mortality or survival of the pups (F1 generation).
The number of viable pups on PND0, PND4 and PND13 as well as pup survival indices on PND0, PND4 and PND13 were comparable to control values in each dose group except of the Mid dose group. However, in the Mid dose group the significantly higher than control values were originated from the ethical euthanasia of pups of the two dams not surviving of the delivery.
There were no significant differences or effects that could be ascribed to the test item treatment on the pre-natal, post-natal or total mortality values (litter mean and %) in the Low and High dose groups (100 and 1000 mg/kg bw/day, respectively). As discussed above, when compared to the control data the significantly lower post-natal mortality in the Mid dose group (300 mg/kg bw/day) observed in the period of PND 0-4 was due to the euthanasia of the living pups of dead / pre-terminally euthanized dams, and was considered as an incidental finding, not related to the test item.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There were test item related differences in the offspring body weights or body weight gains in the Mid and High dose groups (300 and 1000 mg/kg bw/day, respectively) when compared to the control values.
Reduced body weight compared to Control was recorded on PND 13 in the Mid and High dose groups (by 8.0% and 19.9%, respectively, statistically significant at p<0.01 in both cases); while reduced body weight gain was recorded in the period of PND 0-4 in the High dose group (statistically significant at p<0.05) and in the periods of PND 4-13 and PND 0-13 in the Mid and High dose groups (statistically significant at p<0.01 in all those cases). However, in case of the Mid dose group, the observed values were within the historical control range. It should be noted that the results of the Control group of this study were higher than expected (near or slightly above the upper range of the HC database), this is considered to have contributed in the statistical difference. Since the Mid dose data were fully in line with extensive historical control data, it was considered that there was no test item related adverse effect on pup body weight or growth in this dose group. As the measured values were clearly outside the historical control range in case of the High dose group, those changes were considered as a test item related adverse effect.

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

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No test item effect was observed on nipple retention during the study.
No nipples/areolae were present in any of the male pups on PND13.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In case of a High dose pup (#4505/10) terminated for blood sampling on PND4, the following macroscopic findings were recorded: large left kidney, absent left renal papilla, dilated renal pelvis, and abnormal amount (large) / consistency of fluid in the renal pelvis. All those findings were considered as incidental, not related to the test item administration.

Histopathological findings:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid hormone analysis:
In case of PND13 pups, there were no statistically significant thyroid hormone concentration levels recorded when compared to the control.
The thyroid gland weights of the PND13 pups were also comparable with the control value in the Low and Mid dose groups, but higher relative (to body weight) was noted in the High dose group. However, it was a consequence of the significant lower body weight of the High dose pups, not a direct test item effect on thyroids. In summary, there were no effects on the thyroid hormone levels or on the thyroid gland weights in the PND13 pups that were ascribed to the test item.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEL

Remarks:

pups development and survival

Generation:

F1

Effect level:

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

Based on:

test mat.

Remarks:

99.42% purity

Sex:

male/female

Basis for effect level:

body weight and weight gain

Reproductive effects observed:

no

Conclusions:

Based on the results of this study, the following No-Observed-Adverse-Effect Levels (NOAELs) were considered:
The NOAEL for systemic toxicity of the parental generation: 300 mg/kg bw/day (based on body weight and food consumption effects, and stomach microscopic findings).
The NOAEL for reproductive effects of the parental generation: 1000 mg/kg bw/day (based on the lack of relevant findings).
The NOAEL for pups’ (F1 generation) development and survival: 1000 mg/kg bw/day (based on the lack of evidence of a direct effect on the pups at 1000 mg/kg bw/day, where treatment related reduced pup growth and body weight were seen, but were attributed as most probably due to maternal toxicity (~20% lower dam body weight gain and ~20% reduced food intake, associated with local gastric irritation in the lactating dams). No treatment related effect on pup growth was seen at 300 mg/kg/day).

Executive summary:

The purpose of this OECD No. 422 study was to obtain information on the possible toxic effects of Sodium 1,4-diisotridecyl sulphonatosuccinate (CAS 55184-72-0, EC 259-515-6) test item following repeated (daily) administration by oral gavage to Wistar (Crl:WI) rats at 3 dose levels. A control group received the vehicle only (propylene glycol).

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

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

 

Experimental design:

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

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 (Day 28 for males, PPD (Post-partum Day) 14 for females), 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 PND (Post-natal Day) 13 pups and parental males were also determined.

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

Dosing formulation were analysed for concentration and/or homogeneity on four occasions during the study. Overall, the formulations were considered adequate for the study.

RESULTS

In summary, under the conditions of this study the daily administration of Sodium 1,4-diisotridecyl sulphonatosuccinate (CAS 55184-72-0, EC 259-515-6) by oral gavage to Wistar rats at dose levels of 100, 300 or 1000 mg/kg bw/day (Low, Mid and High dose groups, respectively) did not result in test item related mortality or clinical signs. Test item related adverse effect was observed on body weight parameters and food consumption in High dose (1000 mg/kg bw/day) males and females (for females during gestation and lactation periods, no effect was seen in the pre-mating period). At the functional observation battery (FOB) and locomotor activity measurement, there were no changes in animal behaviour, general physical condition or in the reactions to different type of stimuli in test item treated groups when compared to control. No test item-related adverse effects were seen in the clinical pathology parameters. Test item related non-adverse changes (increased Alanine aminotransferase activity in the Mid dose males and High dose males and females, as well as increased Alkaline phosphate activity in High dose females) were considered to be linked to the adaptive response seen in the liver during microscopic examination (hepatocellular hypertrophy and vacuolation which was observed only in High dose animals, but not in Mid or Low dose animals).
No test item related changes were noted in the reproductive parameters during mating and gestation, delivery and post-partum/lactation period until PPD14. The number of implantation or liveborn pups, as well as the pre-natal, post-natal or total mortality values were comparable with the control values in all test item treated dose groups. No test item related macroscopic findings were recorded for F1 pups at necropsy.
There were test item-related differences on the offspring body weights or body weight gains in the Mid and High dose groups (300 and 1000 mg/kg bw/day, respectively) when compared to the control values. As the measured values were clearly outside the historical control range iin case of the High dose group, the changes observed in that dose group were considered as a test item related adverse effect.
No test item-related effects were observed in the organ weights of the test item treated animals compared to controls. Hyperkeratosis of the non-glandular gastric mucosa of the stomach was identified at the High dose level (1000 mg/kg bw/day), as test item-related adverse change.
Mid and Low dose stomach samples of both sexes were also evaluated the get additional information for study interpretation, no test item-related changes were seen in those animals.
No indication of an endocrine disruptor effect was observed in the study based on collected oestrus cycle data, anogenital distance measurement, nipple retention, thyroid hormone measurement, thyroid weight and histopathology and external reproductive organs analysis.
Based on the results of this study, the following No-Observed-Adverse-Effect Levels (NOAELs) were considered: The NOAEL for systemic toxicity of the parental generation: 300 mg/kg bw/day (based on body weight and food consumption effects, and stomach microscopic findings).The NOAEL for reproductive effects of the parental generation: 1000 mg/kg bw/day (based on the lack of relevant findings). The NOAEL for pups’ (F1 generation) development and survival: 1000 mg/kg bw/day (based on the lack of evidence of a direct effect on the pups at 1000 mg/kg bw/day, where treatment related reduced pup growth and body weight were seen, but were attributed as most probably due to maternal toxicity (~20% lower dam body weight gain and ~20% reduced food intake, associated with local gastric irritation in the lactating dams). No treatment related effect on pup growth was seen at 300 mg/kg/day).

 

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Klimisch 1-2

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Reproductive toxicity data are generated for the registered substance.

A Combined repeated dose toxicity with reproduction/developmental toxicity screening test in Wistar rats was conducted under GLP conditions with the registered substance by oral gavage administration at dose levels at 0, 100, 300 and 1000 mg/kg bw/day (Hargitai, 2021). The study did not result in test item related mortality or clinical signs. Test item related adverse effect was observed on body weight parameters and food consumption in High dose (1000 mg/kg bw/day) males and females (for females during gestation and lactation periods, no effect was seen in the pre-mating period). At the functional observation battery (FOB) and locomotor activity measurement, there were no changes in animal behaviour, general physical condition or in the reactions to different type of stimuli in test item treated groups when compared to control. No test item-related adverse effects were seen in the clinical pathology parameters. Test item related non-adverse changes (increased Alanine aminotransferase activity in the Mid dose males and High dose males and females, as well as increased Alkaline phosphate activity in High dose females) were considered to be linked to the adaptive response seen in the liver during microscopic examination (hepatocellular hypertrophy and vacuolation which was observed only in High dose animals, but not in Mid or Low dose animals). No test item related changes were noted in the reproductive parameters during mating and gestation, delivery and post-partum/lactation period until PPD14. The number of implantation or liveborn pups, as well as the pre-natal, post-natal or total mortality values were comparable with the control values in all test item treated dose groups. No test item related macroscopic findings were recorded for F1 pups at necropsy. There were test item-related differences on the offspring body weights or body weight gains in the Mid and High dose groups (300 and 1000 mg/kg bw/day, respectively) when compared to the control values. The difference in the Mid dose group was not considered as an adverse effect of treatment since the values were well within the historic control range, and hence the pup growth was considered to be normal. The measured values of growth and weight were clearly outside the historical control range in case of the High dose group pups, the changes observed in this dose group were considered as a test item related adverse effect (however, significantly lower maternal body weight gain and lower food intake by these lactating dams i.e. maternal toxicity, is considered to be an important factor in, or the direct cause of, the High dose pup effects). No test item-related effects were observed in the organ weights of the test item treated animals compared to controls.Hyperkeratosis of the non-glandular gastric mucosa of the stomach was identified at the High dose level (1000 mg/kg bw/day), as test item-related adverse change. Mid and Low dose stomach samples of both sexes were also evaluated the get additional information for study interpretation, no test item-related changes were seen in those animals. No indication of an endocrine disruptor effect was observed in the study based on collected oestrus cycle data, anogenital distance measurement, nipple retention, thyroid hormone measurement, thyroid weight and histopathology and external reproductive organs analysis. Based on the results of this study, the following No-Observed-Adverse-Effect Levels (NOAELs) were considered:

The NOAEL for systemic toxicity of the parental generation: 300 mg/kg bw/day (based on body weight and food consumption effects, and stomach microscopic findings); the NOAEL for reproductive effects of the parental generation: 1000 mg/kg bw/day (based on the lack of relevant findings); the NOAEL for pups’ (F1 generation) development and survival: 1000 mg/kg bw/day (based on the lack of evidence of a direct effect on the pups at 1000 mg/kg bw/day, where treatment related reduced pup growth and body weight were seen, but were attributed as most probably due to maternal toxicity (~20% lower dam body weight gain and ~20% reduced food intake, associated with local gastric irritation in the lactating dams). No treatment related effect on pup growth was seen at 300 mg/kg/day).

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.

Multigeneration studies

-A key study for reproductive toxicity was available for read across substance Docusate sodium as a 3-generation toxicity study at dietary dose levels of 0.1, 0.5 and 1.0% in the diet (MacKenzie, 1986). The study was conducted according to OECD 416 and GLP guidelines, and was considered to be reliable, adequate and relevant. The test substance caused a reduction in body weights at the dose levels of 0.5 and 1% in the diet for parental males of all generations and for F1 and F2 females. Pup weights at the 0.5% and 1.0% dose levels were lower than those of the control in all three generations. However, the reduced body weight did not interfere with growth and development or reproductive performance, and the test substance had no adverse effects at levels on the reproductive function of either sex in any generation up to 1%. There were no other effects on parental or reproductive parameters. Based on the results of this study, when Docusate sodium was administered in the diet to three successive generations of rats, the no-observable-effect level (NOEL) for body weights of parental animals and offspring was 0.1%; the NOEL for reproductive parameters was 1.0% Docusate sodium, which was considered to correspond with approximately 750 mg/kg bw/day.

- In a supporting successive 2-generation study in rats, dietary doses of Docusate sodium given were 0.5 and 1% (Levinskas & Shaffer, 1970). In the first mating of the F0 generation and the second mating of the F2 generation, pups were weaned directly onto the diets which were being fed to their parents. In the other 3 matings of this study, dams were given a control diet on the day before they were expected to cast their litters to avoid a bitter taste of the milk. When they were weaned, pups of these 3 litters were given diets containing the same levels of test material that their progenitors had been receiving. When pups were 3-4 months of age, they were in turn mated, and the process was repeated with their pups for a total of 3 generations. Pups of all litters, including those which died before weaning, were examined for gross defects. Autopsies were performed, however, only on pups from the first mating of the F2 animals. Those pups were killed at weaning. Immediately after death, the 2 males and 2 females which were the smallest or appeared least healthy of each litter were set aside, while the others were autopsied. Portions of all major organs were taken for histopathology processing and examination from one male and female from each litter. The other male and female were skinned and eviscerated, and the carcasses cleared, and the skeletons stained and examined for defects. In both the first mating of the F0 generation and the second mating of the F2 generation, the fertility and gestation indices were high and comparable. The viability index was good, albeit slightly down for the F3b pups, while the lactation index was depressed for both of these matings. In addition, the mean weight of the pups at weaning decreased with increasing concentrations of test material in the diet of the dams. In the second mating of the F0 animals, the viability and lactation indices and the mean weight of the test pups at weaning still showed decreases relative to the control values. However, in the 2 subsequent matings, all indices for the dosed animals were numerically high and compared favorably with the corresponding control values. Also, the mean weight of the pups at weaning was essentially similar for all groups. Consequently, it is concluded that diets containing 1% or less had no adverse effect on the reproduction and lactation performance of rats. The lowering of the survival rate and the mean body weight of the F1a and F3b pups is attributed to an impairment of nutrition as a result of the taste which is believed to have been secreted into the milk of the dams.

Microscopic study of tissues showed findings which were similar in all groups. In processing the skeletons,the presence of an extra sternebrae in the sternum between the 5th and 6th sternebrae was not considered to parental exposure of test material.

It is concluded that feeding of test material to rats from weaning through reproductive age for successive generations at levels of 1%, or less, did not produce lesions or anomalies in the offspring which could be attributed to the compound.

 

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.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

See attached read-across justification

Reason / purpose for cross-reference:

read-across source

Species:

rat

Strain:

Sprague-Dawley

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

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

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

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

Number of abortions:

no effects observed

Pre- and post-implantation loss:

not specified

Total litter losses by resorption:

effects observed, treatment-related

Description (incidence and severity):

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

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

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

Dead fetuses:

no effects observed

Description (incidence and severity):

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

Changes in pregnancy duration:

not examined

Description (incidence and severity):

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

Changes in number of pregnant:

not examined

Details on maternal toxic effects:

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

Key result

Dose descriptor:

NOAEC

Effect level:

1 other: %

Based on:

act. ingr.

Remarks:

in the diet

Basis for effect level:

body weight and weight gain

early or late resorptions

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Basis for effect level:

body weight and weight gain

early or late resorptions

Fetal body weight changes:

no effects observed

Description (incidence and severity):

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

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

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

Changes in sex ratio:

not specified

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

Description (incidence and severity):

There is no postnatal evaluation in an OECD 414 study.

External malformations:

effects observed, treatment-related

Description (incidence and severity):

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

Skeletal malformations:

no effects observed

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

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

Other effects:

effects observed, treatment-related

Description (incidence and severity):

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

Details on embryotoxic / teratogenic effects:

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

Key result

Dose descriptor:

NOAEC

Effect level:

1 other: %

Based on:

act. ingr.

Remarks:

diet

Sex:

male/female

Basis for effect level:

external malformations

visceral malformations

other:

Remarks on result:

other: secondary to high maternally toxic dose

Key result

Dose descriptor:

NOAEL

Effect level:

1 074 mg/kg bw/day

Based on:

act. ingr.

Remarks:

diet

Sex:

male/female

Basis for effect level:

external malformations

visceral malformations

other: skeletal variations

Abnormalities:

effects observed, treatment-related

Localisation:

external: cranium

skeletal: skull

skeletal: rib

visceral/soft tissue: central nervous system

visceral/soft tissue: eye

Description (incidence and severity):

only at 2.0% dietary level.

Developmental effects observed:

yes

Lowest effective dose / conc.:

2 other: %

Treatment related:

yes

Relation to maternal toxicity:

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

Dose response relationship:

yes

Relevant for humans:

no

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

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

* Significantly different from control (p< 0.05)

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

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

    

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

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

¹ Fetuses may have more than one defect

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

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

 

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

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

¹Fetuses may have more than one defect

^aExcludes 1 fetus lost

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

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

 

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

¹Fetuses may have more than one defect

^aExcludes 1 fetus destroyed during cleaning process

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

 

Conclusions:

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

Executive summary:

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

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

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 074 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Klimisch 2

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

No test data were available for current substance, however read across data were available from Docusate sodium (CAS 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

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

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

 

Conclusion

Prenatal developmental toxicity was tested by dietary administration of read across substance Docusate sodium in rats from day 6 to 15 of gestation. 1% in the diet was a maternal and developmental NOAEL corresponding to 1074 mg/kg bw, whereas at 2% in the diet visceral and skeletal anomalies were observed, which 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 developmental findings in the teratogenicity study, and taking into account that the same metabolic and toxicological behavior can be expected for structural similar substances,no further testing is needed.

Justification for classification or non-classification

Based on these results and according to the EC Directive (No.93/21/EEC) and 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.