Sodium 1,4-dicyclohexyl sulphonatosuccinate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Reproductive toxicity screening was performed with the registered substance in Wistar rats by oral gavage at 0 (distilled water), 100, 300 and 1000/600 mg solid/kg bw/day in key combined repeated dose/reproductive toxicity study (OECD TG No. 422). On day 28 of the dosing period the high dose was reduced to 600 mg/kg bw/day due to mortalities and increasing incidence and severity of noisy/laboured respiration. The mortalities and laboured and noisy respiration were related to the very irritant effect of small amounts of test item reaching the upper respiratory tract via reflux or contamination during dosing (as seen during the preliminary study). This is considered as a local effect of the test item. The NOAEL for reproductive effects of the parental generation was considered to be >=600 mg solid/kg bw/day. The NOAEL for pup development and survival was considered to be >=600 mg solid/kg bw/day.

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

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7 September 2020 to 21 December 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:

2016

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI

Details on species / strain selection:

The test system and the number of animals used in the study were in compliance with the relevant OECD No. 422 guideline. The guideline is designed for use with the rat, which is the preferred rodent species for reproduction toxicity testing. Wistar rat was selected due to experience of the Test Facility with this strain of rat in toxicity and reproduction toxicity studies and its known fertility.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, (Address: Sandhofer Weg 7, D-97633, Sulzfeld, Germany) from SPF colony
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation:
Parental Generation (P): Young adult rats, 11/12 weeks old (females/males) at start of the experiment and 13/14 weeks old (females/males) at mating.
(F1): Not applicable: F1 offspring (no F1 parenteral generation; screening study)
- Weight at study initiation:
Parental Generation (P): Males: 402-481 g, females: 231-291 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.
(F1): Not applicable: F1 offspring (no F1 parenteral generation; screening study)
- Housing: Rodents were group-housed, up to 2 animals of the same sex and dose group/cage, with the exception of the mating and gestation, delivery, lactation period, when they were paired or individually housed (with pups), respectively. Animals were housed in animal room 639 / 609 in Type II, III and/or IV polycarbonate cages. SAFE 3/4-S Hygienic Animal Bedding (Batch number: 03027200710, Expiry date: 10 July 2023) and SAFE Crinklets Natural nesting material (Batch number: 05072200405, Expiry date: 05 April 2023) produced by J. Rettenmaier & Söhne GmbH+Co.KG (Address: Holzmühle 1, D-73494 Rosenberg, Germany) were used in the study. Group housing allowed social interaction. Deep wood sawdust bedding allowed digging and other normal rodent activities. Nesting material allowed normal nesting behaviour. Certified cardboard hiding tunnels (GLP Mini Fun Tunnels, LBS (Serving Biotechnology) Ltd, Address: Unit 20, Gatwick Business Park, Kennel Lane, Hookwood, Surrey, RH6 0AH UK) were also provided to the animals.
- Diet (e.g. ad libitum): The animals received ssniff® SM R/M “Autoclavable complete diet for rats and mice – breeding and maintenance” (Batch number: 560 65984 / 713 70882, Expiry date: 31 October 2020 / 30 April 2021) produced by ssniff Spezialdiäten GmbH (Address: Ferdinand-Gabriel Weg 16, D-59494 Soest, Germany), ad libitum.
- Water (e.g. ad libitum): The animals received tap water from the municipal supply, as for human consumption from a 400- or 500-mL bottle, ad libitum.
- Acclimation period: Environmental acclimation period for the study was 5 days.

DETAILS OF FOOD AND WATER QUALITY:
A sample (approximately 100 g) of batch of diet used in the study was retained and kept under appropriate environmental conditions until the finalization of the study report.
The food was routinely analysed and considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
The quality control analysis of the water was performed once every three months and microbiological assessment was performed monthly, by Veszprém County Institute of State Public Health and Medical Officer Service (H-8200 Veszprém, József Attila u. 36., Hungary). Copies of the relevant Certificates of Analysis were included in the raw data and were 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): 20.0-23.7℃ (target range: 19-25°C)
- Humidity (%): 24-76% (target range: 30-70%)
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, light from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES:
From: Start of in life phase: 15 September 2020 (first vaginal smear sampling)
To: End of in-life phase: 06 December 2020 (last necropsy)

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

distilled

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
As agreed with the Sponsor, total solid content (test item) of the supplied product was taken into consideration during formulation (a conversion factor of 2.87 was used for this purpose). Concentrations and all dose levels in the study were expressed as solid matter as requested by the Sponsor.
The test item was formulated in the selected vehicle (distilled water), as a visibly stable homogenous solution at the appropriate concentrations according to the dose level and volume selected in the Pharmacy of the Test Facility. The formulations were ultrasonicated and 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 to allow their use according to stability assessment results of the analytical method validation study (Study code: 20/032-316AN).
The appropriate amount of the test item was weighed into a clean, calibrated glass container and then mixed properly (with ultrasonication and manual shaking and/or magnetic stirring) with the calculated amount of vehicle to reach homogeneity by visual observation. During the formulation sonication was applied as necessary to aid dissolution.
After filling the syringe with the calculated amount to be given to each individual rat, the outside of the gavage tube was cleaned with a wetted tissue first (using the vehicle of the study) and then with a dry tissue, to reduce any potential surface contamination to an absolute minimum. A constant volume of 5 mL/kg bw was administered to all animals. The actual volume to be administered was calculated and adjusted based on each animal’s most recent body weight. The time of the gavage process was prolonged, to be really sure the rat was well relaxed, and the total amount of gavage liquid was administered slowly into the stomach (‘short’ gavage to the lower oesophagus was avoided).

VEHICLE: distilled water
- Concentration in vehicle: 0, 20, 60 and 200/120 mg solid/mL
- Amount of vehicle (if gavage): Dose formulation volume = 5 mL/ kg bw

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: Females remained with the same male until copulation occurred, for up to 5 days.
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- Unsuccessful pairing: replacement of first male by another male with proven fertility. For one High dose female (#4501) the pairing with the first male (#4001) was not successful because of the dead of the male, hence another male (#4003) was chosen for mating. With this male the mating was successful and in the tables only these data were indicated.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Sample collection was performed on four occasions (preferably during the first*, second and last weeks and approximately midway during the treatment period). Samples were collected immediately after formulation preparation in the Pharmacy of the Test Facility by a responsible member of the Analytical Department.
*Note: The first analytical occasion (07 October 2020) was repeated on 14 October 2020, due to analytical technical error. The measurements could not be evaluated properly, therefore, the results could not be accurately reported. In agreement with the Study Director, the analytical occasion was repeated following week.
On each sampling occasion, top, middle and bottom duplicate samples was taken from test item formulations for concentration and homogeneity measurement, one set to analyse (which can be collected in replicates as practical) and one set as a back-up. Similarly, duplicate samples were taken from the middle of the vehicle control formulation for concentration measurement.
After the analytical sampling, the collected formulation samples were stored at room temperature until measurement.
Analysis of control (vehicle) and test item formulations for concentration and/or homogeneity was performed in the Analytical Laboratory of the Test Facility. Representative samples of control (vehicle) and/or test item formulations were analysed at four times during the study (during the first, second and last weeks and once approximately midway during the treatment period).
Any sample not required for analysis was discarded following acceptance of the results of the formulation analysis by the Contributing Scientist #1 (Analyst) and Study Director.
The formulation analysis was conducted within the determined stability period under the control of the responsible Contributing Scientist #1 (Analyst) in compliance with the analytical method validation and the relevant SOPs of the Test Facility.
Analysis of the formulations for concentration and/or homogeneity of test item was performed using a validated analytical HPLC-UV method (High Performance Liquid Chromatography with ultraviolet detection) in the Analytical Department of the Test Facility (Study code: 20/032-316AN). The density of the formulations was determined at the first analytical sampling by using three parallels in the Analytical Department of the Test Facility, as it was deemed necessary by the Contributing Scientist #1 (Analyst) and Study Director.
Acceptance criteria of the concentration analysis was 100 ± 10% of the nominal concentration.
Acceptance criteria of the homogeneity was that the RSD (relative standard deviation) of replicates (top, middle and bottom of test item formulations) must be less than 10%.
The measured concentrations of the test item in the different formulations varied between 95% and 106% of the nominal concentrations. All test item formulations were shown to be homogeneous. The relative standard deviation (RSD) was below 10% in each case.
Formulations were considered to be adequately stable under the study conditions.

Duration of treatment / exposure:

Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating period), then were euthanized and subjected to necropsy examination.
Females were dosed for 14 days pre-mating, for up to 14 days mating period, through gestation and up to and including the day before necropsy (13 days post-partum dosing).

Frequency of treatment:

daily on a 7 days/week basis

Details on study schedule:

- F1 parental animals = Not applicable: screening study
- Selection of parents from F1 generation = Not applicable: screening study
- Age at mating of the mated animals in the study: 13/14 weeks old (females/males) at mating (Parental Generation(P))

Dose / conc.:

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

Remarks:

solid

Dose / conc.:

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

Remarks:

solid

Dose / conc.:

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

Remarks:

solid; reduced to 600 mg/kg bw/day (actual dose received) at 4 weeks due to adverse clinical effects (local irritation) and mortality.

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected by the Sponsor in consultation with the Study Director based on the results of a Dose Range Finding (DRF) study (Study code: 20/032-220PE, [3]), 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), noisy respiration, laboured respiration, increased salivation, decreased activity, rooting of bedding, hunched back and piloerection were recorded. No test item related adverse effect was seen on body weight, food consumption, clinical pathology (including haematology and clinical chemistry). There were changes: e.g. gastrointestinal irritation, but this is no sign of systemic toxicity, therefore, the levels were acceptable. It was considered that 1000 mg solid /kg bw/day dose level was acceptable for the High dose level of this study. Lower doses were spaced with a factor of approximately 3.
- Rationale for animal assignment (if not random): All adult/parental (P) male and female animals 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 before the first treatment (prior to the start of the treatment).
- Fasting period before blood sampling for clinical biochemistry: Yes, overnight period of food deprivation

Parental animals: Observations and examinations:

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

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

BODY WEIGHT: Yes
- Time schedule for examinations: All adult animals were weighed with accuracy of 1 g weekly during the pre-exposure period, 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 (i.e. within 24 hours after parturition), 4, 7, 10 and 13, and at termination. The body weight of the female animals measured on GD3, GD10, GD17 and PPD10 were measurements only as an aid for the calculation of accurate treatment volumes, these data were 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). Food consumption was measured during mating. Food consumption was measured more frequently during the lactation period (on PPD0, 4, 7, 10 and 13).
Mean daily food consumption per animal was calculated for each interval.

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

OTHER:
OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes. Blood smears were prepared for all selected animals but not examined.
- Time schedule for collection of blood: Blood samples were collected by cardiac puncture under pentobarbital anaesthesia, immediately prior to scheduled necropsy.
- Anaesthetic used for blood collection: Yes: Euthanimal 40% (400 mg/mL sodium pentobarbital solution) was used by intraperitoneal injection
- Animals fasted: Yes overnight period of food deprivation, in case of females this happened after the litter had been culled
- How many animals: 5 males and 5 females/group randomly selected.
- Parameters examined:
RBC: Red Blood Cell (erythrocyte) count
WBC: White Blood Cell (leukocyte) count
Hgb: Haemoglobin concentration
Hct: Haematocrit (relative volume of erythrocytes)
MCV: Mean Corpuscular (erythrocyte)
MCH: Mean Corpuscular (erythrocyte) Haemoglobin
MCHC Mean Corpuscular (erythrocyte) Haemoglobin Concentration
RDW: Red Cell Distribution width
Plt: Platelet (thrombocyte) count
MPV: Mean Platelet Thrombocyte volume
RETIC %: Reticulocyte count
NE %: Neutrophil
LY %: Lymphocyte
MO %: Monocyte
BA %: Basophil
EO %: Eosinophil
LUC %: Large Unstained Cells

Coagulation:
APTT: Activated Partial Thromboplastin Time
PT Prothrombin Time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected by cardiac puncture under pentobarbital anaesthesia, immediately prior to scheduled necropsy.
- Animals fasted: Yes overnight period of food deprivation, in case of females this happened after the litter had been culled
- How many animals: 5 males and 5 females/group randomly selected
- Parameters examined:
Glucose: Blood sugar concentration
T-BIL: Total Bilirubin concentration
Urea: Urea concentration
Chol.: Cholesterol concentration
Creat.: Creatinine concentration
Phos.: Phosphorus concentration
Na+ : Sodium concentration
K+: Potassium concentration
Ca++: Calcium concentration
Cl-: Chloride concentration
Tot. Prot. : Total Protein concentration
Alb.: Albumin concentration
A/G: Alb/glob ration
AST/GOT: Aspartate Aminotransferase activity
ALT/GPT: Alanine Aminotransferase activity
GGT: Gamma-Glutamyl transferase activity
ALKP: Alkaline Phosphatase activity
BA: Bile acids

SERUM HORMONES: Yes
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 one non-pregnant adult female at termination,
• from all adult males at termination.
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. Timing are 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 150 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.

URINALYSIS: Yes
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes for approximately 16 hours
- Animals fasted: Yes overnight period of food deprivation, in case of females this happened after the litter had been culled
- Parameters examined:
LEU / Leukocyte
NIT / Nitrite
pH
PRO / Protein
GLU / Glucose
UBG / Urobilinogen
BIL / Bilirubin
KET / Ketones
BLD / ERY Blood/Erythrocytes
SG / Specific Gravity
SED / Sediment
VOL / Volume
Colour/Appearance

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
Assessment of any potential test item related neurotoxicity was performed during the last exposure week (males on Day 26 am, females on PPD9-10 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).
- Dose groups that were examined: Five males and five females/group were randomly selected
- Battery of functions tested: sensory activity / grip strength / motor activity / other:
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.

IMMUNOLOGY: No

Oestrous cyclicity (parental animals):

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

Sperm parameters (parental animals):

Parameters examined in male parental generations:
For the adult animals, detailed histological examination was performed on the retained reproductive organs (testes, epididymides, prostate gland, seminal vesicles with coagulation gland of all animals of the Control and High dose groups, and of all males that failed to sire.
Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were killed and discarded.
Pups to be culled within each litter were selected randomly. In litters of insufficient size where the number of males or female pups was less than 5, adjustment of the selection process was made to assure 10 pups were retained. Culling was not performed on litter sizes less (or equal) than 10.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups) and the presence of gross abnormalities. Any abnormal behaviour of the offspring was recorded.
Live pups were counted, sexed, weighed individually within 24 hours of parturition (PND0), and on PND4 and PND13, with accuracy of 0.01 g.
All the litters were checked and recorded daily for the number of viable and dead pups; clinical signs and any abnormal behaviour or appearance of the pups (external abnormalities) were also recorded on each day. All observed abnormalities were recorded.
The anogenital distance (AGD) of each pup was measured at the time of the first weighing (PND0). The anogenital distance was normalized to a measure of pup size (the cube root of body weight) for statistical analysis.
Number of nipples/areolae in male pups were recorded on PND13.
One male and one female pup per litter (if possible) were previously selected for culling for blood sampling on PND4.
All pups were necropsied on PND13.


GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.
All culled pups were subjected to necropsy with detailed macroscopic external and internal examination for any abnormalities. The pups found dead and intact (not cannibalized) were subjected to necropsy with macroscopic examination and the cause of death was identified if possible.

Postmortem examinations (parental animals):

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

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

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

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at Post-natal Day 13. (F1 offspring (one male and one female pup per litter if possible) selected for blood sampling were terminated on PND4 due to technical reason). In order to allow for overnight fasting of dams prior necropsy on PPD14, offspring was euthanized on PPD/PND13, and the dams on PPD/PND14.
- These animals were subjected to postmortem examinations (macroscopic examination).

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

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

Statistics:

See under "Any information on materials and methods incl. tables".

Reproductive indices:

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

-Survival Index %: [Number of live pups (at designated time) / Number of pups born] x 100
Survival index on PND13 was calculated from number of pups after culling on PND4 instead of number of pups born.
-Pre-implantation mortality %: [(Number of Corpora lutea – Number of implantations) / (Number of Corpora lutea)] x 100
-Intrauterine mortality %: [(Number of implantations – Number of liveborns) / Number of implantations] x 100
-Total mortality %: [(Number of implantations-Number of viable pups (PND0/4/13)) /Number of implantations] x 100
-Sex ratio% (females): [Number of female pups (PND0/4/13) / Number of viable pups (PND0/4/13)] x 100

Offspring viability indices:

-Survival Index %: [Number of live pups (at designated time) / Number of pups born] x 100
Survival index on PND13 was calculated from number of pups after culling on PND4 instead of number of pups born.
-Pre-implantation mortality %: [(Number of Corpora lutea – Number of implantations) / (Number of Corpora lutea)] x 100
-Intrauterine mortality %: [(Number of implantations – Number of liveborns) / Number of implantations] x 100
-Total mortality %: [(Number of implantations-Number of viable pups (PND0/4/13)) /Number of implantations] x 100
-Sex ratio% (females): [Number of female pups (PND0/4/13) / Number of viable pups (PND0/4/13)] x 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The initial High dose level of 1000 mg solid/kg bw/day was reduced to 600 mg solid/kg bw/day at 4 weeks due to adverse clinical effects (local irritation) and mortality.

Mortality:

mortality observed, treatment-related

Description (incidence):

Test item related mortality and clinical signs at 1000 mg/kg bw/day. The initial High dose level of 1000 mg solid/kg bw/day was reduced to 600 mg solid/kg bw/day at 4 weeks due to adverse clinical effects (local irritation) and mortality.
No test item related systemic-toxicity mortality was observed in the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

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

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

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

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Statistically significantly decreased red blood count (p<0.01), haemoglobin concentration (p<0.01), haematocrit (p<0.01) and increased red cell distribution width (p<0.05) and relative reticulocyte % (p<0.01) were observed in the High dose male group. These were considered to be related to treatment, although females were normal.
Statistically significantly decreased basophils relative % (p<0.05) in the High and Mid dose male groups and increased platelet volumes in Low (p<0.01) and Mid dose (p<0.05) females were in the normal range and were considered to be sporadic differences unrelated to treatment.
Data of red cell distribution width and haemoglobin concentration was out of the historical control range hence these changes are considered as test item related effects. These changes indicate a very slight anaemia, correlating with increased reticulocytes which indicates compensation. The degree of the changes seen is not sufficient to represent a clearly adverse effect.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no biologically relevant effects on the serum chemistry that could be ascribed to as test item related adverse effect.
Statistically significantly increased sodium level (p<0.05) was observed in the Mid dose male group. Statistically significantly decreased calcium level (p<0.05) and bile acid level was observed in the High dose male (p<0.01) and female (p<0.05) group, but the data were within the historical control range.

Endocrine findings:

no effects observed

Description (incidence and severity):

Compared to the control, there were no statistically significant thyroid hormone concentration levels recorded in any of the adult males.
The thyroid gland weights of the males were also statistically not different from the Control group. In summary, there were no effects on the thyroid hormone levels or on the thyroid gland weights in the adult males that were ascribed to the test item.
The measurement of the thyroid hormone levels in the adult females were not performed as it was not deemed necessary by the Study Director.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related changes were observed in the urinalysis parameters in male and female animals of any dose groups when compared to control.
Statistically significantly decreased pH (p<0.05) was measured in the High dose female group but this finding was within the normal range and not considered as test item related adverse effect. The HC range for pH is 6-7, hence the pH of the other group was outside of the HC range but the High dose female group.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

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

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

NON-PREGNANT FEMALES / Parental Generation: There was one non-pregnant female in the study (#2510). No microscopic changes were observed in the non-pregnant females and the male mating pair.
FOUND DEAD ANIMAL / Parental Generation: One High Dose male (#4001) died on Day 18 and one High Dose female (#4511) died on Day 50 of the Study, laboured and noisy respiration were observed right before the death, hence it is considered that a small amount of test item got into the respiratory system. Microscopically agonal congestion/ hemorrhage was seen in the lungs of the male, in the female increased number of atretic follicles was observed and was considered as incidental. The causes of death could not be determined by histopathology.
PRE-TERMINAL EUTHANASIA / Parental Generation: One High Dose female (#4503) was pre-terminally euthanised due to missgavage and laboured/noisy respiration on Day 12 of the Study. Microscopically acute inflammation of the trachea and decreased cellularity in the thymus was observed and was considered as incidental.
TERMINAL EUTHANASIA / Parental Generation: No test item-related findings were seen at a dose level of 1000 / 600 mg/kg bw/day. The liver weight increase (15% relative to body weight) was not found histologically; the increased weight was ascribed to a normal adaptive hypertrophy, less than is visible histologically. In mutual agreement with the pathologist, no further histopathology analysis was needed at the low and mid dose groups.
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.

Histopathological findings: neoplastic:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

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

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Histopathological evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure did not reveal any test item-related findings at a dose level of 1000 / 600 mg/kg bw/day.

Reproductive performance:

no effects observed

Description (incidence and severity):

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

-Mortality and morbidity:
No test item related systemic-toxicity mortality was observed in the study.
One High dose female (#4511) found dead on Day 50 (short after delivery) and one High dose male (#4001) found dead on Day 18. One High dose female (#4503) was pre-terminally euthanised on Day 13.
- Clinical observation:
No test item related clinical signs were observed in the Low dose groups.
Laboured respiration (Day 12) and noisy respiration (Day 34) were sporadically observed in animal found dead (#4511). Laboured and noisy respiration were recorded prior to death in animal found dead (#4001). Noisy and laboured respiration was observed prior to death in animal preterminal euthanised (#4503).
Noisy respiration was also observed in case of 1/12 Mid dose male on Day 13 and Day 14, another 3/11 High dose males from Day 3 until Day 26. Noisy respiration was observed for 4/12 Mid dose females from Day 2 to necropsy, the longevity of the observation was 21 days and another 4/12 High dose females from Day 1 to necropsy, the symptom was recorded at a maximum of 15 day long.
Laboured respiration was observed for another 1/12 High dose males on Day 2 and 3 and 2/12 High dose females on Day 12 and from Day 17 to Day 19, respectively.
Piloerection was observed for 1/12 High dose female on Day 2 and Day 3.
The above findings were probably related to the very irritant effect of small amounts of test item reaching the upper respiratory tract via reflux or contamination during dosing (as seen during the preliminary study). This is considered as a local effect of the test item.
Tonic convulsion (whole body) was observed for one Low dose female (#2508) on Day 36, Day 40, Day 43 and Day 46. Nodule around the left axillary was observed for one Mid dose female (#3508) from Day 38 till necropsy, the longevity of the observation was 15 days. These findings were considered incidental, not test item related adverse effect.
-Body weight and weight changes:
No test item related adverse effect on body weight or body weight gain was detected in mean group Low, Mid and High dose animals (males or females).
-Food consumption and compound intake (no feeding study):
No adverse effect on food consumption was seen in male and female animals in any test item treated group. Statistically significantly increased (by 7.8%) mean food consumption was observed for the High dose males from Day 21 to Day 27.
-Neurological assessment:
There were no changes in animal behaviour, general physical condition or in the reactions to different type of stimuli in the control or test groups.
There was no test item related effect of treatment noted in the Irwin test or during the landing foot splay. The average grip strength for the forelimb was statistically significantly increased (p<0.05) in the High dose female group, but the data was within the historical control range and did not follow a dose response.
All dose groups of males and females had a normal locomotor activity. In all cases, the initial activity was high, with reduced activity in each 5-minute period to an approximate plateau by about 20-30 minutes. There was no statistical significance between the test item treated animals (males and females) and the Control when evaluating the overall total travelled distance (0-60 minutes). The test item did not increase or decrease the normal locomotor activity, all treated groups had a profile of activity the same as historical control data.
-Haematology:
Statistically significantly decreased red blood count (p<0.01), haemoglobin concentration (p<0.01), haematocrit (p<0.01) and increased red cell distribution width (p<0.05) and relative reticulocyte % (p<0.01) were observed in the High dose male group. These were considered to be related to treatment, although females were normal.
Statistically significantly decreased basophils relative % (p<0.05) in the High and Mid dose male groups and increased platelet volumes in Low (p<0.01) and Mid dose (p<0.05) females were in the normal range and were considered to be sporadic differences unrelated to treatment.
Data of red cell distribution width and haemoglobin concentration was out of the historical control range hence these changes are considered as test item related effects. These changes indicate a very slight anaemia, correlating with increased reticulocytes which indicates compensation. The degree of the changes seen is not sufficient to represent a clearly adverse effect.
-Clinical chemistry:
There were no biologically relevant effects on the serum chemistry that could be ascribed to as test item related adverse effect.
Statistically significantly increased sodium level (p<0.05) was observed in the Mid dose male group. Statistically significantly decreased calcium level (p<0.05) and bile acid level was observed in the High dose male (p<0.01) and female (p<0.05) group, but the data were within the historical control range.
-Urinalysis:
No test item-related changes were observed in the urinalysis parameters in male and female animals of any dose groups when compared to control.
Statistically significantly decreased pH (p<0.05) was measured in the High dose female group but this finding was within the normal range and not considered as test item related adverse effect. The HC range for pH is 6-7, hence the pH of the other group was outside of the HC range but the High dose female group.
-Oestrus cycle evaluation in females:
>Pre-exposure period
Each female selected for the study showed acceptable cycles (mean cycle length of 3.98-4.04 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 4.00, 4.15, 4.10 and 4.15 days in the Control, Low dose, Mid dose and High dose groups, respectively).
Prolonged oestrus was recorded for 4 Low dose females (#2501, #2502, #2504 and #2510), but it did not affect mating or pregnancy (from these animals only one female (#2510) was non-pregnant). This fact was considered as being an occasional finding, not being a test item related effect.
No prolonged dioestrus was noted in any of the groups.
-Reproductive ability assessment and indices:
There were no statistical differences between the control and test item treated groups with regard to reproductive ability, mating or gestation indices, and no effects considered adverse or toxicologically significant in correlation with the administration of the test item. The mating index was 100% in treated groups (males and females), the fertility index was 100% for the Control, Mid and High dose group and 92% in case of the Low dose group. The gestation index was also 100% in all groups.
Test item administration was considered to have no impact on the duration of the mating period. Successful coitus (sperm positive vaginal smears and/or vaginal plugs) occurred mostly within 5 days of pairing (cohabitation). The mean duration of mating was 3.00, 2.58, 2.92 and 3.33 days in the Control, Low, Mid and High dose groups, respectively.
For one High dose female (#4501) the pairing with the first male (#4001) was not successful because of the dead of the male, hence another male (#4003) was chosen for mating. With this male the mating was successful and in the tables only these data were indicated.
-Evaluation of the gestation, parturition and post-partum period: The mean duration of pregnancy was comparable in the Control and test item treated groups.
As far as it could be observed during the study, the parturition was normal for all animals, no abnormal delivery was noted.
The number of implantation sites was comparable to the control mean in all dose groups, no statistically significant or biologically relevant differences were noted.
There was statistically significant decrease (p<0.05) on the number of pups born in the High dose group, but the value was well within the normal range (the control group had an unusually high average number).
-Evaluation of the gestation, parturition and post-partum period:
The mean duration of pregnancy was comparable in the Control and test item treated groups.
As far as it could be observed during the study, the parturition was normal for all animals, no abnormal delivery was noted.
The number of implantation sites was comparable to the control mean in all dose groups, no statistically significant or biologically relevant differences were noted.
There was statistically significant decrease (p<0.05) on the number of pups born in the High dose group, but the value was well within the normal range (the control group had an unusually high average number).
-Organ weights:
> Parental Males
No test item-related effects were observed in the liver weights of the test item treated male animals compared to controls.
There were no statistically significant differences in the terminal body weight of the test item treated males compared to control males. The body-related and brain-related weight of the liver was increased statistically significantly (by 15.0% and by 11.7%, respectively) in the High dose group compared to Control. Absolute liver was increased by 11.8%, compared to Control, however not statistically significant.
> Parental Females
No test item-related effects were observed in the organ weights of the test item treated female animals compared to controls.
Terminal body weights of test item treated females were not different from control females. The brain-related weight of kidneys was increased statistically significantly (by 11.2%) in the Mid dose group compared to control, but there was no dose response and data were within the historical control range, hence this is not considered as test item related effect.
There were no other statistically significant and biologically relevant differences among groups in the weights of organs measured when compared to Controls.
-Pathology evaluation:
>NON-PREGNANT FEMALES / Parental Generation: There was one non-pregnant female in the study (#2510).
Macroscopic Findings: Necropsy examination did not show any test item related change in the non-pregnant females and their male mating pairs.
Microscopic Findings: No microscopic changes were observed in the non-pregnant females and the male mating pair.
>FOUND DEAD ANIMAL / Parental Generation: One High Dose male (#4001) died on Day 18 and one High Dose female (#4511) died on Day 50 of the Study, laboured and noisy respiration were observed right before the death, hence it is considered that a small amount of test item got into the respiratory system.
Macroscopic Findings: No test item-related macroscopic changes were observed at necropsy, In the male the stomach and the intestines were dilated with gas, the non-glandular mucosa of the stomach showed multifocal thickness and the lungs were dark red and non-collapsed. In the female the lungs were enlarged and dark red at necropsy.
Microscopic Findings: Microscopically agonal congestion/ hemorrhage was seen in the lungs of the male, in the female increased number of atretic follicles was observed and was considered as incidental. The causes of death could not be determined by histopathology.
>PRE-TERMINAL EUTHANASIA / Parental Generation: One High Dose female (#4503) was pre-terminally euthanised due to missgavage and laboured/noisy respiration on Day 12 of the Study.
Macroscopic Findings: No test item-related macroscopic changes were observed. In the lungs focal dark red discoloration was seen.
Microscopic Findings: Microscopically acute inflammation of the trachea and decreased cellularity in the thymus was observed and was considered as incidental.
TERMINAL EUTHANASIA / Parental Generation:
Macroscopic Findings: No test item-related macroscopic findings were observed up to the dose level of 1000/600 mg solid/kg bw/day.
All observed changes were considered incidental or a common background.
Microscopic Findings: No test item-related findings were seen at a dose level of 1000 / 600 mg/kg bw/day. The liver weight increase (15% relative to body weight) was not found histologically; the increased weight was ascribed to a normal adaptive hypertrophy, less than is visible histologically. In mutual agreement with the pathologist, no further histopathology analysis was needed at the low and mid dose groups.
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.
-Thyroid Hormone Analysis:
Compared to the control, there were no statistically significant thyroid hormone concentration levels recorded in any of the adult males.
The thyroid gland weights of the males were also statistically not different from the Control group. In summary, there were no effects on the thyroid hormone levels or on the thyroid gland weights in the adult males that were ascribed to the test item.
The measurement of the thyroid hormone levels in the adult females were not performed as it was not deemed necessary by the Study Director.

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive effects of parental generation

Effect level:

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

Based on:

other: solid

Sex:

male/female

Basis for effect level:

other: highest dose tested

Clinical signs:

no effects observed

Description (incidence and severity):

There was no test item effect on mortality or survival of the pups (F1 generation).
The mean number of pups born was statistically significantly decreased in the High dose group (p<0.05) but when analysed per litter it was clear that all data were in the normal range and there was no adverse effect of treatment. Pup survival indices on PND0 and PND13 were comparable to control values.
There were no other significant differences or effects that could be ascribed to treatment on the pre-natal, post-natal or total mortality values (litter mean and %) in any of the dose groups.
Evidence of suckling was recorded for all live born pups in the study.

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 mean number of pups born was statistically significantly decreased in the High dose group (p<0.05) but when analysed per litter it was clear that all data were in the normal range and there was no adverse effects of treatment. Pup survival indices on PND0 and PND13 were comparable to control values.
There were no other significant differences or effects that could be ascribed to treatment on the pre-natal, post-natal or total mortality values (litter mean and %) in any of the dose groups.
Evidence of suckling was recorded for all live born pups in the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no differences in the offspring body weights or weight gains.

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

Description (incidence and severity):

screeening study

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

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

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

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

Organ weight findings including organ / body weight ratios:

not examined

Description (incidence and severity):

TERMINAL / F1 Generation (PND13): No test item-related macroscopic findings were observed up to the dose level of 1000/600 mg solid/kg bw/day.

Histopathological findings:

not examined

Description (incidence and severity):

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

Other effects:

no effects observed

Description (incidence and severity):

Thyroid Hormone Analysis:
Compared to the control, there were no statistically significant thyroid hormone concentration levels recorded in any of the PND13 pups.
The thyroid gland weights of the PND13 pups were also statistically not different from the Control group. In summary, there were no effects on the thyroid hormone levels or on the thyroid gland weights in the PND13 pups that were ascribed to the test item.
The measurement of the thyroid hormone levels in the PND4 pups were not performed as it was not deemed necessary by the Study Director.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

-Mortality and clinical observations:
There was no test item effect on mortality or survival of the pups (F1 generation).
The mean number of pups born was statistically significantly decreased in the High dose group (p<0.05) but when analysed per litter it was clear that all data were in the normal range and there was no adverse effect of treatment. Pup survival indices on PND0 and PND13 were comparable to control values.
There were no other significant differences or effects that could be ascribed to treatment on the pre-natal, post-natal or total mortality values (litter mean and %) in any of the dose groups.
Evidence of suckling was recorded for all live born pups in the study.
-Body weight and body weight gain:
There were no differences in the offspring body weights or weight gains.
-Anogenital distance, nipple retention:
No test item effect was observed on anogenital distance or nipple retention during the study.
No statistically significant changes in the anogenital distance measured on PND 0 were noted for test item treated male and female pups when litter mean values were compared to control.
There was no nipples/areolae presence seen in any of the male pups on PND13.
-Pathology evaluation:
>TERMINAL / F1 Generation (PND13)
Macroscopic Findings: No test item-related macroscopic findings were observed up to the dose level of 1000/600 mg solid/kg bw/day.
Microscopic Findings: No histopathological examination was performed on pups (F1 generation).
-Thyroid Hormone Analysis
Compared to the control, there were no statistically significant thyroid hormone concentration levels recorded in any of the PND13 pups.
The thyroid gland weights of the PND13 pups were also statistically not different from the Control group. In summary, there were no effects on the thyroid hormone levels or on the thyroid gland weights in the PND13 pups that were ascribed to the test item.
The measurement of the thyroid hormone levels in the PND4 pups were not performed as it was not deemed necessary by the Study Director.

Key result

Dose descriptor:

NOAEL

Remarks:

pup development and survival

Generation:

F1

Effect level:

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

Based on:

other: solid

Sex:

male/female

Basis for effect level:

other: highest dose tested

Key result

Reproductive effects observed:

no

Conclusions:

Under the experimental conditions of this study the NOAEL for reproductive effects of the parental generation was considered to be >=600 mg solid/kg bw/day.

Executive summary:

The purpose of this OECD No. 422 study was to obtain information on the possible toxic effects of dicyclohexyl sodium sulfosuccinate (CAS 23386-52-9; EC 245-629-3) test item following repeated (daily) administration by oral gavage to Wistar (Crl:WI) rats at 3 dose levels. A control group received the vehicle only (distilled water).

Male and female Wistar rats were treated for 2 weeks pre-mating and then during the mating/post-mating periods. This was 28 days in total for males. Females were treated throughout gestation and up to and including postpartum/lactation day (PPD) 13.

Parameters measured during the study included signs of morbidity and mortality twice daily, daily general and/or weekly detailed observation of clinical signs, weekly body weight and food consumption, and clinical pathology evaluation (including haematology, coagulation, clinical chemistry and urinalysis). Neurological assessments, including a functional observation battery (FOB) and measurements of the landing foot splay, grip strength and motor activity were performed during the last week of the treatment. 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 post-natal day (PND13). At termination, necropsy with macroscopic examination was performed. Weights of selected organs were recorded, and representative tissues/organs were sampled and preserved in appropriate fixatives from the adult animals and/or offspring. The thyroxine (T4) levels in the Day 13 (PPD13) pups and adult males were also assessed. For the adult animals, a detailed histological examination was performed on the selected list of retained organs in the Control and High dose groups.

The dose levels were selected by the Sponsor in consultation with the Study Director based on the results of a Dose Range Finding (DRF) study. Based on the results of the DRF study, 1000 mg solid/kg bw/day was selected as the High dose for this study but was reduced to 600 mg solid/kg bw/day at 4 weeks due to adverse clinical effects (local irritation) and mortality.

Experimental design:

Group Number	Group designation	Dose level (mg solid/kg bw/day)	Dose formulation concentration (mg solid/mL)	Dose formulation volume (mL/kg bw)	Animal numbers
					Male	Female
1	Control	0	0	5	1001-1012	1501-1512
2	Low Dose	100	20		2001-2012	2501-2512
3	Mid Dose	300	60		3001-3012	3501-3512
4	High Dose	1000/600	200/120		4001-4012	4501-4512

Note: On 27 October 2020 (day 28 of the dosing period) the High dose was reduced to 600 mg/kg bw/day due to mortalities and increasing incidence and severity of noisy/laboured respiration at the high dose.

Results:

In summary, under the conditions of this study the daily administration of dicyclohexyl sodium sulfosuccinate (CAS 23386-52-9; EC 245-629-3) by oral gavage to Wistar rats at dose levels of 100, 300 or 1000 / 600 mg solid/kg bw/day (Low, Mid and High dose groups, respectively) did result in test item related mortality and clinical signs at 1000 mg/kg bw/day.

The initial High dose level of 1000 mg solid/kg bw/day was reduced to 600 mg solid/kg bw/day at 4 weeks due to adverse clinical effects (local irritation) and mortality.

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

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

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

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

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

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

No test item related macroscopic or microscopic changes were recorded at necropsy or at histopathology evaluation of routine organs/tissues or in any reproductive organs.

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

The NOAEL for local toxicity of the parental generation: 300 mg solid/kg bw/day. (based on noisy/laboured respiration at 600 mg solid/kg bw/day).

The NOAEL for systemic toxicity of the parental generation was considered to be >=600 mg solid/kg bw/day.

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

The NOAEL for pup development and survival was considered to be >=600 mg solid/kg bw/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

600 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Klimisch 1

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A key OECD No. 422 study was conducted with the registered substance in Wistar rats (12/sex/group by oral gavage at 0 (distilled water), 100, 300 and 1000 mg solid/kg bw/day (Szalóki, 2021). Based on the results of the DRF study, 1000 mg solid/kg bw/day was selected as the High dose for this study but was reduced to 600 mg solid/kg bw/day at 4 weeks due to adverse clinical effects (local irritation) and mortality. Male and female Wistar rats were treated for 2 weeks pre-mating and then during the mating/post-mating periods. This was 28 days in total for males. Females were treated throughout gestation and up to and including postpartum/lactation day (PPD) 13. The systemic toxicity parameters are reported under the Section 7.5. The reproductive performance, pregnancy, parturition and postpartum/lactation period were monitored in the adult animals, and viability, clinical signs and development were evaluated in their F1 offspring until post-natal day (PND13). At termination, necropsy with macroscopic examination was performed. Weights of selected organs were recorded, and representative tissues/organs were sampled and preserved in appropriate fixatives from the adult animals and/or offspring. The thyroxine (T4) levels in the Day 13 (PPD13) pups and adult males were also assessed.

No test item effect on oestrus cycle of parental females was noted. No test item related changes were noted in the reproductive parameters during mating and gestation, delivery and post-partum/lactation period until PPD14. There were no adverse effects on the F1 offspring viability, clinical signs, physical or sexual development. No test item related macroscopic finding was recorded for F1 pups at necropsy. No test item related changes were noted in the reproductive parameters during mating and gestation, delivery and post-partum/lactation period until PPD 14. No test item related macroscopic or microscopic changes were recorded at necropsy or at histopathology evaluation of routine organs/tissues or in any reproductive organs. Under the experimental conditions of this study and based on the results of thyroid hormone measurement, thyroid weights, nipple retention, anogenital distance and external reproductive organs analysis, histopathology and reproductive performance, there was no evidence for any endocrine effects. Based on the results of this study, the following No-Observed-Adverse-Effect Levels (NOAELs) were considered: The NOAEL for reproductive effects of the parental generation was considered to be >=600 mg solid/kg bw/day. The NOAEL for pup development and survival was considered to be >=600 mg solid/kg bw/day.

 

Conclusion

Reproductive toxicity screening was performed with the registered substance in Wistar rats by oral gavage at 0 (distilled water), 100, 300 and 1000/600 mg solid/kg bw/day in key combined repeated dose/reproductive toxicity study (OECD No. 422). The NOAEL for reproductive effects of the parental generation was >= 600 mg solid/kg bw/day and the NOAEL for pups development and survival was >= 600 mg solid/kg bw/day. There were no adverse effects on reproductive organs or tissues or other concerns in relation with reproductive toxicity, therefore further testing data are not required.

Effects on developmental toxicity

Description of key information

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

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

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

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

No further testing is considered needed based on these data.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

Read across argumentation is provided in Section 13.

Reason / purpose for cross-reference:

read-across source

Strain:

Abyssinian

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 acros data were available from Docusate sodium. Justification for read across within the category of Di-ester sulphosuccinates is documented in a separate document attached in Section 13.

Teratogenicity testing in first species

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

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

- As doscusate sodium has a long history of pharmaceutical and dietary supplement use, a 3-generation toxicity study in rat was requested by FDA and JECFA, which confirmed that docusate sodium would pose no reproduction or teratological problems. The data were considered to adequately support safety in combination with information from prenatal developmental toxicity testing of docusate sodium and docusate calcium, therefore a study in a second species was not considered necessary. In addition, the rabbit was not considered to be an adequate model due to its high gastro-intetinal sensitivity to surfactant properties, as demonstrated by diarrhoea followed by mortality in a repeated dose toxicity study. In conclusion, based on the evaluation of the outcome of the first species and other supporting informatoin, a second species for developmental toxicity was not considered necessary and waived according to REACH Annex IX section 8.7.2 column 2.

 

Conclusion

Prenatal developmental toxicity was tested by dietary administration of read across substance Docusate sodium 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, no further testing is needed.

 

Teratogenicity testing in second species

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

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

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


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


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


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

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

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

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

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

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

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

Conclusion

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

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

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

Justification for classification or non-classification

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