Sodium 1,4-diisodecyl sulphonatosuccinate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

An OECD 408 (90-day study) has been started 2016 based on ECHA Communication number CCH-D-2114321066-61-01/D.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

Adopted 21st September 1998

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Docusate sodium

EC Number:

209-406-4

EC Name:

Docusate sodium

Cas Number:

577-11-7

Molecular formula:

C20H38O7S.Na

IUPAC Name:

sodium 1,4-bis[(2-ethylhexyl)oxy]-1,4-dioxobutane-2-sulfonate

Test material form:

other: Rolls of white wax-like plastic sheets

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: SD6073132, Manufacturer: Cytec-Solvay Group Willow Island Plant, #1 Heilman Avenue, Willow Island, WV 26134, USA
- Expiration date of the lot/batch: July 31, 2019 (3 years from date of manufacturing)
- Purity test date: August 4, 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At +10°C to +25°C
- Stability under test conditions: The analysis of the left-over-diet (food which has been available to the animals for 6 to 7 days) revealed a stability of the test item in the test item-diet mixture. The measured concentrations ranged from 88.4% to 110.1% (low dose), from 82.0% to 102.5% (intermediate dose) and from 87.1% to 112.9% (high dose) in test weeks 1, 4, 8 and 13.

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

Selected because of its proven suitability in toxicology studies and to comply with regulatory requirements for testing in a rodent animal species.

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, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age (at 1st dosing): Males: 65 days; Females: 65 days
- Body weight (at 1st dosing): Males: 282.6 – 332.8 g; Females: 207.0 - 260.5 g
- Housing: The animals were kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 18 cm. Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany) was used as bedding material for the cages. The cages were changed and cleaned once a week.
- Diet (e.g. ad libitum): Commercial ssniff®-R/M-H V1530 diet (ssniff® Spezialdiäten GmbH, 59494 Soest, Germany) served as food.This diet was offered without Docusate Sodium to group 1 and with Docusate Sodium to groups 2, 3 and 4 ad libitum. food residue was removed and weighed weekly.
- Water (e.g. ad libitum): Drinking water was offered ad libitum.
- Acclimation period: 6 days

DETAILS OF FOOD AND WATER QUALITY:
Periodic analysis of the food for contaminants based on EPA/USA1 is conducted at least twice a year by LUFA-ITL (Landwirtschaftliche Untersuchungs- und Forschungsanstalt, Institut für Tiergesundheit und Lebensmittelqualität GmbH, 24107 Kiel, Germany.). Certificates of analysis of the composition and for contaminants were provided by the manufacturer and are included in the raw data.
Samples of drinking water are taken periodically by the Wasserwerk Wankendorf and periodic analyses are performed by LUFA-ITL according to the 'Deutsche Trinkwasserverordnung 2001' [German Regulations on drinking water 2001] (Version from August 02, 2013, revised on November 18, 2015.
In addition, drinking water samples taken at LPT are analysed by LUFA-ITL once a year for means of bacteriological investigations according to the 'Deutsche Trinkwasserverordnung 2001, Anlage 1' [German Regulations on drinking water 2001, Addendum 1].

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3°C (maximum range)
- Humidity (%): 55% ± 10% (maximum range). Deviations from the maximum range caused for example during cleaning procedures are dealt with in SOPs.
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
From: Start of the exposure period May 23, 2017
To: Termination of the in-life period:September 18, 2017
Dissection of main study animals August 21, 2017
Dissection of recovery animals September 18, 2017

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

According to expected route of exposure and international guidelines.

Vehicle:

unchanged (no vehicle)

Remarks:

in diet

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): The test item-diet mixtures were freshly prepared once a week.
- Mixing appropriate amounts with (Type of food): The appropriate amount of pre-cooled (+2°C to +8°C) test item (based on the most recently recorded food consumption and body weights) was chopped or shredded into small pieces and weighed into a glass container. Some of the test item and of also pre-cooled diet were mixed with an impact mill to produce a premix. This process was repeated until the whole quantity of test compound was distributed in the diet. Then the premix was added to the diet, mixed with a mixer (Röhnradmischer; Typ ELTE 650; J. Engelsmann AG, Ludwigshafen, Germany) for 15 minutes and then transferred to a closable bucket. Each bucket was labelled with study number, group number, sex, and dose.
To maintain a constant dose level in relation to the animals body weight, the concentration in the diet was adjusted based on the mean group food consumption per sex. The concentration was adjusted weekly using the food consumption values from the previous week.
The total weekly intake of the test item was calculated and reported as daily average for the respective week.
- Storage temperature of food: stored at +10°C to +25°C.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For the determination of the Docusate Sodium concentration in the exposure diets (groups 1 to 4), samples of approximately 10 g were taken at the following times and stored at -20°C or colder until analysis at LPT.
Exposure diet analysis:
Determination Test week Handling of samples per Number of samples
sex and dosage (in total 3 (including control)
dosages and 1 control) per sex# total
Concentration 1 One sample from each of 10 20
and 4 three areas (top, middle and 10 20
homogeneity 8 bottom) of the bucket## 10 20
13 10 20

Concentration 1 Left-over diet food which 4 8
and stability 4 has been available to the 4 8
(left-over diet) 8 animals for 7 days 4 8
13 Left-over diet food which 4 8
has been available to the
animals for 6 days
Total number of samples: 56 112
#: Due to gender dependent body weight gain, the test item concentrations were calculated and mixed separately for males and females.
##: Only one sample from the control.

The samples were labelled with study number, test species, sex, type of sample, concentration, test week, location (top/middle/bottom), and date.
The samples were analysed for Docusate Sodium according to the HPLC-UV method for the determination of the test item in diet-mixture samples validated at LPT (LPT validation study no. 34903). The results of the validation confirmed that the method employed was suitable for the determination and quantification of Docusate Sodium in diet-mixture samples (diet: ssniff® R/M-H V1530).
The mean test item intake per week ranged from 84 to 117 and 78 to 117 mg/kg bw/day for the male and female animals treated with a nominal dose level of 100 mg Docusate Sodium/kg bw/day via the diet, respectively, from 250 to 351 and 237 to 350 mg/kg bw/day for the male and female animals treated with a nominal dose level of 300 mg Docusate Sodium/kg bw/day via the diet, respectively, and from 854 to 1239 and 811 to 1369 mg/kg bw/day for the male and female animals treated with a nominal dose level of 1000 mg Docusate Sodium/kg bw/day via the diet, respectively.
The results of the analysis revealed that the test item-diet mixtures were correctly prepared and were very homogenous. The measured concentrations ranged from 81.6% to 119.4% (low dose), from 83.4% to 115.8% (intermediate dose) and from 91.9% to 111.6% (high dose) in test weeks 1, 4, 8 and 13.
The results were within the admissible limits of 80% to 120% for a test item-diet mixture preparation.
The analysis of the left-over-diet (food which has been available to the animals for 6 to 7 days) revealed a stability of the test item in the test item-diet mixture. The measured concentrations ranged from 88.4% to 110.1% (low dose), from 82.0% to 102.5% (intermediate dose) and from 87.1% to 112.9% (high dose) in test weeks 1, 4, 8 and 13.
No Docusate Sodium could be detected in any of the control samples.

Duration of treatment / exposure:

90 test days, 28 additional days for the animals scheduled for the recovery period

Frequency of treatment:

daily in diet

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control (Group 1; standard diet)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Docusate Sodium (Group 2)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Docusate Sodium (Group 3)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Docusate Sodium (Group 4)

No. of animals per sex per dose:

Main study: 10 per sex per group (80 in total)
Recovey animals: 5 per sex per group for groups 1, 3 and 4 (30 in total)
Additionally, 10 spare animals (5 males and 5 females) were reserved for possible replacement during the adaptation period, however, not used.

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dose levels for this study were selected in agreement with the Sponsor based on available data and the results of a preliminary 14-day dose-range finding study
(LPT study report no. 34271):
The doses administered via the diet in the dose-range finding study 34271 ranged from 821 to 1414 mg/kg bw/day, depending on the daily food intake of the animals. No deaths and no signs of toxicity were noted. No influence was observed on the body weight, the body weight gain, and the food and drinking consumption. No test item-related findings were noted at macroscopic inspection at necropsy. The male animals revealed increased kidney and liver weights on test day 15.
After consideration of these data nominal dose levels of 100, 300 and 1000 mg Docusate Sodium/kg bw/day via the diet were selected for this 90-day toxicity study.
- Rationale for animal assignment (if not random): The animals were allocated to the 4 test groups based on body weight by means of a computerised randomisation program. Animals with a body weight at the extremes of the weight distribution were excluded and replaced by healthy spare animals. Animal replacement after the first dosing was not planned nor did it occur.
- Rationale for selecting satellite groups: 5 animals/sex/group were allocated to groups 1, 3 and 4 at the start of the experiment for 28 days recovery after 90 days of dosing
- Post-exposure recovery period in satellite groups: 28 days
- Section schedule rationale (if not random): Starting on test day 91 the main study animals were dissected following a randomisation scheme. Animals allocated to the recovery period were dissected on test day 119.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The animals were observed individually at least once daily.
Animals were checked regularly throughout the working day from 7.00 a.m. to 3.45 p.m. On Saturdays and Sundays animals were checked regularly from 7.00 a.m. to 11.00 a.m. with a final check performed at approximately 3.30 p.m.
- Cage side observations included any signs of behavioural changes, reaction to treatment or illness: yes.
skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns.
The onset, intensity and duration of any signs observed were recorded.
Dated and signed records of appearance, change and disappearance of clinical signs were maintained on clinical history sheets for individual animals.
Checks were made early in the morning and again in the afternoon of each working day to look for dead or moribund animals. This would have allowed post mortem examination to be carried out during the working period of that day. On Saturdays and Sundays a similar procedure was followed except that the final check was carried out at approximately 4:00 p.m.
However, no premature deaths occurred in this study.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Additionally, once before the first exposure (to allow for within-subject comparisons) and once a week thereafter (always on the first day of the test week), detailed clinical observations were made in all animals. In test week 13 these observations were performed prior to any laboratory investigations. These observations were made outside the home cage in a standard arena and at the same time of day, each time.
Signs noted included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, pilo-erection, pupil size, 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) or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each rat was recorded at the time of group allocation, on the day of commencement of treatment and once a week thereafter always on the same day of the week throughout the experimental period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The quantity of food left by individual animals was recorded on a weekly basis throughout the experimental period. The food intake per rat (g/rat/week) was calculated using the total amount of food given to and left by each rat in each group on completion of a treatment week.
The relative food consumption (in g/kg bw/day) was calculated as follows:

Relative food consumption = Total food given (g) - Total food left (g)
(g/kg bw/day) Number of animal days# × Body weight (kg)

# The term 'animal days' counts one animal day for each animal alive for a whole day; it is assumed that on the day of death an animal does not eat.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
Individual test item intake was calculated on a weekly basis throughout the experimental period based on concentration in the diet, individual food intake and body weight.

FOOD EFFICIENCY: no (not required)

WATER CONSUMPTION AND COMPOUND INTAKE INTAKE (no drinking water study): Yes
- Time schedule for examinations: The drinking water consumption was monitored daily by visual appraisal throughout the study.

OPHTHALMOSCOPIC EXAMINATION: Yes.
- Time schedule for examinations: Examinations were performed on all animals at study initiation (on test day 7), at the end of the main study (on test day 87 in test week 13) and at the end of the
recovery period (on test day 116 in test week 17).
- Dose groups that were examined: on all animals
The eyes were examined with a HEINE ophthalmoscope.
After examination of the pupillary reflex, mydriasis was produced by instillation of STULLN®10 eye drops onto the cornea. The following ocular structures were then examined: Adnexa oculi (i.e. lids, lacrimal apparatus), conjunctiva; Cornea, anterior chamber; Lens, vitreous body, fundus (retina, optic disc)

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
At the end of the treatment period (on the first day of dissection, test day 91) for all animals
At the end of the recovery period (on the day of dissection, test day 119) for all recovery animals
- Anaesthetic used for blood collection: Yes, light ether anaesthesia
- Animals fasted: Yes, fasted overnight
- How many animals: all animals/all recovery animals
- Parameters examined: Haemoglobin content, Erythrocytes, Leucocytes, Reticulocytes, Platelets, Haematocrit, Differential blood count (relative), Differential blood count (absolute), Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
At the end of the treatment period (on the first day of dissection, test day 91) for all animals
At the end of the recovery period (on the day of dissection, test day 119) for all recovery animals
- Animals fasted: Yes, fasted overnight
- How many animals: all animals/all recovery animals
- Parameters examined: Albumin, Globulin, Albumin/globulin ratio, Bile acids, Bilirubin (total), Cholesterol (total), Creatinine, Glucose, Protein (total), Triglycerides, Urea (in blood), Calcium, Chloride, Potassium, Sodium, Alanine amino-transferase (ALAT), Alkaline phosphatase (aP), Aspartate dehydrogenase (ASAT), Lactate dehydrogenase (LDH), Gamma-glutamyl-transferase (Gamma-GT), coagulation (TPT, aPTT).

URINALYSIS: Yes
- Time schedule for collection of urine:
At the end of the treatment period (on test days 90 to 91) for all animals
At the end of the recovery period (on test days 118 to 119) for all recovery animals
- Metabolism cages used for collection of urine: Yes. The urine was collected for 16 hours in an URIMAX funnel cage. The collection of urine was terminated immediately prior to starting the blood withdrawals for the haematological and clinical chemistry examinations.
- Animals fasted: Yes, fasted overnight
- Parameters examined: Volume, Osmolality, pH, Specific gravity.
Protein, Glucose, Bilirubin, Urobilinogen, Ketones, Haemoglobin (approx. Values), Nitrite.
Microscopic examinations of urine samples were carried out by centrifuging samples and spreading the resulting deposit on a microscopic slide. The deposits were examined for the presence of the following parameters: Epithelial cells, Leucocytes, Erythrocytes, Organisms, Further constituents (i.e. sperm, casts), Crystalluria.
The colour and turbidity of the urine were examined visually.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
Neurological screening: In test weeks 13 and 17 (before any blood sampling for laboratory examinations), screening of sensory reactivity to stimuli of different types (e.g. auditory, visual and proprioceptive stimuli) (based on GAD), as well as the assessment of grip strength (MEYER) and motor activity assessment were conducted in all animals approximately 2 hours after dosing outside the home cage.
- Dose groups that were examined: all animals
- Battery of functions tested:

Observational screening
*Righting reflex: The animal was grasped by its tail and flipped in the air (approx. 60 cm) above the cart surface so that it turned head over heels. The normal animal should land squarely on its feet, in this case zero (0) points were scored. If it landed on its side, 1 point was scored; if it landed on its back, 2 points were scored. This test was repeated five times and the total scores were recorded.
*Body temperature: An electronic probe thermometer (with a blunt probe) was used to take a rectal temperature, being allowed to equilibrate for 30 seconds before the reading was recorded.
*Salivation: The animals were observed for discharge of clear fluid from mouth, most frequently seen as beads of moisture on lips in rats. Normal state is to see none, in which case a zero (0) was recorded. If present, a plus sign (+) was recorded.
*Startle response: With the animal on the cart, the metal cage was struck with the blunt probe. The normal animal should exhibit a marked but short-lasting response, in which case a zero (0) was recorded. If there was no response, a plus sign (+) was recorded.
*Respiration: While at rest on the cart, the animal's respiration cycle was observed and evaluated in terms of a scale from 1 (reduced) to 5 (increased), with 3 being normal.
*Mouth breathing: Rats are normally obligatory nose-breathers. Each animal was observed, whether or not it was breathing through its mouth. If the rat was breathing through its nose, a zero (0) was recorded. Mouth breathing was documented by a plus sign (+).
*Urination: When an animal was removed from its cage, the pan beneath the animal's cage was examined while returning the animal to its cage. The signs of urination were evaluated on a scale of 0 (lacking) to 5 (polyurea) with 3 being normal.
*Convulsions: If clonic or tonic convulsions were observed, their intensity was graded on a scale of 1 (minor) to 5 (marked) and the type was recorded. In the normal animal no, convulsions should be observed, in which case a score of zero (0) was recorded.
*Pilo-erection: The fur of the animal's back was observed, whether it was raised or elevated. In the normal animal no pilo-erection should be observed and a score of zero (0) was recorded. If pilo-erection was present, a plus sign (+) was recorded.
*Diarrhoea: In examining the pan beneath an animal's cage, it was noted if there were any signs of loose or liquid stools. Normal state is for there to be none (0), in case of diarrhoea the intensity was recorded on a scale of 1 (slight) to 5 (much increased).
*Pupil size: It was determined if the pupils were constricted or dilated and the observations were evaluated in terms of a scale from 1 (constricted) to 5 (dilated), with 3 being normal.
*Pupil response: The beam of light from the pen light was played across the eyes of the animal and the changes in pupil size were noted. In the normal animal, the pupil is constricted when the beam is on it and then dilates back to normal when the light is removed, whereby a score of zero (0) was recorded. If there was no pupil response, a minus sign was recorded.
*Lacrimation: The animal was observed for the secretion and discharge of tears. In rats the tears contain a reddish pigment. No discharge is normal, whereby a score of zero (0) was recorded. If a discharge was present, a plus sign was recorded.
*Impaired gait: The occurrence of abnormal gait was evaluated. The most frequent impairments are waddling (W), hunched gait (H), or ataxia (A, the inability of all the muscles to act in unison). The extent of any impairment was recorded on a scale of 1 (slight) to 5 (marked). A normal gait was documented by a score of zero (0).
*Stereotypy
Each animal was evaluated for stereotypic behaviour (isolated motor acts or partial sequences of more complex behavioural patterns, occurring out of context and with an abnormal high frequency). These were graded on a scale of 0 (absent = normal) to 5 (marked).
*Toe pinch: The blunt probe was used to bring pressure to bear on one of the digits of the hind limb. This should evoke a response from the normal animal. The response or lack thereof was graded on a scale from 1 (absent) to 5 (exaggerated) with 3 being the normal response.
*Tail pinch: The toe pinch procedure was utilized with the animal's tail instead of its hind limb and was graded on the same scale of 1 to 5 with 3 being the normal response.
*Wire maneuver
The animal was placed on the metal rod suspended parallel to the cart approximately 60 cm above the cart's surface. The animal's ability to move along the rod was evaluated. If impaired, a score of from 1 (slightly impaired) to 5 (unable to stay on wire) was recorded. Normal movement was documented by a score of zero (0).
*Hind leg splay: Using an ink pad, the hind paws were marked with ink. The rat was then held 30 cm above a sheet of blotting paper on the cart. The animal was dropped and the distance between the prints of the two hind paws was measured.
*Positional passivity: The animal was observed after being placed in an awkward position, such as on the edge of the top of the wire-bottomed cage on the cart surface. If the animal immediately moved into a normal position, a score of zero (0) was recorded. If not, a score was recorded on a scale of 1 (slightly impaired) to 5 (cataleptic).
*Tremors: Periods of continued fine movements, usually starting in the limbs (and perhaps limited to them). The normal case is to have none, in which case a score of zero (0) was recorded. If tremors were present, they were graded on a scale of 1 (slight and infrequent) to 5 (continuous and marked).
*Positive geotropism: The animal was placed on the inclined (at an angle of approx. 30°) top surface of the wire cage with its head facing downward. It should turn 180° and face 'uphill', in which case a score of zero (0) was recorded. If this did not occur, a negative sign was recorded.
*Limb rotation: One of the animal's hind limbs was taken and moved through its normal plane of rotation. In the normal state, it should rotate readily but there should be some resistance. The variations from normal were from no resistance (1) to markedly increased resistance or rigidity (5), with 3 being normal.
*Auditory function: Each animal was placed in a container and observed for Preyer's reflex (twitching of the pinna) in response to a high frequency sound stimulus. The stimulus was repeated, if necessary, up to 3 times. A normal response was recorded with a plus sign (+). If there was no response a zero (0) was recorded.

Functional tests
*Grip strength: Prior to testing, the gauge (Chatillon, Model DPP - 1.0 kg) was calibrated with a set of known weights and the apparatus was adjusted for the size of the animal (about 1 cm clearance on both sides of the animal). After the strain gauge had been zeroed and set in the record mode, the animal was placed into the trough with the forepaws inside the triangular grasping ring. Using one hand, the animal was grasped about 2.5 cm of the way up toward the base of the tail and steadily pulled (approx. 2.5 cm/sec) away from the ring until the grip was broken. The animal continued to be pulled along the trough until the hind limbs grasp the T-bar.
The trial was completed when grip of the hind limbs was broken. Three successive readings were taken for each animal with an intertrial interval long enough to record the data and zero both meters for the next trial.
*Locomotor activity: The motility was measured using the TSE InfraMot system8. The infrared sensor was mounted on top of the home cage and any movements were measured for the duration of 12 minutes by sensing the body heat image, i.e. the infrared radiation, and its spatial displacement over time.
Any movements within the cage, even brief movement events of only a few milliseconds duration, were detected and included in the activity data.

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Starting on test day 91 the main study animals were dissected following a randomisation scheme. Main study animals not dissected on test day 91 were dosed on test day 91 and dissected on test day 92. Animals allocated to the recovery period were dissected on test day 119.
The animals were euthanized by carbon dioxide (CO2) inhalation, exsanguinated by cutting the aorta abdominalis, weighed, dissected and inspected macroscopically under the direction of a pathologist.
All superficial tissues were examined visually and by palpation and the cranial roof was removed to allow observation of the brain, pituitary gland and cranial nerves.
After ventral midline incision and skin reflection, all subcutaneous tissues were examined. The condition of the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart.
The abdominal viscera were examined before and after removal, the urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole and the stomach and caecum were incised and examined.
The lungs were removed and all pleural surfaces examined under suitable illumination. The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenal glands, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.

HISTOPATHOLOGY: Yes.
The following organs or parts of organs with the exception of the eyes and testes of all animals were fixed in 7% buffered formalin. The eyes were preserved in Davidson's solution and the testes in modified Davidson's solution for optimum fixation.
Adrenal gland (2), Aorta abdominalis, Bone (os femoris with joint), Bone marrow (os femoris), Brain (3 levels: cerebrum, cerebellum,medulla/pons), Caecum, Epididymis (2), Eye with optic nerve and Harderian gland (2), Gross lesions observed, Heart (left and right ventricle, septum), Intestine, large (colon, rectum), Intestine, small (duodenum, jejunum, ileum, including Peyer´s patches), Swiss roll Method, Kidney and ureter (2), Liver (2 lobes), Lungs (with mainstem bronchi and bronchioles (preserved by inflation with fixative and then immersion)), Lymph node (1, cervical), Lymph node (1, mesenteric), Mammary gland), Muscle (skeletal, leg), Nerve (sciatic), Ovary and oviducts (2), Pancreas, Pituitary, Prostate and seminal vesicles with coagulating glands, Salivary glands (mandibular, parotid, sublingual), Skin (left flank), Spinal cord (3 sections: cervical, midthoracic, lumbar), Spleen, Stomach, Testis (2), Thymus, Thyroid (2) (including parathyroids), Tissue masses or tumours, (including regional lymph nodes), Tongue (including base), Trachea (including larynx), Urinary bladder, Uterus (including cervix), Vagina
The afore-listed organs of all groups 1 and 4 main study and recovery animals were examined histologically after preparation of haematoxylin-eosin stained paraffin sections. In addition, frozen sections of the heart, liver and one kidney were made, stained with Oil Red O and examined as well.
Parathyroids cannot always be identified macroscopically. They were examined microscopically if in the plane of section and in all cases where they were noted as grossly enlarged.

Other examinations:

ORGAN WEIGHTS:
The weights of the following organs of all animals were determined before fixation:
Adrenal gland (2), Brain, Epididymis (2), Heart, Kidney (2), Liver, Ovary (2), Spleen, Testicle (2), Thymus, Uterus (including cervix), As a whole: Prostate and seminal vesicles with coagulating glands.
Paired organs were weighed individually and identified as left or right.
The relative organ weight [g/kg bw] was calculated as follows:

relative organ weight [g/kg bw] = absolute organ weight [g]
animal weight at dissection [kg]

Statistics:

Data for toxicology and pathology were captured, as far as possible; using the departmental computerized systems (Provantis® Integrated Preclinical Software, version 9.4.0, Instem LSS Ltd., Stone, Staffordshire ST15 0SD, United Kingdom).
Raw data not fully compatible with the computerized systems were maintained on paper according to appropriate SOPs.
The test item groups 2 to 4 were compared to the control group 1.
The following statistical methods were used:
Multiple t-test based on DUNNETT, C. W. New tables for multiple comparisons with a control. Biometrics, 482-491 (September 1964): Body weight / Food consumption /Haematology / Coagulation /Clinical chemistry / Urinalysis / Relative and absolute organ weights (p ≤ 0.05 and p ≤ 0.01)

Exact test of R. A. FISHER (if applicable): Histopathology (p ≤ 0.05)

The following settings were used for the statistical evaluation of the parametrical values captured by Provantis:
Homogeneity of variances and normality of distribution were tested using the BARTLETT’s and SHAPIRO-WILKS test. In case of heterogeneity and/or nonnormality of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), intergroup comparisons with the control group was made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).

The following statistical methods were used for the data not captured with the Provantis system:
STUDENT's t-test:
Numerical functional tests: Body temperature / Hind leg splay / Grip strength / Spontaneous motility (p ≤ 0.05 and p ≤ 0.01)
The following limits were used:
p = 0.05 / 0.01 ^ t = 2.0484 / 2.7633 (for 28 degrees of freedom)
p = 0.05 / 0.01 ^ t = 2.0687 / 2.8073 (for 23 degrees of freedom)
p = 0.05 / 0.01 ^ t = 2.3060 / 3.3554 (for 8 degrees of freedom)
These statistical procedures were used for all data.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

All parameters of the detailed clinical observations of all animals scheduled for the control or treatment groups were in the normal range at pre-dose examination on test day 1. All male and female control animals revealed normal values for each parameter set examined throughout the course of the study. None of the animals treated with nominal dose levels of 100, 300 or 1000 mg Docusate Sodium/kg bw/day via the diet for 90 days revealed any changes in external appearance, body posture, movement and coordination capabilities in test weeks 1 to 13.

Mortality:

no mortality observed

Description (incidence):

None of the male and female rats died or had to be sacrificed prematurely after repeated oral administration of nominal dose levels of 100, 300 or 1000 mg Docusate Sodium/kg bw/day via the diet during the 90-day treatment period or during the 28-day treatment-free recovery period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight of the male animals treated with a nominal dose level of 1000 mg Docusate Sodium/kg bw/day via the diet for 90 days was slightly reduced by 6% to 11% compared to the control group as of test day 36. The body weight gain from start (test day 1) to the end of test day 90 was 59% compared to 75% of the animals of the control group. Body weight at autopsy was slightly reduced by 12% compared to the control group. The female animals were not affected.
No test item-related influence was noted on the body weight, body weight gain and body weight at autopsy of the male and female animals previously treated with nominal dose levels of 300 or 1000 mg Docusate Sodium/kg bw/day via the diet for 90 days compared to the control group during the 28-day treatment-free recovery period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The relative food consumption of the male and female rats treated with a nominal dose level of 1000 mg Docusate Sodium/kg bw/day via the diet for 90 days was increased compared to the control group starting in test week 2. The increase versus control group reached 51% in males and 21% in females.
The relative food consumption of the male and female rats previously treated with a nominal dose level of 1000 mg Docusate Sodium/kg bw/day via the diet for 90 days dropped again to or slightly above the values consumed by the control group during the 28-day treatment-free recovery period, however, the male animals still revealed a slightly increased food consumption by 11% at the end of the recovery period compared to the control group.
The calculation of the test item intake via the diet revealed a dose-dependent exposure of the male and female animals to the test item. The mean test item intake per week ranged from 84 to 117 and 78 to 117 mg/kg bw/day for the male and female animals treated with a nominal dose level of 100 mg Docusate Sodium/kg bw/day via the diet, respectively, from 250 to 351 and 237 to 350 mg/kg bw/day for the male and female animals treated with a nominal dose level of 300 mg Docusate Sodium/kg bw/day via the diet, respectively, and from 854 to 1239 and 811 to 1369 mg/kg bw/day for the male and female animals treated with a nominal dose level of 1000 mg Docusate Sodium/kg bw/day via the diet, respectively.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

The visual appraisal of the drinking water consumption did not reveal any test item-related influence in any of the dose groups.

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related changes were noted. The changes noted in the left eye of 1 of 10 male animals treated with a nominal dose level of 100 mg Docusate Sodium/kg bw/day via the diet (slight congestion of iris vessels, slight myosis and no pupil reflex) in test week 1 are considered to be incidental findings due to the low number of animals affected. No changes of the eyes were noted for this animal in test week 13.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related changes were noted. Statistically significant differences in haematological parameters compared to the control which are not considered to be test item-related were: decreased reticulocytes (high dose males), decreased platelets (mid dose males), increased thromboplastin time (high dose females), decreased thromboplastin time (high dose males), decreased activated partial thromboplastin time (high dose males). The slight alteration in comparison to control animals is without any biological relevance for all these parameters.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related changes were noted. Statistically significant differences in clinical chemistry parameters compared to
the control which are not considered to be test item-related were: decreased albumin (high dose females; test day 119), decreased bilirubin (mid and high dose males and females; test day 91); decreased protein (high dose females; test day 119), increased ALAT (mid and high dose males and increased Gamma-GT (high dose females; test day 119). The slight alteration in comparison to control animals is without any biological relevance for all these above parameters. The increased bile acids (mid and low dose females) were due to the relative low or high value observed for the control group.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related changes were noted. The statistical significance of the differences in urinary parameters compared to
the control which are not considered to be test item-related were: increased specific gravity ( mid dose males; test day 91) and decreased pH (mid dose males; test day 91). Both parameters were lacking dose dependence and the slight alteration in comparison to control animals is without any biological relevance. For the increased urine volume in the high dose females on test day 119, the slight alteration in comparison to control animals is without any biological relevance.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

The neurological screening performed at the end of the treatment period in test week 13 did not reveal any test item-related influence in the male and female rats treated with nominal dose levels of 100, 300 or 1000 mg Docusate Sodium/kg bw/day via the diet, neither on any of the parameters examined during the functional observation tests nor on the fore- and hind limb grip strength or on the spontaneous motility. Furthermore, no test item-related influence was noted in the rats previously treated with 1000 mg Docusate Sodium/kg bw/day via the diet at the end of the recovery period in test week 17.
Statistically significant differences in neurological parameters compared to the control which are not considered to be test item-related are: increased body temperature (high dose males), increased forelimb grip strength (mid dose and high dose females), increased hind limb grip strength (mid dose and high dose females). The slight alteration in comparison to control animals is without any biological relevance and/or lacking dose dependence.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Slightly increased relative and/or absolute liver weights were noted for the male and female animals treated with a nominal dose level of 1000 mg Docusate Sodium/kg bw/day via the diet for 90 days compared to the control group on test day 91/92 (up to 18% increase in relative liver weiight n males and females) . The organ weight changes are considered to be test item-related.
Treatment period: Statistically significant differences in relative and absolute organ weights compared to the control which are not considered to be test item-related were: increased relative brain weight high dose males; test day 91/92), decreased absolute heart weight (high dose males; test day 91/92), decreased absolute right ovary weight ( high dose females; test day 91/92) and decreased absolute spleen weight (high dose males; test day 91/92). For all the above parameters the slight alteration in comparison to control animals is without any biological relevance. The increased absolute thymus weight (mid dose femlaes; test day 91/92) was lacking dose dependence.
Recovery period: The liver weights of the male and female animals previously treated with a nominal dose level of 1000 mg Docusate Sodium/kg bw/day via the diet were not test item-relatedly increased compared to the control group at the end of the 28-day treatment-free recovery period. Statistically significant differences in relative and absolute organ weights compared to the control which are not considered to be test item-related (lacking dose dependence) were: increased relative right adrenal gland weight, increased absolute right adrenal gland weight and increased absolute brain weight in the mid dose females on test day 119.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related changes were noted.
Treatment period: Macroscopic changes were noted in the duodenum (dilated), kidneys (cysts) and thymus (reddened / red foci) in individual animals of the test item-treated groups. These changes are considered to be incidental findings due to the low number of animals affected.
Recovery period: Macroscopic changes were noted in the kidneys (cyst), lungs (partly reddened), ovary (cyst filled with clear liquid) and thymus (reddened) in individual animals of the previously test item-treated groups. These changes are considered to be incidental findings due to the low number of animals affected.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

The histological examination of rat organs did not reveal any morphological changes in the animals treated with a nominal dose level of 1000 mg Docusate
Sodium/kg bw/day via the diet for 90 days which are considered to be related to the administration of the test item. No test item-related changes were noted in the rat organs at the end of the 28-day treatment-free recovery period.
A few minor microscopic changes were recorded for the organs and tissues examined in this study. However, the type, incidence and severity of all
microscopic findings observed did not indicate any relationship to the treatment with the test item. All changes noted are regarded as spontaneous and to be within the normal background pathology commonly seen in rats of this strain and age.

Histopathological findings: neoplastic:

no effects observed

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

act. ingr.

Remarks:

Docusate Sodium

Sex:

male/female

Basis for effect level:

other: No haematological, biochemical or histopathological changes or any other noteworthy changes were observed

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The experimental no-observed-adverse-effect level (NOAEL) was above 1000 mg Docusate Sodium/kg bw/day (nominal dose), by daily oral administration via the diet, in particular, as no haematological, biochemical or histopathological changes or any other noteworthy changes were observed.

Executive summary:

The aim of this repeated dose toxicity study was to obtain information on the toxicity of Docusate Sodium administered daily via the diet to rats for 90 consecutive days and to assess the reversibility of any effects at the end of a 28-day recovery period.

The test item supplied was mixed to the diet by LPT. Rats were treated with Docusate Sodium at nominal dose levels of 100, 300 and 1000 mg/kg bw/day. The control animals received standard diet only. Mean actual dose levels of 105, 313 and 1066 mg Docusate Sodium/kg bw/day for the low, intermediate and high dosed males and 107, 319 and 1069 mg Docusate Sodium/kg bw/day for the low, intermediate and high dosed females were achieved during the 90-day treatment period. None of the animals died or had to be sacrificed prematurely.

The body weight of the male animals treated with a nominal dose level of 1000 mg Docusate Sodium/kg bw/day via the diet for 90 days was slightly reduced compared to the control group. The female animals were not affected. The relative food consumption of the male and female rats treated with a nominal dose level of 1000 mg Docusate Sodium/kg bw/day via the diet for 90 days was increased compared to the control group starting in test week 2. The relative and/or absolute liver weights of the male and female animals treated with a nominal dose level of 1000 mg Docusate Sodium/kg bw/day via the diet were slightly increased compared to the control group at the end of the 90-day treatment period.

No test item-related changes were observed for the behaviour or external appearance of the animals, the detailed clinical observations, the neurological screening, the drinking water consumption, for any of the haematological, clinical chemical and urinary parameters, the eyes or optic region, the auditory acuity, and at macroscopic inspection at necropsy at any dose level. The histopathological examination of the animals treated with a nominal dose level of 1000 mg Docusate Sodium/kg bw/day via the diet did not reveal any test item-related morphological changes, neither at the end of the 90-day treatment period nor at the end of the 28-day treatment-free recovery period.

At the end of the 28-day treatment-free recovery period, the values for relative food consumption of the previously high dosed animals were in the range or slightly above the values consumed by the control group, the body weight of the previously high dosed male animals had completely recovered to the values of the control group, and the liver weights of the previously high dosed male and female animals were not test item-relatedly increased compared to the control group.

The experimental NOAEL was above 1000 mg Docusate Sodium/kg bw/day (nominal dose), by daily oral administration via the diet, in particular, as no haematological, biochemical or histopathological changes or any other noteworthy changes were observed.