Sodium 1,4-diisodecyl sulphonatosuccinate

Endpoint summary

Description of key information

Subacute toxicity was tested with the registered substance in Wistar rats by oral gavage at 0 (propylene glycol), 100, 300 and 1000 mg /kg bw/day in a supporting 14-day dose range finding and at 0 (propylene glycol), 100, 300 and 600 mg /kg bw/day in a key combined repeated dose/reproductive toxicity study (OECD No. 422). The NOAEL for local toxicity of the parental generation was <100 mg/kg bw/day (based on local gastric histological effects in Low, Mid and High dose animals). The NOAEL for systemic toxicity of the parental generation was considered to be 300 mg/kg bw/day (based on body weight, body weight gain and food consumption changes at 600 mg/kg bw/day, although these effects were probably secondary to local gastric irritation). The irritating properties of the substance was affecting the forestomach of the rats, which is not present in human, therefore the effect is not relevant for humans and the NOAEL was considered too conservative.

Subchronic toxicity testing with registered substance is ongoing and will be updated later when results are available (currently waived in the dossier).  Repeated dose toxicity was further tested with read-across substances in various species, including rats, dogs, rabbits and monkeys. The oral NOAEL of 1000 mg/kg bw/day was obtained in the key dietary subchronic toxicity study in rats with read-across substance Docusate sodium (CAS 577 -11 -7); other data from other species were of limited reliability and relevance and therefore not taken into account. The NOAEL of 1000 mg/kg bw/day  is considered more appropriate for DNEL derivation awaiting data from a dietary  subchronic toxicity study with registered substance which is ongoing.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

See attached read-across justification

Reason / purpose for cross-reference:

read-across source

Clinical signs:

no effects observed

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

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

Critical effects observed:

not specified

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 read-across test item 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.

Reference 2

Endpoint:

repeated dose toxicity: oral, other

Remarks:

Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 December 2020 to 07 March 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Paris, 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI

Details on species / strain selection:

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

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, (Address: Sandhofer Weg 7, D-97633, Sulzfeld, Germany) from SPF colony
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: Young adult rats, 9-10/10 weeks old (females/males) at start of the experiment and 11-12/12 weeks old (females/males) at mating.
- Weight at study initiation: Males: 354-436 g, females: 206-275 g (at the start of the treatment). The body weights did not exceed ± 20% of the mean weight for each sex at start of treatment.
- Housing: Rodents were group-housed, up to 2 animals of the same sex and dose group/cage, with the exception of the mating and gestation, delivery, lactation period, when they were paired or individually housed (with pups), respectively. Animals were housed in Type II, III and/or IV polycarbonate cages. SAFE 3/4-S Hygienic Animal Bedding (Batch number: 03027201022 / 03027201024, Expiry date: 22 October 2023 / 24 October 2023) and SAFE Crinklets Natural nesting material (Batch number: 05072200405 / 05072200824, Expiry date: 05 April 2023 / 24 August 2023) produced by J. Rettenmaier & Söhne GmbH+Co.KG (Address: Holzmühle 1, D-73494 Rosenberg, Germany) were used in the study. Group housing allowed social interaction. Deep wood sawdust bedding allowed digging and other normal rodent activities, while nesting material allowed normal nesting behaviour. Certified cardboard hiding tunnels (GLP Mini Fun Tunnels, Batch number: A/123/B) produced by LBS (Serving Biotechnology) Ltd. (Address: Unit 20, Gatwick Business Park, Kennel Lane, Hookwood, Surrey, RH6 0AH UK) were also provided to the animals.
- Diet (e.g. ad libitum): The animals received ssniff® SM R/M “Autoclavable complete diet for rats and mice –breeding and maintenance” (Batch number: 713 70882, Expiry date: 28 April 2021) produced
by ssniff Spezialdiäten GmbH (Address: Ferdinand-Gabriel Weg 16, D-59494 Soest, Germany), ad libitum.
- Water (e.g. ad libitum): The animals received tap water from the municipal supply, as for human consumption from a 400- or 500-mL bottle, ad libitum.
- Acclimation period: Environmental acclimation period for the study was 20 days.

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

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1-24.9℃ (target range: 19-25°C)
- Humidity (%):31-69% (target range: 30-70%)
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES:
From: Start of in-life phase: 16 December 2020 (first vaginal smear sampling)
To: End of in-life phase: 26 February 2021 (last necropsy)

Route of administration:

oral: gavage

Details on route of administration:

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:

propylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the selected vehicle (propylene glycol), as a visibly stable homogenous solution at the appropriate concentrations according to the dose level and volume selected in the Pharmacy of the Test Facility. The formulations were stirred with a magnetic stirrer from the preparation until completion of each treatment.
Formulations were prepared a maximum of 4 days prior to administration to animals according to stability assessment results of the analytical method validation study (Study code: 20/029-901AN). Based on those results, the test item formulation in the 2 and 200 mg/mL concentration range were stable for at least 9 days when stored at room temperature.
The calculated amount test item was weighed into a clean, calibrated glass container and then mixed properly (with ultrasonication and magnetic stirring) with the needed amount of vehicle to reach homogeneity by visual observation. During the formulation sonication was applied to aid dissolution.
After filling the syringe with the calculated amount to be given to each individual rat, the outside of the gavage tube was cleaned with a wetted tissue first (using the vehicle of the study) and then with a dry tissue, to reduce any potential surface contamination to an absolute minimum. A constant volume of 5 mL/kg bw was administered to all animals. The actual volume to be administered was calculated and adjusted based on each animal’s most recent body weight.

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

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Formulations were considered to be adequately stable under the study conditions.
Overall, the formulations were considered adequate for the study.

Duration of treatment / exposure:

Dosing of both sexes began after the acclimatisation (20 days) and pre-exposure period (14 days), and it was performed 2 weeks before mating, during the mating, and was continued up to and including the day before the necropsy.
The first day of dosing of each animal was regarded as Day 0.
Males were dosed for 32 days (14 days pre-mating and 18 days mating/post-mating period), then were euthanized and subjected to necropsy examination.
Females were dosed for 14 days pre-mating, for up to 18 days mating period, through gestation and up to and including the day before necropsy (13 days post-partum dosing).

Frequency of treatment:

daily on a 7 days/week basis

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

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

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected by the Sponsor in consultation with the Study Director based on the results of a Dose Range Finding (DRF) study (Study code: 20/029-220PE), with the aim of inducing toxic effects but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose.
In the DRF study (after a 14-day treatment period), decreased activity, ataxia and hunched back, piloerection, splayed gait and soft faeces were recorded with very significant body weight loss and low food consumption, without clearly adverse effects on clinical pathology were seen in the High dose groups. Macroscopic findings were observed in the glandular and nonglandular stomach at all dose levels in males and High dose females indicating a dose related local gastric irritation. The changes seen at 1000 mg/kg bw/day dose level were not life threatening but the body weight effect was greater than is acceptable for a longer term study, but no significant body weight effects were seen at 300 mg/kg/day. The High dose level of the main study should be >300 and <1000 mg/kg/day. The dose of 600 mg/kg/day is selected for the High dose. Lower doses were spaced with a factor of approximately 3.
- Rationale for animal assignment (if not random): All adult/parental (P) male and female animals were sorted according to body weight by computer and divided into weight ranges. There were an equal number of animals from each weight group randomly assigned to each dose group to ensure that animals of all test groups were as nearly as practicable of a uniform weight.
This process was controlled by the software PROVANTIS v.9, to verify the homogeneity/variability between/within the groups. Males and females were randomized separately to the dose groups on the day of the first treatment (prior to the start of the treatment).
- Fasting period before blood sampling for clinical biochemistry: overnight period of food deprivation in case of females this happened after the litter had been necropsied)

Observations and examinations performed and frequency:

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

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

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

FOOD CONSUMPTION AND COMPOUND INTAKE (no feeding study):
-Animal food consumption was determined by weighing the non-consumed diet with a precision of 1 g at least weekly and on Day 0. Food consumption was measured during mating. Food consumption was measured more frequently during the lactation period (on Gestation Day (GD) 0, 3, 7, 10, 14, 17 and 20 and on PPD0, 4, 7, 10 and 13 during the lactation period).
Main daily food consumption was calculated for each interval.

FOOD EFFICIENCY: Not specified

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- 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) Volume
MCH Mean Corpuscular (erythrocyte) Haemoglobin
MCHC Mean Corpuscular (erythrocyte) Haemoglobin Concentration
RDW Red Cell (erythrocyte) 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

PLASMA/SERUM HORMONES/LIPIDS: Yes
- Time of blood sample collection: 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 two pups per litter on PPD 14 (females) / PND13 (pups),
• 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 is documented in the raw data.
Blood samples were kept on ice from sampling until centrifugation (within approximately 30 minutes of collection), then centrifuged rapidly (1600 g / approx. 3000 rpm, 10 minutes, 4°C). The resulting plasma was divided in at least two aliquots (volume target was at least 150 μL for the first aliquot and at least 75 μL for the second aliquot, any remaining sample (if any) was kept as a third aliquot) and stored in an ultra-freezer (-80±10°C) until analysis.

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:
The evaluation of the urine samples was performed by using of Medi-Test URYXXON® Stick 10 Urinalysis-strips:
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 27 am, females on PPD13 am). Selected animals were subjected to the functional observation battery, including Irwin test and measurements of the landing foot splay and fore/hind grip strength.
In order to avoid hypothermia of pups, dams were removed from the pups for not more than approximately 30-40 minutes during FOB or 90 minutes during locomotor activity measurement (SMART).
- 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

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Gross necropsy was performed on each adult animal. Terminally (one day after the last treatment), animals were sacrificed under anaesthesia by exsanguination; anaesthetic product was diluted for pups’ euthanasia as required.
After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities was opened, and the appearance of the tissues and organs were observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate. Special attention was paid to the organs of the reproductive system.
Vaginal smears were prepared and examined for each female on the day of necropsy to determine the stage of oestrus cycle and allow correlation with histopathology of the reproductive organs.
The number of implantation sites and of corpora lutea was recorded in the females as applicable.
Dead pups and pups killed on PND4 and/or PND13 were carefully examined externally for
gross abnormalities. After the external observation, the sex determined at birth was confirmed by observation of the internal reproductive organs. Presence of nipples/areolae in the PND13 male pups was also recorded.

HISTOPATHOLOGY: Yes
In case microscopic examination was needed for a tissue or organ, the retained tissues and organs required for histopathology (below) was embedded in paraffin wax; sections were cut at 4-6 μm by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope.

For the adult animals, detailed histological examination was performed as follows:
• on the selected list of retained tissues and organs (as above) in the Control and High dose groups (selected 5 animals/sex/group, for Control females 6 animals were selected),
• all macroscopic findings (abnormalities), except of minor order from all animals,
• on the retained reproductive organs (testes, epididymides, prostate gland, seminal vesicles with coagulation gland for males and uterus, cervix, ovary, oviduct and vagina for females) of all animals of the Control and High dose groups.
Additional histology on the stomach (glandular and non-glandular), the liver of the Control, Low, Mid and High dose animals (males and females) and on the kidney of the Low and Mid dose males were performed as agreed by the Study Pathologist, the Study Director and the Sponsor.
Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.
Special attention was paid to the organ weight, appearance and histopathology of immunesystem tissues for any evidence of immunotoxicity (spleen, thymus, lymph nodes, bone marrow, and blood smears if examined).
Special attention was paid to the central and peripheral nervous system tissues for any evidence of neurotoxicity.
No histopathological examination was performed on pups (F1 generation).

Statistics:

see under “any information on materials and methods incl. tables”

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

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

Mortality:

no mortality observed

Description (incidence):

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

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

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

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

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

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

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

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

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

Endocrine findings:

no effects observed

Description (incidence and severity):

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

Urinalysis findings:

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.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

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

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

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

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

TERMINAL EUTHANASIA / Parental Generation: macroscopic findings: Test item-related changes were observed in the stomach, as multifocal, white or brown discoloration and/or multifocal/diffuse thickness of the non-glandular mucosa in High and Mid dose animals. The dark red discoloration of the mandibular lymph nodes was considered as procedure related (due to the sublingual blood collection), the multifocal white discoloration of the gastric glandular mucosa was without histological relevance and was considered as incidental.
All other changes were considered incidental or a common background.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

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

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Pre-exposure period
Each female selected for the study showed acceptable cycles (mean cycle length of 4.03-4.05 days was observed in the different groups) before starting the treatment period.
Exposure period (pre-mating and mating periods)
No indication of test item related effect was seen in the oestrus cycle data, collected during the pre-mating and mating periods (mean cycle length was 4.01, 4.08, 4.08 and 4.18 days in the Control, Low dose, Mid dose and High dose groups, respectively).
Prolonged oestrus was recorded in some cases for all dosed groups, a total of 2 Low females,
2 Mid dose females and 1 High dose females were affected (#2508, #2510, #3501, #3509 and
#4510), but as it did not affect mating or pregnancy, this fact was considered as being an
occasional finding, not being a test item related effect.
Prolonged dioestrus was noted for one Low dose female (#2507) and one High dose female
(#4508), but this frequency (1/12) was in line with the normal, expected range and did not
affect the mating or pregnancy.

Details on results:

-Mortality and morbility:
No test item related mortality was observed in the study.
Two deaths of females on days 5 and 6 were considered as accidental and not related to systemic toxicity of the test item, animals were replaced; data for only the replacement animals is given in the report results section.
-Clinical observations:
No test item related systemic clinical signs were observed in the study.
Tonic convulsions were recorded during the lactation period (Day 45, 49 and 51) for a Control female (#1507), on Day 15, 24, 31, 37, 42, 45, 48 and 51 for a Low dose female (#2503), on Day 49 and 51 for one Mid dose female (#3510) and on Day 43 and 50 for one High dose female (#4506) on Day 43 and Day 50.
Red liquid in the oral cavity was observed for one Low dose male (#2002) on Day 14.
Noisy respiration was observed for one Mid dose male (#3008) from Day 16 to 21 and one Mid dose female (#3504) on Day 2 and one High dose female (#4503) on Day 15 and Day 16. These sporadic cases of noisy respiration in a few animals ascribed to reflux or minor exposure to test item in the region of the upper respiratory tract.
Hunched back was observed in one High dose female (#4512) from Day 2 until Day 16.
Piloerection was observed in one High dose female (#4512) from Day 2 until the end of the treatment (there was no symptom between Day 12-34).
Red discharge around nose was observed in one High dose female (#4512) on Day 3.
Alopecia for both fore paws and fur thin on the chest was observed for one Low dose female (#2501) form Day 27 until death. Alopecia for both fore paws was observed for one Mid dose female (#3501) from Day 41 until death.
Broken/missing incisor was observed for one High dose male (#4003) from Day 12 to 18.
Increased respiratory rate was observed in one Control female animal (#1511) on Day 54.
Malocclusion was observed for one Control female animal (#1504) from Day 8 until the end of the treatment.
Scar around the nose / snout was observed for one High dose male (#4012) from Day 14 to 25 and scar on the head (1-2 cm) for one Control female (#1511) was observed on Day 54.
These findings were all considered as being incidental or minor reflux type events, not related to toxicity of the test item administration.
-Body weight and weight changes:
No effect on body weight was observed in the Low, Mid (male and female) and High dose male
groups or body weight gain in the Low and Mid (male and female) dose groups.
Test item related adverse effect was observed on body weight gain in High dose (600 mg/kg bw/day) males. Statistically significantly reduced body weight gain (p< 0.05) was observed in High dose males in the first week of treatment only when compared to control animals. From the end of the first week, the growth rate of High dose males was similar to the other groups (with small statistical differences above and below the control growth rate). At the end of the treatment period the High dose body weight was not significantly different to control (only 1.9% below).
No effects were observed in females during the premating period. Statistically significantly lower than control body weight (by -8.6%, p<0.01) and/or body weight gain (by -20.4%, p<0.01) was observed in the High dose female group during gestation but a higher weight gain was seen during lactation in this group. A similar but lesser difference (-14%), with some statistical differences (p<0.01), was observed in Mid dose females. However, the values of the High and Mid dose groups were within the historical control range, hence the High and Mid dose female body weights were not considered to have been adversely affected by the test item.
-Food consumption and compound intake (no feeding study):
A test item related reduction of food consumption was observed in High dose males (600 mg/kg bw/day) during the first 7-14 days only (p<0.01) (reflecting the body weight gain data); Mid and Low dose males were not affected. For High dose females, a similarly reduced food intake was seen in the first 7 days of treatment (p<0.01).
Test item related lower food consumption was observed in High dose females (600 mg/kg bw/day) during the gestation and lactation phases with statistically significant differences (p<0.01), in the Mid dose females a similar trend was observed, with occasional statistical differences. The percentage differences reflected the body weight gain differences reported above, but although the body weight data of the High and Mid groups were in line with the historic control data, the food intake mean values were below the expected range, suggesting a moderate treatment effect.
The food intake effects were considered to be secondary to local gastric irritation caused by the test item and associated with the lower body weight gains.
-neurological assessment:
There were no changes in animal behaviour, general physical condition or in the reactions to different type of stimuli in the control or test groups.
There was no effect of treatment noted in the Irwin test or during the assessment of grip strength and landing foot splay.
All dose groups of males and females had a normal locomotor activity. In all cases, the initial activity was high, with reduced activity in each 5-minute period to an approximate plateau by about 20-30 minutes. There was no statistical significance between the test item treated animals (males and females) and the Control when evaluating the overall total travelled distance (0-60 minutes). The test item did not increase or decrease the normal locomotor activity, all treated groups had a profile of activity the same as historical control data.
-Haematology:
Five male and five female animals from each main group (6 females in control group for urine analysis) were selected for clinical pathology sampling and analysis at termination (on Day 33 in males, on PPD 14 in females).
No test item-related adverse changes were detected in the test item treated animals (males and females) in any dosed groups when comparing haematology parameters to the relevant Control data.
Statistically significantly decreased red cell distribution width % was observed in the High dose male (p<0.05) and female (p<0.01) group. Although the RDW parameter was statistically significant higher in High dose male animals and lower in female animals (when compared to Control group) it was considered an incidental finding, as any kind of discrepancies were not observed in values of red blood cells (RBC), haemoglobin and mean cell volume (MCV), neither in High dose males nor females.
-Clinical chemistry:
Five male and five female animals from each main group (6 females in control group for urine analysis) were selected for clinical pathology sampling and analysis at termination (on Day 33 in males, on PPD 14 in females).
There were no significant changes or biologically relevant effects on the serum chemistry that could be ascribed to the test item administration in the Low and Mid dose groups for both sexes.
Glucose was statistically significantly decreased (p<0.05 and p<0.01) in the Mid and High dose male groups. Potassium was statistically significantly increased (p<0.05) in the High dose male group. Alkaline phosphatase activity was statistically significantly increased in the High dose female group. All the above data were inside of the historical control data and in the absence of any associated changes, they are not considered as adverse test item related effects.
Alanine aminotransferase (ALT/GPT) activity was significantly increased in the High dose males (p<0.01) when compared to control animals. The observed value was outside of the historical control range, and a similar but not statistically significant difference was seen in females. However, based on the order of magnitude and the lack of any adverse hepatic histopathological changes, this fact was considered as being a test item related slight effect and not considered as adverse.
-Urinalysis:
Five male and five female animals from each main group (6 females in control group for urine analysis) were selected for clinical pathology sampling and analysis at termination (on Day 33 in males, on PPD 14 in females).
No test item-related changes were observed in the urinalysis parameters in male and female animals of any dose groups when compared to control.
Statistically significantly increased urine volume was observed in the Mid and High dose female group (p<0.05). The cause of the high volume of the urine is that the animals very often play with the small ball in the water bottle, and the water and the urine will be collected together. The data were outside of the historical control range but because of the abovementioned cause it is not considered as test item related effect.
Statistically significantly decreased specific gravity was observed in the Low (p<0.05) and High dose (p<0.01) female group and decreased protein level was observed in the High dose (p<0.01) female group. The data were inside of the historical control range.
-Organ weights:
Parental Males
Test item-related changes were observed in the organ weights of the liver of Mid and High dose male animals compared to controls.
The relative to body weight of the liver was increased statistically significantly by 16.8%, in the Mid Dose group and by 17.8% in the High Dose group, the relative to brain weight increased by 18.8% in the High Dose group compared to Controls, correlated with microscopic findings.
Statistically significantly decreased absolute (p<0.05) and body related (p<0.01) weight of the heart was measured in the High dose male group only, not in females. Both values together with the brain related weight (not statistically significant) were outside of the historical control range hence it is considered as test item related effect. However, in the absence of any histopathological changes, the weight difference is not considered to be adverse.
Statistically significantly decreased absolute, body and brain related spleen weights were measured in the High dose group (p<0.05), but all the data were within the historical control range and no histological changes were seen, therefore it is not considered as test item related effect.

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

Key result

Dose descriptor:

NOAEL

Remarks:

local toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: these effects were probably secondary to local gastric irritation

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Validation of Analytical Method for the Determination of Sodium 1,4-diisodecyl sulphonatosuccinate (CAS 29857-13-4, EC 249-894-6) from Gavage Formulation:

The purpose was to validate the implemented analytical method under the study code: FPBSTUDY-222-IMP1 (CRL-VES study code 20/029-901AN) for the quantitative determination of Sodium 1,4-diisodecyl sulphonatosuccinate.

The procedure was found to be suitable for the analysis of the test item from gavage formulations.

The validation parameters met the requirements.

Summary of the validated method parameters is presented in the table below.

 

Table.      Results of the method validation

Validated parameter	Acceptance criteria	Measured values
Specificity	Interference in blank samples must be less than 20.0% of the lowest concentration of the calibration curve.	Interfering components conform to less than 20% of the 10 ng/mL (LOQ) criteria
Repeatability (7 injections)	Areas and retention times of reinjected sample should be RSD < 5.0%	2.14% RSD for Retention time of test item 4.98% RSD for Area of test item
Detector response in 10 – 200 ng/mL concentration range	Correlation coefficient is above of 0.99	R^2 ≥ 0.9933
	Re-calculated concentration values above LOQ level are in 85.0 – 115.0% of the nominal concentrations	85.4 – 107.2
	Re-calculated concentration values at LOQ level are in 80.0 – 120.0% of the nominal concentrations	86.0 – 112.9%
Limit of Quantification (3 injections)	Re-calculated concentration values at LOQ level are in 80.0 – 120.0% of the nominal concentrations	10 ng/mL in calibration solution 0.2 mg/mL in formulation (20000x dilution) 94.7 – 106.0%
	CV% < 20.0%	6.05%
Recovery and Precision
Concentration (mg/mL)		2	20	200
Recovery (%)	90.0 – 110.0%	94.5	95.7	94.1
Precision (%)	less than 10.0% CV	1.81	2.41	1.84
Stability
Stability of analytical sample in autosampler for 11 h 14 min (%)	Considered stable if the measured concentration is 90 – 110% of the initial concentration.	95.9
Concentration (mg/mL)		2	20	200
Stability at 20±5°C – 9 days (%)	Considered stable if the measured concentration is 90 – 110% of the initial concentration.	103.5	100.4	100.8
Stability under transportation conditions 20±5°C – 18 days (%)		101.4	102.6	102.7

 

 

 

Conclusions:

The NOAEL for local toxicity of the parental generation was considered to be <100 mg/kg bw/day (based on local gastric histological effects in Low, Mid and High dose animals)
The NOAEL for systemic toxicity of the parental generation was considered to be 300 mg/kg bw/day. (based on body weight, body weight gain and food consumption changes at 600 mg/kg bw/day, although these effects were probably secondary to local gastric irritation).

Executive summary:

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

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

The dose levels were selected by the Sponsor in consultation with the Study Director based on the results of a Dose Range Finding (DRF) study. Based on the results of the DRF study, 600 mg/kg bw/day was selected as the High dose for this study.

Experimental design:

 

Group Number	Group designation	Dose level (mg/kg bw/day)	Concentration (mg/mL)	Dose volume (mL/kg bw)	Animal numbers
					Male	Female
1	Control	0	0	5	12	12
2	Low Dose	100	20		12	12
3	Mid Dose	300	60		12	12
4	High Dose	600	120		12*	12*

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

RESULTS

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

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

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

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

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

 

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

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

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

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

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

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

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

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Reliable (Klimisch 1)

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Subacute toxicity

A supporting 14-day dose range finding study was conducted with the registered substance in Wistar rats (4/sex/group) by oral gavage at 0 (propylene glycol), 100, 300 and 1000 mg/kg bw for up to 14 days in order to determine the dose levels for a subsequent OECD No. 422 study (Szalóki, 2021). All dose formulations were homogenous. There was no mortality in the study. In the High dose, clinical signs related with treatment included decreased activity, ataxia, hunched back, noisy respiration, splayed gait, piloerection and soft faeces. No test item related effect on body weight, body weight gain or food intake was observed in the Mid or Low groups. Body weight loss with food intake reduction at the High dose males was probably related to gastric effects observed at necropsy; both sexes had a lower body weight at day 14 than the study start weight. No clearly adverse test item-related effects were seen on the examined haematology or clinical chemistry parameters, except that liver enzymes were out of the historical control range in the High dose groups. Macroscopic treatment dose related findings at necropsy were observed with high incidences at the high dose in the glandular and non-glandular stomach at all dose levels in males and High dose females, indicating local irritation. Statistically significantly increased liver weights were seen in both sexes (~20% and 60% in males and females respectively for liver adjusted for bodyweight) was considered to be a treatment effect. Decreased spleen weights in the High dose male group is not a treatment effect (values were within the historical control range) and was not seen in females. In conclusion, based on this 14-day Dose Range Finding (DRF) toxicity study, the changes seen at 1000 mg/kg bw/day dose level were not life threatening but the body weight effect was greater than is acceptable for a longer term study but no significant body weight effects were seen at 300 mg/kg/day. The High dose level of 600 mg/kg/day was suggested for the OECD 422 study.

 

A key OECD No. 422 study was conducted with the registered substance in Wistar rats (12/sex/group by oral gavage at 0 (propylene glycol), 100, 300 and 600 mg/kg bw/day (Szalóki, 2022). Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating period), then were euthanized and subjected to necropsy examination. Females were dosed for 14 days pre-mating, for up to 14 days mating period, through gestation and up to and including the day before necropsy (13 days post-partum dosing). The day of birth (when parturition was complete) was defined as Day 0 post-partum. Females not delivered were sacrificed as practical (27 days after the last day of the mating period). Parameters measured during the study included twice a day mortality checking, daily routine and weekly detailed observation of clinical signs, weekly body weight and food consumption measurements and clinical pathology evaluation (including haematology, coagulation, clinical chemistry and urinalysis). Neurological assessment (Functional Observation Battery (FOB) including measurements of the landing foot splay, grip strength as well as locomotor activity measurement) was performed during the last week of the treatment for each sex. Reproductive performance is reported under Section 8. Weights of selected organs were recorded, and representative tissues/organs were sampled and preserved in appropriate fixatives from the adult animals or F1 animals. The thyroxine (T4) levels in the PND13 pups and parental males were also determined. For the adult animals, a detailed histological examination was performed on the selected list of retained organs of 5 animals/sex in the Control and High dose groups, all found dead animals and all those male / female mating pairs where no liveborn pups were achieved. Dosing formulations were analysed for concentration and/or homogeneity on three occasions during the study. All test item formulations were shown to be homogeneous. The measured concentrations of the test item in the different formulations varied between 93% and 101% of the nominal concentrations. Overall, the formulations were considered adequate for the study. The reproductive toxicity parameters are reported under the Section 7.8.

In summary, the test substance did not result in test item related mortality or adverse clinical signs. Test item related adverse effects were observed on body weight gain parameters and food consumption in High dose (600 mg/kg bw/day) males and moderate effects were observed on body weight and body weight gain in the gestation and lactation period for High dose females. It is most likely these effects were related to local gastric irritation. There were no adverse changes in animal behaviour, general physical condition or in the reactions to different type of stimuli or in clinical pathology in test item treated groups when compared to control. There was no test item effect on mortality or survival of the pups. Test item-related effects were observed in the liver (increased relative organ weights in Mid and High dose males and females) associated with centrilobular hepatocellular hypertrophy in the High dose. Hepatic changes were considered to be adaptive and nonadverse. Test item related effect was observed in the organ weight of the heart (decreased absolute and relative organ weight) of the High dose males (not in females) compared to controls. However, there were no histopathological findings which indicates any adversity. Histologically, test item-related changes were observed in the stomach in High and Mid dose animals with squamous hyperplasia, hyperkeratosis and ulcer of the non-glandular gastric mucosa observed at 300 and 600 mg/kg bw/day dose level in females and at 100, 300 and 600 mg/kg bw/day dose level in males, as test item-related adverse local changes. Sporadic cases of noisy respiration in a few animals ascribed to reflux or minor exposure to test item in the region of the upper respiratory tract. Also, centrilobular hepatocellular hypertrophy was observed at 600 mg/kg bw/day dose level as a non-adverse adaptive change (associated with liver weight increase). Minimal to mild multifocal eosinophilic droplets/globules were seen in the renal tubules in the High dose males and were considered as test item-related but non-adverse. The NOAEL for local toxicity of the parental generation was <100 mg/kg bw/day (based on local gastric histological effects in Low, Mid and High dose animals).

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

 

Subchronic and chronic toxicity

Subchronic toxicity testing with registered substance is ongoing and will be update later when results are available (currently waived in the dossier).

Additional data on subacute toxicity were available from followingsource chemicals:

A key dietary 90 -day subchronic toxicity study with read-across substance Docusate Sodium was performed in rats, followed by a 28 -day recovery period (Hansen, 2017). Mean actual dose levels of 105, 313 and 1066 mg /kg bw/day in males and 107, 319 and 1069 mg bw/day in females were achieved for the low, intermediate and high dose levels, respectively. 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/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.

 

A dose-range-finding study (Hansen, 2018) was done to select the route and dose levels for a study for the above study. A nominal dose level of 1000 mg Docusate Sodium USP/kg bw was administered orally once daily either via gavage or via the diet. The actual doses for the oral administration via the diet 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 (up to 14% increase) and liver weights (up to 32% increase) on test day 15. Oral administration via gavage of 1000 mg Docusate Sodium USP/kg bw/day led to the premature death of 4 of 5 male and 2 of 5 female animals. The remaining animals were prematurely sacrificed on test day 5 (1 male animal) or on test day 10 (3 female animals) for animal welfare reasons. All deceased male and female animals revealed premortal symptoms in form of piloerection, slight to moderate salivation, and slightly to extremely reduced motility. Further, a hemorrhagic nose/snout, breathing sounds, pultaceous feces, and a moisted anus were observed for individual animals. Similar findings were noted for the prematurely sacrificed animals. The macroscopic inspection at necropsy revealed a hemorrhagic or reddened nose and/or snout, a moistened anus and genital area, and reddened walls in the gastrointestinal tract for all prematurely deceased male and female animals. Further findings included undescended testes, a dark stained liver, a reddened thymus, yellowish stained indurations in the urinary bladder, and a greyish/greenish covering of the stomach wall in individual animals. Similar findings were noted for the prematurely sacrificed animals. The body weight of the male animals was severely reduced in comparison to the control group. The female animals were slightly less affected. All animals revealed a body weight loss compared to their respective start body weight. The food intake was reduced compared to the mean value of the control group.

 

In supporting studies withread-across substance Docusate Sodium, groups of 10 (5 male & 5 female) Wistar rats were treated for 6 months at concentrations of 0.25, 0.5, 0.75, 1.0 and 1.25 g/kg diet, corresponding to doses of 190, 370, 550, 750 and 870 mg/kg bw/day. Occasional spells of diarrhea occurred in some animals, particularly at the higher doses. Neither the total red cell, total white cell, nor the differential counts of rats was affected by the continued administration . The dose level of 750 mg/kg was confirmed as NOAEL (Literature, Benaglia et al. 1943). Other studies were also available from literature in various species (Literature, Benaglia et al. 1943 and Case et al., 1977) in dogs, rabbits and Rhesus monkeys. The other species were considered to be less appropriate due to the gastrointestinal tensioactive local irritation by which systemic effects could not be fully evaluated.

 

Conclusion

- The most conservative NOAEL for systemic toxicity of the parental generation was 300 mg/kg bw/day in a rat key combined repeated dose/reproductive toxicity study (OECD No. 422).The gross and histological changes in the non-glandular stomach (forestomach) are considered to be due to repeated irritation, and not relevant for humans, therefore the NOAEL was too conservative. 

- The oral NOAEL of 1000 mg/kg bw/day was obtained in the key dietary subchronic toxicity study in rats with read-across substance Docusate sodium (CAS 577 -11 -7). A subchronic toxicity with registered substance is ongoing.The NOAEL is considered more appropriate for DNEL derivation awaiting data from a dietary  subchronic toxicity study with registered substance which is ongoing. 

- Further information supporting the safety of the test substance is provided in the read across justification for the Diester category (justification with data matrix separately attached in Section 13).

Justification for classification or non-classification

As there were no changes observed below 300 mg/kg bw/day in the subacute study or below 100 mg/kg bw in the subchronic toxicity studies, classification is not warranted according to CLP regulation (No. 1272/2008 of 16 December 2008).