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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
repeated dose toxicity: oral, other
Remarks:
OECD 422 Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
6 July 2020 to May 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
Paris, 29 July 2016
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

Test material

1
Chemical structure
Reference substance name:
sodium bis[C12-14 (branched) alkyl] sulfosuccinate
IUPAC Name:
sodium bis[C12-14 (branched) alkyl] sulfosuccinate
Test material form:
other: White viscous solid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL: see confidential details

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL: see confidential details

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

OTHER SPECIFICS: see confidential details

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:WI (Wistar)
Details on species / strain selection:
The test system and the number of animals used in the study were in compliance with the relevant OECD No. 422 guideline. The guideline is designed for use with the rat, which is the preferred rodent species for reproduction toxicity testing. Wistar rat was selected due to experience of the Test Facility with this strain of rat in toxicity and reproduction toxicity studies and its known fertility.
The minimum number of animals was used, corresponding to the regulatory guidelines being followed, but taking into consideration the scientific reliability of the collected information.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, (Address: Sandhofer Weg 7, D-97633, Sulzfeld, Germany) from SPF colony
- Females nulliparous and non-pregnant
- Age at study initiation: Young adult rats, 10/11 weeks old (females/males) at start of the experiment and 12/13 weeks old (females/males) at mating.
- Weight at study initiation: Males: 390-451 g, females: 226-268 g (at the start of the treatment). The body weights did not exceed ± 20% of the mean weight for each sex at start of treatment.
- Fasting period before study: not applicable
- Housing: Rodents were group-housed, up to 2 animals of the same sex and dose group/cage, with the exception of the mating and gestation, delivery, lactation period, when they were paired or individually housed (with pups), respectively. Type II, III and/or IV polycarbonate cages were used. SAFE 3/4-S Hygienic Animal Bedding and SAFE Crinklets Natural (nesting material) produced by J. Rettenmaier & Söhne GmbH+Co.KG (Address: Holzmühle 1, D-73494 Rosenberg, Germany), were used in the study. Group housing allowed social interaction. Deep wood sawdust bedding allowed digging and other normal rodent activities, while nesting material allowed normal nesting behaviour. Certified cardboard hiding tunnels (GLP Mini Fun Tunnels, Batch number: A123) produced by LBS (Serving Biotechnology) Ltd. (Address: Unit 20, Gatwick Business Park, Kennel Lane, Hookwood, Surrey, RH6 0AH UK) were also provided to the animals.
- Diet (e.g. ad libitum): The animals received ssniff® SM R/M “Autoclavable complete diet for rats and mice – breeding and maintenance” (Batch number: 560 65984, Expiry date: 31 October 2020) produced by ssniff Spezialdiäten GmbH (Address: Ferdinand-Gabriel Weg 16, D-59494 Soest, Germany), ad libitum.
- Water (e.g. ad libitum): The animals received tap water from the municipal supply, as for human consumption from a 400- or 500-mL bottle, ad libitum.
- Acclimation period: 5 days and pre-exposure (14 days)

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

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

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

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral route was selected as it is one of the possible routes of human exposure and was requested by ECHA as most appropriate route (ECHA Decision number: CCH-D-2114489557-29-01/F; Helsinki, 14 November 2019). The way of test item and vehicle control item administration was in compliance with the relevant OECD No. 422 guideline.
The dose levels were selected by the Sponsor in consultation with the Study Director based on the results of a Dose Range Finding (DRF) study (Study code: 20/027-220PE, with the aim of inducing toxic effects but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose.
In the DRF study (after a 14-day treatment period), no clinical signs were recorded and no test item related adverse effect were seen on body weight, food consumption, clinical pathology (including haematology and clinical chemistry), gross necropsy and organ weight determination, except of significantly increased serum GPT (ALT) level at 1000 and 300 mg/kg/bw/day dose groups.
Overall, 1000 mg/kg bw/day, was considered as acceptable for the High dose level of this study. Lower doses were spaced with a factor of approximately 3.
Vehicle:
propylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
As agreed with the Sponsor, no correction for purity of the test item was applied during formulation.
The test item was formulated in the selected vehicle (propylene glycol), as a visibly stable homogenous solution at the appropriate concentrations according to the dose level and volume selected in the Pharmacy of the Test Facility. The formulations were stirred with manual shaking and a magnetic stirrer from the preparation until completion of each treatment.
Formulations were prepared a maximum of 5 days prior to administration to animals according to stability assessment results of the analytical method validation study (Study code: 20/027-316AN). Based on those results, the test item formulation in the 5 and 250 mg/mL concentration range were stable for at least 7 days when stored at room temperature.


VEHICLE propylene glycol
- Justification for use and choice of vehicle (if other than water): Based on the available information provided by the Sponsor as well as results of a trial formulation and the Dose Rage Finding (DRF) study (Study code: 20/027-220PE) performed at the Test Facility, propylene glycol (abbreviated as PG in the raw data and study documents) was selected as vehicle for this study in agreement with the Sponsor. Propylene glycol was considered as being an acceptable vehicle based on the scientific literature and practice of the Test Facility.
- Concentration in vehicle: Dose formulation concentration 0, 20, 60, 200 mg/mL
- Amount of vehicle (if gavage): A constant dosing volume of 5 mL/kg bw was administered to all animals. The actual volume to be administered was calculated and adjusted based on each animal’s most recent body weight.
- Lot/batch no. (if required): 1920944 / STBJ4817 (ThermoFisher / Sigma-Aldrich Co.)
- Purity:100%
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
*Sample Collection
Sample collection was performed at a total of four occasions (during the first and last weeks and twice approximately midway during the treatment period+). Samples were collected immediately after formulation preparation in the Pharmacy of the Test Facility by a responsible member of the Analytical Department.
+Notes: On three occasions (sampling #1, #2 and #4, i.e. during the first and last weeks and approximately midway during the treatment period) samples for all dose formulations were collected. Additional sample was collected for confirmatory purposes from the High dose formulation only near midway of the treatment period (sampling #3).
On each sampling occasion, top, middle and bottom duplicate samples were taken from the dedicated test item formulation(s) for concentration and homogeneity measurement, one set to analyse (which was collected in replicates as practical) and one set as a back-up, if required for any confirmatory analyses. Similarly, duplicate samples were taken on three occasions from the middle of the vehicle control formulation for concentration measurement.
After the analytical sampling, the collected formulation samples were stored at room temperature until measurement.
*Formulation Analysis
Analysis of control (vehicle) and test item formulations for concentration and/or homogeneity was performed in the Analytical Laboratory of the Test Facility. Representative samples of control (vehicle) and/or test item formulation(s) were analysed at four times during the study (during the first and last weeks and twice approximately midway during the treatment period).
*Analytical Method
Analysis of the formulations for concentration and/or homogeneity of test item was performed using a validated analytical HPLC-UV method (High Performance Liquid Chromatography with ultraviolet detection) in the Analytical Department of the Test Facility by using a validated analytical method (Study code: 20/027-316AN [5]). The density of the formulations was determined at the first analytical sampling by using three parallels in the Analytical Department of the Test Facility, as it was deemed necessary by the Contributing Scientist #1 (Analyst) and Study Director.
Acceptance criteria of the concentration analysis was 100 ± 10% of the nominal concentration.
Acceptance criteria of the homogeneity was that the RSD% (relative standard deviation) of replicates (top, middle and bottom of test item formulations) must be less than 10%.
Duration of treatment / exposure:
Dosing of both sexes began after the acclimatisation (5 days) and pre-exposure period (14 days), and it was performed 2 weeks before mating, during the mating, and was continued up to and including the day before the necropsy.
The first day of dosing of each animal was regarded as Day 0.
Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating period), then were euthanized and subjected to necropsy examination.
Females were dosed for 14 days pre-mating, for up to 14 days mating period, through gestation and up to and including the day before necropsy (13 days post-partum dosing). The day of birth (when parturition was complete) was defined as Day 0 post-partum. Females not delivered were sacrificed as practical (first day of dam’s necropsy).
Frequency of treatment:
The dosing solutions were administered to the test item or vehicle-treated (control) animals daily on a 7 days/week basis, in each morning, by oral gavage using a tipped gavage needle attached to a syringe (the surface of the needle was carefully cleaned before every dosing).
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
test material 99.42% purity
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
test material 99.42% purity
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
test material 99.42% purity
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: DRF study
- Rationale for animal assignment (if not random): All adult/parental (P) male and female animals were sorted according to body weight by computer and divided into weight ranges. There were an equal number of animals from each weight group randomly assigned to each dose group to ensure that animals of all test groups were as nearly as practicable of a uniform weight.
This process was controlled by the software PROVANTIS v.9, to verify the homogeneity/variability between/within the groups. Males and females were randomised separately to the dose groups on the day of the first treatment (prior to the start of the treatment).
- Fasting period before blood sampling for clinical biochemistry: Yes (overnight period of food deprivation, in case of females this happened after the litter has had been culled)

Examinations

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.
During these processes the principles and criteria summarized in the OECD Humane Endpoints Guidance Document No. 19 was taken into consideration.
Any clinical sign noted during dosing or at any other occasions (if any) was recorded at the time seen.

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

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

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

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

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

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

HAEMATOLOGY: Yes.
- Time schedule for collection of blood: immediately prior to scheduled necropsy.
- Anaesthetic used for blood collection: Yes pentobarbital anaesthesia
- Animals fasted: Yes (overnight period of food deprivation, in case of females this happened after the litter has had been culled).
- How many animals: All randomly selected animals (5M and 5F per group) *Note: Blood sample was collected but could not be analysed due to insufficient sample volume or incorrect sample for #1509, #2004 and #2507.
- Parameters examined:
Red Blood Cell (erythrocyte) count
White Blood Cell (leukocyte) count
Haemoglobin concentration
Haematocrit (relative volume of erythrocytes)
Mean Corpuscular (erythrocyte) Volume
Mean Corpuscular (erythrocyte) Haemoglobin
Mean Corpuscular (erythrocyte) Haemoglobin Concentration
Red Cell (erythrocyte)distribution width
Platelet (thrombocyte) count
Mean Platelet Thrombocyte volume (fL)
Reticulocyte count (absolute and %)
Neutrophil (absolute and %)
Lymphocyte (absolute and %)
Monocyte (absolute and %)
Basophil (absolute and %)
Eosinophil (absolute and %)
Large Unstained Cells (absolute and %)
Blood smears were prepared for all selected animals but not examined. The smears will be archived at the Test Facility.
COAGULATIONS
Activated Partial Thromboplastin Time
Prothrombin Time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: immediately prior to scheduled necropsy.
- Animals fasted: Yes (overnight period of food deprivation, in case of females this happened after the litter has had been culled).
- How many animals: All randomly selected animals (5M and 5F per group)
Note: No blood sample was taken for #2004.
- Parameters examined:
Blood sugar concentration
Total Bilirubin concentration
Urea concentration
Cholesterol concentration
Creatinine concentration
Phosphorus concentration
Sodium concentration
Potassium concentration
Calcium concentration
Chloride concentration
Total Protein concentration
Albumin concentration
Alb/glob ration
Aspartate Aminotransferase activity
Alanine Aminotransferase activity
Gamma-Glutamyl transferase activity
Alkaline Phosphatase activity
Bile acids

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

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

IMMUNOLOGY: No (Special attention was paid to the organ weight, appearance and histopathology of immune-system tissues for any evidence of immunotoxicity (spleen, thymus, lymph nodes, bone marrow). No additional flow cytometry or other specific examinations were performed additional to the basic histopathological procedure.)

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

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

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



Sacrifice and pathology:
GROSS PATHOLOGY: Yes
*Gross necropsy was performed on each adult animal irrespective of the date of death. Terminally (one day after the last treatment), animals were sacrificed under anaesthesia by exsanguination; anaesthetic product was diluted for pups’ euthanasia as required.
After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities was opened and the appearance of the tissues and organs were observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate. Special attention was paid to the organs of the reproductive system. Vaginal smearsa 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, if possible. Presence of nipples/areolae in the PND13 pups was also recorded.
*Organ Weights:
At the time of termination, body weight and weight of the following organs of all adult animals was determined:
• With a precision of 0.01 g: uterus (including cervix), testes, epididymides, prostate, seminal vesicles with coagulating glands, brain, heart, kidneys, liver, spleen and thymus
• With a precision of 0.001 g: adrenals, ovaries, thyroids with parathyroids
Testes and epididymides were weighed individually. Individual and/or paired absolute organ weight is reported for each animal and adjusted for the body and brain weights. Paired organ weights as applicable were summarised. Relative organ weight (to body and brain weight) was calculated and reported.

HISTOPATHOLOGY: Yes
The weighed organs and all organs showing macroscopic lesions of all adult animals were preserved. The eyes with the optic nerve, testes and epididymides were retained in modified Davidson’s fixative, all other organs in 10% buffered formalin solution.
In addition, on completion of the macroscopic examination the following tissues and organs were retained from all surviving animals:
Gross findings
Adrenal gland
Animal identification (Fixation and preservation only)
Aorta (Aorta thoracic and abdominal)
Brain (7 section according to the NTP recommendations)
Epididymis
Eye with the optic nerve (If applicable, parathyroids and optic nerves was examined histologically only if present in routine sections)
Oesophagus
Femur with marrow
Heart (Section including both ventricles and atria, septum with papillary muscle)
Kidney
Large intestine (Caecum, colon and rectum)
Extraorbital lachrymal gland
Harderian gland
Liver (Liver, 3 lobes, left lateral, right medial, caudate)
Lungs with bronchi (Lungs of euthanized animals was infused with formalin; 3 lobes, left, right cranial, right caudal)
Lymph node (Mandibular and mesenteric)
Ovary
Oviduct
Pancreas
Pituitary
Prostate
Salivary gland (including mandibular, sublingual and parotid glands)
Sciatic nerve
Seminal vesicle with coagulating gland
Skin, subcutis with mammary gland (inguinal)
Skeletal muscle (quadriceps)
Small intestine (Duodenum, ileum and jejunum with Peyer’s patches)
Spinal cord (Transverse sections, 3 levels: cervical, thoracic and lumbar)
Spleen
Sternum with marrow
Stomach
Testis
Thymus
Thyroid with parathyroid gland
Tongue
Trachea
Urinary bladder
Uterus (Horns, body and cervix)
Vagina
Additionally, thyroid glands from one male and one female PND13 pup from each litter was preserved in 10% buffered formalin solution. In this case, the thyroid weight (pooled) was determined after fixation. Trimming was done very carefully and only after fixation to avoid tissue damage.

The retained tissues and organs required for histopathology (below) were embedded in paraffin wax; sections were cut at 4-6 µm by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope.
For the adult animals, detailed histological examination was performed as follows:
• on the selected list of retained tissues and organs (as above) in the Control and High dose groups (selected 5 animals/sex/group)
• any animals found dead or euthanized pre-terminally during the study in all groups
• all macroscopic findings (abnormalities), except of minor order from all animals
• on the retained reproductive organs (testes, epididymides, prostate gland, seminal vesicles with coagulation gland for males and uterus, cervix, ovary, oviduct and vagina for females) of all animals of the Control and High dose groups, and of all females that failed to deliver healthy pups in the Mid dose group (documented by a memo in the raw data)*
• the liver and stomach of all animals.
*Notes: Only two non-pregnant females were observed in the study, both females belonged to the Control group (#1506 and #1512). Two Mid dose females did not survive the delivery (#351 was found dead, #3505 was pre-terminally euthanized).

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 is paid to the organ weight, appearance and histopathology of immunesystem tissues for any evidence of immunotoxicity (spleen, thymus, lymph nodes, bone marrow, and blood smears if examined).
Special attention was paid to the central and peripheral nervous system tissues for any evidence of neurotoxicity.



Statistics:
See under "Any other information on material and mathods incl. tables": Data collection and statistics.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test item related clinical signs were observed in the study.
Tonic convulsions were recorded at the end of the lactation period on the day of termination (Day 52) for a Control female (#1503).
A small wound (<1 cm) and later a crust was observed for one Mid dose male (#3010) in the neck dorsal area from Day 3 to Day 22.
These findings were considered incidental, not related to the test item administration.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One Mid dose female (#3501) was found dead on Day 43 (on the day of delivery), this fact was most probably related to a difficult delivery process, not related to the test item administration as no test item-related macroscopic or microscopic changes were observed.
For another Mid dose female (#3505) hunched back, piloerection and extreme vaginal prolapse were recorded at delivery (GD 22, Day 36), this animal was pre-terminally euthanized due to ethical reason on the same day. No test item-related macroscopic or microscopic changes were observed.
These two cases were considered incidental (most probably related to the difficult delivery), not related to the test item administration.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Test item related adverse effect was observed on body weight parameters in High dose (1000 mg/kg bw/day) males and females during gestation and lactation periods (no effect was seen in the pre-mating period). No test item related adverse effect on body weight or body weight gain was detected in Mid or Low dose (300 or 100 mg/kg bw/day, respectively) animals (males or females). Slightly lower body weight (not significant) and reduced body weight gain (statistically significant at p<0.05) was observed in High dose males on the first week of treatment when compared to control animals
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Test item related adverse effect on food consumption was observed in High dose females (1000 mg/kg bw/day) during the gestation and lactation phases, no effect was observed in the High dose males or Mid and Low dose groups (300 and 100 mg/kg bw/day, respectively) of both sexes. No adverse effect on food consumption was seen in male animals in any test item treated group and no effect was observed in test item treated female animals during the pre-mating period
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No test item-related adverse changes were detected in the test item treated animals (males and females) when comparing haematology parameters to the relevant Control data.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
No test item-related adverse changes were detected in the test item treated animals (males and females) when comparing serum chemistry parameters to the relevant Control data.
Alanine aminotransferase (ALT/GPT) activity was significantly increased in the Mid dose males (p<0.05) and High dose males (p<0.05) and females (p<0.01) when compared to control animals. Furthermore, increased Alkaline phosphate (AP) activity were recorded in High dose females (by 83%, statistically significant at p<0.01), but the observed values were within the historical control range and no similar trend was noted in High dose males. All these increased values were considered as test item related effect; however, in the absence of any indication of adverse histopathology findings in the livers, they were considered as non-adverse.
Reduced cholesterol concentration was observed in High dose females (by 61%, significant at p<0.01), but this fact was most probably related to the significantly reduced food consumption of High dose females during the gestation and lactation periods, not being a direct effect.
Endocrine findings:
no effects observed
Description (incidence and severity):
No indication of an endocrine disruptor effect was observed in the study based on collected oestrus cycle data, anogenital distance measurement, nipple retention, thyroid hormone measurement, thyroid weight and histopathology and external reproductive organs analysis.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No test item-related changes were observed in the urinalysis parameters in male and female animals of any dose groups when compared to control.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no changes in animal behaviour, general physical condition or in the reactions to different type of stimuli in the control or test groups.
Immunological findings:
no effects observed
Description (incidence and severity):
There were no effects observed in the haematological and histopathological examinations on immunological organs and tissues.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related effects were observed in the organ weights of the test item treated male animals compared to controls. There were no statistically significant differences in the terminal body weight of the test item treated males compared to control males. The body-related weight of the epididymides was decreased statistically significantly (by 8.4%) in the Mid dose group compared to Control, but based on the lack of dose response and as there were no similar change in case of the absolute or brain-related weights, this difference was considered as incidental.
No test item-related effects were observed in the organ weights of the test item treated female animals compared to controls. Terminal body weights of test item treated females were not significantly different from control females. The absolute and relative (to body and brain) weights of the heart decreased statistically significantly (by 21.7%, 19.4% and 22.6%, respectively) in the High dose group compared to Controls without histological relevance. The absolute and relative (to body and brain) weights of ovaries were statistically significantly lower than Control in the Low (by 14.8% (p<0.01), 15.3% (p<0.01) and 16.5% (p<0.01), respectively) and High dose females (by 18.1% (p<0.01), 15.5% (p<0.01) and 18.6% (p<0.01), respectively), but there was no dose response and no histological relevance.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test item-related changes were observed in the stomach, as multifocal, white discoloration and/or focal/diffuse thickness of the non-glandular mucosa in 3/12 High dose males and in 3/12 High dose females. All other observed changes were considered incidental or a common background.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related minimal/mild multifocal hyperkeratosis of the non-glandular gastric mucosa were observed in 9/12 High dose males and 9/12 High dose females. Low and mid dose groups were also inveestigated but did not show histopathologcial findings.
In the liver, minimal hepatocellular hypertrophy was seen in 4/12 High dose males and in 2/12 High dose females, additionally minimal/mild, periportal or diffuse hepatocellular vacuolation was present in 5/12 High dose females. Those changes were considered as non-adverse adaptive changes. Low and mid dose groups were also inveestigated but did not show histopathologcial findings.
Histopathological findings: neoplastic:
no effects observed
Details on results:
-Body weight and weight changes
Test item related adverse effect was observed on body weight parameters in High dose (1000 mg/kg bw/day) females. No test item related adverse effect on body weight or body weight gain was detected in Mid or Low dose (300 or 100 mg/kg bw/day, respectively) animals (males or females).
Slightly lower body weight (by 2%, statistically not significant) and reduced body weight gain (by 56%, statistically significant at p<0.05) was observed in High dose males on the first week of treatment when compared to control animals. This difference still could be observed at the end of the treatment period (body weight lower by 3% and body weight gain lower by 18% compared to control, not statistically significant). This High dose body weight (gain) difference, principally in the first week, was considered as a test item related effect.
No effect on body weight parameters was seen in Mid and Low dose males.
In case of females, the observed body weight or body weight gain values were comparable to the control level at the end of the pre-mating period in all dose groups. But the High dose females showed reduced body weight / body weight gain at the end of the gestation period (body weight was lower by approximately 9%, body weight gain by 20%; the difference to control was statistically significant at p<0.01 in both cases). Reduced body weight of the High dose females compared to control (by approximately 7-8%) was also observed at the beginning of the lactation period (PPD 0, 4 and 7, significant at p<0.01), but the difference was smaller by the end of the lactation period (by approximately 7% lower body weight and by 6% lower body weight gain) and none of them gained statistical difference at that point. However, the body weight gain calculated for the entire treatment period (Day 0 – PPD13) was still statistically significantly lower for High dose females than Control (by 17%, significant at p<0.01). These facts were considered as being a test item related adverse effect.
No effect on body weight parameters was seen in Mid and Low dose females.

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

-Endocrine findings
*Oestrus cycle evaluation in females
Pre-exposure period
Each female selected for the study showed acceptable cycles (mean cycle length of 4.03-4.21 days was observed in the different groups) before starting the treatment period.
Exposure period (pre-mating and mating periods)
No indication of test item related effect was seen in the oestrus cycle data, collected during the pre-mating and mating periods (mean cycle length was 4.17, 3.98, 4.19 and 3.97 days in the Control, Low dose, Mid dose and High dose groups, respectively).
Prolonged oestrus was recorded in some cases for all groups, a total of 4 Control females, 2 Mid dose females and 1 High dose female were affected (#1503, #1504, #1507, #1512, #3510, #3512 and #4509), but as it did not affect mating or pregnancy (from these animals only one Control female became non-pregnant) and as the incidence was lower in the test item treated groups than in the Control group; this fact was considered as being an occasional finding, not being a test item related effect.
Prolonged dioestrus was noted for one Low dose female (#2502) and one Mid dose female (#3501), but this frequency (1/12) was in line with the normal, expected range and did not affect the mating or pregnancy.
*Anogenital distance, nipple retention
No test item effect was observed on anogenital distance or nipple retention during the study.
No statistically significant changes in the anogenital distance measured on PND 0 were noted for test item treated male and female pups when litter mean values were compared to control.
No nipples/areolae were present in any of the male pups on PND13.
*Thyroid Hormone Analysis
No test item effect was noted in any dose groups based on the results the T4 hormone measurement, thyroid gland weights and histopathology evaluation.
Statistically significant increase in the T4 thyroid hormone concentration (by 28% and 27% respectively, significant at p<0.01) was detected in Low and High dose parental males (100 and 1000 mg/kg bw/day) when compared to control. However, there was no dose response and the observed results were in line with the results of Control groups of other contemporaneous studies**. Furthermore, the thyroid weights (absolute and body related) were comparable with the Control values for all males of all dose groups. Histopathology results were also taken into consideration, and no microscopic findings were detected in any test item treated male animals. Therefore, the observed statistical difference in the T4 concentrations were not considered to reflect a test item related effect.
**Note: In two other contemporaneous studies (Study codes: 20/028-220P and 20/030-220P, respectively) a mean value of 57.0 ng/mL (range: 44-73 ng/mL) and mean value of 50.7 ng/mL (range: 38-70 ng/mL) were observed.
No relevant changes were noted in the absolute thyroid or relative (to body) thyroid weights of the female animals in any dose groups. No histopathology (microscopic) findings were detected in any test item treated female animals except of minimal amount of multinucleated giant cells in the thyroid sample of a High dose female (#4504) which was considered as an incidental finding.
In case of PND13 pups, there were no statistically significant thyroid hormone concentration levels recorded when compared to the control.
The thyroid gland weights of the PND13 pups were also comparable with the control value in the Low and Mid dose groups, but higher relative (to body weight) was noted in the High dose group. However, it was a consequence of the significant lower body weight of the High dose pups, not a direct test item effect on thyroids. In summary, there were no effects on the thyroid hormone levels or on the thyroid gland weights in the PND13 pups that were ascribed to the test item.

-Gross pathological findings
NON-PREGNANT FEMALES / Parental Generation
Two non-pregnant Control females were observed in the study (#1506 and #1512).
Necropsy examination did not show any test item related change in the non-pregnant females and their male mating pairs.
FOUND DEAD ANIMAL / Parental Generation
One Mid dose female (#3501) was found dead at delivery.
No test item-related macroscopic changes were observed at necropsy, but the uterine horns and cervix was enlarged, the lungs and the thymus were dark red, the stomach was dilated with gas at necropsy.
PRE-TERMINAL EUTHANASIA / Parental Generation
One Mid dose female (#3505) was pre-terminally euthanized due to extreme vaginal prolapse on the day of delivery.
No test item-related macroscopic changes were observed. The uterine horns and cervix were enlarged and dark red, the vagina was enlarged, the thyroids were pale at necropsy.
TERMINAL EUTHANASIA / Parental Generation
Test item-related changes were observed in the stomach, as multifocal, white discoloration and/or focal/diffuse thickness of the non-glandular mucosa in 3/12 High dose males and in 3/12 High dose females.
All other observed changes were considered incidental or a common background.

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

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

Effect levels

Key result
Dose descriptor:
NOAEL
Remarks:
Parental generation systemic toxicity
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
99.42% purity
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
gastrointestinal tract
Organ:
stomach
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no

Any other information on results incl. tables

Method Validation (20/027-316AN)

The purpose of this study was to validate a High Performance Liquid Chromatography method with UV detection (HPLC-UV) in order to determine the concentration of AEROSOL TR-70 E Lyophilized in formulation, primarily to support OECD No. 422 study (Study code: 20/027‑220P). A secondary purpose was to determine the stability of the test item in the required formulation under its conditions of use and storage. The procedure was found to be suitable for the analysis. A summary of the method parameters is presented in Table 1.

Table 1. Results of the Method Validation (20/027-316AN)

Selectivity

The column used provides good separation with a characteristic peak selective for the test item. The UV absorption at 210 nm has a background of above zero (at ~30% of the LOQ); although the Study Plan specified 20% of the LOQ as the maximum for interfering compounds, this background peak is not considered to be an interfering compound in the test item formulations.

Since the calibration graph is corrected with the observed background signal, it is considered that the selectivity is adequate hence there is no impact on the results or integrity of the study.

Reinjection repeatability (7 injections)

RSD%≤ 3.3%

Linear range

0.2 – 2 mg/mLof the test item

Limit of Quantification

0.2 mg/mLof the test item

[= 1 mg/mL (test iteminPG)]

Recovery of thetest itemfromPG(~5 and~250 mg/mL)

96.0 and 97.6%

Precision inPG(~5 and~250 mg/mL)

0.4 and 0.2%

Stability of thetest iteminPGat~5 and~250 mg/mL at room temperature (1 day)

97 and 93%

Stability of thetest iteminPGat~5 and~250 mg/mL at room temperature (3 days)

98 and 93%

Stability of thetest iteminPGat~5 and~250 mg/mL at room temperature (7 days)

95 and 90%

Stability of thetest iteminPGat~5 and~250 mg/mL at 2-8°C (7 days)

98 and 93%

Stability of the samples for at least 24 hours in the autosampler

97 and 97%

Stock solution stability for at least 7 days at 5±3 °C

96%

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, the following No-Observed-Adverse-Effect Levels (NOAELs) were considered:
The NOAEL for systemic toxicity of the parental generation: 300 mg/kg bw/day (based on body weight and food consumption effects, and stomach microscopic findings).
Executive summary:

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

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

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

 

Experimental design:

Group Number

Group designation
Dose level
(mg/kg bw/day)

Dose formulation concentration

(mg/mL)

Dose formulation volume

(mL/kg bw)

Number of animals

Male

Female

1

Control

0

0

5

12

12

2

Low dose

100

20

12

12

3

Mid dose

300

60

12

12

4

High dose

1000

200

12

12

Parameters measured during the study included twice a day mortality checking, daily routine and weekly detailed observation of clinical signs, weekly body weight and food consumption measurements and clinical pathology evaluation (including haematology, coagulation, clinical chemistry and urinalysis). Neurological assessment (Functional Observation Battery (FOB) including measurements of the landing foot splay, grip strength as well as locomotor activity measurement) was performed during the last week of the treatment for each sex. In addition, the reproductive performance, pregnancy, parturition and postpartum/lactation period were monitored in the adult animals, and viability, clinical signs and development were evaluated in their F1 offspring until PND13. At termination (Day 28 for males, PPD (Post-partum Day) 14 for females), necropsy with macroscopic examination was performed. Weights of selected organs were recorded, and representative tissues/organs were sampled and preserved in appropriate fixatives from the adult animals or F1 animals. The thyroxine (T4) levels in the PND (Post-natal Day) 13 pups and parental males were also determined.

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

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

RESULTS (repeated dose section only)

In summary, under the conditions of this study the daily administration of Sodium 1,4-diisotridecyl sulphonatosuccinate (CAS 55184-72-0, EC 259-515-6) by oral gavage to Wistar rats at dose levels of 100, 300 or 1000 mg/kg bw/day (Low, Mid and High dose groups, respectively) did not result in test item related mortality or clinical signs.

Test item related adverse effect was observed on body weight parameters and food consumption in High dose (1000 mg/kg bw/day) males and females during gestation and lactation periods (no effect was seen in the pre-mating period).

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

No test item-related adverse effects were seen in the clinical pathology parameters. Test item related non-adverse changes (increased Alanine aminotransferase activity in the Mid dose males and High dose males and females, as well as increased Alkaline phosphate activity in High dose females) were considered to be linked to the adaptive response seen in the liver during microscopic examination (hepatocellular hypertrophy and vacuolation which was observed only in High dose animals, but not in Mid or Low dose animals).

No test item-related effects were observed in the organ weights of the test item treated animals compared to controls.

Hyperkeratosis of the non-glandular gastric mucosa of the stomach was identified at the High dose level (1000 mg/kg bw/day), as test item-related adverse change. Mid and Low dose stomach samples of both sexes were also evaluated the get additional information for study interpretation, no test item-related changes were seen in those animals.

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

The NOAEL for systemic toxicity of the parental generation: 300 mg/kg bw/day (based on body weight and food consumption effects, and stomach microscopic findings).

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