Butanedioic acid, sulfo-, 4-C12-14 (even numbered)-alkyl esters, disodium salts

Endpoint summary

Description of key information

Subacute toxicity was tested with the registered substance in Wistar rats by oral gavage at 0 (distilled water), 100, 300,  600 and 1000 mg/kg bw (low, mid, mid-high and high dose groups, respectively) in a supporting 11 to 14-day dose range finding and at 0 (distilled water), 60, 150 and 450 mg/kg bw/day in a key combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD No. 422). The NOAEL for systemic toxicity of the parental generation was 150 mg/kg bw/day.

A 90-day study was also available for the read across substance CAS No. 37294-49-8 (Disodium C-isodecyl sulphonato- succinate) in the mono-ester subgroup, given to rats at 0.25, 1 and 4% in the diet. This study showed a NOAEL of 1% in the diet, corresponding with 750 mg/kg bw. For risk assessment, the lowest NOAEL of 150 mg/kg bw in the OECD 422 study with the registered substance was selected as this approach was considered most conservative.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9 February 2021 (study plan) – 26 October 2022 (final report)

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:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Crl:WI (Wistar)

Details on species / strain selection:

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, 10/11 weeks old (males/females)
- Weight at study initiation: Males: 372-436 g, females: 233-279 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: no information
- Housing: Rodents were group-housed, up to 2 animals of the same sex and group/cage in type II, III and/or IV polycarbonate cages. During the mating and gestation, delivery, lactation period, they were paired or individually housed (with pups), respectively. SAFE 3/4-S Hygienic Animal Bedding (Batch number: 03027201024 / 03027201208, Expiry date: 24 October 2023 / 08 December 2023) and SAFE Crinklets Natural nesting material (Batch number: 05072200824, Expiry date: 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: 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: 71370882 / 18776795, Expiry date: 30 April
2021 / 31 August 2021) produced by ssniff Spezialdiäten GmbH (Address: Ferdinand-Gabriel Weg 16, D-59494 Soest, Germany), ad libitum. The supplier provided an analytical certificate for the batch used.
- 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: 8 days

DETAILS OF FOOD AND WATER QUALITY:
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 Public Health and Medical Offer Service (H-8200 Veszprém, József Attila u. 36., Hungary). 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): 19.6 - 24.6°C (target: 22 ± 3°C)
- Humidity (%): 20 - 75% (target: 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: 12 February 2021 (start of in life phase) To: 01 May 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:

other: distilled water

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 (distilled water), as a visibly stable homogenous suspension 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 at preparation until completion of each treatment.
Formulations were prepared fresh every day prior to administration to animals according to stability assessment results of the analytical method development and method validation studies (Study code: 20/125-316ANE and 20/125-316AN). Based on those results, the test item formulation in the 5 and 100 mg/mL concentration range were stable for at least 3 days when stored at room temperature.
Formulations were prepared in clean glass containers. The appropriate amount test item was weighed into a clean, calibrated glass container and then mixed properly (with manual shaking and magnetic stirring) with the needed amount of vehicle to reach homogeneity by visual observation. During the formulation, approximately 1-hour ultrasound sonication was applied for proper homogenisation. Formulations were stored in a closed container at room temperature until use.

VEHICLE: distilled water
- Concentration in vehicle: 0, 12, 30 and 90 mg/mL for the dose levels group of 0, 60, 150 and 450 mg/kg bw/day, respectively.
- Amount of vehicle (if gavage): A constant volume of 5 mL/kg bw was administered to all animals
- Lot/batch no.: 202011110

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Sample collection was performed at a total of three occasions (during the first and last weeks and once 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.
On each sampling occasion, top, middle and bottom duplicate samples were taken from the dedicated test item formulations 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.
Analysis of control (vehicle) and test item formulations for concentration and/or homogeneity was performed in the Analytical Laboratory of the Test Facility. Representative samples of control (vehicle) and/or test item formulations were analysed at three times during the study (during the first and last weeks and once approximately midway during the treatment period).
The formulation analysis was conducted within the determined stability period.
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: 20/125-316AN). The density of the formulations was determined at the first analytical sampling by using three parallels in the Analytical Department of the Test Facility, as it was deemed necessary by the Contributing Scientist #1 (Analyst) and Study Director.
Acceptance criterion of the concentration analysis was 100 ± 15% of the nominal concentration.
Acceptance criterion of the homogeneity was that the RSD% (relative standard deviation) of replicates (top, middle and bottom of test item formulations) must be less than 10%.
The measured concentrations of the test item in the different formulations varied between 96.1% and 100.2% of the nominal concentrations, all of those values were within the acceptance criterion (100 ± 15% of the nominal concentration).
All test item formulations were shown to be homogeneous. The relative standard deviation (RSD) was below 10% in each case.
Formulations were considered to be adequately stable under the study conditions.
Overall, the formulations were considered adequate for the study.

Duration of treatment / exposure:

Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating period), then were euthanized and subjected to necropsy examination.
Females were dosed for 14 days pre-mating, for up to 14 days mating period, through gestation and up to and including the day before necropsy (13 days post-partum dosing). The day of birth (when parturition was complete) was defined as Day 0 post-partum.

Frequency of treatment:

daily

Dose / conc.:

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

Remarks:

low dose

Dose / conc.:

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

Remarks:

mid dose

Dose / conc.:

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

Remarks:

high dose

No. of animals per sex per dose:

12 animals/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The oral route was requested by ECHA as the most appropriate route of human exposure (ECHA decision number: CCH-D-2114489557-29-01/F, Helsinki, 14 November 2019; information provided by the Sponsor). 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: 20/125-220PE), 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, the test item was formulated in distilled water and administered by oral gavage to Wistar rats for 14 consecutive days at dose levels of 100, 300 or 1000 mg/kg body weight/day using a dose volume of 8 mL/kg body weight or at dose level of 600 mg/kg body weight/day using the same dose volume for 10 consecutive days. The following findings were recorded in the DRF study:
Mortality / morbidity (a total of four males and four females) was observed in the 1000 mg/kg bw/day dose group within the first four days of treatment.
On Day 7, body weight of the animals in the 600 mg/kg bw/day dose group was significantly lower than controls (-23.6% and -3.1% in males/females). Food consumption on Day 7 was also significantly lower than control (-65.4% and -27.7% versus control). Hunched back, noisy respiration and soft faeces were also observed in this dose group for males and females, while decreased activity, piloerection, partially closed eyelids, incontinence, increased salivation and red discharge (nose/snout) were observed for males of this dose group. Statistically significant changes were observed for increased neutrophils, decreased lymphocytes in males dosed at 600 mg/kg bw/day. The dose at 600 mg/kg bw/day was considered to exceed the MTD (Maximum Tolerated Dose).
On Day 14, body weight of the animals in the 100 (0.4% and -2.6% in males/females) and 300 mg/kg bw/day (-3.7% and -1.1% in males/females) dose groups was not significantly lower than controls. Food consumption day 0-13 was not significantly lower in the males dosed at 100 and 300 mg/kg bw (4.2% and -5.4%) and females dosed at 300 mg/kg bw/day (-4.2%) but it was significant in females dosed at 100 mg/kg bw (-7.2% versus controls). Noisy respiration was observed in the 300 mg/kg bw/day male animals (1/4) and in the 100 mg/kg bw/day females (1/4) as well.
Test item related increase in the weights of adrenal and stomach and decrease in the weights of thymus were observed in the male animals of the 600 mg/kg bw/day dose group; macroscopic changes in adrenal, stomach and thymus correlated with the organ weight changes. A statistically significant increased weights of liver and stomach were observed in female animals of the 600 and 300 mg/kg bw/day dose groups, macroscopic changes only in the stomach with correlating organ weight changes.
Since the dose level of 600 mg/kg bw/day exceeded the MTD and the effects at 300 mg/kg bw/day were less than the effects intended for a High dose level, it was considered that selecting a High dose level of above 450 mg/kg/day for the main study would be likely to cause mortality or severe toxic effects. A High dose of 450 mg/kg bw/day was considered to be suitable for the main 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 v9.3, 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).
Any unused, spare animals were moved back to the stock colony after they were not needed for the study after the second week of treatment.
- Fasting period before blood sampling for clinical biochemistry: yes, overnight period of food deprivation, in case of females this happened after the litter had been culled

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. There was no such a case in the study.


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 (on PPD0, 4, 7, 10 and 13).
Main daily food consumption was calculated for each interval..

FOOD EFFICIENCY: no

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

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

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At study termination, euthanasia was performed under pentobarbital anaesthesia followed by exsanguination.
- 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: randomly selected animals (7 males* and 5 females/group), *Note: 2 additional male animals were sampled in each group to allow replacement for occasional clotted samples, as all males were terminated on the same day, thus additional sampling was not achievable later
- Parameters examined.
RBC Red Blood Cell (erythrocyte) count, (10^12/L) M/µL
WBC White Blood Cell (leukocyte) count, (10^9/L) K/µL
Hgb Haemoglobin concentration, (g/dL)
Hct Haematocrit (relative volume of erythrocytes) (%)
MCV Mean Corpuscular (erythrocyte) Volume (fL)
MCH Mean Corpuscular (erythrocyte) Haemoglobin, (pg)
MCHC Mean Corpuscular (erythrocyte) Haemoglobin Concentration, (g/dL)
RDW Red Cell (erythrocyte) volume (%)
Plt Platelet (thrombocyte) count (10^9/L) K/µL
MPV Mean Platelet Thrombocyte volume (fL)
RETIC % Reticulocyte count (absolute and %)
NE % Neutrophil (absolute and %)
LY % Lymphocyte (absolute and %)
MO % Monocyte (absolute and %)
BA % Basophil (absolute and %)
EO % Eosinophil (absolute and %)
LUC % Large Unstained Cells (absolute and %)
Coagulation parameters:
APTT Activated Partial Thromboplastin Time (sec)
PT Prothrombin Time (sec)

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: randomly selected animals (7 males* and 5 females/group), *Note: 2 additional male animals were sampled in each group to allow replacement for occasional clotted samples, as all males were terminated on the same day, thus additional sampling was not achievable later
- Parameters examined:
Glucose Blood sugar concentration (mmol/L)
T-BIL Total Bilirubin concentration (μmol/L)
Urea nitrogen Urea concentration (mmol/L)
Chol. Cholesterol concentration (mmol/L)
Creat. Creatinine concentration (μmol/L)
Phos. Phosphorus concentration (mmol/L)
Na+ Sodium concentration (mmol/L)
K+ Potassium concentration (mmol/L)
Ca++Calcium concentration (mmol/L)
Cl- Chloride concentration (mmol/L)
Tot. Prot. Total Protein concentration (g/L)
Alb. Albumin concentration (g/L)
A/G Alb/glob ration
AST/GOT Aspartate Aminotransferase activity (U/L)
ALT/GPT Alanine Aminotransferase activity (U/L)
GGT Gamma-Glutamyl transferase activity (U/L)
ALKP Alkaline Phosphatase activity (U/L)
BA Bile acids (µmol/L)

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,
•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.
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 in the morning 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.
- Animals fasted: Yes : overnight period of food deprivation, in case of females this happened after the litter had been culled

URINALYSIS: yes
- Time schedule for collection of urine: Urine sampling was performed prior to necropsy by placing the selected animals in metabolic cages for approximately 16 hours.
- Metabolism cages used for collection of urine: Yes
- 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 as indicated below:
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: Functional Observational Battery and locomotor activity measurement was performed in the study. Assessment of any potential test item related neurotoxicity was performed during the last exposure week (males on Day 25 am, females on PPD 10/11 am). 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:
Selected animals were subjected to the functional observation battery, including Irwin test and measurements of the landing foot splay and fore/hind grip strength.
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
-method of euthanasia
At study termination, euthanasia was performed under pentobarbital anaesthesia followed by exsanguination.
Euthanimal 40% (400 mg/mL sodium pentobarbital solution) was used by intraperitoneal injection.

-unscheduled euthanasia
Gross necropsy was performed on each animal irrespective of the date of death, including the animals found dead. No organ weight measurement was performed, but tissue or organ samples were retained for those animals.

-scheduled euthanasia
Surviving animals were euthanized at termination. Gross necropsy was performed on each animal. Weight of selected organs were measured, and selected organs and tissues were retained.

-necropsy:
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 were opened, and the appearance of the tissues and organs was 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.

-organ weights:
At the time of termination, body weight and weight of the following organs of all surviving adult animals were determined:
•With a precision of 0.01 g: uterus (including cervix), testes, epididymides, prostate, seminal vesicles with coagulating glands, brain, heart, kidneys, liver, spleen and thymus
•With a precision of 0.001 g: adrenals, ovaries, thyroids with parathyroids
Testes and epididymides were weighed individually. Individual and/or paired absolute organ weight was reported for each animal and adjusted for the body and brain weights. Paired organ weights as applicable were summarised. Relative organ weight (to body and brain weight) was calculated and reported.

-tissue collection and preservation
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
Adrenals
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 (If applicable, parathyroids and optic nerves was examined histologically only if present in routine sections)
Tongue
Trachea
Urinary bladder
Uterus (Horns, body and cervix.)
Vagina


HISTOPATHOLOGY: yes
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),
• one Low dose animal found dead during the study,
• all macroscopic findings (abnormalities), except of minor order from all animals,
• stomach and thymus samples of all the remaining Control, Low, Mid and High dose animals (males / females),
• 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 including the mating pair which failed to deliver healthy pups*.
*Note: Only one non-pregnant female (#4508) was observed in the study. As that female belonged to the High dose group, reproductive organs (and also for her mating pair, #4008) were examined at the first instance.
Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.
Special attention was paid to the organ weight, appearance and histopathology of immune-system tissues for any evidence of immunotoxicity (spleen, thymus, lymph nodes, bone marrow).
Special attention was paid to the central and peripheral nervous system tissues for any evidence of neurotoxicity.
No histopathological examination was performed on pups (F1 generation).

Statistics:

see "Any other information on materials and method incl. tables"

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test item related clinical signs were observed in the study.
Noisy respiration was detected for a High dose male (#4002) on Days 24-25.
A nodule (1-2 cm) was observed for one Control female (#1502) in the abdominal area from Day 38 until termination.
Alopecia was observed on both forelimbs of another Control female (#1510) from Day 38 until termination.
These findings were considered incidental, not related to the test item administration.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No test item related mortality was observed in the study. One Low dose male (#2011) was found dead on Day 23, no clinical signs were shown by this animal prior to death. This case was considered incidental, not related to the test item administration as no test item-related macroscopic or microscopic changes were observed.

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 (450 mg/kg bw/day) males. No test item related adverse effect on body weight or body weight gain was detected in High dose females, or in the Mid or Low dose (150 or 60 mg/kg bw/day, respectively) animals (males or females).
In the first week of treatment, the High dose males had almost no growth, compared to Control males. The difference in body weight compared to control reached statistical significance by the end of the treatment period (body weight lower by 7% at p<0.05 and body weight gain lower by 47% at p<0.01). These results of the High dose males were considered as a test item related adverse effect (and suitable for a High dose in an OECD No. 422 study).
No effect on body weight parameters was seen in Mid and Low dose males.
No clearly adverse effect on body weight parameters was seen in the test item treated females when compared to concurrent controls. The High dose weight gain was lower than the pre-treatment period and lower than typical historical control data for the pre-mating period, although in this study the control weight gain was similar to the High dose. The observed body weight or body weight gain values were comparable to the control level at the end of the pre-mating period, gestation or lactation in all dose groups.
No effect on body weight parameters was seen in Mid and Low dose females.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Test item related adverse effect on food consumption was observed in High dose males
(450 mg/kg bw/day) during the treatment period, no effect was observed in the High dose females, or Mid and Low dose groups (150 and 60 mg/kg bw/day, respectively) of both sexes.
In case of High dose males, reduced values compared to control were recorded during the entire treatment period, statistical significance reached (p<0.01). Based on the body weight values, the observed values were considered as a test item related adverse effect (probably secondary to gastric irritation caused by the test item).
No effect on food consumption was considered in Mid and Low dose male animals. Although the mean food consumption calculated for the entire treatment period in the Mid dose males was statistically significantly lower than Control (by 5.6%, p<0.01), but the difference originated from a transient reduced food consumption of the first week (no statistically significant differences were observed on the other three weeks thereafter), this it was considered as a palatability issue, not being a test item related effect.
No statistically significant effect was observed in test item treated female animals. Although slightly lower than control-level food consumption was recorded for High dose females during the pre-mating (11% below control in the first week), gestation (~5% for GD0-GD20) or lactation period (6.2% during the last part of the lactation period i.e. maximal milk production period), although the differences were not great, the trends indicate some effect which may be below the level considered as adverse.

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. The sporadic statistical differences in the ratio of reticulocytes and eosinophils as well as the prothrombin time in High dose males were not outside the normal control range, based on historic control data.

Clinical biochemistry findings:

effects observed, non-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.
Increased bile acid concentration was observed in High dose males (by >300%, at p<0.01) and High dose females (by 65%, statistically not significant), although a statistically significant increase was also seen in Low dose males (by >100%, at p<0.01) without dose response . The differences between groups are within the ranges seen in similar study control groups, the dose response was not consistent, there were no adverse histological hepatic findings or any other evidence in clinical pathology for hepatic effects of the test item; hence the apparent elevation in some groups is not considered to reflect an adverse effect of treatment.

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. Occasional differences with statistical significance but without dose response were considered as animal variability.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

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

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Males:
Test item-related effect was observed in the thymus weight of High dose males compared to control animals. Terminal body weights of High dose males were statistically significantly lower than Control (by approx. -8%, the difference was statistically significant at p<0.01). Treatment-related decrease was observed in the thymus weights of Mid and High dose males and there was a microscopic correlate for the weight reduction in High dose males. The thymus changes are very common in stressed animals where markedly reduced body weight growth occurs in the first week of treatment (as in this study); since the effect is considered to be related to body weight and not a direct effect on the thymus, it is not considered as an adverse effect of treatment.

There were statistically significant differences among groups in the weights of a few other organs (brain, heart, seminal vesicle and liver), either in the absolute and/or body weight / brain weight related value when compared to Control. However, in the absence of any macroscopic or microscopic correlate, these weight changes were considered as not related to test item administration.

Females:
Test item-related effects were observed in the thymus 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.
Weight differences were observed in the thymus and liver weights . There was macroscopic and microscopic correlate for the thymus weight reduction in females, however, there was no correlate for the weight increase in the liver.
In males a reduced thymus weight and cellularity was ascribed to a general stress effect rather than a direct effect of the test item on the thymus; in High dose females, a clear gastric irritation seen at histology and non-statistical trend for lower food intake are considered to be related to a non-specific stress which is a common cause of these thymic changes. Liver hypertrophy is common in animals treated with xenobiotics, but when the effect is below about 15% (of increase liver weight relative to body weight) the change is usually too small to be detectable by histology examination. The liver weight difference in the absence of histology changes is considered to be non-adverse.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

-FOUND DEAD ANIMAL / Parental Generation (Low dose male (#2011) was found dead on Day 23): Macroscopic Findings: Necropsy revealed diffuse dark red discolouration and enlargement of all lobes of liver and lungs.
- TERMINAL EUTHANASIA / Parental Generation: Macroscopic Findings: Treatment related focal/multifocal thickness of the wall of non-glandular stomach was observed in 9/12 High dose males. Small thymus was observed in 2/11 High dose females.
All other observed changes were considered incidental or a common background.
- NON-PREGNANT FEMALES / Parental Generation (one high dose female #4508): macroscopic findings: Necropsy examination did not show any relevant test item related change in the non-pregnant female and their male mating pair in the High dose group (only multifocal thickness of the non-glandular stomach was observed in both animals).

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

-FOUND DEAD ANIMAL / Parental Generation (Low dose male (#2011) was found dead on Day 23): Microscopic Findings: Microscopically, mild congestion correlated with the macroscopic findings in the liver and lungs. Minimal extramedullary haematopoiesis was observed in the spleen. The histopathological factor contributing to the death of this found dead animal was not determined.
- TERMINAL EUTHANASIA / Parental Generation: Microscopic Findings: Test item-related findings were observed in the stomach and thymus in both sexes.
Stomach: Minimal to moderate focal erosion/ulcer of the non-glandular stomach was observed in 5/12 High dose males. Mild to marked multifocal/diffuse squamous cell hyperplasia of the non-glandular stomach was observed in 12/12 High dose males, and at minimal to mild severity in 7/11 High dose females. 5/12 Mid dose males had minimal multifocal squamous hyperplasia of the non-glandular stomach and in 5/12 Mid dose females at minimal or mild severity. The incidence of minimal squamous cell hyperplasia of the non-glandular stomach in 2/12 Low dose females is considered comparable to the incidence of 1/12 in Control females and not related to test item administration. Submucosal focal/multifocal, mixed/neutrophilic inflammatory cell infiltrate was observed in the non-glandular stomach of 6/12 High dose males. Mucosal focal/multifocal neutrophilic inflammatory cell infiltrate was observed in the non-glandular stomach of 3/12 High dose males.
Thymus: Minimal reduced cellularity of the cortex was observed in the thymus in 3/12 High dose males and 4/11 High dose females. In the Mid dose females reduced cellularity of the thymus was observed in 4/12 animals. The incidence of minimal decreased cellularity in 2/12 Low dose females is considered as within the normal background.
All other changes are commonly seen in control and/or treated animals, or were without meaningful differences in severity and incidence, therefore were regarded as incidental, procedure-related or a common background.
- NON-PREGNANT FEMALES / Parental Generation (one high dose female #4508): microscopic findings: Organs from the reproductive system (ovary, oviduct, uterus, cervix and vagina from female; testes, epididymis, prostate, seminal vesicles and coagulating glands from males) were microscopically examined from the non-pregnant High dose female and its mating partner but did not reveal any test item-related changes.

Histopathological findings: neoplastic:

no effects observed

Details on results:

-mortality and morbidity:
No test item related mortality was observed in the study.
One Low dose male (#2011) was found dead on Day 23, no clinical signs were shown by this animal prior to death. This case was considered incidental, not related to the test item administration as no test item-related macroscopic or microscopic changes were observed.
-clinical observations:
No test item related clinical signs were observed in the study.
Noisy respiration was detected for a High dose male (#4002) on Days 24-25.
A nodule (1-2 cm) was observed for one Control female (#1502) in the abdominal area from Day 38 until termination.
Alopecia was observed on both forelimbs of another Control female (#1510) from Day 38 until termination.
These findings were considered incidental, not related to the test item administration.
-body weight and body weight gain:
Test item related adverse effect was observed on body weight parameters in High dose (450 mg/kg bw/day) males. No test item related adverse effect on body weight or body weight gain was detected in High dose females, or in the Mid or Low dose (150 or 60 mg/kg bw/day, respectively) animals (males or females).
In the first week of treatment, the High dose males had almost no growth, compared to Control males. The difference in body weight compared to control reached statistical significance by the end of the treatment period (body weight lower by 7% at p<0.05 and body weight gain lower by 47% at p<0.01). These results of the High dose males were considered as a test item related adverse effect (and suitable for a High dose in an OECD No. 422 study).
No effect on body weight parameters was seen in Mid and Low dose males.
No clearly adverse effect on body weight parameters was seen in the test item treated females when compared to concurrent controls (Table 2). The High dose weight gain was lower than the pre-treatment period and lower than typical historical control data for the pre-mating period, although in this study the control weight gain was similar to the High dose. The observed body weight or body weight gain values were comparable to the control level at the end of the pre-mating period, gestation or lactation in all dose groups.
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 males
(450 mg/kg bw/day) during the treatment period, no effect was observed in the High dose females, or Mid and Low dose groups (150 and 60 mg/kg bw/day, respectively) of both sexes.
In case of High dose males, reduced values compared to control were recorded during the entire treatment period, statistical significance reached (p<0.01). Based on the body weight values, the observed values were considered as a test item related adverse effect (probably secondary to gastric irritation caused by the test item).
No effect on food consumption was considered in Mid and Low dose male animals. Although the mean food consumption calculated for the entire treatment period in the Mid dose males was statistically significantly lower than Control (by 5.6%, p<0.01), but the difference originated from a transient reduced food consumption of the first week (no statistically significant differences were observed on the other three weeks thereafter), this it was considered as a palatability issue, not being a test item related effect.
No statistically significant effect was observed in test item treated female animals. Although slightly lower than control-level food consumption was recorded for High dose females during the pre-mating (11% below control in the first week), gestation (~5% for GD0-GD20) or lactation period (6.2% during the last part of the lactation period i.e. maximal milk production period), although the differences were not great, the trends indicate some effect which may be below the level considered as adverse.
-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
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. The sporadic statistical differences in the ratio of reticulocytes and eosinophils as well as the prothrombin time in High dose males were not outside the normal control range, based on historic control data.
-Clinical chemistry
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.
Increased bile acid concentration was observed in High dose males (by >300%, at p<0.01) and High dose females (by 65%, statistically not significant), although a statistically significant increase was also seen in Low dose males (by >100%, at p<0.01) without dose response
The differences between groups are within the ranges seen in similar study control groups, the dose response was not consistent, there were no adverse histological hepatic findings or any other evidence in clinical pathology for hepatic effects of the test item; hence the apparent elevation in some groups is not considered to reflect an adverse effect of treatment.
-Urinalysis
No test item-related changes were observed in the urinalysis parameters in male and female animals of any dose groups when compared to control. Occasional differences with statistical significance but without dose response were considered as animal variability.
-organ weights:
Parental Males
Test item-related effect was observed in the thymus weight of High dose males compared to control animals.
Terminal body weights of High dose males were statistically significantly lower than Control (by approx. -8%, the difference was statistically significant at p<0.01). Treatment-related decrease was observed in the thymus weights of Mid and High dose males and there was a microscopic correlate for the weight reduction in High dose males. The thymus changes are very common in stressed animals where markedly reduced body weight growth occurs in the first week of treatment (as in this study); since the effect is considered to be related to body weight and not a direct effect on the thymus, it is not considered as an adverse effect of treatment.
There were statistically significant differences among groups in the weights of a few other organs (brain, heart, seminal vesicle and liver), either in the absolute and/or body weight / brain weight related value when compared to Control. However, in the absence of any macroscopic or microscopic correlate, these weight changes were considered as not related to test item administration.
Parental Females
Test item-related effects were observed in the thymus 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.
Weight differences were observed in the thymus and liver weights. There was macroscopic and microscopic correlate for the thymus weight reduction in females, however, there was no correlate for the weight increase in the liver.
In males a reduced thymus weight and cellularity was ascribed to a general stress effect rather than a direct effect of the test item on the thymus; in High dose females, a clear gastric irritation seen at histology and non-statistical trend for lower food intake are considered to be related to a non-specific stress which is a common cause of these thymic changes. Liver hypertrophy is common in animals treated with xenobiotics, but when the effect is below about 15% (of increase liver weight relative to body weight) the change is usually too small to be detectable by histology examination. The liver weight difference in the absence of histology changes is considered to be non-adverse.
There were no other statistically significant or biologically relevant differences among groups in the weights of organs measured when compared to Controls in females.
-pathology evaluation:
FOUND DEAD ANIMAL / Parental Generation
One Low dose male (#2011) was found dead on Day 23 (no clinical signs were shown by this animal prior to death).
Macroscopic Findings
Necropsy revealed diffuse dark red discolouration and enlargement of all lobes of liver and lungs.
Microscopic Findings
Microscopically, mild congestion correlated with the macroscopic findings in the liver and lungs. Minimal extramedullary haematopoiesis was observed in the spleen. The histopathological factor contributing to the death of this found dead animal was not determined.

TERMINAL EUTHANASIA / Parental Generation
Macroscopic Findings
Treatment related focal/multifocal thickness of the wall of non-glandular stomach was observed in 9/12 High dose males. Small thymus was observed in 2/11 High dose females.
All other observed changes were considered incidental or a common background.
Microscopic Findings
Test item-related findings were observed in the stomach and thymus in both sexes.
Stomach: Minimal to moderate focal erosion/ulcer of the non-glandular stomach was observed in 5/12 High dose males. Mild to marked multifocal/diffuse squamous cell hyperplasia of the non-glandular stomach was observed in 12/12 High dose males, and at minimal to mild severity in 7/11 High dose females. 5/12 Mid dose males had minimal multifocal squamous hyperplasia of the non-glandular stomach and in 5/12 Mid dose females at minimal or mild severity. The incidence of minimal squamous cell hyperplasia of the non-glandular stomach in 2/12 Low dose females is considered comparable to the incidence of 1/12 in Control females and not related to test item administration. Submucosal focal/multifocal, mixed/neutrophilic inflammatory cell infiltrate was observed in the non-glandular stomach of 6/12 High dose males. Mucosal focal/multifocal neutrophilic inflammatory cell infiltrate was observed in the non-glandular stomach of 3/12 High dose males.
Thymus: Minimal reduced cellularity of the cortex was observed in the thymus in 3/12 High dose males and 4/11 High dose females. In the Mid dose females reduced cellularity of the thymus was observed in 4/12 animals. The incidence of minimal decreased cellularity in 2/12 Low dose females is considered as within the normal background.
All other changes are commonly seen in control and/or treated animals, or were without meaningful differences in severity and incidence, therefore were regarded as incidental, procedure-related or a common background.

NON-PREGNANT FEMALES / Parental Generation
One non-pregnant High dose female was identified in the study (#4508).
Macroscopic Findings
Necropsy examination did not show any relevant test item related change in the non-pregnant female and their male mating pair in the High dose group (only multifocal thickness of the non-glandular stomach was observed in both animals).
Microscopic Findings
Organs from the reproductive system (ovary, oviduct, uterus, cervix and vagina from female; testes, epididymis, prostate, seminal vesicles and coagulating glands from males) were microscopically examined from the non-pregnant High dose female and its mating partner but did not reveal any test item-related changes.

-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.
No statistically significant or biologically relevant changes in the T4 thyroid hormone concentration was detected in the test item treated parental males when compared to control.
No relevant changes were noted in the absolute thyroid or relative (to body) thyroid weights of male and female animals in any dose groups. Furthermore, no histopathology (microscopic) findings were detected in any test item treated animals (the bilateral enlargement recorded in a Low dose female (#2505) at necropsy was not confirmed by histopathology).

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

150 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

Key result

Dose descriptor:

NOAEL

Remarks:

local toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

150 mg/kg bw (total dose)

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

A GLP Validation of the Analytical Method Study of Disodium Lauryl Sulfosuccinate (EC 939-638-8)

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 TENSOMILD H026 in formulations, primarily to support OECD No. 422 study (Study code: 20/125-220P).

The procedure was found to be suitable for the analysis. A summary of the method parameters is presented in the  table below. All validation parameters passed versus acceptance criteria.

 

Table. Results of the Method Validation (20/125-316AN)

Selectivity	No interfering component was observed with the control matrix
Reinjection repeatability (7 injections)	RSD% ≤ 2.3%
Linear range	100 – 1000 µg/mL of the supplied test item
Limit of Quantification of the method (LOQ)	~100 µg/mL of the supplied test item
Theoretical quantification limit from the vehicle	~0.625 mg/mL of the supplied test item
Recovery of the test item from vehicle (at ~5 and ~100 mg/mL conc. in ultrapure water)	99% and 101%
Precision (RSD%) of formulations from vehicle (at ~5 and ~100 mg/mL conc. in ultrapure water)	1.3% and 1.4%
Stability of the diluted samples in the autosampler for 360 and 800 µg/mL	At least 46 hours (98 and 96%)
Stock solution (~2 mg/mL) stability at 5 ± 3°C	At least 3 days (95%)
Formulation stability at room temperature (at ~5 and ~100 mg/mL conc. in ultrapure water)	At least 3 days (94% and 93%)
Formulation stability at 5 ± 3°C (at ~5 and ~100 mg/mL conc. in ultrapure water)	At least 3 days (92% and 93%)

Conclusions:

Based on the existing results of this study, the following No-Observed-Adverse-Effect Levels (NOAELs) were considered:
The NOAEL for systemic toxicity of the parental generation: 150 mg/kg bw/day (based on body weight and food consumption effects in males at 450 mg/kg bw/day).
The NOAEL for local toxicity of the parental generation: 60 mg/kg bw/day (based on the local irritation in the stomach in both sexes in the 150 and 450 mg/kg bw/day dose groups).

Executive summary:

The purpose of this OECD No. 422 study was to obtain information on the possible toxic effects of TENSOMILD H026 (containing Disodium lauryl sulfosuccinate (EC 939-638-8) as active ingredient) test item following repeated (daily) administration by oral gavage to Wistar (Crl:WI) rats at 3 dose levels. A control group received the vehicle only (distilled water).

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 Day 13 post-partum.

The dose levels were selected by the Sponsor based on the results of the Dose Range Finding (DRF) study of the test item performed at the Test Facility (Study code: 20/125-220PE) and information from an OECD No. 422 study of a similar monoester compound (available at Sponsor). Based on those results, 450 mg/kg bw/day was selected as the High dose of this study. The experimental design is shown in the table below:

Group Number	Group designation	Dose level (mg/kg bw/day)	Concentration (mg/mL)	Dose volume (mL/kg bw)	Animal numbers
					Males	Females
1	Control	0	0	5	1001-1012	1501-1512
2	Low dose	60	12		2001-2012	2501-2512
3	Mid dose	150	30		3001-3012	3501-3512
4	High dose	450	90		4001-4012	4501-4512

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, the retained reproductive organs of all Control and High dose animals, stomach and thymus from all animals, the found dead animal or all organs with relevant macroscopic changes, and the male / female mating pair where no liveborn pups were achieved.

RESULTS

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

In summary, under the conditions of this study the daily administration of TENSOMILD H026 (containing Disodium lauryl sulfosuccinate (EC 939-638-8) as active ingredient) by oral gavage to Wistar rats at dose levels of 60, 150 or 450 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 (450 mg/kg bw/day) males.

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.

There were no differences between the control and test item treated groups with regard to reproductive ability, mating or gestation.

The number of implantation or liveborn pups, as well as the pre-natal, post-natal or total mortality values were comparable with the control values in all test item treated dose groups. No test item related macroscopic findings were recorded for F1 pups at necropsy.

There were test item-related differences on the offspring body weights or body weight gains in the High dose group (450 mg/kg bw/day) when compared to the study control values, and the High dose pup weights and weight gain were slightly below the contemporaneous historical control range. However, those differences were considered to be related to maternal toxicity (secondary effect), they were not considered as a test item related direct adverse effect.

Increased liver weights in High dose females (450 mg/kg bw/day) were not confirmed at histopathology, but adaptive hypertrophy of 11-14% as seen in this study is often not visible at histopathology; the organ weight difference was considered as non-adverse.

Test item-related changes were observed in the non-glandular stomach in both sexes of the Mid and High dose groups (150 and 450 mg/kg bw/day, respectively) with a dose dependent trend in incidence and severity, indicating local irritation by the test item; males were more affected.

The changes in the non-glandular stomach observed in this study were considered due to the irritant nature of the test item and is considered as a local effect rather than systemic toxicity of the test item. Such changes are commonly associated with lower food intake, lower weight gain (often transient, in the first week of the study) and non-specific stress effects in rats.

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

 

 

Based on the existing results of this study, the following No-Observed-Adverse-Effect Levels (NOAELs) were considered:

The NOAEL for systemic toxicity of the parental generation: 150 mg/kg bw/day (based on body weight and food consumption effects in males at 450 mg/kg bw/day).

The NOAEL for local toxicity of the parental generation: 60 mg/kg bw/day (based on the local irritation in the stomach in both sexes in the 150 and 450 mg/kg bw/day dose groups).

The NOAEL for reproductive effects of the parental generation: 450 mg/kg bw/day (based on the lack of relevant findings).

The NOAEL for pups’ (F1 generation) development and survival: 450 mg/kg bw/day (based on the lack of evidence of a direct effect on the pups at 450 mg/kg bw/day; although pup growth and body weight were lower than the concurrent control, but they were considered to be related to maternal toxicity).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

150 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Klimisch 1

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Organ:

kidney

liver

stomach

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 11 to 14-day dose range finding study was conducted with the registered substance in Wistar rats by oral gavage at 0 (distilled water), 100, 300, 600 and 1000 mg/kg bw/day (low, mid, mid-high and high dose groups, respectively) in order to determine the dose levels for a subsequent OECD No. 422 study (Hargitai, 2022). The vehicle-control, low, mid, and high dose groups were treated in the study for 14 consecutive days; the treatment period was only 11 days for the mid-high dose group. All dose formulations were homogenous. The measured test item concentration in the dosing formulations varied between 93% to 101% of the nominal concentration. Test item related mortality was observed: four female and three male animals were found dead in the High dose group (1000 mg/kg bw/day) and one High dose male was pre-terminally euthanised at the beginning of the study (Days 0-3). The overall pattern of necropsy observations indicated significant gastric effects and stress-related changes in the High dose group. Hunched back, noisy respiration and soft faeces were observed in the Mid-High dose (600 mg/kg bw/day) males and females; decreased activity, piloerection, partially closed eyelids, increased salivation and red discharge (nose/snout) were observed only in male animals of this dose group. Noisy respiration was also observed in the Mid dose (300 mg/kg bw/day) male animals and in the Low dose (100 mg/kg bw/day) female animals. Significant body weight loss was seen in the Mid-High dose males and females on the first week of treatment. Males gained some weight thereafter, but females did not start to gain weight on the second week. No test item related adverse effect was observed on food consumption (the statistically significantly lower food consumption observed on the first week in Mid-High dose males and all test item treated females showed recovery on the second week). There were no clearly adverse findings in haematology or clinical chemistry. Decreased weights of thymus and increase weights of adrenal, were seen in males of the Mid-High dose group. Increased relative liver weights were observed in Mid and Mid-High dose females. Increased stomach weights (absolute and relative to body/brain weight) were recorded in Mid-High dose males and females and in Mid dose females. Necropsy observations correlated with organ weight changes, with indications of effects compatible with stress (thymus and adrenals) in the Mid-High dose group and gastric effects suggesting irritation in the Mid and Mid-High dose groups. In conclusion, the dose levels of 1000 and 600 mg/kg bw/day were above the Maximum Tolerable Dose based on mortality, clinical signs, body weight changes and necropsy findings. At 300 mg/kg bw/day dose level, there were no evident effects in males, but gastric changes in weight and macroscopic irritation and increased liver weight changes were observed in females. Therefore, a dose level of approximately 400-450 mg/kg bw/day seems to be acceptable for the High dose level of the upcoming OECD No. 422 study.

A key subacute OECD No. 422 study was conducted with the registered substance in Wistar rats (12/sex/group) by oral gavage at 0 (distilled water), 60, 150 and 450 mg/kg bw/day (Hargitai, 2022). 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 7.8. 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, the retained reproductive organs of all Control and High dose animals, stomach and thymus from all animals, the found dead animal or all organs with relevant macroscopic changes, and the male / female mating pair where no liveborn pups were achieved. Dosing formulations were analysed for concentration and/or homogeneity on three occasions during the study. Overall, the formulations were considered adequate for the study. In summary, under the conditions of this study the daily administration of TENSOMILD H026 (containing Disodium lauryl sulfosuccinate (EC 939-638-8) as active ingredient) by oral gavage to Wistar rats at dose levels of 60, 150 or 450 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 (450 mg/kg bw/day) males. 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. Increased liver weights in High dose females (450 mg/kg bw/day) were not confirmed at histopathology, but adaptive hypertrophy of 11-14% as seen in this study is often not visible at histopathology; the organ weight difference was considered as non-adverse. Test item-related changes were observed in the non-glandular stomach in both sexes of the Mid and High dose groups (150 and 450 mg/kg bw/day, respectively) with a dose dependent trend in incidence and severity, indicating local irritation by the test item; males were more affected. The changes in the non-glandular stomach observed in this study were considered due to the irritant nature of the test item and is considered as a local effect rather than systemic toxicity of the test item. Based on the existing results of this study, the following No-Observed-Adverse-Effect Levels (NOAELs) were considered: The NOAEL for systemic toxicity of the parental generation: 150 mg/kg bw/day (based on body weight and food consumption effects in males at 450 mg/kg bw/day). The NOAEL for local toxicity of the parental generation: 60 mg/kg bw/day (based on the local irritation in the stomach in both sexes in the 150 and 450 mg/kg bw/day dose groups).

Subchronic toxicity

A 90-day study was not available for the registered substance, however read across data were available from a category member, CAS No. 37294-49-8 (Disodium C-isodecyl sulphonatosuccinate)  and further subchronic toxicity testing is  planned and will be updated later when results are available (currently waived in the dossier).

- For this read across substance, a 14-day dose rang finding study was also conducted as supporting study (Hansen, 2013c). Rats were treated once daily with a test item (47% purity) at 100, 300 and 1000mg act. ingr./kg bw/day by oral gavage administration. None of the animals died prematurely. The body weight of the male rats treated orally at 1000 mg/kg bw/day was slightly decreased on test days 8 and 15 compared to the control group. Body weight gain and body weight at autopsy changed accordingly. None of the male and female rats treated orally at 100, 300 or 1000 mg/kg bw/day revealed any test item-relatedchanges in behaviour, external appearance or faeces. No changes in the relative food consumption were noted at any of the tested dose levels. At macroscopic inspection at necropsy, test item-related changes in the stomach (detachment of mucosa, haemorrhagic foci, mucosa thickened/swollen, ulcer and cardia thickened) were noted for the animals treated with 1000 mg /kg bw/day. Further, the absolute kidney weights of the high dosed animals were decreased. In conclusion, 300 mg/kg body weight can be considered as NOAEL. This was comparable to the registered substance, therefore read across was considered valid.

- A 90-day toxicity study was performed in rats with this read across test item containing ca. 50% active ingredient (Tegeris and Underwood, 1975a) at 0.25%, 1.00% and 4.00% in the diet (corresponding to mean calculated test article intake of 188, 750 and 3000 mg act.ingr./kg bw/day). At the highest dose levels, it caused a significant difference in body weight gain, most probably related to an effect by the compound during absorption from the gastro-intestinal tract. There was one mortality in the high dosed females. Haematology and serum analysis also indicated toxicity at the high dose, whereas at the medium dose, only some organ-to-body weight changes were observed (e.g. liver weight increases in both sexes). Other organ-to-body weight changes were also observed at the high dose group (e.g. slightly decreased gonad weights in males; decreased gonad weight in females); increased relative kidney weights were observed in all female dose groups. Chronic renal disease (mild) was observed in 1/20 males and 6/20 females of the high dose groups. The high dose was considered to be toxic, whereas the changes of the medium dose may be considered adaptive. Therefore 1% (750  mg/kg bw/day) may be considered as NOAEL and 0.25 (174 mg/kg bw/day) as NOEL.

- A 90-day toxicity study in dogs was performed with test item containing ca. 50% active ingredient (Tegeris and Underwood, 1975b) at 0.12%, 0.50% and 2.00% in the diet (corresponding to mean calculated test article intake of 30, 125 and 500 mg act. ingr. /kg bw/day). There was a decreased feed consumption, feed efficiency and corresponding weight loss in the high dose (2.00%) group of dogs. Also testicular atrophy and hepatic fatty infiltration were observed in the same group without functional impairment of the liver. These observations were not considered to be direct toxic manifestations of the test item, as disturbed fat absorption is known as a physiological action of the sulfosuccinate class of compounds. Effects at the highest dose were considered to be secondary manifestations to disturbed fat absorption at these high multiples of human intake. The dose of 500 mg/kg bw (2% in the diet) was considered NOAEL.

- The dog species was considered to be less appropriate due to the disturbed fat absorption, by which systemic effects could not be correctly interpreted. The NOAEL of 750 mg/kg bw/day in rats was selected as main endpoint; this was also consistent with the selection of the rat as main species and NOAELs from the Di-ester substances.

- In conclusion for the subchronic toxicity a NOAEL of 1% in the diet, corresponding with 750 mg/kg bw, was obtained in a 90 -day study with read across substance CAS No. 37294 -49 -8.

Conclusion

- For risk characterisation, the paternal/maternal NOAEL of 150 mg/kg bw in the OECD 422 study with the registered substance was selected as most conservative value.

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

 

Justification for classification or non-classification

As there were no relevant findings below classification threshold of 100 mg/kg bw, classification for repeated dose toxicity is not warranted according to CLP (No. 1272/2008 of 16 December 2008)..