Butanedioic acid, 2-sulfo-, mono(C16-18 and C18-unsatd. alkyl) esters, ammonium sodium salts

Additional toxicological data

Administrative data

Endpoint:

additional toxicological information

Cross-reference

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

repeated dose toxicity: oral, other

Remarks:

DRF (14 days) for OECD 422

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

30 November 2020 to 11 July 2022

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Dose range finding study for OECD 422

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: This non-GLP preliminary Dose Range Finding study did not follow a specific OECD guideline, but was designed to allow selection of appropriate dose levels for the upcoming OECD No. 422 study. This study was conducted in accordance with a written Study Plan; it was authorized by the Sponsor and Test Facility Management and followed the applicable Standard Operating Procedures.
- Short description of test conditions: Four male and four female Wistar rats per group were treated for 3, 8 or 14 consecutive days in order to obtain preliminary information on the potential toxicity of the test item following repeated administration at 4 dose levels. The control group was treated with the vehicle only (propylene glycol).
The first day of dosing was designated as Day 0 for each animal. Based on the results of the first three days of dosing (mortalities in the High Dose group), the High dose level was decreased to 600 mg/kg bw/day. An additional dose group (High dose 2 group, 600 mg/kg bw/day, 4 animals/sex) was started on 14 December 2020 to provide additional data for dose selection for the upcoming OECD No. 422 study (Study code: 20/127-220P). Then, based on the clinical symptoms and serious body weight loss in the Mid and High dose 2 groups, the Mid and High dose 2 animals were not treated from Day 8 to get information on the recovery of gastric effect.
All surviving animals were terminated on Day 14.
- Parameters analysed / observed: Mortality/Morbidity Checks, Clinical Observations, Cage Side Observations, Detailed Clinical Observations, Body Weights, Food Consumption, Clinical Pathology (Haematology, Clinical Chemistry), Gross Pathology, Organ Weights.
Histopathological examination was performed on the liver in case of the Control and Low dose animals.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI (Wistar)

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)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Adult rats, males 9-10 weeks and females 8-9 weeks old at the start
- Weight at study initiation: Males: 360-409 g; females: 225-265 g at the start of the treatment. The weight difference did not exceed ± 20% of the mean weight for each sex at onset of treatment.
- Fasting period before study: Not provided.
- Housing: Rodents were group housed (2 animals/sex/group per cage) in type II and/or III polycarbonate cages. "SAFE 3/4-S" bedding (Batch No.: 03027200710 and 03027201022, Expiry date: 10 July 2023 and 22 October 2023) and "SAFE crinklets natural" nesting material (Batch No.: 05072200405 and 05072200824, Expiry date: 05 April 2023 and 24 August 2023) produced by J. Rettenmaier & Söhne GmbH+Co.KG (Address: Holzmühle 1, D-73494 Rosenberg, Germany) allowed digging and other normal rodent activities. Nesting material and certified cardboard hiding tubes (Fun tunnel; supplied by LBS (Serving Biotechnology) Ltd, Unit 20, Gatwick Business Park, Kennel Lane, Hookwood, Surrey, RH6 0AH, United Kingdom) were also provided to the animals.
- Diet (e.g. ad libitum): Animals received ssniff® SM R/M "Autoclavable complete diet for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH (Address: Ferdinand-Gabriel-Weg 16, D-59494 Soest, Germany), ad libitum.
- Water (e.g. ad libitum): Animals received tap water from the municipal supply, as for human consumption from a 500-mL bottle, ad libitum.
- Acclimation period: 6 days

DETAILS OF FOOD AND WATER QUALITY: The standard content of the diet and Certificates of Analysis (Batch No.: 713 70882, Expiry date: April 2021) as provided by the Supplier were included in the raw data and will be archived.
The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
Water quality control analysis was performed at least once every three months and microbiological assessment was performed monthly, by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, Address: H-8200 Veszprém, József Attila u. 36., Hungary). The quality control results were retained in the Archives of the Test Facility, one certified copy was included in the raw data and will be archived.
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): 21.2-24.9°C (target: 22 ± 3 °C)
- Humidity (%): 26-51 % (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: 09 December 2020 (males/females) To: 23 December 2020 (last scheduled necropsy)

Route of administration:

oral: gavage

Details on route of administration:

A constant dose volume of 5 mL/kg bw was administered daily to all animals by oral gavage using a disposable (plastic) gavage tube attached to a syringe. The individual volume of the treatment was based on the most recent individual body weight of the animals.
The formulations were continuously stirred with a magnetic stirrer until completion of treatment.

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 taken into consideration 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 a magnetic stirrer from the preparation until completion of each treatment.
Formulations were prepared fresh every day prior to administration to animals, this allowed their proper use.
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 magnetic stirring) with the needed amount of vehicle to reach homogeneity by visual observation.

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 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.
- Concentration in vehicle: 0,20, 60, 200/120 and 120 mg/mL for the Control, Low dose, Mid dose, High dose and High dose 2 groups respectively
- Amount of vehicle (if gavage): Dose volume = 5 mL/kg bw
- Lot/batch no. (if required): 1920944
- Purity: anionic matter (method: UNE 55-520-91) 5,710% (Standard 4,000 – 6,000%); residual sulfite (method 8607) 0.2 MEG (Standard 0,0 – 0.9 MEG)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Sample collection was performed on one occasion immediately after formulation preparation in the Pharmacy of the Test Facility by a responsible member of the Analytical Department.
On the sampling occasion, top, middle and bottom duplicate samples were taken from test item formulations for homogeneity and concentration 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 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.
Any remaining samples (back-up set) were discarded following acceptance of the results of the formulation analysis by the Contributing Scientist (Analyst) and Study Director.
Test item content and the homogeneity of gavage formulations were determined with the developed HPLC-MS/MS method. Formulation analysis was performed according to this validated method (Study code: 20/127-901ANE).
Formulation samples were kept at room temperature until shipment. Samples to be analysed were shipped to the Test Site as soon as practical after collection for concentration measurements:
Fumoprep Ltd., H-1044 Budapest, Ipari Park u. 10.,Hungary.
The number and date of shipments were agreed with the Principal Investigator (PI).
The formulation analysis was conducted under the control of the Principal Investigator in compliance with the relevant SOPs of the Test Site for analytical work.
Analysis of the formulations for homogeneity and/or concentration of test item was performed using in the Analytical Department of the Test Facility on one occasion.
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) had to be less than 10%.
All test item formulations were found to be in 100 ±10% of nominal concentrations, thus they were considered acceptable. Formulations were also shown to be homogeneous (the relative standard deviation was in the 1.05-5.32 % range, which was in line with the criterion of being less than 10%).
No test item was identified in the vehicle control sample.

Duration of treatment / exposure:

Treatment period was Days 0-13 in case of Control and Low dose groups, Days 0-7 in case of Mid dose group and Days 0-2 in case of High dose 2 group. The dose level for High dose group was 1000 mg/kg bw/day for the first two days, then it was decreased to 600 mg/kg bw/day. The animals died on Day 2 or Day 3.

Frequency of treatment:

once daily

Dose / conc.:

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

Remarks:

Control group: Treatment period was Days 0-13

Dose / conc.:

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

Remarks:

Low dose group: Treatment period was Days 0-13

Dose / conc.:

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

Remarks:

Mid dose group : Treatment period was Days 0-7

Dose / conc.:

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

Remarks:

High dose: The dose level for High dose group was 1000 mg/kg bw/day for the first two days, then it was decreased to 600 mg/kg bw/day. The animals died on Day 2 or Day 3.

Dose / conc.:

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

Remarks:

High dose 2 group: Treatment period was Days 0-2

No. of animals per sex per dose:

4

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Four male and four female Wistar rats per group were treated for 3, 8 or 14 consecutive days in order to obtain preliminary information on the potential toxicity of the test item following repeated administration at 4 dose levels. The control group was treated with the vehicle only (propylene glycol).
The first day of dosing was designated as Day 0 for each animal. Based on the results of the first three days of dosing (mortalities in the High Dose group), the High dose level was decreased to 600 mg/kg bw/day. An additional dose group (High dose 2 group, 600 mg/kg bw/day, 4 animals/sex) was started on 14 December 2020 to provide additional data for dose selection for the upcoming OECD No. 422 study (Study code: 20/127-220P). Then, based on the clinical symptoms and serious body weight loss in the Mid and High dose 2 groups, the Mid and High dose 2 animals were not treated from Day 8 to get information on the recovery of gastric effect.
All surviving animals were terminated on Day 14.
- Rationale for animal assignment (if not random): At the end of the acclimatisation period (Day -1), the animals were assigned to their respective dose groups by randomisation based on body weights. All animals were within 20% of the overall mean at the start of the study (for each sex). Animals were randomly allocated to the control and dose groups based on the most recent actual body weight; PROVANTIS v.9 software was used in order to verify homogeneity/variation among/within groups. Males and females were randomised separately.
- Fasting period before blood sampling for clinical biochemistry: Yes, an overnight period of food deprivation
- Rationale for selecting satellite groups: See explanation under dose selection rationale
- Post-exposure recovery period in satellite groups: See explanation under dose selection rationale
- Section schedule rationale (if not random): Gross necropsy was performed on each animal irrespective of the date of death, including the animals found dead or euthanized pre-terminally in extremis. Organ weight measurement was performed in case of pre-terminal animals but found dead animals, no organs or tissues were retained.
All surviving animals were euthanized at termination on Day 14. Gross necropsy was performed on each animal. Weight of selected organs was measured, and selected organs and tissues were retained.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day). The principles and criteria summarized in the OECD Humane Endpoints Guidance Document No. 19 were taken into consideration.
Any clinical sign noted during dosing or at any other occasions were recorded at the time seen.
General (routine) clinical observations were made twice daily, once in the morning (am) and once in the afternoon (pm). No general observation was made in the morning on those days when detailed clinical observation is scheduled.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made on all animals outside the home cage in a standard arena prior to the first treatment (to allow for within-subject comparisons) and daily thereafter. It was performed after treatment (1-2 hours after dosing as no peak period of effects was observed on Day 0 after dosing).
Any clinical signs, pertinent behavioural changes and all signs of toxicity including mortality were recorded including onset, degree and duration of signs as applicable. Signs evaluated included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), bizarre behaviour (e.g. self-mutilation, walking backwards) were recorded. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. Any signs of neurotoxicity were specifically documented and reported (no such a case occurred).

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each animal was recorded with a precision of 1 g at randomisation (pre-treatment period, Day -1), on the first day of treatment (Day 0, prior to start of treatment), then weekly, including on Day 13 (last treatment day) and prior to necropsy (fasted, on Day 14). Additional daily body weight measurements were performed from Day 9 until the end of the study in the Mid and High Dose 2 groups.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No feeding study
The determination of food consumption was performed for all groups weekly. The remaining, non-consumed food was weighed with a precision of 1 g. Daily food consumption was calculated for reporting purposes.

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

OPHTHALMOSCOPIC EXAMINATION: No
No ophthalmoscopy was conducted in this DRF study.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the treatment period, prior to scheduled necropsy on Day 14, clinical pathology investigations (haematology and clinical chemistry) was conducted.
- Anaesthetic used for blood collection: Yes (pentobarbital anaesthesia)
- Animals fasted: Yes (after an overnight period of food deprivation)
- How many animals: all animals
- Parameters examined:
RBC: Red Blood Cell (erythrocyte) count
WBC: White Blood Cell (leukocyte) count
Hgb: Haemoglobin concentration
Hct: Haematocrit (relative volume of erythrocytes)
MCV: Mean Corpuscular (erythrocyte) Volume
MCH: Mean Corpuscular (erythrocyte) Haemoglobin
MCHC: Mean Corpuscular (erythrocyte) Haemoglobin Concentration
RDW: Red Cell (erythrocyte) volume
Plt: Platelet (thrombocyte) count
MPV: Mean Platelet Thrombocyte volume
RETIC %: Reticulocyte count
NE %: Neutrophil
LY %: Lymphocyte
MO %: Monocyte
BA %: Basophil
EO %: Eosinophil
LUC %: Large Unstained Cells
No measurement of coagulation parameters was performed in the study.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the treatment period, prior to scheduled necropsy on Day 14, clinical pathology investigations (haematology and clinical chemistry) was conducted.
- Animals fasted: Yes (after an overnight period of food deprivation)
- How many animals: all animals
- 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)
Tot. Prot.: Total Protein concentration (g/L)
Alb. : Albumin concentration (g/L)
A/G: Albumin/globulin 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)

PLASMA/SERUM HORMONES/LIPIDS: No
No thyroid hormone analysis was conducted in this DRF study.

URINALYSIS: No
No urinary analysis was conducted in this DRF study.

NEUROBEHAVIOURAL EXAMINATION: No
No neurological assessment (Functional Observational Battery and SMART) was performed in this DRF study.

IMMUNOLOGY: No

OTHER: No vaginal smear was collected or examined in this DRF study.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Gross necropsy was performed on each animal irrespective of the date of death, including the animals found dead or euthanized pre-terminally in extremis. Organ weight measurement was performed in case of pre-terminal animals but found dead animals, no organs or tissues were retained.
All surviving animals were euthanized at termination on Day 14. Gross necropsy was performed on each animal. Weight of selected organs was measured, and selected organs and tissues were retained.
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.

The following organs were trimmed of fat and weighed in all animals after completion of the 14-day treatment:
With precision of 0.01g: Brain, Epididymides, Heart, Kidneys, Liver, Prostate, Seminal vesicles with coagulating glands, Spleen, Stomach, Testes, Thymus, Uterus including cervix
With precision of 0.001g: Adrenals, Ovaries, Thyroids with parathyroid glands
Paired organs were weighed together. Absolute organ weights were measured, and relative organ weights to the body and brain weights were calculated and reported. Significant difference in size was noted between paired organs, an individual weight of each organ was recorded.

HISTOPATHOLOGY: Yes (liver of Control and Low dose animals))
On completion of the macroscopic examination the following tissues and organs were retained from all animals:
Gross findings
Adrenals
Animal identification (Fixation and preservation only)
Aorta thoracic and abdominal
Brain (Section according to the STP recommendations)
Epididymis
Eye with the optic nerve (If applicable, parathyroid glands and optic nerves were 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 were 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 (1Duodenum, ileum and jejunum with Peyer’s patches)
Spinal cord (Transverse sections, 3 levels -cervical, thoracic and lumbar)
Spleen
Sternum with marrow
Stomach
Testis
Thymus
Thyroid with parathyroid gland 4
Tongue
Trachea
Urinary bladder
Uterus (Horns, body and cervix)
Vagina
The eyes with the optic nerve, testes and epididymides were retained in modified Davidson’s fixative. All other organs were retained in 10% buffered formalin solution.

Histopathological examination was performed on the liver in case of the Control and Low dose animals in order to determine the High dose level for the OECD 422 study, following consultation with the Sponsor. After finalization of the study report, the preserved organs and tissues will be discarded.

Optional endpoint(s):

Optional endpoints: Yes, based on the clinical symptoms and serious body weight loss in the Mid and High dose 2 groups, the surviving Mid and High dose 2 animals were not treated from Day 8 to get information on the recovery of gastric effect.

Statistics:

The normality and heterogeneity of variance between groups were checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests showed no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test was carried out. If the obtained result was positive, Dunnett (Multiple Range) test was used to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01 as appropriate. This parametric analysis is the better option when the normality and heterogeneity assumptions implicit in the tests were adequate.
If either of the Shapiro-Wilk or Levene tests showed significance on the data, then the ANOVA type approach was not valid, and a non-parametric analysis was required. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons were performed using Dunn test; identifying differences of <0.05 or <0.01 as appropriate.
For non-continuous data, the Cochran-Armitage test for trend was applied and the Chi-squared test was used for statistical differences relative to control.
For pathology data (macroscopic data) the Cochran-Armitage test for trend was applied, then if appropriate, the Chi-squared test homogeneity test. If significance was plausible based on a user-defined value (0.05), a pairwise test of each treatment group versus the control group was made. If the group size was <5 then Fisher’s Exact Test was used, if the group sizes were bigger then the Chi-squared test was used; identifying differences of <0.05, <0.01 or <0.001 as appropriate.
Note that the small group sizes meant that statistical comparisons alone were not appropriate to determine treatment effects but might be useful in data interpretation. Where appropriate, the Study Director evaluated if effects were toxicologically significant by comparison with historical control data.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

A range of symptoms (hunched back, piloerection, decreased activity, faeces liquid / soft, noisy respiration, red discharge around nose, wasted and general poor condition) were seen in Mid dose, High dose and High dose 2 animals indicating adverse effects; with no treatment effects in the Low groups.

Mortality:

mortality observed, treatment-related

Description (incidence):

Three animals were found dead and fourteen animals were pre-terminal euthanised during the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The Low dose group had reduced growth or weight loss in the first week, with a near normal growth rate thereafter. The Mid to High dose group survivors lost significant weight in the treatment phase, and continued to have weight loss even after treatment was halted The measured significant mean food consumption values for the Mid dose animals were outside of the historical control range therefore the effect is considered as test item related adverse effect.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Statistically significantly decreased food consumption was observed in the Low dose male (p<0.01) onDay 0-7 and Mid dose (p<0.01) male group on Day 0-7 and Day 0-13 and Mid dose female (p<0.01) group on Day 0-7 and Low dose female group (p<0.01) on Day 0-13.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The Low group had minor haematological differences of equivocal relationship with treatment.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Low dose males had no substantial effects in clinical chemistry; Low dose females had changes in serum enzymes suggestive of hepatic effects, but not indicative of a severe effect.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related effects were observed in the adrenal gland weights in Mid and High dose 2 males and liver weights in Low and High dose 2 females. The Low dose males had no evident changes in organ weight.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Other groups data for clinical pathology was either at euthanasia or after a period without test item treatment, hence are not useful for dose setting, although no pattern was seen to suggest any specific toxic mechanism.
Test item-related bilateral enlargement of the adrenal glands (indicative of stress) and all lobes of liver was observed in High dose 2 male. Multifocal red discolouration of the glandular stomach and multifocal thickness of non-glandular stomach (indicative of gastric irritation) was observed in two Mid dose males, one High dose 2 male and one High dose 2 female.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Mild to moderate periportal hepatocellular vacuolation was seen in Low dose females; it is considered as non-adverse in the context of this study, but it may possibly progress with longer exposure.

Details on results:

- Clinical observations:
Pre-terminal
Hunched back, piloerection, moderately decreased activity, liquid / soft faeces, wasted and red discharge (nose) were observed in one Mid dose male (#3002) from Day 78 to Day 12, the longevity of the symptom was 5, 5, 2, 3 and 5 days, respectively. Hunched back, piloerection, slight to moderate decreased activity, wasted and soft faeces (3/4) were observed in 4/4 Mid dose females and red discharge (nose or eyes) in 3/4 Mid dose females from Day 3 until pre-terminal euthanasia, the longevity of the symptoms was 9, 6, 3, 5 and 3 days, respectively.
From slight to extreme decreased activity, hunched back, piloerection and soft faeces were observed 4/4 animals in the High dose male group on Day 2. Hunched back (3/4), piloerection (2/4) and soft faeces (1/4) were observed on Day 2; hunched back (3/4), slight to extreme decreased activity (3/4), piloerection (3/4), red discharge (nose) (1/4), soft faeces (1/4) and liquid faeces (3/4) were observed in the High dose female group on Day 3.
Piloerection, liquid/soft faeces and red discharge (nose) were observed in 2/4 and slightly decreased activity and hunched back were observed in 1/4 High dose 2 males from Day 1 until pre-terminal euthanasia.
Surviving animals
No clinical signs were observed in the Low dose group during the study.
Piloerection was observed in 3/4 Mid dose males from Day 8 and Day 11. Red discharged (nose) was observed in 3/4 Mid dose males from Day 7 until death. Liquid faeces was observed in 3/4 and soft faeces was seen in 1/4 Mid dose males from Day 10 to Day 13, the longest period was 4 days. Hunched back was observed in 3/4 Mid dose males from Day 8 to Day 13, the longevity of the symptoms was 6 days. Slight to moderate decreased activity was observed in 3/4 Mid dose males from Day 11 to Day 13, the longest period was 3 days.
Soft/liquid faeces, decreased activity, hunched back, piloerection, wheal (left inguinal), crust (both fore paws) and red discharge (nose) were observed in 1/4 High dose 2 male from Day 3 until death. The longevity of the symptoms was 1,3 4, 4, 6, 3 and 4, respectively. Hunched back (2/4), piloerection (3/4), decreased activity (2/4), noisy respiration (1/4) and soft faeces (2/4) were observed in High dose 2 female animals from Day 4 until death.
In summary, a range of symptoms were seen in Mid dose, High dose and High dose 2 animals indicating adverse effects, with no treatment effects in the Low groups.
- Mortality: One animal was found dead in the High dose female group (#4504) on Day 3, hunched back, piloerection and soft faeces were observed prior to death.
One High dose 2 male (#4006) was found dead on Day 8 (1), soft faeces was observed. One High dose 2 female (#4508) animal was found dead on Day 8 (1), no abnormalities was found.
In the High dose group, all males (4/4) and three female (3/4) animal received pre-terminal euthanasia on Day 2 and Day 3, respectively. In the High dose 2 group, two males (2/4) received pre-terminal euthanasia on Day 8 (1) and Day 12 (5). In the Mid dose group one male # 3002 (1/4) on Day 12 and all female (4/4) animal received pre-terminal euthanasia on Day 7-9 and Day 12.
- Body weight and weight changes:
The Low dose males and females had reduced weight gain or weight loss (particularly in females) in the first week, however they had approximately normal weight growth thereafter (indicating a suitable dose level for a repeat dose main study). The Mid to High dose group survivors lost significant weight in the treatment phase and continued to have weight loss even after treatment was halted (these dose levels were >MTD).
- Food consumption (no feeding study):
Statistically significantly decreased food consumption was observed in the Low dose male (p<0.01) on Day 0-7 and Mid dose (p<0.01) male group on Day 0-7 and Day 0-13 and Mid dose female (p<0.01) group on Day 0-7 and Low dose female group (p<0.01) on Day 0-13.
The measured significant mean food consumption values for the Mid dose animals were outside of the historical control range therefore the effect is considered as test item related adverse effect.
- Haematological findings:
Data for the preterminal animals were not evaluated based on the quality of the blood samples because of the bad condition of the animals.
Statistically significant decrease was observed for reticulocytes% (p<0.01) in case of the Low dose male group. Value for the reticulocytes was outside the historical control range, but the relationship to treatment is equivocal.
At the Mid dose male group, there were statistical differences, but none appeared to indicate adverse effects of the test item (other than haemoconcentration in euthanised, poor condition animals). Most of the data were outside of the historical control data range.
At the High dose 2 groups, biologically significant differences were observed but data were not comparable to the Control because of the delayed start of the group.
- Clinical biochemistry findings:
Data for the preterminal animals were not evaluated based on the quality of the blood samples because of the poor condition of the animals.
At the Low dose female and male groups, statistically significantly decreased cholesterol (p<0.01 and p<0.05) was observed, both values were outside of the historical control range. Statistically significantly increased A/G ratio (p<0.05), alanine aminotransferase (p<0.01) and alkaline phosphatase (p<0.05) were observed in the Low dose male group and alkaline phosphatase (p<0.05) were observed in the Low dose female group. The majority of the data were outside of the historical control range therefore they considered as test item related effect, probably related to a hepatotoxicity.
At the Mid dose male group, there were statistical differences, but none appeared to indicate adverse effects of the test item (other than related to poor condition of the surviving animals). Most data were outside of the historical control data range.
At the High dose 2 groups, biologically significant differences were observed but data were not comparable to the Control because of the delayed start of the group.
- Organ weight findings including organ/body weight ratios:
In comparison to Controls, terminal body weights of animals were significantly different between males and females given 600 mg/kg bw /day and males given 300 mg/kg bw/day. Terminal weights were lower by -42.5 % (p<0.01) in terminal males given 300 mg/kg bw/day. In animals given 600 mg/kg bw/day, body weights were lower by – 37.9 % in males and – 34.9% in females.
Treatment-related effects were observed in the adrenal gland weights in males and liver weights in females.
In males given 300 mg/kg bw/day, absolute adrenal gland weights were larger by 17.8 %, related to body weights by 104% (p<0.01) and relative to brain weight by 25.1 % (p<0.05) and absolute liver weight was smaller by 27.8% compared to Control. In males given 600 mg/kg bw/day, absolute adrenal gland weight was larger by 54.2 %, related to body weights by 147.5% and relative to brain weight by 73.1%.
In females given 100 mg/kg bw/day, absolute liver weights were larger by 43.8 % (p<0.01), related to body weights by 46.5% (p<0.01) and relative to brain weight by 44.0 % (p<0.01). In females given 600 mg/kg bw/day, absolute liver weight was larger by 29.0 % (p<0.01), related to body weights by 97.6% (p<0.01) and relative to brain weight by 37.4% (p<0.01) and adrenal glands relative to body weight was larger by 55.9% (p<0.01) compared to Control.
Lower organ weights, including with statistical significance were observed in various organs and it is considered as a reflection of terminal body weight loss rather than the direct effect of the test item.
- Gross pathological findings:
Found dead
One female given 1000 m/kg bw/day (No.4504) was found dead on Day 3 of treatment. One male (No.4006) and one female (No. 4508) given 600 mg/kg bw/day were found dead on Day 8 of the treatment.
Soft faeces, decreased activity, hunched back and piloerection were the clinical signs shown by animal No. 4504 prior to death. Soft faeces were observed in animal No. 4006 prior to death and no clinical signs were observed in animal No. 4508 prior to death.
In animal No. 4504, necropsy revealed bilateral enlargement of the adrenal gland, non-collapsed lungs, diffuse dark red discolouration of the mesenteric lymph node, thin walled non-glandular region of the stomach with clear mucoid/foamy material in the stomach and diffuse dark red discoloration of the thymus.
In animal No. 4508, necropsy revealed small spleen and multifocal red discolouration of the glandular wall of the stomach.
In animal No. 4006, necropsy revealed small accessory sex glands (coagulating gland, prostate and seminal vesicles) and diffuse dark red discolouration of the thymus.
Cause of death of the found dead animals were not determined.
Pre-terminal euthanasia
Four males and three females given 1000 mg/kg bw/day, two males given 600 mg/kg bw /day and one male, and four females given 300 mg/kg bw/day were pre terminally euthanised.
Necropsy revealed bilateral enlargement of adrenal in 1/4 males and 3/3 females, enlargement of all lobes of liver in 2/3 females, pale multifocal discolouration of all lobes of liver in 3/3 females, red diffuse discolouration of all lobes of lungs in 2/4 males, thin wall glandular stomach in 2/4 males and 2/3 females, thin wall non glandular stomach and red diffuse mucosa of the glandular stomach in 2/4 males and 3/3 females given 1000 mg/kg bw /day. Enlargement of the stomach was observed in 2/4 males given 1000 mg/kg bw/day.
Small spleen was seen in 3/4 males and 3/3 females given 1000 mg/kg bw/day, 2/2 males given 600 mg/kg bw/day, 1/1 males and 4/4 females given 300 mg/kg bw /day.
Thymus was small in 2/4 males given 1000 mg/ kg bw/day, 1/2 males given 600 mg/kg bw/day, 1/1 male and 4/4 female given 300 mg/kg bw/day.
Thyroid and parathyroid was small in 1/4 males and 1/3 females given 1000 mg/kg bw/day.
Terminal euthanasia
Test item-related bilateral enlargement of the adrenal glands and all lobes of liver was observed in 1/1 male given 600 mg/kg bw /day.
Multifocal red discolouration of the glandular stomach was observed in 2/3 males given 300 mg/kg bw/day and 1/1 males and 1/3 females given 600 mg/kg bw/day. Multifocal thickness of non-glandular stomach was observed in 1/1 male and 1/3 females given 600 mg/ kg bw/day.
All other observed changes were considered secondary to bad condition or related to body weight loss.
- Histopathological findings: non-neoplastic:
Mild to moderate periportal hepatocellular vacuolation was seen in 4/4 Low dose females; it is considered as non-adverse in the context of this study, but it may possibly progress with longer exposure.

Dose descriptor:

other: At 100 mg/kg bw/day there was transient reduced growth and in females an increased liver weight with clinical pathology changes suggesting hepatotoxicity

Remarks:

The Low dose effects were compatible with longer repeat dose treatment

Effect level:

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

Based on:

act. ingr.

Sex:

female

Basis for effect level:

body weight and weight gain

serum/plasma biochemistry

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Conclusions:

Butanedioic acid, sulfo-, mono (C16-18 and C18-unsatd. alkyl) esters, ammonium sodium salts (CAS 147993-66-6; EC 604-617-1) formulated in propylene glycol was administered by oral gavage to Wistar rats for 3, 8 or 14 consecutive days at dose levels of 100, 300 or 600 or 1000 mg/kg body weight/day (Low, Mid, High dose 2 and High dose groups, respectively) at a dose volume of 5 mL/kg body weight. Exposure resulted in in body weight loss, clinical signs of poor condition and mortality in the High dose, High dose 2 and Mid dose groups.
The dose levels of 1000, 600 and 300 mg/kg bw/day were above the MTD. At 100 mg/kg bw/day there was transient reduced growth and in females an increased liver weight with clinical pathology changes suggesting hepatotoxicity. The Low dose effects were compatible with longer repeat dose treatment. The High dose level of the upcoming OECD No. 422 study should be up to approximately 100 mg/kg/day the suggested High dose.

Executive summary:

The objective of the study was to obtain preliminary information on the toxicity of the test item when administered daily to Wistar rats by oral gavage at four dose levels for up to 14 days in order to determine the dose levels for a subsequent OECD No. 422 study. Young adult animals were used in this study. Control animals were treated with vehicle only (propylene glycol). The dose levels were selected by the Sponsor in consultation with the Study Director based on the available data and information.

Female and male Wistar rats (8-10 weeks old animals) were treated in the study for 3, 8 or 14 consecutive days as shown below. The first day of dosing was designated as Day 0 for each animal. Based on the results of the first three days of dosing (mortalities in the High Dose group), the dose level was decreased. An additional dose group (High dose 2 group, 600 mg/kg bw/day, 4 animals/sex) was started on 14 December 2020 to provide additional data for dose selection for the upcoming OECD No. 422 study (Study code: 20/127-220P). Then, based on the clinical symptoms and serious body weight loss in the Mid and High dose 2 groups, the surviving Mid and High dose 2 animals were not treated from Day 8 to get information on the recovery of gastric effect.

 

Treatment Days (Days 0-13)#
Group No.	Group Designation	Dose level (mg/kg bw/day)	Concentration (mg/mL)	Dose volume (mL/kg bw)	Number of males	Number of females
1	Control	0	0	5	4	4
2	Low dose	100	20		4	4
3	Mid dose	300	60		4	4
4	High dose*	1000/600	200/120		4	4
5	High dose 2	600	120		4	4

Notes:

*The dose level for High dose group was 1000 mg/kg bw/day for the first two days, then it was decreased to 600 mg/kg bw/day. The animals reived pre-terminal euthanasia on Day 2 or Day 3.

#Treatment period was Days 0-13 in case of Control and Low dose groups, Days 0-7 in case of Mid dose group and Days 0-2 in case of High dose and High dose 2 group.

 

Mortality checking and clinical observations were performed twice daily. Body weight and food consumption were measured for all animals at least on Days 0, 7, and 13 or 9 (in case of Group 5), and for body weight measurement, prior to scheduled necropsy, fasted, on Day 14 or Day 10 (in case of Group 5). Following the daily repeated dose administration for 14 days, blood samples were collected for clinical pathology at necropsy from all animals. Gross macroscopic examination was performed at necropsy in each animal. Selected organs were weighed, and selected tissues were preserved in fixative.

Results:

All dose formulations were considered suitable for the study purposes.

Three animals were found dead and fourteen animals were pre-terminal euthanised during the study.

A range of symptoms (hunched back, piloerection, decreased activity, faeces liquid / soft, noisy respiration, red discharge around nose, wasted and general poor condition) were seen in Mid dose, High dose and High dose 2 animals indicating adverse effects; with no treatment effects in the Low groups.

The Low dose group had reduced growth or weight loss in the first week, with a near normal growth rate thereafter. The Mid to High dose group survivors lost significant weight in the treatment phase, and continued to have weight loss even after treatment was halted

The Low group had minor haematological differences of equivocal relationship with treatment. Low dose males had no substantial effects in clinical chemistry; Low dose females had changes in serum enzymes suggestive of hepatic effects, but not indicative of a severe effect. Other groups data for clinical pathology was either at euthanasia or after a period without test item treatment, hence are not useful for dose setting, although no pattern was seen to suggest any specific toxic mechanism.

Test item-related bilateral enlargement of the adrenal glands (indicative of stress) and all lobes of liver was observed in High dose 2 male. Multifocal red discolouration of the glandular stomach and multifocal thickness of non-glandular stomach (indicative of gastric irritation) was observed in two Mid dose males, one High dose 2 male and one High dose 2 female. Mild to moderate periportal hepatocellular vacuolation was seen in Low dose females; it is considered as non-adverse in the context of this study, but it may possibly progress with longer exposure.

Treatment-related effects were observed in the adrenal gland weights in Mid and High dose 2 males and liver weights in Low and High dose 2 females. The Low dose males had no evident changes in organ weight.

 

Butanedioic acid, sulfo-, mono (C16-18 and C18-unsatd. alkyl) esters, ammonium sodium salts (CAS 147993-66-6; EC 604-617-1) formulated in propylene glycol was administered by oral gavage to Wistar rats for 3, 8 or 14 consecutive days at dose levels of 100, 300 or 600 or 1000 mg/kg body weight/day (Low, Mid, High dose 2 and High dose groups, respectively) at a dose volume of 5 mL/kg body weight. Exposure resulted in in body weight loss, clinical signs of poor condition and mortality in the High dose, High dose 2 and Mid dose groups.

The dose levels of 1000, 600 and 300 mg/kg bw/day were above the MTD. At 100 mg/kg bw/day there was transient reduced growth and in females an increased liver weight with clinical pathology changes suggesting hepatotoxicity. The Low dose effects were compatible with longer repeat dose treatment. The High dose level of the upcoming OECD No. 422 study should be up to approximately 100 mg/kg/day the suggested High dose.