Sodium 1,2-diisobutoxycarbonylethanesulphonate

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 and 1000 mg solid/kg bw/day in a supporting 14-day dose range finding and a key combined repeated dose/reproductive toxicity study (OECD No. 422). The NOAEL for systemic toxicity of the parental generation was 1000 mg solid/kg bw/day.

Repeated dose toxicity was further tested in various species, including rats, dogs, rabbits and monkeys. The NOAEL of 750 mg/kg bw/day obtained in the key subchronic 90-day dietary toxicity study in rats was confirmed to be consistent with data from docusate sodium and category members in supporting studies in rats; other data from other species were tested with read-across substance and were of limited reliability and relevance and therefore not taken into account.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1969

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was conducted according to internationally accepted test guidelines and is considered relevant, adequate and reliable. There were some deviations from the study guidelines, however these did not affect the conclusions and the validity of the study.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

only one dose per test substance

GLP compliance:

no

Limit test:

yes

Species:

rat

Strain:

other: Charles River, albino

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Charles River Breeding Laboratories, North Wilmington, Mass.
- Age at study initiation: Not provided
- Weight at study initiation: Day0: mean body weight males: 121 g; mean body weight females: 101 g
- Housing: Individually in standard wire-bottomed steel rat cages
- Diet: Standard rat ration blended with the appropriate amount of test material in a Hobart Mixer. Fresh diets were prepared each week. Each rat was offered an amount of diet sufficient for one week ‘ad libitum’ feeding. However, checks were made periodically to ensure that the food jars were not empty
- Water: No data provided
- Acclimation period: Not provided

ENVIRONMENTAL CONDITIONS
Not provided

IN-LIFE DATES: From: To: Not provided

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: 1.0% in the feed
Taking into account a mean body weight of 250 g and a mean food consumption of 20g/rat/day
(Derelanko M.J., 2008, The Toxicologist's Pocket Handbook, Informa).
1% in the diet = 10000 mg/kg diet corresponds with 10 mg/g diet
20 g feed/rat (250g bw)/day = 80 g feed/kg bw/day = 0.8 g active ingredient/kg bw/day = 800 mg/kg bw/day.
A higher feed intake is possible, e.g. 1000 mg/kg at higher body weight and feed intake, but from a conservative viewpoint 750 mg/kg bw is taken.

DIET PREPARATION
- Rate of preparation of diet (frequency): Fresh diets were prepared each week
- Mixing appropriate amounts with (Type of food): standard rat ration

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
1.0%
Basis:
nominal in diet

No. of animals per sex per dose:

For Aerosol IB: 20 male and 20 female at 1.0% dietary level + 20 male and 20 female as control

Control animals:

yes, concurrent no treatment

Details on study design:

Experimental Animals
The animals employed in the study were Charles River strain (Charles River Breeding Laboratories, North Wilmington, Mass.) albino rats. Two hundred and eighty rats (140 males and 140 females) were selected for the experiment, ear-punched with the animal number assigned and housed individually in standard wire-bottomed steel rat cages. Each cage bore a color-coded card identifying the animal with respect to project number, test material assignment, individual animal number and sex.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: day 0, 15, 30, 45, 60, 75 and 90.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined : Yes
and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 84 days
- Anesthetic used for blood collection No data
- Animals fasted: Yes (fasted serum glucose concentration)
- How many animals: 5 rats of each sex (=10) and 10 control
- Parameters checked in table [No.IV and V] were examined.
Hematocrit Value
Erythrocyte Count
Hemoglobin Concentration
Total Leukocyte Count
Differential Leukocyte Count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 84 days
- Animals fasted: Yes(fasted serum glucose concentration)
- How many animals: 5 rats of each sex (=10) and 10 control
- Parameters checked in table [NoVI and VII] were examined.
Blood Urea Nitrogen (BUN) Concentration
Serum Alkaline Phosphatase (SAP) Activity
Serum Glutamic-Pyruvic Transaminase (SGPT) Activity
Fasted Serum Glucose Concentration

URINALYSIS: Yes
- Time schedule for collection of urine: after 84 days
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes
- Parameters checked in table [No.VIII] were examined.
Glucose Concentration
Albumin Concentration
Microscopic Elements Examination
pH
Specific Gravity

NEUROBEHAVIOURAL EXAMINATION: Yes , but in general, not specific
- Time schedule for examinations: abnormal reactions and death were recorded daily during the investigation
- Dose groups that were examined: control and 1.0% dose
- Battery of functions tested: sensory activity / grip strength / motor activity / other:No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes , no findings
HISTOPATHOLOGY: Yes , no findings

Other examinations:

No

Statistics:

Statistical analyses were conducted upon the absolute organ weights and their corresponding ratios to the weight of the body. An Analysis of
Variance was conducted first and any significant effects disclosed by that treatment were further studied by “t” –tests.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

smaller absolute liver weights among male rats

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Remarks:

1% in the diet

Sex:

male/female

Basis for effect level:

other: No toxicological findings (only a slight significant absolute liver weight decrease in males)

Critical effects observed:

not specified

Table 1. Organ Weight and Ratio:  Summary of Mean Values -    Organ: Liver

	Organ Weight (g)		Organ/Body Weight Ratio (g/100g)
Group	Males	Females	Males	Females
Control	19.2	11.2	4.21	4.03
1% in diet	15.8*	9.81	3.76	3.93

*Statistically significant difference at the 95% confidence level

Conclusions:

The comparisons of final body weights and total weight gains revealed no statistically significant differences between test and control animals.
No outstanding differences in food consumption were noted between test rats and control rats.
No deaths or abnormal behavioral reactions were noted among any of the animals employed in the study.
No outstanding differences between test and control rats were noted with respect to any of the blood parameters studied.
No significant differences between the urine of test rats and control rats were observed.
No outstanding differences between test and control rats were noted at the time of gross pathological examination.
The only statistically significant difference noted were smaller absolute liver weights among male rats fed 1.0% Aerosol IB.
There were no significant differences between the tissues of test and control rats observed upon histopathological examination.

Executive summary:

Six groups of 40 albino rats (20 male, 20 female Charles River Strain) plus 1 control group (20 male, 20 female) were fed with various test items mixed into the diet. The various test items were category members of the Sulfosuccinates Di-ester Group including Butanedioic acid, sulfo-, 1,4-bis (2-methylpropyl)ester, sodium salt. After 84 days 5 hematological values, 4 blood chemical values, 5 urinalysis values were measured for all animals. 40 tissues have been examined pathologically at the conclusion of the 90-days test period. Organ to body weight and organ to brain weight ratios were calculated. Slightly lower absolute liver weights were observed in males, however no significant differences in clinical blood chemistry studies and absolute organ weights have been detected. Body weights, organ to body weight ratios, hematologic studies and urinalysis were not different between test and control animals. No deaths or abnormal behavioral reactions occurred; no gross pathological or histological findings were noted.
 Administration of category members at 1% in the diet (10000 ppm equivalent to 750 mg/kg body weight/day on average basis ) for 90 days in rats did not result in any relevant changes in the subchronic toxicity study. The NOAEL was therefore considered to be 750 mg/kg bw/day. The IBT Report, supplemented by the Intox report and the Validation Report of October 15, 1983, may be considered a valid study and the data and conclusions relied upon.

Reference 2

Endpoint:

repeated dose toxicity: oral, other

Remarks:

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 July 2020 to 28 February 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Paris, 2016

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Paris, 2008

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI

Details on species / strain selection:

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

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, (Address: Sandhofer Weg 7, D-97633, Sulzfeld, Germany) from SPF colony
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Young adult rats, 8/9 weeks old (females/males) at start of the experiment and 12/13 weeks old (females/males) at mating.
- Weight at study initiation: Males: 392-472 g, females: 218-259 g (at the start of the treatment). The body weights did not exceed ± 20% of the mean weight for each sex at start of treatment.
- Housing: Rodents were group-housed, up to 2 animals of the same sex and dose group/cage, with the exception of the mating and gestation, delivery, lactation period, when they were paired or individually housed (with pups), respectively. Animals were housed in animal room 506 in type II, III and/or IV polycarbonate cages. SAFE 3/4-S Hygienic Animal Bedding (Batch number: 03027200428 / 03027200710, Expiry date: 28 April 2023 / 10 July 2023) and SAFE Crinklets Natural nesting material (Batch number: 05072191028 / 05072200405, Expiry date: 28 October 2022 / 05 April 2023) produced by J. Rettenmaier & Söhne GmbH+Co.KG (Address: Holzmühle 1, D-73494 Rosenberg, Germany) were used in the study. Group housing allowed social interaction. Deep wood sawdust bedding allowed digging and other normal rodent activities, while nesting material allowed normal nesting behaviour. Certified cardboard hiding tunnels (GLP Mini Fun Tunnels, Batch number: A/123) produced by LBS (Serving Biotechnology) Ltd. (Address: Unit 20, Gatwick Business Park, Kennel Lane, Hookwood, Surrey, RH6 0AH UK) were also provided to the animals.
- Diet (e.g. ad libitum): The animals received ssniff® SM R/M “Autoclavable complete diet for rats and mice –breeding and maintenance” (Batch number: 560 65984, Expiry date: 31 October 2020) produced by ssniff Spezialdiäten GmbH (Address: Ferdinand-Gabriel Weg 16, D-59494 Soest, Germany), ad libitum.
- Water (e.g. ad libitum): The animals received tap water from the municipal supply, as for human consumption from a 400- or 500-mL bottle, ad libitum.
- Acclimation period: Environmental acclimation period for the study was 5 days.

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

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.5 -24.5°C (target range: 19-25°C)
- Humidity (%):26-67% (target range: 30-70%)
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES:
From: Start of in life phase: 21 July 2020 (first vaginal smear sampling)
To: End of in-life phase: 28 September 2020 (last necropsy)

Route of administration:

oral: gavage

Details on route of administration:

The time of the gavage process was prolonged, to ensure that 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:

water

Remarks:

distilled

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: As agreed with the Sponsor, total solid content (test item) of the supplied product was taken into consideration during formulation (a conversion factor of 2.17 was applied used for this purpose). Concentrations and all dose levels in the study (including raw data and study report) were expressed as solid matter as requested by the Sponsor.
The test item was formulated in the selected vehicle (distilled water), as a visibly stable homogenous formulation (Mid or High dose formulation was opalescent in some cases) at the appropriate concentrations according to the dose levels and volume selected in the Pharmacy of the Test Facility. For the formulation step (to measure the amount needed), the bulk test item was heated up to 40°C for a short period. The formulations were stirred with manual shaking and/or a magnetic stirrer from the preparation until completion of each treatment.
Formulations were prepared a maximum of 4 days prior to administration to animals according to stability assessment results of the analytical method development and method validation studies (Test Facility Study codes: 20/028-316ANE and 20/028-316AN).
The calculated amount test item was weighed into a clean, calibrated glass container and then mixed properly (with manual shaking and/or magnetic stirring) with the needed amount of vehicle to reach homogeneity by visual observation. During the formulation sonication was applied as necessary to aid dissolution. Formulations were stored in a closed container at room temperature until use.
After filling the syringe with the calculated amount to be given to each individual rat, the outside of the gavage tube was cleaned with a wetted tissue first (using the vehicle of the study) and then with a dry tissue, to reduce any potential surface contamination to an absolute minimum. A constant volume of 5 mL/kg bw was administered to all animals. The actual volume administered was calculated and adjusted based on each animal’s most recent body weight.

VEHICLE
- Concentration in vehicle: 0, 20, 60 and 200 mg solid/mL
- Amount of vehicle (if gavage): Dose formulation volume = 5 mL/kg bw
- Lot/batch no. (if required): 202007068 / 202003032

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Sample collection was performed on three occasions (on the first day* and last week and 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.
*Note: At the first sampling, opalescence was noticed in the test item formulations by visual inspection (which was not detected in the DRF study), therefore a second set of test item formulations was additionally prepared and measured to avoid any potential preparation error. Both sets showed acceptable results, the second set was used for dosing.
On each sampling occasion, top, middle and bottom duplicate samples were taken from 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 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 (on the first day* and last week and once approximately midway during the treatment period).
*Note: Due to a dilution error on the first day, the diluted sample of the Mid dose formulations did not fit into the calibrated range (the concentration was higher than the highest concentration calibrator). Therefore, the back-up samples were diluted properly and analysed on the subsequent day.
Any sample not required for analysis was discarded following acceptance of the results of the formulation analysis by the Contributing Scientist #1 (Analyst) and Study Director.
The formulation analysis was conducted within the determined stability period under the control of the responsible Contributing Scientist #1 (Analyst) in compliance with the analytical method validation and the relevant SOPs of the Test Facility.
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: 20/028-316AN). 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 criteria of the concentration analysis was 100 ± 10% of the nominal concentration.
Acceptance criteria of the homogeneity was that the CV (coefficient of variation) of replicates (top, middle and bottom of test item formulations) had to be less than 10%.
The measured concentrations of the test item in the different formulations varied between 93% and 101% of the nominal concentrations. The results were considered to be acceptable.
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.

Duration of treatment / exposure:

Dosing of both sexes began after the acclimatisation (5 days) and pre-exposure period (14 days), and it was performed 2 weeks before mating, during the mating, and was continued up to and including the day before the necropsy.
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).

Frequency of treatment:

daily on a 7 days/week basis

Dose / conc.:

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

Remarks:

solid

Dose / conc.:

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

Remarks:

solid

Dose / conc.:

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

Remarks:

solid

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected by the Sponsor in consultation with the Study Director based on the results of a Dose Range Finding (DRF) study (Study code: 20/028-220PE, [3]), with the aim of inducing toxic effects but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose.
In the DRF study (after a 14-day treatment period), no clinical signs were recorded and no test item related adverse effects were seen on body weight, food consumption, clinical pathology (including haematology and clinical chemistry), gross necropsy and organ weight determination at the highest examined dose of 1000 mg solid/kg bw/day, thus it was considered as acceptable for the High dose level of this study. Lower doses were spaced with a factor of approximately 3.
- Rationale for animal assignment (if not random): All adult/parental (P) male and female animals will be sorted according to body weight by computer and divided into weight ranges. There are an equal number of animals from each weight group randomly assigned to each dose group to ensure that animals of all test groups are as nearly as practicable of a uniform weight.
This process will be controlled by the software PROVANTIS v.9 (or other appropriate software), to verify the homogeneity/variability between/within the groups. Males and females will be randomised separately to the dose groups at the start of the treatment (Day 0).
- Fasting period before blood sampling for clinical biochemistry: 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.
Any clinical sign noted during dosing or at any other occasions was recorded at the time seen.
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).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made at the start of the pre-exposure period and one week later (Day -7) and once before the first exposure on Day 0 (to allow for within-subject comparisons), then weekly (in the morning (am), before treatment) and on the day of necropsy.
These observations were made outside the home cage in a standard arena, at similar times as practical. Signs evaluated included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self- mutilation, walking backwards) were also recorded. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
Pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality were recorded including onset, degree and duration of signs as applicable.
On Gestation Day (GD) 13 and/or 14 the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat).
Furthermore, mated females were examined carefully around the time of expected delivery for any signs of difficult or prolonged parturition.

BODY WEIGHT: Yes
- Time schedule for examinations: All adult animals were weighed with accuracy of 1 g weekly during the pre-exposure period, 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 (Postpartum Day) 0, 4, 7, 10 and 13, and at termination. The body weight of the female animals measured on GD 3, GD 10 and GD 17 as well as PPD 10 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 PPD 0, 4, 7, 10 and 13).
Main daily food consumption was calculated for each interval.

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected by cardiac puncture under pentobarbital anaesthesia, immediately prior to scheduled necropsy.
- Anaesthetic used for blood collection: Yes : Euthanimal (40% sodium pentobarbital solution) was used by intraperitoneal injection
- Animals fasted: Yes: overnight period of food deprivation, in case of females this happened after the litter had been culled
- How many animals: 5 males* and 5 females/group randomly selected.
*Note: Due to technical reason (clotted sample) an extra male was sampled in the Control group to provide backup sample in case of technical difficulties during clinical pathology measurement. Finally, the original sample could be measured, thus total results were provided for a total of 6 animals in the Control group.
- Parameters examined:
RBC Red Blood Cell (erythrocyte) count
WBC White Blood Cell (leukocyte) count
Hgb Haemoglobin concentration
Hct Haematocrit (relative volume of erythrocytes)
MCV Mean Corpuscular (erythrocyte) Volume
MCH Mean Corpuscular (erythrocyte) Haemoglobin
MCHC Mean Corpuscular (erythrocyte) Haemoglobin Concentration
RDW Red Cell (erythrocyte) Distribution Width
Plt Platelet (thrombocyte) count
MPV Mean Platelet Thrombocyte volume
RETIC % Reticulocyte count
NE % Neutrophil
LY % Lymphocyte
MO % Monocyte
BA % Basophil
EO % Eosinophil
LUC % Large Unstained Cells
Coagulation:
APTT Activated Partial Thromboplastin Time
PT Prothrombin Time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected by cardiac puncture under pentobarbital anaesthesia, immediately prior to scheduled necropsy.
- Animals fasted: Yes : overnight period of food deprivation, in case of females this happened after the litter had been culled
- How many animals: 5 males* and 5 females/group randomly selected.
*Note: Due to technical reason (clotted sample) an extra male was sampled in the Control group to provide backup sample in case of technical difficulties during clinical pathology measurement. Finally, the original sample could be measured, thus total results were provided for a total of 6 animals in the Control group.
- Parameters examined:
Glucose Blood sugar concentration
T-BIL Total Bilirubin concentration
Urea Urea concentration
Chol. Cholesterol concentration
Creat. Creatinine concentration
Phos. Phosphorus concentration
Na+ Sodium concentration
K+ Potassium concentration
Ca++Calcium concentration
Cl- Chloride concentration
Tot. Prot. Total Protein concentration
Alb. Albumin concentration
A/G Alb/glob ration
AST/GOT Aspartate Aminotransferase activity
ALT/GPT Alanine Aminotransferase activity
GGT Gamma-Glutamyl transferase activity
ALKP Alkaline Phosphatase activity
BA Bile acids

PLASMA/SERUM HORMONES/LIPIDS: Yes
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 (if possible),
• from all dams and 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 of the day in case of sampling on different days. Timing was 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 (150 μL/aliquot, remaining sample (if any) was kept as a third aliquot) and stored in an ultra-freezer (-80±10°C) until analysis.

URINALYSIS: Yes
- Time schedule for collection of urine: prior to necropsy.
- Metabolism cages used for collection of urine: Yes : for approximately 16 hours
- Animals fasted: Yes : overnight period of food deprivation, in case of females this happened after the litter had been culled
- Parameters examined:
The evaluation of the urine samples was performed by using of Medi-Test URYXXON® Stick 10 Urinalysis-strips:
LEU / Leukocyte
NIT / Nitrite
pH
PRO / Protein
GLU / Glucose
UBG / Urobilinogen
BIL / Bilirubin
KET / Ketones
BLD / ERY Blood/Erythrocytes
SG / Specific Gravity
SED / Sediment
VOL / Volume
Colour/Appearance

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Assessment of any potential test item related neurotoxicity was performed during the last exposure week (males on Day 24 am, females on PPD 5-8 am). Selected animals were subjected to the functional observation battery, including Irwin test and measurements of the landing foot splay and fore/hind grip strength.
In order to avoid hypothermia of pups, dams were removed from the pups for not more than approximately 30-40 minutes during FOB or 90 minutes during locomotor activity measurement (SMART).
- Dose groups that were examined: Five males and five females/group were randomly selected
- Battery of functions tested: sensory activity / grip strength / motor activity / other: A modified Irwin test was performed when sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity was conducted, and the general physical condition and behaviour of animals was tested. Parameters including body position, locomotor activity, respiration rate, respiration type, piloerection, head searching, compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna reflex, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex, and/or vocalisation were evaluated.
To measure the landing foot splay, the fore/hind paws of the rat were painted with ink and the rat was dropped from a horizontal position onto the appropriate record sheet covering the examination table. This was repeated 3 times for each animal. The distance between the two resulting ink spots of the hind limbs was measured.
Fore/hind grip strength was measured using a grip strength meter (Model GS3, Bioseb, Chaville, France), an instrument designed to quantify objectively rodent muscular strength, in order to identify and assess quantitatively any potential effect of test item. The rats were held appropriately such that the fore limbs were allowed to grip the support bar and gently pulled back until they released the bar; the device measures the maximum grip strength. This was performed 3 times for each animal on each test day. The procedure was repeated with the hind limbs with the appropriate grip support. The results are tabulated with individual and mean data.
Locomotor activity assessment was conducted using Automatic Monitoring System of rat locomotor activity SMART v. 2.5 (Harvard Apparatus GmbH, March-Hugstetten, Germany). Locomotor activity was monitored by placing each animal individually into an open-field for 1-hour observation time, when DVD recording of movement was made. Recording was made for a duration of 60 minutes, under dim-light and undisturbed conditions. The DVD was analysed with “SMART” software after all recordings were made to produce the appropriate parameters. Data was evaluated for distance travelled in 5-minute segments. The data from the 5-minute segments was presented graphically with the intention of showing plateau activity in controls and comparing the treatment groups.

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Gross necropsy was performed on each adult animal irrespective of the date of death. Terminally (one day after the last treatment), animals were sacrificed under anaesthesia by exsanguination; anaesthetic product was diluted for pups’ euthanasia as required.
After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities was opened, and the appearance of the tissues and organs were observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate. Special attention was paid to the organs of the reproductive system.
Vaginal smears were prepared and examined for each female on the day of necropsy to determine the stage of oestrus cycle and allow correlation with histopathology of the reproductive organs.
The number of implantation sites and of corpora lutea was recorded in the females as applicable.
Dead pups and pups terminated on PND 4 and/or PND 13 were carefully examined externally for gross abnormalities. After the external observation, the sex determined at birth was confirmed by observation of the internal reproductive organs, if possible. Presence of nipples/areolae in the PND 13 male pups was also recorded.

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 found dead animal (High dose female of #4503),
• all macroscopic findings (abnormalities), except of minor order from all animals,
• on the retained reproductive organs (testes, epididymides, prostate gland, seminal vesicles with coagulation gland for males and uterus, cervix, ovary, oviduct and vagina for females) of all animals of the Control and High dose groups, and of all males that failed to sire and all females that failed to deliver healthy pups (documented by a memo in the raw data)*.
*Notes: Two non-pregnant females were observed in the Control group (#1507 and #1509), another two in the Low dose group (#2505 and #2506) and one in the High dose group (#4511). One female in the High dose group (#4505) had total intrauterine mortality.
Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.
Special attention was paid to the organ weight, appearance and histopathology of immunesystem tissues for any evidence of immunotoxicity (spleen, thymus, lymph nodes, bone marrow).
Special attention was paid to the central and peripheral nervous system tissues for any evidence of neurotoxicity.
No histopathological examination was performed on pups (F1 generation).

Statistics:

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

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Daily administration did not result in test item related adverse clinical signs. Slightly noisy respiration was observed for two High dose males and three High dose females; however, the finding was transiently or intermittently noted mostly (not longer than consecutive 7 days), and there were no other observations in those animals. This clinical sign might be related to local irritative effect of the test item but was not considered as a test item related adverse effect.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Daily administration did not result in test item related mortality. One High dose animal died during gestation due to gavage accident.

Description (incidence and severity):

The bodyweight, body weight gain of the test item treated groups did not show any test item related effect.

Description (incidence and severity):

The food consumption of the test item treated groups did not show any test item related effect.

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No test item related findings were seen in the clinical pathology parameters

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No test item related findings were seen in the clinical pathology parameters

Endocrine findings:

no effects observed

Description (incidence and severity):

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

Urinalysis findings:

no effects observed

Description (incidence and severity):

No test item-related changes were observed in the urinalysis parameters in male and female animals of any dose groups when compared to control.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

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

Immunological findings:

not examined

Description (incidence and severity):

No test item-related effects were observed in the organ weights of the test item treated male or female animals compared to controls.
Terminal body weights of animals were not statistically significantly different between the groups. There were no treatment-related statistically significant differences among groups of males and females in the weights of organs measured when compared to Controls. Due to the lack of dose response, the occasional statistically significant difference in the adrenal weight (absolute and brain related) of Low dose males compared to Control was considered as animal variability, not related to test item administration.

Description (incidence and severity):

No test item-related macroscopic findings were observed in evaluated males and females from the High dose group.

Description (incidence and severity):

No test item-related microscopic findings were observed in evaluated males and females from the High dose group.

Description (incidence and severity):

No test item-related microscopic findings were observed in evaluated males and females from the High dose group.

Details on results:

-Mortality and morbility:
No test item related mortality was observed in the study.
One High dose female (#4503) was found dead on Day 29 (GD 13). The animal had no clinical signs previously and based on the necropsy findings a gavage accident was considered as possible cause of death for this animal.
-Clinical observations:
No test item related adverse clinical signs were observed in the study.
Fur thin was observed for two Control males (#1011 on Days 27-28, and #1012 from Day 18 to Day 28) and one Low dose male (#2005 from Day 7 to Day 28). These findings were considered incidental.
Slightly noisy respiration was observed for two High dose males (#4003 on Day 20 and #4012 on Day 23) and three High dose females (#4505 on Days 5-9, #4507 on Days 2-6 and #4509 on Days 5-6 and 36-42). These findings might be related to local irritative effect of the test item but was not considered as a test item related adverse effect.
Tonic convulsions were observed for one High dose female (#4512) on Days 35, 39-40, 45-47 and 49. As these observations were seen near the delivery (Day 39 was the day of delivery) and early lactation, they might be related to a difficult delivery and the loss of pups (all the pups died by PND 2 in this litter). Based on the isolated occurrence this fact was considered as biological variability, not related to the test item administration.
-Body weight and weight changes:
No test item related effect on body weight or body weight gain was detected in the study (males or females).
-Food consumption and compound intake (no feeding study):
No test item related adverse effect on food consumption was seen in any test item treated group (males and females). Statistically significant changes (increases) in the food consumption were recorded for High dose males or Mid dose females in some periods, but the observed values were still within the historical control range for this strain and age of rats, thus no test item related adverse effect was concluded.
-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), any observed statistically significant difference in case of Mid dose males for a short period was without dose response, and had no relevance for the overall total pattern, thus considered as not being related. 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 changes were detected in the test item treated animals (males and females) when comparing haematology parameters to the relevant Control data.
Statistically significant differences were observed in some cases for Low dose males, but there was no dose dependent relationship and all recorded group mean values were within the historical control range. These differences were considered to not reflect an effect of the test item but being incidental.
-Clinical chemistry:
There were no test item related changes or biologically relevant effects on the serum chemistry that could be ascribed to the test item administration.
Glucose was significantly decreased in the Low dose females (p<0.05) and High dose females (p<0.01) when compared to control animals. However, there was no dose response, the observed values were within the historic control range and no similar trend was seen in males. Thus, those findings were considered incidental, not related to the test item.
-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.
-Organ weights:
No test item-related effects were observed in the organ weights of the test item treated male or female animals compared to controls.
Terminal body weights of animals were not statistically significantly different between the groups. There were no treatment-related statistically significant differences among groups of males and females in the weights of organs measured when compared to Controls. Due to the lack of dose response, the occasional statistically significant difference in the adrenal weight (absolute and brain related) of Low dose males compared to Control was considered as animal variability, not related to test item administration.
-Pathology evaluation
NON-PREGNANT OR NOT DELIVERED FEMALES / Parental Generation
Five non-pregnant females were observed in the study (#1507, #1509, #2505, #2506 and #4511). Furthermore, on High dose female (#4505) had total intrauterine mortality.
Macroscopic Findings
Necropsy examination did not show any test item related change in those females or in their male mating pairs.
Microscopic Findings
Organs from the reproductive system were microscopically examined. No microscopic changes were observed in the non-pregnant or not delivered females or in their male mating pairs.
FOUND DEAD ANIMAL / Parental Generation
One High dose female (#4503) was found dead on Day 29. Gavage accident was considered as possible cause of death for this animal.
Macroscopic Findings
No test item-related macroscopic changes were observed at necropsy. Necropsy examination revealed dark red diffuse discolouration of the non-collapsed lungs, 1 mL of clear liquid within the thoracic cavity, dilatation of the stomach with gas, dark red diffuse discolouration of the ovary and dilatation/dark red multifocal discoloration of the uterus.
Microscopic Findings
No test item-related microscopic changes were observed. The microscopic evaluation correlates for the macroscopic changes were observed in following organs including the lungs with mild multifocal haemorrhage, ovary with mild bilateral congestion (considered as postmortem change) and uterus with presence of placenta. In addition, mild extramedullary haematopoiesis in the spleen and mild intrasinusoidal erythrocytes in mandibular lymph nodes were microscopically seen.
TERMINAL EUTHANASIA / Parental Generation
Macroscopic Findings
No test item-related macroscopic findings were observed up to the High dose level (1000 mg solid/kg bw/day).
All changes were incidental or a common background.
Microscopic Findings
No test item-related findings were seen at the High dose level (1000 mg solid/kg bw/day). All changes were incidental or a common background.
-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.
In parental males, the measured T4 thyroid hormone concentration as well as thyroid weights (absolute and body / brain-related) in the test item treated groups were comparable to Control values. No histopathology (microscopic) findings were detected in the thyroids of any High dose animals except of one male (#4002) where minimal unilateral ectopic tissue (from thymus) was noted and another male (#4012) where minimal unilateral multinucleated giant cells were recorded. Those findings were considered as incidental, not reflecting a test item related effect.
No relevant changes were noted in the absolute or relative (to body / brain) thyroid weights of the parental female animals in any dose groups. No histopathology (microscopic) findings were detected in any High dose females.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

other: solid

Sex:

male/female

Basis for effect level:

other: based on the lack of adverse findings

Key result

Critical effects observed:

no

The purpose was to validate a High Performance Liquid Chromatography method with UV detection (HPLC-UV) in order to determine the concentration (of the active ingredient or the total solid content) of Exodiss IB in formulations, primarily to support OECD No. 422 study (Study code: 20/028-220P).

The method was found to be suitable for the analysis. A summary of the method parameters is presented in Table 1.

Table 1. Results of the Method Validation (20/028-316AN)

Selectivity	No interfering component was observed with the control matrices
Reinjection repeatability (7 injections)	RSD% ≤ 2.5%
Linear range	20 – 1000 μg/mL of the supplied test item (~9.2 – 460 μg/mL of total solid content)
Limit of Quantification of the method (LOQ)	~20 μg/mL of the supplied test item (~9.2 μg/mL of total solid content)
Theoretical quantification limit from the vehicle	0.18 mg/mL of total solid content
Recovery of the test item from vehicle (~4.6 and ~210 mg/mL of total solid in ultrapure water)	98 and 96%
Precision of formulations from vehicle (~4.6 and ~210 mg/mL of total solid in ultrapure water)	1.1 and 0.8%
Stability of the test item in vehicle (~4.6 and ~210 mg/mL of total solid in ultrapure water) for 6 days at room temperature	101 and 104%
Stability of the samples in the autosampler	At least 41 hours
Stock solution stability at 5 ± 3°C	At least 5 days

Conclusions:

The NOAEL for systemic toxicity of the parental generation: 1000 mg solid/kg bw/day (based on the lack of adverse findings).

Executive summary:

The purpose of this OECD No. 422 study was to obtain information on the possible toxic effects of diisobutyl sodium sulfosuccinate (CAS 127-39-9; EC 204-839-5) 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 in consultation with the Study Director based on the results of a Dose Range Finding (DRF) study. Based on those results, 1000 mg solid/kg bw/day was selected as the High dose for this study. Concentrations and all dose levels in the study (including raw data and study report) were expressed as solid matter as requested by the Sponsor.

Experimental design:

Group number	Group design	Dose level (mg solid/kg bw/day)	Dose formulation concentration (mg solid/mL)	Dose formulation volume (mL/kg bw)	Number of animals
					Male	Female
1	Control	0	0	5	12	12
2	Low dose	100	20		12	12
3	Mid dose	300	60		12	12
4	High dose	1000	200		12	12

 

Parameters measured during the study included twice a day mortality checking, daily routine and weekly detailed observation of clinical signs, weekly body weight and food consumption measurements and clinical pathology evaluation (including haematology, coagulation, clinical chemistry and urinalysis). Neurological assessment (Functional Observation Battery (FOB) including measurements of the landing foot splay, grip strength as well as locomotor activity measurement) was performed during the last week of the treatment for each sex. In addition, the reproductive performance, pregnancy, parturition and postpartum/lactation period were monitored in the adult animals, and viability, clinical signs and development were evaluated in their F1 offspring until PND13. At termination, 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 PND13 pups and parental males were also determined.

 

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

Dosing formulation were analysed for concentration and/or homogeneity on three occasions during the study. All test item formulations were shown to be homogeneous. The measured concentrations of the test item in the different formulations varied between 93% and 101% of the nominal concentrations. Overall, the formulations were considered adequate for the study.

 

RESULTS

In summary, under the conditions of this study the daily administration of diisobutyl sodium sulfosuccinate (CAS 127-39-9; EC 204-839-5) by oral gavage to Wistar rats at dose levels of 100, 300 or 1000 mg solid/kg bw/day (Low, Mid and High dose groups, respectively) did not result in test item related mortality or adverse clinical signs.

One High dose animal died during gestation due to gavage accident.

Slightly noisy respiration was observed for two High dose males and three High dose females; however, the finding was transiently or intermittently noted mostly (not longer than consecutive 7 days), and there were no other observations in those animals. This clinical sign might be related to local irritative effect of the test item but was not considered as a test item related adverse effect.

The bodyweight, body weight gain, and food consumption of the test item treated groups did not show any test item related effect.

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

No test item related findings were seen in the clinical pathology parameters.

No test item effect on oestrus cycle of parental females was noted.

No test item related changes were noted in the reproductive parameters during mating and gestation, delivery and post-partum/lactation period until PPD 14.

There were no test item effects on the F1 offspring viability, clinical signs, physical or sexual development. No test item related macroscopic findings were recorded for F1 pups at necropsy.

No test item-related macroscopic and microscopic findings were observed in evaluated males and females from the High dose group.

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

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

The NOAEL for systemic toxicity of the parental generation: 1000 mg solid/kg bw/day (based on the lack of adverse findings).

The NOAEL for reproductive effects of the parental generation: 1000 mg solid/kg bw/day (based on the lack of adverse findings).

The NOAEL for pups’ (F1 generation) development and survival: 1000 mg solid/kg bw/day (based on the lack of adverse findings).

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Reliable (Klimisch 1)

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Subacute toxicity

A supporting 14-day dose range finding study was conducted with the registered substance in Wistar rats (4/sex/group) by oral gavage at 0 (distilled water), 100, 300 and 1000 mg solid/kg bw for up to 14 days in order to determine the dose levels for a subsequent OECD No. 422 study (Hargitai, 2022). . All dose formulations were homogenous. There was no mortality in the study. No clinical signs were noted during the study. No test item-related effect on body weight, body weight gain or food intake was observed. There were no test item-related effects on haematology or clinical chemistry parameters. No test item-related adverse organ weight changes were seen at necropsy. Kidney weights (relative to body) were slightly increased in case of High dose females. No test-item related macroscopic findings were seen at necropsy. In conclusion, based on this 14-day Dose Range Finding (DRF) study, the 1000 mg solid/kg bw/day dose level was acceptable for the High dose level of the upcoming OECD No. 422 study.

 

A key OECD No. 422 study was conducted with the registered substance in Wistar rats (12/sex/group by oral gavage at 0 (distilled water), 100, 300 and 1000 mg solid/kg bw/day (Hargitai, 2022). Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating period), then were euthanized and subjected to necropsy examination. Females were dosed for 14 days pre-mating, for up to 14 days mating period, through gestation and up to and including the day before necropsy (13 days post-partum dosing). The day of birth (when parturition was complete) was defined as Day 0 post-partum. Females not delivered were sacrificed as practical (27 days after the last day of the mating period). Parameters measured during the study included twice a day mortality checking, daily routine and weekly detailed observation of clinical signs, weekly body weight and food consumption measurements and clinical pathology evaluation (including haematology, coagulation, clinical chemistry and urinalysis). Neurological assessment (Functional Observation Battery (FOB) including measurements of the landing foot splay, grip strength as well as locomotor activity measurement) was performed during the last week of the treatment for each sex. Reproductive performance is reported under Section 8. Weights of selected organs were recorded, and representative tissues/organs were sampled and preserved in appropriate fixatives from the adult animals or F1 animals. The thyroxine (T4) levels in the PND13 pups and parental males were also determined. For the adult animals, a detailed histological examination was performed on the selected list of retained organs of 5 animals/sex in the Control and High dose groups, all found dead animals and all those male / female mating pairs where no liveborn pups were achieved. Dosing formulation were analysed for concentration and/or homogeneity on three occasions during the study. All test item formulations were shown to be homogeneous. The measured concentrations of the test item in the different formulations varied between 93% and 101% of the nominal concentrations. Overall, the formulations were considered adequate for the study.
In summary, the test substance did not result in test item related mortality or adverse clinical signs.
One High dose animal died during gestation due to gavage accident. Slightly noisy respiration was observed for two High dose males and three High dose females; however, the finding was transiently or intermittently noted mostly (not longer than consecutive 7 days), and there were no other observations in those animals. This clinical sign might be related to local irritative effect of the test item but was not considered as a test item related adverse effect. The bodyweight, body weight gain, and food consumption of the test item treated groups did not show any test item related effect. At the functional observation battery (FOB) and locomotor activity measurement, there were no changes in animal behaviour, general physical condition, grip strength, motor activity, or in the reactions to different type of stimuli in the control or test groups. No test item related findings were seen in the clinical pathology parameters. No test item-related macroscopic and microscopic findings were observed in evaluated males and females from the High dose group. The NOAEL for systemic toxicity of the parental generation was 1000 mg solid/kg bw/day (based on the lack of adverse findings).

 

Subchronic and chronic toxicity

Six groups of 40 albino rats (20 male, 20 female Charles River Strain) plus 1 control group (20 male, 20 female) were fed with 1% of various test items mixed into the diet. The various test items were category members of the registered substance (Plank et al., 1969). After 84 days hematological values, blood chemical values, urinalysis values were measured for all animals. Tissues were examined pathologically at the conclusion of the 90-days test period. Organ to body weight and organ to brain weight ratios were calculated. Slightly lower absolute liver weights were observed in males, however no significant differences in clinical blood chemistry studies and absolute organ weights have been detected. Body weights organ to body weight ratios, hematologic studies and urinalysis were not different between test and control animals. No deaths or abnormal behavioral reactions occurred; no gross pathological or histological findings were noted. Administration of category members at 1% in the diet (10000 ppm equivalent to 750 mg/kg body weight/day on average basis) for 90 days in rats did not result in any relevant changes in the subchronic toxicity study. The NOAEL was therefore considered to be 750 mg/kg bw/day.
The validity of the study was supported by additional audits on the raw data and histopathological evaluation.
Although deficiencies were detected compared to current standards, the study was concluded to be valid and reliable.

 

In a supporting study, groups of 10 (5 male & 5 female) Wistar rats were treated for 6 months at concentrations of 0.25, 0.5, 0.75, 1.0 and 1.25 g/kg diet, corresponding to doses of 190, 370, 550, 750 and 870mg/kg bw/day. Occasional spells of diarrhea occurred in some animals, particularly at the higher doses. Neither the total red cell, total white cell, nor the differential counts of rats was affected by the continued administration . The dose level of 750 mg/kg was confirmed as NOAEL (Literature, Benaglia et al. 1943).

 

Other studies were also available from literature in various species (Literature, Benaglia et al. 1943 and Case et al., 1977) in dogs, rabbits and Rhesus monkeys. The other species were considered to be less appropriate due to the gastrointestinal tensioactive local irritation by which systemic effects could not be fully evaluated.

 

Conclusion

- The NOAEL for systemic toxicity of the parental generation was 1000 mg solid/kg bw/day in a rat key combined repeated dose/reproductive toxicity study (OECD No. 422).

- Based on the fact that no relevant target organ changes were seen up to highest tested dose of  up to 1000 mg solid/kg bw in the subacute study and up to the dose of 450-1000 mg/kg bw in diet for 90 days in the subchronic studies, allows to conclude that the substance is safe up to highest tested doses.

- Further information supporting the safety of the test substance is provided in the read across justification for the Diester category, showing that all substances in the group had a NOAEL of at least 750 mg/kg bw (justification with data matrix separately attached in Section 13).

Justification for classification or non-classification

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